U.S. patent application number 17/084082 was filed with the patent office on 2021-10-21 for xten conjugate compositions and methods of making same.
The applicant listed for this patent is Amunix Pharmaceuticals, Inc.. Invention is credited to Sheng Ding, Chen Gu, Bryant McLaughlin, Vladimir Podust, Volker Schellenberger, Bee-Cheng Sim, Chia-Wei Wang.
Application Number | 20210322518 17/084082 |
Document ID | / |
Family ID | 1000005649973 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210322518 |
Kind Code |
A1 |
Schellenberger; Volker ; et
al. |
October 21, 2021 |
XTEN CONJUGATE COMPOSITIONS AND METHODS OF MAKING SAME
Abstract
The present invention relates to extended recombinant
polypeptide (XTEN) compositions, conjugate compositions comprising
XTEN and XTEN linked to cross-linkers useful for conjugation to
pharmacologically active payloads, methods of making highly
purified XTEN, methods of making XTEN-linker and XTEN-payload
conjugates, and methods of using the XTEN-cross-linker and
XTEN-payload compositions.
Inventors: |
Schellenberger; Volker;
(Palo Alto, CA) ; Podust; Vladimir; (Castro
Valley, CA) ; Wang; Chia-Wei; (Santa Clara, CA)
; McLaughlin; Bryant; (Millbrae, CA) ; Sim;
Bee-Cheng; (Mountain View, CA) ; Ding; Sheng;
(Foster City, CA) ; Gu; Chen; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amunix Pharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005649973 |
Appl. No.: |
17/084082 |
Filed: |
October 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16133444 |
Sep 17, 2018 |
10953073 |
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17084082 |
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14381199 |
Aug 26, 2014 |
10172953 |
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PCT/US2013/028116 |
Feb 27, 2013 |
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16133444 |
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61634312 |
Feb 27, 2012 |
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61690187 |
Jun 18, 2012 |
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61709942 |
Oct 4, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/0205 20130101;
A61K 49/0052 20130101; A61K 49/0032 20130101; A61K 49/0056
20130101; A61K 47/64 20170801; A61K 38/24 20130101 |
International
Class: |
A61K 38/24 20060101
A61K038/24; A61K 47/64 20060101 A61K047/64; C07K 5/02 20060101
C07K005/02; A61K 49/00 20060101 A61K049/00 |
Claims
1. A method of producing a composition comprising a substantially
homogenous population of XTEN sequences, the method comprising: a.
providing a polypeptide composition comprising at least one
polypeptide having an amino acid sequence selected from the group
of sequences set forth in Table 6, wherein the amino acid sequence
comprises at least one cleavage sequence, b. treating said
polypeptide composition with trypsin under conditions effective to
cleave the cleavage sequence; and wherein at least 95%, 96%, 97%,
98%, or 99% of individual sequences in the resulting substantially
homogeneous population of XTEN sequences have an identical sequence
length.
2. The method of claim 1, further comprising: a. adsorbing the XTEN
sequences onto a chromatography substrate under conditions
effective to capture the XTEN sequences but not the protease; b.
eluting the XTEN sequences; and c. recovering the XTEN sequences
wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% of individual molecules of the population have an identical
sequence length.
3. The method of claim 2, wherein the chromatography substrate is
an anion exchange substrate.
4. The method of claim 3, wherein the anion exchange substrate is
selected from the group consisting of macrocap Q, capto Q,
superQ-650M, and poros D.
5. The method of any one of claims 1-4, wherein the XTEN sequence
has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to a sequence selected from the group of
amino acid sequences set forth in Table 6.
6. The method of any one of claims 1-5, wherein the XTEN sequence
has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to a sequence selected from the group of
amino acid sequences set forth in Tables 2 or 3.
7. A composition produced by the method of any one of claims
1-6.
8. A composition comprising a substantially homogenous population
of poly peptides comprising an extended recombinant polypeptide
(XTEN), and wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of
individual polypeptide molecules in said population have an
identical sequence length.
9. The composition of claim 8, wherein the XTEN is characterized in
that: a. the XTEN comprises about 36 to about 3000 amino acid
residues; b. the sum of glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) residues constitutes
more than about 90% of the total amino acid residues of the XTEN;
c. the XTEN sequence is substantially non-repetitive such that (i)
the XTEN sequence contains no three contiguous amino acids that are
identical unless the amino acids are serine, (ii) at least about
80% of the XTEN sequence consists of non-overlapping sequence
motifs, each of the sequence motifs comprising about 9 to about 14
amino acid residues, wherein any two contiguous amino acid residues
does not occur more than twice in each of the sequence motifs; or
(iii) the XTEN sequence has a subsequence score of less than 10; d.
the XTEN sequence has greater than 90% random coil formation as
determined by GOR algorithm; e. the XTEN sequence has less than 2%
alpha helices and 2% beta-sheets as determined by Chou-Fasman
algorithm; and f. the XTEN sequence lacks a predicted T-cell
epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE
algorithm prediction for epitopes within the XTEN sequence is based
on a score of -9.
10. The composition of claim 8, wherein the XTEN comprises a
sequence having at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or 100% sequence
identity to a sequence selected from the group consisting of the
sequences set forth in Table 2, Table 3, Table 4 and Tables
22-25.
11. The composition of claim 8, further comprising a first affinity
tag.
12. The composition of claim 11, wherein the first affinity tag has
binding affinity for a chromatography substrate selected from the
group consisting of hydrophobic interaction chromatography (HIC)
substrate, cation exchange substrate, anion exchange substrate,
immobilized metal ion affinity chromatography (IMAC) substrate, and
immobilized antibody substrate.
13. The composition of claim 11, wherein the first affinity tag is
selected from the group consisting of the sequences set forth in
Table 7.
14. The composition of any one of claims 11-13, further comprising
a helper sequence.
15. The composition of claim 14, wherein the helper sequence
comprises a sequence having at least about 90%, or at least about
91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%, or
100% sequence identity to a sequence selected from the group
consisting of the sequences set forth in Table 10.
16. The composition of claim 14, wherein the helper sequence is
selected from the group consisting of a. KNPEQAEEQX1EET wherein X1
is independently S or R; b. ANPEQAEEQX1EET wherein X1 is
independently S or R; c. KNPEQAEEQAEEQX1EET wherein X1 is
independently S or R; d. KX2X3EQAEEQAEEQX1EET wherein X1 is
independently S or R, X2 is independently K or N, and X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S; e.
KX2(X3)10QX1EET wherein X1 is independently S or R, X2 is
independently K or N, and X3 is independently K, N, T, Q, H, P, E,
D, A, R, or S; f. KX2(X3)7AEEQX1EET wherein X1 is independently S
or R, X2 is independently K or N, and X3 is independently K, N, T,
Q, H, P, E, D, A, R, or S; g. KX2X3EQE(X3)3AEEQREET wherein X2 is
independently K or N, and X3 is independently K, N, T, Q, H, P, E,
D, A, R, or S; h. KX2X3EQE(X3)3AEE(X3)5 wherein X2 is independently
K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S;
i. KKQEQEKEQAEEQ(X4X5)2REET wherein X4 is independently A or S and
X5 is independently K, Q, or E; KKQEQEKEQAEEQ(X4X5)4REET wherein X4
is independently A or S and X5 is independently K, Q, or E; j.
KKQEQEKEQAEEQ(Z)4REET, wherein Z is any naturally-occurring L-amino
acid; k. KX2(X3)n, wherein n is an integer from 10-40 and X2 is
independently K or N, and X3 is independently K, N, T, Q, H, P, E,
D, A, R, or S; l. (X3)n wherein n is an integer from 10-50 and X3
is independently K, N, T, Q, H, P, E, D, A, R, or S; m.
KX2QEQEKEQAEEQ(X4X5)nX1EET wherein n is zero or an integer from
1-10 and X1 is independently S or R, X2 is independently K or N, X4
is independently A or S, and X5 is independently K, Q, or E; n.
KX2(X3)n(X4X5)mX1EET, wherein n is an integer from 5-20, m is zero
or an integer from 1-10, X1 is independently S or R, X2 is
independently K or N, X3 is independently K, N, T, Q, H, P, E, D,
A, R, or S, X4 is independently A or S, and X5 is independently K,
Q, or E; and o. KX2(X3)n(Z)mX1EET, wherein n is an integer from
5-20, m is zero or an integer from 1-10, X1 is independently S or
R, X2 is independently K or N, X3 is independently K, N, T, Q, H,
P, E, D, A, R, or S, and Z is any naturally-occurring L-amino
acid.
17. The composition of any one of claims 11-16, further comprising
a first cleavage sequence, wherein the first cleavage sequence is
selected from the group consisting of the sequences set forth in
Table 8 and Table 9.
18. The composition of claim 17, wherein the composition has the
configuration of formula I: (HS)-(AT1)-(CS1)-(XTEN) I wherein a. HS
is the helper sequence; b. AT1 is the first affinity tag; c. CS1 is
the first cleavage sequence; and d. XTEN is the extended
recombinant polypeptide.
19. The composition of claim 17, further comprising a second
cleavage sequence wherein the first and the second cleavage
sequences are capable of being cleaved by the same protease, and
wherein the composition has the configuration of formula II:
(HS)-(CS1)-(XTEN)-(CS2)-(AT1) II wherein a. HS is a helper
sequence; b. AT1 is the first affinity tag; c. CS1 is the first
cleavage sequence; d. CS2 is the second cleavage sequence; and e.
XTEN is the extended recombinant poly peptide.
20. The composition of claim 18 or claim 19, wherein the first
affinity tag comprises the sequence RPRPRPRPRPRPR or HHHHHH.
21. The composition of any one of claims 11-17, further comprising
a second affinity tag and a second cleavage sequence, wherein the
second affinity tag has binding affinity to a different
chromatography substrate than the first affinity tag, wherein the
chromatography substrate is selected from the group consisting of a
HIC substrate, cation exchange substrate, anion exchange substrate,
IMAC substrate, and immobilized antibody substrate, and wherein the
first and the second cleavage sequences are capable of being
cleaved by the same protease.
22. The composition of claim 21, wherein the second affinity tag is
different from the first affinity tag and is the second affinity
tag is selected from the group of sequences set forth in Table
7.
23. The composition of claim 21 or claim 22, wherein the
composition has the configuration of formula III:
(HS)-(AT1)-(CS1)-(XTEN)-(CS2)-(AT2) III wherein a. HS is the helper
sequence; b. AT1 is the first affinity tag; c. CS1 is the first
cleavage sequence; d. CS2 is the second cleavage sequence; e. XTEN
is the extended recombinant polypeptide; and f. AT2 is the second
affinity tag.
24. The composition of claim 23, wherein the first affinity tag
comprises the sequence RPRPRPRPRPRPR and the second affinity tag
comprises the sequence HHHHHH.
25. The composition of claim 23, wherein the first affinity tag
comprises the sequence HHHHHH and the second affinity tag comprises
the sequence RPRPRPRPRPRPR.
26. A composition comprising a substantially homogenous population
of a polypeptide obtained by the process comprising: a. culturing a
host cell that comprises a vector encoding the polypeptide in a
fermentation reaction under conditions effective to express the
polypeptide as a component of a crude expression product of the
host cell, wherein the encoded polypeptide comprises an XTEN, a
first cleavage sequence and a first affinity tag; b. adsorbing the
polypeptide of the crude expression product onto a first
chromatography substrate under conditions effective to capture the
first affinity tag onto the first chromatography substrate; c.
eluting the polypeptide; and d. recovering the polypeptide wherein
at least 90%, 91%, 92%, 93%, 94%, or 95% of the polypeptides of the
population have an identical sequence length.
27. The composition of claim 26, wherein the first chromatography
substrate is selected from the group consisting of a HIC substrate,
cation exchange substrate, anion exchange substrate, and IMAC
substrate.
28. The composition of claim 26, wherein the affinity tag is
selected from the group consisting of the affinity tags of Table
7.
29. The composition of claim 27, wherein the first chromatography
substrate is cation exchange and the first affinity tag comprises
the sequence RPRPRPRPRPRPR.
30. The composition of claim 27, wherein the first chromatography
substrate is IMAC and the first affinity tag comprises the sequence
HHHHHHHH.
31. The composition of claim 26, wherein the vector encodes the
polypeptide of any one of claims 11-19.
32. The composition of any one of claims 26-28, wherein the vector
further encodes a second cleavage sequence and a second affinity
tag wherein the first and the second cleavage sequences are capable
of being cleaved by the same protease and wherein the second
affinity tag has binding affinity to a second, different
chromatography substrate than the first affinity tag, and wherein
the composition is obtained by the process further comprising: a.
adsorbing the polypeptide onto a second chromatography substrate
under conditions effective to capture the second affinity tag onto
the second chromatography substrate; b. eluting the polypeptide;
and c. recovering the polypeptide wherein at least 90%, 91%, 92%,
93%, 94%, or 95% of the polypeptides of the population have an
identical sequence length.
33. The composition of claim 32, wherein the vector encodes the
polypeptide of claim 23.
34. The composition of claim 32, wherein first chromatography
substrate is different from the second chromatography substrate and
each of the first and the second chromatography substrate are
independently selected from the group consisting of HIC, cation
exchange, anion exchange, and IMAC.
35. The composition of claim 32, wherein the first chromatography
substrate is cation exchange and the first affinity tag comprises
the sequence RPRPRPRPRPRPR and the second chromatography substrate
is IMAC and the first affinity tag comprises the sequence
HHHHHHHH.
36. The composition of claim 32, wherein the first chromatography
substrate is IMAC and the first affinity tag comprises the sequence
HHHHHHHH and the second chromatography substrate is cation exchange
and the first affinity tag comprises the sequence
RPRPRPRPRPRPR.
37. The composition of any one of claims 23-28 or 32-34, the
process further comprising: a. treating the composition with a
protease under conditions effective to cleave the cleavage
sequence(s), thereby releasing the XTEN from the affinity tag(s);
b. adsorbing the XTEN onto a chromatography substrate under
conditions effective to capture the XTEN but not the affinity
tag(s) or the protease; c. eluting the XTEN; and d. recovering the
XTEN wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of XTEN have an identical sequence length.
38. The composition of claim 37, wherein the cleavage sequence(s)
are capable of being cleaved by a protease selected from the group
consisting of the proteases of Table 9.
39. The composition of claim 38, wherein the cleavage sequence(s)
are capable of being cleaved by trypsin wherein the sequence is
selected from the group consisting of the sequences set forth in
Table 8, and wherein the protease is trypsin.
40. The composition of claim 37 or 38, wherein the chromatography
substrate is anion exchange.
41. The composition of claim 40, wherein the anion exchange
chromatography substrate is selected from the group consisting of
macrocap Q, capto Q, superQ-650M, and poros D.
42. The composition of any one of claims 23-28 or 32-34, the
process further comprising: a. treating the composition with a
protease under conditions effective to cleave the cleavage
sequence(s), thereby releasing the XTEN from the affinity tag(s);
b. adsorbing the protease onto a chromatography substrate under
conditions effective to capture the protease but not the XTEN; and
c. recovering the XTEN from the eluate wherein at least 90%, 91%,
92%, 93%, 94%, or 95% of the individual molecules of XTEN have an
identical sequence length.
43. The composition of claim 42, wherein the cleavage sequence(s)
are capable of being cleaved by a protease selected from the group
consisting of the proteases of Table 9.
44. The composition of claim 42 or claim 43, wherein the cleavage
sequence(s) are capable of being cleaved by trypsin wherein the
sequence is selected from the group consisting of the sequences set
forth in Table 8, and wherein the protease is trypsin.
45. The composition of any one of claims 42-44, wherein the
chromatography substrate is one or more of cation exchange
substrate, HIC substrate or IMAC substrate.
46. A composition comprising an XTEN sequence, wherein the XTEN
sequence further comprises one or more cleavage sequences capable
of being cleaved by trypsin and wherein treatment with trypsin
under conditions effective to cleave all the cleavage sequences
results in a preparation of XTEN fragments wherein each XTEN
fragment has at least about 95%, 96%, 97%, 98%, or 99% sequence
identity to every other fragment in the preparation.
47. The composition of claim 46, wherein the cleavage sequence has
at least 86% sequence identity to or is identical to the sequence
SASRSA or SASKSA.
48. The composition of claim 46, wherein the cleavage sequence
comprises the sequence RX or KX, wherein X is any L-amino acid
other than proline.
49. The composition of claim 46, wherein the composition has at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to the sequences selected from the group of
sequences set forth in Table 6.
50. A method for producing an XTEN sequence in a host cell, the
method comprising culturing a host cell that comprises a vector
encoding a polypeptide comprising a) the XTEN sequence and b) a
helper sequence, in a fermentation reaction under conditions
effective to express the polypeptide as a component of a crude
expression product at a concentration of more than about 2
grams/liter (g/L), or about 3 g/L, or about 4 g/L, or about 5 g/L,
or about 6 g/L, or about 7 g/L of said polypeptide.
51. A method for producing an XTEN sequence in a host cell, the
method comprising culturing a host cell that comprises a vector
encoding a polypeptide comprising a) the XTEN sequence and b) a
helper sequence, in a fermentation reaction under conditions
effective to express the polypeptide as a component of a crude
expression product at a concentration of more than about 10
milligrams/gram of dry weight host cell (mg/g), or at least about
15 mg/g, or at least about 20 mg/g, or at least about 25 mg/g, or
at least about 30 mg/g, or at least about 40 mg/g, or at least
about 50 mg/g of said polypeptide.
52. A method for producing an XTEN sequence in a host cell, the
method comprising culturing a host cell that comprises a vector
encoding a polypeptide comprising a) the XTEN sequence and b) a
helper sequence, in a fermentation reaction under conditions
effective to express the polypeptide as a component of a crude
expression product at a concentration of more than about 10
milligrams/gram of dry weight host cell (mg/g), or at least about
250 micromoles/L, or about 300 micromoles/L, or about 350
micromoles/L, or about 400 micromoles/L, or about 450 micromoles/L,
or about 500 micromoles/L of said polypeptide.
53. The method of any one of claims 50-52, wherein the
concentration is measured when the fermentation reaction reaches an
optical density of at least 130 at a wavelength of 600 nm.
54. The method of any one of claims 50-52, wherein the helper
sequence of the expressed polypeptide is at the N-terminus of the
polypeptide and wherein the helper sequence has at least about 90%,
91%, 92%, 93%, 94%, or 95% sequence identity or is identical to a
sequence selected from the group consisting of the sequences set
forth in Table 10.
55. The method of claim 54, wherein the vector encodes the
polypeptide of any one of claims 14-17.
56. The method of claim 55, further comprising a. recovering the
crude expression product of the host cell fermentation reaction
mixture; b. adsorbing the polypeptide of the crude expression
product onto a first chromatography substrate under conditions
effective to capture the first affinity tag of the polypeptide onto
the chromatography substrate, wherein the first chromatography
substrate is selected from the group consisting of a HIC substrate,
a cation exchange substrate, an anion exchange substrate, and an
IMAC substrate; c. eluting and recovering the XTEN sequence wherein
at least 90%, 91%, 92%, 93%, 94%, or 95% of the polypeptides have
an identical sequence length.
57. The method of any one of claims 50-52, wherein the vector
encodes the polypeptide of any one of claims 21-23.
58. The method of claim 57, further comprising: a. recovering the
crude expression product of the host cell fermentation reaction
mixture; b. adsorbing the polypeptide onto a first chromatography
substrate under conditions effective to capture the first affinity
tag of the polypeptide onto the chromatography substrate wherein
the first chromatography substrate is selected from the group
consisting of a HIC substrate, a cation exchange substrate, an
anion exchange substrate, and an IMAC substrate; c. eluting the
polypeptide; d. adsorbing the polypeptide onto a second
chromatography substrate under conditions effective to capture the
second affinity tag of the polypeptide onto the chromatography
substrate wherein the second chromatography substrate is selected
from the group consisting of a HIC substrate, a cation exchange
substrate, an anion exchange substrate, and an IMAC substrate; e.
eluting the polypeptide; and f. recovering the XTEN sequence
wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of the
polypeptides have an identical sequence length.
59. The method of claim 56 or claim 58, the method further
comprising: a. treating the polypeptide with a protease under
conditions effective to cleave the cleavage sequence, thereby
releasing the XTEN from the polypeptide; b. adsorbing the XTEN onto
an anion chromatography substrate under conditions effective to
capture the XTEN; c. eluting the XTEN; and d. recovering the XTEN
wherein at least 90%, or at least 91%, or at least 92%, or at least
93%, or at least 94%, or at least 95% of the individual XTEN
molecules have an identical sequence length.
60. The method of claim 59, wherein the anion chromatography
substrate is selected from the group consisting of macrocap Q,
capto Q, superQ-650M, and poros D.
61. The method of claim 59 or claim 60, wherein the protease is
trypsin and wherein the sequence is selected from the group
consisting of the sequences set forth in Table 8.
62. The method of claim 56 or claim 58, the method further
comprising: a. treating the polypeptide with a protease under
conditions effective to cleave the cleavage sequence(s), thereby
releasing the XTEN from the polypeptide; b. adsorbing the protease
onto a chromatography substrate under conditions effective to
capture the protease but not the XTEN; and c. recovering the XTEN
in the eluate wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of
the XTEN have an identical sequence length.
63. The method of claim 62, wherein the protease is trypsin, and
wherein the sequence is selected from the group consisting of the
sequences set forth in Table 8.
64. The method of claim 62 or claim 63, wherein the chromatography
substrate is one or more of a HIC substrate, a cation exchange
substrate, and an IMAC substrate.
65. A solid support comprising immobilized thereon a population of
substantially identical polypeptides, wherein the solid support
comprises a chromatography substrate, and the immobilized
polypeptides each comprise an XTEN, a first affinity tag, and a
second affinity tag, wherein: a. the first affinity tag is joined
to the XTEN by a cleavage sequence at the N-terminus of the XTEN;
b. the second affinity tag is joined to the XTEN by a cleavage
sequence at the C-terminus of the XTEN; c. the second affinity tag
is different from the first affinity tag; d. the chromatography
substrate is capable of binding to either said first or said second
affinity tag but not both; and e. at least 90%, 91%, 92%, 93%, 94%,
or 95% of the immobilized polypeptide molecules have an identical
sequence length.
66. The solid support of claim 65, wherein the immobilized
polypeptide molecules comprise the polypeptides any one of claims
21-25.
67. The solid support of claim 65 or claim 66, wherein the cleavage
sequence has at least about 86% sequence identity to or is
identical to the sequence SASRSA or SASKSA.
68. The solid support of claim 65 or claim 66, wherein the cleavage
sequence comprises the sequence RX or KX, wherein X is any L-amino
acid other than proline.
69. The solid support of any one of claims 65-68, wherein the solid
support is selected from the group consisting of HIC chromatography
resin, cation exchange chromatography resin, anion exchange
chromatography resin, and IMAC chromatography resin.
70. The composition of claim 69, wherein the first affinity tag
comprises the sequence RPRPRPRPRPRPR and the second affinity tag
comprises the sequence HHHHHH.
71. The composition of claim 69, wherein the first affinity tag
comprises the sequence HHHHHH and the second affinity tag comprises
the sequence RPRPRPRPRPRPR.
72. A conjugate composition comprising an XTEN covalently linked to
one or more molecules of at least a first cross-linker, wherein the
cross-linker is selected from the group consisting of the
cross-linkers set forth in Table 13, the alkyne reactants set forth
in Table 15, and the azide reactants set forth in Table 15, and the
XTEN is selected from the group consisting of the XTEN of claim 37,
the XTEN of claim 42, the XTEN of claim 7, the sequences set forth
in Table 2, and the sequences set forth in Table 3.
73. The conjugate composition of claim 72, wherein the first
cross-linker is conjugated to the at least first XTEN at a location
selected from the group consisting of: a. an alpha-amino group of
an N-terminal amino acid residue of the XTEN; b. an epsilon amino
group of each lysine residue of the XTEN; and c. a thiol group of
each cysteine residue of the XTEN.
74. The conjugate composition of claim 72 or claim 73, wherein the
XTEN has at least about 9%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or 100% sequence
identity to a sequence selected from the group of sequences set
forth in Table 2 and Table 3.
75. The conjugate composition of claim 74, wherein the XTEN is
selected from the group consisting of AE144, AE288, AE432, AE576,
AE864, Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187, Seg
188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg 195,
Seg 1%, Seg 197, Seg 198, and Seg 199, and the cross-linker is
conjugated to the alpha amino-group of the N-terminal amino acid of
the XTEN.
76. The conjugate composition of claim 74, wherein the XTEN is
selected from the group consisting of Seg 174, Seg 175, Seg 176,
Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg
192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg
199 set forth in Table 3, and the cross-linker is conjugated to the
thiol group of each cysteine residue of the XTEN.
77. The conjugate of any one of claims 72-76, wherein the first
cross-linker is selected from the group consisting of an
N-maleimide, an iodoacetyl reagent, a pyridyl disulfide reagent, a
vinyl sulfone reagent, 3-propargyloxypropanoic acid,
(oxyethyl)n-acetylene where n is 1-10, dibenzylcyclooctyne (DBCO),
cyclooctyne (COT), 3-azide-propionic acid, 6-azide-hexanoic acid,
and (oxyethyl)n-azide where n is 1-10.
78. The conjugate composition of claim 76, wherein the first
cross-linker is conjugated to the thiol group of each cysteine
residue of the XTEN.sub.1 and the conjugate further comprises a
second cross-linker conjugated to the alpha amino-group of the
N-terminal amino acid of the XTEN wherein the cross-linker is
selected from the group consisting of the cross-linkers set forth
in Table 13, the alkyne reactants of Table 15, and the azide
reactants of Table 15.
79. The conjugate composition of claim 78, having the configuration
of formula V: wherein independently for each occurrence; a. CL1 is
the first cross-linker conjugated to cysteine residues of the XTEN;
b. CL2 is the second cross-linker conjugated to XTEN at the
N-terminus; c. x is an integer of 1 to about 10; d. y is an integer
of 1 with the proviso that x+y is >2; e. XTEN is a cysteine
engineered XTEN comprising x number of cysteine residues.
80. The conjugate composition of any one of claims 72-77, further
comprising a single atom residue of a first payload conjugated to
each first cross-linker wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur.
81. The conjugate composition of claim 80, wherein the first
payload of the single atom residue is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and
21.
82. The conjugate composition of any one of claims 72-77, further
comprising a payload selected from the group consisting of the
payloads set forth in Tables 6, 7, 18, and 21 conjugated to each
first cross-linker.
83. The conjugate composition of claim 78 or claim 79, further
comprising a single atom residue of a first payload conjugated to
each of the first cross-linkers wherein the residue is selected
from the group consisting of carbon, nitrogen, oxy gen and sulfur,
and a single atom residue of a second payload conjugated to the
second cross-linker wherein the residue is selected from the group
consisting of carbon, nitrogen, oxygen and sulfur.
84. The conjugate composition of claim 83, wherein the first
payload of the single atom residue is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and 21 and
the second payload of the single atom residue is a different
payload from the first payload and is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and
21.
85. The conjugate composition of claim 78 or claim 79, further
comprising a first payload conjugated to each of the first
cross-linkers wherein the payload is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and 21,
and a second payload different from the first payload conjugated to
the second cross-linker wherein the second payload is selected from
the group consisting of payloads set forth in Tables 6, 7, 18, and
21.
86. The conjugate composition of claim 78 or claim 79, further
comprising a first payload conjugated to each of the first
cross-linkers wherein the payload is selected from the group
consisting drug moieties of Table 21, and a second payload
different from the first payload conjugated to the second
cross-linker wherein the second payload is selected from the group
consisting of targeting moieties of Table 21.
87. The conjugate composition of claim 85 or claim 86, wherein the
second payload is linked to the N-terminus of the XTEN by the
second cross-linker conjugated by reaction of an alkyne reactant
and an azide reactant selected from the group consisting of the
reactants of Table 15.
88. A conjugate composition comprising at least a first and a
second XTEN, wherein the XTEN are the same or are different and
each is selected from the group consisting of the XTEN of claim 37,
the XTEN of claim 42, the XTEN of claim 7, and the sequences set
forth in Table 3, and in which the first and the second XTEN are
conjugated to each other by the N-termini of the first and the
second XTEN with a cross-linker created by reaction of an alkyne
reactant and an azide reactant selected from the group consisting
of the reactants of Table 15.
89. The conjugate composition of claim 88, wherein the first XTEN
and the second XTEN are different and each independently has at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or 100% sequence identity to a sequence
selected from the group of sequences set forth in Table 3.
90. The conjugate composition of claim 89, wherein the first XTEN
has at least about 90%, or at least about 91%, or at least about
92%, or at least about 93%, or at least about 94%, or at least
about 95%, or at least about 96%, or at least about 97%, or at
least about 98%, or at least about 99%, or 100% sequence identity
to a sequence selected from the group of sequences consisting of
Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187, Seg 188, Seg
189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg 195, Seg 196,
Seg 197, Seg 198, and Seg 199 set forth in Table 3 and the second
XTEN has at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or 100% sequence
identity to a different sequence selected from the group of
sequences consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg
186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193,
Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth
in Table 3.
91. The conjugate composition of claim 88, wherein the first XTEN
and the second XTEN are the same are each has at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, or 100% sequence identity to a sequence selected
from the group of sequences set forth in Table 3.
92. The conjugate composition of claim 91, wherein the first XTEN
and the second XTEN each have at least about 90%, or at least about
91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%, or
100% sequence identity to a sequence selected from the group of
sequences consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg
186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193,
Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth
in Table 3.
93. The conjugate composition of any one of claims 88-92, wherein
the first and the second XTEN each comprises one or more cysteine
residues, and further comprises a first cross-linker conjugated to
each cysteine residue of the first XTEN and a second cross-linker
conjugated to each cysteine residue of the second XTEN, wherein the
first and the second cross-linkers are independently selected from
the group consisting of the cross-linkers set forth in Table
13.
94. The conjugate composition of any one of claims 88-92, wherein
the first and the second XTEN each comprises one or more lysine
residues, and further comprises a cross-linker conjugated to each
lysine residue of the first and/or the second XTEN of the
conjugate, wherein the cross-linker is selected from the group
consisting of the cross-linkers set forth in Table 13.
95. The conjugate composition of claim 93 or claim 94, further
comprising a single atom residue of a first payload conjugated to
each cross-linker of the first XTEN wherein the residue is selected
from the group consisting of carbon, nitrogen, oxygen and sulfur,
and further comprises a single atom residue of a second payload
conjugated to each cross-linker of the second XTEN wherein the
residue is selected from the group consisting of carbon, nitrogen,
oxygen and sulfur.
96. The conjugate composition of claim 95, wherein the first
payload of the single atom residue is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and 21,
and the second payload of the single atom residue is a different
payload from the first payload and is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and
21.
97. The conjugate composition of claim 93 or claim 94, further
comprising a first payload conjugated to each cross-linker of the
first XTEN wherein the first payload is selected from the group
consisting of the payloads set forth in Tables 6, 7, 18, and 21,
and further comprises a second payload different from the first
payload wherein the second payload is conjugated to each
cross-linker of the second XEN wherein the second payload is
selected from the group consisting of the payloads set forth in
Tables 6, 7, 18, and 21.
98. The conjugate composition of claim 93 or claim 94, further
comprising a first payload conjugated to each cross-linker of the
first XTEN wherein the first payload is selected from the group
consisting of the targeting moieties set forth in Table 18, and
further comprises a second payload different from the first payload
wherein the second payload is conjugated to each cross-linker of
the second XTEN wherein the second payload is selected from the
group of toxins set forth in Table 18.
99. The conjugate composition of claim 93 or claim 94, further
comprising a first payload conjugated to each cross-linker of the
first XTEN wherein the first payload is selected from the group
consisting of the targeting moieties set forth in Table 21, and
further comprises a second payload different from the first payload
wherein the second payload is conjugated to each cross-linker of
the second XTEN wherein the second payload is selected from the
group of toxins set forth in Table 21.
100. The conjugate composition of claim 99, wherein the first XTEN
is Seg 176 of Table 3 and the second XTEN is selected from the
group consisting of Seg 176 and Seg 177 set forth in Table 3.
101. A conjugate composition comprising at least a first XTEN, a
second XTEN, and a third XTEN wherein the XTEN are each
independently selected from the group consisting of the XTEN of
claim 37, the XTEN of claim 42, the XTEN of claim 7, and the
sequences set forth in Table 3, wherein the first and the second
and the third XTEN are conjugated to each other at the N-termini
using a trivalent cross-linker selected from the group consisting
of the trivalent cross-linkers set for in Table 13 or Table 14.
102. The conjugate composition of claim 100, wherein the trivalent
cross-linker is selected from the group consisting of
Tris-(2-Maleimidoethyl)amine (TMEA) and amine-reactive
Tris-(succimimidyl aminotricetate) (TSAT).
103. The conjugate composition of any one of claims 100-102,
wherein the first, the second, and the third XTEN are the same and
each has at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about %%, or at least about 97%, or at
least about 98%, or at least about 99%, or 100% sequence identity
to a sequence selected from the group consisting of Seg 174, Seg
175, Seg 176, Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190,
Seg 191, Seg 192, Seg 193, Seg 194, Seg 195, Seg 1%, Seg 197, Seg
198, and Seg 199 set forth in Table 3.
104. The conjugate composition of any one of claims 100-102,
wherein the first and the second XTEN are the same and each has at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or 100% sequence identity to a sequence
selected from the group consisting of Seg 174, Seg 175, Seg 176,
Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg
192, Seg 193, Seg 194, Seg 195, Seg 1%, Seg 197, Seg 198, and Seg
199 set forth in Table 3, and the third XTEN is different from the
first and the second XTEN and has at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%,
or 100% sequence identity to a sequence selected from the group
consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187,
Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg
195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table
3.
105. The conjugate composition of claim 103 or claim 104, wherein
the conjugate further comprises a first cross-linker conjugated to
each cysteine residue of the first XTEN, a second cross-linker
conjugated to each cysteine residue of the second XTEN, and a third
cross-linker conjugated to each cysteine residue of the third XTEN,
wherein the cross-linker is selected from the group consisting of
the cross-linkers set forth in Table 13.
106. The conjugate composition of claim 105, having the
configuration of formula XII: ##STR00026## wherein independently
for each occurrence; a. 3.times.CL is the trivalent cross-linker;
b. CL1 is the first cross-linker conjugated to XTEN1; c. CL2 is the
second cross-linker conjugated to XTEN2; d. CL3 is the third
cross-linker conjugated to XTEN3; e. x is an integer of 1 to about
10; f. y is an integer of 1 to about 10; g. z is an integer of 1 to
about 10 with the proviso that x+y+z is >3; h. XTEN1 is the
first XTEN; i. XTEN2 is the second XTEN; and j. XTEN3 is the third
XTEN.
107. The conjugate composition of claim 105 or claim 106, further
comprising a. a single atom residue of a first payload conjugated
to each first cross-linker of the first XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur, b. a single atom residue of a second payload conjugated to
each second cross-linker of the second XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; and c. a single atom residue of a third payload conjugated
to each third cross-linker of the third XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur.
108. The conjugate composition of claim 105 or claim 106, further
comprising a. a first payload conjugated to each first cross-linker
of the first XTEN selected from the group consisting of the
payloads set forth in Tables 6, 7, 18 and 21; b. a second payload
conjugated to each second cross-linker of the second XTEN selected
from the group consisting of the payloads set forth in Tables 6, 7,
18 and 21, wherein the payload is the same or is different from the
first payload; and c. a third payload conjugated to each third
cross-linker of the third XTEN selected from the group consisting
of the payloads set forth in Tables 6, 7, 18 and 21, wherein the
third payload is the same or is different from the first or the
second payload.
109. The conjugate composition of claim 108, wherein the first
payload is a targeting moiety with specific binding affinity to a
target, wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and the second and the third payloads are a drug, which may be
the same or may be different and wherein the drug is selected from
the group consisting of the drugs set forth in Table 6, Table 18,
and Table 21.
110. The conjugate composition of claim 109, wherein the targeting
moiety is selected from the group consisting of LHRH and folate,
and wherein the drug is selected from the group consisting of
doxorubicin, paclitaxel, auristatin, monomethyl auristatin E
(MMAE), monomethyl auristatin F, maytansine, dolastatin,
calicheamicin, vinca alkaloid, camptothecin, mitomycin C,
epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin.
111. The conjugate composition of claim 108, wherein the targeting
moiety, and the drug moiety correspond to any one of conjugates
1-290 set forth in Table 21.
112. The conjugate composition of claim 111, wherein the conjugate
has the XTEN, the targeting moiety, and the drug moiety
corresponding to conjugate 71 of Table 21.
113. A conjugate composition comprising at least a first and a
second and a third and a fourth XTEN, wherein the XTEN are selected
from the group consisting of the XTEN of claim 37, the XTEN of
claim 42, the XTEN of claim 7, and the sequences set forth in Table
3, wherein the XTEN may be the same or different, and in which the
first and the second and the third and the fourth XTEN are
conjugated to each other by the N-terminus using a tetravalent
cross-linker wherein the tetravalent cross-linker is a tetravalent
maleimide cluster.
114. The conjugate composition of claim 113, wherein the first, the
second, the third, and the fourth XTEN are the same and each has at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or 100% sequence identity to a sequence
selected from the group consisting of Seg 174, Seg 175, Seg 176,
Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg
192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg
199 set forth in Table 3.
115. The conjugate composition of claim 113, wherein the first and
the second XTEN are the same and each has at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or 100% sequence identity to a sequence selected from the
group consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186,
Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg
194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in
Table 3, and the third and the fourth XTEN are the same but are
different from the first and the second XTEN and each has at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99%, or 100% sequence identity to a sequence
selected from the group consisting of Seg 174, Seg 175, Seg 176,
Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg
192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg
199 set forth in Table 3.
116. The conjugate composition of claim 114 or claim 115, wherein
the conjugate further comprises a first cross-linker conjugated to
each cysteine residue of the first XTEN, a second cross-linker
conjugated to each cysteine residue of the second XTEN, a third
cross-linker conjugated to each cysteine residue of the third XTEN,
and a fourth cross-linker conjugated to each cysteine residue of
the fourth XTEN, wherein each cross-linker is selected from the
group consisting of the cross-linkers set forth in Table 13.
117. The conjugate composition of any one of claim 116, having the
configuration of formula XIV ##STR00027## wherein independently for
each occurrence: a. 4.times.CL is the tretravalent cross-linker; b.
CL1 is the first cross-linker conjugated to XTEN1; c. CL2 is the
second cross-linker conjugated to XTEN2; d. CL3 is the third
cross-linker conjugated to XTEN3; e. CL4 is the fourth cross-linker
conjugated to XTEN4; f. v is an integer of 1 to about 10; g. x is
an integer of 1 to about 10; h. y is an integer of 1 to about 10;
i. z is an integer of 1 to about 10 with the proviso that x+y+z is
>4; j. XTEN1 is the first XTEN; k. XTEN2 is the second XTEN; l.
XTEN3 is the third XTEN; and m. XTEN4 is the fourth XTEN.
118. The conjugate composition of claim 116 or claim 117, further
comprising a. a single atom residue of a first payload conjugated
to each first cross-linker of the first XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; b. a single atom residue of a second payload conjugated to
each second cross-linker of the second XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; c. a single atom residue of a third payload conjugated to
each third cross-linker of the third XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; and d. a single atom residue of a fourth payload conjugated
to each fourth cross-linker of the fourth XTEN wherein the residue
is selected from the group consisting of carbon, nitrogen, oxygen
and sulfur.
119. The conjugate composition of claim 116 or claim 117, further
comprising a. a first payload conjugated to each first cross-linker
of the first XTEN selected from the group consisting of the
payloads set forth in Tables 6, 7, 18, and 21; b. a second payload
conjugated to each second cross-linker of the second XTEN selected
from the group consisting of the payloads set forth in Tables 6, 7,
18, and 21, wherein the payload is the same or is different from
the first payload; c. a third payload conjugated to each third
cross-linker of the third XTEN selected from the group consisting
of the payloads set forth in Tables 6, 7, 18, and 21, wherein the
payload is the same or is different from the first or the second
payload; and d. a fourth payload conjugated to each fourth
cross-linker of the fourth XTEN selected from the group consisting
of the payloads set forth in Tables 6, 7, 18, and 21, wherein the
payload is the same or is different from the first or the second or
the third payload.
120. The conjugate composition of claim 119, wherein the first
payload is a targeting moiety with specific binding affinity to a
target wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and at least one other of the second, third, and fourth
payloads is a drug wherein the drug is selected from the group
consisting of the drugs set forth in Tables 6, 18 and 21.
121. The conjugate composition of claim 120, wherein the targeting
moiety is selected from the group consisting of LHRH and folate,
and the drug is selected from the group consisting of doxorubicin,
paclitaxel, auristatin, maytansine, dolastatin, calicheamicin,
vinca alkaloid, camptothecin, mitomycin C, epothilone, hTNF, Il-12,
bortezomib, ranpirnase, pseudomonas exotoxin, SN-38, and
rachelmycin.
122. The conjugate composition of claim 119, wherein the first
payload is a targeting moiety with specific binding affinity to a
target wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and at least one other of the second, third, and fourth
payloads is a drug wherein the drug is selected from the group
consisting of the drugs set forth in Tables 6, 18 and 21, and
wherein the XTEN, the targeting moiety, and the drug moiety
correspond to any one of conjugates 1-290 set forth in Table
21.
123. The conjugate composition of claim 101, having the
configuration of formula XVI: ##STR00028## wherein independently
for each occurrence; a. 3.times.CL is the trivalent cross-linker;
b. CL1 is the first cross-linker conjugated to XTEN1; c. CL2 is the
second cross-linker conjugated to XTEN2; d. x is an integer of 1 to
about 10; e. y is an integer of 1 to about 10 with the proviso that
x+y is >2; f. XTEN1 is the first XTEN; g. XTEN2 is the second
XTEN; and h. XTEN3 is the third XTEN wherein the XTEN is selected
from the group consisting of the sequences set forth in Table
2.
124. The conjugate composition of claim 123, further comprising a.
a single atom residue of a first payload conjugated to each first
cross-linker of the first XTEN wherein the residue is selected from
the group consisting of carbon, nitrogen, oxygen and sulfur; and b.
a single atom residue of a second payload conjugated to each second
cross-linker of the second XTEN wherein the residue is selected
from the group consisting of carbon, nitrogen, oxygen and
sulfur.
125. The conjugate composition of claim 123, further comprising: a.
a first payload conjugated to each first cross-linker of the first
XTEN selected from the group consisting of the payloads set forth
in Tables 6, 7, 18 and 21; and b. a second payload conjugated to
each second cross-linker of the second XTEN selected from the group
consisting of the payloads set forth in Tables 6, 7, 18 and 21,
wherein the payload is the same or is different from the first
payload.
126. The conjugate composition of claim 125, wherein the first
payload is a targeting moiety with specific binding affinity to a
target, wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and the second payload is a drug, which may be the same or may
be different and wherein the drug is selected from the group
consisting of the drugs set forth in Table 6, Table 18, and Table
21.
127. The conjugate composition of claim 126, wherein the targeting
moiety is selected from the group consisting of LHRH and folate,
and wherein the drug is selected from the group consisting of
doxorubicin, paclitaxel, auristatin, monomethyl auristatin E
(MMAE), monomethyl auristatin F, maytansine, dolastatin,
calicheamicin, vinca alkaloid, camptothecin, mitomycin C,
epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin.
128. The conjugate composition of claim 125, wherein the first
payload is selected from the group consisting of the drugs of Table
11 and the proteins of Table 12 and the second payload is different
from the first payload and is selected from the group consisting of
the drugs of Table 11 and the proteins of Table 12.
129. The conjugate composition of claim 125, wherein the first
payload and the second payload are identical and are selected from
the group consisting of the drugs of Table 11 and the proteins of
Table 12.
130. The conjugate composition of claim 101, having the
configuration of formula XVII: ##STR00029## wherein independently
for each occurrence; a. 3.times.CL is the trivalent cross-linker;
b. CL1 is the first cross-linker conjugated to XTEN1; c. x is an
integer of 1 to about 10; d. XTEN1 is the first XTEN wherein the
XTEN is selected from the group consisting of the sequences set
forth in Table 3; e. XTEN2 is the second XTEN wherein the XTEN is
selected from the group consisting of the sequences set forth in
Table 2; and f. XTEN3 is the third XTEN wherein the XTEN is
selected from the group consisting of the sequences set forth in
Table 2.
131. The conjugate composition of claim 130, further comprising a
single atom residue of a first payload conjugated to each first
cross-linker of the first XTEN wherein the residue is selected from
the group consisting of carbon, nitrogen, oxygen and sulfur.
132. The conjugate composition of claim 130, further comprising a
first payload conjugated to each first cross-linker of the first
XTEN selected from the group consisting of the payloads set forth
in Tables 6, 7, 18 and 21.
133. A composition comprising multimeric branched XTEN molecules,
wherein a solution of the composition has a viscosity that is less
than a solution comprising a corresponding linear XTEN having the
same number of amino acids and the same molar concentration.
134. The composition of claim 133, wherein the XTEN molecules have
a trimeric, tetrameric, or pentameric configuration.
135. The composition of claim 133, wherein the XTEN molecules are
selected from the group consisting of the sequences set forth in
Table 2 and Table 3.
136. The composition of any one of claims 133-135, wherein the
viscosity of the solution is reduced by at least 5, 6, 7, 8, 9 or
10 cP in a solution containing .quadrature.100 mg/ml of the
multimeric XTEN compared to a solution containing .quadrature.100
mg/ml of the corresponding linear XTEN of equal molar
concentration.
137. A polypeptide having at least about 90%, or at least about
91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%, or
100% sequence identity to a sequence selected from the group of
sequences set forth in Table 52.
138. A pharmaceutical composition, comprising the conjugate of any
one of claims 85-87, 97-99, 108-112, 118-122, 125-127, 129 or 132
and a pharmaceutically acceptable carrier.
139. The pharmaceutical composition of claim 138 for treatment of a
condition selected from the group of conditions set forth in Table
16.
140. The pharmaceutical composition of claim 138 for use in a
pharmaceutical regimen for treatment of a subject, said regimen
comprising the pharmaceutical composition.
141. The pharmaceutical composition of claim 140, wherein the
pharmaceutical regimen further comprises the step of determining
the amount of pharmaceutical composition needed to achieve a
beneficial effect in a subject having a condition selected from the
group of conditions set forth in Table 16.
142. The pharmaceutical composition of claim 141, wherein the
pharmaceutical regimen for treating the subject comprises
administering the pharmaceutical composition in two or more
successive doses to the subject at an effective amount, wherein the
administration results in at least a 10%, or 20%, or 30%, or 40%,
or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at
least one, two, or three parameters associated with the condition
compared to an untreated subject.
143. A conjugate of any one of claims 85-87, 97-99, 108-112,
118-122, 125-127, 129 or 132 for use in the preparation of a
medicament for treatment of a condition selected from the group of
conditions set forth in Table 16.
144. A method of selecting a combination of payloads linked to XTEN
as a therapeutic agent, the method comprising a. providing a
library of XTENs comprising a plurality of XTEN sequences wherein
each of said XTEN sequences is conjugated to at least a first
payload and at least a second payload which is different from the
first payload; b. from said library, selecting an XTEN sequence as
the therapeutic agent if it exhibits an improved in vitro or in
vivo parameter as compared to that of (1) an XTEN sequence
conjugated to the first payload alone; and (2) an XTEN sequence
conjugated to the second payload alone.
145. The method of claim 144, wherein first payload and second
payload are therapeutically effective for ameliorating a common
disease.
146. The method of claim 144, wherein the first drug and second
drug are therapeutically effective for treating different symptoms
of a common disease.
147. The method of claim 145 or claim 146, wherein the common
disease is selected from cancer, cancer supportive care,
cardiovascular, central nervous system, endocrine disease,
gastrointestinal, genitourinary, hematological, HIV infection,
hormonal disease, inflammation, autoimmune disease, infectious
disease, metabolic disease, musculoskeletal disease, nephrology
disorders, ophthalmologic diseases, pain, and respiratory.
148. The method of claim 144, wherein the first payload and second
payload mediate their therapeutic effect via a common biological
pathway.
149. The method of claim 144, wherein the first payload and second
payload are different drugs selected from the group consisting of
the drugs set forth in Table 6, Table 18 and Table 21.
150. The method of claim 144, wherein the first payload and second
payload are different biologically active proteins selected from
the group consisting of the proteins set forth in Table 7, Table 18
and Table 21.
151. The method of claim 144, wherein the first payload is a drug
selected from the group consisting of the drugs set forth in Table
6, Table 18 and Table 21 and the second payload is a biologically
active protein selected from the group consisting of the proteins
set forth in Table 7, Table 18 and Table 21.
152. An isolated polypeptide comprising an extended recombinant
polypeptide (XTEN) that is linked to an affinity purification tag
via a proteolytic cleavage site having a sequence selected from
SASRSA or SASXSA where X is R or K.
153. A polypeptide comprising an extended recombinant polypeptide
(XTEN) that is linked at its N-terminus to a first affinity
purification tag via a proteolytic cleavage site having a sequence
selected from SASRSA or SASXSA where X is R or K, and at its
C-terminus to a second affinity purification tag via a proteolytic
cleavage site having a sequence selected from SASRSA or SASXSA
where X is R or K.
154. A composition having the structure set forth in FIG. 117.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 16/133,444, filed Sep. 17, 2018, which is a continuation of
U.S. application Ser. No. 14/381,199, filed Aug. 26, 2014, which is
a National Stage Entry (and claims priority to) PCT Application No.
PCT/US2013/028116 filed on Feb. 27, 2013, which claims benefit to
U.S. Provisional Application Ser. No. 61/634,312 filed Feb. 27,
2012, U.S. Provisional Application Ser. No. 61/690,187 filed Jun.
18, 2012, and U.S. Provisional Application Ser. No. 61/709,942
filed Oct. 4, 2012. Each of the applications referenced in this
paragraph are incorporated herein by reference in their
entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 10, 2013, is named 32808-736.601_SL.txt and is 2,639,526
bytes in size.
BACKGROUND
[0003] Extending the half-life a therapeutic agent, whether being a
therapeutic protein, peptide or small molecule, often requires
specialized formulations or modifications to the therapeutic agent
itself. Conventional modification methods such as pegylation,
adding to the therapeutic agent an antibody fragment or an albumin
molecule, suffer from a number of profound drawbacks. While these
modified forms can be prepared on a large scale, these conventional
methods are generally plagued by high cost of goods, complex
process of manufacturing, and low purity of the final product.
Oftentimes, it is difficult, if not impossible, to purify to
homogeneity of the target entity. This is particularly true for
pegylation, where the reaction itself cannot be controlled
precisely to generate a homogenous population of pegylated agents
that carry the same number or mass of polyethylene-glycol. Further,
the metabolites of these pegylated agents can have sever side
effects. For example, PEGylated proteins have been observed to
cause renal tubular vacuolation in animal models (Bendele, A.,
Seely, J., Richey, C., Sennello, G. & Shopp, G. Short
communication: renal tubular vacuolation in animals treated with
polyethylene-glycol-conjugated proteins. Toxicol. Sci. 1998. 42,
152-157). Renally cleared PEGylated proteins or their metabolites
may accumulate in the kidney, causing formation of PEG hydrates
that interfere with normal glomerular filtration. In addition,
animals and humans can be induced to make antibodies to PEG (Sroda,
K. et al. Repeated injections of PEG-PE liposomes generate anti-PEG
antibodies. Cell. Mol. Biol. Lett. 2005.10, 37-47).
[0004] Thus, there remains a considerable need for alternative
compositions and methods useful for the production of highly pure
form of therapeutic agents with extended half-life properties at a
reasonable cost.
SUMMARY
[0005] The present invention addresses this need and provides
related advantages. The compositions and methods disclosed herein
not only are useful as therapeutics but are also particularly
useful as research tools for preclinical and clinal development of
a candidate therapeutic agent. In some aspects, the present
invention addresses this need by, in part, generating extended
recombinant polypeptide (XTEN) reagents that can be purified to
homogeneity with one or a few simple steps, and/or that are
amenable to chemical conjugation with payload peptides, proteins
and small molecules with reactive groups using a wide diversity of
conjugation methods. The use of the XTEN reagents generates
high-yield product of XTEN-linked agent that are superior in one or
more aspects including high homogeneity, high solubility, long
stability, and enhanced terminal half-life compared to unconjugated
product.
[0006] The present invention relates, in part, to novel
compositions comprising substantially homogeneous extended
recombinant polypeptides (XTEN) useful as conjugation partners for
linking to one or more payload pharmacologically- or
biologically-active agents, resulting in XTEN-payload compositions.
In one aspect, the invention provides XTEN engineered for covalent
linking to the one or more payloads either directly or via
cross-linkers, resulting in XTEN-payload composition that comprise
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more molecules of one, two,
three or more types of payloads. It is an object of the present
invention to provide such engineered XTEN polypeptides for use in
creating conjugates with payload agents of interest as compositions
with enhanced pharmaceutical properties, including enhanced
pharmacokinetic properties. The invention provides XTEN that are
substantially homogeneous in length and sequence that are useful
for preparing the conjugates comprising the XTEN linked to one or
more payloads such that the resulting XTEN-payload conjugates have
a high degree of purity. Such conjugates of high purity are useful
in preparing pharmaceutical compositions for subjects having a
medical condition for which the one or more payloads have utility
in the prevention, treatment or amelioration of the condition.
[0007] In a first aspect, the invention provides substantially
homogenous XTEN polypeptide compositions useful as conjugation
partners to create XTEN-cross-linker intermediates and XTEN-payload
compositions. In some embodiments, the invention provides a
substantially homogenous population of polypeptides comprising an
extended recombinant polypeptide (XTEN), and wherein at least 90%,
91%, 92%, 93%, 94%, or 95% of individual polypeptide molecules in
said population have identical sequence length. In one embodiment
of the foregoing, the XTEN is characterized in that: the total XTEN
amino acid residues is at least 36 to about 3000 amino acid
residues; the sum of glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) residues constitutes
more than about 90% of the total amino acid residues of the XTEN;
the XTEN sequence is substantially non-repetitive such that (i) the
XTEN sequence contains no three contiguous amino acids that are
identical unless the amino acids are serine, (ii) at least about
80%, or about 90%, or about 95% of the XTEN sequence consists of
non-overlapping sequence motifs, each of the sequence motifs
comprising about 9 to about 14 amino acid residues, wherein any two
contiguous amino acid residues does not occur more than twice in
each of the sequence motifs; or (iii) the XTEN sequence has a
subsequence score of less than 10; the XTEN sequence has greater
than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than
99% random coil formation as determined by GOR algorithm; the XTEN
sequence has less than 2%, or 3%, or 4%, or 5% alpha helices; the
XTEN sequence has less than 2%, or 3%, or 4%, or 5% beta-sheets as
determined by Chou-Fasman algorithm; and the XTEN sequence lacks a
predicted T-cell epitope when analyzed by TEPITOPE algorithm,
wherein the TEPITOPE algorithm prediction for epitopes within the
XTEN sequence is based on a score of -8, or -9, or -10. In another
embodiment of the foregoing, the XTEN comprises a sequence having
at least about 90%, or at least about 91%, or at least about 92%,
or at least about 93%, or at least about 94%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%, or 100% sequence identity to a
sequence selected from the group consisting of the sequences set
forth in Table 2, Table 3, Table 4 and Tables 22-25.
[0008] In other embodiments, the substantially homogenous XTEN
polypeptide compositions comprise one or more affinity tags. In one
embodiment, the invention provides a substantially homogenous XTEN
polypeptide composition comprising a first affinity tag wherein the
first affinity tag has binding affinity for a chromatography
substrate selected from the group consisting of hydrophobic
interaction chromatography (HIC), cation exchange, anion exchange,
immobilized metal ion affinity chromatography (IMAC), and
immobilized antibody. In one embodiment of the foregoing, the first
affinity tag has at least about 90%, 91%, 92%, 93%, 94%, or at
least about 95% sequence identity to a sequence selected from the
group consisting of the sequences set forth in Table 7. In another
embodiment of the foregoing XTEN and affinity tag, the composition
further comprises one or more helper sequences. In one embodiment,
a helper sequence comprises a sequence having at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, or 100% sequence identity to a sequence selected
from the group consisting of the sequences set forth in Table 10.
In another embodiment, the helper sequence is selected from the
group consisting of: KNPEQAEEQX1EET wherein X1 is independently S
or R (SEQ ID NO: 1); ANPEQAEEQX1EET wherein X1 is independently S
or R (SEQ ID NO: 2): KNPEQAEEQAEEQX1 EET wherein X1 is
independently S or R (SEQ ID NO: 3); KX2X3EQAEEQAEEQX1EET wherein
X1 is independently S or R, X2 is independently K or N, and X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S (SEQ ID NO: 4);
KX2(X3).sub.10QX1EET wherein X1 is independently S or R, X2 is
independently K or N, and X3 is independently K, N, T, Q, H, P, E,
D, A, R, or S (SEQ ID NO: 5); KX2(X3).sub.7AEEQX1EET wherein X1 is
independently S or R, X2 is independently K or N. and X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S (SEQ ID NO: 6);
KX2X3EQE(X3).sub.3AEEQREET wherein X2 is independently K or N, and
X3 is independently K, N, T, Q, H, P, E, D, A, R, or S (SEQ ID NO:
7); KX2X3EQE(X3).sub.3AEE(X3).sub.5 wherein X2 is independently K
or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S
(SEQ ID NO: 8); KKQEQEKEQAEEQ(X4X5).sub.2REET wherein X4 is
independently A or S and X5 is independently K, Q, or E (SEQ ID NO:
9); KKQEQEKEQAEEQ(X4X5).sub.4REET wherein X4 is independently A or
S and X5 is independently K, Q, or E (SEQ ID NO: 10);
KKQEQEKEQAEEQ(Z).sub.4REET, wherein Z is any naturally-occurring
L-amino acid (SEQ ID NO: 11); KX2(X3).sub.n, wherein n is an
integer from 1040 and X2 is independently K or N, and X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S (SEQ ID NO: 12);
(X3).about. wherein n is an integer from 10-50 and X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S (SEQ ID NO: 13);
KX2QEQEKEQAEEQ(X4X5).sub.nX1EET wherein n is zero or an integer
from 1-10 and X1 is independently S or R, X2 is independently K or
N, X4 is independently A or S, and X5 is independently K, Q, or E
(SEQ ID NO: 14); KX2(X3).sub.n(X4X5).sub.mX1EET, wherein n is an
integer from 5-20, m is zero or an integer from 1-10, X1 is
independently S or R, X2 is independently K or N, X3 is
independently K, N, T, Q, H, P, E, D, A, R, or S, X4 is
independently A or S, and X5 is independently K, Q, or E (SEQ ID
NO: 15); and KX2(X3).sub.n(Z).sub.mX1EET, wherein n is an integer
from 5-20, m is zero or an integer from 1-10, X1 is independently S
or R, X2 is independently K or N, X3 is independently K, N, T, Q,
H, P, E, D, A, R, or S, and Z is any naturally-occurring L-amino
acid (SEQ ID NO: 16), and any sequence homologs showing at least
80%, 90%, 95%, 98%, or 99% sequence identity of the foregoing when
optimally aligned.
[0009] In other embodiments of the foregoing substantially
homogenous XTEN, affinity tag, and helper sequence compositions,
the composition further comprises a first cleavage sequence. Where
desired, the cleavage sequence is selected from the group
consisting of the sequences set forth in Table 8 and Table 9. In
one embodiment of the foregoing, the composition has the
configuration of formula I:
(HS)-(AT1)-(CS1)-(XTEN) I
wherein HS is the helper sequence; AT1 is the first affinity tag;
CS1 is the first cleavage sequence; and XTEN is the extended
recombinant polypeptide. In another embodiment of the foregoing
compositions, the composition further comprises a second cleavage
sequence. Where desired, the first and the second cleavage
sequences are capable of being cleaved by the same protease, and
wherein the composition has the configuration of formula II:
(HS)-(CS1)-(XTEN)-(CS2)-(AT1) II
wherein HS is a helper sequence; AT1 is the first affinity tag, CS1
is the first cleavage sequence; CS2 is the second cleavage
sequence; and XTEN is the extended recombinant polypeptide. In
another embodiment of the foregoing compositions, the first
affinity tag comprises the sequence RPRPRPRPRPRPR (SEQ ID NO: 17),
HHHHHH (SEQ ID NO: 18), or any affinity tag known in the art or
disclosed herein.
[0010] In other embodiments of the substantially homogenous XTEN
compositions, the compositions comprise a first and a second
affinity tag, a first and a second cleavage sequence, and a helper
sequence wherein the second affinity tag is different from the
first affinity tag and has binding affinity to a different
chromatography substrate than that of the first affinity tag,
wherein the chromatography substrate is selected from the group
consisting of HIC, cation exchange, anion exchange, IMAC, and
immobilized antibody, and wherein the first and the second cleavage
sequences are capable of being cleaved by the same protease, and
wherein the second affinity tag has at least about 90%, 91%, 920,
93%, 94%, or at least about 95% sequence identity to a sequence
selected from the group consisting of the sequences set forth in
Table 7. In one embodiment of the foregoing composition, the
composition has the configuration of formula III:
(HS)-(AT1)-(CS1)-(XTEN)-(CS2)-(AT2) III
[0011] wherein HS is the helper sequence; AT1 is the first affinity
tag; CS1 is the first cleavage sequence; CS2 is the second cleavage
sequence; XTEN is the extended recombinant polypeptide; and AT2 is
the second affinity tag. In another embodiment of the foregoing
composition, the first affinity tag comprises the sequence
RPRPRPRPRPRPR (SEQ ID NO: 17) and the second affinity tag comprises
the sequence HHHHHH (SEQ ID NO: 18). In another embodiment of the
foregoing composition, the first affinity tag comprises the
sequence HHHHHH (SEQ ID NO: 18) and the second affinity tag
comprises the sequence RPRPRPRPRPRPR (SEQ ID NO: 17). In another
embodiment of the foregoing composition, the first affinity tag
comprises the sequence RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19) and
the second affinity tag comprises the sequence HHHHHHHH (SEQ ID NO:
20).
[0012] In another aspect, the invention provides compositions
comprising a substantially homogenous population of a polypeptide
obtained by a process. In some embodiments, the compositions are
obtained by the process comprising: culturing a host cell that
comprises a vector encoding the polypeptide in a fermentation
reaction under conditions effective to express the polypeptide by a
crude expression product of the host cell, wherein the encoded
polypeptide comprises an XTEN, a first cleavage sequence and a
first affinity tag; adsorbing the polypeptide of the crude
expression product onto a first chromatography substrate under
conditions effective to capture the first affinity tag onto the
first chromatography substrate; eluting the polypeptide; and
recovering the polypeptide. In some embodiments, at least 90%, 91%,
92%, 93%, 94%, or 95% of the polypeptides of the resulting
population have identical sequence length. In one embodiment of the
foregoing composition, the first chromatography substrate is
selected from the group consisting of HIC, cation exchange, anion
exchange, and IMAC. In another embodiment of the foregoing
composition, the affinity tag is selected from the group consisting
of the affinity tags of Table 7. In another embodiment of the
foregoing composition the first chromatography substrate is cation
exchange and the first affinity tag comprises the sequence
RPRPRPRPRPRPR (SEQ ID NO: 17). In another embodiment of the
foregoing composition, the first chromatography substrate is IMAC
and the first affinity tag comprises the sequence HHHHHHHH (SEQ ID
NO: 20). In one embodiment of the foregoing composition, the
encoding vector encodes any of the XTEN embodiments described
herein comprising at least affinity tag, at least a first cleavage
sequence, a helper sequence, and optionally a second cleavage
sequence. In another embodiment of the foregoing composition, the
vector further encodes a second cleavage sequence and a second
affinity tag wherein the first and the second cleavage sequences
are capable of being cleaved by the same protease and wherein the
second affinity tag has binding affinity to a second, different
chromatography substrate than the first affinity tag, and wherein
the composition is obtained by the process further comprising:
adsorbing the polypeptide onto a second chromatography substrate
under conditions effective to capture the second affinity tag onto
the second chromatography substrate; eluting the polypeptide; and
recovering the polypeptide wherein at least 90%, 91%, 92%, 93%,
94%, or 95% of the polypeptides of the population have identical
sequence length. In one embodiment of the foregoing, the first
chromatography substrate is different from the second
chromatography substrate and each of the first and the second
chromatography substrate are independently selected from the group
consisting of HIC, cation exchange, anion exchange, and IMAC. In
another embodiment of the foregoing composition, the first
chromatography substrate is cation exchange and the first affinity
tag comprises the sequence RPRPRPRPRPRPR (SEQ ID NO: 17) or
RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19) and the second
chromatography substrate is IMAC and the first affinity tag
comprises the sequence HHHHHHHH (SEQ ID NO: 20) or HHHHHHHH (SEQ ID
NO: 20). In another embodiment of the foregoing composition, the
first chromatography substrate is IMAC and the first affinity tag
comprises the sequence HHHHHHHH (SEQ ID NO: 20) or HHHHHHHH (SEQ ID
NO: 20) and the second chromatography substrate is cation exchange
and the first affinity tag comprises the sequence RPRPRPRPRPRPR
(SEQ ID NO: 17) or RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19). In
another embodiment, the foregoing compositions comprising a first
or a first and a second affinity tag are further processed by
treating the composition with a protease under conditions effective
to cleave the cleavage sequence(s), thereby releasing the XTEN from
the affinity tag(s); adsorbing the XTEN onto a chromatography
substrate under conditions effective to capture the XTEN but not
the affinity tag(s) or the protease; eluting the XTEN; and
recovering the XTEN. At least 90%, 91%, 92%, 93%, 94%, or 95% of
the individual molecules of XTEN in the resulting composition have
identical sequence length. In one embodiment of the foregoing
composition, the cleavage sequence(s) are capable of being cleaved
by a protease selected from the group consisting of the proteases
of Table 9. In another embodiment of the foregoing composition, the
cleavage sequence(s) are capable of being cleaved by trypsin and
the protease is trypsin. In another embodiment of the foregoing
composition, the chromatography substrate is anion exchange. The
anion exchange substrate can be a substrate selected from the group
consisting of macrocap Q, capto Q, superQ-650M, and poros D.
Alternatively, the foregoing compositions comprising one affinity
tag or two affinity tags are further processed by treating the
composition under conditions effective to cleave the cleavage
sequence(s), thereby releasing the XTEN from the one or two
affinity tags; adsorbing the protease onto a chromatography
substrate under conditions effective to capture the protease and
the affinity tags but not the XTEN; and recovering the XTEN from
the eluate. In some embodiments, at least 90%, 91%, 92%, 93%, 94%,
or 95% of the individual molecules of XTEN of the resulting eluate
have identical sequence length. In one embodiment of the foregoing
composition, the cleavage sequence(s) are capable of being cleaved
by a protease selected from the group consisting of the proteases
of Table 9. In another embodiment of the foregoing composition, the
cleavage sequence(s) are capable of being cleaved by trypsin and
the protease utilized is trypsin. The chromatography substrate can
be selected from one or more of cation exchange, HIC or IMAC.
[0013] In another aspect, the invention relates, in part, to
polypeptide compositions that can be cleaved into XTEN segments of
equal length and sequence. In one embodiment, the invention
provides a composition comprising an XTEN sequence, wherein the
XTEN sequence further comprises one or more cleavage sequences
capable of being cleaved by trypsin and wherein treatment with
trypsin under conditions effective to cleave all the cleavage
sequences results in a preparation of XTEN fragments wherein each
XTEN fragment has at least about 99% sequence identity to every
other fragment in the preparation. In one embodiment of the
composition, the cleavage sequence has at least 86% sequence
identity to or is identical to the sequence SASRSA (SEQ ID NO: 21)
or SASKSA (SEQ ID NO: 22). In another embodiment of the
composition, the cleavage sequence comprises the sequence RX or KX,
wherein X is any L-amino acid other than proline. In one embodiment
of the foregoing compositions, the XTEN composition has at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the sequences selected from the group of
sequences set forth in Table 6.
[0014] In another aspect, the invention relates, in part, to
methods for producing XTEN fragments substantially of equal length
and sequence. In one embodiment, the invention provides a method of
producing a substantially homogenous population of an XTEN, the
method comprising treating a population of polypeptides comprising
a sequence selected from the group of sequences set forth in Table
6 with trypsin under conditions effective to cleave all of the
cleavage sequence(s) resulting in a substantially homogenous XTEN
population wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of
individual molecules of the XTEN fragments have identical sequence
length. In one embodiment of the foregoing method, the method
further comprises adsorbing the XTEN fragments onto a
chromatography substrate under conditions effective to capture the
XTEN fragments but not the protease; eluting the XTEN fragments;
and recovering the XTEN fragments wherein at least 90%, 91%, 92%,
93%, 94%, or 95% of individual molecules of the population have
identical sequence length. In one embodiment of the foregoing
method, the chromatography substrate is anion exchange. The
substrate can be selected from the group consisting of macrocap Q,
capto Q, superQ-650M, and poros D. In another embodiment of the
foregoing method, the XTEN has at least about 90.degree./o, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a
sequence selected from the group of sequences set forth in Table 6.
In another embodiment of the foregoing method, the resulting XTEN
fragment has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to a sequence selected from the group
of sequences set forth in Table 2 or 3. In another embodiment, the
invention provides XTEN compositions made by the process of the
foregoing method embodiments.
[0015] In another aspect, the invention relates, in part, to
methods for producing XTEN at high expression yields from a host
cell. In some embodiments, the invention provides a method
comprising culturing a host cell that comprises a vector encoding a
polypeptide comprising the XTEN and a helper sequence in a
fermentation reaction under conditions effective to express the
polypeptide as a component of a crude expression product at a
concentration of more than about 2 grams/liter (g/L), or about 3
g/L, or about 4 g/L, or about 5 g/L. or about 6 g/L, or about 7 g/L
of said polypeptide. In one embodiment of the foregoing method, the
foregoing expression yields are achieved when the fermentation
reaction reaches an optical density of at least 100, or at least
130, or at least 150 at a wavelength of 600 nm. In another
embodiment, the invention provides a method for comprising
culturing a host cell that comprises a vector encoding a
polypeptide comprising the XTEN and a helper sequence in a
fermentation reaction under conditions effective to express the
polypeptide as a component of a crude expression product at a
concentration of more than about 10 milligrams/gram of dry weight
host cell (mg/g), or at least about 15 mg/g, or at least about 20
mg/g, or at least about 25 mg/g, or at least about 30 mg/g, or at
least about 40 mg/g, or at least about 50 mg/g of said polypeptide.
In one embodiment of the foregoing method, the foregoing high-yield
expression is achieved when the fermentation reaction reaches an
optical density of at least 100, or at least 130, or at least 150
at a wavelength of 600 nm. In another embodiment, the invention
provides a method comprising culturing a host cell that comprises a
vector encoding a polypeptide comprising the XTEN and a helper
sequence in a fermentation reaction under conditions effective to
express the polypeptide as a component of a crude expression
product at a concentration of more than about 10 milligrams/gram of
dry weight host cell (mg/g), or at least about 250 micromoles/L. or
about 300 micromoles/L, or about 350 micromoles/L. or about 400
micromoles/L, or about 450 micromoles/L. or about 500 micromoles/L
of said polypeptide. In one embodiment of the foregoing method, the
foregoing expression yields are achieved when the fermentation
reaction reaches an optical density of at least 100, or at least
130, or at least 150 at a wavelength of 600 nm. In one embodiment
of the foregoing methods, the helper sequence of the expressed
polypeptide is at the N-terminus of the polypeptide, wherein the
helper sequence has at least about 90%, 91%, 92%, 93%, 94%, or 95%
sequence identity or is identical to a sequence selected from the
group consisting of the sequences set forth in Table 10. In another
embodiment of the foregoing methods, expression vector further
encodes a first affinity tag and a cleavage sequence between the
affinity tag and the XTEN, and the method further comprises
recovering the crude expression product of the host cell
fermentation reaction mixture; adsorbing the polypeptide of the
crude expression product onto a first chromatography substrate
under conditions effective to capture the first affinity tag of the
polypeptide onto the chromatography substrate wherein the first
chromatography substrate is selected from the group consisting of
HIC, cation exchange, anion exchange, and IMAC; eluting and
recovering the polypeptide wherein at least 90%, 91%, 92%, 93%,
94%, or 95% of the polypeptides have identical sequence length. In
another embodiment of the foregoing methods, expression vector
further encodes a first affinity tag and a second affinity tag
different from the first tag and a cleavage sequence between each
affinity tag and the XTEN, and the method further comprises
recovering the crude expression product of the host cell
fermentation reaction mixture; adsorbing the polypeptide onto a
first chromatography substrate under conditions effective to
capture the first affinity tag of the polypeptide onto the
chromatography substrate wherein the first chromatography substrate
is selected from the group consisting of HIC, cation exchange,
anion exchange, and IMAC; eluting the polypeptide; adsorbing the
polypeptide onto a second chromatography substrate under conditions
effective to capture the second affinity tag of the polypeptide
onto the chromatography substrate wherein the second chromatography
substrate is selected from the group consisting of HIC, cation
exchange, anion exchange, and IMAC; eluting the polypeptide; and
recovering the polypeptide wherein at least 90%, 91%, 92%, 93%,
94%, or 95% of the polypeptides have identical sequence length. In
one embodiment of the foregoing methods, the methods further
comprise treating the polypeptide with a protease under conditions
effective to cleave the cleavage sequence(s), thereby releasing the
XTEN from the polypeptide; adsorbing the XTEN onto an anion
chromatography substrate under conditions effective to capture the
XTEN; eluting the XTEN; and recovering the XTEN wherein at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least
94%, or at least 95% of the individual XTEN molecules have
identical sequence length. In the foregoing methods, the anion
exchange substrate can be selected from the group consisting of
macrocap Q, capto Q, superQ-650M, and poros D. In one embodiment of
the foregoing methods, the cleavage sequences are capable of being
cleaved by trypsin and the protease is trypsin. In another
embodiment of the foregoing methods, the method further comprises
treating the polypeptide with a protease under conditions effective
to cleave the cleavage sequence(s), thereby releasing the XTEN from
the polypeptide; adsorbing the protease onto a chromatography
substrate under conditions effective to capture the protease and
the affinity tags but not the XTEN; and recovering the XTEN in the
eluate wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of the XTEN
have identical sequence length. In one embodiment of the foregoing
method, the cleavage sequence is capable of being cleaved by
trypsin and the protease utilized is trypsin. In the foregoing
method to capture the protease and the affinity tag, the
chromatography substrate can be selected from one or more of HIC,
cation exchange, and IMAC.
[0016] In another aspect, the invention relates, in part, to a
solid support comprising immobilized thereon a population of
substantially identical XTEN polypeptide molecules. In one
embodiment, the invention provides a solid support comprising
immobilized thereon a population of substantially identical
polypeptide molecule wherein the solid support comprises a
chromatography substrate, immobilized polypeptides each comprising
an XTEN, a first affinity tag, and a second affinity tag wherein
the first affinity tag is joined to the XTEN by a cleavage sequence
at the N-terminus of the XTEN and the second affinity tag is joined
to the XTEN by a cleavage sequence at the C-terminus and wherein
the second affinity tag is different from the first affinity tag,
wherein the chromatography substrate is capable of binding to
either said first or said second affinity tag but not both, and
wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of the immobilized
polypeptide molecules have identical sequence length. In one
embodiment of the XTEN comprises a sequence having at least about
90%, or at least about 91%, or at least about 92%, or at least
about 93%, or at least about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or
at least about 99%, or 100% sequence identity to a sequence
selected from the group consisting of the sequences set forth in
Table 2, Table 3, Table 4 and Tables 22-25, the first and the
second affinity tag each independently have at least about 90%,
91%, 92%, 93%, 94%, or at least about 95% sequence identity to a
sequence selected from the group consisting of the sequences set
forth in Table 7, and the cleavage sequence is selected from the
group consisting of the sequences set forth in Table 8 and Table 9.
In one embodiment of the foregoing the cleavage sequence has at
least about 86% sequence identity to or is identical to the
sequence SASRSA (SEQ ID NO: 21) or SASKSA (SEQ ID NO: 22). In one
embodiment of the foregoing the cleavage sequence comprises the
sequence RX or KX, wherein X is any L-amino acid other than
proline. In one embodiment of the foregoing, the solid support is
selected from the group consisting of HIC chromatography resin,
cation exchange chromatography resin, anion exchange chromatography
resin, and IMAC chromatography resin. In one embodiment of the
foregoing, the first affinity tag comprises the sequence
RPRPRPRPRPRPR (SEQ ID NO: 17) or RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID
NO: 19) and the second affinity tag comprises the sequence HHHHHH
(SEQ ID NO: 18) or HHHHHHHH (SEQ ID NO: 20). In another embodiment
of the foregoing, the first affinity tag comprises the sequence
HHHHHH (SEQ ID NO: 18) or HHHHHHHH (SEQ ID NO: 20) and the second
affinity tag comprises the sequence RPRPRPRPRPRPR (SEQ ID NO: 17)
or RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19).
[0017] In another aspect, the invention relates, in part, to
compositions of XTEN conjugated to cross-linkers. In some
embodiments, the invention provides compositions of any of the XTEN
described herein that is covalently linked to one or more molecules
of at least a first cross-linker, wherein the cross-linker is
selected from the group consisting of the cross-linkers set forth
in Table 13, the alkyne reactants set forth in Table 15, and the
azide reactants set forth in Table 15. In one embodiment of the
conjugate composition, the first cross-linker is conjugated to the
at least first XTEN at a location selected from the group
consisting of: an alpha-amino group of an N-terminal amino acid
residue of the XTEN; an epsilon amino group of each lysine residue
of the XTEN; and a thiol group of each cysteine residue of the
XTEN. Where desired, the XTEN in this embodiment has at least about
90%, or at least about 91%, or at least about 92%, or at least
about 93%, or at least about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or
at least about 99%, or 100% sequence identity to a sequence
selected from the group of sequences set forth in Table 2 and Table
3. In another embodiment of the conjugate composition, the XTEN is
selected from the group consisting of AE144, AE288, AE432, AE576,
AE864, Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187, Seg
188, Seg 189, Seg 190, Seg 191. Seg 192, Seg 193, Seg 194, Seg 195,
Seg 196. Seg 197, Seg 198, and Seg 199, and the cross-linker is
conjugated to the alpha amino-group of the N-terminal amino acid of
the XTEN. In another embodiment of the conjugate composition, the
XTEN is selected from the group consisting of Seg 174. Seg 175, Seg
176, Seg 177, Seg 186, Seg 187. Seg 188, Seg 189, Seg 190, Seg 191,
Seg 192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and
Seg 199 set forth in Table 3, and the cross-linker is conjugated to
the thiol group of each cysteine residue of the XTEN. In another
embodiment of the conjugate composition, the first cross-linker is
selected from the group consisting of N-maleimide, iodoacetyl,
pyridyl disulfide and vinyl sulfone, 3-propargyloxypropanoic acid,
(oxyethyl).sub.n-acetylene where n is 1-10, dibenzylcyclooctyne
(DBCO), cyclooctyne (COT), 3-azide-propionic acid, 6-azide-hexanoic
acid, and (oxyethyl).sub.n-azide where n is 1-10. In the foregoing
embodiments of this paragraph, the conjugate has configuration of
formula IV.
##STR00001##
wherein independently for each occurrence CL.sub.1 is the
cross-linker; x is an integer from 1 to about 100, or 1 to about
50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to
about 5, or is 9, or is 3, or is 2, or is 1. Where desired, the
XTEN in this embodiment comprises a sequence having at least about
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3. In one embodiment of the conjugate of
formula IV, CL1 is a cross-linker selected from Table 13. In other
embodiments of the XTEN-crosslinker conjugate compositions, the
compositions further comprise a single atom residue of a first
payload conjugated to each first cross-linker wherein the residue
is selected from the group consisting of carbon, nitrogen, oxygen
and sulfur. In one embodiment of the foregoing, the first payload
of the single atom residue can be selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, and 21.
In other embodiments of the XTEN-crosslinker conjugate
compositions, the compositions further comprise a payload selected
from the group consisting of the payloads set forth in Tables 11
and 12 conjugated to each first cross-linker.
[0018] In other embodiments of the XTEN-crosslinker conjugate
compositions, the invention provides compositions of an XTEN of the
embodiments described herein covalently linked to one or more
molecules of a first cross-linker and one or more molecules of a
second cross-linker, wherein the first cross-linker is conjugated
to either the thiol groups of each cysteine residue of the XTEN or
to the epsilon amino groups of the each lysine residue of the XTEN,
and the second cross-linker conjugated to alpha amino-group of the
N-terminal amino acid of the XTEN wherein each cross-linker is
independently selected from the group consisting of the
cross-linkers set forth in Table 13, the alkyne reactants of Table
15, and the azide reactants of Table 15. In the foregoing
embodiment, the composition has the configuration of formula V:
##STR00002##
wherein independently for each occurrence; CL1 is the first
cross-linker conjugated to cysteine residues of the XTEN; CL2 is
the second cross-linker conjugated to XTEN at the N-terminus; x is
an integer of 1 to about 10; y is an integer of 1 with the proviso
that x+y is .gtoreq.2; and XTEN is either a cysteine engineered
XTEN comprising x number of cysteine residues or a lysine
engineered XTEN comprising x number of lysine residues. In another
embodiments of the XTEN-cross-linker conjugate compositions, the
compositions further comprise a single atom residue of a first
payload conjugated to each of the first cross-linkers wherein the
residue is selected from the group consisting of carbon, nitrogen,
oxygen and sulfur and a single atom residue of a second payload
conjugated to each of the second cross-linkers wherein the residue
is selected from the group consisting of carbon, nitrogen, oxygen
and sulfur. In one embodiment of the foregoing, the first payload
of the single atom residue can be selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, and 21
and the second payload of the single atom residue can be
independently selected from the group consisting of the payloads
set forth in Tables 11, 12, 18, and 21. In some embodiments of the
XTEN-cross-linker-payload residue composition, the composition has
the configuration of formula VI:
##STR00003##
wherein independently for each occurrence P.sub.R1 is a single atom
residue of a payload, wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur; CL.sub.1
is a cross-linker; x is an integer from 1 to about 100, or 1 to
about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or
1 to about 5, or is 3, or is 2, or is 1. Where desired, the XTEN in
this embodiment comprises a sequence having at least about 80%, or
at least about 90%, or at least about 91%, or at least about 92%,
or at least about 93%, or at least about 94%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%, or having 100% sequence identity
to a sequence selected from the group of sequences set forth in
Tables 2 and 3. In one embodiment of the conjugate of formula VI,
the single atom residue of a payload is from a payload selected
from the group consisting of the payloads set forth in Tables 11,
12, 18, 19, and 21. In one embodiment of the conjugate of formula
VI, CL.sub.1 is a cross-linker selected from Table 13. In one
embodiment of the conjugate of formula VI, each cross-linker is
linked to a cysteine sulfur of the XTEN. In another embodiment of
the conjugate of formula VI, each cross-linker is linked to a
lysine epsilon amino group of the XTEN. In another embodiment of
the conjugate of formula VI, x is 1 and the cross-linker is linked
to the N-terminal amino group of the XTEN. In another embodiment of
the conjugate of formula VI, CL.sub.1 is the reaction product of a
first and a second click chemistry reactant selected from Table 15.
In another embodiment, the invention provides a preparation of the
conjugate of formula VI in which at least about 80%, or at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95% of
the XTEN molecules of the preparation of the conjugate have
identical sequence length. In other embodiments of the
XTEN-crosslinker conjugate compositions, the compositions further
comprise a first payload conjugated to each of the first
cross-linkers wherein the payload is selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, and 21,
and a second payload different from the first payload conjugated to
the second cross-linker wherein the second payload is selected from
the group consisting of payloads set forth in Tables 11, 12, 18,
and 21. In one embodiment of the XTEN-crosslinker-payload conjugate
composition, the composition comprises a first payload conjugated
to each of the first cross-linkers wherein the payload is selected
from the group consisting drug moieties of Table 21, and a second
payload different from the first payload conjugated to the second
cross-linker wherein the second payload is selected from the group
consisting of targeting moieties of Table 21. In one embodiment of
the XTEN-crosslinker-payload conjugate composition with a first and
a second payload, a single second payload is linked to the
N-terminus of the XTEN by the second cross-linker conjugated by
reaction of an alkyne reactant and an azide reactant selected from
the group consisting of the reactants of Table 15. In some
embodiments of the XTEN-cross-linker-payload composition, the
composition has the configuration of formula VII:
##STR00004##
wherein independently for each occurrence: P.sub.1 is a payload
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18, 19, and 21; CL.sub.1 is a cross-linker; x is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1; and XTEN is a sequence having at least about 80%,
or at least about 90%, or at least about 91%, or at least about
92%, or at least about 93%, or at least about 94%, or at least
about 95%, or at least about 96%, or at least about 97%, or at
least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3. In one embodiment of the conjugate of
formula VII, CL.sub.1 is a cross-linker selected from Table 13. In
one embodiment of the conjugate of formula VII, each cross-linker
is linked to a cysteine sulfur of the XTEN. In another embodiment
of the conjugate of formula VII, each cross-linker is linked to an
lysine epsilon amino group of the XTEN. In another embodiment of
the conjugate of formula VII, x is 1 and the cross-linker is linked
to the N-terminal amino group of the XTEN. In one embodiment, the
conjugate of formula VII is selected from the group consisting of
the conjugates set forth in Table 21. In another embodiment of the
conjugate of formula VII, CL.sub.1 is the reaction product of a
first and a second click chemistry reactant selected from Table 15.
It will be understood by one of skill in the art that the
compositions of the foregoing embodiments comprising the payload
conjugated to an XTEN-cross-linker using the specified components
represents the reaction product of the reactants and thus differs
from the precise composition of the reactants. In another
embodiment, the invention provides a preparation of the conjugate
of formula VII in which at least about 80%, or at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95% of the XTEN
molecules of the preparation of the conjugate have identical
sequence length.
[0019] In another aspect, the invention relates, in part, to
compositions of a first and a second XTEN conjugated to each other.
In some embodiments, the conjugate composition comprises a first
and a second XTEN, wherein the XTEN are the same or they are
different and each independently has at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or 100% sequence identity to a sequence selected from the
group of sequences set forth in Table 3, and in which the first and
the second XTEN are conjugated to each other by the N-termini of
the first and the second XTEN with a cross-linker created by
reaction of an alkyne reactant and an azide reactant selected from
the group consisting of the reactants of Table 15, resulting in a
dimeric XTEN conjugate. In one embodiment of the dimeric XTEN
composition, at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the first XTEN have identical
sequence length and at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the second XTEN have identical
sequence length. In one embodiment of the dimeric XTEN conjugate,
the first XTEN has at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or 100% sequence
identity to a sequence selected from the group of sequences
consisting of Seg 174, Seg 175, Seg 176, Seg 177. Seg 186, Seg 187,
Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg
195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table 3
and the second XTEN has at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98%, or at least about 99%, or 100%
sequence identity to a different sequence selected from the group
of sequences consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg
186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193,
Seg 194, Seg 195. Seg 196, Seg 197, Seg 198, and Seg 199 set forth
in Table 3. In another embodiment of the dimeric XTEN conjugate,
the first XTEN and the second XTEN are the same and each has at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or 100% sequence identity to a sequence
selected from the group of sequences set forth in Table 3. In
another embodiment of the dimeric XTEN conjugate, the first XTEN
and the second XTEN are the same are each has at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, or 100% sequence identity to a sequence selected
from the group of sequences consisting of Seg 174, Seg 175, Seg
176, Seg 177, Seg 186, Seg 187. Seg 188, Seg 189, Seg 190, Seg 191.
Seg 192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and
Seg 199 set forth in Table 3. In another embodiment of the dimeric
XTEN conjugate, the first and the second XTEN each comprises one or
more cysteine residues, and further comprises a first cross-linker
conjugated to each cysteine residue of the first XTEN and a second
cross-linker conjugated to each cysteine residue of the second
XTEN, wherein the first and the second cross-linkers are
independently selected from the group consisting of the
cross-linkers set forth in Table 13. In another embodiment of the
dimeric XTEN conjugate, the first and the second XTEN each
comprises one or more lysine residues, and further comprises a
cross-linker conjugated to each lysine residue of the first and the
second XTEN of the conjugate, wherein the cross-linker is selected
from the group consisting of the cross-linkers set forth in Table
13. In another embodiment of the dimeric XTEN conjugated to
cross-linkers, the conjugate further comprises a single atom
residue of a first payload conjugated to each cross-linker of the
first XTEN wherein the residue is selected from the group
consisting of carbon, nitrogen, oxygen and sulfur, and further
comprises a single atom residue of a second payload conjugated to
each cross-linker of the second XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur. In the foregoing embodiment, the first payload of the
single atom residue can be selected from the group consisting of
the payloads set forth in Tables 11, 12, 18, and 21, and the second
payload of the single atom residue is a different payload from the
first payload and can be selected from the group consisting of the
payloads set forth in Tables 11, 12, 18, and 21. In some
embodiments of the dimeric XTEN-cross-linker-payload residue
composition, the composition has the configuration of formula X
##STR00005##
wherein independently for each occurrence P.sub.R1 is a single atom
residue of a first payload wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur; P.sub.R2
is a single atom residue of a second payload wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; CL.sub.1 is a cross-linker; x is an integer from 1 to about
100, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to
about 10, or 1 to about 5, or is 9, or is 3, or is 2, or is 1;
CL.sub.2 is a cross-linker that is different from CL.sub.1; y is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1, with the proviso that x+y is .gtoreq.2;
2.times.CL is alternatively a divalent cross-linker or the reaction
product of a first and a second click chemistry reactant selected
from Table 15; XTEN.sub.1 is a polypeptide having at least 80%, or
at least about 90%, or at least about 91%, or at least about 92%,
or at least about 93%, or at least about 94%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%, or having 100% sequence identity
to a sequence selected from the group of sequences set forth in
Tables 2 and 3; and XTEN.sub.2 is a polypeptide having at least
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3. In one embodiment of the conjugate of
formula X, CL.sub.1 and CL.sub.2 are each selected from the group
of cross-linkers set forth in Table 13. In another embodiment of
the conjugate of formula X, x is 1 and CL.sub.1 is linked to the
N-terminal amino group of the XTEN. In another embodiment of the
conjugate of formula X, CL.sub.1 is the reaction product of a first
and a second click chemistry reactant selected from Table 15. In
another embodiment of the conjugate of formula X, C.sub.2 is the
reaction product of a first azide and a second alkyne click
chemistry reactant selected from Table 15. In another embodiment of
the conjugate of formula X, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN.sub.1 and each CL.sub.2 is linked to a cysteine
sulfur of XTEN.sub.2. In another embodiment of the conjugate of
formula X, each CL.sub.1 is linked to a lysine epsilon amino group
of the XTEN.sub.1 and each CL.sub.2 is linked to a lysine epsilon
amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula X, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN.sub.1 and each CL.sub.2 is linked to a lysine
epsilon amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula X, XTEN.sub.1 and XTEN.sub.2 are identical. In
another embodiment of the conjugate of formula X, XTEN.sub.1 and
XTEN.sub.2 are different. In another embodiment, the invention
provides a preparation of the conjugate of formula X in which at
least about 80%, or at least about 90%, or at least about 91%, or
at least about 92%, or at least about 93%, or at least about 94%,
or at least about 95% of the XTEN molecules of the preparation of
the conjugate have identical sequence length. In another embodiment
of the dimeric XTEN conjugated to cross-linkers, the composition
further comprises a first payload conjugated to each cross-linker
of the first XTEN wherein the first payload is selected from the
group consisting of the payloads set forth in Tables 11, 12, 18,
and 21, and further comprises a second payload different from the
first payload wherein the second payload is conjugated to each
cross-linker of the second XEN wherein the second payload is
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18, and 21. In another embodiment of the dimeric
XTEN conjugated to cross-linkers, the composition further comprises
a first payload conjugated to each cross-linker of the first XTEN
wherein the first payload is selected from the group consisting of
the targeting moieties set forth in Table 18 or Table 21, and
further comprises a second payload different from the first payload
wherein the second payload is conjugated to each cross-linker of
the second XTEN wherein the second payload is selected from the
group of toxins set forth in Table 18 or Table 21. In another
embodiment of the dimeric XTEN conjugated to cross-linkers and a
first and a second payload, the first XTEN is Seg 176 set forth in
Table 3 and the second XTEN is selected from the group consisting
of Seg 176 and Seg 177 set forth in Table 3. In some embodiments of
the dimeric XTEN-cross-linker-payload composition, the composition
has the configuration of formula XI
##STR00006##
wherein independently for each occurrence P.sub.1 is a first
payload selected from the group of payloads set forth in Tables 11,
12, 18, 19, and 21; P.sub.2 is a second payload selected from the
group of payloads set forth in Tables 11, 12, 18, 19, and 21 and
that is different from P.sub.1; CL.sub.1 is a cross-linker; x is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1; CL.sub.2 is a cross-linker that is different from
CL.sub.1; y is an integer from 1 to about 100, or 1 to about 50, or
1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about 5,
or is 9, or is 3, or is 2, or is 1, with the proviso that x+y is
.gtoreq.2; 2.times.CL is alternatively a divalent cross-linker or
the reaction product of a first and a second click chemistry
reactant selected from Table 15; XTEN.sub.1 is a first
substantially homogeneous XTEN having at least 80%, or at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99%, or having 100% sequence identity to a
sequence selected from the group of sequences set forth in Tables 2
and 3; and XTEN.sub.2 is a first substantially homogeneous having
at least 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or having 100%
sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3. In one embodiment of the
conjugate of formula XI, CL.sub.1 and CL.sub.2 are each selected
from the group of cross-linkers set forth in Table 13. In another
embodiment of the conjugate of formula XI, x is 1 and CL.sub.1 is
linked to the N-terminal amino group of the XTEN. In another
embodiment of the conjugate of formula XI, CL.sub.1 is the reaction
product of a first and a second click chemistry reactant selected
from Table 15. In another embodiment of the conjugate of formula
XI, C.sub.2 is the reaction product of a first and a second click
chemistry reactant selected from Table 15. In another embodiment of
the conjugate of formula XI, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN.sub.1 and each CL.sub.2 is linked to a cysteine
sulfur of XTEN.sub.2. In another embodiment of the conjugate of
formula XI, each CL.sub.1 is linked to a lysine epsilon amino group
of the XTEN.sub.1 and each CL.sub.2 is linked to a lysine epsilon
amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula XI, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN.sub.1 and each CL.sub.2 is linked to a lysine
epsilon amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula XI, XTEN.sub.1 and XTEN.sub.2 are identical.
In another embodiment of the conjugate of formula XI, XTEN.sub.1
and XTEN.sub.2 are different. In one embodiment, the conjugate of
formula XI is selected from the group consisting of the conjugates
set forth in Table 21. In another embodiment, the invention
provides a preparation of the conjugate of formula XI in which at
least about 80%, or at least about 90%, or at least about 91%, or
at least about 92%, or at least about 93%, or at least about 94%,
or at least about 95% of the respective XTEN.sub.1 and XTEN.sub.2
molecules of the preparation of the conjugate have identical
sequence length.
[0020] In another aspect, the invention relates, in part, to
compositions of a first and a second and a third XTEN conjugated to
each other, resulting in trimeric conjugate compositions. In some
embodiments, the conjugate compositions comprise a first and a
second and a third XTEN wherein the XTEN may be the same or they
may be different, and in which the first and the second and the
third XTEN are conjugated to each other by the N-terminus using a
trivalent cross-linker selected from the group consisting of the
trivalent cross-linkers set for in Table 13 or Table 14. In one
embodiment of the trimeric conjugate, the first and the second and
the third XTEN are identical or are different and each has at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99%, or 100% sequence identity to a sequence
selected from the group of sequences set forth in either Table 2 or
Table 3. In another embodiment of the trimeric conjugate, the first
and the second and the third XTEN are identical or are different
and at least 90%, 91%, 92%, 93%, 94%, or 95% of the individual
molecules of each of the first XTEN have identical sequence length
and at least 90%, 91%, 92%, 93%, 94%, or 95% of the individual
molecules of each of the second XTEN have identical sequence length
and at least 90%, 91%, 92%, 93%, 94%, or 95% of the individual
molecules of each of the third XTEN have identical sequence length.
In another embodiment of the trimeric conjugate the trivalent
cross-linker is selected from the group consisting of
Tris-(2-Maleimidoethyl)amine (TMEA) and amine-reactive
Tris-(succimimidyl aminotricetate) (TSAT). In another embodiment of
the trimeric conjugate, the first and the second and the third XTEN
are identical and each has at least about 90%, or at least about
91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%, or
100% sequence identity to a sequence selected from the group
consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187,
Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg
195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table 3.
In another embodiment of the trimeric conjugate, the first and the
second and the third XTEN are identical and each has at least about
90%, or at least about 91%, or at least about 92%, or at least
about 93%, or at least about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or
at least about 99%, or 100% sequence identity to a sequence
selected from the group consisting of Seg 174, Seg 175, Seg 176.
Seg 177, Seg 186, Seg 187, Seg 188, Seg 189. Seg 190, Seg 191, Seg
192, Seg 193, Seg 194. Seg 195. Seg 196, Seg 197, Seg 198, and Seg
199 set forth in Table 3, and the third XTEN is different from the
first and the second XTEN and has at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%4,
or 100% sequence identity to a sequence selected from the group
consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186. Seg 187,
Seg 188, Seg 189, Seg 190, Seg 191. Seg 192, Seg 193, Seg 194, Seg
195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table 3.
In another embodiment of the trimeric conjugate, each XTEN
comprises at least a first cysteine residue and the conjugate
further comprises a first cross-linker conjugated to each cysteine
residue of the first XTEN, a second cross-linker conjugated to each
cysteine residue of the second XTEN, and a third cross-linker
conjugated to each cysteine residue of the third XTEN, wherein the
cross-linker is selected from the group consisting of the
cross-linkers set forth in Table 13. In some embodiments of the
trimeric conjugate, the composition has the configuration of
formula XII:
##STR00007##
wherein independently for each occurrence; 3.times.CL is the
trivalent cross-linker, CL1 is the first cross-linker conjugated to
XTEN.sub.1; CL2 is the second cross-linker conjugated to
XTEN.sub.2; CL3 is the third cross-linker conjugated to XTEN.sub.3;
x is an integer of 1 to about 10; y is an integer of 1 to about 10;
z is an integer of 1 to about 10 with the proviso that x+y+z is
.gtoreq.3; XTEN.sub.1 is the first XTEN; XTEN.sub.2 is the second
XTEN; and XTEN.sub.3 is the third XTEN. In another embodiment of
the trimeric conjugate, the conjugate further comprises a single
atom residue of a first payload conjugated to each first
cross-linker of the first XTEN wherein the residue is selected from
the group consisting of carbon, nitrogen, oxygen and sulfur; a
single atom residue of a second payload conjugated to each second
cross-linker of the second XTEN wherein the residue is selected
from the group consisting of carbon, nitrogen, oxygen and sulfur;
and a single atom residue of a third payload conjugated to each
third cross-linker of the third XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxy gen and
sulfur. In another embodiment of the trimeric conjugate
composition, the composition further comprises a first payload
conjugated to each first cross-linker of the first XTEN selected
from the group consisting of the payloads set forth in Tables 11,
12, 18 and 21; a second payload conjugated to each second
cross-linker of the second XTEN selected from the group consisting
of the payloads set forth in Tables 11, 12, 18 and 21, wherein the
payload is the same or is different from the first payload; and a
third payload conjugated to each third cross-linker of the third
XTEN selected from the group consisting of the payloads set forth
in Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first or the second payload. In one embodiment
of the trimeric XTEN-payload conjugate composition, the first
payload is a targeting moiety with specific binding affinity to a
target, wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and the second and the third payloads are a drug, which may be
the same or may be different and wherein the drug is selected from
the group consisting of the drugs set forth in Table 11, Table 18,
and Table 21. In one embodiment of the trimeric XTEN-payload
conjugate composition wherein the first payload is a targeting
moiety with specific binding affinity to a target and the second
payload and the third payload is a drug, the targeting moiety is
selected from the group consisting of LHRH and folate and the drug
is selected from the group consisting of doxorubicin, paclitaxel,
auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin
F, maytansine, dolastatin, calicheamicin, vinca alkaloid,
camptothecin, mitomycin C, epothilone, hTNF, Il-12, bortezomib,
ranpirnase, pseudomonas exotoxin, SN-38, and rachelmycin. In one
embodiment of the trimeric XTEN-payload conjugate composition
wherein the first payload is a targeting moiety with specific
binding affinity to a target and the second payload and the third
payload is a drug, the targeting moiety, and the drug moiety
correspond to any one of conjugates 1-290 set forth in Table 21. In
another embodiment of the trimeric XTEN-payload conjugate
composition wherein the first payload is a targeting moiety with
specific binding affinity to a target and the second payload and
the third payload is a drug, the conjugate has the XTEN, the
targeting moiety, and the drug moiety corresponding to conjugate 71
of Table 21. In another embodiment of the trimeric XTEN-payload
conjugate composition, the composition has the configuration of
formula XIII
##STR00008##
wherein independently or each occurrence 3.times.CL is the
trivalent cross-linker is selected from the group of trivalent
cross-linkers set forth in Tables 13 and 14; P.sub.1 is conjugated
to each cross-linker of the first XTEN and is selected from the
group consisting of the payloads set forth in Tables 11, 12, 18 and
21, P2 is a second payload conjugated to each cross-linker of the
second XTEN and is selected from the group consisting of the
payloads set forth in Tables 11, 12, 18 and 21, wherein the payload
is the same or is different from the first payload, and P.sub.3 is
a third payload conjugated to each cross-linker of the third XTEN
and is selected from the group consisting of the payloads set forth
in Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first or the second payload; CL.sub.1 is the
first cross-linker, x is an integer from 1 to about 100, or 1 to
about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or
1 to about 5, or is 9, or is 3, or is 2, or is 1; CL2 is a second
cross-linker, y is an integer from 1 to about 100, or 1 to about
50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to
about 5, or is 9, or is 3, or is 2, or is 1; and z is an integer
from 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to
about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3, or
is 2, or is 1, with the proviso that x+y+z is .gtoreq.3; XTEN.sub.1
is the first XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3; XTEN.sub.2 is
the second XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3; and XTEN.sub.3
is the third XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3 wherein
XTEN.sub.1, XTEN2, and XTEN.sub.3 are the same or are different
XTEN sequences. In some embodiments, the conjugate of formula XIII
further comprises a first payload wherein the payload is a
targeting moiety with specific binding affinity to a target,
wherein the targeting moiety is selected from the group consisting
of the targeting moieties set forth in Tables 17-19 and 21, and at
least one other of the payloads is a drug wherein the drug is
selected from the group consisting of the drugs set forth in Table
11, Table 19, and Table 21. In one embodiment of the foregoing, the
targeting moiety is LHRH or folate and the drug is selected from
doxorubicin, paclitaxel, auristatin, monomethyl auristatin E
(MMAE), monomethyl auristatin F, maytansine, dolastatin,
calicheamicin, vinca alkaloid, camptothecin, mitomycin C,
epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin. In another embodiment of the
trimeric XTEN conjugate composition, the composition has the
configuration of formula XIV:
##STR00009##
wherein independently for each occurrence; 3.times.CL is the
trivalent cross-linker, CL1 is the first cross-linker conjugated to
XTEN.sub.1; CL2 is the second cross-linker conjugated to
XTEN.sub.2; x is an integer of 1 to about 10; y is an integer of 1
to about 10 with the proviso that x+y is .gtoreq.2; XTEN.sub.1 is
the first XTEN; XTEN.sub.2 is the second XTEN; and XTEN.sub.3 is
the third XTEN wherein the XTEN is selected from the group
consisting of the sequences set forth in Table 2. In one embodiment
of the trimeric XTEN conjugate composition of formula XVI, the
composition further comprises a single atom residue of a first
payload conjugated to each first cross-linker of the first XTEN
wherein the residue is selected from the group consisting of
carbon, nitrogen, oxygen and sulfur; and a single atom residue of a
second payload conjugated to each second cross-linker of the second
XTEN wherein the residue is selected from the group consisting of
carbon, nitrogen, oxygen and sulfur. In another embodiment of the
trimeric XTEN conjugate composition of formula XVI, the composition
further comprises a first payload conjugated to each first
cross-linker of the first XTEN selected from the group consisting
of the payloads set forth in Tables 11, 12, 18 and 21; and a second
payload conjugated to each second cross-linker of the second XTEN
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first payload. In one embodiment of the
foregoing, the first payload is a targeting moiety with specific
binding affinity to a target, wherein the targeting moiety is
selected from the group consisting of the targeting moieties set
forth in Tables 17-19 and 21, and the second payloads is a drug
selected from the group consisting of the drugs set forth in Table
6, Table 18, and Table 21. In another embodiment of the foregoing,
the first payload is a targeting moiety is selected from the group
consisting of LHRH and folate, and the second payload is a drug is
selected from the group consisting of doxorubicin, paclitaxel,
auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin
F, maytansine, dolastatin, calicheamicin, vinca alkaloid,
camptothecin, mitomycin C, epothilone, hTNF, Il-12, bortezomib,
ranpirnase, pseudomonas exotoxin, SN-38, and rachelmycin. In one
embodiment of the foregoing, the first payload is a drug selected
from the group consisting of the drugs of Table 11 and the proteins
of Table 12 and the second payload is different from the first
payload and is selected from the group consisting of the drugs of
Table 11 and the proteins of Table 12. In another embodiment of the
foregoing, the first payload and the second payload are identical
and are selected from the group consisting of the drugs of Table 11
and the proteins of Table 12. In another embodiment of the trimeric
XTEN conjugate composition, the composition has the configuration
of formula XV:
##STR00010##
wherein independently for each occurrence; 3.times.CL is a
trivalent cross-linker linking XTEN.sub.1, XTEN.sub.2, XTEN.sub.3;
CL1 is the first cross-linker conjugated to XTEN.sub.1; x is an
integer of 1 to about 10; XTEN.sub.1 is the first XTEN wherein the
XTEN is selected from the group consisting of the sequences set
forth in Table 3; XTEN.sub.2 is the second XTEN wherein the XTEN is
selected from the group consisting of the sequences set forth in
Table 2; and XTEN.sub.3 is the third XTEN wherein the XTEN is
selected from the group consisting of the sequences set forth in
Table 2. In one embodiment of the trimeric XTEN conjugate
composition configured as formula XVII, the composition further
comprises a single atom residue of a first payload conjugated to
each first cross-linker of the first XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur. In one embodiment of the trimeric XTEN conjugate
composition configured as formula XVII, the composition further
comprises a first payload conjugated to each first cross-linker of
the first XTEN selected from the group consisting of the payloads
set forth in Tables 11, 12, 18 and 21.
[0021] In another aspect, the invention relates, in part, to
compositions of a first, a second, a third and a fourth XTEN
conjugated to each other, resulting in tetrameric conjugate
compositions. In some embodiments, the conjugate compositions
comprise a first and a second and a third and a fourth XTEN wherein
the XTEN are selected from the group consisting of the sequences
set forth in Table 3, wherein the XTEN may be the same or they may
be different, and in which the first and the second and the third
and the fourth XTEN are conjugated to each other by the N-terminus
using a tretravalent cross-linker wherein the tetravalent
cross-linker is a tetravalent maleimide cluster. In one embodiment
of the tetrameric conjugate, the first and the second and the third
and the fourth XTEN are identical or are different and each has at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or 100% sequence identity to a sequence
selected from the group of sequences set forth in either Table 2 or
Table 3. In another embodiment of the tetrameric conjugate, the
first and the second and the third XTEN are identical or are
different and at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the first XTEN have identical
sequence length and at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the second XTEN have identical
sequence length and at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the third XTEN have identical
sequence length and at least 90%, 91%, 92%, 93%, 94%, or 95% of the
individual molecules of each of the fourth XTEN have identical
sequence length. In another embodiment of the tetrameric conjugate
the first, the second, the third, and the fourth XTEN are the same
and each has at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or 100% sequence
identity to a sequence selected from the group consisting of Seg
174, Seg 175. Seg 176, Seg 177, Seg 186, Seg 187, Seg 188. Seg 189,
Seg 190. Seg 191, Seg 192, Seg 193, Seg 194, Seg 195. Seg 196, Seg
197, Seg 198, and Seg 199 set forth in Table 3. In another
embodiment of the tetrameric conjugate, the first and the second
XTEN are the same and each has at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%,
or 100% sequence identity to a sequence selected from the group
consisting of Seg 174, Seg 175, Seg 176. Seg 177. Seg 186, Seg 187,
Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg
195, Seg 196, Seg 197. Seg 198, and Seg 199 set forth in Table 3,
and the third and the fourth XTEN are the same but are different
from the first and the second XTEN and each has at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, or 100% sequence identity to a sequence selected
from the group consisting of Seg 174, Seg 175, Seg 176. Seg 177,
Seg 186, Seg 187, Seg 188, Seg 189. Seg 190, Seg 191, Seg 192, Seg
193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set
forth in Table 3. In another embodiment of the tetrameric
conjugate, each XTEN comprises at least a first cysteine residue
and the conjugate further comprises a first cross-linker conjugated
to each cysteine residue of the first XTEN, a second cross-linker
conjugated to each cysteine residue of the second XTEN, a third
cross-linker conjugated to each cysteine residue of the third XTEN,
and a fourth cross-linker conjugated to each cysteine residue of
the fourth XTEN, wherein each cross-linker is selected from the
group consisting of the cross-linkers set forth in Table 13. In
some embodiments of the tetrameric conjugate compositions, the
composition has the configuration of formula XVI
##STR00011##
wherein independently for each occurrence: 4.times.CL is the
tetravalent cross-linker, CL1 is the first cross-linker conjugated
to XTEN.sub.1; CL2 is the second cross-linker conjugated to
XTEN.sub.2; CL3 is the third cross-linker conjugated to XTEN.sub.3;
CL4 is the fourth cross-linker conjugated to XTEN.sub.4; v is an
integer of 1 to about 10; x is an integer of 1 to about 10; y is an
integer of 1 to about 10; z is an integer of 1 to about 10 with the
proviso that x+y+z is .gtoreq.4; XTEN.sub.1 is the first XTEN;
XTEN.sub.2 is the second XTEN; XTEN.sub.3 is the third XTEN; and
XTEN.sub.3 is the fourth XTEN. In another embodiment of the
tetrameric conjugate composition, the composition further comprises
a single atom residue of a first payload conjugated to each first
cross-linker of the first XTEN wherein the residue is selected from
the group consisting of carbon, nitrogen, oxygen and sulfur; a
single atom residue of a second payload conjugated to each second
cross-linker of the second XTEN wherein the residue is selected
from the group consisting of carbon, nitrogen, oxygen and sulfur; a
single atom residue of a third payload conjugated to each third
cross-linker of the third XTEN wherein the residue is selected from
the group consisting of carbon, nitrogen, oxygen and sulfur; and a
single atom residue of a fourth payload conjugated to each fourth
cross-linker of the fourth XTEN wherein the residue is selected
from the group consisting of carbon, nitrogen, oxygen and sulfur.
In another embodiment of the tetrameric conjugate composition, the
composition further comprises a first payload conjugated to each
first cross-linker of the first XTEN selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, and 21;
a second payload conjugated to each second cross-linker of the
second XTEN selected from the group consisting of the payloads set
forth in Tables 11, 12, 18, and 21, wherein the payload is the same
or is different from the first payload; a third payload conjugated
to each third cross-linker of the third XTEN selected from the
group consisting of the payloads set forth in Tables 11, 12, 18,
and 21, wherein the payload is the same or is different from the
first or the second payload; and a fourth payload conjugated to
each fourth cross-linker of the fourth XTEN selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, and 21,
wherein the payload is the same or is different from the first or
the second or the third payload. In one embodiment of the
tetrameric XTEN-payload conjugate composition, the first payload is
a targeting moiety with specific binding affinity to a target
wherein the targeting moiety is selected from the group consisting
of the targeting moieties set forth in Tables 17-19 and 21, and at
least one other of the second, third, and fourth payloads is a drug
wherein the drug is selected from the group consisting of the drugs
set forth in Tables 11, 18 and 21. In one embodiment of the
tetrameric XTEN-payload conjugate composition, the first payload is
a targeting moiety wherein the targeting moiety is selected from
the group consisting of LHRH and folate, and at least one of the
second, third and fourth payload is a drug selected from the group
consisting of doxorubicin, paclitaxel, auristatin, maytansine,
dolastatin, calicheamicin, vinca alkaloid, camptothecin, mitomycin
C, epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin. In another embodiment of the
tetrameric XTEN-payload conjugate composition, the first payload is
a targeting moiety with specific binding affinity to a target
wherein the targeting moiety is selected from the group consisting
of the targeting moieties set forth in Tables 17-19 and 21, and at
least one other of the second, third, and fourth payloads is a drug
wherein the drug is selected from the group consisting of the drugs
set forth in Tables 11, 18 and 21, and wherein the XTEN, the
targeting moiety, and the drug moiety correspond to any one of
conjugates 1-290 set forth in Table 21.
[0022] In another aspect, the invention relates, in part, to
compositions comprising multimeric XTEN molecules configured in a
branched manner, wherein a solution of the composition has a
reduced. In one embodiment, the invention provides a composition
comprising a solution that comprises a multimeric XTEN having at
least three XTEN fragments linked together in a branched manner
(e.g. trimeric manner) wherein the viscosity of the solution is
reduced by at least 5, 6, 7, 8, 9 or 10 cP in a solution containing
.quadrature.100, 130, or 150 mg/ml of the trimeric XTEN preparation
compared to a solution containing .quadrature.100, 130, or 150
mg/ml of the corresponding linear XTEN of equal molar
concentration. In another embodiment, the invention provides a
composition comprising a solution that comprises a multimeric XTEN
having at least four XTEN fragments linked together in a branched
manner (e.g. tetrameric manner) wherein the composition has a
viscosity that is less than a solution comprising a corresponding
linear XTEN having the same number of amino acids and the same
molar concentration, wherein the viscosity of the solution is
reduced by at least 5, 6, 7, 8, 9 or 10 cP in a solution containing
.quadrature.100, 130, or 150 mg/ml of the trimeric XTEN preparation
compared to a solution containing .quadrature.100, 130, or 150
mg/ml of the corresponding linear XTEN of equal molar
concentration. In another embodiment, the invention provides a
composition comprising a solution that comprises a multimeric XTEN
having at least five XTEN fragments linked together in a branched
manner (e.g. pentameric manner) wherein the composition has a
viscosity that is less than a solution comprising a corresponding
linear XTEN having the same number of amino acids and the same
molar concentration, wherein the viscosity of the solution is
reduced by at least 5, 6, 7, 8, 9 or 10 cP in a solution containing
.quadrature.100, 130, or 150 mg/ml of the trimeric XTEN preparation
compared to a solution containing .quadrature.100, 130, or 150
mg/ml of the corresponding linear XTEN of equal molar
concentration. In the foregoing embodiments of this paragraph, the
individual XTEN of the multimeric configurations are selected from
the group consisting of the sequences set forth in Table 2 and
Table 3.
[0023] In another embodiment, the invention provides compositions
of a polypeptide having at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98%, or at least about 99%, or 100%
sequence identity to a sequence selected from the group of
sequences set forth in Table 52.
[0024] In another embodiment, the invention provides a
pharmaceutical composition, comprising the conjugate of any one of
the XTEN-payload conjugate embodiments described herein, and a
pharmaceutically acceptable carrier. In one embodiment, the
foregoing pharmaceutical composition has utility in the treatment
of a condition selected from the group of conditions set forth in
Table 16. In another embodiment, the foregoing pharmaceutical
composition has utility for use in a pharmaceutical regimen for
treatment of a subject, said regimen comprising the pharmaceutical
composition. In another embodiment, the foregoing pharmaceutical
regimen further comprises the step of determining the amount of
pharmaceutical composition needed to achieve a beneficial effect in
a subject having a condition selected from the group of conditions
set forth in Table 16. In another embodiment, the foregoing
pharmaceutical regimen used for treating the subject comprises
administering the pharmaceutical composition in two or more
successive doses to the subject at an effective amount, wherein the
administration results in at least a 10%, or 20%, or 30%, or 40%,
or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at
least one, two, or three parameters associated with the condition
compared to an untreated subject.
[0025] In another embodiment, the invention provides a conjugate of
any one of the XTEN-payload conjugate embodiments described herein
for use in the preparation of a medicament for treatment of a
condition selected from the group of conditions set forth in Table
16.
[0026] In some embodiments, the invention provides methods of
selecting a combination of payloads linked to XTEN as a therapeutic
agent, the method comprising providing a library of XTENs
comprising a plurality of XTEN sequences wherein each of said XTEN
sequences is conjugated to at least a first payload and at least a
second payload which is different from the first payload; from said
library, selecting an XTEN sequence as the therapeutic agent if it
exhibits an improved in vitro or in vivo parameter as compared to
that of (1) an XTEN sequence conjugated to the first payload alone;
and (2) an XTEN sequence conjugated to the second payload alone. In
one embodiment of the method, the first payload and second payload
are therapeutically effective for ameliorating a common disease
(e.g. a disease to which both the first and second payload
targets). In one embodiment of the method, the first drug and
second drug are therapeutically effective for treating different
symptoms of a common disease. In one embodiment of the method, the
common disease is selected from cancer, cancer supportive care,
cardiovascular, central nervous system, endocrine disease,
gastrointestinal, genitourinary, hematological, HIV infection,
hormonal disease, inflammation, autoimmune disease, infectious
disease, metabolic disease, musculoskeletal disease, nephrology
disorders, ophthalmologic diseases, pain, and respiratory. In one
embodiment of the method, the first payload and second payload
mediate their therapeutic effect via a common biological pathway.
In one embodiment of the method, the first payload and second
payload are different drugs selected from the group consisting of
the drugs set forth in Table 11, Table 18 and Table 21. In one
embodiment of the method, the first payload and second payload are
different biologically active proteins selected from the group
consisting of the proteins set forth in Table 12, Table 18 and
Table 21. In one embodiment of the method, the first payload is a
drug selected from the group consisting of the drugs set forth in
Table 11, Table 18 and Table 21 and the second payload is a
biologically active protein selected from the group consisting of
the proteins set forth in Table 12, Table 18 and Table 21.
[0027] In another embodiment, the invention provides an isolated
polypeptide comprising an extended recombinant polypeptide that is
linked to an affinity purification tag via a proteolytic cleavage
site having a sequence selected from SASRSA (SEQ ID NO: 21) or
SASXSA (SEQ ID NO: 23) where X is R or K.
[0028] In another embodiment, the invention provides an isolated
polypeptide comprising a polypeptide comprising an XTEN that is
linked at its N-terminus to a first affinity purification tag via a
proteolytic cleavage site having a sequence selected from SASRSA
(SEQ ID NO: 21) or SASXSA (SEQ ID NO: 23) where X is R or K, and at
its C-terminus to a second affinity purification tag via a
proteolytic cleavage site having a sequence selected from SASRSA
(SEQ ID NO: 21) or SASXSA (SEQ ID NO: 23) where X is R or K.
[0029] In another aspect, the invention relates to a method of
treating a condition in a subject with an XTEN-payload conjugate
composition. In one embodiment, the invention provides a method of
treating a condition in a subject comprising administering an
effective amount of the conjugate of any one of the XTEN-payload
embodiments described herein to a subject in need thereof. In
another embodiment, the invention provides a method of treating a
condition in a subject comprising administering an effective amount
of the conjugate of the group consisting of the conjugates set
forth in Table 21 to a subject in need thereof. In the foregoing
embodiments of this paragraph, the condition to be treated
includes, but is not limited to, the conditions set forth in Table
13. In another embodiment, the invention provides a pharmaceutical
composition comprising any of the XTEN-payload conjugate
embodiments described herein and a pharmaceutically acceptable
carrier for use in a treatment regimen, the regimen comprising
administering two or more consecutive doses of the pharmaceutical
composition.
[0030] In one embodiment, the invention provides the use of a
conjugate of any one of the XTEN-payload embodiments described
herein for the preparation of a medicament for treatment of a
condition selected from the group of conditions set forth in Table
16. In another embodiment, the invention provides a pharmaceutical
composition for treatment of a condition selected from the group of
conditions set forth in Table 16. comprising an effective amount of
a conjugate of any one of the XTEN-payload embodiments described
herein.
[0031] In another embodiment, the invention provides a composition
having the structure set forth in FIG. 117.
[0032] It is specifically contemplated that the conjugate
embodiments can exhibit one or more or any combination of the
properties disclosed herein. In addition, any of the XTEN
compositions disclosed herein can be utilized in any of the methods
disclosed herein.
INCORPORATION BY REFERENCE
[0033] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The features and advantages of the invention may be further
explained by reference to the following detailed description and
accompanying drawings that sets forth illustrative embodiments
[0035] FIGS. 1A-IE show schematics of XTEN suitable for conjugation
with payloads. FIG. 1A shows unmodified XTEN. FIG. 1B shows a
cysteine-engineered XTEN with an internal cysteine with a thiol
side chain; below is an XTEN with an a reactive N-terminal amino
group; below is an XTEN with an N-terminal cysteine with a thiol
reactive group. FIG. 1C shows cysteine-engineered XTEN with
multiple internal cysteines. FIG. 1D shows two variations of a
cysteine-engineered XTEN with an internal cysteine with a thiol
side chain and a reactive N-terminal amino group and, at the
bottom, a shows a cysteine- and lysine-engineered XTEN with
internal cysteines and internal lysines. FIG. 1E is a schematic of
another embodiment.
[0036] FIG. 2 shows a conjugation reaction utilizing NHS-esters and
their water soluble analogs sulfo-NHS-esters) reacting with a
primary amino group to yield a stable amide XTEN-payload
product.
[0037] FIG. 3 shows a conjugation reaction utilizing thiol groups
and an N-maleimide. The maleimide group reacts specifically with
sulfhydryl groups when the pH of the reaction mixture is between pH
6.5 and 7.5, forming a stable thioether linkage that is not
reversible.
[0038] FIG. 4 shows a conjugation reaction utilizing haloacetyls.
The most commonly used haloacetyl reagents contain an iodoacetyl
group that reacts with sulfhydryl groups at physiological pH. The
reaction of the iodoacetyl group with a sulfhydryl proceeds by
nucleophilic substitution of iodine with a thiol producing a stable
thioether linkage in the XTEN-payload.
[0039] FIG. 5 shows a conjugation reaction utilizing pyridyl
disulfides. Pyridyl disulfides react with sulfhydryl groups over a
broad pH range (the optimal pH is 4-5) to form disulfide bonds
linking XTEN to payloads.
[0040] FIGS. 6A-6B show a conjugation reaction utilizing
zero-length cross-linkers wherein the cross-linkers are used to
directly conjugate carboxyl functional groups of one molecule (such
as a payload) to the primary amine of another molecule (such as an
XTEN).
[0041] FIG. 7 shows different configurations of XTEN precursors
that are multifunctional (or multivalent), including dedrimers.
Non-limiting examples of trifunctional linkers are "Y-shaped"
sulfhydryl-reactive TMEA (Tris-(2-Maleimidoethyl)amine) and
amine-reactive TSAT (Tris-(succimimidyl aminotricetate). Any
combination of reactive moieties can be designed using a scaffold
polymer, either linear (forming a "comb" configuration) or branched
(forming a "dendrimer" configuration), for multivalent display.
[0042] FIG. 8 shows a conjugation reaction utilizing the Huisgen
1,3-dipolar cycloaddition of alkynes to azides to form
1,4-disubsituted-1,2,3-triazoles, as shown.
[0043] FIG. 9 shows a conjugation reaction using thio-ene based
click chemistry that may proceed by free radical reaction, termed
thiol-ene reaction, or anionic reaction, termed thiol Michael
addition.
[0044] FIG. 10 shows a conjugation reaction utilizing click
chemistry based on reactions between hydrazides and aldehydes,
resulting in the illustrated hydrazone linkage in the
XTEN-payload.
[0045] FIG. 11 shows a reaction between a C-terminal acylazide and
a primary amino group resulting in the formation of an amide
bond.
[0046] FIG. 12 shows a conjugation reaction utilizing Native
Chemical Ligation (NCL) involving a C-terminal thioester as an
electrophile and N-terminal Cysteine as a nucleophile. The result
of this reaction is a native amide bond at the ligation site of the
XTEN-payload composition.
[0047] FIG. 13 shows a conjugation reaction utilizing expressed
protein ligation (EPL) methodology. The EPL method is based on
protein splicing, the process in which a protein undergoes an
intramolecular rearrangement resulting in the extrusion of an
internal sequence (intein) and the joining of the lateral sequences
(exteins). In the method, the fused protein undergoes an N-S shift
when the side chain of the first cysteine residue of the intein
portion of the precursor protein nucleophilically attacks the
peptide bond of the residue immediately upstream (that is, for
example, the final residue of XTEN) to form a linear thioester
intermediate, followed by a rearrangement to form to form an amide
bond between the XTEN-cross-linker and the payload.
[0048] FIG. 14 shows a conjugation reaction utilizing traceless
Staudinger ligation, like Native Chemical Ligation (NCL), resulting
in a native amide bond at the ligation site
[0049] FIG. 15 shows a conjugation reaction utilizing enzymatic
ligation. Transglutaminases are enzymes that catalyze the formation
of an isopeptide bond between the .gamma.-carboxamide group of
glutamine of a payload peptide or protein and the .epsilon.-amino
group of a lysine in a lysine-engineered XTEN (or an N-terminal
amino group), thereby creating inter- or intramolecular cross-links
between the XTEN and payload.
[0050] FIG. 16 shows enzymatically-created XTEN-payload
compositions utilizing the sortase A transpeptidase enzyme from
Staphylococcus aureus to catalyze the cleavage of a short 5-amino
acid recognition sequence LPXTG (SEQ ID NO: 24) between the
threonine and glycine residues of Protein1 that subsequently
transfers the acyl-fragment to an N-terminal oligoglycine
nucleophile of Protein1. By functionalizing the Protein2 to contain
an oligoglycine, the enzymatic conjugation of the two proteins is
accomplished in a site-specific fashion to result in the desired
XTEN-payload composition. FIG. 16 discloses SEQ ID NOS 1175-1176,
respectively, in order of appearance.
[0051] FIGS. 17A-17B show various XTEN-cross-linker precursor
segments that are used as reactants to link to payloads or to other
XTEN reactants. FIG. 17A is intended to show that the 1B represents
the remaining reactive group of the precursors on the right. FIG.
17B shows similar reactive precurors with either multiple (left) or
single (right) payload A molecules conjugated to the XTEN.
[0052] FIG. 18 shows exemplary permutations of XTEN-cross-linker
precursor segments with two reactive groups of cross-linkers or
reactive groups of an incorporated amino acid that are used as
reactants to link to payloads or to other XTEN reactants. The 1B
and 2B represent reactive groups that will, in other figures, react
with a like-numbered reactive group; 1 with 1 and 2 with 2,
etc.
[0053] FIGS. 19A-19C are intended to show examples of various
reactants and the nomenclature for configurations illustrated
elsewhere in the Drawings. FIG. 19A shows various forms of reactive
XTEN segment precurors, each with a different reactive group on the
N-terminus. FIG. 19B shows various cross-linkers with 2, 3 or 4
reactive groups. In the first case, the divalent cross-linker is a
heterofunctional linker that reacts with two different types of
reactive groups, represented by "2" and "1". In the case of the
trivalent and tetravalent cross-linker, each reacts with only one
type of reactive group, represented by "1". FIG. 19C illustrates
the nomenclature of the reaction products of two XTEN segment
precursors. In the top version, a 1A was reacted with a 1B to
create a dimeric XTEN linked at the N-termini, with the residue of
the cross-linker indicated by 1A.sub.R-1B.sub.R, while the bottom
version is also a dimeric XTEN linked at the N-termini, with the
residue of the cross-linker indicated by 2A.sub.R-2B.sub.R.
[0054] FIGS. 20A-20B illustrate the creation of various XTEN
precursor segments. FIG. 20A shows the steps of making an XTEN
polypeptide, followed by reaction of the N-terminus with the
cross-linker with 2B-1A reactive groups, with the 1A reacting with
the N-terminal 1B (e.g., an alpha amino acid) to create the XTEN
precursor 2 with the reactive group 2B. FIG. 20B shows the
sequential addition of two cross-linkers with 2A reactive groups to
2B reactive groups of the XTEN, resulting in XTEN precuror 4, which
is then reacted with a cross-linker at the N-terminus between a
reactive 1B and the 1A of a cross-linker, resulting in XTEN
precuror 5, with reactive groups 4B and 3B. In such case, the
XTEN-precurors 5 then could serve as a reactant with two different
payloads or XTEN.
[0055] FIGS. 21A-21B illustrate examples of multimeric conjugates.
FIG. 21A illustrates how three molecules of an XTEN with a
conjugated payload A can be conjugated to a trimeric cross-linker,
resulting in a trimeric XTEN-payload conjugate with three A
payloads. FIG. 21B illustrates how three molecules of a polypeptide
with an A payload can be conjugated to a trimeric cross-linker,
resulting in a trimeric XTEN-payload conjugate with three
polypeptides with A payloads.
[0056] FIGS. 22A-22D illustrate examples of multivalent XTEN
conjugates that can originate from XTEN precursors with a single
cysteine. The amino group in the XTEN precursor acts as reactive
group 2B and the thiol group as reactive group 1B. The XTEN
precursor can be cross-linked using cross-linker that can react
with group 1B. The valency of the cross-linker controls the valency
of the resulting intermediate. This cross-linked intermediate can
be reacted with a payload carrying a reactive group 2A that can
react with the amino group forming the conjugation link 2A-BR. FIG.
22A is an XTEN precursor with single thiol group. FIG. 22B is a
divalent conjugate. FIG. 22C is a trimeric conjugate. FIG. 22D is a
tetrameric conjugate.
[0057] FIGS. 23A-23B illustrate examples of multivalent XTEN
conjugates that can originate from XTEN precursors with a single
cysteine. The amino group in the XTEN precursor acts as reactive
group 1B and the thiol group as reactive group 2B. The XTEN
precursor can be cross-linked using cross linker that can react
with group 1B. The valency of the cross linker controls the valency
of the resulting intermediate. This cross linked intermediate can
be reacted with a payload carrying a reactive group 2A that can
react with the thiol group forming the conjugation link 2A-BR. FIG.
23A illustrates the thiol group located close to the C-terminus of
XTEN. As a result the payload is located at the distal ends of the
final trimeric conjugate. FIG. 23B illustrates that the thiol group
is located close to the N-terminus of XTEN. As a result the payload
is located at the proximal ends of the final conjugate resulting in
increased payload shielding by XTEN.
[0058] FIGS. 24A-24C illustrate an example of the creation of a
"comb" configuration. FIG. 24A is a XTEN-payload precursor
comprising linker reactive group 1A. The payload can be
recombinantly fused to XTEN or it can be conjugated. FIG. 24B
illustrates an XTEN-precursor with the comb-like cross-linkers.
This can be an XTEN that carries a multiple reactive groups B. FIG.
24C shows the final product in the "comb" configuration, with five
Payload A. Valency is controlled by the number of reactive groups
in the Comb-like precursor.
[0059] FIGS. 25A-25B illustrate various configurations of
bispecific conjugates with two payloads. FIG. 25A illustrates
configurations with one molecule each of two payloads, while FIG.
25B illustrates various configurations with multiple copies of one
or both payloads.
[0060] FIGS. 26A-26B illustrate various examples of conjugates with
high valency. Conjugations sites of payloads can grouped (FIG. 26A)
or interspersed (FIG. 26B).
[0061] FIGS. 27A-27B illustrate the preparation of bispecific
conjugates from an XTEN precursor carrying both amino and thiol
groups in which many chemistries can be used and the order of
payload addition can vary. One can generate linker-conjugates as
precursors. FIG. 27A shows the creation of a single XTEN precursor
to which two different payloads are attached. FIG. 27B shows a
segment approach starting from two XTEN precursor molecules. This
approach allows one to conjugate both payloads to XTEN using the
same type of linker chemistry. In this case, the figure shows thiol
as the group to which payloads are conjugated, and then the
N-terminus of each segment is modified with a cross-linker to
enable head-to-head segment conjugation, resulting in a dimeric,
bispecific conjugate final product.
[0062] FIGS. 28A-28C show examples of multivalent conjugates
combining an antibody, XTEN, and a payload. Such constructs can
have different valencies and provide many benefits in that the XTEN
can have a cleavable linker, XTEN can provides solubility to the
composition, and it can allow adjustment of the drug load per IgG,
and the XTEN can be pre-conjugated with drug to simplify
manufacturing. FIG. 28A illustrates two XTENs conjugated to IgG at
Cys residues in the hinge region. FIG. 28B illustrates four XTEN
conjugated to IgG using Cys in the hinge region. FIG. 28C
illustrates XTEN conjugated outside of hinge. This can be done by
inserting Cys to control conjugation site or by random conjugation
to Lys side chains.
[0063] FIG. 29 shows examples of the construction of conjugates
combining an antibody, XTEN, and a payload. The antibody can have
one or multiple reactive groups 1B. XTEN can be conjugated to one
or multiple Payloads A. In addition XTEN can carry a reactive group
1A that preferentially reacts with the reactive group 1B on the
antibody. The location of reactive groups 1B in the antibody
controls the number and location of XTENs that are conjugated to
the antibody, resulting in the final product.
[0064] FIGS. 30A-30C show examples of conjugates comprising a
targeting moiety, XTEN, and a payload. Targeting moieties can be
peptides, peptoids, or receptor ligands. FIG. 30A shows
1.times.(1.times.3) conjugate. FIG. 30B shows 1.times.2(1.times.3)
conjugate. FIG. 30C shows a 3.times.1(1.times.3) conjugate.
[0065] FIG. 31 shows examples of conjugates comprising multiple
different targeting moieties, XTEN, and a payload. Targeting
moieties can be peptides, peptoids, receptor ligands.
[0066] FIG. 32 shows examples of conjugates comprising a targeting
moiety, XTEN, and a multiple different payloads.
[0067] FIG. 33 shows examples of combinatorial XTEN conjugates.
Payloads A, B, C, and D carry a reactive group 2A that reacts with
reactive group 2B on the XTEN precursor. In the next step, Payloads
E and F carry a reactive group 1A that reacts with reactive group
1B on XTEN, resulting in a library of different permutations of
bispecific conjugates. In this case, the reactive groups 1B and 2B
are thiol- and amino-groups, respectively.
[0068] FIG. 34 shows an example of the creation of a combinatorial
XTEN conjugate library. Payloads A, B, C are conjugated to XTEN
carrying reactive group 1A, resulting in one set of XTEN-precursor
segments. Payloads E, F, and G are conjugated to XTEN carrying
reactive group 1B, resulting in a second set of XTEN-precursor
segments. These segments are subjected to combinatorial conjugation
and then are purified from reactants. This enables the formation of
combinatorial products that can be immediately subjected to in
vitro and in vivo testing. In this case, reactive groups 1A and 1B
are the alpha-amino groups of XTEN with or without a bispecific
cross-linker. In one example, the 1A is an azide and 1B is an
alkyne or vice versa, while the payloads are attached to XTEN via
thiol groups in XTEN.
[0069] FIG. 35 shows an example of the creation of a combinatorial
XTEN conjugate library that optimizes the ratio between two
payloads. Each library member carries a different ratio of payload
A and payload E.
[0070] FIG. 36 shows an example of the creation of a combinatorial
XTEN conjugate library that creates combinations of targeting
moieties and payloads. The targeting moieties 1, 2, and 3 are
conjugated to XTEN carrying reactive group 1A. Payloads E, F, and G
are conjugated to XTEN carrying reactive group 1B. These segments
are subjected to combinatorial conjugation, enabling the formation
of combinatorial products where each library member comprises
targeting moieties and payloads. All XTEN segments carrying
payloads and conjugation groups can be purified as combinatorial
products that can be immediately subjected to in vitro and in vivo
testing.
[0071] FIG. 37 shows an example of an XTEN conjugate comprising
targeting moieties and payloads that exert selective action on the
surface of a target cell, such as a tumor cell. The particular
design of the dimeric XTEN conjugate comprises LHRH and
doxorubicin. This conjugate binds to the LHRH-receptor on that is
over-expressed on many cancer cells. Receptor binding results in
internalization followed by proteolytic break down and the
intracellular liberation of doxorubicin, which is toxic to the
cell.
[0072] FIG. 38 is a schematic flowchart of representative steps in
the assembly, production and the evaluation of a XTEN.
[0073] FIG. 39 is a schematic flowchart of representative steps in
the assembly of an XTEN polynucleotide construct encoding a fusion
protein. Individual oligonucleotides 501 are annealed into sequence
motifs 502 such as a 12 amino acid motif ("12-mer"), which is
ligated to additional sequence motifs from a library to create a
pool that encompasses the desired length of the XTEN 504, as well
as ligated to a smaller concentration of an oligo containing BbsI,
and KpnI restriction sites 503. The resulting pool of ligation
products is gel-purified and the band with the desired length of
XTEN is cut, resulting in an isolated XTEN gene with a stopper
sequence 505. The XTEN gene is cloned into a stuffer vector. In
this case, the vector encodes an optional CBD sequence 506 and a
GFP gene 508. Digestion is then performed with BbsI/HindIII to
remove 507 and 508 and place the stop codon. The resulting product
is then cloned into a BsaI/HindIII digested vector, resulting in
gene 500 encoding an XTEN.
[0074] FIG. 40 is a schematic flowchart of representative steps in
the assembly of a gene encoding XTEN, its expression, conjugation
with a payload and recovery as an XTEN-paylad, and its evaluation
as a candidate product.
[0075] FIGS. 41A, 41B, and 41C show generalized XTEN with either N-
or C-terminal tags or N- and C-terminal sequences optimized for
purification using methods illustrated in FIG. 42.
[0076] FIG. 42 shows a generalized scheme for purification of XTEN
with, in this illustrative embodiment, two tags in which a two-step
purification method to capture first one tag and then the second
can be utilized to remove truncated XTEN from fermentation,
resulting in the highly puritified target XTEN entity.
[0077] FIG. 43 shows an SDS-PAGE gel of the CBD-TEV site-XTEN_AE864
and CBD-TEV site-XTEN_AE864-GFP constructs expressed in E. coli
BL21 DE3 rne-131 and E. coli BL21 DE3 cells from shake flask
cultures as described in Example 10. Gel lane samples with MW
markers and expressed proteins from constructs are: 1) MW marker;
2-5) lysates from 4 independent flasks expressing CBD-TEV
site-XTEN_AE864-GFP fusion protein in E. coli BL21 DE3; 6-9)
lysates from 4 independent flasks expressing CBD-TEV
site-XTEN_AE864-GFP fusion protein in E. coli BL21 DE3 rne-131;
10-13) lysates from 4 independent flasks expressing CBD-TEV
site-XTEN_AE864 fusion protein in E. coli BL21 DE3; 14-17) lysates
from 4 independent flasks expressing CBD-TEV site-XTEN_AE864 fusion
protein in E. coli BL21 DE3 rne-131. Full-length protein spots
appear within the outline box. Bands of lower molecular weight are
host-cell proteins.
[0078] FIG. 44 shows relative GFP fluorescence of the CBD-TEV
site-XTEN_AE864-GFP expressed in E. coli BL21 DE3 rne-131 and E.
coli BL21 DE3 cells from shake flask cultures as described in
Example 10.
[0079] FIG. 45 shows an SDS-PAGE gel of the CBD-R-C-XTEN_AE864-RH8
("H8" disclosed as SEQ ID NO: 20) (EC682) and CBD-R-XTEN_AE864-RH8
("H8" disclosed as SEQ ID NO: 20) (EC683) constructs expressed in
E. coli fermentations as described in Example 17. Gel lane samples
with MW markers and expressed proteins from constructs are: 1) MW
marker, E. coli fermentation #EC682 clarified soluble lysates time
points after inoculation 2) 16 hours, 3) 24 hours, 4) 40 hours, 5)
45 hours; E. coli fermentation #EC683 clarified soluble lysates at
time points after inoculation 6) 16 hours, 7) 24 hours, 8) 40
hours, 9) 45 hours; Purified CBD-R-XTEN_AE864-RH8 ("H8" disclosed
as SEQ ID NO: 20) reference standard 10) 1 microgram, 11) 2
micrograms, and 12) 4 micrograms. For the E. coli fermentation
clarified soluble lysates each lane represents 3 microliters of the
fermenter culture. Full-length protein spots appear within the
outline box. Bands of lower molecular weight are host-cell
proteins.
[0080] FIG. 46 shows the trace output of Toyopearl Phenyl 650 M
Hydrophobic Interaction Chromatography, as described in Example
18.
[0081] FIG. 47 shows a non-reducing 4-12% Bis-Tris SDS-PAGE
analysis of Toyopearl Phenyl 650 M Hydrophobic Interaction
Chromatography fractions, as indicated in the figure and as
described in Example 18. The materials per lane are: Lane 1:
Marker; Lane 2: Load 7.5 .mu.l; Lane 3: Flow-through 1; Lane 4:
Flow-through 2; Lane 5: Elution fraction E1; Lane 6: Elution
fraction E2; Lane 7: Elution fraction E3; Lane 8: Elution fraction
E4; Lane 9: Elution fraction E5; Lane 10: Elution fraction E6; Lane
11: Elution fraction E7; Lane 12: Elution fraction E8.
[0082] FIGS. 48A and 48B show a non-reducing 4-12% Bis-Tris
SDS-PAGE analysis of Toyopearl IMAC Chromatography flow through,
wash (FIG. 48A) and elution fractions (FIG. 48B)(non-reducing) as
described in Example 18.
[0083] FIG. 49 shows a non-reducing SDS-PAGE analysis of the
trypsin-digested IMAC pool described in Example 18. FIG. 49
discloses "H8" as SEQ ID NO: 20.
[0084] FIG. 50 shows the elution profile of the MacroCap Q
Chromatography described in Example 18.
[0085] FIGS. 51A-51C show a 4-12% Bis-Tris SDS-PAGE analysis of the
MacroCap Q elution fractions, as described in Example 18. FIG. 51A,
flow-through, Coomassie staining. FIG. 51B, elution fractions,
Coomassie staining. FIG. 51C, elution fractions, silver
staining.
[0086] FIG. 52 shows the traces from C18 RP-HPLC analysis of
MacroCap Q elution fractions, as described in Example 18.
[0087] FIG. 53 shows a trace from a C18 RP-HPLC of the MacroCap Q
Elution Pool, as described in Example 18.
[0088] FIG. 54 shows a non-reducing SDS-PAGE analysis of the
Toyopearl Phenyl 650 M Hydrophobic Interaction Chromatography
fractions, as described in Example 19.
[0089] FIG. 55 shows a non-reducing SDS-PAGE analysis of Toyopearl
IMAC Chromatography fractions, as described in Example 19.
[0090] FIG. 56 shows a non-reducing 4-12% Bis-Tris SDS-PAGE/silver
staining analysis of the MacroCap Q Elution fractions as described
in Example 19.
[0091] FIG. 57 shows traces from C18 RP-HPLC analysis of MacroCap Q
elution fractions, as described in Example 19.
[0092] FIG. 58 shows the trace from C18 RP-HPLC analysis of the
MacroCap Q elution pool described in Example 19.
[0093] FIG. 59 shows an SDS-PAGE analysis of XTEN constructs with
experimental tags after expression in E. coli as described in
Example 20. Soluble lysates were loaded on the 4-12% Bis-Tris
polyacrylamide gel, with amounts loaded per lane equivalent to 36
.mu.l of cell culture suspension. The gel was stained with
Coomassie Blue stain using standard methods.
[0094] FIG. 60 shows an SDS-PAGE analysis of the RP11-XTEN-His8
("His8" disclosed as SEQ ID NO: 20) construct expressed in E. coli,
as described in Example 20. Heat-treated soluble lysates were
loaded on the 4-12% Bis-Tris polyacrylamide gel with amounts
equivalent to 1 or 2 .mu.l of cell culture suspension,
respectively. The gel was stained with Coomassie Blue stain. The
gel demonstrates that essentially all the expressed RP11-XTEN-His8
("His8" disclosed as SEQ ID NO: 20) protein was found in the
pelleted fraction. FIG. 60 discloses "H8" as SEQ ID NO: 20.
[0095] FIG. 61 shows an SDS-PAGE analysis of the MacroCap SP
purification of RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO: 20)
polypeptide described in Example 21. Fractions were analyzed by
4-12% SDS-PAGE followed by Coomassie staining.
[0096] FIG. 62 shows an SDS-PAGE analysis of the IMAC purification
of the RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO: 20)
polypeptide described in Example 21. Fractions were analyzed by
4-12% SDS-PAGE followed by Coomassie staining.
[0097] FIGS. 63A-63B show an SDS-PAGE analysis of the trypsin
digestion of RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO: 20)
protein purified by two chromatographic steps (SP+IMAC) described
in Example 21. Preparations were analyzed by 4-12% SDS-PAGE
followed by Coomassie staining (FIG. 63A) and silver staining (FIG.
63B).
[0098] FIGS. 64A-64C show the results of the analysis of the
conjugation reaction of DBCO-Mal to the 3.times.Thiol-XTEN as
described in Example 23. FIG. 64A shows the C18 RP-HPLC analysis of
the reaction mixture. A 20 .mu.g protein sample was loaded on a
Phenomenex Jupiter C185 uM 300 A 4.6 mm.times.150 mm column. The
proteins were eluted with a 5-50% gradient of acetonitrile in 0.1%
trifluoroacetic acid. FIG. 64B shows the HIC purification of
DBCO-XTEN reaction product. FIG. 64C shows the C18 RP-HPLC analysis
of the HIC-purified DBCO-XTEN reaction product.
[0099] FIGS. 65A-65B show results from trypsin cleavage of a double
tagged precursor XTEN, as described in Example 24. FIG. 65A shows
a4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 2
.mu.g per lane. The gel was stained with an Invitrogen SimplyBlue
SafeStain. FIG. 65B shows a4-12% Bis-Tris SDS-PAGE analysis of
protein samples loaded at 0.5 .mu.g per lane. The gel was stained
with a Pierce Silver Stain Kit.
[0100] FIGS. 66A-66C show results of an SDS-PAGE analysis of
MacroCap Q purification of trypsin digested double tagged
precursor, as described in Example 24. FIG. 66A shows a 4-12%
Bis-Tris SDS-PAGE analysis of protein samples loaded at 3 .mu.g per
lane. The gel was stained with Invitrogen SimplyBlue SafeStain.
FIG. 66B shows a4-12% Bis-Tris SDS-PAGE analysis of protein samples
loads at 0.5 .mu.g per lane. The gel was stained with a Pierce
Silver Stain Kit. FIG. 66C shows a 4-12% Bis-Tris SDS-PAGE analysis
of protein samples loaded at 0.5 .mu.g per lane. The gel was
stained with a Pierce Silver Stain Kit.
[0101] FIGS. 67A-67C show results from a C18 RP-HPLC test for
residual trypsin activity. FIG. 67A is the trace output of analysis
of synthetic [G2]GLP2 peptide in intact form. FIG. 67B is the trace
output of analysis of synthetic [G2]GLP2 peptide digested with
bovine trypsin. FIG. 67C is the trace output of analysis of
XTEN_AE869_Am1,C2 spiked with [G2]GLP2 and incubated overnight at
37.degree. C., as described in Example 24.
[0102] FIGS. 68A-68C show preparation of GLP2-XTEN conjugate from
GLP2-Cys peptide and 1.times.Amino-XTEN as described in Example 26.
20 .mu.g protein samples were loaded on Phenomenex Jupiter C185 uM
300 A 4.6 mm.times.150 mm column. Proteins were eluted with 5-50%
gradient of acetonitrile in 0.1% trifluoroacetic acid and detected
by absorbance at 214 nm (left panels A-C). 100 .mu.g protein
samples were desalted using NanoSep 3K Omega centrifugal devices
(Pall Corp.). Protein solutions in 50% acetonitrile, 0.5% formic
acid were infused into high-resolution mass spectrometer at flow
rate 10 ul/min. ESI-MS spectra were acquired in 800-1600 amu range
and reconstructed into zero-charge spectra using Bayesian Protein
Reconstruction Software (right panels A-C). FIG. 68A: initial
1.times.Amino-XTEN protein. FIG. 68B: product of the reaction
between 1.times.Amino-XTEN and sulfo-SMCC cross-linker. FIG. 68C:
purified GLP2-XTEN conjugate after reaction between GLP2-Cys and
N-Mal-XTEN.
[0103] FIGS. 69A-69B show preparation of GLP2-XTEN conjugate from
GLP2-Mal peptide and 1.times.Thiol-XTEN as described in Example 27.
20 .mu.g protein samples were loaded on Phenomenex Jupiter C185 uM
300 A 4.6 mm.times.150 mm column. Proteins were eluted with 5-50%
gradient of acetonitrile in 0.1% trifluoroacetic acid and detected
by absorbance at 214 nm (left panels A, B). 100 .mu.g protein
samples were desalted using NanoSep 3K Omega centrifugal devices
(Pall Corp.). Protein solutions in 50% acetonitrile, 0.5% formic
acid were infused into high-resolution mass spectrometer at flow
rate 10 ul/min. ESI-MS spectra were acquired in 800-1600 amu range
and reconstructed into zero-charge spectra using Bayesian Protein
Reconstruction Software (right panels A, B). FIG. 69A: initial
1.times.Thiol-XTEN protein. FIG. 69B: product of the reaction
between GLP2-Mal and 1.times.Thiol-XTEN.
[0104] FIGS. 70A-70B show the results of the purification of
GLP2-XTEN using preparative C4 RP-HPLC as described in Example 27.
FIG. 70A shows a chromatography profile of preparative RP-HPLC. A
fraction at 56-62 min was collected and evaporated under vacuum.
FIG. 70B shows an analysis by C18 RP-HPLC for purified
GLP2-XTEN.
[0105] FIGS. 71A-71C show results of the conjugation of DBCO-Mal to
1.times.Thiol-XTEN, as described in Example 28. FIG. 71A shows C18
RP-HPLC analysis of the reaction mixture. A 20 .mu.g protein sample
was loaded on Phenomenex Jupiter C185 uM 300 A 4.6 mm.times.150 mm
column. Proteins were eluted with a 5-50% gradient of acetonitrile
in 0.1% trifluoroacetic acid. FIG. 71B shows the HIC purification
of DBCO-XTEN. FIG. 71C shows the C18 RP-HPLC analysis of the
HIC-purified DBCO-XTEN.
[0106] FIGS. 72A-72B show results of analytical assays of XTEN
conjugated with cross-linked FITC, as described in Example 31. FIG.
72A shows the co-migration in a gel imaged by UV light box to show
the large apparent MW of FITC-containing conjugated species, also
detected by SEC at OD214 (protein signal) and OD495 (FITC signal)
in a SEC column, indicating successful labeling of the XTEN with
minimal free dye contamination. The materials by lane (left to
right, after the MW standards are: labeled FITC-CL-CBD-XTEN;
labeled FITC-CL-XTEN; purified FITC-CL-XTEN; purified FITC-CL-XTEN;
and purified FITC-CL-XTEN. The gel was imaged by UV light box to
show FITC apparent MW of FITC containing species. FIG. 72B shows
the results of SEC analysis of FITC-conjugated XTEN, showing the
overlap of the output of materials detected at OD214 and OD495, and
also the apparent large molecular weight.
[0107] FIG. 73 shows results of SEC analyses of the peak elution
fractions of conjugates of GFP cross-linked to XTEN and free GFP,
as described in Example 32. Cross-linking was confirmed by
co-migration of the OD214 protein signal and OD395 GFP signal in
the SEC column.
[0108] FIG. 74 shows the results of pharmacokinetic assays of
GFP-X-XTEN and FITC-X-XTEN tested in cynomolgus monkeys, as
described in Example 33.
[0109] FIG. 75 shows the pharmacokinetic profile (plasma
concentrations) in cynomolgus monkeys after single doses of
different compositions of GFP linked to unstructured polypeptides
of varying length, administered either subcutaneously or
intravenously, as described in Example 33. The compositions were
GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP.
Blood samples were analyzed at various times after injection and
the concentration of GFP in plasma was measured by ELISA using a
polyclonal antibody against GFP for capture and a biotinylated
preparation of the same polyclonal antibody for detection. Results
are presented as the plasma concentration versus time (h) after
dosing and show, in particular, a considerable increase in
half-life for the XTEN_AD836-GFP, the composition with the longest
sequence length of XTEN. The construct with the shortest sequence
length, the GFP-L288 had the shortest half-life.
[0110] FIGS. 76A-76C show an SDS-PAGE gel of samples from a
stability study of the fusion protein of XTEN_AE864 fused to the
N-terminus of GFP. The GFP-XTEN was incubated in cynomolgus plasma
and rat kidney lysate for up to 7 days at 37.degree. C., as
described in Example 55. In addition, GFP-XTEN administered to
cynomolgus monkeys was also assessed. Samples were withdrawn at 0,
1 and 7 days and analyzed by SDS PAGE followed by detection using
Western analysis and detection with antibodies against GFP. FIG.
76A shows the sequence of XTEN_AE864 showed negligible signs of
degradation over 7 days in plasma. FIG. 76B shows in vivo stability
of the fusion protein was tested in plasma samples wherein the
GFP_AE864 was immunoprecipitated and analyzed by SDS PAGE. Samples
that were withdrawn up to 7 days after injection showed very few
signs of degradation. FIG. 76C shows XTEN_AE864 was rapidly
degraded in rat kidney lysate over 3 days.
[0111] FIG. 77 shows the near UV circular dichroism spectrum of
Ex4-XTEN_AE864, performed as described in Example 56.
[0112] FIG. 78 shows results of a size exclusion chromatography
analysis of glucagon-XTEN construct samples measured against
protein standards of known molecular weight, with the graph output
as absorbance versus retention volume, as described in Example 58.
The glucagon-XTEN constructs are 1) glucagon-Y288; 2)
glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results
indicate an
[0113] FIG. 79 is a schematic of the logic flow chart of the
algorithm SegScore (Example 59). In the figure the following legend
applies: i, j--counters used in the control loops that run through
the entire sequence; HitCount--this variable is a counter that
keeps track of how many times a subsequence encounters an identical
subsequence in a block; SubSeqX--this variable holds the
subsequence that is being checked for redundancy; SubSeqY--this
variable holds the subsequence that the SubSeqX is checked against;
BlockLen--this variable holds the user determined length of the
block; SegLen--this variable holds the length of a segment. The
program is hardcoded to generate scores for subsequences of lengths
3, 4, 5, 6, 7, 8, 9, and 10; Block--this variable holds a string of
length BlockLen. The string is composed of letters from an input
XTEN sequence and is determined by the position of the i counter,
SubSeqList--this is a list that holds all of the generated
subsequence scores.
[0114] FIG. 80 depicts the application of the algorithm SegScore to
a hypothetical XTEN of 11 amino acids in order to determine the
repetitiveness. An XTEN sequence (SEQ ID NO: 1177) consisting of N
amino acids is divided into N-S+1 subsequences of length S (S=3 in
this case). A pair-wise comparison of all subsequences is performed
and the average number of identical subsequences is calculated to
result, in this case, in a subsequence score of 1.89.
[0115] FIG. 81 provides the results of the assay to measure the
fluorescence signal of RP11 clones pSD0107 to pSD0118), as
described in Example 12. One positive control (pLCW970) and two
negative controls (pBr322 and pLCW970+10 mM phosphate) were
included. The GFP expression level was measured using samples from
2-3 shake flasks per construct.
[0116] FIGS. 82A-82F show the screening results of libraries
LCW1157-1159. FIGS. 82A, 82B, and 82C provide the fluorescence
histograms of LCW1157-1159, showing the number of colonies
identified for each fluorescence signal region, as described in
Example 12. The average fluorescence reading of the negative
control (black arrow) and positive pSD0116 (white arrow) are marked
in the figures. FIGS. 82D, 82E, and 82F provide the correlation
between the fluorescence reading in the original test and the
retest of the select clones.
[0117] FIG. 83 shows results of the SDS-PAGE analysis of the top 8
expression construct products and controls under unreduced
conditions, as described in Example 12. The desired full length
protein end product RP11-XTEN-GFP is indicated by an arrow, and the
higher band is the dimer of the protein. Lanes: 1-8: top 8
expression constructs (expression level from high to low, based on
fluorescence reading of the retests), 1. LCW1159.004, 2.
LCW1159.006, 3. LCW1158.004, 4. LCW1157.040, 5. LCW1158.003, 6.
LCW1157.039, 7. LCW1157.025, 8. LCW1157.038; C1-C3: Controls: C1.
pSD0114, C2. pSD0116, C3. pCW1146 (Negative control).
[0118] FIG. 84 shows the SDS-PAGE evaluation of the MacroCap SP
capture efficiency for the top 4 expression construct products
under non-reducing conditions, as described in Example 12. Lanes
1-4: load, flow through, wash and elution of LCW1159.004, 2. Lanes
5-8: load, flow through, wash and elution of LCW1159.006. Lanes
9-12: load, flow through, wash and elution of LCW1158.004. 13-16:
load, flow through, wash and elution of LCW1157.040. Lanes
17-201-4: load, flow through, wash and elution of negative control.
Unmarked lanes are molecular weight standards.
[0119] FIG. 85 shows the summary of library LCW1163 screening
results with a comparison of the fluorescence signal of the top 4
expression products and the controls in the retests, as described
in Example 12. Each sample had 4 replicates, represented by 4
individual dots in the figure.
[0120] FIG. 86 shows the summary of library LCW1160 screening
results, as described in Example 12. Fluorescence histogram of
LCW1157-1159, showing the number of colonies identified for each
fluorescence signal region; average fluorescence reading of
negative control (black arrow), pSD0116 (white arrow), and
LCW1159.004 (high expression candidates from screening
LCW1157-1159, grey arrow) were marked in the figures.
[0121] FIGS. 87A-87B show 4-12% SDS-PAGE/silver staining analysis
of MacroCap Q fractions as described in Example 14. FIG. 87A: Batch
2, lane 1: molecular weight standard; lanes 2-5: MacroCap Q flow
through fractions 1-4, respectively; lanes 6-16: MacroCap Q elution
fractions 1-11, respectively. FIG. 87B: Batch 1, lane 1: molecular
weight standard; lanes 2-6: MacroCap Q flow through fractions 1-5,
respectively; lanes 7-16: MacroCap Q elution fractions 1-10,
respectively.
[0122] FIGS. 88A, 88B, 88C, 88D, and 88E show results from the
analyses of intermediates and final product during the preparation
of 1.times.DBCO,3.times.LHRH-XTEN, as described in Example 34.
[0123] FIGS. 89A-89C show results of analyses of reaction mixtures
from the preparation of conjugates to
1.times.Azide,3.times.MMAE-XTEN analyzed by C18-RP-HPLC and mass
spectroscopy, as described in Example 35. FIG. 89A is analysis of
the initial 1.times.Amino,3.times.Thiol-XTEN reactant. FIG. 89B is
analysis of the protein modification with MMAE-Maleimide, showing
the mass increase corresponding to modifications of three cysteines
with MMAE-Mal. FIG. 89C shows the analysis of the protein
modification with Azide-PEG4-NHS ester, with mass increases
corresponding to the single addition of the azide-PEG4 moiety.
[0124] FIGS. 90A-90B show analyses of the reaction products in
conjugates of 3.times.LHRH,3.times.MMAE-XTEN as described in
Example 36. FIG. 90A: SDS-PAGE analysis of the click conjugate. 0.5
.mu.g of proteins were loaded per lane on 12% Bis-Tris NuPAGE mini
gel (Life Technologies). The gel was stained with Pierce Silver
Stain Kit (Thermo Scientific, cat. #24612). Lane1,
1.times.Azide,3.times.MMAE-XTEN; lane 2,
1.times.DBCO,3.times.LHRH-XTEN; lane 3, products of click chemistry
reaction. The conjugation product band is indicated by the arrow.
FIG. 90B: C4 RP-HPLC analysis of the click conjugate reactants and
products--(1) 1.times.DBCO,3.times.LHRH-XTEN; (2)
1.times.Azide,3.times.MMAE-XTEN; (3) products of click chemistry
reaction.
[0125] FIGS. 91A-91F show a flow chart of the reaction during
preparation of conjugates of 1.times.LHRH,3.times.MMAE-XTEN, as
described in Example 37. FIG. 91A: initial
1.times.Amino,3.times.Thiol-XTEN; FIG. 91B: protein modification
with 2,2'-Dipyridyl disulfide; FIG. 91C: protein modification with
DBCO-sulfo-NHS; FIG. 91D: deprotection of cysteines with TCEP; FIG.
91E: Modification of three cysteines with MMAE-Mal; FIG. 91F:
Conjugation of LHRH-azide to N-terminal DBCO.
[0126] FIGS. 92A-92C show a flow chart of the reaction during
preparation of conjugates of 1.times.Mal,3.times.PTX-XTEN reactant,
as described in Example 41. FIG. 92A: Initial
1.times.Amino,3.times.Thiol-XTEN; FIG. 92B: Protein modification
with PTX-Mal; FIG. 92C: Protein modification with Sulfo-SMCC.
[0127] FIGS. 93A-93C show results of analyses of reaction mixtures
from the preparation of iodoacetyl-XTEN, as described in Example
42. FIG. 93A: 1.times.Amino-XTEN analyzed by C18-RP-HPLC before and
after incubation with 10.times. excess of SIA.FIG. 93B: ESI-MS
analysis of 1.times.Amino-XTEN modified with SIA. FIG. 93C: Samples
analyzed by C18 RP-HPLC-Bottom profile--HCKFWW (SEQ ID NO: 25)
peptide. Medium profile--IA-XTEN. Upper profile--reaction of
IA-XTEN with 5.times. excess of HCKFWW (SEQ ID NO: 25) peptide.
[0128] FIGS. 94A-94D show the results of screening libraries
LCW1171, 1172, 1203, and 1204, as described in Example 14. FIG.
94A-D: Fluorescence histogram of LCW1171, 1172, 1203, 1204, showing
the number of colonies identified for each fluorescence signal
region; average fluorescence reading of negative control (black
arrow) and pSD0116 (white arrow) when screening LCW1171-1172 were
marked in the FIGS. 94A and 94B; average fluorescence reading of
negative control (black arrow), pSD0116 (white arrow), and CBD
control (grey arrow) when screening LCW1203-1204 are marked in
FIGS. 94C and 94D.
[0129] FIGS. 95A, 95B, and 95C show the results of screening
libraries LCW1208-1210, as described in Example 12. FIGS. 95A-95C:
Fluorescence histograms of LCW1208-1210, showing the number of
colonies identified for each fluorescence signal region; average
fluorescence reading of negative control (black arrow) and CBD
control (grey arrow) are marked in the figures.
[0130] FIGS. 96A and 96B illustrated the production of XTEN
segments from a precursor that contains three repeat copies of XTEN
of identical length and sequence. In FIG. % A, the XTEN precursor
comprises three identical copies of XTEN that are flanked by
identical protease cleavage sites. In FIG. 96B, the XTEN precursor
further comprises N- and C-terminal affinity purification tags to
facilitate purification of full-length precursor molecules.
Following purification of the precursor it is cleaved by protease
that acts on all the incorporated cleavage sequences to release the
tags from the XTEN, which is followed by purification to separate
the individual units of XTEN, facilitating the high-yield
production of XTENs with short and intermediate lengths from
long-chain precursor molecules.
[0131] FIGS. 97A-97G illustrate different embodiments of trimeric,
branched XTEN-payload conjugates in which all conjugates shown can
be prepared from the identical XTEN molecules via conjugation to
its N-terminal amino group and a functional group, such as the
thiol of cysteine, that is located close to the C-terminus. FIGS.
97A and 97B illustrates conjugates having a single payload
molecule, with FIG. 97A using a 4-arm cross-linker with all the
XTEN conjugated in close proximity to the payload, resulting in
significant shielding of payload interactions with other molecules.
FIG. 97B illustrates a configuration in where the payload is
conjugated to a single XTEN arm that is branched at the distal end
of the configuration, resulting in reduced payload shielding
compared to the configuration of FIG. 97A. FIG. 97C illustrates a
conjugate with two payloads that can result in increased avidity or
increased potency. FIGS. 97D and 97E illustrate configurations with
three identical payloads to further increase potency and/or
avidity. FIG. 97F illustrates a configuration with one payload A
and two identical copies of payload B for high-avidity binding or
interactions. FIG. 97G illustrates a configuration with 3 different
payloads enabling the inclusion of three different functions into a
single XTEN conjugate.
[0132] FIG. 98 illustrates a scheme for synthesis of a conjugate
between a branched XTEN and a single payload molecule. Initially,
the thiol group in XTEN is blocked by reaction with iodoacetamide
(alternatively, one can start the synthesis using XTEN which lacks
a thiol group). Next, a DBCO group is added to the alpha-amino
group of XTEN, then is reacted with a tetrafunctional cross linker
that comprises one iodoacetyl group and three azide groups. The
resulting XTEN is next reacted with a payload that carries a free
thiol group resulting in the final XTEN-payload conjugate.
[0133] FIG. 99 illustrates a scheme for synthesis of a conjugate
between a branched XTEN and a single payload molecule. An
intermediate is produced by reacting XTEN with a trifunctional
linker comprising two azide functions and an NHS function followed
by the addition of payload A to the thiol group via maleimide
chemistry (the order of these two steps can be inverted). A second
intermediate is produce by reacting XTEN with a cysteine with
iodoacetamide to block the free thiol group followed by addition of
DBCO to the alpha-amino group via NHS activation (the order of
these two steps can be inverted). Subsequently, the two
intermediate molecules are conjugated using a click chemistry
reaction, resulting in the final XTEN-payload conjugate.
[0134] FIG. 100 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and two identical payload molecules. An
intermediate is produced by adding a DBCO group to the alpha-amino
group of an XTEN via NHS chemistry. A second intermediate is
produced by blocking the free thiol group of an XTEN with
iodoacetamide followed by addition of a trifunctional cross-linker
(2 N-maleimide groups and a carboxyl group that is activated by
NHS) to the alpha amino-group (the order of these two steps can be
inverted). The two intermediates are reacted resulting in the
branched conjugate, and then two payload A molecules are added via
click chemistry reaction resulting in the final product
XTEN-payload conjugate.
[0135] FIG. 101 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and three identical payload molecules. An
intermediate is produced by adding a DBCO group to the alpha-amino
group of an XTEN via NHS chemistry. Another intermediate is
produced by conjugating payload A to the thiol group of an XTEN via
a N-maleimide functional group. The three molecules are linked
together via a trifunctional cross-linker comprising three azide
functions, resulting in the final XTEN-payload conjugate.
[0136] FIG. 102 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and three identical payload molecules. An
intermediate is produced by adding a DBCO group to the thiol group
of an XTEN A via N-maleimide chemistry. In the next step, payload A
is conjugated to the alpha amino-group of the XTEN intermediate via
NHS chemistry. Three molecules of the resulting XTEN are linked via
a trifunctional cross-linker comprising three azide functions,
resulting in the final XTEN-payload conjugate.
[0137] FIG. 103 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and two Payload A and one Payload B
molecules per conjugate. An intermediate is produced by adding
Payload A to the thiol group of an XTEN using an N-maleimide
functional group, followed by the addition of a trifunctional cross
linker (two azide groups and a carboxyl group that is activated by
NHS) to the alpha amino-group (the order of these two steps can be
inverted). A second intermediate is produced by adding DBCO to the
alpha amino-group of an XTEN via NHS activation followed by the
addition of Payload B to the free thiol group of the XTEN using an
N-maleimide group (the order of these two steps can be inverted).
Two molecules of the second intermediate are reacted with one
molecule of the first intermediate to form the final XTEN-payload
conjugate.
[0138] FIG. 104 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and three different payloads. An
intermediate is produced by adding Payload A to the thiol group on
an XTEN using an N-maleimide functional group followed by the
addition of a trifunctional cross linker (one azide group, one
N-maleimide group and one carboxyl group that is activated by NHS)
to the alpha amino-group (the order of these two steps can be
inverted). A second intermediate is produced by adding Payload B to
the alpha amino-group of XTEN via NHS chemistry. A third
intermediate is produced by adding DBCO to the alpha amino-group of
an XTEN via NHS activation followed by the addition of payload C to
the free thiol group of the XTEN using an N-maleimide group (the
order of these two steps can be inverted). The three intermediates
are reacted with each other to form the final XTEN-payload
conjugate.
[0139] FIG. 105 illustrates a scheme for synthesis of a conjugate
having a dimeric or tetrameric branched XTEN and Payload A
molecules. An intermediate is produced by adding DBCO to the thiol
group of an XTEN using N-maleimide functional group followed by the
addition Payload A to the amino group of the XTEN using NHS (the
order of these two steps can be inverted). Subsequently the
intermediate is multimerized by addition of azide cross-linkers.
Use of a divalent cross-linker yields the dimeric configuration,
and a tetravalent cross-linker yields the tetrameric configuration
of the final product.
[0140] FIG. 106 illustrates a scheme for synthesis of a conjugate
having a branched XTEN and three different payloads. An
intermediate is produced by adding Payload A to the thiol group of
an XTEN using N-maleimide functional group, followed by the
addition of a trifunctional cross linker (one azide group, one
N-maleimide group and one carboxyl group that is activated by NHS)
to the alpha amino-group (the order of these two steps can be
inverted). A second intermediate is produced by adding Payload B to
the free thiol group of an XTEN via an N-maleimide functional
group. A third intermediate is produced by adding DBCO to the alpha
amino-group of an XTEN via NHS activation followed by the addition
of Payload C to the free thiol group using a N-maleimide group (the
order of these two steps can be inverted). The three intermediates
are reacted with each other to form the final XTEN-payload
conjugate.
[0141] FIGS. 107A-107C shows results of analyses of reaction
mixtures from the preparation of conjugates to
1.times.DBCO,3.times.FA(.gamma.)-XTEN analyzed by C18-RP-HPLC and
mass spectroscopy, as described in Example 38. FIG. 107A is
analysis of the initial 1.times.Amino,3.times.Thiol-XTEN reactant.
FIG. 107B is analysis of the protein modification with
Folate-gamma-Maleimide, showing the mass increase corresponding to
modifications of three cysteines with FA(.gamma.)-Mal. FIG. 107C
shows the analysis of the protein modification with DBCO-sulfo-NHS
ester, with mass increases corresponding to the single addition of
the DBCO moiety.
[0142] FIG. 108 shows C4 RP-HPLC analyses of the click conjugate
reactants and product 3.times.FA(.gamma.),3.times.MMAE-XTEN, as
described in Example 39. (1) 1.times.DBCO,3.times.FA(.gamma.)-XTEN;
(2) 1.times.Azide,3.times.MMAE-XTEN; (3) products of click
chemistry reaction.
[0143] FIGS. 109A-109C show analyses of final
3.times.FA(.gamma.),3.times.MMAE-XTEN product purified by
preparative RP-HPLC, as described in Example 39. FIG. 109A shows
size exclusion chromatography analysis (Phenomenex
BioSep-SEC-s4000600.times.7.80 mm column, 50 mM Sodium Phosphate pH
6.5, 300 mM NaCl buffer, flow rate 0.5 ml/min, isocratic elution 70
min). FIG. 109B shows RP-HPLC analysis (Phenomenex Jupiter C185
.mu.M 300 .ANG. 150.times.4.60 mm column, Buffer A: 0.1% TFA in
H2O, Buffer B: 0.1% TFA in CAN, flow rate 1 ml/min, gradient 5% to
50% B in 45 min). FIG. 109C shows ESI-MS analysis (QSTAR-XL,
calculated MW 85,085.4 Da, experimental MW 85,091 Da).
[0144] FIG. 110 shows the results of GPCR Ca.sup.2+ mobilization
activity of recombinant GLP2-2 G-XTEN (filled squares) and
conjugate GLP2-2G-XTEN (filled circles), performed as described in
Example 62.
[0145] FIG. 111 shows the results of an in vitro human plasma
stability of recombinant GLP2-2G-XTEN (filled squares) and
conjugate GLP2-2G-XTEN (filled triangles) at various time points at
37.degree. C., performed as described in Example 63.
[0146] FIG. 112 shows the results of the pharmacokinetic profile of
recombinant GLP2-2G-XTEN (filled squares) and conjugate
GLP2-2G-XTEN (filled triangles) in rats, performed as described in
Example 64.
[0147] FIGS. 113A-113D. FIG. 113A shows the SEC-HPLC analysis of
the reaction products between Tris-[2-maleimidoethyl]amine and
1.times.Amino,1.times.Thiol-XTEN432: FIG. 113A-conjugation mixture:
peak 1--trimeric XTEN, peak 2--dimeric XTEN, peak 3--unreacted
monomeric XTEN; FIG. 113B--linear XTEN_1296 control; FIG.
113C--linear XTEN_864 control; FIG. 113D--linear XTEN_432
control.
[0148] FIGS. 114A-114B. FIG. 114A shows the C18 RP-HPLC analysis of
DBCO-sulfo-NHS conjugation to 1.times.Amino-XTEN_288, as described
in Example 65. Unreacted XTEN eluted at 19 min.
1.times.DBCO-XTEN_288 eluted at 27 min. DBCO-sulfo-NHS reagent and
product of its hydrolysis eluted at 41.5 min and 38.5 min,
respectively. FIG. 114B shows C18 RP-HPLC analysis of
Azido-PEG4-NHS ester conjugation to Tris(2-aminoethyl)amine.
3.times.Azide-PEG4-TAEA was identified by MALDI-TOF MS and ESI-MS
as a product with MW of 966 Da.
[0149] FIG. 115 shows the SEC-HPLC analysis, as described in
Example 66, of the reaction products between
3.times.Azide-PEG4-TAEA and 1.times.DBCO-XTEN_288: (trace A)
conjugation mixture: peak 1--trimeric XTEN, peak 2--dimeric XTEN,
peak 3--unreacted monomeric XTEN, peak 4--low molecular weight
compounds; (trace B) linear XTEN_864 control; (trace C) linear
XTEN576 control; (trace D) linear XTEN_288 control.
[0150] FIG. 116 shows results of a killing assay demonstrating
selective cytotoxicity of 3.times.FA(.gamma.),3.times.MMAE-XTEN on
KB cells, as described in Example 69. The inhibitory dose response
curves are shown for the groups of free MMAE (filled circles);
3.times.MMAE-XTEN (filled, inverted triangles) and
3.times.FA(.gamma.),3.times.MMAE-XTEN in the presence (filled
triangles) and absence (filled squares) of folic acid competitor on
KB cells.
[0151] FIGS. 117A-117C shows the structure of the XTEN-payload
conjugate 3.times.FA(.gamma.),3.times.MMAE-XTEN. FIG. 117A shows
the two XTEN (SEQ ID NOS 1178-1179, respectively, in order of
appearance) linked by the reaction of the azide
1-azido-3,6,9,12-tetraoxapentadecan-15-oic acid,
N-hydroxysuccinimide ester and the alkyne
6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid,
N-hydroxysuccinimide (or N-hydroxysulfosuccinimide) ester. FIG.
117B shows the X residue of Cys modified with
folate-.gamma.-aminopentyl-maleimide. FIG. 117C shows the Z residue
of Cys modified with
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylaur-
istatin E.
DETAILED DESCRIPTION
[0152] Before the embodiments of the invention are described, it is
to be understood that such embodiments are provided by way of
example only, and that various alternatives to the embodiments of
the invention described herein may be employed in practicing the
invention. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention.
[0153] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. Numerous
variations, changes, and substitutions will now occur to those
skilled in the art without departing from the invention.
Definitions
[0154] In the context of the present application, the following
terms have the meanings ascribed to them unless specified
otherwise:
[0155] As used throughout the specification and claims, the terms
"a", "an" and "the" are used in the sense that they mean "at least
one", "at least a first", "one or more" or "a plurality" of the
referenced components or steps, except in instances wherein an
upper limit is thereafter specifically stated. Therefore, a
"payload", as used herein, means "at least a first payload" but
includes a plurality of payloads. The operable limits and
parameters of combinations, as with the amounts of any single
agent, will be known to those of ordinary skill in the art in light
of the present disclosure.
[0156] The terms "polypeptide", "peptide", and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified, for example, by disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component.
[0157] As used herein, the term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including but
not limited to both the D or L optical isomers, and amino acid
analogs and peptidomimetics. Standard single or three letter codes
are used to designate amino acids.
[0158] A "pharmacologically active" agent includes any drug,
compound, composition of matter or mixture desired to be delivered
to a subject, e.g. therapeutic agents, diagnostic agents, or drug
delivery agents, which provides or is expected to provide some
pharmacologic, often beneficial, effect that can be demonstrated in
vivo or in vitro. Such agents may include peptides, proteins,
carbohydrates, nucleic acids, nucleosides, oligonucleotides, and
small molecule synthetic compounds, or analogs thereof.
[0159] The term "natural L-amino acid" means the L optical isomer
forms of glycine (G), proline (P), alanine (A), valine (V), leucine
(L), isoleucine (I), methionine (M), cysteine (C), phenylalanine
(F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K),
arginine (R), glutamine (Q), asparagine (N), glutamic acid (E),
aspartic acid (D), serine (S), and threonine (T).
[0160] The term "non-naturally occurring," as applied to sequences
and as used herein, means polypeptide or polynucleotide sequences
that do not have a counterpart to, are not complementary to, or do
not have a high degree of homology with a wild-type or
naturally-occurring sequence found in a mammal. For example, a
non-naturally occurring polypeptide or fragment may share no more
than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid
sequence identity as compared to a natural sequence when suitably
aligned.
[0161] The terms "hydrophilic" and "hydrophobic" refer to the
degree of affinity that a substance has with water. A hydrophilic
substance has a strong affinity for water, tending to dissolve in,
mix with, or be wetted by water, while a hydrophobic substance
substantially lacks affinity for water, tending to repel and not
absorb water and tending not to dissolve in or mix with or be
wetted by water. Amino acids can be characterized based on their
hydrophobicity. A number of scales have been developed. An example
is a scale developed by Levitt, M, et al., J Mol Biol (1976)
104:59, which is listed in Hopp, T P, et al., Proc Natl Acad Sci
USA (1981) 78:3824. Examples of "hydrophilic amino acids" are
arginine, lysine, threonine, alanine, asparagine, and glutamine. Of
particular interest are the hydrophilic amino acids aspartate,
glutamate, and serine, and glycine. Examples of "hydrophobic amino
acids" are tryptophan, tyrosine, phenylalanine, methionine,
leucine, isoleucine, and valine.
[0162] A "fragment" when applied to a biologically active protein,
is a truncated form of a the biologically active protein that
retains at least a portion of the therapeutic and/or biological
activity. A "variant," when applied to a biologically active
protein is a protein with sequence homology to the native
biologically active protein that retains at least a portion of the
therapeutic and/or biological activity of the biologically active
protein. For example, a variant protein may share at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence
identity compared with the reference biologically active protein.
As used herein, the term "biologically active protein variant"
includes proteins modified deliberately, as for example, by site
directed mutagenesis, synthesis of the encoding gene, insertions,
or accidentally through mutations and that retain activity.
[0163] The term "sequence variant" means polypeptides that have
been modified compared to their native or original sequence by one
or more amino acid insertions, deletions, or substitutions.
Insertions may be located at either or both termini of the protein,
and/or may be positioned within internal regions of the amino acid
sequence. A non-limiting example is insertion of an XTEN sequence
within the sequence of the biologically-active payload protein.
Another non-limiting example is substitution of an amino acid in an
XTEN with a different amino acid. In deletion variants, one or more
amino acid residues in a polypeptide as described herein are
removed. Deletion variants, therefore, include all fragments of a
payload polypeptide sequence. In substitution variants, one or more
amino acid residues of a polypeptide are removed and replaced with
alternative residues. In one aspect, the substitutions are
conservative in nature and conservative substitutions of this type
are well known in the art.
[0164] The term "moiety" means a component of a larger composition
or that is intended to be incorporated into a larger composition,
such as a functional group of a drug molecule or a targeting
peptide joined to a larger polypeptide.
[0165] As used herein, "terminal XTEN" refers to XTEN sequences
that have been fused to or in the N- or C-terminus of the payload
when the payload is a peptide or polypeptide.
[0166] The term "XTEN release site" refers to a cleavage sequence
in XTEN-payload that can be recognized and cleaved by a protease,
effecting release of an XTEN or a portion of an XTEN from the
XTEN-payload polypeptide. As used herein, "mammalian protease"
means a protease that normally exists in the body fluids, cells or
tissues of a mammal. XTEN release sites can be engineered to be
cleaved by various mammalian proteases (a.k.a. "XTEN release
proteases") such as trypsin, FXIa, FXIIa, kallikrein, FVIIIa,
FVIIIa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17,
MMP-20, or any protease that is present in a subject. Other
equivalent proteases (endogenous or exogenous) that are capable of
recognizing a defined cleavage site can be utilized. The cleavage
sites can be adjusted and tailored to the protease utilized.
[0167] The term "within", when referring to a first polypeptide
being linked to a second polypeptide, encompasses linking that
connects the N-terminus of the first or second polypeptide to the
C-terminus of the second or first polypeptide, respectively, as
well as insertion of the first polypeptide into the sequence of the
second polypeptide. For example, when an XTEN is linked "within" a
payload polypeptide, the XTEN may be linked to the N-terminus, the
C-terminus, or may be inserted between any two amino acids of the
payload polypeptide.
[0168] "Activity" as applied to form(s) of a XTEN-payload
composition provided herein, refers to an action or effect,
including but not limited to receptor binding, antagonist activity,
agonist activity, a cellular or physiologic response, or an effect
generally known in the art for the payload, whether measured by an
in vitro, ex vivo or in vivo assay or a clinical effect.
[0169] As used herein, the term "ELISA" refers to an enzyme-linked
immunosorbent assay as described herein or as otherwise known in
the art.
[0170] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for the subject vectors such
as those described herein. Host cells include progeny of a single
host cell. The progeny may not necessarily be completely identical
(in morphology or in genomic of total DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
vector of this invention.
[0171] "Isolated" when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. As
is apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody, or
fragments thereof, does not require "isolation" to distinguish it
from its naturally occurring counterpart. In addition, a
"concentrated", "separated" or "diluted" polynucleotide, peptide,
polypeptide, protein, antibody, or fragments thereof, is
distinguishable from its naturally occurring counterpart in that
the concentration or number of molecules per volume is generally
greater than that of its naturally occurring counterpart. In
general, a polypeptide made by recombinant means and expressed in a
host cell is considered to be "isolated."
[0172] An "isolated" nucleic acid is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the polypeptide-encoding nucleic acid. For example, an
isolated polypeptide-encoding nucleic acid molecule is other than
in the form or setting in which it is found in nature. Isolated
polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-encoding nucleic acid
molecule as it exists in natural cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal or extra-chromosomal location
different from that of natural cells.
[0173] A "chimeric" protein contains at least one fusion
polypeptide comprising at least one region in a different position
in the sequence than that which occurs in nature. The regions may
normally exist in separate proteins and are brought together in the
fusion polypeptide; or they may normally exist in the same protein
but are placed in a new arrangement in the fusion polypeptide. A
chimeric protein may be created, for example, by chemical
synthesis, or by creating and translating a polynucleotide in which
the peptide regions are encoded in the desired relationship.
[0174] "Fused," and "fusion" are used interchangeably herein, and
refers to the joining together of two or more peptide or
polypeptide sequences by recombinant means.
[0175] "Operably linked" means that the DNA sequences being linked
are contiguous, and in reading phase or in-frame. An "in-frame
fusion" refers to the joining of two or more open reading frames
(ORFs) to form a continuous longer ORF, in a manner that maintains
the correct reading frame of the original ORFs. For example, a
promoter or enhancer is operably linked to a coding sequence for a
polypeptide if it affects the transcription of the polypeptide
sequence. Thus, the resulting recombinant fusion protein is a
single protein containing two or more segments that correspond to
polypeptides encoded by the original ORFs (which segments are not
normally so joined in nature).
[0176] "Crosslinking," "conjugating," "link," "linking" and "joined
to" are used interchangeably herein, and refer to the covalent
joining of two different molecules by a chemical reaction. The
crosslinking can occur in one or more chemical reactions, as
described more fully, below.
[0177] The term "conjugation partner" as used herein, refers to the
individual components that can be linked or are linked in a
conjugation reaction.
[0178] The term "conjugate" is intended to refer to the
heterogeneous molecule formed as a result of covalent linking of
conjugation partners one to another, e.g., a biologically active
payload covalently linked to a XTEN molecule or a cross-linker
covalently linked to a reactive XTEN.
[0179] "Cross-linker" and "linker" and "cross-linking agent" are
used interchangably and in their broadest context to mean a
chemical entity used to covalently join two or more entities. For
example, a cross-linker joins two, three, four or more XTEN, or
joins a payload to an XTEN, as the entities are defined herein. A
cross-linker includes, but is not limited to, the reaction product
of small molecule zero-length, homo- or hetero-bifunctional, and
multifunctional cross-linker compounds, the reaction product of two
click-chemistry reactants. It will be understood by one of skill in
the art that a cross-linker can refer to the covalently-bound
reaction product remaining after the crosslinking of the reactants.
The cross-linker can also comprise one or more reactants which have
not yet reacted but which are capable to react with another
entity.
[0180] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminus direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide. A "partial sequence" is a linear sequence of part
of a polypeptide that is known to comprise additional residues in
one or both directions.
[0181] "Heterologous" means derived from a genotypically distinct
entity from the rest of the entity to which it is being compared.
For example, a glycine rich sequence removed from its native coding
sequence and operatively linked to a coding sequence other than the
native sequence is a heterologous glycine rich sequence. The term
"heterologous" as applied to a polynucleotide, a polypeptide, means
that the polynucleotide or polypeptide is derived from a
genotypically distinct entity from that of the rest of the entity
to which it is being compared.
[0182] The terms "polynucleotides", "nucleic acids", "nucleotides"
and "oligonucleotides" are used interchangeably. They refer to a
polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof.
Polynucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: coding or non-coding
regions of a gene or gene fragment, loci (locus) defined from
linkage analysis, exons, introns, messenger RNA (mRNA), transfer
RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and
primers. A polynucleotide may comprise modified nucleotides, such
as methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component.
[0183] The term "complement of a polynucleotide" denotes a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence, such that
it could hybridize with a reference sequence with complete
fidelity.
[0184] "Recombinant" as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of
recombination steps which may include cloning, restriction and/or
ligation steps, and other procedures that result in expression of a
recombinant protein in a host cell.
[0185] The terms "gene" and "gene fragment" are used
interchangeably herein. They refer to a polynucleotide containing
at least one open reading frame that is capable of encoding a
particular protein after being transcribed and translated. A gene
or gene fragment may be genomic or cDNA, as long as the
polynucleotide contains at least one open reading frame, which may
cover the entire coding region or a segment thereof. A "fusion
gene" is a gene composed of at least two heterologous
polynucleotides that are linked together.
[0186] "Homology" or "homologous" or "sequence identity" refers to
sequence similarity or interchangeability between two or more
polynucleotide sequences or between two or more polypeptide
sequences. When using a program such as BestFit to determine
sequence identity, similarity or homology between two different
amino acid sequences, the default settings may be used, or an
appropriate scoring matrix, such as blosum45 or blosum80, may be
selected to optimize identity, similarity or homology scores.
Preferably, polynucleotides that are homologous are those which
hybridize under stringent conditions as defined herein and have at
least 70%, preferably at least 80%, more preferably at least 90%,
more preferably 95%, more preferably 97%, more preferably 98%, and
even more preferably 99% sequence identity compared to those
sequences. Polypeptides that are homologous preferably have
sequence identities that are at least 70%, preferably at least 80%,
even more preferably at least 90%, even more preferably at least
95-99% identical.
[0187] "Ligation" as applied to polynucleic acids refers to the
process of forming phosphodiester bonds between two nucleic acid
fragments or genes, linking them together. To ligate the DNA
fragments or genes together, the ends of the DNA must be compatible
with each other. In some cases, the ends will be directly
compatible after endonuclease digestion. However, it may be
necessary to first convert the staggered ends commonly produced
after endonuclease digestion to blunt ends to make them compatible
for ligation.
[0188] The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a
polynucleotide will hybridize to its target sequence, to a
detectably greater degree than other sequences (e.g., at least
2-fold over background). Generally, stringency of hybridization is
expressed, in part, with reference to the temperature and salt
concentration under which the wash step is carried out. Typically,
stringent conditions will be those in which the salt concentration
is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short
polynucleotides (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long polynucleotides (e.g., greater than 50
nucleotides)--for example, "stringent conditions" can include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and three washes for 15 min each in 0.1.times.SSC/1% SDS at
60.degree. C. to 65.degree. C. Alternatively, temperatures of about
65.degree. C., 60.degree. C., 55.degree. C., or 42.degree. C. may
be used. SSC concentration may be varied from about 0.1 to
2.times.SSC, with SDS being present at about 0.1%. Such wash
temperatures are typically selected to be about 5.degree. C. to
20.degree. C. lower than the thermal melting point for the specific
sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An
equation for calculating Tm and conditions for nucleic acid
hybridization are well known and can be found in Sambrook, J. et
al., "Molecular Cloning: A Laboratory Manual," 3.sup.rd edition,
Cold Spring Harbor Laboratory Press, 2001. Typically, blocking
reagents are used to block non-specific hybridization. Such
blocking reagents include, for instance, sheared and denatured
salmon sperm DNA at about 100-200 .mu.g/ml. Organic solvent, such
as formamide at a concentration of about 35-50% v/v, may also be
used under particular circumstances, such as for RNA:DNA
hybridizations. Useful variations on these wash conditions will be
readily apparent to those of ordinary skill in the art.
[0189] The terms "percent identity," percentage of sequence
identity," and "% identity," as applied to polynucleotide
sequences, refer to the percentage of residue matches between at
least two polynucleotide sequences aligned using a standardized
algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in the sequences being compared in order to
optimize alignment between two sequences, and therefore achieve a
more meaningful comparison of the two sequences. Percent identity
may be measured over the length of an entire defined polynucleotide
sequence, or may be measured over a shorter length, for example,
over the length of a fragment taken from a larger, defined
polynucleotide sequence, for instance, a fragment of at least 45,
at least 60, at least 90, at least 120, at least 150, at least 210
or at least 450 contiguous residues. Such lengths are exemplary
only, and it is understood that any fragment length supported by
the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to describe a length over which percentage
identity may be measured. The percentage of sequence identity is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of matched positions
(at which identical residues occur in both polypeptide sequences),
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. When sequences of different length are to be compared,
the shortest sequence defines the length of the window of
comparison. Conservative substitutions are not considered when
calculating sequence identity.
[0190] "Percent (%) sequence identity," with respect to the
polypeptide sequences identified herein, is defined as the
percentage of amino acid residues in a query sequence that are
identical with the amino acid residues of a second, reference
polypeptide sequence or a portion thereof, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity,
thereby resulting in optimal alignment. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve optimal alignment over the full
length of the sequences being compared. Percent identity may be
measured over the length of an entire defined polypeptide sequence,
or may be measured over a shorter length, for example, over the
length of a fragment taken from a larger, defined polypeptide
sequence, for instance, a fragment of at least 15, at least 20, at
least 30, at least 40, at least 50, at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is
understood that any fragment length supported by the sequences
shown herein, in the tables, figures or Sequence Listing, may be
used to describe a length over which percentage identity may be
measured.
[0191] "Repetitiveness" used in the context of polynucleotide
sequences refers to the degree of internal homology in the sequence
such as, for example, the frequency of identical nucleotide
sequences of a given length. Repetitiveness can, for example, be
measured by analyzing the frequency of identical sequences.
[0192] A "vector" is a nucleic acid molecule, preferably
self-replicating in an appropriate host, which transfers an
inserted nucleic acid molecule into and/or between host cells. The
term includes vectors that function primarily for insertion of DNA
or RNA into a cell, replication of vectors that function primarily
for the replication of DNA or RNA, and expression vectors that
function for transcription and/or translation of the DNA or RNA.
Also included are vectors that provide more than one of the above
functions. An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide(s). An "expression system" usually
connotes a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0193] "Serum degradation resistance," as applied to a polypeptide,
refers to the ability of the polypeptides to withstand degradation
in blood or components thereof, which typically involves proteases
in the serum or plasma. The serum degradation resistance can be
measured by combining the protein with human (or mouse, rat,
monkey, as appropriate) serum or plasma, typically for a range of
days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about
37.degree. C. The samples for these time points can be run on a
Western blot assay and the protein is detected with an antibody.
The antibody can be to a tag in the protein. If the protein shows a
single band on the western, where the protein's size is identical
to that of the injected protein, then no degradation has occurred.
In this exemplary method, the time point where 50% of the protein
is degraded, as judged by Western blots or equivalent techniques,
is the serum degradation half-life or "serum half-life" of the
protein.
[0194] The terms "t.sub.1/2", "half-life", "terminal half-life",
"elimination half-life" and "circulating half-life" are used
interchangeably herein and, as used herein means the terminal
half-life calculated as ln(2)/K.sub.e1. K.sub.e1 is the terminal
elimination rate constant calculated by linear regression of the
terminal linear portion of the log concentration vs. time curve.
Half-life typically refers to the time required for half the
quantity of an administered substance deposited in a living
organism to be metabolized or eliminated by normal biological
processes.
[0195] "Active clearance" means the mechanisms by which a protein
is removed from the circulation other than by filtration, and which
includes removal from the circulation mediated by cells, receptors,
metabolism, or degradation of the protein.
[0196] "Apparent molecular weight factor" and "apparent molecular
weight" are related terms referring to a measure of the relative
increase or decrease in apparent molecular weight exhibited by a
particular amino acid or polypeptide sequence. The apparent
molecular weight is determined using size exclusion chromatography
(SEC) or similar methods by comparing to globular protein
standards, and is measured in "apparent kD" units. The apparent
molecular weight factor is the ratio between the apparent molecular
weight and the actual molecular weight; the latter predicted by
adding, based on amino acid composition, the calculated molecular
weight of each type of amino acid in the composition or by
estimation from comparison to molecular weight standards in an SDS
electrophoresis gel. Determination of both the apparent molecular
weight and apparent molecular weight factor for representative
proteins is described in the Examples.
[0197] The terms "hydrodynamic radius" or "Stokes radius" is the
effective radius (R.sub.h in nm) of a molecule in a solution
measured by assuming that it is a body moving through the solution
and resisted by the solution's viscosity. In the embodiments of the
invention, the hydrodynamic radius measurements of the XTEN
polypeptides correlate with the "apparent molecular weight factor"
which is a more intuitive measure. The "hydrodynamic radius" of a
protein affects its rate of diffusion in aqueous solution as well
as its ability to migrate in gels of macromolecules. The
hydrodynamic radius of a protein is determined by its molecular
weight as well as by its structure, including shape and
compactness. Methods for determining the hydrodynamic radius are
well known in the art, such as by the use of size exclusion
chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and
7,294,513. Most proteins have globular structure, which is the most
compact three-dimensional structure a protein can have with the
smallest hydrodynamic radius. Some proteins adopt a random and
open, unstructured, or `linear` conformation and as a result have a
much larger hydrodynamic radius compared to typical globular
proteins of similar molecular weight.
[0198] "Physiological conditions" refers to a set of conditions in
a living host as well as in vitro conditions, including
temperature, salt concentration, pH, that mimic those conditions of
a living subject. A host of physiologically relevant conditions for
use in in vitro assays have been established. Generally, a
physiological buffer contains a physiological concentration of salt
and is adjusted to a neutral pH ranging from about 6.5 to about
7.8, and preferably from about 7.0 to about 7.5. A variety of
physiological buffers are listed in Sambrook et al. (2001).
Physiologically relevant temperature ranges from about 25.degree.
C. to about 38.degree. C., and preferably from about 35.degree. C.
to about 37.degree. C.
[0199] A "single atom residue of a payload" means the atom of a
payload that is chemically linked to XTEN after reaction with the
subject XTEN or XTEN-linker compositions; typically a sulfur, an
oxygen, a nitrogen, or a carbon atom. For example, an atom residue
of a payload could be a sulfur residue of a cysteine thiol reactive
group in a payload, a nitrogen molecule of an amino reactive group
of a peptide or polypeptide or small molecule payload, a carbon or
oxygen residue or a reactive carboxyl or aldehyde group of a
peptide, protein or a small molecule or synthetic, organic
drug.
[0200] A "reactive group" is a chemical structure that can be
coupled to a second reactive group. Examples of reactive groups are
amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups,
aldehyde groups, azide groups. Some reactive groups can be
activated to facilitate conjugation with a second reactive group,
either directly or through a cross-linker. As used herein, a
reactive group can be a part of an XTEN, a cross-linker, an
azide/alkyne click-chemistry reactant, or a payload so long as it
has the ability to participate in a chemical reaction. Once
reacted, a conjugation bond links the residues of the payload or
cross-linker or XTEN reactants.
[0201] "Controlled release agent", "slow release agent", "depot
formulation" and "sustained release agent" are used interchangeably
to refer to an agent capable of extending the duration of release
of a polypeptide of the invention relative to the duration of
release when the polypeptide is administered in the absence of
agent. Different embodiments of the present invention may have
different release rates, resulting in different therapeutic
amounts.
[0202] The term "payload" as used herein refers to any protein,
peptide sequence, small molecule, drug or composition of matter
that has a biological, pharmacological or therapeutic activity or
beneficial effect when administered in a subject or that can be
demonstrated in vitro. Payload also includes a molecule that can be
used for imaging or in vivo diagnostic purposes. Examples of
payloads include, but are not limited to, cytokines, enzymes,
hormones, blood coagulation factors, and growth factors,
chemotherapeutic agents, antiviral compounds, toxins, anti-cancer
drugs, radioactive compounds, and contrast agents, as well as
targeting peptides, proteins, antibodies, antibody fragments, or
compounds used to bind to receptors or ligands.
[0203] The terms "antigen", "target antigen" and "immunogen" are
used interchangeably herein to refer to the structure or binding
determinant that an antibody fragment or an antibody fragment-based
therapeutic binds to or has specificity against.
[0204] The term "antagonist", as used herein, includes any molecule
that partially or fully blocks, inhibits, or neutralizes a
biological activity of a native polypeptide disclosed herein.
Methods for identifying antagonists of a polypeptide may comprise
contacting a native polypeptide with a candidate antagonist
molecule and measuring a detectable change in one or more
biological activities normally associated with the native
polypeptide. In the context of the present invention, antagonists
may include proteins, nucleic acids, carbohydrates, antibodies or
any other molecules that decrease the effect of a biologically
active protein.
[0205] A "defined medium" refers to a medium comprising nutritional
and hormonal requirements necessary for the survival and/or growth
of the cells in culture such that the components of the medium are
known. Traditionally, the defined medium has been formulated by the
addition of nutritional and growth factors necessary for growth
and/or survival. Typically, the defined medium provides at least
one component from one or more of the following categories: a) all
essential amino acids, and usually the basic set of twenty amino
acids plus cysteine; b) an energy source, usually in the form of a
carbohydrate such as glucose; c) vitamins and/or other organic
compounds required at low concentrations; d) free fatty acids; and
e) trace elements, where trace elements are defined as inorganic
compounds or naturally occurring elements that are typically
required at very low concentrations, usually in the micromolar
range. The defined medium may also optionally be supplemented with
one or more components from any of the following categories: a) one
or more mitogenic agents; b) salts and buffers as, for example,
calcium, magnesium, and phosphate; c) nucleosides and bases such
as, for example, adenosine and thymidine, hypoxanthine; and d)
protein and tissue hydrolysates.
[0206] The term "agonist" is used in the broadest sense and
includes any molecule that mimics a biological activity of a native
polypeptide disclosed herein. Suitable agonist molecules
specifically include agonist antibodies or antibody fragments,
fragments or amino acid sequence variants of native polypeptides,
peptides, small organic molecules, etc. Methods for identifying
agonists of a native polypeptide may comprise contacting a native
polypeptide with a candidate agonist molecule and measuring a
detectable change in one or more biological activities normally
associated with the native polypeptide.
[0207] "Inhibition constant", or "Ki", are used interchangeably and
mean the dissociation constant of the enzyme-inhibitor complex, or
the reciprocal of the binding affinity of the inhibitor to the
enzyme.
[0208] As used herein, "treat" or "treating," or "palliating" or
"ameliorating" are used interchangeably and mean administering a
drug or a biologic to achieve a therapeutic benefit, to cure or
reduce the severity of an existing condition, or to achieve a
prophylactic benefit, prevent or reduce the likelihood of onset or
severity the occurrence of a condition. By therapeutic benefit is
meant eradication or amelioration of the underlying condition being
treated or one or more of the physiological symptoms associated
with the underlying condition such that an improvement is observed
in the subject, notwithstanding that the subject may still be
afflicted with the underlying condition.
[0209] A "therapeutic effect" or "therapeutic benefit," as used
herein, refers to a physiologic effect, including but not limited
to the mitigation, amelioration, or prevention of disease in humans
or other animals, or to otherwise enhance physical or mental
wellbeing of humans or animals, resulting from administration of a
polypeptide of the invention other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
the biologically active protein. For prophylactic benefit, the
compositions may be administered to a subject at risk of developing
a particular disease, condition or symptom of the disease (e.g., a
bleed in a diagnosed hemophilia A subject), or to a subject
reporting one or more of the physiological symptoms of a disease,
even though a diagnosis of this disease may not have been made.
[0210] The terms "therapeutically effective amount" and
"therapeutically effective dose", as used herein, refer to an
amount of a drug or a biologically active protein, either alone or
as a part of a polypeptide composition, that is capable of having
any detectable, beneficial effect on any symptom, aspect, measured
parameter or characteristics of a disease state or condition when
administered in one or repeated doses to a subject. Such effect
need not be absolute to be beneficial. Determination of a
therapeutically effective amount is well within the capability of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0211] The term "therapeutically effective dose regimen", as used
herein, refers to a schedule for consecutively administered
multiple doses (i.e., at least two or more) of a biologically
active protein, either alone or as a part of a polypeptide
composition, wherein the doses are given in therapeutically
effective amounts to result in sustained beneficial effect on any
symptom, aspect, measured parameter or characteristics of a disease
state or condition.
I). General Techniques
[0212] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, J. et al., "Molecular Cloning: A
Laboratory Manual," 3.sup.rd edition, Cold Spring Harbor Laboratory
Press, 2001; "Current protocols in molecular biology", F. M.
Ausubel, et al. eds., 1987; the series "Methods in Enzymology,"
Academic Press, San Diego, Calif.; "PCR 2: a practical approach",
M. J. MacPherson, B. D. Hames and G. R. Taylor eds., Oxford
University Press, 1995; "Antibodies, a laboratory manual" Harlow,
E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; "Goodman
& Gilman's The Pharmacological Basis of Therapeutics,"
11.sup.th Edition, McGraw-Hill, 2005; and Freshney, R I., "Culture
of Animal Cells: A Manual of Basic Technique," 4.sup.th edition,
John Wiley & Sons, Somerset, N J, 2000, the contents of which
are incorporated in their entirety herein by reference.
[0213] Host cells can be cultured in a variety of media.
Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing
eukaryotic cells. In addition, animal cells can be grown in a
defined medium that lacks serum but is supplemented with hormones,
growth factors or any other factors necessary for the survival
and/or growth of a particular cell type. Whereas a defined medium
supporting cell survival maintains the viability, morphology,
capacity to metabolize and potentially, capacity of the cell to
differentiate, a defined medium promoting cell growth provides all
chemicals necessary for cell proliferation or multiplication. The
general parameters governing mammalian cell survival and growth in
vitro are well established in the art. Physicochemical parameters
which may be controlled in different cell culture systems are,
e.g., pH, pO.sub.2, temperature, and osmolarity. The nutritional
requirements of cells are usually provided in standard media
formulations developed to provide an optimal environment. Nutrients
can be divided into several categories: amino acids and their
derivatives, carbohydrates, sugars, fatty acids, complex lipids,
nucleic acid derivatives and vitamins. Apart from nutrients for
maintaining cell metabolism, most cells also require one or more
hormones from at least one of the following groups: steroids,
prostaglandins, growth factors, pituitary hormones, and peptide
hormones to proliferate in serum-free media (Sato, G. H., et al. in
"Growth of Cells in Hormonally Defined Media", Cold Spring Harbor
Press, N.Y., 1982). In addition to hormones, cells may require
transport proteins such as transferrin (plasma iron transport
protein), ceruloplasmin (a copper transport protein), and
high-density lipoprotein (a lipid carrier) for survival and growth
in vitro. The set of optimal hormones or transport proteins will
vary for each cell type. Most of these hormones or transport
proteins have been added exogenously or, in a rare case, a mutant
cell line has been found which does not require a particular
factor. Those skilled in the art will know of other factors
required for maintaining a cell culture without undue
experimentation.
[0214] Growth media for growth of prokaryotic host cells include
nutrient broths (liquid nutrient medium) or LB medium (Luria
Bertani). Suitable media include defined and undefined media. In
general, media contains a carbon source such as glucose needed for
bacterial growth, water, and salts. Media may also include a source
of amino acids and nitrogen, for example beef or yeast extract (in
an undefined medium) or known quantities of amino acids (in a
defined medium). In some embodiments, the growth medium is LB
broth, for example LB Miller broth or LB Lennox broth. LB broth
comprises peptone (enzymatic digestion product of casein), yeast
extract and sodium chloride. In some embodiments, a selective
medium is used which comprises an antibiotic. In this medium, only
the desired cells possessing resistance to the antibiotic will
grow.
II). XTEN Protein Polymer and Conjugate Compositions
[0215] The present invention relates, in part, to substantially
homogeneous compositions comprising extended recombinant
polypeptides (XTEN). In a first aspect, the invention provides XTEN
compositions that are substantially homogeneous in length. Such
compositions are useful as reagent conjugation partners to create
XTEN-cross-linker intermediates and XTEN-payload compositions.
Additionally, it is an object of the present invention to provide
methods to create the substantially homogeneous XTEN compositions.
The present invention also provides methods to create such
substantially homogeneous XTEN compositions at high yield.
[0216] In a second aspect, the invention provides XTEN. For
example, the XTENs capable of linking to one or more payload
conjugation partners, resulting in payload-XTEN conjugates are
specifically engineered to incorporate defined numbers of reactive
amino acids for linking to the payloads either directly or via
cross-linkers or azide/alkyne reactants. The present invention also
provides methods to create such engineered XTEN polymers for use in
creating conjugates with payload agents of interest as compositions
with enhanced pharmaceutical properties, including enhanced
pharmacokinetic and pharmacologic properties, as well as reduced
toxicity.
[0217] In another aspect, the invention provides substantially
homogeneous XTEN polymers comprising defined numbers of
cross-linkers or azide/alkyne reactants as reactant conjugation
partners in monomeric and multimeric configurations and methods of
the preparation of such reactants. The XTEN derivatives comprising
cross-linkers or azide/alkyne reactants are used as reactants in
the conjugation of payload agents to result in XTEN-payload
conjugate exhibiting the desired physical, pharmaceutical, and
pharmacological properties.
[0218] In another aspect, the invention provides compositions of
XTEN-payload in which one or more XTEN are chemically linked to one
or more payloads, including combinations of different payloads, in
defined numbers in either monomeric or multimeric configurations to
provide compositions with enhanced pharmaceutical, pharmacokinetic,
and pharmacologic properties. Such compositions linked to such
payloads may have utility, when administered to a subject, in the
prevention, treatment or amelioration of diseases or conditions due
to a pharmacologic or biologic effect of the payload.
[0219] 1. XTEN: Extended Recombinant Polypeptides
[0220] In one aspect, the invention provides substantially
homogeneous XTEN polypeptide compositions that are useful as
conjugation partners to link to one or more payloads, either
directly or via a cross-linker reactant resulting in an
XTEN-payload conjugate.
[0221] XTEN are polypeptides with non-naturally occurring,
substantially non-repetitive sequences having a low degree or no
secondary or tertiary structure under physiologic conditions. XTEN
typically have from about 36 to about 3000 amino acids, of which
the majority or the entirety are small hydrophilic amino acids. As
used herein, "XTEN" specifically excludes whole antibodies or
antibody fragments (e.g. single-chain antibodies and Fc fragments).
XTEN polypeptides have utility as a conjugation partners in that
they serve in various roles, conferring certain desirable
properties when linked to a payload. The resulting XTEN-payload
conjugates have enhanced properties, such as enhanced
pharmacokinetic, physicochemical, pharmacologic, and pharmaceutical
properties compared to the corresponding payload not linked to
XTEN, making them useful in the treatment of certain conditions for
which the payload is known in the art to be used.
[0222] The unstructured characteristic and physicochemical
properties of the XTEN result, in part, from the overall amino acid
composition that is disproportionately limited to 4-6 types of
hydrophilic amino acids, the linking of the amino acids in a
quantifiable non-repetitive design, and the length of the XTEN
polypeptide. In an advantageous feature common to XTEN but uncommon
to native polypeptides, the properties of XTEN disclosed herein are
not tied to absolute primary amino acid sequences, as evidenced by
the diversity of the exemplary sequences of Table 2 that, within
varying ranges of length, possess similar properties, many of which
are documented in the Examples. Accordingly, XTEN have properties
more like non-proteinaceous, hydrophilic polymers than they do
proteins. The XTEN of the present invention exhibit one or more of
the following advantageous properties: conformational flexibility,
reduced or lack of secondary structure, high degree of aqueous
solubility, high degree of protease resistance, low immunogenicity,
low binding to mammalian receptors, a defined degree of charge, and
increased hydrodynamic (or Stokes) radii; properties that are
similar to certain hydrophilic polymers (e.g., polyethylene glycol)
that make them particularly useful as conjugation partners.
[0223] The XTEN component(s) of the subject conjugates are designed
to behave like denatured peptide sequences under physiological
conditions, despite the extended length of the polymer. "Denatured"
describes the state of a peptide in solution that is characterized
by a large conformational freedom of the peptide backbone. Most
peptides and proteins adopt a denatured conformation in the
presence of high concentrations of denaturants or at elevated
temperature. Peptides in denatured conformation have, for example,
characteristic circular dichroism (CD) spectra and are
characterized by a lack of long-range interactions as determined by
NMR. "Denatured conformation" and "unstructured conformation" are
used synonymously herein. In some embodiments, the invention
provides XTEN sequences that, under physiologic conditions,
resemble denatured sequences that are largely devoid of secondary
structure. In other cases, the XTEN sequences are substantially
devoid of secondary structure under physiologic conditions.
"Largely devoid," as used in this context, means that less than 50%
of the XTEN amino acid residues of the XTEN sequence contribute to
secondary structure as measured or determined by the means
described herein. "Substantially devoid," as used in this context,
means that at least about 60%, or about 70%, or about 80%, or about
90%, or about 95%, or about 97%, or at least about 99% of the XTEN
amino acid residues of the XTEN sequence do not contribute to
secondary structure, as measured or determined by the methods
described herein.
[0224] A variety of methods and assays are known in the art for
determining the physicochemical properties of the subject XTEN.
Such properties include but are not limited to secondary or
tertiary structure, solubility, protein aggregation, stability,
absolute and apparent molecular weight, purity and uniformity,
melting properties, contamination and water content. The methods to
measure such properties include analytical centrifugation, EPR,
HPLC-ion exchange, HPLC-size exclusion chromatography (SEC),
HPLC-reverse phase, light scattering, capillary electrophoresis,
circular dichroism, differential scanning calorimetry,
fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR,
Raman spectroscopy, refractometry, and UV/Visible spectroscopy. In
particular, secondary structure can be measured
spectrophotometrically, e.g., by circular dichroism spectroscopy in
the "far-UV" spectral region (190-250 nm). Secondary structure
elements, such as alpha-helix and beta-sheet, each give rise to a
characteristic shape and magnitude of CD spectra, as does the lack
of these structure elements. Secondary structure can also be
predicted for a polypeptide sequence via certain computer programs
or algorithms, such as the well-known Chou-Fasman algorithm (Chou,
P. Y., et al. (1974) Biochemistry, 13: 222-45) and the
Garnier-Osguthorpe-Robson algorithm ("Gor algorithm") (Gamier J,
Gibrat J F, Robson B. (1996), GOR method for predicting protein
secondary structure from amino acid sequence. Methods Enzymol
266:540-553), as described in US Patent Application Publication No.
20030228309A1. For a given sequence, the algorithms can predict
whether there exists some or no secondary structure at all,
expressed as the total and/or percentage of residues of the
sequence that form, for example, alpha-helices or beta-sheets or
the percentage of residues of the sequence predicted to result in
random coil formation (which lacks secondary structure).
Polypeptide sequences can be analyzed using the Chou-Fasman
algorithm using sites on the world wide web at, for example,
fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 and the
Gor algorithm at
npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.p1?page=npsa_gor4.html (both
accessed on Sep. 5, 2012). Additional methods are disclosed in
Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.
[0225] In one embodiment, the XTEN sequences used in the subject
conjugates have an alpha-helix percentage ranging from 0% to less
than about 5% as determined by the Chou-Fasman algorithm. In
another embodiment, the XTEN sequences have a beta-sheet percentage
ranging from 0% to less than about 5% as determined by the
Chou-Fasman algorithm. In one embodiment, the XTEN sequences of the
conjugates have an alpha-helix percentage ranging from 0% to less
than about 5% and a beta-sheet percentage ranging from 0% to less
than about 5% as determined by the Chou-Fasman algorithm. In one
embodiment, the XTEN sequences of the conjugates have an
alpha-helix percentage less than about 2% and a beta-sheet
percentage less than about 2%. The XTEN sequences of the conjugate
compositions have a high degree of random coil percentage, as
determined by the GOR algorithm. In some embodiments, an XTEN
sequence has at least about 80%, more preferably at least about
90%, more preferably at least about 91%, more preferably at least
about 92%, more preferably at least about 93%, more preferably at
least about 94%, more preferably at least about 95%, more
preferably at least about 96%, more preferably at least about 97%,
more preferably at least about 98%, and most preferably at least
about 99% random coil, as determined by the GOR algorithm. In one
embodiment, the XTEN sequences of the conjugate compositions have
an alpha-helix percentage ranging from 0% to less than about 5% and
a beta-sheet percentage ranging from 0% to less than about 5% as
determined by the Chou-Fasman algorithm and at least about 90%
random coil, as determined by the GOR algorithm. In another
embodiment, the XTEN sequences of the disclosed compositions have
an alpha-helix percentage less than about 2% and a beta-sheet
percentage less than about 2% at least about 90% random coil, as
determined by the GOR algorithm. In another embodiment, the XTEN
sequences of the compositions are substantially lacking secondary
structure as measured by circular dichroism.
[0226] The selection criteria for the XTEN to be linked to the
payload used to create the conjugate compositions generally relate
to attributes of physicochemical properties and conformational
structure of the XTEN that is, in turn, used to confer enhanced
pharmaceutical, pharmacologic, and pharmacokinetic properties to
the compositions.
1. Non-Repetitive Sequences
[0227] It is specifically contemplated that the subject XTEN
sequences included in the subject conjugate composition embodiments
are substantially non-repetitive. In general, repetitive amino acid
sequences have a tendency to aggregate or form higher order
structures, as exemplified by natural repetitive sequences such as
collagens and leucine zippers. These repetitive amino acids may
also tend to form contacts resulting in crystalline or
pseudocrystaline structures. In contrast, the low tendency of
non-repetitive sequences to aggregate enables the design of
long-sequence XTENs with a relatively low frequency of charged
amino acids that would otherwise be likely to aggregate if the
sequences were repetitive. The non-repetitiveness of a subject XTEN
can be observed by assessing one or more of the following features.
In one embodiment, a substantially non-repetitive XTEN sequence has
no three contiguous amino acids in the sequence that are identical
amino acid types unless the amino acid is serine, in which case no
more than three contiguous amino acids are serine residues. In
another embodiment, as described more fully below, a substantially
non-repetitive XTEN sequence in which 80-99% of the sequence is
comprised of motifs of 9 to 14 amino acid residues wherein the
motifs consist of 3, 4, 5 or 6 types of amino acids selected from
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
and proline (P), and wherein the sequence of any two contiguous
amino acid residues in any one motif is not repeated more than
twice in the sequence motif.
[0228] The degree of repetitiveness of a polypeptide or a gene can
be measured by computer programs or algorithms or by other means
known in the art. According to the current invention, algorithms to
be used in calculating the degree of repetitiveness of a particular
polypeptide, such as an XTEN, are disclosed herein, and examples of
sequences analyzed by algorithms are provided (see Examples,
below). In one embodiment, the repetitiveness of a polypeptide of a
predetermined length can be calculated (hereinafter "subsequence
score") according to the formula given by Equation I:
.times. Subsequence .times. .times. score = i = 1 m .times. count i
m ##EQU00001## wherein .times. : .times. .times. m = ( amino
.times. .times. acid .times. .times. length .times. .times. of
.times. .times. polypeptide ) - ( amino .times. .times. acid
.times. .times. length .times. .times. of .times. .times.
subsequence ) + 1 ; and ##EQU00001.2## Count i - cumulative .times.
.times. number .times. .times. of .times. .times. occurrences
.times. .times. of .times. .times. each .times. .times. unique
.times. .times. subsequence .times. .times. within .times. .times.
sequence i ##EQU00001.3##
[0229] An algorithm termed "SegScore" was developed to apply the
foregoing equation to quantitate repetitiveness of polypeptides,
such as an XTEN, providing the subsequence score wherein sequences
of a predetermined amino acid length "n" are analyzed for
repetitiveness by determining the number of times (a "count") a
unique subsequence of length "s" appears in the set length, divided
by the absolute number of subsequences within the predetermined
length of the sequence. FIG. 79 depicts a logic flowchart of the
SegScore algorithm, while FIG. 80 portrays a schematic of how a
subsequence score is derived for a fictitious XTEN with 11 amino
acids and a subsequence length of 3 amino acid residues. For
example, a predetermined polypeptide length of 200 amino acid
residues has 192 overlapping 9-amino acid subsequences and 1983-mer
subsequences, but the subsequence score of any given polypeptide
will depend on the absolute number of unique subsequences and how
frequently each unique subsequence (meaning a different amino acid
sequence) appears in the predetermined length of the sequence.
[0230] In the context of the present invention, "subsequence score"
means the sum of occurrences of each unique 3-mer frame across 200
consecutive amino acids of the cumulative XTEN polypeptide divided
by the absolute number of unique 3-mer subsequences within the 200
amino acid sequence. Examples of such subsequence scores derived
from 200 consecutive amino acids of repetitive and non-repetitive
polypeptides are presented in Example 45. In one embodiment, the
invention provides a XTEN-payload comprising one XTEN in which the
XTEN has a subsequence score less than 12, more preferably less
than 10, more preferably less than 9, more preferably less than 8,
more preferably less than 7, more preferably less than 6, and most
preferably less than 5. In another embodiment, the invention
provides XTEN-cross-linker conjugates comprising an XTEN in which
the XTEN have a subsequence score of less than 10, more preferably
less than 9, more preferably less than 8, more preferably less than
7, more preferably less than 6, and most preferably less than 5. In
another embodiment, the invention provides XTEN-click-chemistry
conjugates comprising an XTEN in which the XTEN have a subsequence
score of less than 10, more preferably less than 9, more preferably
less than 8, more preferably less than 7, more preferably less than
6, and most preferably less than 5. In yet another embodiment, the
invention provides XTEN conjugate compositions comprising at least
two linked XTEN in which each individual XTEN has a subsequence
score of less than 10, or less than 9, or less than 8, or less than
7, or less than 6, or less than 5, or less. In yet another
embodiment, the invention provides XTEN conjugate compositions
comprising at least three linked XTEN in which each individual XTEN
has a subsequence score of less than 10, or less than 9, or less
than 8, or less than 7, or less than 6, or less than 5, or less. In
the embodiments of the XTEN compositions described herein, an XTEN
with a subsequence score of 10 or less (i.e., 9, 8, 7, etc.) is
characterized as substantially non-repetitive.
[0231] In one aspect, the non-repetitive characteristic of XTEN of
the present invention together with the particular types of amino
acids that predominate in the XTEN, rather than the absolute
primary sequence, confers one or more of the enhanced
physicochemical and biological properties of the XTEN and the
resulting XTEN-payload conjugates. These enhanced properties
include a higher degree of expression of the XTEN protein in the
host cell, greater genetic stability of the gene encoding XTEN, a
greater degree of solubility, less tendency to aggregate, and
enhanced pharmacokinetics of the resulting conjugate compared to
payloads not conjugated to XTEN or payloads conjugated to proteins
having repetitive sequences. These enhanced properties permit more
efficient manufacturing, greater uniformity of the final product,
lower cost of goods, and/or facilitate the formulation of
XTEN-comprising pharmaceutical preparations containing extremely
high protein concentrations, in some cases exceeding 100 mg/ml. In
some embodiments, the XTEN polypeptide sequences of the conjugates
are designed to have a low degree of internal repetitiveness in
order to reduce or substantially eliminate immunogenicity when
administered to a mammal. Polypeptide sequences composed of short,
repeated motifs largely limited to only three amino acids, such as
glycine, serine and glutamate, may result in relatively high
antibody titers when administered to a mammal despite the absence
of predicted T-cell epitopes in these sequences. This may be caused
by the repetitive nature of polypeptides, as it has been shown that
immunogens with repeated epitopes, including protein aggregates,
cross-linked immunogens, and repetitive carbohydrates are highly
immunogenic and can, for example, result in the cross-linking of
B-cell receptors causing B-cell activation. (Johansson, J., et al.
(2007) Vaccine, 25:1676-82; Yankai, Z., et al. (2006) Biochem
Biophys Res Commun, 345:1365-71; Hsu, C. T., et al. (2000) Cancer
Res, 60:3701-5); Bachmann M F, et al. Eur J Immunol. (1995)
25(12):3445-3451).
2. Exemplary Sequence Motifs
[0232] The present invention encompasses XTEN used as conjugation
partners that comprise multiple units of shorter sequences, or
motifs, in which the amino acid sequences of the motifs are
substantially non-repetitive. The non-repetitive property can be
met even using a "building block" approach using a library of
sequence motifs that are multimerized to create the XTEN sequences,
as shown in FIGS. 18-19. While an XTEN sequence may consist of
multiple units of as few as four different types of sequence
motifs, because the motifs themselves generally consist of
non-repetitive amino acid sequences, the overall XTEN sequence is
designed to render the sequence substantially non-repetitive.
[0233] In one embodiment, an XTEN has a substantially
non-repetitive sequence of greater than about 36 to about 3000, or
about 100 to about 2000, or about 144 to about 1000 amino acid
residues, wherein at least about 80%, or at least about 85%, or at
least about 90%, or at least about 95%, or at least about 97%, or
about 99% to about 100% of the XTEN sequence consists of
non-overlapping sequence motifs, and wherein each of the motifs has
about 9 to 36 amino acid residues. As used herein,
"non-overlapping" means that the individual motifs do not share
amino acid residues but, rather, are fused to other motifs or amino
acid residues in a linear fashion. In other embodiments, at least
about 80%, or at least about 85%, or at least about 90%, or at
least about 95%, or at least about 97%, or about 99% to about 100%
of the XTEN sequence consists of non-overlapping sequence motifs
wherein each of the motifs has 9 to 14 amino acid residues. In
still other embodiments, at least about 80%, or at least about 85%,
or at least about 90%, or at least about 95%, or at least about
97%, or about 99% to about 100% of the XTEN sequence consists of
non-overlapping sequence motifs wherein each of the motifs has 12
amino acid residues. In these embodiments, it is preferred that the
sequence motifs are composed of substantially (e.g., 90% or more)
or exclusively small hydrophilic amino acids, such that the overall
sequence has an unstructured, flexible characteristic. Examples of
amino acids that are included in XTEN are, e.g., arginine, lysine,
threonine, alanine, asparagine, glutamine, aspartate, glutamate,
serine, and glycine. In one embodiment, XTEN sequences have
predominately four to six types of amino acids selected from
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
or proline (P) that are arranged in a substantially non-repetitive
sequence that is about 36 to about 3000, or about 100 to about
2000, or about 144 to about 1000 amino acid residues in length. In
some embodiment, an XTEN sequence is made of 4, 5, or 6 types of
amino acids selected from the group consisting of glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) or proline
(P). In some embodiments, XTEN have sequences of about 36 to about
1000, or about 100 to about 2000, or about 400 to about 3000 amino
acid residues wherein at least about 80% of the sequence consists
of non-overlapping sequence motifs wherein each of the motifs has 9
to 36 amino acid residues and wherein at least 90%, or at least
91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or 100% of each of the
motifs consists of 4 to 6 types of amino acids selected from
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
and proline (P), and wherein the content of any one amino acid type
in the full-length XTEN does not exceed 30%. In other embodiments,
at least about 90% of the XTEN sequence consists of non-overlapping
sequence motifs wherein each of the motifs has 9 to 36 amino acid
residues wherein the motifs consist of 4 to 6 types of amino acids
selected from glycine (G), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P), and wherein the content of any one
amino acid type in the full-length XTEN does not exceed 40%, or
about 30%, or 25%, or about 17%, or about 12%, or about 8%. In
other embodiments, at least about 90% of the XTEN sequence consists
of non-overlapping sequence motifs wherein each of the motifs has
12 amino acid residues consisting of 4 to 6 types of amino acids
selected from glycine (G), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P), and wherein the content of any one
amino acid type in the full-length XTEN does not exceed 40%, or
30%, or about 25%, or about 17%, or about 12%, or about 8%. In yet
other embodiments, at least about 90%, or about 91%, or about 92%,
or about 93%, or about 94%, or about 95%, or about %%, or about
97%, or about 98%, or about 99%, to about 100% of the XTEN sequence
consists of non-overlapping sequence motifs wherein each of the
motifs has 12 amino acid residues consisting of glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P).
[0234] In some embodiments, the invention provides XTEN-payload,
XTEN-cross-linker, and XTEN-click-chemistry reactant conjugates
comprising one, or two, or three, or four or more substantially
non-repetitive XTEN sequence(s) of about 36 to about 1000 amino
acid residues, or cumulatively about 100 to about 3000 amino acid
residues wherein at least about 80%, or at least about 90%, or
about 91%, or about 92%, or about 93%, or about 94%, or about 95%,
or about %%, or about 97%, or about 98%, or about 99% to about 100%
of the sequence consists of multiple units of four or more
non-overlapping sequence motifs selected from the amino acid
sequences of Table 1, wherein the overall sequence remains
substantially non-repetitive. In some embodiments, the XTEN
comprises non-overlapping sequence motifs in which about 80%, or at
least about 85%, or at least about 90%, or about 91%, or about 92%,
or about 93%, or about 94%, or about 95%, or about 96%, or about
97%, or about 98%, or about 99% or about 100% of the sequence
consists of multiple units of non-overlapping sequences selected
from a single motif family selected from Table 1, resulting in a
family sequence. Family, as applied to motifs, means that the XTEN
has motifs selected from a single motif category from Table 1;
i.e., AD, AE, AF, AG, AM, AQ, BC, or BD. In other embodiments, the
XTEN comprises multiple units of motif sequences from two or more
of the motif families of Table 1 selected to achieve desired
physicochemical characteristics, including such properties as net
charge, hydrophilicity, lack of secondary structure, or lack of
repetitiveness that may be conferred by the amino acid composition
of the motifs, described more fully below. In the embodiments
hereinabove described in this paragraph, the motifs or portions of
the motifs incorporated into the XTEN can be selected and assembled
using the methods described herein to achieve an XTEN of about 36,
about 42, about 72, about 144, about 288, about 576, about 864,
about 1000, about 2000 to about 3000 amino acid residues, or any
intermediate length. Non-limiting examples of XTEN family sequences
useful for incorporation into the subject conjugates are presented
in Table 2. It is intended that a specified sequence mentioned
relative to Table 2 has that sequence set forth in Table 2, while a
generalized reference to an AE144 sequence, for example, is
intended to encompass any AE sequence having 144 amino acid
residues; e.g., AE144_1A, AE144_2A, etc., or a generalized
reference to an AG144 sequence, for example, is intended to
encompass any AG sequence having 144 amino acid residues, e.g.,
AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, etc.
TABLE-US-00001 TABLE 1 XTEN Sequence Motifs of 12 Amino Acids and
Motif Families Motif MOTIF SEQ ID Family* SEQUENCE NO: AD
GESPGGSSGSES 26 AD GSEGSSGPGESS 27 AD GSSESGSSEGGP 28 AD
GSGGEPSESGSS 29 AE, AM GSPAGSPTSTEE 30 AE, AM, AQ GSEPATSGSETP 31
AE, AM, AQ GTSESATPESGP 32 AE, AM, AQ GTSTEPSEGSAP 33 AF, AM
GSTSESPSGTAP 34 AF, AM GTSTPESGSASP 35 AF, AM GTSPSGESSTAP 36 AF,
AM GSTSSTAESPGP 37 AG, AM GTPGSGTASSSP 38 AG, AM GSSTPSGATGSP 39
AG, AM GSSPSASTGTGP 40 AG, AM GASPGTSSTGSP 41 AQ GEPAGSPTSTSE 42 AQ
GTGEPSSTPASE 43 AQ GSGPSTESAPTE 44 AQ GSETPSGPSETA 45 AQ
GPSETSTSEPGA 46 AQ GSPSEPTEGTSA 47 BC GSGASEPTSTEP 48 BC
GSEPATSGTEPS 49 BC GTSEPSTSEPGA 50 BC GTSTEPSEPGSA 51 BD
GSTAGSETSTEA 52 BD GSETATSGSETA 53 BD GTSESATSESGA 54 BD
GTSTEASEGSAS 55 *Denotes individual motif sequences that, when used
together in various permutations, results in a "family
sequence"
TABLE-US-00002 TABLE 2 XTEN Polypeptides SEQ XTEN ID Name Amino
Acid Sequence NO: AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS
56 AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 57 AE42_2
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 58 AE42_3
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 59 AG42_1
GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP 60 AG42_2
GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP 61 AG42_3
SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 62 AG42_4
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 63 AE48
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS 64 AM48
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS 65 AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS 66
EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144_lA
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 67
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_2A
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP 68
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSESATPESGPG AE144_2B
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP 69
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSESATPESGPG AE144_3A
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 70
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_3B
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 71
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_4A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP 72
ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_4B
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP 73
ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_5A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP 74
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG AE144_6B
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG 75
SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPG AF144
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESP 76
SGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGT
SPSGESSTAPGTSPSGESSTAPGTSPSGESSTAP AG144_1
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS 77
PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASP AG144_2
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSA 78
STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AG144_A
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSG 79
ATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP AG144_B
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG 80
ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP AG144_C
GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS 81
TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGS
STPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AG144_F
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG 82
ATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AG144_3
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS 83
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS
SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP AG144_4
GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS 84
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGS
SPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP AE288_1
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT 85
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG
PGTSTEPSEGSAP AE288_2
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS 86
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGTSTEPSEGSAP AG288_1
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG 87
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS
SPGSSTPSGATGS AG288_2
GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 88
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSST
PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSP AF504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSG 89
ATGSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS PGTSSTGSP
AF540 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTA 90
ESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS
TSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESS
TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTS
ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSAS
PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES
PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPG
TSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPS
GTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTS
PSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGS 91
SEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGS
SESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSG
SESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGG
EPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGS
SGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGG
SSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSG
SEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSE
SGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSS
ESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGE
SSGSSESGSSEGGPGSEGSSGPGESS AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS 92
EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS
TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST
EEGTSESATPESGPGTSTEPSEGSAP AF576
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTA 93
ESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS
TSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESS
TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTS
ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSAS
PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSES
PSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPG
TSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPS
GTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTS
PSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESP
GPGTSTPESGSASPGTSTPESGSASP AG576
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS 94
GATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST
GTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGS
SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGS AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGS 95
PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSES
ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE
SATPESGPGTSTEPSEGSAP AD836
GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGS 96
SGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGE
SPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSE
GGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGG
EPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGES
SGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEP
SESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPG
SGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGP
GESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGES
PGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGS
SESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPG
ESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGG
EPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGG
PGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEP
SESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESG SGGEPSESGSS
AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS 97
EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS
TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST
EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP
TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS
EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG
SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AF864
GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPES 98
GSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGS
TSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS
TAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSAS
PGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE
SGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPG
TSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAP
STXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTA
PGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSES
PSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPG
STSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPS
GTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTS
PSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESP
GPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AG864_2
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSG 99
ATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS
STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPES 100
GSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS
EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPS
GATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPG
SEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAP
STGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTS
ESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG
PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSST
AESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG
STSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGT
GPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPES 101
GSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS
EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPS
GATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPG
SEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPA
PSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
EGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSES
PSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATG
SPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASAS
GAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS
TGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA
SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSP
GSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTS
STGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP BC864
GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATS 102
GTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGS
EPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEP
GSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGA
SEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGS
AGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPAT
SGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPG
TSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPT
STEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTS
TEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTE
PSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTE
PSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGA
GSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATS
GTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGS
EPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEP
GSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA BD864
GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATS 103
GSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGS
ETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSE
SGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSET
ATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSA
SGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESA
TSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAG
SETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSG
SETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTS
TEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETST
EAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSES
ATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETA
GTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATS
GSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGS
ETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGS
ETAGTSESATSESGAGTSESATSESGAGSETATSGSETA AE948
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS 104
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
SAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEP
ATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSA
PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS
TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGP AE1044
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSP 105
TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS
EPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEG
SAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEP
SEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGS
ETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
PTSTEEGTSESATPESGPGTSESATPESGPGTST AE1140
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESAT 106
PESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG
SAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEP
SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP
AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTE
PSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGT
SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE
SGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEP
ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
PGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA AE1236
GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS 107
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTS
TEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSET
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
TSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTE
PSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGP
GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGT
SESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
ETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEP
ATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP AE1332
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATS 108
GSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESG
PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP
SEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSG
SETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS
TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSES
ATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT
SESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGS
ETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSET
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESA
TPESGPGTSESATPESGPGTSTEPSEGSAPGTST AE1428
GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESAT 109
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSE
TPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSES
ATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG
SAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTST
EPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSESATPESGPGSPA AE1524
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP 110
TSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS
TEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEP
SEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEG
TSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSE
PATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATS
GSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS
TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTST
EPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGSPA AE1620
GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESAT 111
PESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTS
TEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSE
PATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE
GTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGS
EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGS
ETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST AE1716
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATS 112
GSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGT
SESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGS
ETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTE
EGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS
PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTS
TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
PGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSE AE1812
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATS 113
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
TEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPG
TSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT
STEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATS
GSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEG
SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG
PGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP AE1908
GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS 114
EGSAPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGS
EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPAT
SGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTS
ESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSES
ATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS
TEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEP AE2004A
GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS 115
GSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPE
SGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA
PGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEP
SEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
ESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAG
SPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS
EGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG
SAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE AG948
GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGT 116
ASSSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGS
SPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGAT
GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSS
TGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGT
GPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPS
ASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP
GTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT
ASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGS
SPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGASPGTSST
GSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP
GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSP AG1044
GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTS 117
STGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGA
SPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS
GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA
SSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGAS
PGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGS
SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPG
SGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGT
SSTGSPGSSTPSGATGSPGTPGSGTASSSPGSST AG1140
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTS 118
STGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTG
PGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSA
STGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTA
SSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGAS
PGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
GTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGS
STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST
GSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGSST AG1236
GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 119
ASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGT
PGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSST
PSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSS
PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSS
TGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTP
GSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSP
GTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG
ATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGS
SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTAS
SSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASP AG1332
GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTS 120
STGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGS
SPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG
TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG
SSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTA
SSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSS
PSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS
SPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGP
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTAS
SSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPG
SGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSS
PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG AG1428
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGT 121
ASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGS
SPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGT
SSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPG
ASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGA
TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSS
PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS
SPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPS
ASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGS
PGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGTPGSGTASSSPGASP AG1524
GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG 122
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGT
PGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTG
TGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSAST
GTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATG
SPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSP
GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTS
STGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSP
SASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPG AG1620
GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGT 123
ASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGAT
GSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA
SSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTP
GSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTP
SGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSP
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG
ATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGS
SPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTG
TGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPG
SGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG
TASSSPGTPGSGTASSSPGSSTPSGATGSPGSST AG1716
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGT 124
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGT
PGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTG
TGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTG
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPS
GATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP
GTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGT
PGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSP
SASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGS
PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA
STGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPG
TPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSS
TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS
SPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPS
ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG AG1812
GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT 125
ASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGS
PGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPS
GATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGT
ASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGA
SPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSS
TGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS
TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGASPGTSSTGSPGSSTPSGATGSPGASP AG1908
GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSAS 126
TGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGT
PGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTG
TGPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGS
PGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGT
SSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPG
ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGAS
PGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGT
GPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT
ASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS
SPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTG
TGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGS
PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGSSPSASTGTGPGSSP AG2004A
GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG 127
ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGT
PGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSS
PGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS
GATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS
TGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGAS
PGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATG
SPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPG
SGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG
TASSSPGSSPSASTGTGPGSSPSASTGTGPGASP AE72B
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP 128
ESGPGSEPATSGSETPG AE72C
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT 129
STEEGTSTEPSEGSAPG AE108A
TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST 130
EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS AE108B
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT 131
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS 132
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 133
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP
AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS 134
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGSEPATS AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 135
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS 136
ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGTSESATPESGPGTSTEPSE AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG 137
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS
TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE
TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA
TSGSETPGTSESA AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT 138
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS 139
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA
TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG
TSTEPSEGSAPGSEPATSGSETPGTSESAT AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS 140
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
SPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT 141
SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPE
SGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG
TSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 142
SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA
GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS
PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP
ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESAT AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGS 143
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPES GPGTSTEPS
AE540A TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG 144
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGS
ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG 145
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGSEPATSGSETPGTSESA AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS 146
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESAT
AE648A PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT 147
STEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT 148
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG
SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG
SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGSEPATS AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP 149
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTE
AE756A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP 150
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT 151
SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG
SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS
EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGS
ETPGTSESATPESGPGTSTEPS AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT 152
SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET
PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS
PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSE SAT AG72A
GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG 153
TSSTGSPGTPGSGTASS AG72B
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS 154
STGSPGTPGSGTASSSP AG72C
SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 155
GSPGSSTPSGATGSPGA AG108A
SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG 156
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP AG108B
PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS 157
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS AG144A
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSA 158
STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AG144B
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG 159
PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASP AG180A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 160
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGS AG216A
TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGA 161
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG AG252A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 162
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPG AG288A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 163
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGTPGS AG324A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP 164
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGS
SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG360A
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP 165
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS
SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST
PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGSSTPSGATGSPGSSTPSGATGSPGASPG AG396A
GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG 166
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGASPGT
AG432A GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG 167
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSS
TGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS
TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS AG468A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP 168
GASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPG AG504A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP 169
GASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA SSSPGSSTP
AG540A TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP 170
GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTS
STGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGS
SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSG
TASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAST
GTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG576A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP 171
GSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA
TGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGASPG AG612A
STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS 172
STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGAT
GSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA
SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GASPGTS
AG648A GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP 173
GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGT
GPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG684A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP 174
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGS
SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG
SPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGASPG AG720A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP 175
GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG
ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSST
PSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSG
TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GASPG
AG756A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 176
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG AG792A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 177
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPG AG828A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP 178
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGS STP AE869
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 179
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSET
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS
TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG
PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR AE144_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT 180
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSESATPESGPGTESASR AE288_R1
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG 181
PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPSASR AE432_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT 182
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTESASR AE576_R1
SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA 183
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGT
SESATPESGPGTSTEPSEGSAPSASR AE864_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT 184
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR AF864_R1
SAGSPGSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGS 185
TSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG
TAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSP
SGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSAS
PGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPE
SGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPG
STSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESG
SASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGST
SESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESP
GPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSS
TAESPGPGTSTPESGSASPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAP
GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGE
SSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGT
SPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSG
TAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTS
ESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSSASR AG864_R1
SAGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGS 186
STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASSASR
[0235] In some embodiments wherein the XTEN has less than 100% of
its amino acids consisting of 4, 5, or 6 types of amino acid
selected from glycine (G), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P), or less than 100% of the sequence
consisting of the sequence motifs from Table 1 or the XTEN
sequences of Tables 2, 3 and 22-25 the other amino acid residues of
the XTEN are selected from any of the other 14 natural L-amino
acids, but are preferentially selected from hydrophilic amino acids
such that the XTEN sequence contains at least about 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic
amino acids. An individual amino acid or a short sequence of amino
acids other than glycine (G), alanine (A), serine (S), threonine
(T), glutamate (E) and proline (P) may be incorporated into the
XTEN to achieve a needed property, such as to permit incorporation
of a restriction site by the encoding nucleotides, or to facilitate
linking to a payload component, or incorporation of a cleavage
sequence. As one exemplary embodiment, described more fully below,
the invention provides XTEN that incorporates from 1 to about 20,
or 1 to about 15, or 1 to about 10, or 1 to 5 lysine residues
wherein the reactive lysines are utilized for linking to
cross-linkers or payloads, as described herein. In another
embodiment, described more fully below, the XTEN incorporates from
1 to about 20, or 1 to about 15, or 1 to about 10, or 1 to 5
cysteine residues wherein the reactive cysteines are utilized for
linking to cross-linkers or payloads, as described herein. In
another embodiment, the XTEN incorporates from 1 to about 20
cysteine and lysine residues wherein the lysines and cysteines are
utilized for linking to different cross-linkers or payloads, as
described herein. In another embodiment, the XTEN incorporations 1,
2, 3, 4, 5 or more arginine residues that are not followed by
proline residues to provide cleavage sequences that can be cleaved
by trypsin to create XTEN segments, described more fully herein,
below. The XTEN amino acids that are not glycine (G), alanine (A),
serine (S), threonine (T), glutamate (E) and proline (P) are either
interspersed throughout the XTEN sequence, are located within or
between the sequence motifs, or are concentrated in one or more
short stretches of the XTEN sequence such as at or near the N- or
C-terminus. As hydrophobic amino acids impart structure to a
polypeptide, the invention provides that the content of hydrophobic
amino acids in the XTEN utilized in the conjugation constructs will
typically be less than 5%, or less than 2%, or less than 1%
hydrophobic amino acid content. Hydrophobic residues that are less
favored in construction of XTEN include tryptophan, phenylalanine,
tyrosine, leucine, isoleucine, valine, and methionine.
Additionally, one can design the XTEN sequences to contain less
than 5% or less than 4% or less than 3% or less than 2% or less
than 1% or none of the following amino acids: methionine (to avoid
oxidation), asparagine and glutamine (to avoid desamidation). In
other embodiments, the amino acid content of methionine and
tryptophan in the XTEN component used in the conjugation constructs
is typically less than 5%, or less than 2%, and most preferably
less than 1%. In other embodiments, the XTEN of the subject XTEN
conjugates will have a sequence that has less than 10% amino acid
residues with a positive charge, or less than about 7%, or less
that about 5%, or less than about 2% amino acid residues with a
positive charge, the sum of methionine and tryptophan residues will
be less than 2%, and the sum of asparagine and glutamine residues
will be less than 5% of the total XTEN sequence.
3. Cysteine- and Lysine-Engineered XTEN Sequences
[0236] In another aspect, the invention provides XTEN with defined
numbers of incorporated cysteine or lysine residues;
"cysteine-engineered XTEN" and "lysine-engineered XTEN",
respectively. It is an object of the invention to provide XTEN with
defined numbers of cysteine and/or lysine residues to permit
conjugation between the thiol group of the cysteine or the epsilon
amino group of the lysine and a reactive group on a payload or a
cross-linker to be conjugated to the XTEN backbone. In one
embodiment of the foregoing, the XTEN of the invention has between
about 1 to about 100 lysine residues, or about 1 to about 70 lysine
residues, or about 1 to about 50 lysine residues, or about 1 to
about 30 lysine residues, or about 1 to about 20 lysine residues,
or about 1 to about 10 lysine residues, or about 1 to about 5
lysine residues, or 1 to about 3 lysine residues, or alternatively
only a single lysine residue. In another embodiment of the
foregoing, the XTEN of the invention has between about 1 to about
100 cysteine residues, or about 1 to about 70 cysteine residues, or
about 1 to about 50 cysteine residues, or about 1 to about 30
cysteine residues, or about 1 to about 20 cysteine residues, or
about 1 to about 10 cysteine residues, or about 1 to about 5
cysteine residues, or 1 to about 3 cysteine residues, or
alternatively only a single cysteine residue. In another embodiment
of the foregoing, the XTEN of the invention has about 1 to about 10
lysine residues and about 1 to about 10 cysteine residues. Using
the foregoing lysine- and/or cysteine-containing XTEN, conjugates
can be constructed that comprise XTEN, an optional cross-linker,
plus a payload useful in the treatment of a condition in a subject
wherein the maximum number of molecules of the payload agent linked
to the XTEN component is determined by the numbers of lysines,
cysteines or other amino acids with a reactive side group (e.g., a
terminal amino or thiol) incorporated into the XTEN.
[0237] In one embodiment, the invention provides
cysteine-engineered XTEN where nucleotides encoding one or more
amino acids of an XTEN are replaced with a cysteine amino acid to
create the cysteine-engineered XTEN gene. In another embodiment,
the invention provides cysteine-engineered XTEN where nucleotides
encoding one or more cysteine amino acids are inserted into an-XTEN
encoding gene to create the cysteine-engineered XTEN gene. In other
cases, oligonucleotides encoding one or more motifs of about 9 to
about 14 amino acids comprising codons encoding one or more
cysteines are linked in frame with other oligos encoding XTEN
motifs or full-length XTEN to create the cysteine-engineered XTEN
gene. In one embodiment of the foregoing, where the one or more
cysteines are inserted into an XTEN sequence during the creation of
the XTEN gene, nucleotides encoding cysteine can be linked to
codons encoding amino acids used in XTEN to create a cysteine-XTEN
motif with the cysteine(s) at a defined position using the methods
described herein (see Example 61 and FIGS. 40-41), or by standard
molecular biology techniques, and the motifs subsequently assembled
into the gene encoding the full-length cysteine-engineered XTEN. In
such cases, where, for example, nucleotides encoding a single
cysteine are added to the DNA encoding a motif selected from Table
1, the resulting motif would have 13 amino acids, while
incorporating two cysteines would result in a motif having 14 amino
acids, etc. In other cases, a cysteine-motif can be created de novo
and be of a pre-defined length and number of cysteine amino acids
by linking nucleotides encoding cysteine to nucleotides encoding
one or more amino acid residues used in XTEN (e.g., G, S, T, E, P,
A) at a defined position, and the encoding motifs subsequently
assembled by annealing with other XTEN-encoding motif sequences
into the gene encoding the full-length XTEN, as described herein
and illustrated in FIGS. 7-8. In cases where a lysine-engineered
XTEN is utilized to make the conjugates of the invention, the
approaches described above would be performed with codons encoding
lysine instead of cysteine. Thus, by the foregoing, a new XTEN
motif can be created that could comprise about 9-14 amino acid
residues and have one or more reactive amino acids; i.e., cysteine
or lysine. Non-limiting examples of motifs suitable for use in an
engineered XTEN that contain a single cysteine or lysine are:
TABLE-US-00003 (SEQ ID NO: 187) GGSPAGSCTSP (SEQ ID NO: 188)
GASASCAPSTG (SEQ ID NO: 189) TAEAAGCGTAEAA (SEQ ID NO: 190)
GPEPTCPAPSG (SEQ ID NO: 191) GGSPAGSKTSP (SEQ ID NO: 192)
GASASKAPSTG
However, the invention contemplates motifs of different lengths,
such as those of Table 5 and Table 11, for incorporation into
XTEN.
[0238] In such cases where a gene encoding an XTEN with one or more
cysteine and/or lysine motifs is to be constructed from existing
XTEN motifs or segments, the gene can be designed and built by
linking existing "building block" polynucleotides encoding both
short- and long-length XTENs; e.g., AE48, AE144, AE288, AE432,
AE576, AE864, AM48, AM875, AE912, AG864, or the nucleotides
encoding the 36'mers of Examples 1-4, and Tables 22-25, which can
be fused in frame with the nucleotides encoding the cysteine-
and/or lysine-containing motifs or, alternatively, the cysteine-
and/or lysine-encoding nucelotides can be PCR'ed into an existing
XTEN sequence (as described more fully below and in the Examples)
using, for example, nucleotides encoding the islands of Tables 4
and 5 to build an engineered XTEN in which the reactive cysteine
and/or lysines are placed in one or more designed locations in the
sequence in the desired quantity. Non-limiting examples of such
engineered XTEN are provided in Table 3. Thus, in one embodiment,
the invention provides an XTEN sequence having at least about 80%
sequence identity, or at least about 90%, or about 91%, or about
92%, or about 93%, or about 94%, or about 95%, or about 96%, or
about 97%, or about 98%, or about 99% sequence identity, or is
identical to a sequence or a fragment of a sequence selected from
of Table 3, when optimally aligned. However, application of the
cysteine- or lysine-engineered methodology to create XTEN
encompassing cysteine or lysine residues is not meant to be
constrained to the precise compositions or range of composition
identities of the foregoing embodiments. As will be appreciated by
those skilled in the art, the precise location and numbers of
incorporated cysteine or lysine residues in an XTEN can be varied
without departing from the invention as described.
TABLE-US-00004 TABLE 3 Cysteine- and lysine-engineered XTEN SEQ ID
Name Amino Acid Sequence NO: Seg 1
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 193
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPR
Seg 2 ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 194
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPTAEAAGCGTAEAAR Seg 3
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 195
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 4
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 196
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 5
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 197
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPT
AEAAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG
SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAE
AAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAA
GCGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC GTAEAAR
Seg 6 ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 198
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTETAEAAGCGTAEAAPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSTAEAAGCGTAEAAAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSTAEAAGCGTAEAAPAGSPT
STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATTAEAAGC
GTAEAAPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS
TEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPTAEAAGCGTAEAAR Seg 7
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 199
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAG
CGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEETAEAAGCGTAEAAGTSTEPSEG
SAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 8
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 200
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTAEAAGCGTAEAAESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSTAEAAGCGTAEAAE
GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSTAEAAGCGTAEAAAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTTAEAAGCGTAEAASTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATAEAAGCG
TAEAATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPETAEAAGCGTAEAASGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 9
ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 201
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPR
Seg 10 AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG
202 SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC GTAEAAR
Seg 11 ATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
203 TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPR Seg 12
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 204
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTTAEAAG
CGTAEAAEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATT
AEAAGCGTAEAASGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPR Seg 13
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 205
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPTAEAAGCGTAEAAR Seg 14
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 206
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPR Seg 15
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 207
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGG KPGR Seg
16 AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 208
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APGGKPGR
Seg 17 AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST
209 EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGGKPGGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGGKPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAPGGKPGR Seg 18
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 210
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGGKPGGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGGKPGGSPAGSPTSTEEGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGGKPGR Seg 19
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 211
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEGGKPGPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSGGKPGAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSGGKPGPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATGGKPGPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG
SAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGGKPGR Seg 20
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 212
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGKPGGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGGKPGGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGGKPGGTSTE
PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE
PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGGKPGR Seg 21
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 213
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSGGKPGESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSGGKPGEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSGGKPGAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTGGKPGSTE
PSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SEPAGGKPGTSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST
EPSEGSAPGTSESATPEGGKPGSGPGTSESATPESGPGTSESATPESGPGSEPATSGS
ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 22
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 214
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPR Seg 23
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 215
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 24
AGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 216
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APR Seg
25 AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 217
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTGGKPGE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATGGKPGSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APR Seg
26 AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 218
SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGG KPGR Seg
27 AEATTAAGGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE 219
EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPR Seg 28
AEATTAAGGATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 220
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG
TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 29
AEATTAAGGATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 221
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG
TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGC
GTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR
Seg 30 AEATTAAGGATAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 222
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG
TSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEA AR Seg 31
ATGTATSEGSPEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST 223
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPR Seg 32
ATGTATSEGSPETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSE 224
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 33
ATGTATSEGSPETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSE 225
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAG
CGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 34
ATGTATSEGSPETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSE 226
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEA AR Seg 35
EPTAATTGESAGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST 227
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPR Seg 36
EPTAATTGESAGTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPS 228
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 37
EPTAATTGESAGTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPS 229
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAG
CGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 38
EPTAATTGESAGTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPS 230
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEA AR Seg 39
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 231
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPR Seg 40
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 232
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 41
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 233
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAA
GCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 42
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 234
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC GTAEAAR
Seg 43 ATGTATSEGSPEEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
235 TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPR Seg 44
ATGTATSEGSPEEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTE 236
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 45
ATGTATSEGSPEEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTE 237
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAA
GCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 46
ATGTATSEGSPEEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTE 238
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC GTAEAAR
Seg 47 AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
239 STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPRPRPRPRP Seg 48
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 240
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAARPRPRPRP Seg 49
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 241
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAA
GCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAARPRPRPRP Seg 50
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 242
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC
GTAEAARPRPRPRP Seg 51
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 243
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA
TSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPR Seg 52
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 244
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 53
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 245
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAA
GCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 54
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 246
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGC GTAEAAR
Seg 55 AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 247
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGGKPGGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 56
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 248
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 57
AEATTAAGGAEEEGGKPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP 249
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGGKPGGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGGKPGGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 58
AEATTAAGGAEEETAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEP 250
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGGKPGGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 59
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 251
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPTAEAAGCGTAEAAR Seg 60
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 252
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 61
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 253
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPTAEAAGCGTAEAAR Seg 62
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 254
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 63
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 255
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPTAEAAGCGTAEAAR Seg 64
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 256
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 65
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 257
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPTAEAAGCGTAEAAR Seg 66
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 258
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAR Seg 67
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 259
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGGKPGR Seg 68
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 260
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGR Seg 69
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 261
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGGKPGR Seg 70
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 262
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 71
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 263
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGGKPGR Seg 72
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 264
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGGKPGR Seg 73
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 265
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGGKPGR Seg 74
AEATTAAGGAEEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 266
STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGKPGR Seg 75
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 267
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPR Seg 76
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 268
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPTAEAAGCGTAEAAR Seg 77
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 269
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 78
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 270
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGSSTPS
GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSST
PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP
GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS
SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 79
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 271
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
TAEAAGCGTAEAAGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTA
EAAGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATG
SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPTAE
AAGCGTAEAAGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAR Seg 80
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 272
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTAEAAGCGTAEAATSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGSSPSASTGTTAEAAGCGTAEAAGPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTTAEAAGCGTAEAAPGS
GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSTAEA
AGCGTAEAASTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPTAEAAGCGTAEAAR Seg 81
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 273
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAA
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA
TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSG
TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEA
AGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPTAEAAGCGTAEAAGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS
STPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 82
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 274
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASTAEAAGCGTAEAAPGTSSTGSPGASPGTSSTGSP
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGTAEAAGCGTAEA
AATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA
SPGTSSTGTAEAAGCGTAEAASPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG
SPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA
TGSPGSSTPSGATGSPGSSPSASTGTGPGATAEAAGCGTAEAASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPTAEAAGC
GTAEAASGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTAEAAGCGTAEAATGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 83
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 275
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPR Seg 84
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 276
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAR Seg 85
ATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG 277
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPR Seg 86
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 278
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTTAEAA
GCGTAEAAPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
STAEAAGCGTAEAAGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPR Seg 87
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 279
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPTAEAAGCGTAEAAR Seg 88
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 280
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS PR Seg
89 AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 281
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS PGGKPGR
Seg 90 AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS
282 TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG
TSSTGSPGGKPGR Seg 91
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 283
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGGKPGGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGT
SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGGKPGGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGGKPGR Seg 92
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 284
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGGKPGGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGGKPGGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS
TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP
SGATGSPGASPGTSSTGSPGGKPGR Seg 93
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 285
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGGGKPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGGKPGGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS
PGASPGTSSTGSPGTGGKPGPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSGGKPGSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSP
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGS
SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP
GSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 94
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 286
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPS
GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGGKPGGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPG
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGG
KPGGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 95
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 287
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASGGKPGPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGGGKPGATGSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGGGKPGSPGASPGTSSTGS
PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAGGKP
GSPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPGGKPGSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS
SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGGGKPGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTP
SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 96
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 288
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PR Seg 97
AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 289
GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKP GR Seg 98
AGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSS 290
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPR
Seg 99 AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
291 GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTGGKPG
PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGGKPGGA
TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGT
SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGT SSTGSPR
Seg 100 AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
292 GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS PGGKPGR
Seg 101 AEATTAAGGAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS
293 PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP
GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA
STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSST
PSGATGSPGASPGTSSTGSPR Seg 102
AEATTAAGGATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTG 294
TGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPG
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS
TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP
SGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 103
AEATTAAGGATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTG 295
TGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAG
CGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGS
SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA
TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST
PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 104
AEATTAAGGATAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSASTG 296
TGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCG TAEAAR
Seg 105 ATGTATSEGSPEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS
297 SSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSPR Seg 106
ATGTATSEGSPETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAST 298
GTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 107
ATGTATSEGSPETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAST 299
GTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAA
GCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 108
ATGTATSEGSPETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAST 300
GTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGC GTAEAAR
Seg 109 EPTAATTGESAGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS
301 SSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS
PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSPR Seg 110
EPTAATTGESAGTAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAS 302
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 111
EPTAATTGESAGTAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAS 303
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAA
GCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 112
EPTAATTGESAGTAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSAS 304
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGC GTAEAAR
Seg 113 AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT
305 ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPR Seg 114
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 306
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS
STPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 115
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 307
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS
STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 116
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 308
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGT
SSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGC GTAEAAR
Seg 117 ATGTATSEGSPEEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG
309 TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSS
TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGT
PGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPR Seg 118
ATGTATSEGSPEEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPS 310
ASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 119
ATGTATSEGSPEEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPS 311
ASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAE
AAGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGS
PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 120
ATGTATSEGSPEEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPS 312
ASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATG
SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAR Seg 121
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 313
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPRPRPRPRP Seg 122
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 314
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS
STPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAARPRPRPRP Seg 123
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 315
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS
STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAARPRPRPRP Seg 124
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 316
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGT
SSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGC
GTAEAARPRPRPRP Seg 125
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 317
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPR Seg 126
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 318
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS
STPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 127
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 319
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEA
AGCGTAEAAGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS
STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 128
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 320
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGT
SSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGC GTAEAAR
Seg 129 AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA
321 STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 130
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 322
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPGASPGT
SSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 131
AEATTAAGGAEEEGGKPGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGT 323
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGSSTPSGATGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS
PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 132
AEATTAAGGAEEETAEAAGCGTAEAAGASPGTSSTGSPGSSPSASTGTGPGSSPSA 324
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGGKPGGTPGSGTASSSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAAGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 133
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 325
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPTAEAAGCGTAEAAR Seg 134
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 326
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 135
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 327
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPTAEAAGCGTAEAAR Seg 136
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 328
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAA R Seg 137
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 329
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPTAEAAGCGTAEAAR Seg 138
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 330
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPTAEAAGCGTAEAAR Seg 139
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 331
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPTAEAAGCGTAEAAR Seg 140
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 332
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPTAEAAGCGTAEAA R Seg 141
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 333
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGGKPGR Seg 142
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 334
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 143
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 335
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGGKPGR Seg 144
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 336
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGGKPGR Seg 145
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 337
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGGKPGR Seg 146
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 338
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGGKPGR Seg 147
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 339
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGGKPGR Seg 148
AEATTAAGGAEEEGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT 340
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGGKPGR Seg 149
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS 341
APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSCTSPGGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG Seg 150
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTS 342
TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP
ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGASASCAPSTGGGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG
SEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPG Seg 151
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 343
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGGSPA
GSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPG
Seg 152 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGPEPTCP
344 APSGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG Seg 153
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES 345
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGG
GSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGGGSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPG Seg 154
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGASASCA 346
PSTGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GGASASCAPSTGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG Seg 155
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGGSPAGS 347
CTSPGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPG
Seg 156 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASCA
348 PSTGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP
SGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTS
ESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAP
GTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGT
GPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS
TGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESAT
PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPG
TSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTA
PGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG Seg 157
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGPEPTCP 349
APSGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPG
Seg 158 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGPEPTCP
350 APSGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGGPEPTCPAPSGGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGS
STPSGATGSPG Seg 159
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS 351
APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSKTSPGGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG Seg 160
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTS 352
TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP
ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGASASKAPSTGGGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG
SEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPG Seg 161
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 353
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGGSPA
GSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPG
Seg 162 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGASASKA
354 PSTGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG Seg 163
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES 355
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGG
GSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPG Seg 164
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGS 356
KTSPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GGGSPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTS
TEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG Seg 165
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGGSPAGS 357
KTSPGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPG
Seg 166 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKA
358 PSTGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP
SGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTS
ESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAP
GTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGT
GPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS
TGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESAT
PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPG
TSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTA
PGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG Seg 167
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKA 359
PSTGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPG
Seg 168 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKA
360 PSTGGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGGASASKAPSTGGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPG
SSTPSGATGSPG Seg 169
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES 361
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGG
GSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPG Seg 170
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGS 362
CTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTE
PSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSCTSPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGGGSPAGSCTSPGGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGG
SPAGSCTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG Seg 171
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGS 363
KTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTE
PSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSKTSPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGGGSPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGG
SPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG Seg 172
SAGSPTAEAAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 364
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPTAEAAGCGTAEAASASR Seg 173
SAGSPGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 365
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAGPTKPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPTKPGTSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE
GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAEAAGCGTAEAASASR Seg 174
SAGSPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP 366
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAA
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS
TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAASASR Seg 175
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 367
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGTAEAAGCGTAEAASTEPSEGSAPGTSESATPES
GPGSPAGSPTSTEEGSPATAEAAGCGTAEAASPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSESATTAEAAGCGTAEAASETPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TTAEAAGCGTAEAAAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESTAEA
AGCGTAEAATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGTAEAAGCGT
AEAATEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAEAAGCGTAEAAS ASR Seg
176 SAGSPTAEAAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 368
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPTAEAAGCGTAEAASASR Seg 177
SAGSPTAEAAGCGTAEAAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT 369
AEAAGCGTAEAASTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPATAEAAG
CGTAEAASPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATTAEAAGCGTAEA
ASETPGTSESATPESGPGSEPATSGSETPGTSESATPESGTAEAAGCGTAEAAGSPA
GSPTSTEEGTSESATPESGPGSEPATSGSETPGTTAEAAGCGTAEAAAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGTAEAAGCGTAEAATEEGTSTEPSEGSAPGTSTEPS
EGSAPGSEPATSGSETPTAEAAGCGTAEAASASR Seg 178
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 370
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR Seg 179
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 371
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR Seg 180
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 372
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGKPGTAEAAGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAPGK Seg 181
SAGSPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP 373
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPSASR Seg 182
CGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP 374
SEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR Seg 183
MKNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSAGSPTGPGSEPA 375
TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS
APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASRSAHHHHHHHH Seg 184
MKNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSAGSPTAEAAGC 376
GTAEAAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTAEAAGCGTAEA
ASTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPATAEAAGCGTAEAASPTST
EEGTSESATPESGPGTSTEPSEGSAPGTSESATTAEAAGCGTAEAASETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGTAEAAGCGTAEAAGSPAGSPTSTEEGTSE
SATPESGPGSEPATSGSETPGTTAEAAGCGTAEAAAGSPTSTEEGSPAGSPTSTEEG
TSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSESATPESGPGSEPATSGSE
TPGSEPATSGTAEAAGCGTAEAATEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS
GSETPTAEAAGCGTAEAASASRSAHHHHHHHH Seg 185
MKNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSAGSPGSPAGSPT 377
STEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPTAEAAGCGTAEAAPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTAEAAGCGTAEAASTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE
GSPATAEAAGCGTAEAASPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATTA
EAAGCGTAEAASETPGTSESATPESGPGSEPATSGSETPGTSESATPESGTAEAAG
CGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTTAEAAGCGTAE
AAAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGTAEAAGCGTAEAATEEGTSTEPS
EGSAPGTSTEPSEGSAPGSEPATSGSETPTAEAAGCGTAEAASASRSAHHHHHHH H Seg 186
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 378
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGKPGTAEAAGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAPGK Seg 187
SAGSPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP 379
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPSASR Seg 188
SAGSPTEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSESATPESGPGS 380
EPATSGSETPGSEPATSGTAEAAGCGTAEAATEEGTSTEPSEGSAPGTSTEPSEGSA
PGSEPATSGSETPTAEAAGCGTAEAASASR Seg 189
SAGSPTPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS 381
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPTAEAAGCGTAEAASASR Seg 190
SAGSPTGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST 382
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG
SEPATSGSETPTAEAAGCGTAEAASASR Seg 191
SAGSPTPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS 383
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSESATPESGPGSEPA
TSGSETPGSEPATSGTAEAAGCGTAEAATEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPTAEAAGCGTAEAASASR Seg 192
SAGSPGSTSSTAESPGPGSTSSTAESPGPGCTSESPSGTAPGSTSSTAESPGPGSTSST 384
AESPGPGTSTPESGSASPGSTSCSPSGEAPGTSPSGESSTAPGSTSESPSGTAPGSTSE
SPSGTAPETSPSGESCTAPGSTSASR Seg 193
SAGSPGTPGSGTASSSPGSSTPSGATGSPGCAGSGTASSSPGSSTPSGATGSPGTPG 385
SGTASSSPGSSTPSGATGSPGSSTCSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA
SPGTSSTGSPGTPGSGTACSSPGSSSASR Seg 194
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE 386
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASK Seg 195
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 387
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTACSEGSAPSASR Seg 196
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 388
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSCASASR Seg 197
SAGSPGSCAGSPTSTEEGTSESACPESGPGTSTEPSEGSCPGSPAGSPTSTEEGTCTE 389
PSEGSAPGTSTEPCSGSAPGTSESATPESCPGSEPATSGSETPGSCPATSGSETPGSP
AGSCTSTEEGTSESATPESCPGTESASR Seg 198
SAGSPTGCGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 390
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSCTPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSCGSAPSASR Seg 199
SAGSPTGCGSEPATSGSETPGTSESATPESGPGSEPATSGSCTPGTSESATPESGPGT 391
STEPSEGSAPGSPAGSPCSTEEGTSESATPESGPGSEPATSGSETPGTSESCTPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSCTPSEGSAPGTSESATPESGPGTSESATPES
GPGCSESATPESGPGSEPATSGSETPGSEPATSGSETCGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGCAPGSEPATSGSETPGTSESATPESGPGTSTEPSCGSAPSASR
[0239] In another embodiment, the invention provides inserts of
lysine as part of a longer sequence defined as a lysine island.
Examples of lysine island are shown in Table 4. The benefit of
flanking all lysine residues in an XTEN with similar or identical
sequence is that it results in a more uniform chemical reactivity
for each lysine. Another benefit results from the ability to
perform peptide mapping to measure the degree of payload linking.
Examples include the islands I_L6, I_L7, and I_L8 of Table 4. These
islands comprise glutamate residues that facilitate peptide mapping
using GluC protease. In another embodiment, the invention provides
inserts of cysteine as part of a longer sequence defined as a
cysteine island. Examples of cysteine island are shown in Table 5.
The benefit of flanking all cysteine residues in an XTEN with
similar or identical sequence is that it results in a more uniform
chemical reactivity for each cysteine. Another benefit results from
the ability to perform peptide mapping to measure the degree of
payload conjugation. Examples include islands I_C4, I_C7, I_C8, and
I_C9 of Table 5. These islands comprise glutamate residues that
facilitate peptide mapping using GluC protease. The islands can be
inserted into constructs encoding the existing XTEN by conventional
PCR methods, as described above and in the Examples.
Oligonucleotides encoding the islands can be inserted into
constructs encoding the existing XTEN by conventional PCR methods.
For example, in one embodiment, where an existing full-length XTEN
gene is to be modified with nucleotides encoding one or more
reactive cysteine or lysine residues, an oligonucleotide can be
created that encodes a cysteine or lysine and that exhibits partial
homology to and can hybridize with one or more short sequences of
the XTEN, resulting in a recombination event and substitution of a
cysteine or the lysine codon for an existing codon of the XTEN gene
(see, e.g., Examples 6 and 7 for a description of the general
methods). In one exemplary embodiment, the recombination results in
a replacement with the amino acid sequence GGSPAGSCTSP (SEQ ID NO:
187) of the I_C1 island. However, the oligonucleotides can be
designed to place the cysteine (or lysine) in a different location
in the motif or to include a second cysteine (or lysine) in the
motif. The cysteine- or lysine-encoding oligonucleotides can be
designed to hybridize with a given sequence segment at different
points along the known XTEN sequence to permit their insertion into
an XTEN-encoding gene. Thus, the invention contemplates that
multiple XTEN gene constructs can be created with cysteines or
lysines inserted at different locations within the XTEN sequence by
the selection of restriction sites within the XTEN sequence and the
design of oligonucleotides appropriate for the given location and
that encode a cysteine or lysine, including use of designed
oligonucleotides that result in multiple insertions in the same
XTEN sequence. By the design and selection of one or more such
oligonucleotides in consideration of the known sequence of the
XTEN, and the appropriate use of the methods of the invention, the
potential number of substituted reactive cysteine or lysine
residues inserted into the full-length XTEN can be estimated and
then confirmed by sequencing the resulting XTEN gene.
TABLE-US-00005 TABLE 4 Examples of lysine islands Designator Amino
Acid Sequence SEQ ID NO: I_L1 GGSPAGSKPTSP 392 I_L2 GASASKPAPSTG
393 I_L3 PKP I_L4 PPKPP 394 I_L5 GGKPG 395 I_L6 EGGKPGES 396 I_L7
EGGSPAGSKPTSPE 397 I_L8 EGASASKPAPSTGE 398
TABLE-US-00006 TABLE 5 Examples of cysteine islands Designator
Sequence SEQ ID NO: I_C1 GGSPAGSCTSP 399 I_C2 GASASCAPSTG 400 I_C3
GPEPTCPAPSG 401 I_C4 TAEAAGCGTAEAA 402 I_C5 GECEP 403 I_C6 GRPCRP
404 I_C7 GETSPAGSCTSPTET 405 I_C8 TESGRPCRPSET 406 I_C9
GPEPTCPAPSEG 407
[0240] XTEN can be designed to comprise both lysine and cysteine
residues for conjugation as illustrated in FIG. 1D. This enables
one to conjugate two different payloads to the same XTEN polymer
using conjugations methods tailored to react with the functional
group or linker attached to the cysteine or lysine. Such mixed
payloads can have additive and/or synergistic pharmacologic effects
when administered to a subject in a single composition.
Alternatively, the mixed payloads can be a combination of a
targeting moiety and an active payload in order to deliver the
pharmacophore to a desired location in the subject. By controlling
the number and position of lysine and cysteine residues one can
control the number and position of conjugated payloads. This
enables one to adjust the relative potency or selectivity of the
payloads in the resulting XTEN-payload conjugate.
[0241] The design, selection, and preparation methods of the
invention enable the creation of engineered XTEN that are reactive
with electrophilic functionality. The methods to make the subject
conjugates provided herein enable the creation of XTEN-payload
conjugates, XTEN-cross-linker conjugates, and XTEN-azide/alkyne
reactant conjugates with the linker or payload molecules added in a
quantified fashion at designated sites, as illustrated
schematically in FIGS. 1A-1E. Payloads, cross-linkers, and
azide/alkyne reactants may be site-specifically and efficiently
linked to the N- or C-terminus of XTEN, to cysteine-engineered XTEN
with a thiol-reactive reagent, or to lysine-engineered XTEN of the
invention with an amine-reactive reagent, and to an alpha amino
group at the N-terminus of XTEN, as described more fully, below,
and then are purified and characterized as shown schematically in
FIG. 40 using, for example, the non-limiting methods described more
specifically in the Examples.
4. Length of Sequence
[0242] In another aspect, the invention provides XTEN of varying
lengths for incorporation into the compositions wherein the length
of the XTEN sequence(s) are chosen based on the property or
function to be achieved in the composition. Depending on the
intended property or function, the XTEN-payload conjugates comprise
short or intermediate length XTEN or longer XTEN sequences, or
multimers of short, intermediate or longer XTEN that can serve as
carriers. While not intended to be limiting, the XTEN or fragments
of XTEN include short segments of about 6 to about 99 amino acid
residues, intermediate lengths of about 100 to about 399 amino acid
residues, and longer lengths of about 400 to about 1000 and up to
about 3000 amino acid residues. Thus, the XTEN utilized as
conjugation partners for incorporation into the subject conjugates
encompass XTEN or fragments of XTEN with lengths of about 6, or
about 12, or about 36, or about 40, or about 48, or about 72 or
about 96, or about 144, or about 288, or about 400, or about 432,
or about 500, or about 576, or about 600, or about 700, or about
800, or about 864, or about 900, or about 1000, or about 1500, or
about 2000, or about 2500, or up to about 3000 amino acid residues
in length. In other cases, the XTEN sequences can be about 6 to
about 50, about 50 to about 100, about 100 to 150, about 150 to
250, about 250 to 400, about 400 to about 500, about 500 to about
900, about 900 to 1500, about 1500 to 2000, or about 2000 to about
3000 amino acid residues in length. The precise length of an XTEN
incorporated into the subject XTEN-payload conjugates can vary
without adversely affecting the biological activity of the
conjugate. In one embodiment, one or more of the XTEN may be
selected from one of the XTEN family sequences; e.g., AD, AE, AF,
AG, AM, AQ, BC, or BD. In some embodiments, the XTEN utilized to
create the subject conjugates comprise XTEN selected from any one
of the sequences in Table 2, Table 3, and Tables 22-25, which may
be linked to the payload component directly or via cross-linkers
disclosed herein. In other embodiments, the one or more XTEN
utilized to create the subject conjugates individually comprise an
XTEN sequence having at least about 80% sequence identity, or
alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity compared to an XTEN selected from Tables 2, 3, 22-25 or a
fragment thereof, when optimally aligned with a sequence of
comparable length. In some embodiments, the subject conjugates
comprise 2, 3, 4, or more XTEN sequence, wherein the cumulative
length of the residues in the XTEN sequences is greater than about
100 to about 3000, or about 400 to about 2000, or about 800 to 1000
amino acid residues and the XTEN can be identical or they can be
different in sequence or in length. As used herein, cumulative
length is intended to encompass the total length, in amino acid
residues, when more than one XTEN is incorporated into the
conjugate.
[0243] As described more fully below, methods are disclosed in
which the XTEN-payload conjugates are designed by selecting the
length of the XTEN and a method of linking with a cross-linker
reactant or the payload to confer a physicochemical property (e.g.,
stability or solubility) or to result in a target half-life or
retention of activity when an XTEN-payload conjugate is
administered to a subject.
[0244] XTEN are used as a carrier in the compositions, the
invention taking advantage of the discovery that increasing the
length of the non-repetitive, unstructured polypeptides enhances
the unstructured nature of the XTENs and correspondingly enhances
the physicochemical and pharmacokinetic properties of constructs
comprising the XTEN carrier. In general, XTEN as monomers or as
multimers with cumulative lengths longer that about 400 residues
incorporated into the conjugates result in longer half-life
compared to shorter cumulative lengths, e.g., shorter than about
280 residues. As described more fully in the Examples, proportional
increases in the length of the XTEN, even if created by a repeated
order of single family sequence motifs (e.g., the four AE motifs of
Table 1), result in a sequence with a higher percentage of random
coil formation, as determined by GOR algorithm, or reduced content
of alpha-helices or beta-sheets, as determined by Chou-Fasman
algorithm, compared to shorter XTEN lengths. In addition,
increasing the length of the unstructured polypeptide fusion
partner, as described in the Examples, results in a construct with
a disproportionate increase in terminal half-life compared to
polypeptides with unstructured polypeptide partners with shorter
sequence lengths. In some embodiments, where the XTEN serve
primarily as a carrier, the invention encompasses XTEN conjugate
compositions comprising two, three, four or more XTEN wherein the
cumulative XTEN sequence length of the XTEN proteins is greater
than about 100, 200, 400, 500, 600, 800, 900, or 1000 to about 3000
amino acid residues, wherein the construct exhibits enhanced
pharmacokinetic properties when administered to a subject compared
to a payload not linked to the XTEN and administered at a
comparable dose. In one embodiment of the foregoing, the two or
more XTEN sequences each exhibit at least about 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, or 98% or more identity to a sequence
selected from any one of Table 2, Table 3, or Tables 22-25, and the
remainder, if any, of the carrier sequence(s) contains at least 90%
hydrophilic amino acids and less than about 2% of the overall
sequence consists of hydrophobic or aromatic amino acids or
cysteine. The enhanced pharmacokinetic properties of the
XTEN-payload conjugate, in comparison to payload not linked to
XTEN, are described more fully, below.
5. XTEN Segments from XTEN Precursors
[0245] In another aspect, the invention provides methods to create
XTEN of short or intermediate lengths from longer "donor" XTEN
sequences, wherein the longer donor XTEN sequence is truncated at
the N-terminus, or the C-terminus, or segments are created by
protealysis of XTEN comprising cleavage sequences, thereby
resulting in a short or intermediate length XTEN. In non-limiting
examples, an AG864 sequence of 864 amino acid residues can be
truncated to yield an AG144 with 144 residues, an AG288 with 288
residues, an AG576 with 576 residues, or other intermediate
lengths, while the AE864 sequence can be truncated to yield
multiple AE144 sequences, an AE288 sequence or an AE576 sequence
with 288 or 576 residues or other shorter or intermediate lengths.
Similarly, the DNA encoding the longer "donor" sequences can be
manipulated to incorporate cysteine or lysine residues intended for
use in conjugates with short or intermediate length XTEN. It is
specifically contemplated that such an approach can be utilized
with any of the XTEN embodiments described herein or with any of
the sequences listed in Tables 2, 3, 21 and 22 to result in XTEN of
a desired length.
[0246] In another aspect, the invention provides XTEN with cleavage
sequences incorporated internal to the sequence at defined
intervals such that the XTEN can be processed by cleavage into 2,
3, 4, 5, or 6 shorter XTEN of uniform lengths. As illustrated in
FIG. % A, a monomeric XTEN is designed with two internal cleavage
sequences that, when treated with a protease under conditions
effective to result in the cleavage of all cleavage sequences,
results in three XTEN segments of uniform length. In addition, the
XTEN are designed with a sequence such that the resulting XTEN
segments also have the identical amino acid sequence, inclusive of
the residual cleavage sequence. In one embodiment, the invention
provides an XTEN with a defined, sequence comprising 1, 2, 3, 4, or
5 arginine (R) residues internal to the XTEN sequence and spaced at
uniform intervals along the XTEN sequence bridging identical XTEN
segments wherein treatment with trypsin results in cleavage of the
XTEN into XTEN segments to having an identical length and sequence.
In the foregoing embodiment, the arginine residue does not have a
proline residue at the adjacent P1' position. Thus, by treatment of
the foregoing with trypsin, an XTEN with 1 internal arginine would
result in 2 identical XTEN segments, an XTEN with 2 internal
argninines would result in 3 identical XTEN segments, etc. In
another embodiment, each arginine of the foregoing embodiments is
replaced with lysine residues. In another embodiment, the invention
provides an XTEN with a defined sequence comprising 1, 2, 3, 4, or
5 cleavage sequences internal to the XTEN sequence and spaced at
uniform intervals along the XTEN sequence, wherein each cleavage
sequence is SASRSA, and wherein treatment with trypsin results in
cleavage of the XTEN into XTEN segments to having an identical
length and sequence. In another embodiment, the invention provides
an XTEN with at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99% sequence identity to a
sequence selected from the group of sequences set forth in Table 6.
In another embodiment, the invention provides an XTEN with at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99% sequence identity to a sequence selected from
the group of sequences set forth in Table 6, wherein the XTEN
further comprises a first and a second affinity tag wherein each
affinity tags are linked to the XTEN by a cleavage sequence at the
N- and C-termini of the XTEN, respectively, wherein each cleavage
sequence is capable of being cleaved by trypsin, and wherein the
first affinity tag is different from the second affinity tag and
each is independently selected from the group consisting of the
affinity tags set forth in Table 7. The foregoing embodiment is
illustrated in FIG. 96B, wherein the treatment with protease of the
XTEN with two internal cleavage sequences and an N-terminal and a
C-terminal affinity tag each linked to the XTEN by cleavage
sequence results in cleavage of the construct into three XTEN
segments of uniform length and liberation of the two affinity tags,
the resulting preparation of which can be subsequently processed
into substantially homogeneous XTEN as described herein, below.
Variations of XTEN comprising such uniform cleavage sequences and
their distribution in the sequence are contemplated by the
invention.
TABLE-US-00007 TABLE 6 Precursor XTEN with Internal Cleavage
Sequences SEQ XTEN ID Name Amino Acid Sequence NO: AE864_R2
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST 408 (2x
AE432_R1) EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESAT
PESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTESASRSAGSPGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTESASR
AE864_R3 SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
409 (3x AE288_R1)
TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPS
ASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
APSASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPSASR
AE864_R6 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST
410 (6x AE144_R1)
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
SPAGSPTSTEEGTSESATPESGPGTESASRSAGSPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTES
ASRSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTESASRSAGSPGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
TESASRSAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSESATPESGPGTESASRSAGSPGSPAGSPTSTEEGTSE
SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGTESASR
Seg 200 SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
411 (3x Seg195)
TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTACSEGSAPS
ASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTACSEGS
APSASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTACS EGSAPSASR
Seg 201 SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
412 (3x Seg196)
TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSCAS
ASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
CASASRSAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
EGSCASASR
6. Net Charge
[0247] In other embodiments, the XTEN polypeptides have an
unstructured characteristic imparted by incorporation of amino acid
residues with a net charge and containing a low percentage or no
hydrophobic amino acids in the XTEN sequence. The overall net
charge and net charge density is controlled by modifying the
content of charged amino acids in the XTEN sequences, either
positive or negative, with the net charge typically represented as
the percentage of amino acids in the polypeptide contributing to a
charged state beyond those residues that are cancelled by a residue
with an opposing charge. In some embodiments, the net charge
density of the XTEN of the conjugates may be above +0.1 or below
-0.1 charges/residue. By "net charge density" of a protein or
peptide herein is meant the net charge divided by the total number
of amino acids in the protein. In other embodiments, the net charge
of an XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%,
about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about
11%, about 12%, about 13%, about 14%, about 15%, about 16%, about
17%, about 18%, about 19%, or about 20% or more. Based on the net
charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In one
embodiment, the XTEN will have an isoelectric point between 1.5 and
4.5 and carry a net negative charge under physiologic
conditions.
[0248] Since most tissues and surfaces in a human or animal have a
net negative charge, in some embodiments the XTEN sequences are
designed to have a net negative charge to minimize non-specific
interactions between the XTEN containing compositions and various
surfaces such as blood vessels, healthy tissues, or various
receptors. Not to be bound by a particular theory, an XTEN can
adopt open conformations due to electrostatic repulsion between
individual amino acids of the XTEN polypeptide that individually
carry a net negative charge and that are distributed across the
sequence of the XTEN polypeptide. In some embodiments, the XTEN
sequence is designed with at least 90% to 95% of the charged
residues separated by other non-charged residues such as serine,
alanine, threonine, proline or glycine, which leads to a more
uniform distribution of charge, better expression or purification
behavior. Such a uniform distribution of net negative charge in the
extended sequence lengths of XTEN also contributes to the
unstructured conformation of the polymer that, in turn, can result
in an effective increase in hydrodynamic radius. In preferred
embodiments, the negative charge of the subject XTEN is conferred
by incorporation of glutamic acid residues. Generally, the glutamic
residues are spaced uniformly across the XTEN sequence. In some
cases, the XTEN can contain about 10-80, or about 15-60, or about
20-50 glutamic residues per 20 kDa of XTEN that can result in an
XTEN with charged residues that would have very similar pKa, which
can increase the charge homogeneity of the product and sharpen its
isoelectric point, enhance the physicochemical properties of the
resulting XTEN-payload for, and hence, simplifying purification
procedures. For example, where an XTEN with a negative charge is
desired, the XTEN can be selected solely from an AE family
sequence, which has approximately a 17% net charge due to
incorporated glutamic acid, or can include varying proportions of
glutamic acid-containing motifs of Table 1 to provide the desired
degree of net charge. Non-limiting examples of AE XTEN include, but
are not limited to the AE36, AE42, AE144, AE288, AE432, AE576,
AE624, AE864, and AE912 sequences of Tables 2 and 21 or fragments
thereof. In one embodiment, an XTEN sequence of Tables 2 or 3 can
be modified to include additional glutamic acid residues to achieve
the desired net negative charge. Accordingly, in one embodiment the
invention provides XTEN in which the XTEN sequences contain about
1%, 2%, 4%, 8%, 10%, 15%, 17%, 20%, 25%, or even about 30% glutamic
acid. In some cases, the XTEN can contain about 10-80, or about
15-60, or about 20-50 glutamic residues per 20 kDa of XTEN that can
result in an XTEN with charged residues that would have very
similar pKa, which can increase the charge homogeneity of the
product and sharpen its isoelectric point, enhance the
physicochemical properties of the resulting XTEN conjugate
composition, and hence, simplifying purification procedures. In one
embodiment, the invention contemplates incorporation of up to 5%
aspartic acid residues into XTEN in addition to glutamic acid in
order to achieve a net negative charge.
[0249] Not to be bound by a particular theory, the XTEN of the
XTEN-payload conjugates with the higher net negative charge are
expected to have less non-specific interactions with various
negatively-charged surfaces such as blood vessels, tissues, or
various receptors, which would further contribute to reduced active
clearance. Conversely, it is believed that the XTEN of the
XTEN-payload conjugates with a low (or no) net charge would have a
higher degree of interaction with surfaces that can potentiate the
activity of the associated conjugate in the vasculature or
tissues.
[0250] In other embodiments, where no net charge is desired, the
XTEN can be selected from, for example, AG XTEN components, such as
the AG motifs of Table 1, or those AM motifs of Table 1 that have
no net charge. Non-limiting examples of AG XTEN include, but are
not limited to 36, 42, 144, 288, 576, and 864 AG family sequences
of Tables 2 and 22, or fragments thereof. In another embodiment,
the XTEN can comprise varying proportions of AE and AG motifs in
order to have a net charge that is deemed optimal for a given use
or to maintain a given physicochemical property.
[0251] The XTEN of the conjugates of the present invention
generally have no or a low content of positively charged amino
acids. In some embodiments, the XTEN may have less than about 10%
amino acid residues with a positive charge, or less than about 7%,
or less than about 5%, or less than about 2%, or less than about 1%
amino acid residues with a positive charge. However, the invention
contemplates constructs where a defined number of amino acids with
a positive charge, such as lysine, are incorporated into XTEN to
permit conjugation between the epsilon amine of the lysine and a
reactive group on a payload or a cross-linker to be conjugated to
the XTEN backbone. In one embodiment of the foregoing, the XTEN of
the subject conjugates has between about 1 to about 100 lysine
residues, or about 1 to about 70 lysine residues, or about 1 to
about 50 lysine residues, or about 1 to about 30 lysine residues,
or about 1 to about 20 lysine residues, or about 1 to about 10
lysine residues, or about 1 to about 5 lysine residues, or about 1
to about 3 lysine residues, or alternatively only a single lysine
residue. Using the foregoing lysine-containing XTEN, conjugates can
be constructed that comprise XTEN, an optional linker, plus a
payload useful in the treatment of a condition in a subject wherein
the maximum number of molecules of the payload agent linked to the
XTEN component is determined by the numbers of lysines with a
reactive side group (e.g., a terminal amine) incorporated into the
XTEN.
7. Low Immunogenicity
[0252] In another aspect, the invention provides XTEN compositions
having a low degree of immunogenicity or are substantially
non-immunogenic. Several factors can contribute to the low
immunogenicity of XTEN, e.g., the non-repetitive sequence, the
unstructured conformation, the high degree of solubility, the low
degree or lack of self-aggregation, the low degree or lack of
proteolytic sites within the sequence, and the low degree or lack
of epitopes in the XTEN sequence.
[0253] Conformational epitopes are formed by regions of the protein
surface that are composed of multiple discontinuous amino acid
sequences of the protein antigen. The precise folding of the
protein brings these sequences into a well-defined, stable spatial
configurations, or epitopes, that can be recognized as "foreign" by
the host humoral immune system, resulting in the production of
antibodies to the protein or the activation of a cell-mediated
immune response. In the latter case, the immune response to a
protein in an individual is heavily influenced by T-cell epitope
recognition that is a function of the peptide binding specificity
of that individual's HLA-DR allotype. Engagement of a MHC Class II
peptide complex by a cognate T-cell receptor on the surface of the
T-cell, together with the cross-binding of certain other
co-receptors such as the CD4 molecule, can induce an activated
state within the T-cell. Activation leads to the release of
cytokines further activating other lymphocytes such as B cells to
produce antibodies or activating T killer cells as a full cellular
immune response.
[0254] The ability of a peptide to bind a given MHC Class II
molecule for presentation on the surface of an APC (antigen
presenting cell) is dependent on a number of factors; most notably
its primary sequence. In one embodiment, a lower degree of
immunogenicity is achieved by designing XTEN sequences that resist
antigen processing in antigen presenting cells, and/or choosing
sequences that do not bind MHC receptors well. The invention
provides XTEN-payload, XTEN-cross-linker, and XTEN-click-chemistry
reactant conjugates with substantially non-repetitive XTEN
polypeptides designed to reduce binding with MHC II receptors, as
well as avoiding formation of epitopes for T-cell receptor or
antibody binding, resulting in a low degree of immunogenicity.
Avoidance of immunogenicity can attribute to, at least in part, a
result of the conformational flexibility of XTEN sequences; i.e.,
the lack of secondary structure due to the selection and order of
amino acid residues. For example, of particular interest are
sequences having a low tendency to adapt compactly folded
conformations in aqueous solution or under physiologic conditions
that could result in conformational epitopes. The administration of
polypeptides comprising XTEN, using conventional therapeutic
practices and dosing, would generally not result in the formation
of neutralizing antibodies to the XTEN sequence, and also reduce
the immunogenicity of the payload in the conjugates.
[0255] In one embodiment, the XTEN sequences utilized in the
subject polypeptides can be substantially free of epitopes
recognized by human T cells. The elimination of such epitopes for
the purpose of generating less immunogenic proteins has been
disclosed previously; see for example WO 98/52976, WO 02/079232,
and WO 00/3317 which are incorporated by reference herein. Assays
for human T cell epitopes have been described (Stickler, M., et al.
(2003) J Immunol Methods, 281: 95-108). Of particular interest are
peptide sequences that can be oligomerized without generating T
cell epitopes or non-human sequences. This is achieved by testing
direct repeats of these sequences for the presence of T-cell
epitopes and for the occurrence of 6 to 15-mer and, in particular,
9-mer sequences that are not human, and then altering the design of
the XTEN sequence to eliminate or disrupt the epitope sequence. In
some embodiments, the XTEN sequences are substantially
non-immunogenic by the restriction of the numbers of epitopes of
the XTEN predicted to bind MHC receptors. With a reduction in the
numbers of epitopes capable of binding to MHC receptors, there is a
concomitant reduction in the potential for T cell activation as
well as T cell helper function, reduced B cell activation or
upregulation and reduced antibody production. The low degree of
predicted T-cell epitopes can be determined by epitope prediction
algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999)
Nat Biotechnol, 17: 555-61), as shown in Example 46. The TEPITOPE
score of a given peptide frame within a protein is the log of the
K.sub.d (dissociation constant, affinity, off-rate) of the binding
of that peptide frame to multiple of the most common human MHC
alleles, as disclosed in Sturniolo, T. et al. (1999) Nature
Biotechnology 17:555). The score ranges over at least 20 logs, from
about 10 to about -10 (corresponding to binding constraints of
10e.sup.10 K.sub.d to 10e.sup.-10 K.sub.d), and can be reduced by
avoiding hydrophobic amino acids that serve as anchor residues
during peptide display on MHC, such as M, I, L, V, F. In some
embodiments, an XTEN component incorporated into either a
XTEN-payload, XTEN-cross-linker, or XTEN-click-chemistry reactant
conjugate does not have a predicted T-cell epitope at a TEPITOPE
threshold score of about -5, or -6, or -7, or -8, or -9, or at a
TEPITOPE score of -10. As used herein, a score of "-9" is a more
stringent TEPITOPE threshold than a score of -5.
8. Increased Hydrodynamic Radius
[0256] In another aspect, a subject XTEN useful as a fusion partner
has a high hydrodynamic radius; a property that confers a
corresponding increased apparent molecular weight to the
XTEN-payload composition compared to the payload without the XTEN.
As detailed in Example 26, the linking of XTEN to therapeutic
protein sequences results in compositions that can have increased
hydrodynamic radii, increased apparent molecular weight, and
increased apparent molecular weight factor compared to a
therapeutic protein not linked to an XTEN. For example, in
therapeutic applications in which prolonged half-life is desired,
compositions in which one or more XTEN with a high hydrodynamic
radius are conjugated to a payload can effectively enlarge the
hydrodynamic radius of the conjugate beyond the glomerular pore
size of approximately 3-5 nm (corresponding to an apparent
molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic
and biodistribution properties of poly(ethylene glycol)-protein
conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced
renal clearance of circulating proteins with a corresponding
increase in terminal half-life and other enhanced pharmacokinetic
properties. The hydrodynamic radius of a protein is conferred by
its molecular weight as well as by its structure, including shape
or compactness. Not to be bound by a particular theory, the XTEN
can adopt open conformations due to the electrostatic repulsion
between individual charges of incorporated charged residues in the
XTEN as wells as because of the inherent flexibility imparted by
the particular amino acids in the sequence that lack potential to
confer secondary structure. The open, extended and unstructured
conformation of the XTEN polypeptide has a greater proportional
hydrodynamic radius compared to polypeptides of a comparable
sequence length and/or molecular weight that have secondary or
tertiary structure, such as typical globular proteins. Methods for
determining the hydrodynamic radius are well known in the art, such
as by the use of size exclusion chromatography (SEC), as described
in U.S. Pat. Nos. 6,406,632 and 7,294,513. Example 26 demonstrates
that increases in XTEN length result in proportional increase in
the hydrodynamic radius, apparent molecular weight, and/or apparent
molecular weight factor, and thus permit the tailoring of an
XTEN-payload to desired cut-off values of apparent molecular
weights or hydrodynamic radii. Accordingly, in certain embodiments,
the XTEN-payload can be configured with an XTEN such that the
resulting conjugate can have a hydrodynamic radius of at least
about 5 nm, or at least about 8 nm, or at least about 10 nm, or
about 12 nm, or about 15 nm, or about 20 nm, or about 30 nm or
more. In the foregoing embodiments, the large hydrodynamic radius
conferred by the XTEN in a XTEN-payload conjugate can lead to
reduced clearance of the resulting conjugate, an increase in
terminal half-life, and an increase in mean residence time. As
described in the Examples, when the molecular weights of the
XTEN-containing compositions are derived from size exclusion
chromatography analyses, the open conformation of the XTEN due to
the low degree of secondary structure results in an increase in the
apparent molecular weight of the conjugates into which they are
incorporated. In one embodiment, the present invention makes use of
the discovery that the increase in apparent molecular weight can be
accomplished by the linking not only of a single XTEN of a given
length, but also by the linking of 2, 3, 4 or more XTEN of
proportionally shorter lengths, either in linear fashion or as a
trimeric or tetrameric, branched configuration, as described more
fully, below. In some embodiments, the XTEN comprising a payload
and one or more XTEN exhibits an apparent molecular weight of at
least about 400 kD, or at least about 500 kD, or at least about 700
kD, or at least about 1000 kD, or at least about 1400 kD, or at
least about 1600 kD, or at least about 1800 kD, or at least about
2000 kD. Accordingly, the XTEN-payload conjugate exhibits an
apparent molecular weight that is about 1.3-fold greater, or about
2-fold greater, or about 3-fold greater or about 4-fold greater, or
about 8-fold greater, or about 10-fold greater, or about 12-fold
greater, or about 15-fold, or about 20-fold greater than the actual
molecular weight of the conjugate. In one embodiment, the isolated
XTEN-payload conjugate of any of the embodiments disclosed herein
exhibit an apparent molecular weight factor under physiologic
conditions that is greater than about 1.3, or about 2, or about 3,
or about 4, or about 5, or about 6, or about 7, or about 8, or
about 10, or greater than about 15. In another embodiment, the
XTEN-payload has, under physiologic conditions, an apparent
molecular weight factor that is about 3 to about 20, or is about 5
to about 15, or is about 8 to about 12, or is about 9 to about 10
relative to the actual molecular weight of the conjugate.
Generally, the increased apparent molecular weight of the subject
XTEN-payload conjugates enhances the pharmacokinetic properties of
the composition by a combination of factors, which include reduced
active clearance, reduced renal clearance, and reduced loss through
capillary and venous junctions.
9. Compositions and Methods of Purifying XTEN as Substantially
Homogeneous Preparations
[0257] It is an object of the invention to provide compositions of
XTEN and methods of making preparations comprising XTEN with a high
level of purity and uniformity in the length and composition of the
XTEN described herein.
[0258] The expression of recombinant XTEN protein or a recombinant
fusion protein comprising XTEN in a host cell normally, like any
globular protein, results in a mixture of different compounds in
which a portion are truncated versions of the desired protein
length. The truncation can be the result of early termination of
translation, mRNA instability, or proteolysis in the host cell.
Because globular proteins generally have efficient or complete
folding into their three-dimensional structure while truncated
versions do not, typical purification and recovery processes can
successfully separate and remove the truncated versions such that a
high level of product homogeneity is achieved in a given
preparation of globular proteins. However, protein polymers such as
XTEN are unique in that, given their unstructured nature, generally
lack three-dimensional structures. It has been difficult to obtain
a homogeneous preparation of full-length XTENs due to one or more
of the above-mentioned reasons. This is because incomplete or
truncated XTEN chains differ only slightly in their physicochemical
properties from the desired full-length sequences such that
traditional processes that would be sufficient for purification of
globular proteins are not effective in the removal of truncated
XTEN from the expression product in order to obtain a substantially
homogeneous preparation of full-length sequences. While the subject
XTEN of the invention, including XTEN linked to payload, can be
purified to a moderate degree of homogeneity by conventional means
used for proteins, such as salt fractionation, ion exchange
chromatography, size exclusion chromatography, hydroxyapatite
adsorption chromatography, hydrophobic interaction chromatography
or gel electrophoresis, these methods alone do not result in
preparations wherein the XTEN are substantially homogeneous in
sequence length.
[0259] The subject methods provided herein permit production of
substantially homogenous preparation of XTENs via one or a few
simple purification steps. In one embodiment, the practice of any
of such methods of the present invention can utilize an XTEN
designed to further comprise, as a fusion protein, affinity tags
located at either or both of the N- and C-termini of the XTEN such
that the expressed product can be subject to purification methods
to selectively capture the full-length expressed polypeptide,
thereby removing truncated XTEN by-products (see FIGS. 41-42).
Non-limiting examples of affinity tags that can be added to the
termini of XTEN are presented in Table 7. Non-limiting examples of
methods of the design, expression, and purification methods to
achieve substantially homogeneous XTEN are described in the
Examples.
[0260] In some embodiments, the invention provides substantially
homogeneous polypeptide compositions with XTEN fused directly to
one affinity tag (such as, but not limited to the tags of Table 7)
linked to either the N- or C-terminus of the XTEN. In other
embodiments, the invention provides substantially homogeneous
polypeptide compositions with XTEN fused to one affinity tag (such
as, but not limited to the tags of Table 7) by a cleavage sequence
linked to either the N- or C-terminus of the XTEN. In other
embodiments, the invention provides substantially homogeneous
polypeptide compositions with XTEN fused directly to one or two
different affinity tags (such as, but not limited to the tags of
Table 7) linked to the N- and/or C-termini of the XTEN, as shown in
FIG. 41. In other embodiments, the invention provides substantially
homogeneous compositions with XTEN fused to one or two cleavage
sequences (such as, but not limited to the cleavage sequences of
Table 8 or Table 9) which, in turn, are each fused to different
affinity tags (such as, but not limited to the tags of Table 7)
linked to the N- or C-termini or both the N- and C-termini of the
XTEN, as shown in FIG. 41. In yet other embodiments, the invention
provides substantially homogeneous polypeptide compositions with
XTEN fused directly to one or two different affinity tags (such as,
but not limited to the tags of Table 7) linked to the N- and/or
C-termini of the XTEN that further comprise a helper sequence (such
as, but not limited to the sequences of Table 12) fused to the
N-terminus of the protein. As used in the context of the proteins
described herein, "substantially homogeneous" means that at least
about 85%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, 96%, 97%, 98%, 99% or even higher of the
polypeptide sequences of the preparation have identical sequence
length. The percent values are based on the area percentage of the
chromatogram of the preparation analyzed by HPLC or the area
percentage of the scan of the preparation analyzed by SDS-PAGE, or
by other such standard procedures known in the art for assessing
the purity of proteins.
[0261] In one embodiment, the invention provides a substantially
homogenous polypeptide having the configuration of formula I:
(HS)-(AT1)-(CS1)-(XTEN) I
wherein HS is the helper sequence, AT1 is the first affinity tag,
CS1 is the first cleavage sequence, and XTEN is the extended
recombinant polypeptide.
[0262] In another embodiment, the invention provides a
substantially homogenous polypeptide having the configuration of
formula II
(HS)-(CS1)-(XTEN)-(CS2)-(AT1) II
wherein HS is the helper sequence, AT1 is the first affinity tag,
CS1 is the first cleavage sequence, CS2 is the second cleavage
sequence and XTEN is the extended recombinant polypeptide.
[0263] In another embodiment, wherein the composition has the
configuration of formula III:
(HS)-(AT1)-(CS1)-(XTEN)-(CS2)-(AT2) III
wherein HS is the helper sequence, AT1 is the first affinity tag,
AT2 is the second affinity tag, CS1 is the first cleavage sequence,
CS2 is the second cleavage sequence and XTEN is the extended
recombinant polypeptide.
[0264] The polypeptide constructs comprising affinity tags have the
advantageous property, compared to XTEN not linked to affinity
tags, of being able to be purified to substantially homogeneous
length by use of chromatography substrates to which the affinity
tags will bind. In some embodiments, the categories of
chromatography substrates used in the method of purification are
selected from the chromatography substrates set forth in Table 7,
which are utilized for the purification of XTEN linked to the
corresponding the indicated affinity tag in the tables. As will be
appreciated by one of skill in the art, the categories of
chromatography substrate can encompass different chemical groups
linked to different matrices or resins; e.g., anion exchange
substrates include quaternary trimethylammonium and
diethylaminoethyl bound to resins, cation exchange substrates
include sulfo or sulfopropyl or carboxymethyl or phosphate groups
bound to resins, HIC substrates include ethyl, isopropyl, butyl,
phenyl or octyl groups bound to resins, and IMAC substrates include
iminodiacetic acid and nitriloacetic acid groups bound to resins.
The foregoing substrates are listed for illustrative purposes and
are not intended to limit the scope of substrates that can be
employed to practice the invention.
[0265] In some embodiments, the invention provides substantially
homogeneous XTEN prepared from the a polypeptide comprising an XTEN
fused to a first or a first and a second affinity tag by cleavage
sequences (such as, but not limited to the cleavage sequences of
Table 8) capable of being cleaved by a protease, wherein the
preparation is treated with the protease to cleave the cleavage
sequences to release the XTEN from the polypeptide, followed by a
chromatography step to bind and then elute, and then recover the
substantially homogeneous XTEN. In one embodiment of the foregoing,
the protease is trypsin and the cleavage sequences are capable of
being cleaved by trypsin, non-limiting examples of which are listed
in Tables 11 and 15. In another embodiment of the foregoing, the
protease is TEV and the cleavage sequences are capable of being
cleaved by TEV. In another embodiment of the foregoing, the cleaved
XTEN is purified by binding to MacoCap SP chromatography substrate
followed by elution with a salt or buffer solution such as, but not
limited to, sodium phosphate/NaCl, resulting in the substantially
homogenous XTEN. As used in the context of XTEN and/or polypeptides
comprising XTEN, a preparation that is "substantially purified"
means that at least about 85%, and more preferably at least about
90%, and more preferably at least about 91%, and more preferably at
least about 92%, and more preferably at least about 93%, and more
preferably at least about 94%, and more preferably at least about
95% or more of the individual molecules of a given preparation have
identical sequence length; in other words, the same number of amino
acids. The methods that can be utilized to assay for homogeneity of
length include mass spectroscopy, size exclusion
chromatography/HPLC, or SDS-PAGE followed by silver staining; the
methods can be used individually or collectively to quantitate the
degree of homogeneity. A generalized scheme for purification of
polypeptides comprising XTEN with affinity tag sequences optimized
for purification is shown in FIGS. 41-42. After purification the
tags can be proteolytically cleaved (FIG. 41B) or retained (FIG.
41C). The XTEN can be purified from contaminants due to the unique
amino acid composition of XTEN, as illustrated in FIG. 42.
[0266] In one embodiment, the invention provides a method to
produce a substantially purified preparation of a polypeptide
comprising an XTEN, comprising the steps of designing a gene
encoding an XTEN and a first affinity tag, creating an expression
vector suitable for transforming a host cell comprising the
encoding gene operably linked to control sequences, transforming
the host cell with the expression vector, culturing the host cell
under conditions suitable for the expression of the XTEN with
linked affinity tag, subjecting the crude expression product to a
purification process that comprises an affinity purification step
wherein the crude expression product is loaded onto a first
chromatography substrate that selectively binds the first affinity
tag, washing the chromatography substrate to elute material not
bound to the chromatography substrate, eluting the retained protein
under appropriate conditions and recovering the eluate wherein the
recovered polypeptide is substantially homogeneous in length. In
another embodiment, the invention provides a method to produce a
substantially homogeneous preparation of a polypeptide comprising
an XTEN, comprising the steps of designing a gene encoding an XTEN
comprising a first and a second affinity tag, creating an
expression vector suitable for transforming a host cell comprising
the encoding gene operably linked to control sequences,
transforming the host cell with the expression vector, culturing
the host cell under conditions suitable for the expression of the
polypeptide, subjecting the crude expression product to a
purification process that comprises an affinity purification step
wherein the lysate is loaded onto a first chromatography substrate
that selectively binds the first affinity tag, washing the
chromatography substrate to elute material not bound to the
chromatography substrate, eluting the retained protein under
appropriate conditions and recovering the eluate, loading the
recovered XTEN polypeptide onto a second chromatography substrate
under conditions effective to capture the polypeptide with the
second affinity tag onto the chromatography substrate, washing the
chromatography substrate to elute material not bound to the
chromatography substrate, eluting the XTEN polypeptide under
conditions effective to elute the XTEN polypeptide with the second
affinity tag, recovering the eluate containing the polypeptide
comprising the XTEN polypeptide with the first and the second
affinity tag wherein the recovered polypeptide is substantially
homogeneous in length. In yet another embodiment, the invention
provides a method to produce a substantially homogeneous
preparation of a polypeptide comprising an XTEN, comprising the
steps of designing a gene encoding an XTEN comprising a first
affinity tag linked by a cleavage sequence to the N-terminus of the
encoded XTEN and a second affinity tag linked by a cleavage
sequence to the C-terminus of the encoded XTEN, creating an
expression vector suitable for transforming a host cell comprising
the encoding gene operably linked to control sequences,
transforming the host cell with the expression vector, culturing
the host cell under conditions suitable for the expression of the
polypeptide, subjecting the crude expression product to a
purification process that comprises an affinity purification step
wherein the lysate is loaded onto a first chromatography substrate
that selectively binds the first affinity tag, washing the
chromatography substrate to elute material not bound to the
chromatography substrate, eluting the retained protein under
appropriate conditions and recovering the eluate, loading the
recovered polypeptide onto a second chromatography substrate under
conditions effective to capture the polypeptide with the second
affinity tag onto the chromatography substrate, washing the
chromatography substrate to elute material not bound to the
chromatography substrate, eluting the polypeptide under conditions
effective to elute the polypeptide with the second affinity tag,
then treating the recovered polypeptide with a protease under
conditions effective to release the XTEN from the polypeptide and
loading the material onto a chromatography substrate capable of
capturing the XTEN but not the affinity tags, washing the
chromatography substrate to elute material not bound to the
chromatography substrate, eluting the XTEN, recovering the eluate
containing the XTEN polypeptide wherein the recovered XTEN is
substantially homogeneous in length. In one embodiment of the
foregoing methods described in this paragraph, the first and second
affinity tags are selected from the group of affinity tags set
forth in Table 7. In one embodiment of the method, the first
affinity tag linked to the XTEN as a fusion protein comprises the
sequence RPRPRPRPRPRPR (SEQ ID NO: 17) and the chromatography
substrate used to bind the polypeptide is MacroCap SP. In another
embodiment of the foregoing methods, the first affinity tag linked
to a first terminus of the XTEN as a fusion protein comprises the
sequence RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19), the second
affinity tag linked to a second terminus of the XTEN comprises the
sequence HHHHHHHH (SEQ ID NO: 20), the first chromatography
substrate used to bind the polypeptide is MacroCap SP, and the
second chromatography substrate used to bind the polypeptide is a
immobilized metal on affinity (IMAC) substrate. In another
embodiment of the foregoing methods, the first affinity tag fused
to a cleavage sequence fused to a first terminus of the XTEN as a
fusion protein comprises the sequence RPRPRPRPRPRPR (SEQ ID NO: 17)
or RPRPRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 19), the second affinity tag
fused to a cleavage sequence to a second terminus of the XTEN
comprises the sequence HHHHHH (SEQ ID NO: 18) or HHHHHHHH (SEQ ID
NO: 20), the first chromatography substrate used to bind the
polypeptide is MacroCap SP, the second chromatography substrate
used to bind the polypeptide is a immobilized metal on affinity
(IMAC) substrate, the cleavage sequences comprise an arginine or
lysine (including, but not limited to the sequences of Tables 8 and
9) and are cleaved by trypsin, and Macrocap Q is the chromatography
substrate used to bind the XTEN freed from the affinity tags or, in
the alternative, the freed XTEN is captures as flow-through by
passing the protease-treated preparation through one or more of
cation exchange, HIC and/or IMAC to capture the cleavage products
and protease, leaving the XTEN in the flow-through, which is then
recovered as a substantially homogeneous preparation.
[0267] It will be appreciated by one of skill in the art that the
order and specific conditions of the steps of the method will vary
depending on the composition of the XTEN-affinity tag polypeptide
as well as the starting expression level and degree of
contamination of truncated contaminants. For example, with certain
XTEN compositions, the use of a single affinity tag linked to the
XTEN will be sufficient to achieve a preparation in which the
polypeptide molecules are substantially homogeneous in length. In
such cases, in one embodiment the single affinity tag is selected
from the affinity tags set forth in Table 7. With other XTEN
compositions, the use of a first and a second affinity tag will be
sufficient to achieve a preparation in which the polypeptide
molecules are substantially homogeneous in length and in such
cases, in one embodiment, the first and second affinity tags are
different and each is selected from the affinity tags set forth in
Table 7. It will be further appreciated by one of skill in the art
that once the polypeptides comprising cleavage sequences are
purified, the recovered polypeptide can be subsequently treated by
proteolysis to release the one or two affinity tags, followed by
passing the treated XTEN through a chromatography substrate to
recover the XTEN without linked affinity tags. A schematic of the
method is illustrated in FIG. 42 and exemplary methodologies are
described in the Examples. Many different proteases can be utilized
for the release of terminal purification tags, depending on the
sequence linking the affinity tag to the XTEN, including but not
limited to a protease selected from Table 9. In one embodiment, the
protease is selected from the group consisting of trypsin,
chymotrypsin, tobacco etch mosaic virus protease (TEV), FXa, and
enterokinase. In another embodiment, the cleavage sequence
incorporated into the polypeptide comprises an arginine residue
that can be cleaved and the affinity tag removed by treatment with
trypsin, thereby releasing the XTEN that is subsequently recovered
in substantially purified form by chromatography such as, by
capture using anion exchange (including but not limited to,
MacroCap Q) or recovered as flow-through wherein the non-XTEN
cleavage products and protease are captured by one or more of HIC,
cation exchange, or IMAC chromatography, leaving substantially
homogeneous XTEN in the flow-through.
TABLE-US-00008 TABLE 7 Affinity Tags and Chromatography Substrate
Categories that Bind Affinity Tags SEQ ID Chromatography Affinity
Tag Amino Acid Sequence NO: Substrate LYPYPYP, LYYYPP, WPWP, FPFPFP
413-416 HIC (Y).sub.n, (W).sub.n, (YP).sub.n, (WP).sub.n,
(FP).sub.n, (LP)n with n = 3-20 417-422 HIC (RP).sub.n, (KP).sub.n,
(HP).sub.n, (H).sub.n, (R).sub.n with n = 3-20 423-427 Cation
exchange, IMAC (E).sub.n, (D).sub.n, (ED).sub.n, (EP).sub.n, (DP)n
with n = 3-20 428-432 Anion exchange RPRPRPRPRP 433 Cation exchange
RPRPRPRPRPGR 434 Cation exchange RPRPRPRPRPRPRP 435 Cation exchange
RPRPRPRPRPRPRPGR 436 Cation exchange KPKPKPKPKP 437 Cation exchange
KPKPKPKPKPGR 438 Cation exchange RPRPRPRPRPRPRPRPRP 439 Cation
exchange RPRPRPRPRPRPRPRPRPGR 440 Cation exchange
RPRPRPRPRPRPRPRPRPRPRP 441 Cation exchange RPRPRPRPRPRPRPRPRPRPRPGR
442 Cation exchange RPRPRPRPRPRPRPRPRPRPRPRPRPRP 443 Cation
exchange RPRPKPRPKPRPKPRPKP 444 Cation exchange PRPKPRPKPRPKPRPKPGR
445 Cation exchange RPRPKPRPKPRPKPRPKPRPKP 446 Cation exchange
RPRPKPRPKPRPKPRPKPRPKPGR 447 Cation exchange GSPYGYPYSYS,
GSPWGSPTSTE, GSPAGSPTSTE, 448-450 HIC GSPXGXPXSXS, GSPSGXPXSXS,
GSPSGTPXSXS 451-453 HIC where X = Ile, Leu, Val, Phe, Trp, or Tyr
GSPXGXPXSXS, GSPSGXPXSXS, GSPSGTPXSXS 454-456 Cation exchange,
where X = Arg, Lys, or His IMAC HHHHHH, HHHHHHHH 18 and 20 IMAC
STRPSRRSRRG, STRRGTRRGTRRG, 457-458 Cation exchange STRPSRGRARG,
STRPSRRARG, STRPSRRRRG, 459-461 Cation exchange STEPSEESEEG,
STEEGTEEGTEEG, 462-463 Anion exchange STEPSEGEAEG, STEPSEEAEG,
STEPSEEEEG, 464-466 Anion exchange
TABLE-US-00009 TABLE 8 Trypsin Cleavage Sequences P4 P3 P2 P1 P1'
P2' SEQ ID NO: S A S R S A 467 S A S K S A 468 G S G R A T 469 E A
A R H H 470 A P G R H H 471 G S G R G S 472 R X* K X* *X = any
L-amino acid other than proline
TABLE-US-00010 TABLE 9 Proteases and Protease Cleavage Sequences
Exemplary SEQ SEQ Protease Acting Cleavage ID ID Upon Sequence
Sequence NO: Cleavage Sequences* NO: FXIa KLTR.dwnarw.AET 473
KD/FL/T/R.dwnarw.VA/VE/GT/GV FXIa DFTR.dwnarw.VVG 474
KD/FL/T/R.dwnarw.VA/VE/GT/GV FXIIa TMTR.dwnarw.IVGG 475 Kallikrein
SPFR.dwnarw.STGG 476 -/-/FL/RY.dwnarw.SR/RT/-/- FVIIa
LQVR.dwnarw.IVGG 477 FIXa PLGR.dwnarw.IVGG 478
-/-/G/R.dwnarw.-/-/-/- FXa IEGR.dwnarw.TVGG 479
IA/E/GFP/R.dwnarw.STI/VFS/-/G FIIa (thrombin) LTPR.dwnarw.SLLV 480
-/-/PLA/R.dwnarw.SAG/-/-/- Elastase-2 LGPV.dwnarw.SGVP 481
-/-/-/VIAT.dwnarw.-/-/-/- Granzyme-B VAGD.dwnarw.SLEE 482
V/-/-/D.dwnarw.-/-/-/- MMP-12 GPAG.dwnarw.LGGA 483
G/PA/-/G.dwnarw.L/-/G/- 491 MMP-13 GPAG.dwnarw.LRGA 484
G/P/-/G.dwnarw.L/-/GA/- 492 MMP-17 APLG.dwnarw.LRLR 485
-/PS/-/-.dwnarw.LQ/-/LT/- MMP-20 PALP.dwnarw.LVAQ 486 TEV
ENLYFQ.dwnarw.G 487 ENLYFQ.dwnarw.G/S 493 Enterokinase
DDDK.dwnarw.IVGG 488 DDDK.dwnarw.IVGG 488 Protease 3C
LEVLFQ.dwnarw.GP 489 LEVLFQ.dwnarw.GP 489 (PreScission .TM.)
Sortase A LPKT.dwnarw.GSES 490 L/P/KEAD/T.dwnarw.G/-/EKS/S 494
Trypsin K.dwnarw.X** or R.dwnarw.X K/X or R/X Trypsin R.dwnarw.X**
SASRSA 21 .dwnarw.indicates cleavage site *the listing of multiple
amino acids before, between, or after a slash indicate alternative
amino acids that can be substituted at the position; ''-''
indicates that any amino acid may be substituted for the
corresponding amino acid indicated in the middle column **x is any
L-amino acid other than proline
[0268] In another embodiment, XTEN can be designed such that one or
both affinity tags linked to the termini and used to facilitate
purification can remain part of the final product, eliminating the
requirement for a protease release step. If purification tags are
designed to remain a part of a drug product, then tag sequences are
selected that do not elicit a pronounced immune response.
Immunogenicity can be predicted using computational prediction
algorithms or experimental assays. Sequence, that avoid T-cell and
B-cell epitopes are preferred. Non-limiting examples of sequences
incorporated into the terminus of XTEN sequences that facilitate
capture and that may optionally remain associated with the
conjugate constructs are provided in Table 7.
10. Compositions for Increased Expression of XTEN
[0269] In another aspect, the invention provides constructs
comprising polynucleic acid sequences encoding XTEN and methods of
making the XTEN for use in the subject conjugates in which
additional encoding polynucleotide helper sequences are added to
the 5' end of polynucleotides encoding the XTEN or are added to the
5' end of sequences encoding an affinity tag linked to the 5' end
of sequences encoding an XTEN to enhance and facilitate the
expression of the XTEN or XTEN with cleavage sequences linked to
affinity tag polypeptides in transformed host cells, such as
bacteria. Examples of such encoded helper sequences are given in
Table 10 and in the Examples. In one embodiment, the invention
provides a polynucleotide sequence construct encoding a polypeptide
comprising a helper sequence having at least about 80%, or at least
about 90%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%
sequence identity to a sequence selected from Table 10 linked to
the N-terminus of a first affinity tag selected from the group of
sequences set forth in Table 7 that, in turn, is either linked to a
cleavage sequence described herein or directly to the N-terminus of
an XTEN having at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%
sequence identity, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99% to
a sequence selected from the group of sequences set forth in Tables
2 and 3. The invention provides expression vectors encoding the
constructs useful in methods to produce substantially homogeneous
preparations of polypeptides and XTEN at high expression levels. In
some embodiments, the invention provides methods for producing a
substantially homogenous population of polypeptides comprising an
XTEN and a first and a second affinity tag and a helper sequence,
the method comprising culturing in a fermentation reaction a host
cell that comprises a vector encoding a polypeptide comprising an
XTEN and the first and second affinity tag under conditions
effective to express the polypeptide such that more than about 2
g/L, or more than about 3 g/L, or more than about 4 g/L, or more
than about 5 g/L, or more than about 6 g/L, or more than about 7
grams per liter (7 g/L) of the polypeptide is produced as a
component of a crude expression product of the host cell when the
fermentation reaction reaches an optical density of at least 130 at
a wavelength of 600 nm. In one embodiment, the method further
comprises the steps of adsorbing the polypeptide onto a first
chromatography substrate under conditions effective to capture the
first affinity tag of the polypeptide onto the chromatography
substrate; eluting and recovering the polypeptide; adsorbing the
polypeptide onto a second chromatography substrate under conditions
effective to capture the second affinity tag of the polypeptide
onto the chromatography substrate; eluting the polypeptide; and
recovering the substantially homogeneous polypeptide preparation.
In one embodiment of the foregoing method, the vector further
comprises nucleotides encoding a helper sequence at the N-terminus
of the encoded polypeptide wherein the helper sequence has at least
80%, or at least 90%, or at least 95% sequence identity to a
sequence set forth in Table 10. In other embodiments, the invention
provides methods for producing a substantially homogenous
population of polypeptides comprising an XTEN and a first and a
second affinity tag and a helper sequence, the method comprising
culturing in a fermentation reaction a host cell that comprises a
vector encoding a polypeptide comprising an XTEN and the first and
second affinity tag under conditions effective to express the
polypeptide product at a concentration of more than about 10
milligrams/gram of dry weight host cell (mg/g), or at least about
250 micromoles/L, or about 300 micromoles/L, or about 350
micromoles/L, or about 400 micromoles/L, or about 450 micromoles/L,
or about 500 micromoles/L of said polypeptide when the fermentation
reaction reaches an optical density of at least 130 at a wavelength
of 600 nm. In one embodiment of the foregoing, the method further
comprises the steps of adsorbing the polypeptide onto a first
chromatography substrate under conditions effective to capture the
first affinity tag of the polypeptide onto the chromatography
substrate; eluting and recovering the polypeptide; adsorbing the
polypeptide onto a second chromatography substrate under conditions
effective to capture the second affinity tag of the polypeptide
onto the chromatography substrate; eluting the polypeptide; and
recovering the substantially homogeneous polypeptide preparation.
In one embodiment of the foregoing method, the vector further
comprises nucleotides encoding a helper sequence at the N-terminus
of the encoded polypeptide wherein the helper sequence has at least
80%, or at least 90%, or at least 95% sequence identity to a
sequence set forth in Table 10. In other embodiments, the invention
provides methods for producing a substantially homogenous
population of polypeptides comprising an XTEN and a first and a
second affinity tag and a helper sequence, the method comprising
culturing in a fermentation reaction a host cell that comprises a
vector encoding a polypeptide comprising an XTEN and the first and
second affinity tag under conditions effective to express the
polypeptide product at a concentration of more than about 10
milligrams/gram of dry weight host cell (mg/g), or at least about
15 mg/g, or at least about 20 mg/g, or at least about 25 mg/g, or
at least about 30 mg/g, or at least about 40 mg/g, or at least
about 50 mg/g of said polypeptide when the fermentation reaction
reaches an optical density of at least 130 at a wavelength of 600
nm. In one embodiment of the foregoing, the method further
comprises the steps of adsorbing the polypeptide onto a first
chromatography substrate under conditions effective to capture the
first affinity tag of the polypeptide onto the chromatography
substrate; eluting and recovering the polypeptide; adsorbing the
polypeptide onto a second chromatography substrate under conditions
effective to capture the second affinity tag of the polypeptide
onto the chromatography substrate; eluting the polypeptide; and
recovering the substantially homogeneous polypeptide preparation.
In one embodiment of the foregoing method, the vector further
comprises nucleotides encoding a helper sequence at the N-terminus
of the encoded polypeptide wherein the helper sequence has at least
80%, or at least 90%, or at least 95% sequence identity to a
sequence set forth in Table 10. In another embodiment, the
constructs of the foregoing methods of the paragraph further
comprise nucleotides encoding protease cleavage sequences between
the affinity tags and the XTEN and the method provides that the
recovered polypeptides of the preparation are treated with a
protease capable of cleaving the cleavage sequences, such as but
not limited to trypsin, thereby releasing the XTEN from the
polypeptide; the XTEN is adsorbed onto a chromatography substrate
under conditions effective to capture the XTEN; the XTEN is then
eluted and recovered as a substantially homogeneous XTEN.
TABLE-US-00011 TABLE 10 Examples of helper sequences to facilitate
protein expression, secretion and processing in bacteria SEQ ID
Amino Acid at Position** Amino Acid Sequence* NO: X1 X2 X3 X4 X5 Z
ADAQKAADNKKP 495 KTLVYCSEGSPE 496 ENNAQTTNESAG 497 KDTIALVVSTLN 498
APKDNTWYTGA 499 ADVPAGVTLAEK 500 KIEEGKLVIWIN 501 AEATTAAGGA 502
ATGTATSEGSPE 503 EPTAATTGESAG 504 AETTAPAGST 505 APTEATAGTGA 506
AETPAGATGAE 507 APEEGTAGGA 508 SLSTEATAKIVSEFGRDAN 509
ANPEQLEEQREETRLIIEE 510 SASTEATAKAASEAGRDAN 511 ANPEQAEEQREETR 512
ANPEQAEEQREET 513 ANPEQAEEQSEET 514 KNPEQAEEQREET 515 KNPEQAEEQSEET
516 KNPEQAEEQAEEQREET 517 KNPEQAEEQAEEQSEET 518 KNHEQAEEQAEEQSEET
519 KKHEQAEEQAEEQSEET 520 KKPEQAEEQAEEQREET 521 KNHEQEKEKAEEQSEET
522 KKQEQEEKKAEEQREET 523 KNHEKDEKKAEEQSEET 524 KKQEQEKEQAEEQREET
525 KNPEQEKEKAEEQREET 526 KKPEQEEKQAEEQREET 527
KKQEQEKEQAEEQAESEREET 528 KKQEQEKEQAEEQSQSQREET 529
KKQEQEKEQAEEQSESEREET 530 KKQEQEKEQAEEQAKAESEAEREET 531
KKQEQEKEQAEEQSKSQAEAEREET 532 KKQEQEKEQAEEQAQAQAEDEREET 533
KKQEQEKEQAEEQSKSKAEDEREET 534 KKQEQEKEQAEEQPEVQREET 535
KKQEQEKEQAEEQVENPREET 536 KKQEQEKEQAEEQELCEREET 537
KKQEQEKEQAEEQGIDTREET 538 KNPEQAEEQX1EET 1 S/R ANPEQAEEQX1EET 2 S/R
KNPEQAEEQAEEQX1EET 3 S/R KX2X3EQAEEQAEEQX1EET 4 S/R K/N
K/N/T/Q/H/P/E/D/A/R/S KX2(X3).sub.10QX1EET 5 S/R K/N
K/N/T/Q/H/P/E/D/A/R/S KX2(X3).sub.7AEEQX1EET 6 S/R K/N
K/N/T/Q/H/P/E/D/A/R/S KX2X3EQE(X3).sub.3AEEQREET 7 K/N
K/N/T/Q/H/P/E/D/A/R/S KX2X3EQE(X3).sub.3AEE(X3).sub.5 8 K/N
K/N/T/Q/H/P/E/D/A/R/S KKQEQEKEQAEEQ(X4X5).sub.2REET 9 A/S K/Q/E
KKQEQEKEQAEEQ(X4X5).sub.4REET 10 A/S K/Q/E
KKQEQEKEQAEEQ(Z).sub.4REET 11 any KX2(X3).sub.n, where n = 10-40 12
K/N K/N/T/Q/H/P/E/D/A/R/S (X3).sub.n, where n = 10-50 13
K/N/T/Q/H/P/E/D/A/R/S KX2QEQEKEQAEEQ(X4X5).sub.nX1EET, 14 S/R K/N
A/S K/Q/E where n = 0-10 KX2(X3).sub.n(X4X5).sub.n,X1EET, where n =
15 S/R K/N K/N/T/Q/H/P/E/D/A/R/S A/S K/Q/E 5-20 and m = 0-10
KX2(X3).sub.n(Z).sub.n,X1EET, where n = 5-20 16 S/R K/N
K/N/T/Q/H/P/E/D/A/R/S any and m = 0-10 *where n or m = 0, the
adjoining amino acids are contiguous **indicates the amino acid(s)
that can be utilized at the given position in the amino acid
sequence entries, with the alternatives separated by ''/''
III). Payloads
[0270] The present invention relates in part to XTEN conjugates
linked to one or more payload molecules. It is contemplated that
XTEN can be linked to a broad diversity of payload molecules,
including biologically active peptides, proteins, polymers,
pharmacologically active small-molecules, polynucleic acids,
targeting peptides and proteins, targeting small molecules,
antibodies and antibody fragments, and imaging small-molecule
payloads, as well as combinations of these types of payloads
resulting in compositions with 2, 3, 4 or more types of payloads.
The invention addresses a long-felt need in increasing the terminal
half-life of exogenously administered therapeutic and diagnostic
payloads to a subject in need thereof, as well as combinations of
payloads that may include a therapeutic component and a targeting
component.
[0271] Non-limiting examples of functional classes of
pharmacologically active payload agents for use in linking to an
XTEN of the invention may be any one or more of the following:
hypnotics and sedatives, psychic energizers, tranquilizers,
respiratory drugs, anticonvulsants, muscle relaxants, antiparkinson
agents (dopamine antagnonists), analgesics, anti-inflammatories,
antianxiety drugs (anxiolytics), appetite suppressants,
antimigraine agents, muscle contractants, anti-infectives
(antibiotics, antivirals, antifungals, vaccines), antiarthritics,
antimalarials, antiemetics, anepileptics, bronchodilators,
coagulation factors, cytokines, chemokines, interleukins, growth
factors, growth hormones, endocrine hormones, exocrine hormones,
insulin, glucose-regulating peptides, anti-cancer agents,
antithrombotic agents, antihypertensives, cardiovascular drugs,
antiarrhythmics, antioxicants, anti-asthma agents, hormonal agents
(including contraceptives), sympathomimetics, diuretics, lipid
regulating agents, antiandrogenic agents, antiparasitics,
anticoagulants, neoplastics, antineoplastics, hypoglycemics,
nutritional agents and supplements, growth supplements,
antienteritis agents, vaccines, antibodies, diagnostic agents,
contrasting agents, and radioactive imaging agents.
[0272] More particularly, the active payload may fall into one of a
number of structural classes, including but not limited to small
molecule drugs, biologically active proteins (peptides,
polypeptides, proteins, recombinant proteins, antibodies, and
glycoproteins), steroids, nucleotides, oligonucleotides,
polynucleotides, fats, electrolytes, and the like. For the
XTEN-payload conjugation compositions, it is specifically
contemplated that a payload can be a pharmacologically active agent
that possesses a suitably reactive functional group, including, but
not limited to a native amino group, a sulfydryl group, a carboxyl
group, an aldehyde group, a ketone group, an alkene group, an
alkyne group, an azide group, an alcohol group, a heterocycle, or,
alternatively, is modified to contain at least one of the foregoing
reactive groups suitable for coupling to either an XTEN,
XTEN-cross-linker, or XTEN-click-chemistry reactant of the
invention using any of the conjugation methods described herein or
are otherwise known to be useful in the art for conjugating such
reactive groups. Specific functional moieties and their
reactivities are described in Organic Chemistry, 2nd Ed. Thomas
Sorrell, University Science Books, Herndon, Va. (2005). Further, it
will be understood that any payload containing a reactive group or
that is modified to contain a reactive group will also contain a
residue after conjugation to which either the XTEN, the
XTEN-cross-linker, or the XTEN-click-chemistry reactant is
linked.
[0273] Exemplary payloads suitable for covalent attachment to
either an XTEN polymer, XTEN-cross-linker, or XTEN-click-chemistry
reactant include biologically active proteins and pharmacologically
active small molecule drugs with activity. Exemplary drugs suitable
for the inventive compositions can be found as set forth in the
official United States Pharmacopeia, official Homeopathic
Pharmacopeia of the United States, or official National Formulary,
in the Physician's Desk Reference (PDR) and in the Orange Book
maintained by the U.S. Food and Drug Administration (FDA).
Preferred drugs are those having the needed reactive functional
group or those that can be readily derivatized to provide the
reactive functional group for conjugation and will retain at least
a portion of the pharmacologic activity of the unconjugated payload
when conjugated to XTEN.
[0274] 1. Drugs as Payloads
[0275] In some embodiments, the drug payload for conjugation to
either the subject XTEN, the XTEN-cross-linkers, or the
XTEN-click-chemistry reactants described herein is one or more
agents described herein or selected from the payloads of Table 11,
or a pharmaceutically acceptable salt, acid or derivative or
agonist thereof. In one embodiment, the drug is derivatized to
introduce a reactive group for conjugation to the subject XTEN, the
XTEN-cross-linkers, or the XTEN-click-chemistry reactants described
herein. In another embodiment, the drug for conjugation is
derivatized to introduce a cleavable linker such as, but not
limited to, valine-citrulline-PAB, wherein the linker is capable of
being cleaved by a circulating or an intracellular protease after
administration to a subject, thereby freeing the drug from the
conjugate.
TABLE-US-00012 TABLE 11 Drugs for Conjugation to XTEN Drugs
Erlotinib; Bortezomib; Alitretinoin, Allopurinol, arsenic trioxide,
clofarabine, dexrazoxane, Fulvestrant; Sutent (SU11248), Letrozole;
Imatinib mesylate; PTK787/ZK 222584; Bendamustine; Romidepsin;
Pralatrexate; Cabazitaxel (XRP-6258); Everolimus (RAD-001);
Abirateron; Oxaliplatin; 5- FU (5-fluorouracil), leucovorin,
rapamycin; lapatinib; lonafarnib; sorafenib; gefitinib;
cyclosphosphamide; busulfan; improsulfan; piposulfan; benzodopa;
carboquone; meturedopa; uredopa; altretamine; triethylenemelamine;
triethylenephosphoramide; triethylenethiophosphoramide;
trimethylomelamine; bullatacin; bullatacinone; camptothecin;
topotecan; bryostatin; callystatin; CC-1065; adozelesin;
calicheamycin; auristatin; monomethyl auristatin E (MMAE);
monomethyl auristatin F (MMAF); (valine- citrulline-PAB)-monomethyl
auristatin E; (valine-citrulline-PAB)-monomethyl auristatin F;
carzelesin; bizelesin; cryptophycins (particularly cryptophycin 1
and cryptophycin 8); dolastatin; duocarmycin; eleutherobin;
pancratistatin; sarcodictyin; spongistatin; chlorambucil;
chlornaphazine; cholophosphamide; estramustine; ifosfamide;
mechlorethamine; mechlorethamine oxide hydrochloride; melphalan;
novembichin; phenesterine; prednimustine; trofosfamide; uracil
mustard; carmustine; chlorozotocin; fotemustine; lomustine;
nimustine; ranimnustine; calicheamicin; dynemicin; dynemicin A;
clodronate; esperamicin; neocarzinostatin chromophore;
aclacinomysins, actinomycin; anthramycin; azaserine; bleomycin;
cactinomycin; carabicin; carminomycin; carzinophilin;
chromomycinis; dactinomycin; daunorubicin; detorubicin;
6-diazo-5-oxo-L-norleucine; doxorubicin; morpholino-doxorubicin;
lenalidomide, cyanomorpholino-doxorubicin;
(valine-citrulline-PAB)-doxorubicin; 2-pyrrolino- doxorubicin and
deoxydoxorubicin; epirubicin; esorubicin; idarubicin;
marcellomycin; mitomycin C; mycophenolic acid; nogalamycin;
olivomycin; peplomycin; potfiromycin; puromycin; quelamycin;
rodorubicin; streptonigrin; streptozocin; tubercidin; ubenimex;
zinostatin; zorubicin; 5-fluorouracil (5-FU); fdenopterin;
methotrexate; pteropterin; trimetrexate; fludarabine;
6-mercaptopurine; thiamiprine; ancitabine; azacitidine;
6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine;
enocitabine; meclorethamine, floxuridine; calusterone;
dromostanolone propionate; epitiostanol; mepitiostane;
testolactone; aminoglutethimide; trilostane; frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansine; ansamitocins; mitoguazone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; methoxsalen, podophyllinic acid; 2-ethylhydrazide;
procarbazine; razoxane; rhizoxin; ribavirin; zidovudine; acyclovir;
gangcyclovir; vidarabine; idoxuridine; trifluridine; foscarnet;
amantadine; rimantadine; saquinavir; indinavir; ritonavir;
alpha-interferons and other interferons; AZT; sizofuran;
spirogermanium; tenuazonic acid; triaziquone; 2;
2',2''-trichlorotriethylamine; T-2 toxin; verracurin A; roridin A;
anguidine); urethan; vindesine; dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); cyclophosphamide; thiotepa; taxoids; epaclitaxel;
paclitaxel; docetaxel; doxetaxel; irinotecan; pemetrexed;
chloranbucil; gemcitabine; 6- thioguanine; cisplati; carboplatin;
vinblastine; platinum; etoposide, VP-16; ifosfamide; mitoxantrone;
novantrone, teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoic acid; capecitabine; mesna,
lidocaine; bupivacaine; memantine; quinacrine, donepezil;
rivastigmine; galantamine; morphine; oxycodone; hydromorphone;
oxymorphone; metopon; apomorphine; normorphine; etorphine;
buprenorphine; meperidine; lopermide; anileridine; ethoheptazine;
piminidine; betaprodine; diphenoxylate; fentanil; sufentanil;
alfentanil; remifentanil; levorphanol; dextromethorphan;
phenazocine; pentazocine; cyclazocine; methadone; isomethadone;
propoxyphene; naloxone; naltrexone; treprostinil; N-
methylnaloxone; 6-amino-14-hydroxy-17-allylnordesomorphine;
naltrendol;, N-methylnaltrexone; nalbuphine; butorphanol;
cyclazocine; pentazocine,; nalmephene; naltrindole;
nor-binaltorphimine; oxilorphan; 6-amino-6-desoxo-naloxone;
pentazocine; levallorphanmethylnaltrexone; buprenorphine;
cyclorphan; levalorphan; cyclosporine; cyclosporine A;
mycophenylate mofetil; sirolimus; tacrolimus; prednisone;
azathioprine; cyclophosphamide; prednisone; aminocaproic acid;
chloroquine; hydroxychloroquine; dexamethasone; chlorambucil;
danazol; bromocriptine; Nilotinib (AMN107); Nelarabine, amifostine,
amiodarone, aminocaproic acid, aminohippurate sodium,
aminoglutethimide, aminolevulinic acid, aminosalicylic acid,
amsacrine, anagrelide, anastrozole, asparaginase, anthracyclines,
bexarotene, bicalutamide, bleomyein, buserelin, busulfan,
cabergoline, capecitabine, carboplatin, carmustine, chlorambucin,
cilastatin sodium, cisplatin, cladribine, clodronate,
cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis
retinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,
daunorubicin, deferoxamine, dexamethasone, diclofenac,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin,
estramustine, etoposide, exemestane, fexofenadine, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, epinephrine, L-Dopa, hydroxyurea, idarubicin,
ifosfamide, imatinib, irinotecan, itraconazole, goserelin acetate,
letrozole, leucovorin, levamisole, lisinopril, lovothyroxine
sodium, mechlorethamine, medroxyprogesterone, megestrol, melphalan,
metaraminol bitartrate, metoclopramide, mexiletine, mitomycin,
mitotane, naloxone, nicotine, nilutamide, octreotide, pamidronate,
pilcamycin, porfimer, prednisone, prochlorperazine, ondansetron,
raltitrexed, sirolimus, tacrolimus, tamoxifen, temozolomide,
testosterone, tetrahydrocannabinol, thalidomide, thioguanine,
topotecan, tretinoin, valrubicin, vincristine, vindesine,
vinorelbine, dolasetron, granisetron; formoterol, fluticasone,
leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,
nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides
such as erythromycin, oleandomycin, troleandomycin, roxithromycin,
clarithromycin, davercin, azithromycin, flurithromycin,
dirithromycin, josamycin, spiromycin, midecamycin, leucomycin,
miocamycin, rokitamycin, andazithromycin, and swinolide A;
fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,
trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,
grepafloxacin, sunitinib, gatifloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and
sitafloxacin; aminoglycosides such as gentamicin, netilmicin,
paramecin, tobramycin, amikacin, kanamycin, neomycin, and
streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin,
colistin, daptomycin, gramicidin, colistimethate; polymixins such
as polymixin B, capreomycin, bacitracin, penems; penicillins
including penicllinase-sensitive agents like penicillin G,
penicillin V; penicllinase- resistant agents like methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram
negative microorganism active agents like ampicillin, amoxicillin,
and hetacillin, cillin, and galampicillin; antipseudomonal
penicillins like carbenicillin, ticarcillin, azlocillin,
mezlocillin, and piperacillin; cephalosporins like cefpodoxime,
cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin,
cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole,
cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin,
cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile,
cefepime, cefixime, cefonicid, cefoperazone, cefotetan,
cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams
like aztreonam; and carbapenems such as imipenem, meropenem,
pentamidine isethiouate, albuterol sulfate, lidocaine,
metaproterenol sulfate, beclomethasone diprepionate, triamcinolone
acetamide, budesonide acetonide, fluticasone, ipratropium bromide,
flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such
as paclitaxel; SN-38, tyrphostines, 20-epi-1,25 dihydroxy
vitaminD3, 5- ethynyluracil, abiraterone, Acivicin, Aclarubicin,
Acodazole Hydrochloride, AcrQnine, acylfulvene, adecypenol,
adramycin, Aldesleukin, ALL-TK antagonists, ambamustine, amidox,
Ambomycin, Ametantrone Acetate, amrubicin, andrographolide,
angiogenesis inhibitors, antagonist D, antagonist G, antarelix,
anti-androgen, anti-dorselizing morphogenetic protein-1,
anti-estrogen, antimetabolites, anti- neoplaston, anti-oestrogens,
anti-sense oligonucleotides, anti-venom, aphidicolinglycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ara-CDP-DL-PTBA, arginine deaminase, Asperlin, asulacrine,
atamestane, atrimustine, atrsacrine, axinastatin 1, axinastatin 2,
axinastatin 3, azasetron, azatoxin, azatyrosine, Azptepa:
Azotomycin, baccatin III derivatives, balanol, Batimastat,
BCR/ABLantagonists, benzochlorins, Benzodepa, benzoylstaurosporine,
staurosporine, beta-alethine, betaclamycin B,
betalactamderivatives, betamethasone, betulinic acid,
bFGFinhibitor, Bicalutamide, Bisantrene Hydrochloride,
bisaziridinylspermine, bisnafide, Bisnafide Dimesylate,
bistrateneA, Bleomycin Sulfate, breflate, Brequinar Sodium, bromine
epiandrosterone, Bropirimine, budotitane, buthionine sulfoximine,
calcipotriol, calphostin C, camptothecin derivatives, canarypox
IL-2, capedtabine, Caracemide, Carbetimer,
carboxamide-amino-triazole: carboxyamidotriazole, CaRestM3,
CARN700, cartilage derived inhibitor, Carubicin Hydrochloride,
casein kinase inhibitors(ICOS), castanospermine, cecropin B,
Cedefingol, cetrorelix, chlorins, chloroquinoxaline sulfonamide, ,
chlorotrianisene, cicaprost, Cirolemycin, cis-porphyrin, clomifene
analogues, clotrimazole, collismycin A, collismycin B,
combretastatin A4, combretastatin analogue, conagenin, crambescidin
816, crisnatol, Crisnatol Mesylate, cryptophycin 8, cryptophycin A
derivatives, curacin A, cyclopentanthraquinones, cycloplatam,
cypemycin, cytarabineocfosfate, cytolyticfactor, cytostatin,
cytotoxic agents, Daunorubicin Hydrochloride, Decitabine,
dehydrodidemnin B, deslorelin, dexifosfamide, Dexormaplatin,
dexrmzoxane, dexverapamil, Dezaguanine, Dezaguanine Mesylate, DHEA,
diaziquorie, dicarbazine, didemnin 13, didox, diethylnorspermine,
dihydro-5-azacytidine: dihydrotaxol,9-, dioxamycin,
diphenylspiromustine, docosanol, Doxorubicin Hydrochloride,
Droloxifene, Droloxifene Citrate, Dromostanolone Propionate,
dronabinol, Duazomycin, duocannycin SA, ebselen, ecorustine,
edelfosine, edrocolomab, Eflomithine Hydrochloride, eflornithine,
elemene, Elsamitrucin, emitefur, Enloplatin, Enpromate,
epiandrosterone, Epipropidine, Epirubicin Hydrochloride,
epristeride, Erbulozole, erythrocyte gene therapy, Esorubicin
Hydrochloride, estramustine analogue, estrogen agonists, estrogen
antagonists, Etanidazole, ethinyloestradiol, Ethiodized Oil I131,
Etoposide Phosphate, Etoprine, fadrozole, Fadrozole Hydrochloride,
Fazarabine, fazarabine, fenretinide, Fenretinide: Floxuridine,
finasteride, flavopiridol, flezelastine, Fludarabine Phosphate,
fluorodaunorunicin hydrochloride, Flurocitabine, forfenimex,
formestane, Fosquidone, fostriecin, Fostriecin Sodium,
gadoliniumtexaphyrin, galocitabine, ganirelix, gelatinase
inhibitors: gemcitabine, Gemcitabine Hydrochloride, glutathione
inhibitors, Gold Au198, goserelin, hepsulfam, heregulin,
hexamethylenebisacetamide, hexamethylmelamine, human chorionic
gonadotrophin: monophosphoryl lipid A + myobacterium cell walls k,
hypericin, ibandronic acid, Idarubicin Hydrochloride, idoxifene,
idramantone, Ilmofosine, ilomastat, imidazoacridones, imiquimod,
immuno stimulant peptides: insulin-like growth factor-1 receptor
inhibitor, interferon agonists, Interferon Alfa-2a, Interferon
Alfa-2b, Interferon
Alfa-n1, Interferon Alfa-n3, Interferon Beta-Ia, Interferon
Gamma-Ib, iobenguane, iododoxorubicin, ipomeanol, Iproplatin,
Irinotecan Hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-Ntriacetate, lanreotide, Lanreotide Acetate, leinamycin,
lenograstim, lentinan sulfate, leptolstatin, leukemia inhibiting
factor, leukocyte alpha interferon, Leuprolide Acetate, leuprolide
+ estrogen + progesterone, leuprorelin, liarozole, Liarozole
Hydrochloride, linear polyamine analogue, lipophilic disaccharide
peptide, lipophilic platinum compounds, lissoclinamide7,
lobaplatin, lombricine, lometrexol, Lometrexol Sodium, lonidamine,
Losoxantrone Hydrochloride, lovastatin, loxoribine, luprolide,
lurtotecan, lutetlumtexaphyrin, lysofylline, lyticpeptides,
maitansine, mannostatin A, marimastat, Masoprocol, maspin,
matrilysin inhibitors, matrix metallo proteinase inhibitors,
mecaptopurine, Mechlorethamine Hydrochloride, Megestrol Acetate,
Melengestrol Acetate, Menogaril, merbarone, meterelin,
methioninase, methlorethamine, Metoprine, Meturedepa, microalgal,
mifepristone, MIFinhibitor, miltefosine, mirimostim, mismatched
double stranded RNA, Mitindomide, Mitocarcin, Mitocromin,
Mitogillin, mitoguazone, Mitomalcin, mitomycin analogues,
mitonafide, Mitosper, mitotoxin fibroblast growth factor-saporin,
mofarotene, molgramostim, monoclonal antibody, multiple drug
resistance gene inhibitor, multiple tumor suppressor1-based
therapy, mustard anticancer agent, mutamycin, mycaperoxide B,
mycobacterial cell wall extract, Mycobacterium bovis, myriaporone,
N-acetyldinaline: N-substitutedbenzamides, nafarelin, nagrestip,
naloxone + pentazocine, napavin, naphterpin, nartograstim,
nedaplatin, nemorubicin, neridronic acid, neutral endo peptidase,
nisamycin, nitric oxide modulators, nitrogen mustard derivatives,
nitroxide antioxidant, nitrullyn, Nocodazole: Nogalamycin,
O6-benzylguanine, oestradiol, okicenone, oligonucleotides,
onapristone: ondansetron, ondansetron, oracin, oral cytokine
inducer, Ormaplatin, osaterone, oxaunomycin, Oxisuran, palauamine,
palmitoylrizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine: pegaspargase, Pegaspargase, peldesine:
pentosanpolysulfatesodium, Peliomycin, Pentamustine, pentrozole,
Peplomycin Sulfate, perflubron, Perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, piritrexim, Piroxantrone Hydrochloride,
placetin A, placetin B, plasminogen activator inhibitor, platinum
complex, platinum compounds, platinum-triamine complex, Plicamycin,
Plomestane, Porfimer Sodium, Procarbazine Hydrochloride,
propylbis-acridone, prostaglandin J2, prostatic carcinoma,
proteasome inhibitors, protein A-based immune modulator, protein
kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine
nucleoside phosphorylase inhibitors, Puromycin Hydrochloride,
purpurins, Pyrazofurin, pyrazoloacridine, pyridoxylated hemoglobin
polyoxyethylene conjugate, raf antagonists, ramosetron, rasfarnesyl
protein transferase inhibitors, ras inhibitors: ras-GAP inhibitor,
retelliptine demethylated, rhenium Re186 etidronate, Riboprine,
ribozymes, RII retinamide, RM-131 (ghrelin agonist), RM-493 (agonis
for melanocortin type 4 receptor), Rogletimide, rohitumine,
romurtide, roquinimex, rubiginone B1, ruboxyl, Safingol
Hydrochloride, Safingol, saintopin: Sar CNU, sarcophytol A,
sargramostim, Sdi1 mimetics, Semustine, senescence derived
inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, Simtrazene, single
chain antigen binding protein, sobuzoxane, sodium borocaptate,
sodium phenylacetate, solverol, somatomedin binding protein,
sonermin, Sparfosate Sodium, sparfosic acid, Sparsomycin,
Spirogermanium Hydrochloride, Spiromustine, spiromustine:
splenopentin, Spiroplatin, splcamycin D, squalamine, stem cell
inhibitor, stem-cell division inhibitors, stipiamide, stromelysin
inhibitors, Strontium Chloride Sr89, sulfmosine, Sulofenur,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosamino glycans, Talisomycin,
tallimustine, tamoxifen methiodide, tauromustine, tazarotene,
Tecogalan Sodium, Tegafur, tellurapyrylium, telnoporfin, telomerase
inhibitors, Teloxantrone Hydrochloride, Temoporfin, ternozolomide,
Teroxirone, tetrachlorodecaoxide, tetrazomine, texotere,
thallblastine, thiocoraline, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, Tiazofurin, tinethylotiopurpurin, Tirapazamine, titanocene
dichloride, Topotecan Hydrochloride, topsentin, toremifene,
Toremifene Citrate, totipotent stem cell factor, translation
inhibitors, Trestolone Acetate, triacetyluridine, triciribine,
Triciribine Phosphate: Trimetrexate, Trimetrexate Glucuronate,
Triptorelin, triptorelin: tropisetron, tubulozole hydrochloride,
turosteride, tyrosine kinase inhibitors, UBC inhibitors, urodepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, vector system,
velaresol, venom, veramine, verdins, Verleporfin, verteporfin,
Vinblastine Sulfate, vincristine sulfate, vindesine, Vindesine
Sulfate, Vinepidine Sulfate, Vinglycinate Sulfate, Vinleurosine
Sulfate, vinorelbine tartrate, vinrosidine sulfate, vinxaltine,
Vinzolidine Sulfate, vitaxin, Vorozole, zanoterone, Zeniplatin,
zilascorb, zinostatin stimalamer, Zorubicin Hydrochloride, Bovine
pancreatic RNase, Human pancreatic RNAse, Mammalian pancreatic
RNase, onconase, ranpirnase, pokeweed antiviral protein,
rachelmycin, ricin-A chain, gelonin, everolimus, carfilzomib,
tubulysin, tubulysin B, tubulysin M, maytansinoid DM1, maytansinoid
DM4, triptolide, SJG-136, apaziquone, irofulven, illudin S,
tomaymycin, zoledronate
[0276] 2. Biologically Active Proteins as Payloads
[0277] In another aspect, the invention provides XTEN-payload
compositions in which the payload is a biologically active protein,
either as a peptide or polypeptide. In some embodiments of
XTEN-payload conjugates, the payload is any pharmacologically
active peptide or polypeptide that can be expressed recombinantly
as a fusion protein linked to one or more XTEN. In other
embodiments of XTEN-payload conjugates, the payload is any
pharmacologically active peptide or polypeptide that can be
conjugated to one or more XTEN. The conjugates may be in a
configuration as described herein, below. The exemplary peptide or
polypeptide payloads are meant to encompass analogs, agonists,
antagonists, inhibitors, and isomers. It will be understood that
the subject peptides and proteins encompass synthetic,
semi-synthetic, recombinant, native, glycosylated, and
non-glycosylated forms, as well as biologically active fragments,
sequence variants, species variants, homologs and mutations thereof
as long as the resulting variant protein retains a portion of
activity of the parent or native protein.
[0278] Biologically active protein sequences can be obtained from
publicly available databases, patents, or literature references and
other such sources that are well known in the art. For example,
sequences can be obtained from Universal Protein Resource
(UniProt)/Swiss-Prot, European Bioinformatics Institute (EBI), the
SIB Swiss Institute of Bioinformatics, the Protein Information
Resource (PIR). Chemical Abstracts Services (CAS) Registry Numbers
(published by the American Chemical Society) and/or GenBank
Accession Numbers (e.g., AAA-AZZ, HAA-HZZ, JAA-JZZ), Model Protein
identifiers available through the National Center for Biotechnology
Information (NCBI) webpage, available on the world wide web at
ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or
GenBank database that contain an amino acid sequence of the protein
of interest or of a fragment or variant of the protein. For such
sequence identifiers provided herein, the summary pages associated
with each of these CAS and GenBank and GenSeq Accession Numbers as
well as the cited journal publications (e.g., PubMed ID number
(PMID)) are each incorporated by reference in their entireties,
particularly with respect to the amino acid sequences described
therein.
[0279] In one embodiment, the XTEN-payload composition, whether in
recombinant or conjugate form, comprises one or more molecules of a
biologically active peptide or protein that includes, but is not
limited to a peptide or polypeptide selected from the payloads set
forth in Table 12, or a sequence variant thereof that retains at
least a portion of the activity of the biologically active protein.
By "sequence variant," it is meant that the biologically active
protein exhibits at least about 80%, or 90%, or 91%, or 92%, or
93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99 sequence
identity, when optimally aligned, to that of the known peptide or
polypeptide, such as are listed in Table 12.
TABLE-US-00013 TABLE 12 Biologically Active Proteins for linking to
XTEN Protein/Peptide Name EPO, IFN-.alpha., IFN-.beta.,
IFN-.gamma., consensus IFN, factor VII, factor VIII, factor IX,
IL-1, IL-2, REMICADE (infliximab), RITUXAN (rituximab), ENBREL
(etanercept), SYNAGIS (palivizumab), REOPRO (abciximab), HERCEPTIN
(trastuzimab), tPA, CERIZYME (imiglucerase), Hepatitus-B vaccine,
rDNAse, alpha-1 proteinase inhibitor, C-peptide, fuzeon, G-CSF,
GM-CSF, growth hormone, somatropin, growth hormone releasing
hormone, insulin, insulin analogues, glucagon, GLP-1, GLP-2, FSH,
TNF-receptor, uricase, VEGF, PTH, aspariginase, amdoxovir (DAPD),
antide, becaplermin, calcitonins, cyanovirin, denileukin diftitox,
erythropoietin (EPO), ceredase, cerezyme, alpha- glucosidase,
collagen, cyclosporin, alpha defensins, beta defensins, exendin-4,
granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO),
alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage
colony stimulating factor (GMCSF), fibrinogen, filgrastim, growth
hormones human growth hormone (hGH), growth hormone releasing
hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenic
proteins such as bone morphogenic protein-2, bone morphogenic
protein-6, OP-1; acidic fibroblast growth factor, basic fibroblast
growth factor, CD-40 ligand, heparin, human serum albumin, low
molecular weight heparin (LMWH), interferons such as interferon
alpha, interferon beta, interferon gamma, interferon omega,
interferon tau, consensus interferon; interleukins and interleukin
receptors such as interleukin-1 receptor, interleukin-2,
interluekin-2 fusion proteins, interleukin-1 receptor antagonist,
interleukin-3, interleukin-4, interleukin-4 receptor,
interleukin-6, interleukin-8, interleukin-12, interleukin-13
receptor, interleukin-17 receptor; lactoferrin and lactoferrin
fragments, luteinizing hormone releasing hormone (LHRH), insulin,
pro-insulin, insulin analogues (e.g., mono- acylated insulin as
described in U.S. Pat. No. 5,922,675), amylin, somatostatin,
somatostatin analogs including octreotide, vasopressin, follicle
stimulating hormone (FSH), influenza vaccine, insulin-like growth
factor (IGF), insulintropin, macrophage colony stimulating factor
(M-CSF), plasminogen activators such as alteplase, urokinase,
reteplase, streptokinase, pamiteplase, lanoteplase, and
teneteplase; nerve growth factor (NGF), osteoprotegerin,
platelet-derived growth factor, tissue growth factors, transforming
growth factor-1, vascular endothelial growth factor, leukemia
inhibiting factor, keratinocyte growth factor (KGF), glial growth
factor (GGF), T Cell receptors, CD molecules/antigens, tumor
necrosis factor (INF), monocyte chemoattractant protein-1,
endothelial growth factors, parathyroid hormone (PTH), thymosin
alpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10,
thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,
phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4),
VLA-4 inhibitors, bisphosponates, respiratory syncytial virus
antibody, cystic fibrosis transmembrane regulator (CFTR) gene,
deoxyreibonuclease (Dnase), bactericidal/permeability increasing
protein (BPI), anti-CMV antibody, etanercept, abciximab,
afeliomomab, basiliximab, daclizumab, infliximab, ibritumomab
tiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab
conjugate, olizumab, rituximab, HERCEPTIN (trastuzumab), boxtox,
DYSPORT (abobotulinumtoxinA), alglucosidase alfa, daptomycin, YH-16
, choriogonadotropin alfa, filgrastim, cetrorelix, interleukin-2,
aldesleukin, teceleukin, denileukin diftitox, interferon alfa-n3
(injection), interferon alfa-n1, DL-8234, interferon, Suntory
(gamma-1a), interferon gamma, thymosin alpha 1, tasonermin, DIGIFAB
(digoxin immune Fab), VIPERATAB (purified sheep-derived Vipera
berus common adder antivenom), ECHITAB (monospecific ovine Fab
antivenom), CROFAB (Crotalidae Polyvalent Immune Fab), nesiritide,
abatacept, alefacept, REBIF (interferon beta-1a), eptotermin alfa,
teriparatide (osteoporosis), calcitonin injectable, bone disease),
calcitonin (nasal, osteoporosis), etanercept, hemoglobin glutamer
250 (bovine), drotrecogin alfa, collagenase, carperitide,
recombinant human epidermal growth factor (topical gel, wound
healing), DWP-401 (Nepidermin), darbepoetin alfa, epoetin omega,
epoetin beta, epoetin alfa, desirudin, lepirudin, bivalirudin,
nonacog alpha, Mononine, eptacog alfa (activated), recombinant
Factor VIII + VWF, RECOMBINATE (Antihemophilic Factor), recombinant
Factor VIII, Factor VIII (recombinant), Alphanate, octocog alfa,
Factor VIII, palifermin, Indikinase, tenecteplase, alteplase,
pamiteplase, reteplase, nateplase, monteplase, follitropin alfa,
rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim,
sermorelin, glucagon, exenatide, pramlintide, imiglucerase,
galsulfase, Leucotropin, molgramostim, triptorelin acetate,
histrelin, histrelin acetate, deslorelin, nafarelin, ATRIGEL
(leuprolide sustained release depot), DUROS (leuprolide implant),
somatropin, Eutropin, KP-102 program, somatropin, somatropin,
mecasermin (growth failure), enfuvirtide, Org-33408, insulin
glargine, insulin glulisine, insulin (inhaled), insulin lispro,
insulin detemir, RapidMist (insulin) (buccal), mecasermin
rinfabate, anakinra, celmoleukin, 99mTc-apcitide injection,
myelopid, Betaseron, glatiramer acetate, Gepon, oprelvekin, human
leukocyte-derived alpha interferons, Bilive, insulin (recombinant),
recombinant human insulin, insulin aspart, mecasermin, ROFERON-A
(interferon alpha 2a), interferon-alpha 2, Alfaferone, interferon
alfacon-1, interferon alpha, AVONEX (interferonbeta-1a)'
recombinant human luteinizing hormone, domase alfa, trafermin,
ziconotide, taltirelin, dibotermin alfa, atosiban, becaplermin,
eptifibatide, ZEMAIRA (Alpha1 proteinase inhibitor), CTC-111,
Shanvac-B, HPV vaccine (quadrivalent), NOV-002, octreotide,
lanreotide, ancestim, agalsidase beta, agalsidase alfa, laronidase,
prezatide copper acetate (topical gel), rasburicase, ranibizumab,
ACTIMMUNE (Interferon gamma-1b), PEG-Intron, Tricomin, recombinant
house dust mite allergy desensitization injection, recombinant
human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin
delta, transgenic antithrombin III, Granditropin, Vitrase,
recombinant insulin, interferon-alpha (oral lozenge), GEM-21S,
vapreotide, idursulfase, omapatrilat, recombinant serum albumin,
certolizumab pegol, glucarpidase, human recombinant C1 esterase
inhibitor (angioedema), lanoteplase, recombinant human growth
hormone, BIOJECTOR 2000 (enfuvirtide) (needle-free injection),
VGV-1, interferon (alpha), lucinactant, aviptadil (inhaled,
pulmonary disease), icatibant, ecallantide, omiganan, Aurograb,
pexiganan acetate, ADI-PEG-20, LDI-200, degarelix,
cintredekinbesudotox, FavId, MDX-1379, ISAtx-247, liraglutide,
teriparatide (osteoporosis), tifacogin, AA-4500, T4N5 liposome
lotion, catumaxomab, DWP-413, ART-123, Chrysalin, desmoteplase,
amediplase, corifollitropin alpha, TH-9507, teduglutide, Diamyd,
DWP-412, growth hormone (sustained release injection), recombinant
G-CSF, insulin (inhaled, AIR), insulin (inhaled, Technosphere),
insulin (inhaled, AERx), RGN-303, DiaPep277, interferon beta
(hepatitis C viral infection (HCV)), interferon alfa-n3 (oral),
belatacept, transdermal insulin patches, AMG-531, MBP-8298,
XERECEPT (corticorelin), opebacan, AIDSVAX, GV-1001, LymphoScan,
ranpimase, Lipoxysan, lusupultide, MP52 (beta-tricalciumphosphate
carrier, bone regeneration), melanoma vaccine, sipuleucel-T,
CTP-37, INSEGIA (G17DT Immunogen), vitespen, human thrombin
(frozen, surgical bleeding), thrombin, TransMID, alfimeprase,
Puricase, terlipressin, EUR- 1008M, recombinant FGF-1, BDM-E,
rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhaled, cystic
fibrosis), SCV-07, OPI-45, Endostatin, Angiostatin, ABT-510, Bowman
Birk Inhibitor Concentrate, XMP-629, 99mTc-Hynic-Annexin V,
kahalalide F, CTCE-9908, teverelix (extended release), ozarelix,
romidepsin, BAY-50-4798, interleukin-4, PRX-321, Pepscan,
iboctadekin, rh lactoferrin, TRU-015, IL-21, ATN-161, cilengitide,
ALBUFERON (albinterferon alfa-2b), Biphasix, IRX-2, omega
interferon, PCK-3145, CAP-232, pasireotide, huN901-DM1, ovarian
cancer immunotherapeutic vaccine, SB-249553, Oncovax-CL, OncoVax-P,
BLP-25, CerVax-16, nemifitide, rAAT (inhaled), rAAT
(dermatological), CGRP (inhaled, asthma), pegsunercept, thymosin
beta-4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100,
salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis),
examorelin, capromorelin, Cardeva, velafermin, 131I-TM-601, KK-220,
TP-10, ularitide, depelestat, hematide, Chrysalin (topical),
rNAPc2, recombinant Factor VIII (PEGylated liposomal), bFGF,
PEGylated recombinant staphylokinase variant, V-10153, SonoLysis
Prolyse, NeuroVax, CZEN-002, islet cell neogenesis therapy, rGLP-1,
BIM-51077, LY-548806, exenatide, AVE-0010, GA-GCB, avorelin, AOD-
9604, linaclotide acetate, CETi-1, HEMOSPAN (PEG-conjugated
hemoglobin), VAL (injectable), insulin, recombinant methionyl human
leptin, pitrakinra subcutaneous injection, eczema), pitrakinra
(inhaled dry powder, asthma), MULTIKINE (Leukocyte Interleukin),
RG-1068, MM-093, NBI- 6024, AT-001, PI-0824, Org-39141, Cpn10
(autoimmune iseases/inflammation), talactoferrin (topical), rEV-131
(ophthalmic), rEV-131 (respiratory disease), oral recombinant human
insulin (diabetes), RPI-78M, CYT-99007 CTLA4-Ig, DTY-001,
valategrast, interferon alfa-n3 (topical), IRX-3, RDP-58, TAUFERON
(interferon tau), bile salt stimulated lipase, Merispase, alkaline
phosphatase, EP-2104R, Melanotan-II, bremelanotide, ATL-104,
recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174,
CJC-1008, dynorphinA, SI-6603, LAB GHRH, AER-002, BGC-728, malaria
vaccine (virosomes, PeviPRO), ALTU-135, parvovirus B19 vaccine,
influenza vaccine (recombinant neuraminidase), malaria/HBV vaccine,
anthrax vaccine, Vacc- 5q, Vacc-4x, HIV vaccine (oral), HPV
vaccine, Tat Toxoid, YSPSL, CHS-13340 , PTH(1-34) liposomal cream
(Novasome), Ostabolin-C, PTH analog (topical, psoriasis),
MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85 A vaccine
(tuberculosis), FAR-404, BA-210, recombinant plague F1V vaccine,
AG-702, OxSODro1, rBetV1, Der-p1/Der-p2/Der-p7 allergen-targeting
vaccine (dust mite allergy), PR1 peptide antigen (leukemia), mutant
ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine
(adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5,
CDX- 110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide,
telbermin, rupintrivir, reticulose, rGRF, P1A, alpha-galactosidase
A, ACE-011, ALTU-140, CGX-1160, angiotensin therapeutic vaccine,
D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis),
DRF-7295, ABT-828, ErbB2-specific immunotoxin (anticancer),
DT388IL-3, TST-10088, PRO-1762, Combotox,
cholecystokinin-B/gastrin-receptor binding peptides, 111In-hEGF,
AE-37, trastuzumab- DM1, Antagonist G, IL-12 (recombinant),
PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19
based radioimmunotherapeutics (cancer), Re-188-P-2045, AMG-386,
DC/I540/KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1
vaccine (peptides), NA17.A2 peptides, melanoma vaccine (pulsed
antigen therapeutic), prostate cancer vaccine, CBP-501, recombinant
human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A2
(injectable), ACP-HIP, SUN-11031, peptide YY [3-36] (obesity,
intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, human
parathyroid
hormone 1-34 (nasal, osteoporosis), F-18-CCR1, AT-1001 (celiac
disease/diabetes), JPD-003, PTH(7- 34) liposomal cream (Novasome),
duramycin, CAB-2 , CTCE-0214, erythropoietin, EPO-Fc, CNTO- 528,
AMG-114 , JR-013, Factor XIII, aminocandin, PN-951, 716155,
SUN-E7001, TH-0318, BAY-73-7977 , teverelix (immediate release),
EP-51216, hGH, OGP-I, sifuvirtide, TV-4710, ALG- 889, Org-41259,
rhCC10, F-991, thymopentin (pulmonary diseases), r(m)CRP,
hepatoselective insulin, subalin, L19-IL-2 fusion protein, elafin,
NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor
agonist, AL-108, AL-208, nerve growth factor antagonists (pain),
SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1,
AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043,
hPTH(1-34), 768974, SYN-101, PGN-0052, aviscumine, BIM- 23190,
enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, TNF, desmopressin,
onercept, TP- 9201, AC165198, Activin receptor type IIA, Adenosine
deaminase, Adipotide, Afostase alfa (alkaline phosphatase), Alpha
melanocyte stimulating hormone, Alpha-1 antitrypsin,
Alpha-galactosidase, Angiotensin, Anti-angiopoietin-1 inhibitor,
Anti-angiopoietin-2 inhibitor, Apolipoprotein A1, ARCALYST
(filonacept), Arginine deiminase, Asparaginase, Atilmotin, AZD2820,
Bradykinin receptor antagonist, Calcitonin, Calcitonin gene-related
peptide, Cenderitide, Cholecystokinin, Ciliary nemotropic factor
(CNTF), Ciligenitide, Coagulation factor IX, Coagulation factor
VII, Coagulation factor VIII, Coagulation factor X, Coagulation
factor XIII, Collagenase, Complement C1 esterase inhibitor
(conestat alfa), Complement factor C3 inhibitor, Complement factor
C5 inhibitor, Corticotropin-releasing factor, C-peptide, C-type
natriuretic peptide, Defensins, DiaPep277, Diphenhydramine,
Ecallantide, Endostatin, Eptifibatide, Fibrinogen, Fibroblast
growth factor receptor agonist, Follicle-stimulating hormone (FSH),
Follistatin, FP-1039 (FGF trap), Fuzeon, Gastrin, Ghrelin, Ghrelin
antagonist, GIP-1, GIP-1/GLP-1 dual agonist, Glucagon,
Glucagon-like peptide (GLP) 1, Glucagon-like peptide (GLP) 2,
Glucocerebrosidase (Cerezyme), Glutamate carboxypeptidase
(carboxypeptidase G2), Glutaminase, Granulocyte colony stimulating
factor (GCSF), Growth hormone, Growth hormone releasing hormone
(GHRH), Hematide, Heparinase, Hirudin, Human chorionic gonadotropin
(hCG), Human deoxyribonuclease I, Humanin, Hyaluronidase,
Icatibant, ELAPRASE (Iduronate-2-sulfatase), INGAP (Exsulin),
Insulin, Irisin, KAI-4169, REFLUDAN (Lepirudin), Leukemia
inhibiting factor, L-iduronidase, LRP5 inhibitor, LRP6 inhibitor,
Luteinizing hormone (LH), Macrophage inflammatory protein 2
(GroBeta-T CXC chemokine), Mannose binding lectin, Melanocortin
stimulating hormone, Methioninase (METase), Mirostipen, MUCI
inhibitor, Myostatin inhibitor, N-acetylgalactosamine 4-sulfatase
(Naglazyme), CEREBROLYSIN (Nerve growth factor), Neuropeptide Y2,
Neurophilin, NU206, ONCONASE (Ranpimase), Ontak (IL-2-toxin),
Opioid growth factor, Oxyntomodulin, Oxytocin, Paliperidone,
Pancreatic polypeptide, PanCyte, Parathyroid hormone (PTH),
Parathyroid hormone related protein (PTHrP), Peptide YY (3-36),
Phenylalanine ammonia lyase (PAL), Phenylalanine hydroxylase(PAH),
Pituitary adenylate cyclase-activating polypeptide (PACAP),
Platelet-activating factor acetylhydrolase, POT-4 (APL-1),
Pramlintide, P-Selectin, Relaxin, PULMOZYME (rhDNase), RNase,
Sanvar, Secretin, SN38, Somatostatin (Octreotide, Pasireotide,
Sandostatin etc.), Somavert (human growth hormone receptor
antagonist), Stem cell growth factor, Superoxide dismutase, TACI,
Thrombin inhibitor (direct), Thrombomodulin, Thrombopoietin (TPO),
Thymosin alpha 1 (Thymalfasin), Thyroid stimulating hormone (TSH),
Thyrotropin releasing hormone, Tigapotide, Tissue plasminogen
activator (tPA), TLN-232, Tripeptidyl peptidase 1, Tumour necrosis
factor receptor, Tyrosine kinase receptor (TrkA), UGP281, Urate
oxidase, Uricase, Urocortin 2, Urokinase plasminogen activator,
Vascular endothelial growth factor (VEGF) inhibitor, Vasoactive
intestinal peptide, Vasopressin, von Willebrand Factor (vWF),
PRIALT (Ziconotide), Zinc protoporphyrin, Adrenal corticotrophin
hormone (ACTH), CD25, Interleukin-1 receptor, Interleukin-21,
ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, Aggrecan,
AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2,
ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, APRIL, AR, AZGP1
(zinc-a-glycoprotein), A4 integrin, B7, B7.1, B7.2, BAD, BAFF,
BAG1, BAI1, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BlyS, BMP1, BMP2,
BMP3B (GDF10), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1
(plectin), BRCA1, C19orf10 (IL27w), C3, C4A, C5, C5R1, CANT1,
CASP1, CASP4, CAV1, CCBP2 (D6/JAB61), CCL1 (1-309), CCL11
(eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17
(TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20
(MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC/STC-1), CCL23
(MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26
(eotaxin-3), CCL27 (CTACK/ILC), CCL28, CCL3 (MIP-1a), CCL4
(MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2,
CCND1, CCNE1, CCNE2, CCR1 (CKR1/HM145), CCR2 (mcp- 1RB/RA), CCR3
(CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-
L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9
(GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD164, CD19, CD1C, CD20,
CD200, CD-22, CD24, CD28, CD3, CD37, CD38, CD3E, CD3G, CD3Z, CD4,
CD11a (LFA-1 integrin alphaL), CD40, CD40L, CD44, CD45RB, CD52,
CD69, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD340,
CDH1 (E-cadherin), CDH10, CDH12, CDH13, CDH18, CDH19, CDH20, CDH5,
CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A
(p21Wap1/Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (pl6INK4a),
CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, Chitinase, CHST10,
CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8;
CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); cMET; CMKLR1;
CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP;
CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1
(b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28);
CXCL1 (GRO1); CXCL10(IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1);
CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5
(ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2);
CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP;
DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3;
EFNB2; EGF; EGFR; ELAC2; elastase; ENG; ENO1; ENO2; ENO3; EPHB4;
EPO; ERBB-2 (Her2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL;
FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12;
FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF);
FGF20; FGF21; FGF22; FGF23; FGF3 (int- 2); FGF4 (HST); FGF5; FGF6
(HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1
(EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin);
FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1;
GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2
(CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN
(Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HER2; HGF;
HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA;
HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2;
IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1;
IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB;
IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1;
IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18;
IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6;
IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1;
IL1RAPL2; IL1RL1; IL1RL2; IL1RN; IL2; IL20; IL20RA; IL21R; IL22;
IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29;
IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6;
IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB;
IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAKI; IRAK2;
ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4
integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC
Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5;
KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific
type II keratin); LAMA5; LEP (leptin); LFA3; LIGHT; Lingo-p75;
Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR;
MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF;
MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3
(metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2;
neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66
(Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2;
NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2;
NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2;
NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1;
P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1;
PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG;
PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2;
PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RANKL;
RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; RSV; SI00A2;
SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2
(mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine);
SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1);
SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B
(Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10;
TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1;
TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO;
TIE (Tie-1); TIMP3; tissue factor; TLR10; ILR2; TLR3; TLR4; TLR5;
ILR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3;
TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas);
TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE);
TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L);
TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40
ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand);
TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors (TLR1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12 to TLR-13); TOP2A (topoisomerase Iia);
TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6;
TREM1; TREM2; TRPC6; TSLP; TWEAK; VAP1; VEGF; VEGFB; VEGFC;
versican; VHL C5; VLA-1; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b);
XCR1 (GPR5/CCXCR1); YY1; ZFPM2.
[0280] 3. Exemplary Biologically Active Proteins as Payloads
[0281] Proteinacious compounds that are specifically contemplated
as payloads in the subject compositions are the following peptides
and proteins:
[0282] "C-type Natriuretic peptide" or "CNP" means the human
protein (UniProt No. P23582) encoded by the NPPC gene that is
cleaved to the 22 amino acid peptide C-type natriuretic peptide
(CNP), having the sequence GLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 539),
as well as species and synthetic variations thereof, having at
least a portion of the biological activity of the native peptide.
CNP is a selective agonist for the B-type natriuretic receptor
(NPRB) and is reported to be a potent stimulator of endochondral
bone growth. CNP binds to its receptor, initiates intracellular
signals & ultimately inhibit the overactive FGFR3 pathway. Use
of CNP is indicated for achondroplasia, a common form of skeletal
dysplasia or short-limbed dwarfism, and human disorders caused by
FGFR3 mutations, including syndromes affecting skeletal
development; e.g., hypochondroplasia [HCH], ACH, thanatophoric
dysplasia [TD]), skin (epidermal nevi, seborrhaeic keratosis,
acanthosis nigricans), and cancer (multiple myeloma [MM], prostate
and bladder carcinoma, seminoma) (Foldynova-Trantirkova S. Hum
Mutat. (2012) 33:29). The half-life of CNP-22 is reported to be 2.6
min, being rapidly metabolized by neutral endopeptidase &
cleared by a clearance receptor (Prickett T., 2004, Clinical
Science, 106:535), thereby limiting its utility.
[0283] "Luteinizing hormone-releasing hormone" or "LHRH" means the
human protein (UniProt No. P01148) encoded by the GNRH1 gene that
is processed in the preoptic anterior hypothalamus from a 92-amino
acid preprohormone into the linear decapeptide end-product having
the sequence pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ
ID NO: 540), as well as species and synthetic variations thereof,
having at least a portion of the biological activity of the native
peptide. LHRH plays a pivotal role in the regulation of the
pituitary/gonadal axis, and thus reproduction. LHRH exerts its
effects through binding to high-affinity receptors on the pituitary
gonadotroph cells and subsequent release of FSH and LH. LHRH is
found in organs outside of the hypothalamus and pituitary, and
because a high percentage of certain cancer tissues have LHRH
binding sites and because sex steroids have been implicated in the
development of breast and prostate cancers, hormonal therapy with
LHRH agonists are approved or are considered for the treatment of
sex-steroid-dependent conditions such as estrogen-dependent breast
cancer, ovarian cancer, endometrial cancer, bladder cancer and
androgen-dependent prostate carcinoma. Because the half-life is
reported to be less than 4 minutes. (Redding T W, et al. The
Half-life, Metabolism and Excretion of Tritiated Luteinizing
Hormone-Releasing Hormone (LH-RH) in Man. J Clin Endocrinol. Metab.
(1973) 37:626-631), its utility as a therapeutic is limited.
[0284] "Cilengitide" means the synthetic cyclic RGD pentapeptide
having the sequence Arg-Gly-Asp-Dphe-NmeVal (SEQ ID NO: 541) or the
chemical name
2-[(2S,5R,8S,11S)-5-benzyl-11-{3-[(diaminomethylidene)amino]propyl}--
7-methyl-3,6,9,12,15-pentaoxo-8-(propan-2-yl)-1,4,7,10,13-pentaazacyclopen-
tadccan-2-yl]acetic acid (CAS No. 188968-51-6). Cilengitide is
selective for .alpha.v integrins, which are important in
angiogenesis (forming new blood vessels). The binding of such
ligands activates the integrins to regulate tumor cell invasion,
migration, proliferation, survival & angiogenesis. Hence, the
use of cilengitide is under investigation for the treatment of
glioblastoma by inhibiting angiogenesis (Burke P, et al.
Cilengitide targeting of .alpha.v.beta.33 integrin receptor
synergizes with radioimmunotherapy to increase efficacy and
apoptosis in breast cancer xenografts". Cancer Res (2002) 62(15):
4263-4272). Because cilengitide has a short half-life of 3-5 h, and
poor solubility limiting the maximum drug concentration to 15 mg/mL
(O'Donnell P H. A phase I study of continuous infusion cilengitide
in patients with solid tumors. Invest New Drugs (2012) 30:604), its
utility as a therapeutic is limited.
[0285] "Growth hormone releasing hormone" or "GHRH" (also known as
growth-hormone-releasing factor, GRF, GHRF, somatoliberin or
somatocrinin" means the 44-amino acid peptide hormone produced in
the arcuate nucleus of the hypothalamus having the sequence
YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL (SEQ ID NO: 542), as
well as species and synthetic variations thereof, having at least a
portion of the biological activity of the native peptide, including
the biologically active 1-29 amino acid truncation peptide
YADAIFTNSYRKVLGQLSARKLLQDIMSR (SEQ ID NO: 543). GHRH is released
from neurosecretory nerve terminals and is carried by the
hypothalamo-hypophyseal portal system to the anterior pituitary
gland where it acts on GHRH receptor to stimulate pulsatile growth
hormone release. The GHRH analog tesamorelin is a drug approved for
the treatment of lipodystrophy in HIV patients under highly active
antiretroviral therapy, and is also considered for use in cachexia,
abdominal obesity in growth-hormone deficient patients, muscle
wasting related to certain chronic diseases, mild cognitive
impairment, and growth hormone replacement in growth hormone
deficient patients. Because the half-life is reported to be less
than 15 minutes, (Chapman I M. J Endocrinol (1991) 128:369-374),
its utility as a therapeutic is limited.
[0286] "Peptide YY" and "PYY" mean human peptide YY polypeptide
(UniProt No. P10082), synthetic versions and species and
non-natural sequence variants having at least a portion of the
biological activity of mature PYY. As used herein, "PYY" includes
both major forms of the human full length, 36 amino acid peptide,
PYY.sub.1-36 and the predominant circulating form PYY.sub.3-36
("PYY3-36") which have the PP fold structural motif. PYY3-36 has
the sequence IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH2 (SEQ ID NO:
544). PYY is produced by specialized endocrine cells (L-cells) in
the gut after a person eats and inhibits gastric motility and
increases water and electrolyte absorption in the colon. PYY may
also suppress pancreatic secretion. The naturally occurring PYY3-36
is a nonselective Y.sub.1, Y.sub.2, & Y.sub.5 agonist.
PPY-containing fusion proteins of the invention may find particular
use in the treatment of diabetes for glucose regulation,
insulin-resistance disorders, and obesity. Analogs of PYY have been
prepared, as described in U.S. Pat. Nos. 5,604,203, 5,574,010 and
7,166,575. Because the half-life is reported to be less than 1 h.
(Addison M L. A role for metalloendopeptidases in the breakdown of
the gut hormone. PYY 3-36. Endocrinology (2011) 152(12):4630-4640)
and is typically administered by the intranasal route three times
daily, its utility as a therapeutic is limited.
[0287] "Leptin" means the naturally occurring leptin (UnitProt No.
P41159) encoded by the Ob(Lep) gene, synthetic versions and species
and non-natural sequence variants having at least a portion of the
biological activity of the mature leptin. Leptin has the sequence
VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVY
QQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYST
EVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID NO: 545), and has a disulfide
bridge between residues 97 and 147. Leptin plays a key role in
regulating energy intake and energy expenditure, including
appetite, metabolism, and body weight. Leptin-containing
polypeptides of the invention may find particular use in the
treatment of diabetes for glucose regulation, insulin-resistance
disorders, obesity, congenital/acquired lipodystrophy,
HAART-induced lipodystrophy, hypothalamic amenorrhea. Leptin has
been cloned, as described in U.S. Pat. No. 7,112,659, and leptin
analogs and fragments in U.S. Pat. Nos. 5,521,283, 5,532,336,
PCT/US96/22308 and PCT/US96/01471. Because the commercially
available form, metreleptin has a half-life reported to be 8-30 min
(Klein S., et al. Adipose tissue leptin production and plasma
leptin kinetics in humans. Diabetes (1996) 45:984-987) and the
majority of current leptin therapies require 1.times.-2.times./day
dosing, its utility as a therapeutic is limited.
[0288] "Pramlintide" means the synthetic amylin mimetic having the
sequence KCNTATCATNRLANFLVHSSNNFGPILPPTNVGSNTY-NH2 (SEQ ID NO:
546), and sequence variants having at least a portion of the
biological activity of pramlintide or native amylin. The
pramlintide has a sequence wherein amino acids from the rat amylin
sequence are substituted for amino acids in the human amylin
sequence. Amylin is a 37aa peptide secreted by pancreatic b-cells
that is co-released with insulin in pulsatile fashion, typically in
a molar ratio of 100 insulin to 1 amylin. Amylin functions to
inhibit gastric emptying, glucagon secretion, promote satiety &
meal termination (Kong M F, et al. Infusion of pramlintide, a human
amylin analogue, delays gastric emptying in men with IDDM.
Diabetologia. (1997) 40:82-88). Pramlintide is used as an adjunct
to insulin therapy in T1D and T2D and shows improvement in glycemic
control and reduction in insulin requirements, and also demonstrate
modest reduction in body weight (Neary M T, Batterham R L. Gut
hormones: Implications for the treatment of obesity. Pharmacology
& Therapeutics (2009)124:44-56). Because pramlintide has a
half-life reported to be 20 min (McQueen, J. Pramlintide acetate.
Am. J. Health-System Pharmacy (2005) 22:2363-2372) and requires
2.times.-3.times./day dosing, its utility as a therapeutic is
limited.
[0289] "Oxytocin" means the mammalian hormone peptide (UniProt No.
P01178) having the sequence CYIQNCPLG-NH2 (SEQ ID NO: 547) and a
disulfide bridge between residues 1 and 6, and synthetic versions,
such as pitocin. Oxytocin acts primarily as a neuromodulator in the
brain, having a structure very similar to that of vasopressin,
which are the only known hormones released by the human posterior
pituitary gland to act at a distance. Oxytocin has
uterine-contracting properties mediated by specific, high-affinity
oxytocin receptors expressed in the mammary gland and the uterus;
hence its role in parturition and lactation. Oxytocin-containing
polypeptides of the invention may find particular use in the
treatment of autism, fragile X syndrome, chronic daily headache,
and male infertility.
[0290] "Relaxin" means the protein hormone that is a heterodimer of
two peptide chains of 24 & 29 amino acids linked by disulfide
bridges created from the 185 amino acid precursor protein (UniProt
No. P04090); the B chain having the sequence
DSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 548) and the A chain
having the sequence QLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 549), with
the disulfide bridges between B10-A10 and B23-A24, and includes
synthetic and recombinant versions. Relaxin is produced by the
corpus luteum during the menstrual cycle and pregnancy in women and
by the prostate in men. Relaxin orchestrates many of the maternal
physiological responses to pregnancy, acts as a systemic and renal
vasodilator, is a cardioprotective & antifibrotic agent.
Relaxin binds to relaxin receptor (GPCR), increases cAMP &
activates PKC, PI3K & endothelin type B receptor resulting in
increased nitric oxide production, and also activates MAPK, which
may play a role in relaxin induced VEGF expression.
Relaxin-containing polypeptides of the invention may find
particular use in the treatment of acute decompensated heart
failure (ADHF). Because the reported half-life of relaxin in humans
is less than 10 min (Dschietzig T, et al. Intravenous recombinant
human relaxin in compensated heart failure: a safety, tolerability,
and pharmacodynamic trial. J Card Fail. 2009; 15:182-190), the
utility of the unmodified protein as a therapeutic is limited.
[0291] "Cenderitide" and "CD-NP" means a human C-type natriuretic
peptide-(32-53)-peptide (CNP-22) with eastern green mamba
(Dendroaspis angusticeps) natriuretic peptide-(24-38)-peptide
having the sequence GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID
NO: 550), with disulfide bridges between residues 6 and 22. The
chimeric peptide has vasoprotective and RAAS suppressing actions
via activation of the receptors guanylyl cyclase (GC)-A and GC-B,
and may potentiate renal enhancement and cardiac unloading while
having minimal hypotensive effects. Accordingly, it may have use in
treatment of cardiorenal disease such as acute decompensated heart
failure (ADHF) and acute myocardial infarction (AMI), particularly
during the "post-acute" treatment period.
[0292] "Peginesatide" or "hematide" is a peptide composed of two
synthetic 21 amino-acid peptides having the sequence
GlyGlyLeuTyrAlaCysHisMetGlyProIleThrlNalValCysGlnProLeuArgSarLys
(SEQ ID NO: 551) that are linked at lysine with a branched
polyethylene glycol. Peginesatide is a novel analog of
erythropoietin that has erythropoietic properties and is being
developed for medical use as a treatment for anemia due to chronic
kidney disease (CKD) in patients not on dialysis.
[0293] "Oxyntomodulin" or "OXM" means human oxyntomodulin,
synthetic versions and sequence variants thereof having at least a
portion of the biological activity of mature oxyntomodulin.
Oxyntomodulin is a 37 amino acid peptide having the sequence
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO: 552), is produced
postprandially from intestinal L-cells in the colon and contains
the 29 amino acid sequence of glucagon followed by an 8 amino acid
carboxyterminal extension. Oxyntomodulin is an agonist at both the
glucagon receptor and the GLP-1R, with its anorectic effect
probably mediated via the latter receptor. OXM has been found to
suppress appetite. OXM-containing polypeptides of the invention may
find particular use in the treatment of diabetes for glucose
regulation, insulin-resistance disorders, obesity, and can be used
as a weight loss treatment. As native oxyntomodulin has been
reported to have a half-life of .about.12 min in human plasma
(measured with a cross-reacting glucagon assay; Schjoldager B T.
Oxyntomodulin: a potential hormone from the distal gut.
Pharmacokinetics and effects on gastric acid and insulin secretion
in man. Eur J Clin Invest. (1988) 18(5):499-503), the utility of
the unmodified protein as a therapeutic is limited.
[0294] "POT4" or "APL-1" means the synthetic cyclic peptide having
the sequence
H-Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr-NH2 (SEQ ID
NO: 553). POT4 is a more potent C3 complement inhibitor than
compstatin, which inhibits the cleavage of native C3 to its active
fragments C3a and C3b, and has extended circulating in vivo
half-life of 8 hours. It is considered for use to prevent
inflammation, damage and upregulation of angiogenic factors like
VEGF in diseases like age-related macular degeneration (AMD),
paroxysmal nocturnal hemoglobinuria (PNH), asthma and COPD.
[0295] "Interferon-lambda", "IFN-.lamda.", interleukin-29" and
"IL-29" means the human interleukin (UniProt No. Q81U54 (20-200))
encoded by the IL29 gene having the sequence
GPVPTSKPTITGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDL
RLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAG
PRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPE ST (SEQ
ID NO: 554), recombinant and synthetic versions and sequence
variants thereof having at least a portion of the biological
activity of mature IL-29. A type III interferon, IL-29 signals
through a heterodimer receptor complex (IL-10R2 & IL-28R.alpha.
receptor chains) distinct from type I IFN (IFNAR1/IFNAR2 receptor
complex), and plays an important role in anti-viral immunity.
Notably, the IL-29 receptor is highly expressed on hepatocytes, the
primary site of HCV infection, but is not significantly expressed
on immune or bone marrow cells. Pegylated versions have an
estimated half-life of 50-70 h.
[0296] "Interferon-beta" or "IFN-.beta." means the human protein
encoded by the IFNB1 gene having the sequence
TABLE-US-00014 (SEQ ID NO: 555)
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL
KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR
VEILRNFYFINRLTGYLRN,
and recombinant and synthetic versions and sequence variants
thereof having at least a portion of the biological activity of
mature IFN- . IFN- is produced by various cell types including
fibroblasts & macrophages, and mediates antiviral,
antiproliferative & immunomodulatory activities in response to
viral infection & other biological inducers. The binding of
IFN- to specific receptors on the surface of human cells initiates
a cascade of intracellular events that leads to the expression of
numerous interferon-induced gene products such as 2',
5'-oligoadenylate synthetase. 2-microglobulin, and neopterin. These
gene products are routinely used as biomarkers in clinical setting.
IFN- is used in treatment of various forms of multiple sclerosis
(MS), including elapse remitting MS, secondary progressive MS,
primary progressive MS, juvenile onset MS, and clinically isolated
syndromes suggestive of MS. Commercially-available forms of IFN-
have reported half-lives of 4 to 67 h and require frequent dosing,
such that their utility as a therapeutic is limited.
[0297] "C-peptide" means the human pancreatic protein having the
sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 556), and
recombinant and synthetic versions and sequence variants thereof
having at least a portion of the biological activity of native
C-peptide. C-peptide is the middle segment of proinsulin that is
between the N-terminal B-chain and the C-terminal A-chain, and is
cleaved from preproinsulin as mature insulin is formed and
secreted. Cirulating C-peptide binds to a receptor that is likely
G-protein-coupled, and the signal activates Ca2+-dependent
intracellular signaling pathways such as MAPK, PLC.gamma., and PKC,
leading to upregulation of a range of transcription factors as well
as eNOS and Na+K+ATPase activities. C-peptide is considered for use
in diabetic complications and diabetic nephropathy. Since the
reported half-life is about 30 minutes (Matthews D R The half-life
of endogenous insulin and C-peptide in man assessed by somatostatin
suppression. Clin Endocrinol (Oxf). (1985) 23(1):71-79), the
utility of the unmodified protein as a therapeutic is limited.
[0298] "Ghrelin" means the human hormone having the sequence
GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO: 557), truncated versions,
recombinant and synthetic versions and sequence variants thereof
having at least a portion of the biological activity of native
ghrelin, including the native, processed 27 or 28 amino acid
sequence and homologous sequences. Ghrelin induces satiation, or
species and non-natural sequence variants having at least a portion
of the biological activity of mature ghrelin, including the native,
processed 27 or 28 amino acid sequence and homologous sequences.
Ghrelin is produced mainly by P/D1 cells lining the fundus of the
human stomach and epsilon cells of the pancreas that stimulates
hunger, and is considered the counterpart hormone to leptin.
Ghrelin levels increase before meals and decrease after meals, and
can result in increased food intake and increase fat mass by an
action exerted at the level of the hypothalamus. Ghrelin also
stimulates the release of growth hormone. Ghrelin is acylated at a
serine residue by n-octanoic acid; this acylation is essential for
binding to the GHS1a receptor and for the agonist activity and the
GH-releasing capacity of ghrelin. Ghrelin-containing polypeptides
of the invention may find particular use as agonists; e.g., to
selectively stimulate motility of the GI tract in gastrointestinal
motility disorder, to accelerate gastric emptying, or to stimulate
the release of growth hormone. The invention also contemplates
unacylated forms and sequence variants of ghrelin, which act as
antagonists. Ghrelin analogs with sequence substitutions or
truncated variants, such as described in U.S. Pat. No. 7,385,026,
may find particular use as fusion partners to XTEN for use as
antagonists for improved glucose homeostasis, treatment of insulin
resistance and treatment of obesity, cancen cachexia,
post-operative ileus, bowel disorders, and gastrointestinal
disorders. The isolation and characterization of ghrelin has been
reported (Kojima M, et al., Ghrelin is a growth-hormone-releasing
acylated peptide from stomach. Nature. 1999; 402(6762):656-660) and
synthetic analogs have been prepared by peptide synthesis, as
described in U.S. Pat. No. 6,967,237. As ghrelin has a reported
terminal half-life of 10-30 min (Akamizu T, et al.
Pharmacokinetics, safety, and endocrine and appetite effects of
ghrelin administration in young healthy subjects. Eur J.
Endocrinology (2004)150(4):447-455), the utility of the unmodified
protein as a therapeutic is limited, and analogs with, at position
3, the native serine amino acid with an octyl side group instead of
the native octanoyl side group may confer added resistant to
proteases.
[0299] "Follistatin," also known as "activin-binding protein" or
"FSH-suppressing protein (FSP)," means the protein that, in humans,
is encoded by the FST gene. As used herein, "follistatin" includes
homologs, species variants, sequence variants and fragments
thereof. The mature protein form in humans has 315 amino acids, is
referred to as FS-315 and has been cloned (U.S. Pat. Nos. 5,041,538
and 5,182,375). Follistatin contains two potential N-glycosylation
sites, Asn95 and Asn259, however it has been demonstrated that
mutation at these sites followed by testing of the recombinant
product for their ability to inhibit FSH secretion and to bind
activin resulted in each mutant having a similar property as the
non-mutated recombinant hFS-315, suggesting that glycosylation of
the follistatin molecule has no effect in these functions (Inouye,
S., et al. Site-specific mutagenesis of human follistatin. BBRC
(1991) 179(1):352-358). Porcine follistatin is disclosed in Ueno et
al., PNAS:USA 84:8282-8286 (1987) and bovine follistatin is
disclosed in Robertson et al., Biochem. Biophys. Res. Commun.
149:744-749 (1987). As bone morphogenetic proteins and
growth/differentiation factors such as activin and myostatin have
the ability to induce the growth, formation, differentiation and
maintenance of various tissues, including bone, cartilage,
tendon/ligament, muscle, neural, and various organs, their
neutralization by follistatin and follistatin agonists have
therapeutic value (U.S. Pat. Nos. 5,545,616, 5,041,538, and
AU9675056). As follistatin administered to a subject is rapidly
eliminated from the circulation, with a terminal half-life of just
over 2 hours in rats (Kogure K, et al. Intravenous administration
of follistatin: delivery to the liver and effect on liver
regeneration after partial hepatectomy. Hepatology. (1996)
24(2):361-366), the utility of the unmodified protein as a
therapeutic is limited.
[0300] "Vasoactive intestinal peptide" and "VIP" means the 28 amino
acid peptide hormone (UniProt No. P01282 (125-152)) encoded by the
VIP gene residues having the sequence
HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID NO: 558) and recombinant
and synthetic versions and sequence variants thereof having at
least a portion of the biological activity of native VIP. The VIP
peptide is produced in many tissues, including the gut, pancreas
and suprachiasmatic nuclei of the hypothalamus in the brain. VIP
stimulates contractility in the heart, causes vasodilation,
increases glycogenolysis, lowers arterial blood pressure and
relaxes the smooth muscle of trachea, stomach and gall bladder.
Changes in concentration are associated with myocardial fibrosis,
heart failure, cardiomyopathy and pulmonary hypertension, and its
deficiency in the respiratory system is considered to be a
pathogenetic factor in pulmonary disease (Said S I. 2007,
Circulation, 115: 1260: Said S1, 2008, Ann N Y Acad Sci, 1144:148;
Petkov V et. al., 2003, J Clin Invest, 111:1339). VIP is considered
for use in treating resistant hypertension, primary pulmonary
arterial hypertension (PAH), asthma, COPD, diabetes, erectile
dysfunction, and female sexual dysfunction. As its half-life is
reported to be approximately 1 minute (Domschke S, et al.
Vasoactive intestinal peptide in man: pharmacokinetics, metabolic
and circulatory effects. Gut (1978) 19:1049-1053), the utility of
the unmodified protein as a therapeutic is limited.
[0301] "Fuzeon" means the 36 amino acid peptide derived from the
gp41 of HIV, a viral protein involved in fusion of HIV to CD4+ T
cells, having the sequence YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
(SEQ ID NO: 559), and recombinant and synthetic versions and
sequence variants thereof having at least a portion of the binding
activity of native gp41. Fuzeon and multimers thereof or conjugates
with related peptides are used or are being considered for use in
treating resistant forms of HIV infection. As fuzeon has a
half-life of 3.8 h in patients, requiring frequent injection
administrations, its utility is limited.
[0302] "KAI-4169" means the peptide agonist of the human cell
surface calcium-sensing receptor (CaSR) under development by KAI
Pharma for the treatment of secondary hyperparathyroidism (SHPT) in
kidney disease patients and bone disorder (CKD-MBD) patients.
[0303] "Pasireotide" means the a somatostatin analog having the
chemical name
[(3S,6S,9S,12R,15S,18S,20R)-9-(4-aminobutyl)-3-benzyl-12-(1H-indol-3-
-yhlmethyl)-2,5,8,11,14,17-hexaoxo-15-phenyl-6-[(4-phenylmethoxyphenyl)met-
hyl]-1,4,7,10,13,16-hexazabicyclo[16.3.0]henicosan-20-yl]
N-(2-aminoethyl)carbamate used for the treatment of Cushing's
disease. Pasireotide is a multi-receptor somatostatin analogue with
high binding affinity for somatostatin-R-subtypes R1, 2, 3 & 5
that suppresses growth hormone, IGF-1 and adrenocorticotropic
hormone secretion. In addition to treatment of Cushing's Disease,
it is also considered for use in acromegaly, neuroendocrine
disease, liver disease, symptomatic polycystic liver disease,
neuroendocrine tumor, lympangioleiomyomatosis, congenital
hyperinsulinism, recurrent or progressive meningioma, and other
endocrine disorders. As a commercially-available form has a
reported half-life of 12 to 17 h (Petersenn, S. et al. Tolerability
and Dose Proportional Pharmacokinetics of Pasireotide Administered
as a Single Dose or Two Divided Doses in Healthy Male Volunteers: A
Single-Center, Open-Label, Ascending-Dose Study. Clinical
Therapeutics (2012) 34:677-688), its utility is limited.
[0304] "Irisin" means the clevage product of the protein encoded by
the FNDC5 gene having the sequence
DSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSC
ALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE (SEQ ID NO:
560), and recombinant and synthetic versions and sequence variants
thereof having at least a portion of the biological activity of
native irisin. Irisin mediates beneficial effects of muscular
exercise, and induces browning of white adipose tissue by
up-regulating UCP1 expression through activation of the nuclear
receptor PPARA. Mildly increased irisin levels have been shown to
result in increased energy expenditure, reduced body weight and
improved diet-induced insulin resistance (Bostrom P, 2012, Nature,
481:463). Irisin is considered for use in treating obesity,
diabetes, and metabolic disorders.
[0305] "TXA127" and "PanCyte" are analogs of angiotensin (1-7),
with TXA127 having the sequence NRVYIHP (SEQ ID NO: 561) and
PanCyte is an cyclic analog linking the 4th and 7th residues with
dAla and Ala, respectively, with the result that it is more
resistant to degradation and has a longer half-life. The analogs
bind to MAS receptor and stimulate early hematopoietic precursor
cells in bone marrow, and also have vasodilation, anti-trophic,
antifibrotic, natriuresis, anti-inflammatory, anti-thrombotic
effects. The compounds are considered for use in acceleration of
platelet recovery following stem cell transplant for patients with
hematological cancers such as acute myelogenous leukemia (AML),
myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL),
chronic myelogenous leukemia (CML), chronic lymphocytic leukemia
(CLL), Hodgkin's lymphoma (HL), or non-Hodgkin's lymphoma (NHL),
and multiple myeloma, and use in treating pulmonary fibrosis, acute
lung injury, pulmonary arterial hypertension, and fibrosis of the
kidney and liver.
[0306] "Interleukin-7" and "IL-7" means the human interleukin
(UniProt No. P13232 (26-177)) encoded by the IL-7 gene having the
sequence
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRA
ARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKS
LKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 562), and
recombinant and synthetic versions and sequence variants thereof
having at least a portion of the biological activity of native
IL-7, IL-7 IL-7 stimulates the differentiation of multipotent
(pluripotent) hematopoietic stein cells into lymphoid progenitor
cells, including expansion of CD4/CD8 T cells. IL-7 limits the
production of suppressor regulatory T cells and T cell anergy
through TGF-B antagonism, and supports production of central memory
T cells IL-7 is considered for use in treating lymphopenia in HIV,
oncology, transplant, HBV and HCV infection, as well as treating
minimal residual disease or advanced tumors, and may have roles in
immune reconstitution or enhancement of im-munotherapy. As the
reported half-life of TL-7 in humans is approximately 10 h
(Sportes, C. et al. Phase I Study of Recombinant Human
Interleukin-7 Administration in Subjects with Refractory
Malignancy. Clin Cancer Res 2010; 16:727-735), its utility in
unmodified form is limited.
[0307] "Fibroblast growth factor 18" or "FGF-18" means the human
protein (UniProt No. O76093(28-207)) encoded by the FGF18 gene,
having the sequence
EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQL
LVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFTEKVLENNYTALMSAK
YSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIRP THPA
(SEQ ID NO: 563) and recombinant and synthetic versions and
sequence variants thereof having at least a portion of the
biological activity of native FGF-18 FGF-18 is a protein member of
the fibroblast growth factor (FGF) family. FGF family members
possess broad mitogenic and cell survival activities, and are
involved in a variety of biological processes, including embryonic
development, cell growth, morphogenesis, tissue repair, tumor
growth, and invasion. It has been shown in vitro that this protein
is able to induce neurite outgrowth in PC12 cells. FGF-18
stimulates the proliferation of chondrocyte & osteoblasts
(cells that produce and maintain bone and cartilage) and its use is
considered for the repair and generation of the cartilage, for
example in the knee joints (Ellsworth J L. Fibroblast growth
factor-18 is a trophic factor for mature chondrocytes and their
progenitors. Osteoarthritis Cartilage (2002) 10:308-320).
[0308] "Alpha-Melanocyte Stimulating Hormone" or ".alpha.-MSH" is
the 13-amino acid peptide generated as a proteolyic cleavage
product from ACTH (1-13), which is in turn a cleavage product of
proopiomelanocortin (POMC), having the sequence N-Ac-SYSMGFRWGLPV
(SEQ ID NO: 564), and synthetic versions and sequence variants
thereof having at least a portion of the biological activity of
native .alpha.-MSH. Alpha-MSH is a non-selective agonist of the
melanocortin receptors MC1, MC3, MC4 & MC5 but not MC2 (which
is exclusive for ACTH). Alpha-MSH stimulates melanocytes to produce
& release melanin which has a photo-protective effect: it
signals the brain, which has effects on appetite and sexual
arousal. It is considered for use in treating erythropoietic
protoporphyria (EPP, intolerant to sun), nonsegmental vitilligo
(skin discoloration), actinic keratosis (AK, solar keratosis,
precursor to skin cancer), polymorphous light eruption (PLE/PMLE),
post-surgery kidney damage, erectile dysfunction, and sexual
dysfunction. Because its half-life is mere seconds, its utility in
unmodified form is limited.
[0309] "Endostatin" means the naturally-occurring 20-kDa C-terminal
fragment derived from type XVIII collageh (UniProt. No.
P39060(1572-1754)) having the sequence
HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYS
IVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSV
WHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSF MTASK
(SEQ ID NO: 565), and recombinant and synthetic versions and
sequence variants thereof having at least a portion of the
biological activity of native endostatin. Endostatin is an
angiogenesis inhibitor and may interfere with the pro-angiogenic
action of growth factors such as basic fibroblast growth factor
(bFGF/FGF-2) and VEGF. It is considered for use in certain cancers.
Because its half-life is 13 h (Thomas, J P et al. Phase I
Pharmacokinetic and Pharmacodynamic Study of Recombinant Human
Endostatin in Patients With Advanced Solid Tumors. J. Clin. Oncol.
(2003) 21:223-231), its utility in unmodified form is limited.
[0310] "Humanin" means the peptide (UniProt No. Q8IVG9(1-24))
encoded by the MT-RNR2 gene, having the sequence
MAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 566), and recombinant and
synthetic versions and sequence variants thereof having at least a
portion of the biological activity of native humanin. Humanin has a
role in neuro-protection against cell death associated with
Alzheimer's disease (AD), AD-specific insults, prion induced
apoptosis & chemically induced neuronal damage (Hashimoto, Y, A
rescue factor abolishing neuronal cell death by a wide spectrum of
familial Alzheimer's disease genes and A.beta.. PNAS (2001)
98:6336-6341). More recently, humanin was found to help improve
insulin action and lower blood glucose levels (Muzumdar R H,
Humanin: A Novel Central Regulator of Peripheral Insulin Action.
PLoS One (2009) 4:e6334). Humanin s considered for use in treating
Alzheimer's disease, diabetes, and vascular & cardiovascular
diseases.
[0311] "Glucagon" means the human glucagon glucose regulating
peptide having the sequence HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID
NO: 567), and recombinant and synthetic versions and sequence
variants thereof having at least a portion of the biological
activity of native glucagon. The term "glucagon" as used herein
also includes peptide mimetics of glucagon. Native glucagon is
produced by the pancreas, released when blood glucose levels start
to fall too low, causing the liver to convert stored glycogen into
glucose and release it into the bloodstream. While the action of
glucagon is opposite that of insulin, which signals the body's
cells to take in glucose from the blood, glucagon also stimulates
the release of insulin, so that newly-available glucose in the
bloodstream can be taken up and used by insulin-dependent tissues.
Glucagon-containing polypeptides of the invention may find
particular use in increasing blood glucose levels in individuals
with extant hepatic glycogen stores and maintaining glucose
homeostasis in diabetes. Glucagon has been cloned, as disclosed in
U.S. Pat. No. 4,826,763.
[0312] "Glucagon-like protein-1" or "GLP-1" means human glucagon
like peptide-1 and sequence variants thereof having at least a
portion of the biological activity of native GLP-1. The term
"GLP-1" includes human GLP-1(1-37) having the sequence
HDEFERHAEGTFTSDVSSTLEGQAALEFIAWLVKGRG (SEQ ID NO: 568),
GLP-1(7-37), and GLP-1(7-36)amide. GLP-1 stimulates insulin
secretion, but only during periods of hyperglycemia. The safety of
GLP-1 compared to insulin is enhanced by this property and by the
observation that the amount of insulin secreted is proportional to
the magnitude of the hyperglycemia. The biological half-life of
GLP-1(7-37)OH is a mere 3 to 5 minutes (U.S. Pat. No. 5,118,666).
GLP-1-containing polypeptides of the invention may find particular
use in the treatment of diabetes and insulin-resistance disorders
for glucose regulation. GLP-1 has been cloned and derivatives
prepared, as described in U.S. Pat. No. 5,118,666.
[0313] "Glucagon-like protein-2" or "GLP-2" means, collectively
herein, human glucagon like peptide-2 having the sequence
HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 569), species
homologs of human GLP-2, and non-natural sequence variants having
at least a portion of the biological activity of mature GLP-2
including variants such as, but not limited to, a variant with
glycine substituted for alanine at position 2 of the mature
sequence resulting in HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO:
570) ("2G") as well as Val, Glu, Lys, Arg, Leu or Ile substituted
for alanine at position 2. GLP-2 or sequence variants have been
isolated, synthesized, characterized, or cloned, as described in
U.S. Pat. Nos. 5,789,379; 5,834,428; 5,990,077; 5,994,500;
6,184,201; 7,186,683; 7,563,770; 20020025933; and 20030162703.
[0314] "Insulin" means human insulin or a homolog, species
variants, or sequence variants thereof that includes, but is not
limited to, the mature human insulin protein composed of 51 amino
acids with a molecular weight of 5808 Da and the proinsulin
precursor of 110 amino acids. The precursor protein is processed to
mature insulin that has an A-chain with sequence
GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 571) and a B-chain with sequence
FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 572) bound together by
disulfide bonds.
[0315] "Factor XIII A chain", "FXIIIA" or "F13A" means the
coagulation protein (UniProt No. P00488(2-732)) having the sequence
SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPRGVNLQEFLNVTSVHLFKERWDT
NKVDHHTDKYENNKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIP
VPIVSELQSGKWGAKIVMREDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPE
TDTYILFNPWCEDDAVYLDNEKEREEYVLNDIGVIFYGEVNDIKTRSWSYGQFEDGILDT
CLYVMDRAQMDLSGRGNPIKVSRVGSAMVNAKDDEGVLVGSWDNIYAYGVPPSAWT
GSVDILLEYRSSENPVRYGQCWVFAGVFNTFLRCLGIPARIVTNYFSAHDNDANLQMDIF
LEEDGNVNSKLTKDSVWNYHCWNEAWMTRPDLPVGFGGWQAVDSTPQENSDGMYRC
GPASVQAIKHGHVCFQFDAPFVFAEVNSDLIYITAKKDGTHVVENVDATHIGKLIVTKQI
GGDGMMDITDTYKFQEGQEEERLALETALMYGAKKPLNTEGVMKSRSNVDMDFEVEN
AVLGKDFKLSITFRNNSHNRYTITAYLSANITFYTGVPKAEFKKETFDVTLEPLSFKKEAV
LIQAGEYMGQLLEQASLHFFVTARINETRDVLAKQKSTVLTIPEIIIKVRGTQVVGSDMT
VTVQFTNPLKETLRNVWVHLDGPGVTRPMKKMFREIRPNSTVQWEEVCRPWVSGHRK
LIASMSSDSLRHVYGELDVQIQRRPSM (SEQ ID NO: 573), and recombinant and
synthetic versions and sequence variants thereof having at least a
portion of the biological activity of native FXIIIA. Factor XIII is
the last enzyme in the coagulation cascade and is responsible for
cross-linking fibrin molecules to each other in a newly formed
blood clot. By forming intermolecular covalent bonds between fibrin
monomers and by cross-linking alpha-2 antiplasmin, fibrinogen,
fibronectin, collagen, and other proteins to enhance the mechanical
strength of the fibrin clot, protect from proteolytic degradation,
and provide stability to the extracellular matrix. Plasma FXIII
circulates as a heterotetramer composed of 2 A subunits and 2 B
subunits noncovalently linked together and bound to fibrinogen. The
B subunit, which appears to stabilize the structure of the A
subunit and to protect the A subunit from proteolysis, is normally
present in excess in plasma as free FXIII-B subunit. Most patients
with FXIII deficiency have mutations in the FXIII-A subunit; few
cases of patients with FXIII-B subunit mutations have been reported
(Mikkola, H, 1996, Semin Thromb Hemost, 22:393; Ichinose A, 1996,
Semin Thromb Hemost 22:385). FXIIIA is used or is considered for
use in treating hemophilia and related coagulopathies, congenital
FXIII deficiency, and acquired FXIII deficiency due to chronic
liver disease, inflammatory bowel disease, and post-surgery
bleeding.
[0316] "Factor X" or "FX" means the coagulation protein (UniProt
No. P0074212-488)) having the sequence
GRPLHLVLLSASLAGLLLLGESLFIRREQANNILARVTRANSFLEEMKKGHLERECMEET
CSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGK
NCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLE
RRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVG
GQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQE
EGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMT
QKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDA
CQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLP
KAKSHAPEVITSSPLK (SEQ ID NO: 574), and recombinant and synthetic
versions and sequence variants thereof having at least a portion of
the biological activity of native FX Factor X is activated into
factor Xa by both factor IX (with its cofactor, factor VIII, to
make a complex known as intrinsic Xase) and factor VII with its
cofactor, tissue factor (to make a complex known as extrinsic
Xase). Factor X is the first member of the final common (or
thrombin) pathway. Factor X is used to treat factor X deficiency,
hemophilia A & B using bypass strategies due to FVIII and FIX
patients developing inhibitory antibodies to FVIII and FIX
replacement therapies), emergency treatment of patients with
hemorrhages due to oral anticoagulants overdose or unknown causes
of critical bleeding, and patients who develop acquired FX
deficiency caused by lack of vitamin K, amyloidosis, severe liver
disease & use of anticoagulants (e.g. warfarin). While the
half-life of mature factor X is 40-45 h, the plasma half-life of
activated factor X (Fxa) is <1-2 min ((Bunce M W, 2008. Blood,
117:290), making its utility in unmodified form limited being
rapidly inactivated by anti-thrombin III & TFP1.
[0317] 4. Nucleic Acids as Payloads
[0318] The invention also contemplates the use of nucleic acids as
payloads in the XTEN conjugates. In one embodiment, the invention
provides XTEN-payload conjugates wherein the payload is selected
from the group consisting of aptamers, antisense oligonucleotides,
ribozyme nucleic acids, RNA interference nucleic acids, and
antigene nucleic acids. Such nucleic acids used as therapeutics are
know in the art (Edwin Jarald, Nucleic acid drugs: a novel
approach. African Journal of Biotechnology Vol. 3 (12):662-666,
2004; Joanna B. Opalinska. Nucleic-acid therapeutics: basic
principles and recent applications. Nature Reviews Drug Discovery
1:503-514, 2002).
IV). XTEN-Cross-Linker and XTEN-Payload Conjugates and Methods of
Making Such Conjugates
[0319] The present invention relates in part to highly purified
preparations of XTEN-cross-linker conjugate compositions useful as
conjugation partners to which payloads are conjugated, as described
herein. The invention also relates to highly purified preparations
of payloads linked to one or more XTEN using the XTEN-cross-linker
conjugation partners. The present invention encompasses
compositions and methods of making the XTEN-payload conjugates
formed by linking of any of the herein described XTEN with a
payload, as well as reactive compositions and methods of making the
compositions formed by conjugating XTEN with a cross-linker or
other chemical methods described herein. It is specifically
intended that the terms "XTEN-payload" and "XTEN-cross-linker"
encompass the linked reaction products remaining after the
conjugation of the reactant conjugation partners, including the
reaction products of cross-linkers, click-chemistry reactants, or
other methods described herein.
[0320] In some embodiments, the XTEN utilized to create the subject
conjugates comprise XTEN selected from any one of the sequences in
Table 2, Table 3, and Tables 22-25, which may be linked to the
payload component directly or via cross-linkers disclosed herein.
In other embodiments, the one or more XTEN utilized to create the
subject conjugates individually comprise an XTEN sequence having at
least about 80% sequence identity, or alternatively 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity compared to an XTEN
selected from Tables 2, 3, and 22-25 or a fragment thereof, when
optimally aligned with a sequence of comparable length. In one
embodiment, the subject conjugates are multimeric in that they
comprise a first and a second XTEN sequence, wherein the XTEN are
the same or they are different and wherein each individually
comprises an XTEN sequence having at least about 80% sequence
identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity compared to an XTEN selected from Tables 2, 3,
and 22-25 or a fragment thereof, when optimally aligned with a
sequence of comparable length. In another embodiment, the subject
conjugates are multimeric in that they comprise a first, a second,
and a third XTEN sequence, wherein the XTEN are the same or they
are different and wherein each individually comprises an XTEN
sequence having at least about 80% sequence identity, or
alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity compared to an XTEN selected from Tables 2, 3, 22-25 or a
fragment thereof, when optimally aligned with a sequence of
comparable length. In yet another embodiment, the subject
conjugates are multimeric in that they comprise 3, 4, 5, 6 or more
XTEN sequences, wherein the XTEN are the same or they are different
and wherein each individually comprises an XTEN sequence having at
least about 80% sequence identity, or alternatively 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity compared to an XTEN
selected from Tables 2, 3, and 22-25 or a fragment thereof. In the
multimeric conjugates, the cumulative length of the residues in the
XTEN sequences is greater than about 200 to about 3000 or about 400
to about 1000 amino acid residues, and the XTEN can be identical or
they can be different in sequence or in length. As used herein,
cumulative length is intended to encompass the total length, in
amino acid residues, when more than one XTEN is incorporated into
the conjugate.
[0321] In one aspect, the invention provides compositions of XTEN
covalently linked to a small molecule payload drug, resulting in an
XTEN-drug conjugate ("XTEN-D"). In another aspect, the invention
provides compositions of XTEN covalently linked to a payload
biologically active protein (which encompasses peptides or
polypeptides), resulting in an XTEN-peptide/polypeptide conjugate
("XTEN-P"). In another aspect, the invention provides compositions
of one or more XTEN recombinantly linked to a payload peptide or
polypeptide, resulting in an XTEN-peptide/polypeptide recombinant
fusion protein ("XTEN-PR"). In another aspect, the invention
provides compositions of one or more XTEN linked to payloads of one
or more drugs and one or more proteins that can be biologically
active or can be targeting moieties. In particular, the invention
provides isolated XTEN-D, XTEN-P, XTEN-PR, and XTEN-D-P
compositions useful in the treatment of a condition for which the
administration of a payload drug and/or protein is known in the art
to be useful in the treatment, amelioration or prevention of a
disease or condition in a subject. The XTEN-D conjugates generally
comprise one or more of the following components: 1) XTEN; 2)
cross-linker, and 3) payload to which the XTEN is chemically
conjugated either directly or by use of a cross-linker, such as
commercially-available cross-linkers described herein, or by use of
click-chemistry reactants, or in some cases, may be created by
conjugation between reactive groups in the XTEN and payload without
the use of a linker as described herein. The XTEN-P generally
comprise one or more of the following components: 1) XTEN; 2)
cross-linker, and 3) biologically active protein payload, and are
also generally created by conjugation with the use of a
cross-linker or click-chemistry reactants. The XTEN-PR conjugates
generally comprises one or more of the following components: 1) one
or more XTEN; 2) a spacer sequence and 3) payload. The XTEN-D-P
generally comprise one or more of the following components: 1)
XTEN; 2) optional linker, 3) biologically active protein; and 4)
drug, wherein the payloads are generally created by conjugation
with the use of a cross-linker or click-chemistry reactants, as
described above. However, in some cases of foregoing types of
compositions, the composition can be created without the use of a
cross-linker provided the components are otherwise chemically
reactive.
[0322] The conjugation of XTEN to payloads confers several
advantages on the resulting compositions compared to the payloads
not linked to XTEN. As described more fully below, non-limiting
examples of the enhanced properties include increases in the
overall solubility and metabolic stability, reduced susceptibility
to proteolysis in circulation, reduced immunogenicity, reduced rate
of absorption when administered subcutaneously or intramuscularly,
reduced clearance by the kidney, enhanced interactions with
substrate, reduced toxicity, targeted delivery of payload, and
enhanced pharmacokinetic properties. Enhanced pharmacokinetic
properties of the conjugates compared to payload not linked to XTEN
include longer terminal half-life (e.g., two-fold, three-fold,
four-fold or more), increased area under the curve (AUC) (e.g.,
25%, 50%, 100% or more), lower volume of distribution, slower
absorption after subcutaneous or intramuscular injection (an
advantage compared to commercially-available forms of payload that
must be administered by a similar route) such that the Cmax is
lower, which, in turn, results in reductions in adverse effects of
the payload that, collectively, results in an increased period of
time that a conjugation composition administered to a subject
provides therapeutic activity. In some embodiments, the conjugation
compositions comprise cleavage sequences (described more fully,
below) that permits sustained release of active payload, such that
the administered XTEN-payload acts as a depot when subcutaneously
or intramuscularly administered. It is specifically contemplated
that XTEN-payload conjugates can exhibit one or more or any
combination of the improved properties disclosed herein. As a
result of these enhanced properties, the XTEN-payload conjugates
permit less frequent dosing, more tailored dosing, and/or reduced
toxicity compared to payload not linked to XTEN and administered in
a comparable fashion. Such XTEN-payload conjugates have utility to
treat certain conditions known in the art to be affected,
ameliorated, or prevented by administration of the payload to a
subject in need thereof, as described herein.
[0323] 1. Cross-Linker and Azide/Alkyne Click-Chemistry Reactants
for Conjugation
[0324] In another aspect, the invention relates to XTEN conjugated
to cross-linkers, resulting in XTEN-cross-linker conjugates that
can be utilized to prepare XTEN-payload conjugation compositions.
In particular, the herein-described XTEN-cross-linker conjugate
partners are useful for conjugation to payload agents or surfaces
bearing at least one thiol, amino, carboxyl, aldehyde or alcohol or
any other reactive group available and suitable, as known in the
art, for reaction between the components described herein.
[0325] In another aspect, the invention relates to methods of
making conjugates of XTEN-cross-linker reactants and
XTEN-click-chemistry azide/alkyne reactants, resulting in
conjugates that can be utilized to prepare the subject XTEN-payload
compositions. In particular, the herein-described methods for
making XTEN-cross-linkers and XTEN-azide/alkyne reactants are
useful wherein the payload agent or a reaction surface bears at
least one thiol, amino, carboxyl, aldehyde, alkene, alkyne,
heterocycle, alcohol, or other reactive group available for
reaction.
[0326] Exemplary embodiments of XTEN have been described above,
including preparations of substantially homogeneous XTEN. The
invention provides XTEN that further serve as a platform to which
payloads can be conjugated, such that they serve as a "carrier",
conferring certain desirable pharmacokinetic, chemical and
pharmaceutical properties to the compositions, amongst other
properties described below. In other embodiments, the invention
provides polynucleotides that encode XTEN that can be linked to
genes encoding peptide or polypeptide payloads that can be
incorporated into expression vectors and incorporated into suitable
hosts for the expression and recovery of the subject XTEN-payload
recombinant fusion proteins.
[0327] In some embodiments, the XTEN components as described
herein, above, are engineered to incorporate a defined number of
reactive amino acid residues that can be reacted with cross-linking
agents or can further contain reactive groups that can be used to
conjugate to payloads. In one embodiment, the invention provides
cysteine-engineered XTEN wherein the cysteine, each of which
contains a reactive thiol group, are conjugated to a cross-linker,
resulting in an XTEN-cross-linker conjugate. In another embodiment,
invention provides lysine-engineered XTEN wherein lysine, each of
which contains a positively charged hydrophilic .epsilon.-amino
group, are conjugated to a cross-linker, resulting in an
XTEN-cross-linker conjugate. In the embodiments of
cysteine-engineered XTEN, each comprises about 1 to about 100
cysteine amino acids, or from 1 to about 50 cysteine amino acids,
or from 1 to about 40 cysteine amino acids, or from 1 to about 20
cysteine amino acids, or from 1 to about 10 cysteine amino acids,
or from 1 to about 5 cysteine amino acids, or 9 cysteines, or 3
cysteines, or a single cysteine amino acid that is available for
conjugation. In the embodiments of lysine-engineered XTEN, each
comprises about 1 to about 100 lysine amino acids, or from 1 to
about 50 lysine amino acids, or from 1 to about 40 lysine
engineered amino acids, or from 1 to about 20 lysine engineered
amino acids, or from 1 to about 10 lysine engineered amino acids,
or from 1 to about 5 lysine engineered amino acids, or 9 cysteines,
or 3 cysteines, or a single lysine that is available for
conjugation. In another embodiment, the engineered XTEN comprises
both cysteine and lysine residues of the foregoing ranges or
numbers.
[0328] Generally, XTEN cysteine thiol groups are more reactive,
i.e., more nucleophilic, towards electrophilic conjugation reagents
than amine or hydroxyl groups. In addition, cysteine residues are
generally found in smaller numbers in a given protein; thus are
less likely to result in multiple conjugations within the same
protein. Cysteine residues have been introduced into proteins by
genetic engineering techniques to form covalent attachments to
ligands or to form new intramolecular disulfide bonds (Better et al
(1994) J. Biol. Chem. 13:9644-9650; Bernhard et al (1994)
Bioconjugate Chem. 5:126-132; Greenwood et al (1994) Therapeutic
Immunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci USA
96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214;
Chmura et al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484;
U.S. Pat. No. 6,248,564).
[0329] In one embodiment, the invention provides an isolated
composition comprising a cysteine-engineered XTEN conjugated to a
cross-linker, wherein the cross-linker is selected from
sulfhydryl-reactive homobifunctional or heterobifunctional
cross-linkers. In another embodiment, the invention provides an
isolated composition comprising a lysine-engineered XTEN conjugated
by a cross-linker, wherein the cross-linker is selected from
amine-reactive homobifunctional or heterobifunctional
cross-linkers. Cross-linking is the process of chemically linking
two or more molecules by a covalent bond. The process is also
called conjugation or bioconjugation with reference to its use with
proteins and other biomolecules. For example, proteins can be
modified to alter N- and C-termini, and amino acid side chains on
proteins and peptides in order to block or expose reactive binding
sites, inactivate functions, or change functional groups to create
new targets for cross-linking
[0330] In one aspect, the invention provides methods for the
site-specific conjugation to XTEN polymer, accomplished using
chemically-active amino acid residues or their derivatives (e.g.,
the N-terminal .alpha.-amine group, the .epsilon.-amine group of
lysine, the thiol group of cysteine, the C-terminal carboxyl group,
carboxyl groups of glutamic acid and aspartic acid. Functional
groups suitable for reactions with primary .alpha.- and
.epsilon.-amino groups are chlorocyanurates, dichlorotreazines,
trezylates, benzotriazole carbonates, p-nitrophenyl carbonates,
trichlorophenyl carbonates, aldehydes, mixed anhydrides,
carbonylimidazoles, imidoesters, N-hydroxysuccinimide esters,
N-hydroxysulfosuccinimide esters (Harris, J. M., Herati, R. S.
Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155
(1991); Herman, S., et al. Macromol. Chem. Phys. 195, 203-209
(1994); Roberts, M. J. et. al. Advanced Drug Delivery Reviews, 54,
459-476 (2002)). N-hydroxysuccinimide esters (NHS-esters and their
water soluble analogs sulfo-NHS-esters) are commonly used for
protein conjugation (see FIG. 2). NHS-esters yield stable amide
products upon reaction with primary amines with relatively
efficient coupling at physiological pH. The conjugation reactions
are typically performed in 50-200 mM phosphate,
bicarbonate/carbonate, HEPES or borate buffers (pH between 7 and 9)
at 4.degree. C. to room temperature from 0.5 to 2 hrs. NHS-esters
are usually used at two- to 50-fold molar excess to protein.
Typically, the concentration of the reagent can vary from 0.1-10
mM, while the optimal protein concentration is 50-100 .mu.M.
[0331] In another method, given that XTEN polypeptides possess only
a single N-terminal .alpha.-amino group, the XTEN can be engineered
to contain additional .epsilon.-amino group(s) of intentionally
incorporated lysine residues; exemplary sequences of which are
provided in Table 3. The .alpha.- and .epsilon.-amino groups have
different pKa values: approximately 7.6 to 8.0 for the
.alpha.-amino group of the N-terminal amino acid, and approximately
10-10.5 for the .epsilon.-amino group of lysine. Such a significant
difference in pKa values can be used for selective modification of
amino groups. Deprotonation of all primary amines occurs at pH
above pH 8.0. In this environment, the nucleophilic properties of
different amines determine their reactivity. When deprotonated, the
more nucleophilic .epsilon.-amino groups of lysines are generally
more reactive toward electrophiles than .alpha.-amino groups. On
the other hand, at a lower pH (for example pH 6), the more acidic
.alpha.-amino groups are generally more deprotonated than
.epsilon.-amino groups, and the order of reactivity is inverted.
For example, the FDA-approved drug Neulasta (pegfilgranstim) is
granulocyte colony-stimulating factor (G-CSF) modified by covalent
attachment of 20 kDa PEG-aldehyde. Specific modification of the
protein's N-terminal amino acid was accomplished by exploiting the
lower pKa of .alpha.-amino group as compared to .epsilon.-amino
groups of internal lysines (Molineaux, G. Curr. Pharm. Des. 10,
1235-1244 (2004), U.S. Pat. No. 5,824,784).
[0332] The XTEN polypeptides comprising cysteine residues can be
genetically engineered using recombinant methods described herein
(see, e.g., Examples) or by standard methods known in the art.
Conjugation to thiol groups can be carried using highly specific
reactions, leading to the formation of single conjugate species
joined by cross-linking agents. Functional groups suitable for
reactions with cysteine thiol-groups are N-maleimides, haloacetyls,
and pyridyl disulfides. The maleimide group reacts specifically
with sulfhydryl groups when the pH of the reaction mixture is
between pH 6.5 and 7.5, forming a stable thioether linkage that is
not reversible (see FIG. 3). At neutral pH, maleimides react with
sulfhydryls 1,000-fold faster than with amines, but when the pH is
raised to greater than 8.5, the reaction favors primary amines.
Maleimides do not react with tyrosines, histidines or methionines.
For reaction solutions, thiols must be excluded from reaction
buffers used with maleimides as they will compete for coupling
sites. Excess maleimides in the reaction can be quenched at the end
of a reaction by adding free thiols, while EDTA can be included in
the coupling buffer to minimize oxidation of sulfhydryls.
[0333] In another embodiment, the invention contemplates use of
haloacetyl reagents that are useful for cross-linking sulfhydryls
groups of XTEN or payloads to prepare the subject conjugates. The
most commonly used haloacetyl reagents contain an iodoacetyl group
that reacts with sulfhydryl groups at physiological pH. The
reaction of the iodoacetyl group with a sulfhydryl proceeds by
nucleophilic substitution of iodine with a thiol producing a stable
thioether linkage (see FIG. 4). Using a slight excess of the
iodoacetyl group over the number of sulfhydryl groups at pH 8.3
ensures sulfhydryl selectivity. If a large excess of iodoacetyl
group is used, the iodoacetyl group can react with other amino
acids. Imidazoles can react with iodoacetyl groups at pH 6.9-7.0,
but the incubation must proceed for longer than one week. Histidyl
side chains and amino groups react in the unprotonated form with
iodoacetyl groups above pH 5 and pH 7, respectively. In another
embodiment, cross-linkers useful for sulfhydryls groups are pyridyl
disulfides. Pyridyl disulfides react with sulfhydryl groups over a
broad pH range (the optimal pH is 4-5) to form disulfide bonds
linking XTEN to payloads (see FIG. 5). As a disulfide, conjugates
prepared using these reagents are cleavable. During the reaction, a
disulfide exchange occurs between the molecule's--SH group and the
2-pyridyldithiol group. As a result, pyridine-2-thione is released.
These reagents can be used as crosslinkers and to introduce
sulfhydryl groups into proteins. The disulfide exchange can be
performed at physiological pH, although the reaction rate is
slower.
[0334] The XTEN-payload conjugates comprising active synthetic
peptides or polypeptides can be prepared using chemically active
amino acid residues or their derivatives; e.g., the N-terminal
.alpha.-amino group, the .epsilon.-amino group of lysine, a thiol
group of cysteine, the carboxyl group of the C-terminal amino acid,
a carboxyl group of aspartic acid or glutamic acid. Each peptide
contains N-terminal .alpha.-amino group regardless of a primary
amino acid sequence. If necessary, N-terminal .alpha.-amino group
can be left protected/blocked upon chemical synthesis of the active
peptide/polypeptide. The synthetic peptide/polypeptide may contain
additional .epsilon.-amino group(s) of lysine that can be either
natural or specifically substituted for conjugation. As described
above, .alpha.- and .epsilon.-amino groups can be selectively
modified at different pH. Another approach to selectively modify
either .alpha.- or .epsilon.-amino group in a synthetic peptide is
a reversible protection of amino groups with Di-tert-butyl
dicarbonate (BOC.sub.2). For example, selective BOC protection of
vapreotide peptide (a synthetic somatostatin analog) has been
achieved by modification at pH 6 (.alpha.-group protected) or pH
8.5 (s-group protected). The remaining free amino group was then
specifically modified by PEG-N-hydroxysuccinimide or PEG-aldehyde.
Finally, BOC protection was removed by acidic treatment to yield
mono-modified peptides (Morpurgo, M. et al. Selective Alkylation
and Acylation of .alpha. and .epsilon. Amino Groups with PEG in a
Somatostatin Analogue: Tailored Chemistry for Optimized
Bioconjugates. Bioconjugate Chem. 2002. 13:1238-1243).
[0335] Since cysteines are generally less abundant in natural
peptide and protein sequences than lysines, the use of cysteines as
a site for conjugation reduces the likelihood of multiple
conjugations to XTEN-cross-linker molecules in a reaction. It also
reduces the likelihood of peptide/protein deactivation upon
conjugation. Moreoever, conjugation to cysteine sites can often be
carried out in a well-defined manner, leading to the formation of
single species XTEN polymer-peptide or XTEN polymer-polypeptide
conjugates. In some cases cysteine may be absent in the amino acid
sequence of the peptide to be conjugated. In such a case, cysteine
residue can be added to the N- or C-terminus of the peptide either
recombinantly or synthetically using standard methods.
Alternatively, a selected amino acid can be chemically or
genetically modified to cysteine. As one example, serine
modification to cysteine is considered a conservative mutation.
Another approach to introduce a thiol group in cysteine-lacking
peptides is chemical modification of the lysine .epsilon.-amino
group using thiolating reagents such as 2-iminothiolane (Traut's
reagent), SATA (N-succinimidyl S-acetylthioacetate), SATP
(N-succinimidyl S-acetylthiopropionate), SAT-PEO.sub.4-Ac
(N-Succinimidyl S-acetyl(thiotetraethylene glycol)), SPDP
(N-Succinimidyl 3-(2-pyridyldithio)propionate), LC-SPDP
(Succinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate)
(described more fully, below). Once a unique thiol group is
introduced in the peptide, it can be selectively modified by
compounds containing sufhydryl-reactive such as N-maleimides,
haloacetyls, and pyridyl disulfides, as described above.
[0336] The conjugation between the XTEN polypeptide and a peptide,
protein or small molecule drug payload may be achieved by a variety
of linkage chemistries, including commercially available
zero-length, homo- or hetero-bifunctional, and multifunctional
cross-linker compounds, according to methods known and available in
the art, such as those described, for example, in R. F. Taylor
(1991) "Protein immobilization. Fundamentals and Applications",
Marcel Dekker Inc., N.Y.; G. T. Hermanson et al. (1992)
"Immobilized Affinity Ligand Techniques", Academic Press, San
Diego; G. T. Hermanson (2008) "Bioconjugate Techniques", 2.sup.nd.
ed. Elsevier, Inc., S. S. Wong (1991) "Chemistry of Protein
Conjugation and Crosslinking", CRC Press, Boca Raton. Suitable
cross-linking agents for use in preparing the conjugates of the
disclosure are commercially-available from companies like
Sigma-Aldrich, Thermo Fisher Scientific (Pierce Protein Research
Products), Invitrogen, ProteoChem, G-Biosciences. Preferred
embodiments of cross-linkers comprise a thiol-reactive functional
group or an amino-reactive functional group. A list of exemplary
cross-linkers is provided in Table 13.
TABLE-US-00015 TABLE 13 Exemplary cross-linkers Cross-linker
maleimides, haloacetyls, pyridyl disulfides, haloacetyls, pyridyl
disulfides, ABH (p- Azidobenzoyl hydrazide), AMAS
(N-(.alpha.-Maleimidoacetoxy)-succinimide ester), ANB-NOS (N-
5-Azido-2-nitrobenzyloxy-succinimide), APDP
(N-(4-[p-Azidosalicylamido]butyl)-3'-(2'- pyridyldithio)
propionamide), ASBA (4-(p-Azidosalicylamido)-butylamine), BASED
(Bis (.beta.-[4- azidosalicylamido]ethyl) disulfide), BMB
(1,4-Bis-Maleimidobutane), BMDB (1,4
Bismaleimidyl-2,3-dihydroxybutane), BMH (Bis-Maleimidohexane), BMOE
(Bis- Maleimidoethane), BMPH (N-(.beta.-Maleimidopropionic
acid)hydrazide), BMPS (N-(.beta.- Maleimidopropyloxy)succinimide
ester), BM(PEG).sub.2 (1,8-Bis-Maleimidodiethylene-glycol),
BM(PEG).sub.3 (1,11-Bis-Maleimidotriethyleneglycol), BS.sup.2G (Bis
(sulfosuccinimidyl)glutarate), BS.sup.3 (Sulfo-DSS) (Bis
(sulfosuccinimidyl)suberate), BS[PEG].sub.5 (Bis (NHS)PEG5),
BS(PEG).sub.9 (Bis (NHS)PEG9), BSOCOES
(Bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone), C6-SANH (C6-
Succinimidyl 4-hydrazinonicotinate acetone hydrazone), C6-SFB
(C6-Succinimidyl 4- formylbenzoate), DCC
(N,N-Dicyclohexylcarbodiimide), DFDNB (1-5-Difluoro-2,4-
dinitrobenzene), DMA (Dimethyl adipimidate), DMP (Dimethyl
pimelimidate), DMS (Dimethyl suberimidate), DPDPB
(1,4-Di-(3'-[2'pyridyldithio]propionamido) butane), DSG
(Disuccinimidyl glutarate), DSP (Dithiobis(succimidylpropionate),
Lomant's Reagent), DSS (Disuccinimidyl suberate), DST
(Disuccinimidyl tartarate), DTBP (Dimethyl 3,3'-
dithiobispropionimidate), DTME (Dithiobis-maleimidoethane), DTSSP
(Sulfo-DSP) (3,3'- Dithiobis (sulfosuccinimidylpropionate)), EDC
(1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), EGS
(Ethylene glycol bis(succinimidylsuccinate)), EMCA (N-.epsilon.-
Maleimidocaproic acid), EMCH (N-(.epsilon.-Maleimidocaproic
acid)hydrazide), EMCS (N-(.epsilon.-
Maleimidocaproyloxy)succinimide ester), GMBS
(N-(.gamma.-Maleimidobutyryloxy)succinimide ester), KMUA
(N-.kappa.-Maleimidoundecanoic acid), KMUH
(N-(.kappa.-Maleimidoundecanoic acid)hydrazide), LC-SDA
(NHS-LC-Diazirine), LC-SMCC (Succinimidyl 4-(N- maleimidomethyl)
cyclohexane-1-carboxy-(6-amidocaproate)), LC-SPDP (Succinimidyl
6-(3'- [2-pyridyldithio]propionamido)hexanoate), MBS
(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), MPBH
(4-(4-N-Maleimidophenyl)-butyric acid hydrazide), NHS-ASA (N-
Hydroxysuccinimidyl-4-azidosalicylic acid), PDPH
(3-(2-Pyridyldithio)propionylhydrazide), PMPI
(N-(p-Maleimidophenyl)isocyanate), SADP (Succinimidyl
(4-azidophenyl dithio) propionate), SAED (Succimidyl
2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-
dithiopropionate), SAND
(Succinimidyl-2-(m-azido-o-nitrobenzamido)ethyl 1,3'-
dithiopropionate), SANH (Succinimidyl 4-hydrazinonicotinate acetone
hydrazone), SANPAH (N-Succinimidyl
6-(4'-azido-2'-nitrophenylamino)hexanoate), SASD
(Succinimidyl-2-(p- azidosalycylamido)ethyl-1,3-dithiopropionate),
SBAP (Succinimdyl 3- (bromoacetamido)propionate), SDA
(NHS-Diazirine), SDAD (NHS-SS-Diazirine), SFAD
(Succinimidyl(perfluoroazidobenzamido)ethyl 1,3'-dithiopropionate),
SFB (Succinimidyl 4- formylbenzoate), SHTH (Succinimidyl
4-hydrazidoterephthalate), SIA (N-succinimidyl iodoacetate), SIAB
(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SMPB (Succinimidyl
4-(p- maleimidophenyl) butyrate), SMCC (Succinimidyl
4-(N-maleimido-methyl)cyclohexane-1- carboxylate), SM[PEG].sub.2
(NHS-PEG2-Maliemide), SM[PEG].sub.4 (NHS-PEG4-Maliemide),
SM(PEG).sub.6 (NHS-PEG6-Maleimide), SM[PEG].sub.8
(NHS-PEG8-Maliemide), SM[PEG].sub.12 (NHS- PEG12-Maliemide),
SM(PEG).sub.24 (NHS-PEG24-Maleimide), SMPB (Succinimidyl 4-(p-
maleimido-phenyl)butyrate), SMPH
(Succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate), SMPT
(4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridyldithio)toluene),
SPB (Succinimidyl-(4- psoralen-8-yloxy)butyrate), SPDP
(N-Succinimidyl 3-(2-pyridyldithio)propionate), Sulfo-DST
(Sulfodisuccinimidyl tartrate), Sulfo-EGS (Ethylene glycol bis
(sulfo-succinimidyl succinate)), Sulfo-EMCS
(N-(.epsilon.-Maleimidocaproyloxy)sulfosuccinimide ester),
Sulfo-GMBS (N-(.gamma.- Maleimidobutryloxy)sulfosuccinimide ester),
Sulfo-HSAB (N-Hydroxysulfosuccinimidyl-4- azidobenzoate),
Sulfo-KMUS (N-(.kappa.-Maleimidoundecanoyloxy)sulfosuccinimide
ester), Sulfo- LC-SDA (Sulfo-NHS-LC-Diazirine), Sulfo-LC-SMPT
(Sulfosuccinimidyl 6-(.alpha.-methyl-.alpha.-[2-
pyridyldithio]-toluamido)hexanoate), Sulfo-LC-SPDP
(Sulfosuccinimidyl 6-(3'-[2- pyridyldithio]propionamido)hexanoate),
Sulfo-MBS (m-Maleimidobenzoyl-N- hydroxysulfosuccinimide ester),
Sulfo-NHS-LC-ASA (Sulfosuccinimidyl(4-azido-salicylamido)
hexanoate), Sulfo-SADP (Sulfosuccinimidyl (4-azidophenyl dithio)
propionate), Sulfo-SAED (Sulfosuccimidyl
2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionate),
Sulfo- SAND (Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl
1,3'-dithiopropionate), Sulfo- SANPAH (Sulfosuccinimidyl
6-(4'-azido-2'-nitrophenylamino)hexanoate), Sulfo-SASD
(Sulfosuccinimidyl-2-(p-azidosalycylamido)ethyl-1,3-dithiopropionate),
Sulfo-SDA (Sulfo- NHS-Diazirine), Sulfo-SDAD
(Sulfo-NHS-SS-Diazirine), Sulfo-SFAD
(Sulfosuccinimidyl(perfluoroazidobenzamido)ethyl
1,3'-dithiopropionate), Sulfo-SIAB
(Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate), Sulfo-SMCC
(Sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate), Sulfo-SMPB
(Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate), THPP
(.beta.-(Tris[hydroxymethyl]phosphine)propionic acid (betaine)),
TMEA (Tris-(2-Maleimidoethyl)amine), TSAT (Tris-(succinimidyl
aminotriacetate)), 3- propargyloxypropanoic acid, NHS ester,
acetylene-PEG-NHS ester, dibenzylcyclooctyne, (DBCO)-NHS ester,
DBCO-PEG-NHS ester, cyclooctyne (COT)-NHS ester, COT-PEG-NHS ester,
COT-PEG-pentafluorophenyl (PFP) ester, BCOT-NHS ester, BCOT-PEG-NHS
ester, BCOT-PEG-pentafluorophenyl (PFP) ester,
Acetylene-PEG4-maleimide, DBCO-maleimide, COT-maleimide,
BCOT-maleimide, 3-azide-propionic acid, NHS ester, 6-azide-hexanoic
acid, NHS ester, 3-azide-propionic acid, PFP ester,
6-azide-hexanoic acid, PFP ester, azide-PEG-NHS ester,
azide-PEG-PFP ester, azide-PEG-maleimide,
N-(5-Aminopentyl)maleimide, aminopentyl- maleimide
[0337] Non-limiting examples of cross-linkers are ABH
(p-Azidobenzoyl hydrazide), AMAS
(N-(.alpha.-Maleimidoacetoxy)-succinimide ester), ANB-NOS
(N-5-Azido-2-nitrobenzyloxy-succinimide), APDP
(N-(4-[p-Azidosalicylamido]butyl)-3'-(2'-pyridyldithio)
propionamide), ASBA (4-(p-Azidosalicylamido)-butylamine), BASED
(Bis (.beta.-[4-azidosalicylamido]ethyl) disulfide), BMB
(1,4-Bis-Maleimidobutane), BMDB (1,4
Bismaleimidyl-2,3-dihydroxybutane), BMH (Bis-Maleimidohexane), BMOE
(Bis-Maleimidoethane), BMPH (N-(.beta.-Maleimidopropionic
acid)hydrazide), BMPS (N-(.beta.-Maleimidopropyloxy)succinimide
ester), BM(PEG).sub.2 (1,8-Bis-Maleimidodiethylene-glycol),
BM(PEG).sub.3 (1,11-Bis-Maleimidotriethyleneglycol), BS.sup.2G (Bis
(sulfosuccinimidyl)glutarate), BS.sup.3 (Sulfo-DSS) (Bis
(sulfosuccinimidyl)suberate), BS[PEG].sub.5 (Bis (NHS)PEG5),
BS(PEG), (Bis (NHS)PEG9), BSOCOES
(Bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone), C6-SANH
(C6-Succinimidyl 4-hydrazinonicotinate acetone hydrazone), C6-SFB
(C6-Succinimidyl 4-formylbenzoate), DCC
(N,N-Dicyclohexylcarbodiimide), DFDNB
(1-5-Difluoro-2,4-dinitrobenzene), DMA (Dimethyl adipimidate), DMP
(Dimethyl pimelimidate), DMS (Dimethyl suberimidate), DPDPB
(1,4-Di-(3'-[2'pyridyldithio]propionamido) butane), DSG
(Disuccinimidyl glutarate), DSP (Dithiobis(succimidylpropionate),
Lomant's Reagent), DSS (Disuccinimidyl suberate), DST
(Disuccinimidyl tartarate), DTBP (Dimethyl
3,3'-dithiobispropionimidate), DTME (Dithiobis-maleimidoethane),
DTSSP (Sulfo-DSP) (3,3'-Dithiobis (sulfosuccinimidylpropionate)),
EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride),
EGS (Ethylene glycol bis(succinimidylsuccinate)), EMCA
(N-.epsilon.-Maleimidocaproic acid), EMCH
(N-(.epsilon.-Maleimidocaproic acid)hydrazide), EMCS
(N-(.epsilon.-Maleimidocaproyloxy)succinimide ester), GMBS
(N-(.gamma.-Maleimidobutyryloxy)succinimide ester), KMUA
(N-.kappa.-Maleimidoundecanoic acid), KMUH
(N-(.kappa.-Maleimidoundecanoic acid)hydrazide), LC-SDA
(NHS-LC-Diazirine), LC-SMCC (Succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxy-(6-amidocaproate)), LC-SPDP (Succinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), MBS
(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), MPBH
(4-(4-N-Maleimidophenyl)-butyric acid hydrazide), NHS-ASA
(N-Hydroxysuccinimidyl-4-azidosalicylic acid), PDPH
(3-(2-Pyridyldithio)propionylhydrazide), PMPI
(N-(p-Maleimidophenyl)isocyanate), SADP (Succinimidyl
(4-azidophenyl dithio) propionate), SAED (Succimidyl
2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionate),
SAND (Succinimidyl-2-(m-azido-o-nitrobenzamido)ethyl
1,3'-dithiopropionate), SANH (Succinimidyl 4-hydrazinonicotinate
acetone hydrazone), SANPAH (N-Succinimidyl
6-(4'-azido-2'-nitrophenylamino)hexanoate), SASD
(Succinimidyl-2-(p-azidosalycylamido)ethyl-1,3-dithiopropionate),
SBAP (Succinimdyl 3-(bromoacetamido)propionate), SDA
(NHS-Diazirine), SDAD (NHS-SS-Diazirine), SFAD
(Succinimidyl(perfluoroazidobenzamido)ethyl 1,3'-dithiopropionate),
SFB (Succinimidyl 4-formylbenzoate), SHTH (Succinimidyl
4-hydrazidoterephthalate), SIA (N-succinimidyl iodoacetate), SIAB
(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SMPB (Succinimidyl
4-(p-maleimidophenyl) butyrate), SMCC (Succinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate), SM[PEG].sub.2
(NHS-PEG2-Maliemide), SM[PEG].sub.4 (NHS-PEG4-Maliemide),
SM(PEG).sub.6 (NHS-PEG6-Maleimide), SM[PEG].sub.8
(NHS-PEG8-Maliemide), SM[PEG].sub.12 (NHS-PEG12-Maliemide),
SM(PEG).sub.24 (NHS-PEG24-Maleimide), SMPB (Succinimidyl
4-(p-maleimido-phenyl)butyrate), SMPH
(Succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate), SMPT
(4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridyldithio)toluene),
SPB (Succinimidyl-(4-psoralen-8-yloxy)butyrate), SPDP
(N-Succinimidyl 3-(2-pyridyldithio)propionate), Sulfo-DST
(Sulfodisuccinimidyl tartrate), Sulfo-EGS (Ethylene glycol bis
(sulfo-succinimidyl succinate)), Sulfo-EMCS
(N-(.epsilon.-Maleimidocaproyloxy)sulfosuccinimide ester),
Sulfo-GMBS (N-(.gamma.-Maleimidobutryloxy)sulfosuccinimide ester),
Sulfo-HSAB (N-Hydroxysulfosuccinimidyl-4-azidobenzoate), Sulfo-KMUS
(N-(.kappa.-Maleimidoundecanoyloxy)sulfosuccinimide ester),
Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine), Sulfo-LC-SMPT
(Sulfosuccinimidyl
6-(.alpha.-methyl-.alpha.-[2-pyridyldithio]-toluamido)hexanoate),
Sulfo-LC-SPDP (Sulfosuccinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), Sulfo-MBS
(m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester),
Sulfo-NHS-LC-ASA (Sulfosuccinimidyl(4-azido-salicylamido)
hexanoate), Sulfo-SADP (Sulfosuccinimidyl (4-azidophenyl dithio)
propionate), Sulfo-SAED (Sulfosuccimidyl
2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionate),
Sulfo-SAND (Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl
1,3'-dithiopropionate), Sulfo-SANPAH (Sulfosuccinimidyl
6-(4'-azido-2'-nitrophenylamino)hexanoate), Sulfo-SASD
(Sulfosuccinimidyl-2-(p-azidosalycylamido)ethyl-1,3-dithiopropionate),
Sulfo-SDA (Sulfo-NHS-Diazirine), Sulfo-SDAD
(Sulfo-NHS-SS-Diazirine), Sulfo-SFAD
(Sulfosuccinimidyl(perfluoroazidobenzamido)ethyl
1,3'-dithiopropionate), Sulfo-SIAB
(Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate), Sulfo-SMCC
(Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
Sulfo-SMPB (Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate), THPP
(.beta.-(Tris[hydroxymethyl]phosphine)propionic acid (betaine)),
TMEA (Tris-(2-Maleimidoethyl)amine), TSAT (Tris-(succinimidyl
aminotriacetate)).
[0338] In some embodiments, XTEN-payload conjugates using
cross-linking reagents introduce non-natural spacer arms. However,
in cases where a native peptide bond is preferred, the invention
provides that a reaction can be carried out using zero-length
cross-linkers that act via activation of a carboxylate group. In
the embodiments thereof, in order to achieve reaction selectivity,
the first polypeptide has to contain only a free C-terminal
carboxyl group while all lysine, glutamic acid and aspartic acid
side chains are protected and the second peptide/protein N-terminal
.alpha.-amine has to be the only available unprotected amino group
(requiring that any lysines, asparagines or glutamines be
protected). In such cases, use of XTEN AG family sequences of Table
2 that are without glutamic acid as the first polypeptide in the
XTEN-payload or XTEN-cross-linker is preferred. Accordingly, in one
embodiment, the invention provides XTEN-cross-linker and
XTEN-payload comprising AG XTEN sequences wherein the compositions
are conjugated to payloads using a zero-length cross-linkers.
Exemplary zero-length cross-linkers utilized in the embodiment
include but are not limited to DCC (N,N-Dicyclohexylcarbodiimide)
and EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride) wherein the cross-linkers are used to directly
conjugate carboxyl functional groups of one molecule (such as a
payload) to the primary amine of another molecule, such as a
payload with that functional group (see FIG. 6). Sulfo-NHS
(N-hydroxysulfosuccinimide) and NHS (N-hydroxysuccinimide) are used
as catalysts for conjugation, increasing reaction efficiency
(Grabarek Z, Gergely J. Zero-length crosslinking procedure with the
use of active esters. (1990) Anal. Biochem. 185(1), 131-135). EDC
reacts with carboxylic acid group and activates the carboxyl group
to form an active O-acylisourea intermediate, allowing it to be
coupled to the amino group in the reaction mixture. The
O-acylisourea intermediate is unstable in aqueous solutions, making
it ineffective in two-step conjugation procedures without
increasing the stability of the intermediate using
N-hydroxysuccinimide. This intermediate reacts with a primary amine
to form an amide derivative. The crosslinking reaction is usually
performed between pH 4.5 to 5 and requires only a few minutes for
many applications. However, the yield of the reaction is similar at
pH from 4.5 to 7.5. The hydrolysis of EDC is a competing reaction
during coupling and is dependent on temperature, pH and buffer
composition. 4-Morpholinoethanesulfonic acid (MES) is an effective
carbodiimide reaction buffer. Phosphate buffers reduce the reaction
efficiency of the EDC, but increasing the amount of EDC can
compensate for the reduced efficiency. Tris, glycine and acetate
buffers may not be used as conjugation buffers.
[0339] The invention also provides compositions in which three
XTENs are linked by trivalent cross-linkers, resulting in trimeric
XTEN-cross-linker conjugates. Trimeric cross-linkers can be created
by connecting a symmetric trivalent core such as tertiary amine,
trisubstituted methane or 1,3,5-trisubstituted benzene or
asymmetric trivalent molecule such a LysLys dipeptide or a GluGlu
dipeptide or a AspAsp dipeptide or a CysCysCys tripeptide by
spacers with various reactive side groups described in Table 14,
using standard conjugation techniques. In one embodiment, the
invention provides compositions in which three XTENs are covalently
linked by a trivalent cross-linker selected from the group
consisting of thiol-reactive Tris-(2-Maleimidoethyl)amine (TMEA),
amine-reactive Tris-(succimimidyl aminotricetate) (TSAT) and the
cross-linkers set forth in Table 14.
TABLE-US-00016 TABLE 14 Trivalent Cross-linkers Trivalent
Cross-linker* Trivalent Core Group 1 Group 2 Group 3 Tertiary amine
Azide Azide Azide Trisubstituted Alkyne Alkyne Alkyne methane NHS
NHS NHS 1,3,5- Maleimide Maleimide Maleimide trisubstituted
Iodoacetyl Iodoacetyl Iodoacetyl benzene Azide NHS NHS LysLys Azide
Azide NHS GluGluGlu Azide Iodoacetyl Iodoacetyl AspAspAsp Azide
Azide Iodoacetyl CysCysCys Alkyne NHS NHS Alkyne Alkyne NHS Alkyne
Iodoacetyl Iodoacetyl Alkyne Alkyne Iodoacetyl Alkyne Maleimide
Maleimide Alkyne Alkyne Maleimide NHS Maleimide Maleimide NHS NHS
Maleimide NHS Alkyne Maleimide *One of the trivalent core + any one
of Group 1 + any one of Group 2 + any one of Group 3
[0340] In other embodiments, XTEN and payloads can be conjugated
using a broad group of cross-linkers, including those consisting of
a spacer arm (linear or branched) and two or more reactive ends
that are capable of attaching to specific functional groups (e.g.,
primary amines, sulfhydryls, etc.) on proteins or other molecules.
Linear cross-linkers can be homobifunctional or heterobifunctional.
Homobifunctional cross-linkers have two identical reactive groups
which are used to cross-link proteins in one step reaction
procedure. Non-limiting examples of amine-reactive homobifunctional
cross-linkers are BS2G (Bis (sulfosuccinimidyl)glutarate), BS3
(Sulfo-DSS) (Bis (sulfosuccinimidyl)suberate), BS[PEG]5 (Bis
(NHS)PEG5), BS(PEG)9 (Bis (NHS)PEG9), BSOCOES
(Bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone), DFDNB
(1-5-Difluoro-2,4-dinitrobenzene), DMA (Dimethyl adipimidate), DMP
(Dimethyl pimelimidate), DMS (Dimethyl suberimidate), DSG
(Disuccinimidyl glutarate), DSP (Dithiobis(succimidylpropionate)
(Lomant's Reagent), DSS (Disuccinimidyl suberate), DST
(Disuccinimidyl tartarate), DTBP (Dimethyl
3,3'-dithiobispropionimidate), DTSSP (Sulfo-DSP) (3,3'-Dithiobis
(sulfosuccinimidylpropionate)), EGS (Ethylene glycol
bis(succinimidylsuccinate)), Sulfo-EGS (Ethylene glycol bis
(sulfo-succinimidyl succinate)).
[0341] Additionally, examples of homobifunctional cross-linkers
employed in the compositions and in the methods to create the
XTEN-payload and/or XTEN-cross-linker compositions are
sulfhydryl-reactive agents such as BMB (1,4-Bis-Maleimidobutane),
BMH (Bis-Maleimidohexane), BMDB (1,4
Bismaleimidyl-2,3-dihydroxybutane), BMOE (Bis-Maleimidoethane),
BM(PEG)2 (1,8-Bis-Maleimidodiethylene-glycol), BM(PEG)3
(1,11-Bis-Maleimidotriethyleneglycol), DPDPB
(1,4-Di-(3'-[2'pyridyldithio]propionamido) butane), DTME
(Dithiobis-maleimidoethane).
[0342] For the creation of XTEN-cross-linker conjugates for
subsequent conjugation to payloads, as well as the creation of
XTEN-payload conjugates, heterobifunctional cross-linkers are
preferred as the sequential reactions can be controlled. As
heterobifunctional cross-linkers possess two different reactive
groups, their use in the compositions allows for sequential
two-step conjugation. A heterobifunctional reagent is reacted with
a first protein using the more labile group. In one embodiment, the
conjugation of the heterobifunctional cross-linker to a reactive
group in an XTEN results in an XTEN-cross-linker conjugate. After
completing the reaction and removing excess unreacted cross-linker,
the modified protein (such as the XTEN-cross-linker) can be added
to the payload which interacts with a second reactive group of the
cross-linker, resulting in an XTEN-payload conjugate. Most commonly
used heterobifunctional cross-linkers contain an amine-reactive
group at one end and a sulfhydryl-reactive group at the other end.
Accordingly, these cross-linkers are suitable for use with
cysteine- or lysine-engineered XTEN, or with the alpha-amino group
of the N-terminus of the XTEN. Non-limiting examples of
heterobifunctional cross-linkers are AMAS
(N-(.alpha.-Maleimidoacetoxy)-succinimide ester), BMPS
(N-(.beta.-Maleimidopropyloxy)succinimide ester), EMCS
(N-(.epsilon.-Maleimidocaproyloxy)succinimide ester), GMBS
(N-(.gamma.-Maleimidobutyryloxy)succinimide ester), LC-SMCC
(Succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxy-(6-amidocaproate)), LC-SPDP (Succinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), MBS
(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), SBAP (Succinimdyl
3-(bromoacetamido)propionate), SIA (N-succinimidyl iodoacetate),
SIAB (N-Succinimidyl(4-iodoacetyl)aminobenzoate), SMPB
(Succinimidyl 4-(p-maleimidophenyl) butyrate), SMCC (Succinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate), SM[PEG].sub.2
(NHS-PEG2-Maliemide), SM[PEG].sub.4 (NHS-PEG4-Maliemide),
SM(PEG).sub.6 (NHS-PEG6-Maleimide), SM[PEG].sub.8
(NHS-PEG8-Maliemide), SM[PEG].sub.12 (NHS-PEG12-Maliemide),
SM(PEG).sub.24 (NHS-PEG24-Maleimide), SMPB (Succinimidyl
4-(p-maleimido-phenyl)butyrate), SMPH
(Succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate), SMPT
(4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridyldithio)toluene),
SPDP (N-Succinimidyl 3-(2-pyridyldithio)propionate), Sulfo-EMCS
(N-(.epsilon.-Maleimidocaproyloxy)sulfosuccinimide ester),
Sulfo-GMBS (N-(.gamma.-Maleimidobutryloxy)sulfosuccinimide ester),
Sulfo-KMUS (N-(.kappa.-Maleimidoundecanoyloxy)sulfosuccinimide
ester), Sulfo-LC-SMPT (Sulfosuccinimidyl
6-(.alpha.-methyl-.alpha.-[2-pyridyldithio]-toluamido)hexanoate),
Sulfo-LC-SPDP (Sulfosuccinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), Sulfo-MBS
(m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), Sulfo-SIAB
(Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate), Sulfo-SMCC
(Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
Sulfo-SMPB (Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate). An
example of a heterobifunctional cross-linker that allows covalent
conjugation of amine- and sulfhydryl-containing molecules is
Sulfo-SMCC (SulfoSulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Sulfo-SMCC is a
water soluble analog of SMCC that can be prepared in aqueous
buffers up to 10 mM concentration. The cyclohexane ring in the
spacer arm of this cross-linker decreases the rate of hydrolysis of
the maleimide group compared to similar reagents not containing
this ring. This feature enables XTEN that have been
maleimide-activated with SMCC or Sulfo-SMCC to be lyophilized and
stored for later conjugation to a sulfhydryl-containing molecule.
Thus, in one embodiment, the invention provides an
XTEN-cross-linker having an XTEN having at least about 80% sequence
identity, or at least about 90%, or about 91%, or about 92%, or
about 93%, or about 94%, or about 95%, or about 96%, or about 97%,
or about 98%, or about 99% sequence identity, or is identical to a
sequence or a fragment of a sequence selected from of Table 3, when
optimally aligned, wherein XTEN-cross-linker has one or more
cross-linkers of sulfo-SMCC linked to the .alpha.-amino group of
the XTEN or the .epsilon.-amine of a lysine-engineered XTEN. In
another embodiment, the invention provides an XTEN-cross-linker
having an XTEN having at least about 80% sequence identity, or at
least about 90%, or about 91%, or about 92%, or about 93%, or about
94%, or about 95%, or about 96%, or about 97%, or about 98%, or
about 99% sequence identity, or is identical to a sequence or a
fragment of a sequence selected from of Table 2, when optimally
aligned, wherein the XTEN-cross-linker has one sulfo-SMCC linked to
the amino group of the N-terminus of the XTEN. The foregoing
described heterobifunctional cross-linkers conjugate two molecules
via a single amine and a single cysteine. A special type of
cross-linker was developed for site-specific conjugation to
disulfide bridges in proteins (Balan S. et al. Site-specific
PEGylation of protein disulfide bonds using a three-carbon bridge.
(2007) Bioconjugate Chem. 18, 61-76; Brocchini S. et al. Disulfide
bridge based PEGylation of proteins. (2008) Advanced Drug Delivery
Reviews 60, 3-12). First, the linker is synthesized as an
amine-specific 4-[2,2-bis[p-tolylsulfonyl)methyl]acetyl) benzoic
acid-NHS ester. This molecule can be covalently attached to the
amino group of XTEN yielding XTEN-Bis(sulfone). Incubation of the
latter molecule in 50 mM sodium phosphate buffer, pH 7.8, will
result in elimination of toluene sulfinic acid to generate
XTEN-.alpha.,.beta.-unsaturated .beta.'-monosulfone. The resulting
molecule will react with a disulfide bridge-containing payload
protein in a site-specific manner. In a first step the disulfide
bridge is converted into two thiols by reduction. In a second step,
the XTEN-monosulfone bis-alkylates two cysteines resulting in a
chemically-stable three-carbon bridge. The same
.alpha.,.beta.-unsaturated .beta.'-monosulfone can be used not only
for conjugation to two thiol groups derived from a disulfide bridge
but also for conjugation to polyhistidine tags (Cong Y. et al.
Site-specific PEGylation at histidine tags. (2012) Bioconjugate
Chem. 23, 248-263).
[0343] Conjugation using XTEN-cross-linker compositions with the
sulfo-SMCC is usually performed in a two-step process. In one
embodiment, the amine-containing protein is prepared in conjugation
buffer of, e.g., phosphate-buffered saline (PBS=100 mM sodium
phosphate, 150 mM sodium chloride, pH 7.2) or a comparable amine-
and sulfhydryl-free buffer at pH 6.5-7.5. The addition of EDTA to
1-5 mM helps to chelate divalent metals, thereby reducing disulfide
formation in the sulfhydryl-containing protein. The concentration
of the amine-containing protein determines the cross-linker molar
excess to be used. In general, in protein samples of <1 mg/ml
utilize an 40-80-fold molar excess, protein samples of 1-4 mg/ml
utilize a 20-fold molar excess, and protein samples of 5-10 mg/ml
utilize a 5- to 10-fold molar excess of the cross-linker. The
reaction mixture (amine-containing protein and cross-linker) is
incubated for 30 minutes at room temperature or 2 hours at
4.degree. C. and then the excess cross-linker is removed using a
desalting column equilibrated with conjugation buffer. In the case
of preparing a XTEN-cross-linker, the composition would be held at
that point. In embodiments wherein the XTEN-cross-linker is
conjugated to a payload, the sulfhydryl-containing payload and the
XTEN-cross-linker conjugate are mixed in a molar ratio
corresponding to that desired for the final conjugate (taking into
account the number of expected cross-linkers conjugated to one or
more amino groups per molecule of the XTEN) and consistent with the
single sulfhydryl group that exists on the payload. The reaction
mixture is incubated at room temperature for 30 minutes or 2 hours
at 4.degree. C. Conjugation efficiency can be estimated by SDS-PAGE
followed by protein staining or by appropriate analytical
chromatography technique such as reverse phase HPLC or cation/anion
exchange chromatography.
[0344] In one embodiment, the invention provides XTEN-cross-linker
conjugate compositions created using cross-linkers that are
multivalent, resulting in compositions that have 2, 3, 4, 5, 6 or
more XTEN. In another embodiment, the invention provides
XTEN-cross-linker-payload conjugate compositions created using
cross-linkers that are multivalent, resulting in compositions that
have 2, 3, 4, 5, 6 or more XTEN linked to 1, 2, 3, 4, 5, 6 or more
different payloads. Non-limiting examples of multivalent
trifunctional cross-linkers are "Y-shaped" sulfhydryl-reactive TMEA
(Tris-(2-Maleimidoethyl)amine) and amine-reactive TSAT
(Tris-(succimimidyl aminotricetate). Any combination of reactive
moieties can be designed using a scaffold polymer, either linear or
branched, for multivalent compositions. Examples are shown in FIG.
7, wherein the constructs can have any combination of homo- or
heterofunctional reactive groups. Of particular interest are
trimeric configurations, shown schematically in FIGS. 21-23 and
97-105, and tetrameric configurations, shown in FIGS. 21 and
105-106. Not to be bound by a particular theory, a conjugate
composition having three XTEN linked by a trifunctional linker
(with payloads linked, in turn to XTEN via incorporated lysine or
cysteine residues) can utilize proportionally shorter XTEN for each
"arm" of the construct compared to a monovalent XTEN-payload
composition wherein the same number of payloads are linked to the
incorporated cysteine or lysine or N-terminal amino residues of
each XTEN, and the resulting trimeric XTEN-payload composition will
have a comparable apparent molecular weight and hydrodynamic radius
as the monomeric XTEN-payload composition, yet will have lower
viscosity, aiding administration of the composition to the subject
through small-bore needles, and will provide equal or better
potency from the payloads due to reduced steric hindrance and
increased flexibility of the composition compared to the monomeric
XTEN-payload composition having an equivalent number of XTEN amino
acids.
[0345] In one embodiment, the invention provides a composition
comprising three XTEN linked by a trivalent cross-linker wherein a
solution containing approximately 100 mg/ml of protein of the
composition has a viscosity that is at least about 5 cP, or about 6
cP, or about 7 cP, or about 8 cP, or about 9 cP, or about 10 cP
lower than the corresponding linear XTEN of equal molecular weight
and concentration. In another embodiment, the invention provides a
composition comprising four XTEN linked by a tetravalent
cross-linker wherein a solution containing approximately 100 mg/ml
of protein of the composition has a viscosity that is at least
about 5 cP, or about 6 cP, or about 7 cP, or about 8 cP, or about 9
cP, or about 10 cP lower than the corresponding linear XTEN of
equal molecular weight and concentration.
[0346] Methods to make such compositions using the multivalent
cross-linkers can employ similar reaction conditions as described
herein, above, while an exemplary method and supporting data are
provided in the Examples, below. Additionally, multivalent
cross-linkers can be readily obtained by modification of lysine
oligomers. For instance, the peptide Lys-Lys comprises three amino
groups, one alpha-amino group and two epsilon amino groups at each
Lys residue. These amino groups can be converted into many other
reactive groups by reacting them with Bifunctional cross-linkers
which have one amine-reactive group. For example the reaction of
Lys-Lys with DBCO-NHS cross-linker yields a product that carries
three DBCO groups. The reaction of Lys-Lys with NMal-NHS cross
linker yields product that carries three NMal groups. In similar
way one can obtain tetravalent cross-linkers based on Lys-Lys-Lys
and higher valency cross-linkers by using longer lysine
peptides.
[0347] Cross-linkers can be classified as either "homobifunctional"
or "heterobifunctional" wherein homobifunctional cross-linkers have
two or more identical reactive groups and are used in one-step
reaction procedures to randomly link or polymerize molecules
containing like functional groups, and heterobifunctional
cross-linkers possess different reactive groups that allow for
either single-step conjugation of molecules that have the
respective target functional groups or allow for sequential
(two-step) conjugations that minimize undesirable polymerization or
self-conjugation. In a preferred embodiment, where
XTEN-cross-linkers are prepared and isolated as compositions for
subsequent reaction, the XTEN-cross-linker is linked to a
heterbifunctional cross-linker and has at least one reactive group
available for subsequent reaction.
[0348] In one embodiment, the invention provides XTEN-cross-linkers
and XTEN-payloads that are conjugated utilizing cleavable
cross-linkers with disulfide bonds. Typically, the cleavage is
effected by disulfide bond reducing agents such as
.beta.-mercaptoethanol, DTT, TCEP, however it is specifically
contemplated that such compositions would be cleavable endogenously
in a slow-release fashion by conditions with endogenous reducing
agents (such as cysteine and glutathione). The following are
non-limiting examples of such cross-linkers: APDP
(N-(4-[p-Azidosalicylamido]butyl)-3'-(2'-pyridyldithio)
propionamide), BASED (Bis (.beta.-[4-azidosalicylamido]ethyl)
disulfide), DPDPB (1,4-Di-(3'-[2'pyridyldithio]propionamido)
butane), DSP (Dithiobis(succimidylpropionate) (Lomant's Reagent),
DTBP (Dimethyl 3,3'-dithiobispropionimidate), DTME
(Dithiobis-maleimidoethane), DTSSP (Sulfo-DSP) (3,3'-Dithiobis
(sulfosuccinimidylpropionate)), LC-SPDP (Succinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), PDPH
(3-(2-Pyridyldithio)propionylhydrazide), SDAD (NHS-SS-Diazirine),
SMPT
(4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridyldithio)toluene),
SPDP (N-Succinimidyl 3-(2-pyridyldithio)propionate), Sulfo-LC-SMPT
(Sulfosuccinimidyl
6-(.alpha.-methyl-.alpha.-[2-pyridyldithio]-toluamido)hexanoate),
Sulfo-LC-SPDP (Sulfosuccinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate), Sulfo-SAED
(Sulfosuccimidyl
2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionate),
Sulfo-SAND (Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl
1,3'-dithiopropionate), Sulfo-SDAD (Sulfo-NHS-SS-Diazirine),
Sulfo-SFAD (Sulfosuccinimidyl(perfluoroazidobenzamido)ethyl
1,3'-dithiopropionate. In another embodiment, XTEN-payload
conjugates comprising BSOCOES
(Bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone) can be cleaved
under alkaline conditions. In another embodiment, XTEN-payload
conjugates comprising DST (Disuccinimidyl tartarate) and BMDB (1,4
Bismaleimidyl-2,3-dihydroxybutane) can be cleaved by periodate
oxidation. EGS (Ethylene glycol bis(succinimidylsuccinate)) and
Sulfo-EGS (Ethylene glycol bis (sulfo-succinimidyl succinate)) are
cleaved by hydroxylamine but would be expected to be cleaved
endogenously such that the active payload would be released from
the conjugate.
[0349] In general, the conjugation reagents described above assume
that a cross-linker is reactive with the otherwise stable and inert
groups such as amines, sulfhydryls and carboxyls. In other
embodiments, the invention provides a different approach of
conjugation based on separate modifications of the XTEN and payload
with two functional groups which are stable and inactive toward
biopolymers in general yet highly reactive toward each other.
Several orthogonal reactions have been grouped under the concept of
click chemistry, which provides XTEN-azide/alkyne reactants that
have good stability properties and are therefor particularly suited
as reagents for subsequent conjugation with payloads in a separate
reaction (Kolb H. C., Finn M. G., Sharpless K. B. Click chemistry:
diverse chemical function from a few good reactions. (2001) Angew.
Chem. Int. Ed. Engl. 40(11), 2004-2021). Generally, click chemistry
is used as a reaction concept which embraces reactions involving
(1) alkyne-azide; (2) "ene"-thiol, and (3) aldehyde-hydrazide, and
the invention contemplates use of all three. One example is the
Huisgen 1,3-dipolar cycloaddition of alkynes to azides to form
1,4-disubsituted-1,2,3-triazoles, shown in FIG. 8. Azide and alkyne
moieties can be introduced into peptide/protein or drug payloads or
into XTEN by chemical modification of N-terminal .alpha.-amino
groups, .epsilon.-amino groups of lysine, and sulfhydryl groups of
cysteine. Table 15 provides non-limiting examples of click
chemistry reactants contemplated for use in the making of the
conjugate compositions, wherein one component of the intended
conjugate (and XTEN or a payload) is reacted with a reactant 1 of
the Table and the second component (a payload or an XTEN) is
reacted with a azide reactant 2 of the Table. For example, one
molecule is modified with an alkyne moiety using an amine-reactive
alkyne, such as 3-propargyloxypropanoic acid, NHS ester,
acetylene-PEG4-NHS ester, dibenzylcyclooctyne (DBCO)-NHS ester,
DBCO-PEG4-NHS ester, cyclooctyne (COT)-PEG2-NHS ester, COT-PEG3-NHS
ester, COT-PEG4-NHS ester, COT-PEG2-pentafluorophenyl (PFP) ester,
COT-PEG3-PFP ester, COT-PEG4-PFP ester, BCOT-PEG2-NHS ester,
BCOT-PEG3-NHS ester, BCOT-PEG4-NHS ester, BCOT-PEG2-PFP ester,
BCOT-PEG3-PFP ester, BCOT-PEG4-PFP ester. Alternatively, the
molecule is modified with a sulfhydryl-reactive alkyne such as
acetylene-PEG4-Maleimide, DBCO-Maleimide, or DBCO-PEG4-Maleimide.
The second molecule is modified with azide-PEG2-NHS ester,
azide-PEG3-NHS ester, azide-PEG4-NHS ester, azide-PEG2-PFP ester,
azide-PEG3-PFP ester, azide-PEG4-PFP ester or azide-PEG4-Maleimide.
The azide and alkyne moieties can be used interchangeably; they are
biologically unique, stable and inert towards biological molecules
and aqueous environments. When mixed, the azide and alkyne
reactants form an irreversible covalent bond without any side
reactions (Moses J. E. and Moorhouse A. D. The growing applications
of click chemistry. (2007) Chem. Soc. Rev. 36, 1249-1262;
Breinbauer R. and Kohn M. Azide-alkyne coupling: a powerful
reaction for bioconjugate chemistry. (2003) Chem Bio Chem 4(11),
1147-1149; Rostovtsev V. V., Green L. G., Fokin V. V., Sharpless K.
B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed
regioselective "ligation" of azides and terminal alkynes.(2002)
Angew Chem Int Ed Engl. 41(14), 2596-2599). In one embodiment, the
invention provides a conjugate comprising a first XTEN conjugated
to a second XTEN wherein the first XTEN is linked to a alkyne
reactant 1 from Table 15, the second XTEN is linked to a azide
reactant 2 from Table 15, and then the first XTEN and the second
XTEN are linked under conditions effective to react the alkyne
reactant 1 and the azide reactant 2, resulting in the XTEN-XTEN
conjugate. In another embodiment, the invention provides a
conjugate comprising a first XTEN conjugated to a payload wherein
the XTEN is linked to a alkyne reactant 1 from Table 15, the
payload is linked to a azide reactant 2 from Table 15, and then the
XTEN and the payload are linked under conditions effective to react
the alkyne reactant 1 and the azide reactant 2, resulting in the
XTEN-payload conjugate. In another embodiment, the invention
provides a conjugate comprising a first XTEN conjugated to a
payload wherein the XTEN is linked to a azide reactant 2 from Table
15, the payload is linked to a alkyne reactant 1 from Table 15, and
then the XTEN and the payload are linked under conditions effective
to react the alkyne reactant 1 and the azide reactant 2, resulting
in the XTEN-payload conjugate. In the foregoing embodiments, the
conditions to effect the reactions are those described herein or
are reaction conditions known in the art for the conjugation of
such reactants. The invention also contemplates the various
combinations of the foregoing conjugates; e.g., an XTEN-XTEN
conjugate in which the XTEN are linked by click chemistry reactants
and in which one XTEN further comprises one or more molecules of a
payload conjugated to the XTEN using click chemistry, an XTEN-XTEN
conjugate in which the XTEN are linked by click chemistry reactants
in which one XTEN further comprises one or more molecules of a
first payload conjugated to the XTEN using click chemistry and the
second XTEN further comprises one or more molecules of a second
payload conjugated to the XTEN using click chemistry. Additional
variations on these combinations will be readily apparent to those
of ordinary skill in the art.
TABLE-US-00017 TABLE 15 Alkyne and Azide Click-chemistry Reactants
Alkyne Reactant 1 Attached to: Azide Reactant 2 Attached to:
3-propargyloxy propanoic acid, Amine 3-azide-propionic acid, NHS
Amine NHS ester* ester* acetylene-(oxyethyl).sub.n-NHS Amine
6-azide-hexanoic acid, NHS Amine ester*, where n is 1-10 ester*
dibenzylcyclooctyne (DBCO)- Amine 3-azide-propionic acid, PFP Amine
NHS ester* ester DBCO-(oxyethyl).sub.n- NHS ester*, Amine
6-azide-hexanoic acid, PFP Amine where n is 1-10 ester cyclooctyne
(COT)-NHS ester* Amine azide-(oxyethyl).sub.nNHS ester*, Amine
where n is 1-10 COT-(oxyethyl).sub.n- NHS ester*, Amine
azide-(oxyethyl).sub.n- PFP ester, Amine where n is 1-10 where n is
1-10 COT-(oxyethyl).sub.n- Amine 1-azido-3,6,9,12- Amine
pentafluorophenyl (PFP) ester, tetraoxapentadecan-15-oic where n is
1-10 acid N-hydroxysuccinimide ester BCOT-NHS ester* Amine
azide-(oxyethyl).sub.n- maleimide, Thiol where n is 1-10
BCOT-(oxyethyl).sub.n- NHS ester*, Amine where n is 1-10
BCOT-(oxyethyl).sub.n- Amine pentafluorophenyl (PFP) ester, where n
is 1-10 6-(11,12- Amine didehydrodibenzo[b,f]azocin-
5(6H)-yl)-6-oxohexanoic acid N- hydroxysulfosuccinimide ester
ccetylene-(oxyethyl).sub.n- Thiol maleimide, where n is 1-10
DBCO-maleimide Thiol COT-maleimide Thiol BCOT-maleimide Thiol
*could be either NHS ester or sulfo-NHS ester
[0350] In some embodiments, the XTEN-XTEN conjugates and the
XTEN-payload conjugates are conjugated using thio-ene based click
chemistry that proceeds by free radical reaction, termed thiol-ene
reaction, or anionic reaction, termed thiol Michael addition (see
FIG. 9) (Hoyle C. E. and Bowman C. N. Thiol-ene click chemistry.
(2010) Angew. Chem. Int. Ed 49, 1540-1573). It particular, is
believed that thiol Michael addition is better suited for
XTEN-payload conjugates wherein the payload is a protein (Pounder
R. J. et. al. Metal free thiol-maleimide `Click` reaction as a mild
functionalisation strategy for degradable polymers. (2008) Chem
Commun (Camb). 41, 5158-5160). As at least one molecule needs to
contain a free thiol group, a cysteine-engineered XTEN can be
utilized if the payload does not contain cysteine. Alternatively,
the thiol group can be introduced by chemical modification of
N-terminal .alpha.-amino group or the lysine .epsilon.-amino groups
of either the XTEN or the payload peptide/protein using thiolating
reagents such as 2-iminothiolane (Traut's reagent), SATA
(N-succinimidyl S-acetylthioacetate), SATP (N-succinimidyl
S-acetylthiopropionate), SAT-PEO.sub.4-Ac (N-Succinimidyl
S-acetyl(thiotetraethylene glycol)), SPDP (N-Succinimidyl
3-(2-pyridyldithio)propionate), LC-SPDP (Succinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate). Such methods are
known in the art (Carlsson J. et al. (1978) Biochem. J. 173,
723-737; Wang D. et al. (1997) Bioconjug. Chem. 8, 878-884; Traut
R. R. et al. (1973) Biochemistry 12(17), 3266-3273; Duncan, R. J.
S. et. al. (1983) Anal. Biochem. 132. 68-73; U.S. Pat. No.
5,708,146). The second component of thiol-Michael addition reaction
requires a reagent with electron-deficient carbon-carbon double
bond, such as in (meth)acrylates, maleimides,
.alpha.,.beta.-unsaturated ketones, fumarate esters, acrylonitrile,
cinnamates, and crotonates.
[0351] The N-maleimides are commonly used as sulfhydryl-reactive
functionalities and can be introduced into the payload protein or
the XTEN molecule via N-terminal .alpha.-amino group or Lys
.epsilon.-amino group modification using commercially available
heterobifunctional cross-linkers such as AMAS
(N-(.alpha.-Maleimidoacetoxy)-succinimide ester), BMPS
(N-(.beta.-Maleimidopropyloxy)succinimide ester) and others
described above. The resulting two molecules containing free thiol
and maleimide moieties, respectively, form a stable covalent bond
under mild conditions, resulting in a XTEN-payload linked by
maleimide.
[0352] In other embodiments, XTEN-XTEN conjugates and XTEN-payload
conjugates are created utilizing click chemistry based on reactions
between hydrazides and aldehydes, such as described by Ganguly et
al. and as shown in FIG. 10 (Ganguly T. et al. The
hydrazide/hydrazone click reaction as a biomolecule labeling
strategy for M(CO)3 (M=Re, 99mTc) radiopharmaceuticals. (2011)
Chem. Commun. 47, 12846-12848). For example, an XTEN can be
modified to have a hydrazine or hydrazide that is mixed with a
payload having an aldehyde group to yield the desired XTEN-payload
conjugate. In one embodiment, the invention provides XTEN with at
least one hydrazine or hydrazide introduced in either the
.alpha.-N-terminal amino group or, alternatively one or more lysine
.epsilon.-amino groups are modified to provide an XTEN suitable as
a reagent for conjugation to a target payload as it is considered
to be stable. The resulting bis-arylhydrazones formed from aromatic
hydrazines and aromatic aldehydes are stable to 92.degree. C. and a
wide range of pH values from 2.0-10.0 (Solulink, Inc.,
Protein-Protein Conjugation Kit, Technical Manual, Catalog
#S-9010-1). The leaving group in the reaction is water and no
reducing agents (e.g., sodium cyanoborohydride) are required to
stabilize the bond. Molecules modified with either
hydrazine/hydrazide or aldehyde moieties have good stability in
aqueous environments and remain active without special handling
requirements. The amino group(s) of the XTEN molecule are modified
by NHS-ester/hydrazide, such as SANH (succinimidyl
4-hydrazinonicotinate acetone hydrazone), C6-SANH (C6-Succinimidyl
4-hydrazinonicotinate acetone hydrazone), SHTH (Succinimidyl
4-hydrazidoterephthalate hydrocholoride). In a typical reaction, a
protein is prepared as 1-5 mg/ml solution in modification buffer
(100 mM Phosphate, 150 mM NaCl, pH 7.4) and the modifying agent is
added in a 5- to 20-fold molar excess and the reaction is carried
out for 2 hrs at room temperature. Separately, the payload molecule
is modified with NHS-ester/aldehyde SFB (succinimidyl
4-formylbenzoate) or C6-SFB (C6-Succinimidyl 4-formylbenzoate)
under similar conditions. Both modified molecules are then desalted
into conjugation buffer (100 mM phosphate, 150 mM NaCl, pH 6.0).
The resulting components are mixed together using 1 mole equivalent
of a limiting protein and 1.5-2 mole equivalents of a protein that
can be used in abundance. A catalyst buffer of 100 mM aniline in
100 mM phosphate, 150 mM NaCl, pH 6.0 is added to adjust the final
concentration of aniline to 10 mM and the reaction is carried out
for 2 hrs at room temperature.
[0353] In another embodiment, the XTEN-payload conjugate can be
produced by reaction between an aldehyde and primary amino group
followed by reduction of the formed Schiff base with sodium
borohydride or cyanoborohydride. As a first step in the method, an
XTEN molecule, such as XTEN with a primary .alpha.-amino group or
Lys-containing XTEN with an .epsilon.-amino group, is modified by
NHS-ester/aldehyde SFB (succinimidyl 4-formylbenzoate), C6-SFB
(C6-succinimidyl 4-formylbenzoate) or SFPA (succinimidyl
4-formylphenoxyacetate) using typical amine-NHS chemistry in an
amine-free coupling buffer such as 0.1M sodium phosphate, 0.15M
NaCl, pH 7.2. The resulting modified aldehyde-XTEN can either be
held at this point as an XTEN-cross-linker composition or can be
used as a reagent to create an XTEN-payload conjugate. To make the
XTEN-payload, the modified aldehyde-XTEN is mixed with a payload
with a reactive amino-group and a mild reducing agent such as
20-100 mM sodium cyanoborohydride. The reaction mixture is
incubated up to 6 hours at room temperature or overnight at
4.degree. C. Unreacted aldehyde groups are then blocked with 50-500
mM Tris.HCl, pH 7.4 and 20-100 mM sodium cyanoborohydride,
permitting separation of the conjugated purified XTEN-payload.
[0354] In other embodiments, the invention provides XTEN-payload
conjugates comprising peptides or protein payloads wherein the
payload is conjugated via chemical ligation based on the reactivity
of the peptide/protein C-terminal acyl azide of the payload. As an
example, when the peptide or protein is produced using solid-phase
peptide synthesis (SPPS) with hydroxymethylbenzoic acid (HMBA)
resin, the final peptide can be cleaved from the resin by a variety
of nucleophilic reagents to give access to peptides with diverse
C-terminal functionalities. In one embodiment, the method includes
hydrazinolysis of the peptidyl/protein resins to yield peptide or
protein hydrazides. Nitrosation of resulting acyl hydrazides with
sodium nitrite or tert-butyl nitrite in dilute hydrochloric acid
then results in formation of acyl azides. The resulting carbonyl
azide (or acyl azide) is an activated carboxylate group (esters)
that can react with a primary amine of an XTEN to form a stable
amide bond, resulting in the XTEN-payload conjugate. In alternative
embodiments, the primary amine could be the .alpha.-amine of the
XTEN N-terminus or one or more .epsilon.-amine of engineered lysine
residues in the XTEN sequence. In the conjugation reaction, the
azide function is the leaving group, shown in FIG. 11. The
conjugation reaction with the amine groups occurs by attack of the
nucleophile at the electron-deficient carbonyl group (Meienhofer,
J. (1979) The Peptides: Analysis, Synthesis, Biology. Vol. 1,
Academic Press: N.Y.; ten Kortenaar P. B. W. et. al. Semisynthesis
of horse heart cytochrome c analogues from two or three fragments.
(1985) Proc. Natl. Acad Sci. USA 82, 8279-8283)
[0355] In yet other embodiments, the invention provides
XTEN-cross-linker and XTEN-payload conjugates in which the
conjugation is performed by orthogonal protein ligation in which an
initial chemoselective capture is followed by an intramolecular
acyl rearrangement, as shown in FIG. 12. The chemoselective capture
requires a nucleophile or electrophile proximally placed at an
N-terminal amine and another compatible electrophile or nucleophile
also proximally located at a C-terminal carboxylic ester. In the
embodiment, it is specifically contemplated that the XTEN can serve
as either Protein1 or Protein2 in FIG. 12. Thus, in alternative
embodiments, the XTEN can be reacted with appropriate reagents to
produce the thioester on the C-terminus or introduce a cysteine on
the N-terminus to produce alternative XTEN-cross-linker
compositions. In using the foregoing XTEN-cross-linker conjugates
to make the XTEN-payload, the chemoselective capture of the
nucleophile and electrophile pair forming an ester or a thioester
brings the N-terminal amino group and C-terminal ester of the
respective reactants into such a close proximity to permit a
spontaneous intramolecular acyl transfer to form an amide bond.
Most orthogonal ligation reactions do not require protection of
side-chain groups and take place under mild conditions that are
compatible with biological environments (Tam J. P., Xu J., Eom K.
D. Methods and strategies of peptide ligation. (2001) Biopolymers
(Peptide Science) 60, 194-205).
[0356] In another embodiment, the conjugates can be created by a
method reaction known as Native Chemical Ligation (NCL) involving a
C-terminal thioester as an electrophile and N-terminal cysteine as
a nucleophile. The result of this reaction is a native amide bond
at the ligation site of the XTEN-payload conjugate (Dawson P. E.,
Muir T. W., Clark-Lewis I., Kent S. B. Synthesis of proteins by
native chemical ligation. (1994) Science 266, 776-779; Tam J. P.;
Lu Y.-A.; Liu C. F.; Shao, J. Peptide synthesis using unprotected
peptides through orthogonal coupling methods. (1995) Proc. Natl.
Acad. Sci. USA, 92, 12485-12489; Johnson, E. C. B.; Kent, S. B. H.
J. Insights into the mechanism and catalysis of the native chemical
ligation reaction. (2006) J. Am. Chem. Soc. 128, 6640-6646; Kent S.
B. (2009) Total chemical synthesis of proteins. (2009) Chem. Soc.
Rev. 38:338-351). The first amino acid of the C-terminal component
in NCL reaction (shown as Protein2 in FIG. 12) is cysteine. Such a
protein can be XTEN with cysteine in the first position or any
other protein prepared by conventional recombinant protein
biosynthesis, including a peptide/protein payload. The N-terminal
component (shown as Payload in FIG. 13) is prepared as C-terminal
thioester by chemical synthesis. Examples of thioester synthesis
methods are known and available in the art, such as those
described, for example, in Li X., Kawakami T., Aimoto S., Direct
preparation of peptide thioesters using an Fmoc solid-phase method.
(1998) Tetrahedron Lett., 39, 8660-8672); Ingenito R., Bianchi E.,
Fattori D., Pessi A. Solid-phase synthesis of peptide C-terminal
thioesters by Fmoc/tBu chemistry. (1999) J. Am. Chem. Soc., 121,
11369-11374); Sewing A., Hilvert D. Fmoc-compatible solid-phase
peptide synthesis of long C-terminal peptide thioesters. (2001)
Angew. Chem. Int. Ed 40, 3395-3398; Brask J., Albericio F., Jensen
K. J., Fmoc solid-phase synthesis of peptide thioesters by masking
as trithioorthoesters. (2003) Org. Lett., 2003, 5, 2951-2953;
Ollivier N., Behr J.-B., El-Mahdi O., Blanpain A., Melnyk O.
Fmoc-solid-phase synthesis of peptide thioesters using an
intramolecular N, S-acyl shift. (2005) Org. Lett., 7, 2647-2650.
Usually, .alpha.-alkylthioesters are preferred because of ease of
preparation and storage. However, because they are rather
unreactive, the ligation reaction is catalyzed by in situ
transthioesterification with thiol additives, with the most common
thiol catalysts being 2-mercaptoethanesulfonate (MESNa) or
4-mercaptophenylacetic acid (MPAA). Chemical conjugation is
typically complete in few hours and with high yields. While all 20
natural amino acids are suitable as the last residue of N-terminal
component, the highest ligation rates were reported for glycine and
histidine, making XTEN particularly suited for this reaction as the
exemplary XTEN of Table 2 are nearly all glycine N-terminal
polypeptides (Hackeng T. M. et al. Protein synthesis by native
chemical ligation: expanded scope by using straightforward
methodology. (1999) Proc. Natl. Acad. Sci. USA 96, 10068-10073). In
other embodiments of this conjugation method, orthogonal ligation
reactions include: (1) C-terminal thioacid with N-terminal BrAla or
N-terminal aziridine (Tam J. P.; Lu Y.-A.; Liu C. F.; Shao, J.
Peptide synthesis using unprotected peptides through orthogonal
coupling methods. (1995) Proc. Natl. Acad Sci. USA, 92,
12485-12489); (2) C-terminal thioacid with N-terminal
Cys-perthioester (Liu, C. F., Rao, C., Tam, J. P. (1996)
Tetrahedron Lett., 37, 933-936); (3) C-terminal thioester with
N-terminal Homocysteine (Tam J. P., Yu Q. Methionine ligation
strategy in the biomimetic synthesis of parathyroid hormones.
(1998) Biopolymers 46(5), 319-327); and (4) C-terminal thioacid and
N-terminal His (Zhang L., Tam J. P. (1997) Tetrahedron. Lett. 38,
3-6). In the method, the preparation of C-terminal thioesters by
chemical synthesis constrains the size of N-terminal component in
NCL reaction. However, use of expressed protein ligation (EPL)
methodology overcomes size limitations of peptide .alpha.-thioester
imposed by the need of chemical synthesis (Muir T. W.; Sondhi D.;
Cole P. A. Expressed protein ligation: a general method for protein
engineering. (1998) Proc. Natl. Acad. Sci. USA 95, 6705-6710; Muir
T. W. Semisynthesis of proteins by expressed protein ligation.
(2003) Annu. Rev. Biochem. 72, 249-289). The EPL method is based on
protein splicing, the process in which a protein undergoes an
intramolecular rearrangement resulting in the extrusion of an
internal sequence (intein) and the joining of the lateral sequences
(exteins). The latter process involves a formation of ester or
thioester intermediates. In practicing the invention, the
commercially available Escherichia coli protein expression vectors
allows one to produce proteins of interest, such as XTEN, expressed
in frame fused with an intein-chitin binding domain (CBD) sequence.
In the method, the fused protein undergoes an N-S shift when the
side chain of the first cysteine residue of the intein portion of
the precursor protein nucleophilically attacks the peptide bond of
the residue immediately upstream (that is, for example, the final
residue of XTEN) to form a linear thioester intermediate, as shown
in FIG. 13. The chemical ligation step is initiated by incubating
the protein with thiophenol (or other thiol catalysts such as MESNa
and MPAA) and a cysteine-containing synthetic peptide or protein.
This results in the in situ generation of a highly reactive phenyl
.alpha.-thioester derivative of, for example, the XTEN protein that
then rapidly ligates with the synthetic peptide/protein payload to
result in the desired XTEN-payload conjugate. In another
embodiment, an XTEN-thioester intermediate can be cleaved by 50 mM
2-mercaptoethanesulfonic acid (MESNa) in 20 mM Na-HEPES, pH 8.5,
50-1000 mM NaCl, and 1 mM EDTA (optional) and the resulting
MESNa-tagged protein can be purified and stored -80.degree. C. in 5
mM Bis Tris, pH 6.5, 250 mM NaCl until use as an XTEN-cross-linker
conjugate for NCL reaction as the N-terminal component in the above
described conjugation. The C-terminal component can be a payload
with either a natural or synthetic peptide/protein with an
N-terminal cysteine.
[0357] In yet other embodiments, the invention provides
XTEN-cross-linker and XTEN-payload conjugates in which the
conjugation between the XTEN and payload is performed by traceless
Staudinger ligation, like Native Chemical Ligation (NCL), resulting
in a native amide bond at the ligation site. In an advantage to the
method, a cysteine is not required at the ligation juncture (Saxon,
E.; Armstrong, C. R.; Bertozzi, C. R. A "traceless" Staudinger
ligation for the chemoselective synthesis of amide bonds. (2000)
Org. Lett. 2, 21412143; Nilsson, B. L.; Kiessling, L. L.; Raines,
R. T. Staudinger ligation: a peptide from a thioester and azide.
(2000) Org. Lett. 2, 1939-1941). Instead, an N-terminal Protein 1
is prepared as a C-terminal thioester using
diphenylphosphinemethanethiol (see FIG. 14), while a C-terminal
Protein 2 is prepared as an N-terminal azide that can be generated
via a diazo-transfer reaction (Cavender C. J.; Shiner V. J., Jr.
(1972) J. Org. Chem. 22, 3567-3569; Lundquist J. T., IV, Pelletier
J. C. Improved solid-phase peptide synthesis method utilizing
alpha-azide-protected amino acids. (2001) Org. Lett. 3, 781-783). A
phosphine residue reacts with the azide of Protein2 to form an
iminophosphorane after elimination of nitrogen (Staudinger
reaction). The resulting iminophosphorane with its highly
nucleophilic nitrogen atom can also be regarded as an aza-ylide.
The nucleophilic nitrogen atom of the aza-ylide then attacks the
carbonyl group of Protein1, cleaving the thioester. It is
specifically intended that either XTEN or the payload can be either
Protein1 or Protein2 in this reaction. Hydrolysis of the rearranged
XTEN-payload product finally produces a native amide and liberates
phosphine component as phosphine(V) oxide.
Bis(p-dimethylaminoethylphenyl)phosphinomethanethiol, a
water-soluble variant of diphenylphosphinemethanethiol, mediates
the rapid ligation of equimolar substrates in water (Tam, A.;
Soellner, M. B.; Raines, R. T. Water-soluble phosphinothiols for
traceless Staudinger ligation and integration with Expressed
Protein Ligation. (2007) J. Am. Chem. Soc., 129, 1142111-430).
[0358] In another embodiment, the invention provides XTEN-payload
conjugates prepared by enzymatic ligation. Transglutaminases are
enzymes that catalyze the formation of an isopeptide bond between
the .gamma.-carboxamide group of glutamine of a payload peptide or
protein and the .epsilon.-amino group of a lysine in a
lysine-engineered XTEN, thereby creating inter- or intramolecular
cross-links between the XTEN and payload (see FIG. 15), resulting
in the composition (Lorand L, Conrad S. M. Transglutaminases.(1984)
Mol. Cell Biochem. 58(1-2), 9-35). Non-limiting examples of enzymes
that have been successfully used for ligations are factor XIIIa
(Schense J. C., Hubbell J. A. Cross-linking exogenous bifunctional
peptides into fibrin gels with factor XIIIa. (1999) Bioconjug.
Chem. 10(1):75-81) and tissue transglutaminase (Collier J. H.,
Messersmith P. B. Enzymatic modification of self-assembled peptide
structures with tissue transglutaminase. (2003) Bioconjug. Chem.
14(4), 748-755; Davis N. E., Karfeld-Sulzer L. S., Ding S., Barron
A. E. Synthesis and characterization of a new class of cationic
protein polymers for multivalent display and biomaterial
applications. (2009) Biomacromolecules 10 (5), 1125-1134). The
glutamine substrate sequence GQQQL (SEQ ID NO: 575) is known to
have high specificity toward tissue transglutaminase (Hu B. H.,
Messersmith P. B. Rational design of transglutaminase substrate
peptides for rapid enzymatic formation of hydrogels.(2003) J. Am.
Chem. Soc. 125(47), 14298-14299). Tissue transglutaminase sequence
specificity was less stringent for an acyl acceptor (lysine) than
for acyl donor (glutamine) (Greenberg C. S., Birckbichler P. J.,
Rice R. H. Transglutaminases: multifunctional cross-linking enzymes
that stabilize tissues. (1991) FASEB J. 1991, 5, 3071-3077).
[0359] In an alternative embodiment of an enzymatically-created
XTEN-payload composition, the sortase A transpeptidase enzyme from
Staphylococcus aureus is used to catalyze the cleavage of a short
5-amino acid recognition sequence LPXTG (SEQ ID NO: 24) between the
threonine and glycine residues of Protein1, and subsequently
transfers the acyl-fragment to an N-terminal oligoglycine
nucleophile of Protein1 (see FIG. 16). By functionalizing the
Protein2 to contain the oligoglycine, it is possible to
enzymatically conjugate the two proteins in a site-specific fashion
to result in the desired XTEN-payload composition. The
(poly)peptide bearing the sortase recognition site (LPXTG) (SEQ ID
NO: 24) can be readily made using standard molecular biology
cloning protocols. It is convenient to introduce glutamic acid in
the X position of the recognition site, as this residue is commonly
found in natural substrates of sortase A (Boekhorst J., de Been M.
W., Kleerebezem M., Siezen R. J. Genome-wide detection and analysis
of cell wall-bound proteins with LPxTG-like sorting motifs. (2005)
J. Bacteriol. 187, 4928-4934). A high level of transacylation can
be achieved by placing the sortase cleavage site both at the
C-terminus of the substrate (Popp M. W., Antos J. M., Grotenbreg G.
M., Spooner E., Ploegh H. L. Sortagging: A versatile method for
protein labeling. (2007) Nat. Chem. Biol. 311, 707-708) and in
flexible loops (Popp M. W., Artavanis-Tsakonas K., Ploegh H. L.
Substrate filtering by the active-site crossover loop in UCHL3
revealed by sortagging and gain-of-function mutations. (2009) J.
Biol. Chem. 284(6), 3593-3602). For proteins labeled at the
C-terminus, it is important that the glycine in the minimal LPETG
(SEQ ID NO: 576) tag is not placed at the C-terminus; it must be in
a peptide linkage with at least one further C-terminal amino acid.
In addition, better linkage is achieved by adding an extra glycine
to the C-terminus of the cleavage site to yield LPETGG (SEQ ID NO:
577) (Pritz S., Wolf Y., Kraetke O., Klose J., Bienert M.,
Beyermann M. Synthesis of biologically active peptide nucleic
acid-peptide conjugates by sortase-mediated ligation. (2007) J.
Org. Chem. 72, 3909-3912; Tanaka T., Yamamoto T., Tsukiji S.,
Nagamune T. Site-specific protein modification on living cells
catalyzed by sortase. (2008) Chembiochem 95, 802-807). Nucleophiles
compatible with sortase-mediated transpeptidation have the single
structural requirement of a stretch of glycine residues with a free
amino terminus. Successful transpeptidation can be achieved with
nucleophiles containing anywhere from one to five glycines;
however, in a preferred embodiment, a maximum reaction rate is
obtained when two or three glycines are present.
[0360] While the various embodiments of conjugation chemistry have
been described in terms of protein-protein conjugations, it is
specifically intended that in practicing the invention, the payload
moiety of the XTEN-payload conjugates can be a small molecule drug
in those conjugation methods applicable to functional groups like
amines, sulfhydryls, carboxyl that are present in the target small
molecule drugs. It will be understood by one of ordinary skill in
the art that one can apply even more broad chemical techniques
compared to protein and peptides whose functionalities are usually
limited to amino, sulfhydryl and carboxyl groups. Drug payloads can
be conjugated to the XTEN through functional groups including, but
not limited to, primary amino groups, aminoxy, hydrazide, hydroxyl,
thiol, thiolate, succinate (SUC), succinimidyl succinate (SS),
succinimidyl propionate (SPA), succinimidyl butanoate (SBA),
succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC),
N-hydroxysuccinimide (NHS), p-nitrophenyl carbonate (NPC). Other
suitable reactive functional groups of drug molecule payloads
include acetal, aldehydes (e.g., acetaldehyde, propionaldehyde, and
butyraldehyde), aldehyde hydrate, alkenyl, acrylate, methacrylate,
acrylamide, active sulfone, acid halide, isocyanate,
isothiocyanate, maleimide, vinylsulfone, dithiopyridine,
vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate,
tosylate, and tresylate.
[0361] In another embodiment, the drug payloads can also be
conjugated to XTEN-cross-linker conjugates using a heterocycle ring
system in which one or more ring atoms is a heteroatom, e.g. a
nitrogen, an oxygen, a phosphorus or a sulfur atom. The heterocycle
group comprises at least 1 to as many as 20 carbon atoms and 1 to 3
heteroatoms selected from N, O, P, and S. In the embodiment, the
heterocycle may be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S)
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are
described in Paquette, Leo A. "Principles of Modern Heterocyclic
Chemistry", W. A. Benjamin, New York, (1968); "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28. Non-limiting examples of heterocycles that may be found
in drugs suitable for conjugation include pyridyl, dihydroypyridyl,
tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl,
sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl,
thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl,
thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl,
benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,
2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,
bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl.
[0362] In some embodiments of the XTEN-payload conjugates with
drugs as the payload, the drug molecules are attached to lysine- or
cysteine engineered XTEN (such as the sequences of Table 3) by
cross-linkers having two reactive sites for binding to the drug and
the XTEN. Preferred cross-inker groups are those that are
relatively stable to hydrolysis in the circulation, are
biodegradable and are nontoxic when cleaved from the conjugate. In
addition, the use of cross-linkers can provide the potential for
conjugates with an even greater flexibility between the drug and
the XTEN, or provide sufficient space between the drug and the XTEN
such that the XTEN does not interfere with the binding between the
pharmacophore and its binding site. In one embodiment, a
cross-linker has a reactive site that has an electrophilic group
that is reactive to a nucleophilic group present on an XTEN.
Preferred nucleophiles include thiol, thiolate, and primary amine.
The heteroatom of the nucleophilic group of a lysine- or
cysteine-engineered XTEN is reactive to an electrophilic group on a
cross-linker and forms a covalent bond to the cross-linker unit,
resulting in an XTEN-cross-linker conjugate. Useful electrophilic
groups for cross-linkers include, but are not limited to, maleimide
and haloacetamide groups, and provide a convenient site for
attachment to the XTEN. In another embodiment, a cross-linker has a
reactive site that has a nucleophilic group that is reactive to an
electrophilic group present on a drug such that a conjugation can
occur between the XTEN-cross-linker and the payload drug, resulting
in an XTEN-drug conjugate. Useful electrophilic groups on a drug
include, but are not limited to, hydroxyl, thiol, aldehyde, alkene,
alkane, azide and ketone carbonyl groups. The heteroatom of a
nucleophilic group of a cross-linker can react with an
electrophilic group on a drug and form a covalent bond. Useful
nucleophilic groups on a cross-linker include, but are not limited
to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,
hydrazine carboxylate, and arylhydrazide. The electrophilic group
on a drug provides a convenient site for attachment to a
cross-inker.
[0363] In a particular embodiment, the conjugation of drugs to the
lysine epsilon amino group of a subject lysine-engineered XTEN
makes use of a reactive drug-N-hydroxylsuccinimide reactant, or
esters such as drug-succinimidyl propionate, or drug-succinimidyl
butanoate or other drug-succinimide conjugates. Alternatively,
lysine residues of the subject lysine-engineered XTEN may be used
to introduce free sulfhydryl groups through reaction with
2-iminothiolane. Alternatively, targeting substance lysines of
subject lysine-engineered XTEN may be linked to a
heterobifunctional reagent having a free hydrazide or aldehyde
group available for conjugation with an active drug agent. Reactive
esters can conjugate at physiological pH, but less reactive
derivatives typically require higher pH values. Low temperatures
may also be employed if a labile protein payload is being used.
Under low temperature conditions, a longer reaction time may be
used for the conjugation reaction.
[0364] In another particular embodiment, the invention provides
XTEN-payload conjugates with an amino group conjugation with lysine
residues of a subject lysine-engineered XTEN wherein the
conjugation is facilitated by the difference between the pKa values
of the .alpha.-amino group of the N-terminal amino acid
(approximately 7.6 to 8.0) and pKa of the .epsilon.-amino group of
lysine (approximately 10). Conjugation of the terminal amino group
often employs reactive drug-aldehydes (such as drug-propionaldehyde
or drug-butylaldehyde), which are more selective for amines and
thus are less likely to react with, for example, the imidazole
group of histidine. In addition, amino residues are reacted with
succinic or other carboxylic acid anhydrides, or with
N,N'-Disuccinimidyl carbonate (DSC), N,N'-carbonyl diimidazole
(CDI), or p-nitrophenyl chloroformate to yield the activated
succinimidyl carbonate, imidazole carbamate or p-nitrophenyl
carbonate, respectively. Derivatization with these agents has the
effect of reversing the charge of the lysinyl residues. Conjugation
of a drug-aldehyde to the terminal amino group of a subject XTEN
typically takes place in a suitable buffer performed at a pH which
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and that of the
.alpha.-amino group of the N-terminal residue of the protein. In
the method of the embodiment, the reaction for coupling uses a pH
in the range of from about pH 7 up to about 8. Useful methods for
conjugation of the lysine epsilon amino group have been described
in U.S. Pat. Nos. 4,904,584 and 6,048,720.
[0365] The person with ordinary skill in the art will be aware that
the activation method and/or conjugation chemistry to be used in
the creation of the XTEN-payload conjugates depends on the reactive
groups of the XTEN polypeptide as well as the functional groups of
the drug moiety (e.g., being amino, hydroxyl, carboxyl, aldehyde,
sulfhydryl, alkene, alkane, azide, etc), the functional group of
the drug-cross-linker reactant, or the functional group of the
XTEN-cross-linker reactant. The drug conjugation may be directed
towards conjugation to all available attachment groups on the
engineered XTEN polypeptide such as the specific engineered
attachment groups on the incorporated cysteine residues or lysine
residues. In order to control the reactants such that the
conjugation is directed to the appropriate reactive site, the
invention contemplates the use of protective groups during the
conjugation reaction. A "protecting group" is a moiety that
prevents or blocks reaction of a particular chemically reactive
functional group in a molecule under certain reaction conditions.
The protecting group will vary depending upon the type of
chemically reactive group being protected as well as the reaction
conditions to be employed, as well as the presence of additional
reactive groups in the molecule. Non-limiting examples of
functional groups which may be protected include carboxylic acid
groups, hydroxyl groups, amino groups, thiol groups, and carbonyl
groups. Representative protecting groups for carboxylic acids and
hydroxyls include esters (such as a p-methoxybenzyl ester), amides
and hydrazides; for amino groups, carbamates (such as
tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and
esters; for thiol groups, thioethers and thioesters; for carbonyl
groups, acetals and ketals; and the like. Such protecting groups
are well-known to those skilled in the art and are described, for
example, in T. W. Greene and G. M. Wuts, Protecting Groups in
Organic Synthesis, Third Edition, Wiley, New York, 1999, and
references cited therein. The conjugation may be achieved in one
step or in a stepwise manner (e.g., as described in WO 99/55377),
such as through addition of a reaction intermediate cross-linker,
using the cross-linkers disclosed herein or those known in the art
to be useful for conjugation to cysteine or lysine residues of
polypeptides to be linked to reactive functional groups on drug
molecules.
[0366] In some embodiments of the invention, the method for
conjugating a cross-linker to a cysteine-engineered XTEN may
provide that the XTEN is pre-treated with a reducing agent, such as
dithiothreitol (DTT) to reduce any cysteine disulfide residues to
form highly nucleophilic cysteine thiol groups (--CH.sub.2SH). The
reducing agent is subsequently removed by any conventional method,
such as by desalting. The reduced XTEN thus reacts with drug-linker
compounds, or cross-linker reagents, with electrophilic functional
groups such as maleimide or .alpha.-halo carbonyl, according to,
for example, the conjugation method of Klussman et al. (2004)
Bioconjugate Chemistry 15(4), 765-773. Conjugation of a
cross-linker or a drug to a cysteine residue typically takes place
in a suitable buffer at pH 6-9 at temperatures varying from
4.degree. C. to 25.degree. C. for periods up to about 16 hours.
Alternatively, the cysteine residues can be derivatized. Suitable
derivatizing agents and methods are well known in the art. For
example, cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as iodoacetic
acid or iodoacetamide, to give carboxymethyl or carboxyamidomethyl
derivatives. Cysteinyl residues also are derivatized by reaction
with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(4-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0367] In some instances, the conjugation is performed under
conditions aiming at reacting as many of the available XTEN
attachment groups as possible with drug or drug-linker molecules.
This is achieved by means of a suitable molar excess of the drug in
relation to the polypeptide. Typical molar ratios of activated drug
or drug-linker molecules to polypeptide are up to about 1000-1,
such as up to about 200-1 or up to about 100-1. In some cases, the
ratio may be somewhat lower, however, such as up to about 50-1,
10-1 or 5-1. Equimolar ratios also may be used.
[0368] In the embodiments, the XTEN-payload conjugates of the
disclosure retain at least a portion of the pharmacologic activity
compared to the corresponding payload not linked to XTEN. In one
embodiment, the XTEN-payload retains at least about 1%, or at least
about 5%, or at least about 10%, or at least about 20%, or at least
about 30%, or at least about 40%, or at least about 50%, or at
least about 60%, or at least about 70%, or at least about 80%, or
at least about 90%, or at least about 95% of the pharmacologic
activity of the payload not linked to XTEN.
[0369] In one embodiment, XTEN-payload conjugates can be designed
to release the payload in the body by unspecific or enzymatic
hydrolysis of the linker, including disulfide bond reduction,
pH-dependent release, or by exogenous or endogenous proteases,
including the proteases of Table 9. Macromolecules can be taken up
by the cell either through receptor-mediated endocytosis,
adsorptive endocytosis or fluid phase endocytosis (Jain R. K.
Transport of molecules across tumor vasculature. (1987) Cancer
Metastasis Rev. 6(4), 559-593; Jain R. K. Transport of molecules,
particles, and cells in solid tumors. (1999) Ann. Rev. Biomed. Eng.
1, 241-263; Mukherjee S., Ghosh R. N., Maxfield F. R. Endocytosis.
(1997) Physiol. Rev. 77(3), 759-803). Upon cellular uptake of
XTEN-payload, the payload can be released by low pH values in
endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), as well as by
lysosomal enzymes (e.g., esterases and proteases). Example of
acid-sensitive cross-linker is 6-maleimidodocaproyl hydrazone which
can be coupled to thiol-bearing carriers. The hydrazone linker is
rapidly cleaved at pH values <5 allowing a release of the
payload in the acidic pH of endosomes and lysosomes following
internalization of the conjugate (Trail P. A. et al. Effect of
linker variation on the stability, potency, and efficacy of
carcinoma-reactive BR64-doxorubicin immunoconjugates. (1997) Cancer
Res. 57(1), 100-105; Kratz F. et al. Acute and repeat-dose toxicity
studies of the (6-maleimidocaproyl)hydrazone derivative of
doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the
anticancer agent doxorubicin. (2007) Hum. Exp. Toxicol. 26(1),
19-35). Clinically approved mAb-drug conjugate, gemtuzumab
ozogamicin (Mylotarg.TM.) is a drug-antibody conjugate containing a
humanized mAb P67.6 against CD33, linked chemically to the
cytotoxic antibiotic agent calicheamicin. The linker between the
antibody and the drug incorporates two labile bonds: a hydrazone
and a sterically hindered disulfide. It has been shown that the
acid-sensitive hydrazone bond is the actual cleavage site (Jaracz
S., Chen J., Kuznetsova L. V., Ojima I. Recent advances in
tumor-targeting anticancer drug conjugates. (2005) Bioorg. Med.
Chem. 13(17), 5043-5054).
[0370] For those XTEN-payload conjugates in which the payload is
linked by a disulfide bond, the payload can be released from XTEN
by reduction of disulfide bond within the labile linker. For
example, huN901-DM1 is a tumor-activated immunotherapeutic prodrug
developed by ImmunoGen, Inc. for the treatment of small cell lung
cancer. The prodrug consists of humanized anti-CD56 mAb (huN901)
conjugated with microtubule inhibitor maytansinoid DM1. An average
of 3.5-3.9 molecules of DM1 are bound to each antibody via hindered
disulfide bonds. Although the disulfide link is stable in blood, it
is cleaved rapidly on entering the cell targeted by huM901, thus
releasing active DM1 (Smith S. V. Technology evaluation:
huN901-DM1, ImmunoGen. (2005) Curr. Opin. Mol. Ther. 7(4),
394-401). DM1 has been also coupled to Millennium Pharmaceuticals
MLN-591, an anti-prostate-specific membrane antigen mAb. DM1 is
linked to the antibody via a hindered disulfide bond that provides
serum stability at the same time as allowing intracellular drug
release on internalization (Henry M. D. et al. A prostate-specific
membrane antigen-targeted monoclonal antibody-chemotherapeutic
conjugate designed for the treatment of prostate cancer. (2004)
Cancer Res. 64(21), 7995-8001).
[0371] Release of the payload from the carrier XTEN can be achieved
by creating compositions using short cleavable peptides as linkers
between the payload and XTEN. Example of the conjugate assessed
clinically is doxorubicin-HPMA (N-(2-hydroxypropyl)methacrylamide)
conjugate in which doxorubicin is linked through its amino sugar to
the HPMA copolymer via a tetrapeptide spacer GlyPheLeuGly (SEQ ID
NO: 578) that is cleaved by lysosomal proteases, such as cathepsin
B (Vasey P. A. et al. Phase I clinical and pharmacokinetic study of
PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]:
first member of a new class of chemotherapeutic agents-drug-polymer
conjugates. (1999) Clin. Cancer Res. 5(1), 83-94). Other examples
of carrier-drug conjugates with peptide linkers that reached
clinical stage of development are macromolecular platinum
complexes. Two HPMA-based drug candidates consisted of a HPMA
copolymer backbone to which the complexing aminomalonate platinum
complexes were bound through cathepsin B-cleavable peptide spacer
GlyPheLeuGly (SEQ ID NO: 578) or tripeptide spacer GlyGlyGly
(Rademaker-Lakhai J. M. et al. A Phase I and pharmacological study
of the platinum polymer AP5280 given as an intravenous infusion
once every 3 weeks in patients with solid tumors. (2004) Clin.
Cancer Res. 10(10), 3386-3395; Sood P. et al. Synthesis and
characterization of AP5346, a novel polymer-linked
diaminocyclohexyl platinum chemotherapeutic agent. (2006)
Bioconjugate Chem. 17(5), 1270-1279).
[0372] A highly selective method was developed to target prostate
cancer via prostate-specific antigen (PSA) protease which is almost
exclusively expressed in prostate tissue and prostate carcinomas. A
novel albumin-binding prodrug of paclitaxel,
EMC-ArgSerSerTyrTyrSerLeu-PABC-paclitaxel (SEQ ID NO: 579) (EMC:
.epsilon.-maleimidocaproyl; PABC: p-aminobenzyloxycarbonyl) was
synthesized. This prodrug was water soluble and was bound to
endogenous and exogenous albumin. Albumin-bound form of the prodrug
was cleaved by PSA releasing the paclitaxel-dipeptide
Ser-Leu-PABC-paclitaxel. Due to the incorporation of a PABC
self-eliminating linker, this dipeptide was rapidly degraded to
liberate paclitaxel as a final cleavage product (Elsadek B. et al.
Development of a novel prodrug of paclitaxel that is cleaved by
prostate-specific antigen: an in vitro and in vivo evaluation
study. (2010) Eur. J. Cancer 46(18), 3434-3444).
[0373] Self-immolative spacers have gained significant interest due
to their utility in prodrug delivery systems. Several reports
described linear self-eliminating systems or dendrimeric structures
which can release all of their units through a domino-like chain
fragmentation, initiated by a single cleavage event (Haba K. et al.
Single-triggered trimeric prodrugs. (2005) Angew. Chem., Int. Ed.
44, 716-720; Shabat D. Self-immolative dendrimers as novel drug
delivery platforms. (2006) J. Polym. Sci., Part A: Polym. Chem. 44,
1569-1578. Warnecke A., Kratz F. 2,4-Bis(hydroxymethyl)aniline as a
building block for oligomers with self-eliminating and multiple
release properties. (2008) J. Org. Chem. 73, 1546-1552; Sagi A. et
al. Self-immolative polymers. (2008) J. Am. Chem. Soc. 130,
5434-5435). In one study, a self-immolative dendritic prodrug with
four molecules of the anticancer agent camptothecin and two
molecules of PEG5000 was designed and synthesized. The prodrug was
effectively activated by penicillin-G-amidase under physiological
conditions and free camptothecin was released to the reaction media
to cause cell-growth inhibition (Gopin A. et al. Enzymatic
activation of second-generation dendritic prodrugs: conjugation of
self-immolative dendrimers with poly(ethylene glycol) via click
chemistry. (2006) Bioconjugate Chem. 17, 1432-1440). Incorporation
of a specific enzymatic substrate, cleaved by a protease that is
overexpressed in tumor cells, could generate highly efficient
cancer-cell-specific dendritic prodrug activation systems.
Non-limiting examples of spacer sequences that are cleavable by
proteases are listed in Table 9.
[0374] In some embodiments, the invention provides XTEN-payload
configurations, including dimeric, trimeric, tetrameric and higher
order conjugates in which the payload is attached to the XTEN using
a labile linker as described herein, above. In one embodiment of
the foregoing, the composition further includes a targeting
component to deliver the composition to a ligand or receptor on a
targeted cell. In another embodiment, the invention provides
conjugates in which one, two, three, or four XTEN-payload
compositions are conjugated with labile linkers to antibodies or
antibody fragments, providing soluble compositions for use in
targeted therapy of clinical indications such as, but not limited
to, various treatment of tumors and other cancers wherein the
antibody provides the targeting component and then, when
internalized within the target cell, the labile linker permits the
XTEN-payload to disassociate from the composition and effect the
intended activity (e.g, cytotoxicity in a tumor cell). Hence, the
inventive compositions are a type of immunoconjugate.
[0375] The unstructured characteristics and uniform composition and
charge of XTEN result in properties that can be exploited for
purification of XTEN-payload conjugates following a conjugation
reaction. Of particular utility is the capture of XTEN conjugates
by ion exchange, which allows the removal of un-reacted payload and
payload derivatives. Of particular utility is the capture of
conjugates by hydrophobic interaction chromatography (HIC). Due to
their hydrophilic nature, most XTEN polypeptides show low binding
to HIC resins, which facilitates the capture of XTEN-payload
conjugates due to hydrophobic interactions between the payload and
the column material, and their separation from un-conjugated XTEN
that failed to conjugate to the payload during the conjugation
process. The high purity of XTEN and XTEN-payload conjugates offers
a significant benefit compared to most chemical or natural
polymers, particularly pegylated payloads. Most chemical and
natural polymers are produced by random- or semi-random
polymerization, which results in the generation of many homologs.
Such polymers can be fractionated by various methods to increase
fraction of the target entity in the product. However, even after
enrichment most preparations of natural polymers and their payload
conjugates contain less than 10% target entity. Examples of PEG
conjugates with G-CSF have been described in [Bagal, D., et al.
(2008) Anal Chem, 80: 2408-18]. This publication shows that even a
PEG conjugate that is approved for therapeutic use contains more
than 100 homologs that occur with a concentration of at least 10%
of the target entity.
[0376] The complexity of random polymers, such as PEG, is a
significant impediment for the monitoring and quality control
during conjugation and purification. In contrast, XTEN purified by
the methods described herein have high levels of purity and
uniformity. In addition, the conjugates created as described herein
routinely contain greater than about 80%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% of the intended target and in the
intended configuration, resulting in easy to interpret mass spectra
and chromatograms.
[0377] 2. Monomeric XTEN-Cross-Linker, and XTEN-Payload
Configurations
[0378] In another aspect, the invention provides XTEN-cross-linker
conjugates and XTEN-payload conjugates with a single XTEN, wherein
the conjugate is designed in different configurations. Exemplary
configurations of such conjugates follow.
[0379] In one embodiment, the invention provides a conjugate having
the configuration of formula IV:
##STR00012##
wherein independently for each occurrence CL1 is a cross-linker; x
is an integer from 1 to about 100, or 1 to about 50, or 1 to about
40, or 1 to about 20, or 1 to about 10, or 1 to about 5, or is 9,
or is 3, or is 2, or is 1; and XTEN is a sequence having at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or having 100%
sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3. In one embodiment of the
conjugate of formula IV, CL1 is a cross-linker selected from Table
13. In another embodiment of the conjugate of formula IV, x has the
foregoing ranges and a cross-linker of Table 13 is linked to each
cysteine sulfur of the XTEN. In another embodiment of the conjugate
of formula IV, x has the foregoing ranges and a cross-linker is
linked to a each lysine epsilon amino group of the XTEN. In another
embodiment of the conjugate of formula IV, x is 1 and a
cross-linker of Table 13 is linked to the N-terminal amino group of
the XTEN. It will be understood by one of skill in the art that the
compositions of the foregoing embodiments comprising the
cross-linker conjugated to an XTEN using the specified components
represents the reaction product of the individual reactants and
thus differs from the precise composition of the reactants. In
another embodiment, the invention provides a preparation of the
conjugate of formula IV in which at least about 80%, or at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95% of
the XTEN molecules of the preparation of the conjugate have
identical sequence length.
[0380] In another embodiment, the invention provides a conjugate
having the configuration of formula V:
##STR00013##
wherein independently for each occurrence CL.sub.1 is a
cross-linker; x is an integer from 1 to about 100, or 1 to about
50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to
about 5, or is 9, or is 3, or is 2, or is 1; CL.sub.2 is a
cross-linker that is different from CL.sub.1; y is an integer from
1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to about
20, or 1 to about 10, or 1 to about 5, or is 9, or is 3, or is 2,
or is 1, with the proviso that x+y is .gtoreq.2; and XTEN is a
sequence having at least about 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3. In one
embodiment of the conjugate of formula V, CL.sub.1 and CL.sub.2 are
each selected from the group of cross-linkers set forth in Table
13. In one embodiment of the conjugate of formula V, x has the
foregoing ranges and each CL.sub.1 is linked to an epsilon amino
group of each lysine of the XTEN and y has the foregoing ranges and
each CL.sub.2 is linked to a sulfur group of each cysteine of the
XTEN. In another embodiment of the conjugate of formula V, x is 1
and CL.sub.1 is linked to the N-terminal amino group of the XTEN
and each CL.sub.2 is linked to a cysteine sulfur group of the XTEN.
It will be understood by one of skill in the art that the
compositions of the foregoing embodiments comprising the
cross-linker conjugated to an XTEN using the specified components
represents the reaction product of the reactants and thus differs
from the precise composition of the reactants. In another
embodiment, the invention provides a preparation of the conjugate
of formula V in which at least about 80%, or at least about 90%, or
at least about 91%, or at least about 92%, or at least about 93%,
or at least about 94%, or at least about 95% of the XTEN molecules
of the preparation of the conjugate have identical sequence
length.
[0381] In another aspect, the invention provides XTEN-payload
conjugates having defined configurations. The invention takes
advantage of the reactive XTEN-cross-linker conjugation partner
compositions described herein to which reactive molecules of
payloads can be joined by chemical reaction.
[0382] In one embodiment, the invention provides a conjugate having
the configuration of formula VI:
##STR00014##
wherein independently for each occurrence P.sub.R1 is a single atom
residue of a payload, wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur; CL.sub.1
is a cross-linker; x is an integer from 1 to about 100, or 1 to
about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or
1 to about 5, or is 3, or is 2, or is 1; and XTEN is a sequence
having at least about 80%, or at least about 90%, or at least about
91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99%, or
having 100% sequence identity to a sequence selected from the group
of sequences set forth in Tables 2 and 3. In one embodiment of the
conjugate of formula VI, the single atom residue of a payload is
from a payload selected from the group consisting of the payloads
set forth in Tables 11, 12, 18, 19, and 21. In one embodiment of
the conjugate of formula VI, CL.sub.1 is a cross-linker selected
from Table 13. In one embodiment of the conjugate of formula VI,
each cross-linker is linked to a cysteine sulfur of the XTEN. In
another embodiment of the conjugate of formula VI, each
cross-linker is linked to an lysine epsilon amino group of the
XTEN. In another embodiment of the conjugate of formula VI, x is 1
and the cross-linker is linked to the N-terminal amino group of the
XTEN. In another embodiment of the conjugate of formula VI,
CL.sub.1 is the reaction product of a first and a second click
chemistry reactant selected from Table 15. In another embodiment,
the invention provides a preparation of the conjugate of formula VI
in which at least about 80%, or at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95% of the XTEN molecules of the
preparation of the conjugate have identical sequence length.
[0383] In another embodiment, the invention provides a conjugate
having the configuration of formula VII:
##STR00015##
wherein independently for each occurrence: P.sub.1 is a payload
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18, 19, and 21; CL.sub.1 is a cross-linker; x is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1; and XTEN is a sequence having at least about 80%,
or at least about 90%, or at least about 91%, or at least about
92%, or at least about 93%, or at least about 94%, or at least
about 95%, or at least about 96%, or at least about 97%, or at
least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3. In one embodiment of the conjugate of
formula VII, CL.sub.1 is a cross-linker selected from Table 13. In
one embodiment of the conjugate of formula VII, each cross-linker
is linked to a cysteine sulfur of the XTEN. In another embodiment
of the conjugate of formula VII, each cross-linker is linked to an
lysine epsilon amino group of the XTEN. In another embodiment of
the conjugate of formula VII, x is 1 and the cross-linker is linked
to the N-terminal amino group of the XTEN. In one embodiment, the
conjugate of formula VII is selected from the group consisting of
the conjugates set forth in Table 21. In another embodiment of the
conjugate of formula VII, CL.sub.1 is the reaction product of a
first and a second click chemistry reactant selected from Table 15.
It will be understood by one of skill in the art that the
compositions of the foregoing embodiments comprising the payload
conjugated to an XTEN-cross-linker using the specified components
represents the reaction product of the reactants and thus differs
from the precise composition of the reactants. In another
embodiment, the invention provides a preparation of the conjugate
of formula VII in which at least about 80%, or at least about 90%,
or at least about 91%, or at least about 92%, or at least about
93%, or at least about 94%, or at least about 95% of the XTEN
molecules of the preparation of the conjugate have identical
sequence length.
[0384] In another embodiment, the invention provides a conjugate
having the configuration of formula VIII:
##STR00016##
wherein independently tor each occurrence P.sub.R1 is a single atom
residue of a payload, wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur; P.sub.R2
is a single atom residue of a payload, wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; CL.sub.1 is a cross-linker; x is an integer from 1 to about
100, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to
about 10, or 1 to about 5, or is 9, or is 3, or is 2, or is 1;
CL.sub.2 is a cross-linker that is different from CL.sub.1; y is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1, with the proviso that x+y is .gtoreq.2; and XTEN
is a sequence having at least about 80%, or at least about 90%, or
at least about 91%, or at least about 92%, or at least about 93%,
or at least about 94%, or at least about 95%, or at least about
96%, or at least about 97%, or at least about 98%, or at least
about 99%, or having 100% sequence identity to a sequence selected
from the group of sequences set forth in Tables 2 and 3. In one
embodiment of the conjugate of formula VIII, the single atom
residue of a payload is from a payload selected from the group
consisting of the payloads set forth in Tables 11, 12, 18, 19, and
21. In one embodiment of the conjugate of formula VIII, CL.sub.1
and CL.sub.2 are each selected from the group of cross-linkers set
forth in Table 13. In one embodiment of the conjugate of formula
VIII, each CL.sub.1 is linked to an lysine epsilon amino group of
the XTEN and each CL.sub.2 is linked to a cysteine sulfur of the
XTEN. In another embodiment of the conjugate of formula VIII, x is
1 and CL.sub.1 is linked to the N-terminal amino group of the XTEN
and CL.sub.2 is linked to either a cysteine sulfur or an lysine
epsilon amino group of the XTEN. In another embodiment of the
conjugate of formula VIII, CL.sub.1 is the reaction product of a
first and a second click chemistry reactant selected from Table 15.
In another embodiment of the conjugate of formula VIII, C.sub.2 is
the reaction product of a first and a second click chemistry
reactant selected from Table 15. In another embodiment, the
invention provides a preparation of the conjugate of formula VIII
in which at least about 80%, or at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95% of the XTEN molecules of the
preparation of the conjugate have identical sequence length.
[0385] In another embodiment, the invention provides a conjugate
having the configuration of formula IX:
##STR00017##
wherein independently for each occurrence P.sub.1 is a payload
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18, 19, and 21; P.sub.2 is a payload selected from
the group consisting of the payloads set forth in Tables 11, 12,
18, 19, and 21 and that is different from P.sub.1; CL.sub.1 is a
cross-linker; x is an integer from 1 to about 100, or 1 to about
50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to
about 5, or is 9, or is 3, or is 2, or is 1; CL.sub.2 is a
cross-linker that is different from CL.sub.1; y is an integer from
1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to about
20, or 1 to about 10, or 1 to about 5, or is 9, or is 3, or is 2,
or is 1, with the proviso that x+y is .gtoreq.2; and XTEN is a
sequence having at least about 80% b, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3. In one
embodiment of the conjugate of formula IX, the single atom residue
of a payload is from a payload selected from the group consisting
of the payloads set forth in Tables 11, 12, 18, 19, and 21. In one
embodiment of the conjugate of formula IX, CL.sub.1 and CL.sub.2
are each selected from the group of cross-linkers set forth in
Table 13. In one embodiment of the conjugate of formula IX, each
CL.sub.1 is linked to an lysine epsilon amino group of the XTEN and
each CL.sub.2 is linked to a cysteine sulfur of the XTEN. In
another embodiment of the conjugate of formula IX, x is 1 and
CL.sub.1 is linked to the N-terminal amino group of the XTEN and
CL.sub.2 is linked to either a cysteine sulfur or an lysine epsilon
amino group of the XTEN. In another embodiment of the conjugate of
formula IX, CL.sub.1 is the reaction product of a first and a
second click chemistry reactant selected from Table 15. In another
embodiment of the conjugate of formula IX, C.sub.2 is the reaction
product of a first and a second click chemistry reactant selected
from Table 15. In one embodiment, the conjugate of formula IX is
selected from the group consisting of the conjugates set forth in
Table 21. In another embodiment, the invention provides a
preparation of the conjugate of formula IX in which at least about
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95% of the XTEN molecules of the preparation of the
conjugate have identical sequence length.
[0386] 3. Dimeric, Trimeric, Tetrameric, and Multimeric
Configurations of XTEN-Cross-Linkers and XTEN-Payload
Conjugates
[0387] In one aspect, the invention provides conjugates wherein
different numbers of XTEN or XTEN-payload conjugation partners are
joined by linkers in a numerically-defined configuration; e.g.,
dimeric, trimeric, tetrameric, or multimeric. As used herein,
"precursor" is intended to include components used as reactants in
a conjugation reaction leading to an intermediate or final
composition, and includes but is not limited to XTEN segments of
any length (including the XTEN of Tables 2 and 3 or as depicted in
the various formulae, above), XTEN-crosslinkers,
XTEN-payload-crosslinker segments, payloads with reactive groups,
linkers, and other such components described herein.
[0388] In some embodiments, the invention provides conjugates in
which two XTEN or XTEN-payload precursor segments are linked by a
divalent cross-linker, resulting in a divalent configuration, such
as shown in FIG. 19C and FIG. 27B. In one embodiment of the
divalent XTEN-payload conjugate, each XTEN-payload can be a
monomeric fusion protein comprising a biologically active peptide
or polypeptide, wherein each fusion protein precursor segment is
linked to the divalent linker by the alpha-amino group of the
N-terminus, resulting in the divalent conjugate. In another
embodiment of the divalent XTEN-payload conjugate, each
XTEN-payload precursor segment is a monomeric fusion protein
comprising a biologically active peptide or polypeptide, wherein
each fusion protein is linked to the divalent linker at the
C-terminus, resulting in the divalent conjugate. In another
embodiment of the divalent XTEN-payload conjugate, each XTEN
comprises one or more payloads (that can be a peptide, polypeptide
or a drug) conjugated to the XTEN, wherein each XTEN precursor is
linked to the other XTEN precursor comprising one or more second,
different payload molecules by a divalent linker at the N-terminus,
resulting in the divalent conjugate. In another embodiment of the
divalent XTEN-payload conjugate, each XTEN comprises one or more
payloads (that can be a peptide, polypeptide or a drug) conjugated
to the XTEN, wherein each XTEN precursor is linked to the other
XTEN precursor comprising one or more second, different payload
molecules by a divalent linker by the carboxyl group or a modified
group at the C-terminus (including, but not limited to XTEN
modified by insertion of a cysteine at the C-terminus), resulting
in the divalent conjugate. In the foregoing embodiments of the
paragraph, as would be appreciated by one of ordinary skill in the
art in light of the present disclosure, there are different
approaches to create the precurors to be linked, such as
conjugating a linker to a first precuror XTEN-payload and then
effecting a second reaction to join the precursor to the reactive
group of the terminus the second XTEN-payload precursor.
Alternatively, one or both of the XTEN termini can be modified as
precurors that can then be joined by click chemistry or by other
methods described or illustrated herein, leaving few or no residual
atoms to bridge the intersection of the XTEN. In another
embodiment, two XTEN-payload precuror sequences are linked by a
disulfide bridge using cysteines or thiol groups introduced at or
near the termini of the precursor XTEN-payload reactants, resulting
in a divalent XTEN-payload conjugate. Exemplary configurations of
such divalent conjugates follow.
[0389] In one embodiment, the invention provides a conjugate having
the configuration of formula X
##STR00018##
wherein independently for each occurrence P.sub.R1 is a single atom
residue of a first payload wherein the residue is selected from the
group consisting of carbon, nitrogen, oxygen and sulfur; P.sub.R2
is a single atom residue of a second payload wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur; CL.sub.1 is a cross-linker; x is an integer from 1 to about
100, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to
about 10, or 1 to about 5, or is 9, or is 3, or is 2, or is 1;
CL.sub.2 is a cross-linker that is different from CL.sub.1; y is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1, with the proviso that x+y is .gtoreq.2;
2.times.CL is alternatively a divalent cross-linker or the reaction
product of a first and a second click chemistry reactant selected
from Table 15; XTEN.sub.1 is a polypeptide having at least 80%, or
at least about 90%, or at least about 91%, or at least about 92%,
or at least about 93%, or at least about 94%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%, or having 100% sequence identity
to a sequence selected from the group of sequences set forth in
Tables 2 and 3; and XTEN.sub.2 is a polypeptide having at least
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3. In one embodiment of the conjugate of
formula X, CL.sub.1 and CL.sub.2 are each selected from the group
of cross-linkers set forth in Table 13. In another embodiment of
the conjugate of formula X, x is 1 and CL.sub.1 is linked to the
N-terminal amino group of the XTEN. In another embodiment of the
conjugate of formula X, CL.sub.1 is the reaction product of a first
and a second click chemistry reactant selected from Table 15. In
another embodiment of the conjugate of formula X, C.sub.2 is the
reaction product of a first and a second click chemistry reactant
selected from Table 15. In another embodiment of the conjugate of
formula X, each CL.sub.1 is linked to a cysteine sulfur of the
XTEN, and each CL.sub.2 is linked to a cysteine sulfur of
XTEN.sub.2. In another embodiment of the conjugate of formula X,
each CL.sub.1 is linked to a lysine epsilon amino group of the
XTEN.sub.1 and each CL.sub.2 is linked to a lysine epsilon amino
group of the XTEN.sub.2. In another embodiment of the conjugate of
formula X, each CL.sub.1 is linked to a cysteine sulfur of the
XTEN.sub.1 and each CL.sub.2 is linked to a lysine epsilon amino
group of the XTEN.sub.2. In another embodiment of the conjugate of
formula X, XTEN.sub.1 and XTEN.sub.2 are identical. In another
embodiment of the conjugate of formula X, XTEN.sub.1 and XTEN.sub.2
are different. In another embodiment, the invention provides a
preparation of the conjugate of formula X in which at least about
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95% of the XTEN molecules of the preparation of the
conjugate have identical sequence length.
[0390] In another embodiment, the invention provides a conjugate
having the configuration of formula XI
##STR00019##
wherein independently for each occurrence P.sub.1 is a first
payload selected from the group of payloads set forth in Tables 11,
12, 18, 19, and 21; P.sub.2 is a second payload selected from the
group of payloads set forth in Tables 11, 12, 18, 19, and 21 and
that is different from P.sub.1; CL.sub.1 is a cross-linker; x is an
integer from 1 to about 100, or 1 to about 50, or 1 to about 40, or
1 to about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3,
or is 2, or is 1; CL.sub.2 is a cross-linker that is different from
CL.sub.1; y is an integer from 1 to about 100, or 1 to about 50, or
1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about 5,
or is 9, or is 3, or is 2, or is 1, with the proviso that x+y is
.gtoreq.2; 2.times.CL is alternatively a divalent cross-linker or
the reaction product of a first and a second click chemistry
reactant selected from Table 15; XTEN.sub.1 is a first
substantially homogeneous XTEN having at least 80%, or at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99%, or having 100% sequence identity to a
sequence selected from the group of sequences set forth in Tables 2
and 3; and XTEN.sub.2 is a first substantially homogeneous having
at least 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or having 100%
sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3. In one embodiment of the
conjugate of formula XI, CL.sub.1 and CL.sub.2 are each selected
from the group of cross-linkers set forth in Table 13. In another
embodiment of the conjugate of formula XI, x is 1 and CL.sub.1 is
linked to the N-terminal amino group of the XTEN. In another
embodiment of the conjugate of formula XI, CL.sub.1 is the reaction
product of a first and a second click chemistry reactant selected
from Table 15. In another embodiment of the conjugate of formula
XI, C.sub.2 is the reaction product of a first and a second click
chemistry reactant selected from Table 15. In another embodiment of
the conjugate of formula XI, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN.sub.1 and each CL.sub.2 is linked to a cysteine
sulfur of XTEN.sub.2. In another embodiment of the conjugate of
formula XI, each CL.sub.1 is linked to a lysine epsilon amino group
of the XTEN.sub.1 and each CL.sub.2 is linked to a lysine epsilon
amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula XI, each CL.sub.1 is linked to a cysteine
sulfur of the XTEN, and each CL.sub.2 is linked to a lysine epsilon
amino group of the XTEN.sub.2. In another embodiment of the
conjugate of formula XI, XTEN.sub.1 and XTEN.sub.2 are identical.
In another embodiment of the conjugate of formula XI, XTEN.sub.1
and XTEN.sub.2 are different. In one embodiment, the conjugate of
formula XI is selected from the group consisting of the conjugates
set forth in Table 21. In another embodiment, the invention
provides a preparation of the conjugate of formula XI in which at
least about 80%, or at least about 90%, or at least about 91%, or
at least about 92%, or at least about 93%, or at least about 94%,
or at least about 95% of the respective XTEN.sub.1 and XTEN.sub.2
molecules of the preparation of the conjugate have identical
sequence length.
[0391] In other embodiments, the invention provides XTEN-linker and
XTEN-linker payload conjugates with a trimeric configuration, such
as shown in FIGS. 21-23.
[0392] The invention provides trimeric conjugates in which three
XTEN-cross-linker conjugates are linked by a trivalent linker,
resulting in a trimeric XTEN-cross-linker configuration. In one
embodiment, the invention provides a trimeric XTEN-crosslinker
having the configuration of formula XII
##STR00020##
wherein independently for each occurrence 3.times.CL is the
trivalent cross-linker, CL1 is the first cross-linker conjugated to
XTEN1, CL2 is the second cross-linker conjugated to XTEN2, CL3 is
the third cross-linker conjugated to XTEN3, x is an integer of 1 to
about 10, y is an integer of 1 to about 10, z is an integer of 1 to
about 10 with the proviso that x+y+z is >3, XTEN.sub.1 is the
first XTEN having at least 80%, or at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%,
or having 100% sequence identity to a sequence selected from the
group of sequences set forth in Tables 2 and 3; XTEN.sub.2 is the
second XTEN having at least 80%, or at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%,
or having 100% sequence identity to a sequence selected from the
group of sequences set forth in Tables 2 and 3; and XTEN.sub.3 is
the third XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3 wherein
XTEN.sub.1, XTEN.sub.2, and XTEN.sub.3 are the same or are
different XTEN sequences. In one embodiment of the conjugate of
formula XII, CL1, CL2, and CL3 are each selected from the group
consisting of the cross-linkers set forth in Table 13, and are the
same or are different. In one embodiment, the conjugate of formula
XII further comprises a single atom residue of a first payload
conjugated to each cross-linker of the first substantially
homogeneous XTEN wherein the residue is selected from the group
consisting of carbon, nitrogen, oxygen and sulfur, a single atom
residue of a second payload conjugated to each cross-linker of the
second substantially homogeneous XTEN wherein the residue is
selected from the group consisting of carbon, nitrogen, oxygen and
sulfur, and a single atom residue of a third payload conjugated to
each cross-linker of the third substantially homogeneous XTEN
wherein the residue is selected from the group consisting of
carbon, nitrogen, oxygen and sulfur.
[0393] In another embodiment, the invention provides trimeric
conjugates in which three XTEN-payload precursors are linked by a
trivalent linker, resulting in a trimeric XTEN-payload
configuration, such as shown in FIGS. 21 and 97-106. In one
embodiment, the invention provides a trimeric XTEN-crosslinker
having the configuration of formula XIII
##STR00021##
wherein independently for each occurrence 3.times.CL is the
trivalent cross-linker is selected from the group of trivalent
cross-linkers set forth in Tables 13 and 14; P.sub.1 is conjugated
to each cross-linker of the first XTEN and is selected from the
group consisting of the payloads set forth in Tables 11, 12, 18 and
21, P2 is a second payload conjugated to each cross-linker of the
second XTEN and is selected from the group consisting of the
payloads set forth in Tables 11, 12, 18 and 21, wherein the payload
is the same or is different from the first payload, and P.sub.3 is
a third payload conjugated to each cross-linker of the third XTEN
and is selected from the group consisting of the payloads set forth
in Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first or the second payload; CL.sub.1 is the
first cross-linker, x is an integer from 1 to about 100, or 1 to
about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or
1 to about 5, or is 9, or is 3, or is 2, or is 1; CL2 is a second
cross-linker, y is an integer from 1 to about 100, or 1 to about
50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to
about 5, or is 9, or is 3, or is 2, or is 1; and z is an integer
from 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to
about 20, or 1 to about 10, or 1 to about 5, or is 9, or is 3, or
is 2, or is 1, with the proviso that x+y+z is .gtoreq.3; XTEN.sub.1
is the first XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3; XTEN.sub.2 is
the second XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3; and XTEN.sub.3
is the third XTEN having at least 80%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or
at least about 94%, or at least about 95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about
99%, or having 100% sequence identity to a sequence selected from
the group of sequences set forth in Tables 2 and 3 wherein
XTEN.sub.1, XTEN.sub.2, and XTEN.sub.3 are the same or are
different XTEN sequences. In some embodiments, the conjugate of
formula XIII further comprises a first payload wherein the payload
is a targeting moiety with specific binding affinity to a target,
wherein the targeting moiety is selected from the group consisting
of the targeting moieties set forth in Tables 17-19 and 21, and at
least one other of the payloads is a drug wherein the drug is
selected from the group consisting of the drugs set forth in Table
11, Table 19, and Table 21. In one embodiment of the foregoing, the
targeting moiety is LHRH or folate and the drug is selected from
doxorubicin, paclitaxel, auristatin, monomethyl auristatin E
(MMAE), monomethyl auristatin F, maytansine, dolastatin,
calicheamicin, vinca alkaloid, camptothecin, mitomycin C,
epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin. In another embodiment of the
trimeric XTEN conjugate composition, the composition has the
configuration of formula XIV:
##STR00022##
wherein independently for each occurrence; 3.times.CL is the
trivalent cross-linker, CL1 is the first cross-linker conjugated to
XTEN.sub.1; CL2 is the second cross-linker conjugated to
XTEN.sub.2; x is an integer of 1 to about 10; y is an integer of 1
to about 10 with the proviso that x+y is .gtoreq.2; XTEN, is the
first XTEN; XTEN.sub.2 is the second XTEN; and XTEN.sub.3 is the
third XTEN wherein the XTEN is selected from the group consisting
of the sequences set forth in Table 2. In one embodiment of the
trimeric XTEN conjugate composition of formula XIV, the composition
further comprises a single atom residue of a first payload
conjugated to each first cross-linker of the first XTEN wherein the
residue is selected from the group consisting of carbon, nitrogen,
oxygen and sulfur; and a single atom residue of a second payload
conjugated to each second cross-linker of the second XTEN wherein
the residue is selected from the group consisting of carbon,
nitrogen, oxygen and sulfur. In another embodiment of the trimeric
XTEN conjugate composition of formula XIV, the composition further
comprises a first payload conjugated to each first cross-linker of
the first XTEN selected from the group consisting of the payloads
set forth in Tables 6, 7, 18 and 21; and a second payload
conjugated to each second cross-linker of the second XTEN selected
from the group consisting of the payloads set forth in Tables 6, 7,
18 and 21, wherein the payload is the same or is different from the
first payload. In one embodiment of the foregoing, the first
payload is a targeting moiety with specific binding affinity to a
target, wherein the targeting moiety is selected from the group
consisting of the targeting moieties set forth in Tables 17-19 and
21, and the second payloads is a drug selected from the group
consisting of the drugs set forth in Table 6, Table 18, and Table
21. In another embodiment of the foregoing, the first payload is a
targeting moiety is selected from the group consisting of LHRH and
folate, and the second payload is a drug is selected from the group
consisting of doxorubicin, paclitaxel, auristatin, monomethyl
auristatin E (MMAE), monomethyl auristatin F, maytansine,
dolastatin, calicheamicin, vinca alkaloid, camptothecin, mitomycin
C, epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin. In one embodiment of the
foregoing, the first payload is a drug selected from the group
consisting of the drugs of Table 11 and the proteins of Table 12
and the second payload is different from the first payload and is
selected from the group consisting of the drugs of Table 11 and the
proteins of Table 12. In another embodiment of the foregoing, the
first payload and the second payload are identical and are selected
from the group consisting of the drugs of Table 11 and the proteins
of Table 12. In another embodiment of the trimeric XTEN conjugate
composition, the composition has the configuration of formula
XV:
##STR00023##
wherein independently for each occurrence; 3.times.CL is the
trivalent cross-linker, CL1 is the first cross-linker conjugated to
XTEN.sub.1; x is an integer of 1 to about 10; XTEN.sub.1 is the
first XTEN wherein the XTEN is selected from the group consisting
of the sequences set forth in Table 3; XTEN.sub.2 is the second
XTEN wherein the XTEN is selected from the group consisting of the
sequences set forth in Table 2; and XTEN.sub.3 is the third XTEN
wherein the XTEN is selected from the group consisting of the
sequences set forth in Table 2. In one embodiment of the trimeric
XTEN conjugate composition configured as formula XV, the
composition further comprises a single atom residue of a first
payload conjugated to each first cross-linker of the first XTEN
wherein the residue is selected from the group consisting of
carbon, nitrogen, oxygen and sulfur. In one embodiment of the
trimeric XTEN conjugate composition configured as formula XV, the
composition further comprises a first payload conjugated to each
first cross-linker of the first XTEN selected from the group
consisting of the payloads set forth in Tables 6, 7, 18 and 21.
[0394] In another embodiment of the trimeric XTEN-payload
conjugate, each XTEN-payload can be a monomeric fusion protein
comprising a biologically active peptide or polypeptide, wherein
the fusion protein is linked to the trivalent linker at an amino
group or a thiol group of the XTEN. In another embodiment of the
trimeric XTEN-payload conjugate, each XTEN-payload can be a
conjugate of a payload linked to the XTEN, which can be a
biologically active peptide or polypeptide or a pharmacologically
active small molecule or toxin, linked to the XTEN that, in turn,
is linked to the trivalent linker at the N-terminus of the XTEN. In
the foregoing XTEN-linker-payload embodiments hereinabove described
in this paragraph, the three payloads can be identical or they can
be different. In one embodiment of the trimeric XTEN-payload
conjugate, the conjugate comprises at least one biologically active
protein and at least one drug linked to different XTEN that, in
turn, is linked to the trivalent linker at the N-terminus of the
XTEN. In a particular embodiment of the foregoing configuration,
the at least one biologically active protein is a targeting moiety
and the at least one drug is a toxin including, but not limited to
doxorubicin, paclitaxel, auristatin, monomethyl auristatin E,
monomethyl auristatin F, maytansine, dolastatin, calicheamicin,
vinca alkaloid, camptothecin, mitomycin C, epothilone, hTNF, Il-12,
bortezomib, ranpirnase, pseudomonas exotoxin, SN-38, and
rachelmycin. Depending on the position of the thiol or the epsilon
amino group in the XTEN, one can control if the payload is interior
to (as shown in FIG. 23B) or at the terminus of the cross-linked
XTEN (as shown in FIG. 23A). In the foregoing embodiment,
exemplarily, non-limiting targeting moieties include LHRH, folate,
octreotide, pasireotide, bombesin, monocloncal antibodies, scFV,
centryins, and the antibody fragments, scaffolds and mimetics of
Table 17. Exemplary configurations of such trimeric conjugates
follow.
[0395] The invention further provides XTEN-linker and XTEN-linker
payload conjugates with a tetrameric configuration. In one
embodiment, the invention provides conjugates in which four XTEN
sequences are linked by a tetraravalent linker, resulting in a
tetrarameric XTEN-crosslinker configuration, such as shown in FIG.
22D. Non-limiting examples of tetravalent linkers include a
tetraravalent-thiol, a quadravalent-N-maleimide linker such as
described in U.S. Pat. No. 7,524,821, or an antibody or antibody
fragment with four reactive groups.
[0396] The invention provides conjugates in which four
XTEN-cross-linker precursor sequences are linked by a tetrarvalent
linker, resulting in a tetraravalent XTEN-cross-linker
configuration. In one embodiment, the invention provides a
tetrameric XTEN-crosslinker having the configuration of formula
XVI
##STR00024##
wherein independently for each occurrence: 4.times.CL is the
tetravalent cross-linker; CL.sub.1 is the first cross-linker
conjugated to XTEN.sub.1; CL.sub.2 is the second cross-linker
conjugated to XTEN.sub.2; CL.sub.3 is the third cross-linker
conjugated to XTEN.sub.3; CL.sub.4 is the fourth cross-linker
conjugated to XTEN.sub.4; v is an integer of 1 to about 10; x is an
integer of 1 to about 10; y is an integer of 1 to about 10; z is an
integer of 1 to about 10 with the proviso that x+y+z is .gtoreq.4;
XTEN.sub.1 is the first XTEN having at least 80%, or at least about
90%, or at least about 91%, or at least about 92%, or at least
about 93%, or at least about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or
at least about 99%, or having 100% sequence identity to a sequence
selected from the group of sequences set forth in Tables 2 and 3;
XTEN.sub.2 is the second XTEN having at least 80%, or at least
about 90%, or at least about 91%, or at least about 92%, or at
least about 93%, or at least about 94%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99%, or having 100% sequence identity to a
sequence selected from the group of sequences set forth in Tables 2
and 3; XTEN.sub.3 is the third XTEN having at least 80%, or at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or having 100% sequence identity to a
sequence selected from the group of sequences set forth in Tables 2
and 3 wherein XTEN.sub.1, XTEN.sub.2, and XTEN.sub.3 are the same
or are different XTEN sequences; XTEN.sub.4 is the fourth XTEN
having at least 80%, or at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98%, or at least about 99%, or having
100% sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3 wherein XTEN.sub.1,
XTEN.sub.2, XTEN.sub.3 and XTEN.sub.4 are the same or are different
XTEN sequences.
[0397] The invention provides conjugates in which four XTEN-payload
precursor sequences are linked by a tetrarvalent linker, resulting
in a tetraravalent XTEN-payload configuration as shown in FIGS. 22C
and 105. In one embodiment, the invention provides a tetrameric
XTEN-payload having the configuration of formula XVII
##STR00025##
wherein independently for each occurrence: 4.times.CL is the
tetravalent cross-linker; P.sub.1 is conjugated to each
cross-linker of the first XTEN and is selected from the group
consisting of the payloads set forth in Tables 11, 12, 18 and 21,
P2 is a second payload conjugated to each cross-linker of the
second XTEN and is selected from the group consisting of the
payloads set forth in Tables 11, 12, 18 and 21, wherein the payload
is the same or is different from the first payload, P.sub.3 is a
third payload conjugated to each cross-linker of the third XTEN and
is selected from the group consisting of the payloads set forth in
Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first or the second payload; P.sub.4 is a fourth
payload conjugated to each cross-linker of the fourth XTEN and is
selected from the group consisting of the payloads set forth in
Tables 11, 12, 18 and 21, wherein the payload is the same or is
different from the first, the second or the third payload; CL.sub.1
is the first cross-linker conjugated to XTEN.sub.1; CL.sub.2 is the
second cross-linker conjugated to XTEN.sub.2; CL.sub.3 is the third
cross-linker conjugated to XTEN.sub.3; CL.sub.4 is the fourth
cross-linker conjugated to XTEN.sub.4; v is an integer of 1 to
about 10; x is an integer of 1 to about 10; y is an integer of 1 to
about 10; z is an integer of 1 to about 10 with the proviso that
x+y+z is .gtoreq.4; XTEN.sub.1 is the first XTEN having at least
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%, or having 100% sequence
identity to a sequence selected from the group of sequences set
forth in Tables 2 and 3; XTEN.sub.2 is the second XTEN having at
least 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or having 100%
sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3; XTEN.sub.3 is the third XTEN
having at least 80%, or at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98%, or at least about 99%, or having
100% sequence identity to a sequence selected from the group of
sequences set forth in Tables 2 and 3 wherein XTEN.sub.1,
XTEN.sub.2, and XTEN.sub.3 are the same or are different XTEN
sequences; XTEN.sub.4 is the fourth XTEN having at least 80%, or at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99%, or having 100% sequence identity to a
sequence selected from the group of sequences set forth in Tables 2
and 3 wherein XTEN.sub.1, XTEN.sub.2, XTEN.sub.3 and XTEN.sub.4 are
the same or are different XTEN sequences.
[0398] In another embodiment of the tetraravalent XTEN-payload
conjugate, each XTEN-payload can be a monomeric fusion protein
comprising a biologically active peptide or polypeptide, wherein
the fusion protein is linked to the tetraravalent linker at an
amino group or a thiol group of the XTEN. In another embodiment of
the tetraravalent XTEN-payload conjugate, each XTEN-payload can be
a conjugate of a payload, which can be a biologically active
peptide or polypeptide or a pharmacologically active small molecule
or toxin, linked to the XTEN that, in turn, is linked to the
tetraravalent linker by the N-terminus of the XTEN. In the
foregoing XTEN-linker-payload embodiments hereinabove described in
this paragraph, the four payloads can be identical or they can be
different. In a particular embodiment of the foregoing
configuration, the at least one biologically active protein is a
targeting moiety and the at least one drug is a toxin including,
but not limited to doxorubicin, paclitaxel, auristatin, maytansine,
dolastatin, calicheamicin, vinca alkaloid, camptothecin, mitomycin
C, epothilone, hTNF, Il-12, bortezomib, ranpirnase, pseudomonas
exotoxin, SN-38, and rachelmycin. Depending on the position of the
thiol or the epsilon amino group in the XTEN, one can control if
the payload is interior to or at the terminus of the cross-linked
XTEN.
[0399] 4. Multivalent Configurations with Four or More
XTEN-Payloads
[0400] Using XTEN of Table 3, compositions are contemplated
containing four or more XTEN-payload molecules linked to the
cysteine- or lysine-engineered backbone, resulting in a "comb"
multivalent configuration, or linking multiple branched precurors
to make a "dendrimer" configuration, as illustrated in FIG. 7. In
one embodiment, the multivalent configuration conjugate is created
by reacting a cysteine- or lysine-engineered XTEN with an
XTEN-payload comprising a linker appropriate for reaction with the
cysteine- or lysine-engineered XTEN (as illustrated in FIGS.
24A-24C), resulting in the final product. In another embodiment,
the multivalent configuration conjugate is created by reacting a
cysteine- or lysine-engineered XTEN with linkers with an
XTEN-payload comprising a cysteine or a primary or epsilon amino
group appropriate for reaction with the linker linked to the
cysteine- or lysine-engineered XTEN, resulting in the final
product. In the embodiments, the valency of the final product is
controlled by the number of reactive groups incorporated into the
XTEN, whether a reactive amino acid or linker. Additionally, it is
contemplated that the final product can be designed to locate the
payload either close to the XTEN termini, which improves
interactions with its ligand, or close to the branch points to
shield the payload and reduce the degree of interaction with the
ligand.
[0401] 5. Bispecific Payload Configurations on Monomer XTEN
Backbone
[0402] In another aspect, the invention provides conjugates
containing two different payload molecules linked to a single
cysteine- and lysine-engineered XTEN backbone, as illustrated in
FIG. 27A, resulting in a bivalent conjugate. In one embodiment, the
bivalent configuration conjugate is created by reacting the
engineered XTEN, such as those specifically provided in Table 3,
with a first XTEN-payload comprising a linker appropriate for
reaction with the cysteine-engineered XTEN, followed by a second
reaction with a second XTEN-payload comprising a linker appropriate
for reaction with the lysine-engineered XTEN, resulting in the
final product. The number and location of payloads is controlled by
the design of the engineered XTEN, with the placement of the
reactive thiol or amino group being determinative. In one
embodiment, the bivalent conjugate comprises a single molecule of a
first payload and a single molecule of a second payload linked to
the cysteine-lysine-engineered XTEN by linkers. In another
embodiment, the bivalent conjugate comprises one, or two, or three,
or four, or five molecules of a first payload and a single molecule
of a second payload linked to the cysteine-lysine-engineered XTEN
by linkers. In another embodiment, the bivalent conjugate comprises
one, or two, or three, or four, or five molecules of a first
payload and one, or two, or three, or four, or five molecules of a
second payload linked to the cysteine-lysine-engineered XTEN by
linkers.
[0403] In another embodiment, the bivalent configuration conjugate
is created by reacting the cysteine- and lysine-engineered XTEN,
such as those of Table 3, with a first linker appropriate for
reaction with the cysteine-engineered XTEN, followed by a second
reaction with a a linker appropriate for reaction with the
lysine-engineered XTEN, then reacting the XTEN-crosslinker backbone
with a first payload with a thiol reactive group capable of
reacting with the first linker, followed by a reaction of a second
payload with an amino group capable of reacting with the second
cross-linker, resulting in the final product.
[0404] 6. XTEN-Cross-Linker and XTEN-Payload Conjugates with Spacer
and Release Groups
[0405] In another aspect, the invention provides XTEN-crosslinker
and XTEN-payload conjugates configured with one or more spacers
incorporated into or adjacent to the XTEN that are designed to
incorporate or enhance a functionality or property to the
composition, or as an aid in the assembly or manufacture of the
compositions. Such properties include, but are not limited to,
inclusion of a sequence capable of being proteolytically cleaved or
a labile functional group to permit release of the payload, or a
spacer can be introduced between an XTEN sequence and a payload
component to decrease steric hindrance such that the payload
component may interact appropriately with its target ligand.
[0406] In one embodiment, the one or more spacers are incorporated
into the linkers of the subject conjugates. For spacers and methods
of identifying desirable spacers, see, for example, George, et al.
(2003) Protein Engineering 15:871-879, specifically incorporated by
reference herein. In one embodiment, the spacer comprises one or
more peptide sequences that are between 1-50 amino acid residues in
length, or about 1-25 residues, or about 1-10 residues in length.
Spacer sequences, exclusive of cleavage sites, can comprise any of
the 20 natural L-amino acids, and will preferably have XTEN-like
properties in that the majority of residues will be hydrophilic
amino acids that are sterically unhindered. The spacer can be
polyglycines or polyalanines, or is predominately a mixture of
combinations of glycine, serine and alanine residues. In one
embodiment, the spacer sequence is a sequence from Table 15. In
another embodiment, the spacer sequence is GPEGPS (SEQ ID NO:
580).
[0407] In addition, spacer sequences are designed to avoid the
introduction of T-cell epitopes which can, in part, be achieved by
avoiding or limiting the number of hydrophobic amino acids utilized
in the spacer, the determination of epitopes is described above and
in the Examples.
[0408] In one embodiment, the spacer comprises a release group that
permits the release of the payload from the conjugate. In another
embodiment, the cross-linker comprises a release group that permits
the release of the payload from the conjugate. The release group
may be any labile group providing for such a releasable attachment.
In one embodiment, the release group is a chemically cleavable
linkage or labile chemical linkage. Such linkages may typically be
cleaved by methods that are well known to those of skill in the
art, such as by acid, base, oxidation, reduction, displacement or
elimination. In a particular embodiment, the chemistry cleavable
linkage comprises a modified base, a modified sugar, a disulfide
bond, a chemically cleavable group incorporated into the
cross-linker or spacer. Some examples of these linkages are
described in PCT WO 96/37630 and U.S. Pat. No. 7,132,519,
incorporated herein by reference. Release groups encompassed by the
invention also include groups or linkages cleavable by an enzyme.
Enzymatically-cleavable release groups include phosphodiester or
amide linkages as well as restriction endonuclease recognition
sites. In one embodiment, the invention provides compositions
comprising one or more payloads in which a cleavable linker of
valine-citrulline is between the payload and the XTEN, permitting
cleavage by cathepsin when the composition is internalized
intracellularly; e.g., inside a tumour cell. In another embodiment,
release groups are cleavable by nucleases. These nucleases may
typically be an exonuclease or a restriction endonuclease. Typical
exonucleases include exonucleases specific for both double-stranded
and single-stranded polynucleic acids. Additionally, restriction
endonucleases encompassed by certain embodiments include Type IIS
and Type II restriction endonucleases. In other embodiments the
release group may be a sequence that is cleavable by a protease,
wherein the sequence is selected from the sequences set fort in
Table 9. Typical proteases acting on sequences suitable for
inclusion in the inventive compositions include endoproteinases,
including the proteinases of Table 9.
[0409] 7. Libraries of XTEN-Payload Configurations
[0410] In another aspect, the invention provides libraries of
XTEN-payload precursors, methods to make the libraries, and methods
to combine the library precursors in a combinatorial approach, as
illustrated in FIGS. 34-35, to achieve optimal combinations of, as
well as the optimal ratio of payloads. In one embodiment, the
invention provides a library of individual XTEN each linked to 1,
or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more or
more molecules of a given payload, including those described
herein, to create the library of XTEN-payload precursors. In the
method, a series of XTEN-payload precursors to be linked are
further conjugated to a linker, and then is subsequently mixed and
reacted with the other XTEN-payload precursors capable of reacting
with the linker under conditions to effect the conjugation,
resulting in a library of the various permutations and ratios of
payloads linked to XTEN. Such a library is then screened in an in
vitro or in vivo assay suitable to assess a parameter in a given
clinical indication (e.g., cancer, metabolic disorder, diabetes) in
order to determine those compositions providing the optimal
response. In one exemplary embodiment, one category of payload
precursor includes various targeting modules, such as peptides
(e.g., the targeting moieties of Table 17) with binding affinity to
a tumor-associated antigen of Table 20, and the second category of
precursor is one or more drugs, such as a cytotoxic drug or a drug
chosen from Table 9. Each category of precursor to be linked is
further conjugated to a linker, and, as illustrated in FIG. 36, is
subsequently mixed and reacted with the other XTEN-payload
precursors capable of reacting with the linker under conditions to
effect the conjugation, resulting in a library of the various
targeting moieties and drug permutations in varying ratios to each
other. The XTEN-payload conjugates are designed to permit fixed
ratios of one payload to another, e.g., is 1:1, or 1:1.5, or 1:2,
or 1:3, or 2:3, or 1:4, or 1:5 or 1:9 in the case of two different
payloads. Similar ranges of ratios would be applied for library
conjugates comprising 3, 4, 5 or more payloads.
[0411] In other embodiments, the libraries are constructed using
three or more payloads known to have a beneficial effect in the
treatment of a common disease. In one embodiment, a library
comprises payloads linked to XTEN, wherein each payload is a drug
or biologically effective for ameliorating a common disease. In
another embodiment, a library comprises payloads linked to XTEN,
wherein each drug or biologic is effective for treating different
symptoms of a common disease. In another embodiment, a library
comprises payloads linked to XTEN, wherein each drug or biologic
mediates their therapeutic effect via a common biological pathway.
In the foregoing embodiments of the libraries, the common disease
is selected from cancer, cancer supportive care, cardiovascular,
central nervous system, endocrine disease, gastrointestinal,
genitourinary, hematological, HIV infection, hormonal system,
inflammation, autoimmune disease, infectious diseases, metabolic
disease, musculoskeletal disease, nephrology disorders,
ophthalmologic diseases, pain, and respiratory. With greater
particularity, the disease for which the libraries are constructed
with payloads known to have a beneficial effect is selected from
Table 16. Payloads suitable for use in the treatment or prevention
of such diseases include those described herein (e.g., the payloads
of Tables 11, 12, 18, and 21), or can be found in commonly
accessible databases or would otherwise be known to those of
ordinary skill in the art.
TABLE-US-00018 TABLE 16 Diseases for which Payloads are Indicated
Disease Achondroplasia Acromegaly AIDS Alzheimer's disease Anemia
Arthritis Asthma Atherosclerosis Autism Autoimmune disease Batten
disease Bone & cartilage repair Cachexia Cancer (all types)
Cardiovascular diseases Chemotherapy-induced diseases Chronic
kidney disease-induced complication Coagulation disorder Colitis
Congenital hyperinsulinism Congestive heart failure COPD Crohn's
disease Cystic fibrosis Diabetes Diabetes-induced complication
Diabetic nephropathy Diabetic neuropathy Diagnostic Eating disorder
Erythropoietic protoporphyria Gastrointestinal disorder Gout Growth
hormone deficiency Hemophilia Hepatitis B Hepatitis C Hereditary
emphysema HIV Hyperlipidemia/Dyslipidemia Hyperparathyroidism
Hypertension Hypoglycemia Hypoparathyroidism Hypothyroidism
Infectious diseases Infertility Inflammatory diseases Lipodystrophy
Lymphopenia Macular degeneration Metabolic conditions Mucositis
Multiple sclerosis Muscular dystrophy Musculoskeletal Myocardial
infarction/ischemia Neutropenia Obesity Osteoarthritis Osteoporosis
Pain Parkinsons disease Paroxysmal nocturnal hemoglobinuria
Phenylketonuria Psoriasis Pulmonary arterial hypertension Pulmonary
hypertension Radiotherapy- induced diseases Sepsis Sexual
dysfunction Short bowel syndrome Stroke Thrombocytopenia Thyroid
disease Transplantation Viral infection
[0412] In one embodiment, as illustrated in FIG. 37, the bispecific
conjugate has a drug module linked to the XTEN by a cleavable or
labile linker, wherein the linker can be cleaved or disassociates
after administration to a subject, including upon intracellular
internalization in a cell targeted by the targeting modules. In
another embodiment the drug is linked to XTEN by an non-cleavable
linker but the conjugate remains susceptible to degradation. Upon
internalization the XTEN is cleaved by proteases and the drug
connected to its linker is liberated resulting in cytotoxicity.
[0413] In one exemplary embodiment, the targeting module is
luteinizing hormone-releasing hormone (aka LHRH, GnRH, and
gonadotropin-releasing hormone), the drug is doxorubicin, wherein
the ratio of LHRH to doxorubicin is 1:1, or 1:1.5, or 1:2, or 1:3,
or 1:9, or 2:3, or 3:1, or 3:2, or 2:1, or 1.5:1. The conjugate can
be generated starting from XTEN precursors. One XTEN precursor can
carry 1, 2, or more drug molecules and a reactive cross-linker or
click chemistry reactant or a reactive amino acid. A second XEN
precursor carries 1, 2, or more LHRH domains for targeting and a
reactive cross-linker or click chemistry reactant or a reactive
amino acid. Both precursor segments are then joined by reaction
between reactive groups of the respective XTEN. In one exemplary
embodiment the reactive group is an azide that is conjugated to the
N-terminus of first XTEN segment via a cross-linker, and reactive
group of the second XTEN is an alkyne that is conjugated to the
N-terminus of the second XTEN segment via a cross-linker. In
another embodiment of the LHRH-XTEN-drug conjugate, the drug is
maytansin. In another embodiment of the LHRH-XTEN-drug conjugate
the drug is auristatin.
[0414] 8. Conjugates of XTEN-Payload Linked to Targeting
Moieties
[0415] In another aspect, the present invention provides conjugate
compositions comprising one or more XTEN-payload compositions
linked to targeting moieties. The subject targeted compositions
find use in the treatment of a variety of conditions where
selective delivery of a therapeutic or toxic payload to a cell,
tissue or organ is desired. The invention contemplates a diversity
of targeting moieties for use in the subject compositions,
including antibodies, antibody fragments, and antibody mimetics
including, but not limited to those set forth in Table 17, as well
as peptides and small molecules capable of binding ligands or
receptors associated with disease or metabolic or physiologic
abnormalities. In one embodiment, the invention provides a
conjugate comprising at least one targeting moiety from Tables 17,
18 or 21 linked to at least one XTEN. In another embodiment, the
invention provides a conjugate comprising at least one targeting
moiety from Tables 17, 18 or 21 linked to each of at least two, or
three, or four XTEN. In another embodiment, the invention provides
a conjugate comprising at least one targeting moiety from Tables
17, 18 or 21 linked to at least one XTEN and at least one drug or
biologic payload selected from the group consisting of the payloads
set forth in Table 11, Table 12, Table 18, or Table 21 linked to
the at least one XTEN. In one embodiment, the invention provides
targeting moiety-XTEN-drug conjugate compositions wherein the
composition is selectively delivered to a ligand or receptor on a
targeted cell, which can then be internalized into the cell, as
illustrated in FIG. 37, resulting in a pharmacologic effect know in
the art for the drug component.
[0416] As illustrated in FIGS. 21-24, and 28-32, such conjugate
compositions can have different valencies, with one, two, three, or
four or more XTEN-payload molecules linked to one or more targeting
antibody or targeting moiety. In the case of antibody targeting
moieties, in one embodiment the XTEN-payload is linked by a
cross-linker to a cysteine in a hinge region of the antibody. In
another embodiment, the XTEN-payload is linked by a disulfide
bridge to a cysteine in a hinge region of the antibody.
Accordingly, an antibody-XTEN-payload conjugate can comprise 1, 2,
3, or 4 or more XTEN-payload segments linked to the antibody,
antibody fragment or mimetic. In another embodiment XTEN is
conjugated outside of the hinge region, which includes inserting
cysteine in the antibody to control conjugation sites or by
conjugation to existing lysine side chains. The linking of
XTEN-payload to create the antibody conjugates has many benefits:
a) the XTEN payload serves as a cleavable linker, b) it provides
solubility, c) it allows setting the ratio of drugload per IgG, and
d) it can be pre-conjugated with drug to simplify
manufacturing.
TABLE-US-00019 TABLE 17 Targeting Moieties: Antibody fragments,
scaffolds and mimetics Targeting Moieties ABDURINS
AdNectins/Fibronectin type III domain Adnexins/Fibronectin
Affibodies/Protein Z Affilins AFFINILUTE AFFINIMIP AFIM
Anticalins/Lipocalins Aptabody Aptamers Armadillo repeat proteins
Avimers Azymetric Bispecific diabodies BiTEs Bivalent diabodies
Centyrins DARPins/Ankyrin repeat proteins Diabodies Domain
antibodies/dAbs/human Vh Engineered affinity proteins Evibodies
Fabs Fv Fynomers Glubody Im7/ColE7 immunity protein iMabs
Knottin/Cysteine-knot miniproteins Kunitz domains Maxibodies
Microbodies Minibodies Molecular imprinted polymers (MIPs)
Monobodies Monoclonal T cell receptors (mTCR) MonoLex Nanobodies
Nanofitins Phylomers Shark Vhh SMIPs SOMAmers Stable scFV
Spiegelmers Synbodies TandAbs .RTM. Telobodies Tetrabodies
Tetranectins Tetravalent bispecific antibodies Trans-body
Triabodies
[0417] In some embodiments, the invention provides conjugates
comprising a targeting component as one payload and a toxin as a
second payload, with one or more copies of each payload type linked
to the XTEN of the composition. In a variation of the foregoing,
the conjugate can optionally have the toxin linked to the XTEN with
a labile or a cleavable linker such that the toxin is liberated
when delivered to or is internalized within the target. In another
variation of the foregoing, the targeting component is an antibody
or antibody fragment, with one, two, three, or four XTEN-payload
compositions conjugated with linkers to the antibody (e.g.,
conjugated to cysteines in the hinge region as illustrated in FIGS.
28-29), providing conjugates for use in targeted therapy of
clinical indications such as, but not limited to, various treatment
of tumors and other cancers wherein the antibody provides the
targeting component and the XTEN-payload effects the intended
activity (e.g, cytotoxicity in a tumor cell). Hence, the inventive
conjugates are a type of immunoconjugate. The targeted conjugates
can be designed with targeting components that are derived from
antibodies, or antibody mimetics, or are peptides or small
molecules that bind ligands associated with diseases cells or
organs. Non-limiting examples of categories of antibody fragments,
scaffolds and mimetics are provided in Table 17. Non-limited
examples of specific targeting components, the targets to which
they are directed, and toxins that may be utilized as payloads in
the inventive conjugates are provided in Table 18. It is
specifically contemplated that the targeted conjugate compositions
of the present disclosure include compositions of any given
targeting component that can be used in combination with one or
more of any of the toxins of Table 18 or the payloads provided in
Table 11 or Table 12. It is further contemplated that an
XTEN-payload conjugate can comprise two or more targeting
components, which may be identical or may be different. It is
contemplated that such conjugates can be used in treatment of
conditions such as, but not limited to those set forth in Table
15.
TABLE-US-00020 TABLE 18 Exemplary targeting moieties, toxin
payloads, and targets to which conjugate compositions can be
directed Class Target Targeting Moiety Toxin Peptide LHRHR LHRH
& analogues (e.g. D- doxorubicin Lys-(6)-LHRH) paclitaxel CD13,
Aminopeptidase NGR class (e.g. CNGRC (SEQ auristatin (e.g. ID NO:
581), CNGRCG (SEQ monomethyl ID NO: 582), GNGRG (SEQ auristatin E;
ID NO: 583), KNGRE (SEQ ID monomethyl NO: 584), (GNGRG)2KGK
auristatin F) (SEQ ID NO: 585), maytansine (e.g. CVLNGRMEC (SEQ ID
NO: maytansinoid DM1; 586), NGRAHA (SEQ ID NO: maytansinoid DM4)
587), CNGRCVSGCAGRC dolastatin (SEQ ID NO: 588)) calicheamicin
Folate receptor Folate & analogue (e.g. .gamma.- vinca alkaloid
(e.g. folate, .alpha.-folate; pteroate-gly) desacetylvinblastine
Integrin Cilengitide; RGD-4C; iRGD monohydrazide) LRP receptor
Angiopep-2 camptothecin Somatostatin receptor Somatostatin &
analogues (e.g. mitomycin C octreotide; pasireotide; epothilone
lanreotide; vapreotide, JF-07- hTNF 69) IL-12 Nucleolin F3 peptide
Bortezomib PDGFR-beta RGR Ranpirnase LyP-1 receptor LyP-1;
CGNKRTRGC (SEQ pseudomonas ID NO: 589) exotoxin Chondroitin sulfate
TAASGVRSMH (SEQ ID NO: SN-38 proteoglygan NG2 590); LTLRWVGLMS (SEQ
Rachelmycin ID NO: 591) m-TOR inhibitor VPAC1 and VPAC2 Vasoactive
intestinal peptide rapamycin CCK1 and CCK2 Cholecystokinin
tubulysin (tubulysin Gastrin receptor, CCK1 & Gastrin B;
tubulysin M) CCK2 duocarmycin GRP receptor subtype
Gastrin-releasing peptide Neurotensin receptor Neurotensin
Alpha-MSH receptor Alpha-melanocyte stimulating hormone Oxytocin
receptor Oxytocin Lymphatic vessels LyP-2; CNRRTKAGC (SEQ ID NO:
592) Lymphatic vessels LSD; CLSDGKRKC (SEQ ID NO: 593) Lymphatic
vessels REA; CREAGRKAC (SEQ ID NO: 594) Lymphatic vessels AGR,
CAGRRSAYC (SEQ ID NO: 595) Pericytes & endothelia cells RSR;
CRSRKG (SEQ ID NO: 596) Pericytes & endothelia cells KAA;
CKAAKNK (SEQ ID NO: 597) Blood vessels CSRPRRSEC (SEQ ID NO: 598)
Angiogenic blood vessels & KRK; CGKRK (SEQ ID NO: tumor cells
599) Angiogenic blood vessels CDTRL (SEQ ID NO: 600) Angiogenic
blood vessels & CGTKRKC (SEQ ID NO: 601) tumor cells Protein
DR4, DRS TRAIL Various DARPINS Antibody-like Various Centyrins
scaffold Antibody Lewis-Y-related antigen Br96;
anti-Lewis-Y-related antigen antibody HER2 Trastuzumab; Pertuzumab;
anti-HER2 antibody EGFR Cetuximab; anti-EGFR antibody Nectin-4
anti-nectin-4 antibody CanAg (mucin-type huC242, anti-CanAg
antibody glycoprotein) CD138 anti-CD138 antibody CD19 MDX-1342;
MOR-208; HuB4; anti-CD19 antibody CD22 Epratuzumab; Bectumomab;
Inotuzumab; Moxetumomab, RFB4; anti-CD22 antibody CD23 Lumiliximab,
anti-CD23 antibody CD25 (IL-2 receptor) Daclizumab, anti-CD25
antibody CD30 Xmab2513; cAC10; MDX-060; anti-CD30 antibody CD33
Gemtuzumab; HuM195; huMy9-6; anti-CD33 antibody CD38 Daratumumab,
anti-CD38 antibody CD40 SGN-40; HCD122; anti-CD40 antibody CD56
huN901; anti-CD56 antibody CD70 MDX-1411; anti-CD70 antibody CD74
Milatuzumab; anti-CD74 antibody CD79b anti-CD79b antibody CD80
Galiximab; anti-CD80 antibody Carcinoembiyonic antigen Lapetuzumab,
hCOL-lanti-CEA (CEA) antibody Cripto anti-Cripto antibody cMET
CE-355621, DN30, MetMAb; antagonist anti-cMET antibody EpCAM
Adecatumumab; Edrecolomab; Catumaxomab; anti-EpCAM antibody EphA2
1C1, anti-EphA2 antibody GPNMB (human gylcoprotein glembatumumab,
anti-GPNMB NMB (osteoactivin)) antibody Integrins anti-integrin
antibody MUC-1 (epitope CA6) anti-MUC-1 antibody PSMA MDX-070,
MLN591, anti-PSMA antibody TGFa anti-TGFa antibody TIM1 anti-TIM1
antibody Folate receptor 1 M9346A, Farletuzinnab, anti- folate
receptor antibody IL-13 receptor anti-IL-13 receptor antibody
[0418] In particular embodiments, the invention provides
XTEN-payload conjugates comprising one or more LHRH targeting
components selected from Table 19 and one or more drug components
selected from Table 11. In the foregoing embodiment, the LHRH can
be linked to one XTEN segment that, in turn, is linked to one or
more XTEN segments to which the drug components are conjugated.
Alternatively, the LHRH and drug components can be conjugated to a
monomeric XTEN. Further, the drug components can optionally be
linked to XTEN using labile or cleavable linkers that permit the
drug to be liberated from the conjugate after administration to a
subject.
TABLE-US-00021 TABLE 19 Exemplary LHRH Composition SEQ ID NO:
pGlu-HWSYGLRPG-NH2 602 pGlu-HWSY[D-Lys]LRPG-NH2 603
pGlu-HWSY[D-Trp]LRPG-NH2 604 pGlu-HWSY[D-Leu]LRP-NHEt 605
pGlu-HWSY[D-Ser(tBu)]LRP-NHEt 606 pGlu-HWSY[D-2-Nal]LRPG-NH2 607
pGlu-HWSY[D-His(Bzl)]LRP-NHEt 608
pGlu-HWSY[D-Ser(tBu)]LRP-Azagly-NH2 609 pGlu-HWSY[D-Trp]LRP-NHEt
610 pGlu-HWSHDWLPG-NH2 611
[0419] Additional targets contemplated for which the XTEN-payload
conjugates can be directed include tumor-associated antigens listed
in Table 20. In one embodiment, the invention provides XTEN-payload
conjugates comprising one or more targeting components capable of
binding one or more targets of Table 20.
TABLE-US-00022 TABLE 20 Tumor-associated antigen targets TAA
targets (synonyms) Accession Number and References Her2 (ErbB2)
GenBank accession no. M11730; U.S. Pat. No. 5,869,445;
WO2004048938; WO2004027049; WO2004009622; WO2003081210;
WO2003089904; WO2003016475; US2003118592; WO2003008537;
WO2003055439; WO2003025228; WO200222636; WO200212341; WO200213847;
WO200214503; WO200153463; WO200141787; WO200044899; WO200020579;
WO9630514; EP1439393; WO2004043361; WO2004022709; WO200100244
BMPR1B (bone morphogenetic GenBank accession no. NM_001203;
WO2004063362; protein receptor-type IB) WO2003042661; US
2003134790; WO2002102235; WO2003055443; WO200299122; WO2003029421;
WO2003024392; WO200298358' WO200254940; WO200259377; WO200230268
E16 (LAT1, SLC7A5) GenBank accession no. NM_003486); WO2004048938;
WO2004032842; WO2003042661; WO2003016475; WO200278524; WO200299074;
WO200286443; WO2003003906; WO200264798; WO200014228; US2003224454;
WO2003025138 STEAP1 (six transmembrane GenBank accession no.
NM_012449; WO2004065577; epithelial antigen of prostate)
WO2004027049; EP1394274; WO2004016225; WO2003042661; US2003157089;
US2003185830; US2003064397; WO200289747618; WO2003022995 STEAP2
(six transmembrane GenBank accession no. AF455138; WO2003087306;
epithelial antigen of prostate 2) US2003064397; WO200272596;
WO200172962; WO2003104270; WO2003104270; US2004005598;
WO2003042661; US2003060612; WO200226822; WO200216429 CA125/0772P
(MUC16) GenBank accession no. AF361486; WO2004045553; WO200292836;
WO200283866; US2003124140 megakaryocyte potentiating factor GenBank
accession no. NM_005823; WO2003101283; (MPF, mesothelin)
WO2002102235; WO2002101075; WO200271928; WO9410312 Na/Pi
cotransporter type IIb GenBank accession no. NM_006424;
WO2004022778; (NaPi3b) EP1394274; WO2002102235; EP875569;
WO200157188; WO2004032842; WO200175177 Semaphorin 5b (SEMA5B,
GenBank accession no. AB040878; WO2004000997; SEMAG) WO2003003984;
WO200206339; WO200188133; WO2003054152; WO2003101400 Prostate
cancer stem cell antigen GenBank accession no. AY358628;
US2003129192; (PSCA hlg) US2004044180; US2004044179; US2003096961;
US2003232056; WO2003105758; US2003206918; EP1347046; WO2003025148
ETBR (Endothelin type B receptor) GenBank accession no. AY275463;
WO2004045516; WO2004048938; WO2004040000; WO2003087768;
WO2003016475; WO2003016475; WO200261087; WO2003016494;
WO2003025138; WO200198351; EP522868; WO200177172; US2003109676;
U.S. Pat. No. 6,518,404; U.S. Pat. No. 5,773,223; WO2004001004
TRPV4 (Transient receptor potential US Pat App No. 20090208514
cation channel, subfamily V) CDC45L GenBank Accession NO. AJ223728;
US Pat App No. 20090208514 CRIPTO (CR, CR1, CRGF) GenBank accession
no. NP_003203 or NM_003212; US2003224411; WO2003083041;
WO2003034984; WO200288170; WO2003024392; WO200216413; WO200222808;
U.S. Pat. No. 5,854,399; U.S. Pat. No. 5,792,616 CD21 (CR2
(Complement receptor GenBank accession no. M26004; WO2004045520; 2)
or C3DR (C3d/Epstein Barr virus US2004005538; WO2003062401;
WO2004045520; receptor) WO9102536; WO2004020595 CD79b (CD79B,
CD79.beta., IGb GenBank accession no. NM_000626 or 11038674;
(immunoglobulin-associated beta), WO2004016225; WO2003087768;
US2004101874; B29) WO2003062401; WO200278524; US2002150573; U.S.
Pat. No. 5,644,033; WO2003048202; WO 99/558658, U.S. Pat. No.
6,534,482; WO200055351 FcRH2 (IFGP4, IRTA4, SPAP1A GenBank
accession no. NM _030764, AY358130; (SH2 domain containing
WO2004016225; WO2003077836; WO200138490; phosphatase anchor protein
1a), WO2003097803; WO2003089624 SPAP1B, SPAP1C) NCA (CEACAM6)
GenBank accession no. M18728; WO2004063709; EP1439393;
WO2004044178; WO2004031238; WO2003042661; WO200278524; WO200286443;
WO200260317 MDP (DPEP1) GenBank accession no. BC017023;
WO2003016475; WO200264798 IL20R.alpha. (IL20Ra, ZCYTOR7) GenBank
accession no. AF184971; EP1394274; US2004005320; WO2003029262;
WO2003002717; WO200222153; US2002042366; WO200146261; WO200146232;
WO9837193 BECAN (Brevican core protein) GenBank accession no.
AF229053; US2003186372; US2003186373; US2003119131; US2003119122;
US2003119126; US2003119121; US2003119129; US2003119130;
US2003119128; US2003119125; WO2003016475; WO200202634 EphB2R (DRT,
ERK, Hek5, GenBank accession no. NM_004442; WO2003042661; EPHT3,
Tyro5) WO200053216; WO2004065576 (Claim 1); WO2004020583;
WO2003004529; WO200053216 B7h (ASLG659) GenBank accession no.
AX092328; US20040101899; WO2003104399; WO2004000221; US2003165504;
US2003124140; US2003065143; WO2002102235; US2003091580;
WO200210187; WO200194641; WO200202624; US2002034749; WO200206317;
WO200271928; WO200202587; WO200140269; WO200036107; WO2004053079;
WO2003004989; WO200271928 PSCA (Prostate stem cell antigen GenBank
accession no. AJ297436; WO2004022709; precursor EP1394274;
US2004018553; WO2003008537 (Claim 1); WO200281646; WO2003003906;
WO200140309; US2001055751; WO200032752; WO9851805; WO9851824;
WO9840403 BAFF-R (B cell-activating factor GenBank accession No.
AF116456; WO2004058309; receptor, BLyS receptor 3, BR3)
WO2004011611; WO2003045422; WO2003014294; WO2003035846;
WO200294852; WO200238766; WO200224909 CD22 (B-cell receptor
CD22-.beta.- GenBank accession No. AK026467; WO2003072036 form,
BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) CD79a
(immunoglobulin-associated GenBank accession No. NP_001774.10;
alpha) WO2003088808, US20030228319; WO2003062401; US2002150573;
WO9958658; WO9207574; U.S. Pat. No. 5,644,033 CXCR5 (Burkitt's
lymphoma GenBank accession No. NP_001707.1; receptor 1)
WO2004040000; WO2004015426; US2003105292; U.S. Pat. No. 6,555,339;
WO200261087; WO200157188; WO200172830; WO200022129; WO9928468; U.S.
Pat. No. 5,440,021; WO9428931; WO9217497 HLA-DOB GenBank accession
No. NP _002111.1; WO9958658; U.S. Pat. No. 6,153,408; U.S. Pat. No.
5,976,551; U.S. Pat. No. 6,011,146 P2X5 GenBank accession No.
NP_002552.2; WO2004047749; WO2003072035; WO200222660; WO2003093444;
WO2003087768; WO2003029277 CD72 (B-cell differentiation antigen
GenBank accession No. NP_001773.1; CD72, Lyb-2) WO2004042346;
WO2003026493; WO200075655 CD180 (LY64) GenBank accession No.
NP_005573.1; US2002193567; WO9707198; WO2003083047; WO9744452 FcRH1
(Fc receptor-like protein 1) GenBank accession No. NP_443170.1)
WO2003077836; WO200138490; WO2003089624; EP1347046; WO2003089624
IRTA2 (Immunoglobulin GenBank accession No. Human: AF343662,
AF343663, superfamily receptor translocation AF343664, AF343665,
AF369794, AF397453; associated 2) WO2003024392; WO2003077836;
WO200138490 TENB2 (TMEFF2, tomoregulin, GenBank accession No.
AF179274; AY358907, TPEF, HPP1) CAF85723, CQ782436; WO2004074320;
WO2003042661; WO2003009814; EP1295944; WO200230268; WO200190304;
US2004249130; US2004022727; WO2004063355; US2004197325;
US2003232350; US2004005563; US2003124579 CS1 (CRACC, 19A, APEX-1,
GenBank Accession No. NM 021181; US 20100168397 FOAP12) DLL4
GenBank Accession No. NM 019074; US 20100303812 Lewis Y ADB235860;
U.S. Pat. No. 7,879,983 CD40 (Bp50, CDW40, MGC9013, AL035662.65;
U.S. Pat. No. 6,946,129 TNFRSF5, p50) OBA1 (5T4) GenBank Accession
No. NP_001159864.1; US 20100021483 p97 Woodbury et al., 1980, Proc.
Natl. Acad. Sci. USA 77: 2183-2186; Brown et al., 1981, J. Immunol.
127: 539- 546 carcinoembryonic antigen (CEA) GenBank Accession No.
NP_004354.2; U.S. Pat. No. 6,676,924 TAG-72 U.S. Pat. No. 7,256,004
DNA Neuropilin-1 (NRP1) GenBank Accession No. NP_001019799.1; US
20080213268 A33 GenBank Accession No. NP_005805.1; U.S. Pat. No.
7,579,187 Mucin-1 (MUC1) GenBank Accession No. NP_001018016.1;
NP_001018017.1; U.S. Pat. No. 7,183,388 ED-B fibronectin U.S. Pat.
No. 7,785,591 Thomsen-Friedenreich antigen (TF) U.S. Pat. No.
7,374,755; US 20100297159 Bombesin receptor U.S. Pat. No. 5,750,370
CanAg Carcinoembryonic antigen (CEA) U.S. Pat. No. 4,818,709 CD13
CD138 CD30 CD47 CD56 CD70 Chondroitin sulfate proteoglygan NG2
EphA2 Folate receptor 1 U.S. Pat. No. 5,547,668 gastrin receptor
GPNMB (human gylcoprotein) NMB (osteoactivin) GRP receptor subtype
integrin avb3 US20110166072 LHRHR US20110104074 LRP receptor LyP-1
receptor Nectin-4 Neurotensin receptor U.S. Pat. No. 8,058,230
Nucleolin Somatostatin receptor TIM1 VPAC1 VPAC2 Alpha-MSH receptor
CD25 Interleukin-1 receptor
[0420] In particular embodiments, the invention provides
XTEN-payload conjugates comprising one, two or more targeting
components and one, two or more drug components conjugated to XTEN.
Non-limiting embodiments of specific conjugate compositions are
provided in Table 21, in which the named composition of column 2
has specified components of: i) XTEN sequences of Table 3
designated in the XTEN column of the Table; ii) targeting moiety
payloads specified in the Targeting Moiety column of the Table that
provide targeting capability of the composition (with the number of
moieties specified; e.g., 1.times. or 3.times.); and iii) drug
pharmacophore specified in the Drug Moiety column of the Table
(with the number of drug molecules conjugated to the XTEN; e.g.,
3.times. or 9.times.). As would be appreciated by one of skill in
the art, the invention contemplates other combinations of the
disclosed components, as well as different numbers or ratios of the
respective specified components, as well as different XTEN
sequences to which the payloads are conjugated. For example, the
invention contemplates that the number of drug moieties attached to
a given XTEN can be 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or
9, or 10 or more and that the XTEN would have, for example, the
corresponding number of cysteine or lysine residues to which the
drug moieties would be conjugated. Further, the invention
contemplates that the number of targeting moieties attached to the
conjugate can be 1, or 2, or 3 or more, which would similarly be
linked to XTEN with an N-terminal amino group or a corresponding
number of lysine or cysteine residues.
TABLE-US-00023 TABLE 21 Exemplary conjugates XTEN XTEN linked to
linked to Targeting Drug Targeting Drug Conjugate Conjugate Name*
Moiety** Moiety** Moiety* Moiety* 1 1xLHRH-XTEN-3xDox Seg 174
1xLHRH 3xDoxorubicin 2 1xLHRH-XTEN-9xDox Seg 175 1xLHRH
9xDoxorubicin 3 3xLHRH-XTEN-3xDox Seg 176 Seg 176 3xLHRH
3xDoxorubicin 4 3xLHRH-XTEN-9xDox Seg 176 Seg 177 3xLHRH
9xDoxorubicin 5 XTEN-3xDox Seg 174 3xDoxorubicin 6 XTEN-9xDox Seg
175 9xDoxorubicin 7 1xLHRH-XTEN-3xMMAE Seg 174 1xLHRH 3xMMAE *** 8
1xLHRH-XTEN-9xMMAE Seg 175 1xLHRH 9xMMAE 9 3xLHRH-XTEN-3xMMAE Seg
176 Seg 176 3xLHRH 3xMMAE 10 3xLHRH-XTEN-9xMMAE Seg 176 Seg 177
3xLHRH 9xMMAE 11 XTEN-3xMMAE Seg 174 3xMMAE 12 XTEN-9xMMAE Seg 175
9xMMAE 13 1xLHRH-XTEN-3xMMAF Seg 174 1xLHRH 3xMMAF **** 14
1xLHRH-XTEN-9xMMAF Seg 175 1xLHRH 9xMMAF 15 3xLHRH-XTEN-3xMMAF Seg
176 Seg 176 3xLHRH 3xMMAF 16 3xLHRH-XTEN-9xMMAF Seg 176 Seg 177
3xLHRH 9xMMAF 17 XTEN-3xMMAF Seg 174 3xMMAF 18 XTEN-9xMMAF Seg 175
9xMMAF 19 1xLHRH-XTEN- Seg 174 1xLHRH 3xMertansine 3xMertansine 20
1xLHRH-XTEN- Seg 175 1xLHRH 9xMertansine 9xMertansine 21
3xLHRH-XTEN- Seg 176 Seg 176 3xLHRH 3xMertansine 3xMertansine 22
3xLHRH-XTEN- Seg 176 Seg 177 3xLHRH 9xMertansine 9xMertansine 23
XTEN-3xMertansine Seg 174 3xMertansine 24 XTEN-9xMertansine Seg 175
9xMertansine 25 1xLHRH-XTEN- Seg 174 1xLHRH 3xMaytansinoid
3xMaytansinoid DM4 DM4 26 1xLHRH-XTEN- Seg 175 1xLHRH
9xMaytansinoid 9xMaytansinoid DM4 DM4 27 3xLHRH-XTEN- Seg 176 Seg
176 3xLHRH 3xMaytansinoid 3xMaytansinoid DM4 DM4 28 3xLHRH-XTEN-
Seg 176 Seg 177 3xLHRH 9xMaytansinoid 9xMaytansinoid DM4 DM4 29
XTEN-3xMaytansinoid Seg 174 3xMaytansinoid DM4 DM4 30
XTEN-9xMaytansinoid Seg 175 9xMaytansinoid DM4 DM4 31 1xLHRH-XTEN-
Seg 174 1xLHRH 3xPaclitaxel 3xPaclitaxel 32 1xLHRH-XTEN- Seg 175
1xLHRH 9xPaclitaxel 9xPaclitaxel 33 3xLHRH-XTEN- Seg 176 Seg 176
3xLHRH 3xPaclitaxel 3xPaclitaxel 34 3xLHRH-XTEN- Seg 176 Seg 177
3xLHRH 9xPaclitaxel 9xPaclitaxel 35 XTEN-3xPaclitaxel Seg 174
3xPaclitaxel 36 XTEN-9xPaclitaxel Seg 175 9xPaclitaxel 37
1xLHRH-XTEN-3xSN-38 Seg 174 1xLHRH 3xSN-38 38 1xLHRH-XTEN-9xSN-38
Seg 175 1xLHRH 9xSN-38 39 3xLHRH-XTEN-3xSN-38 Seg 176 Seg 176
3xLHRH 3xSN-38 40 3xLHRH-XTEN-9xSN-38 Seg 176 Seg 177 3xLHRH
9xSN-38 41 XTEN-3xSN-38 Seg 174 3xSN-38 42 XTEN-9xSN-38 Seg 175
9xSN-38 43 1xLHRH-XTEN- Seg 174 1xLHRH 3xGemcitabine 3xGemcitabine
44 1xLHRH-XTEN- Seg 175 1xLHRH 9xGemcitabine 9xGemcitabine 45
3xLHRH-XTEN- Seg 176 Seg 176 3xLHRH 3xGemcitabine 3xGemcitabine 46
3xLHRH-XTEN- Seg 176 Seg 177 3xLHRH 9xGemcitabine 9xGemcitabine 47
XTEN-3xGemcitabine Seg 174 3xGemcitabine 48 XTEN-9xGemcitabine Seg
175 9xGemcitabine 49 1xLHRH-XTEN- Seg 174 1xLHRH 3xCarboplatin
3xCarboplatin 50 1xLHRH-XTEN- Seg 175 1xLHRH 9xCarboplatin
9xCarboplatin 51 3xLHRH-XTEN- Seg 176 Seg 176 3xLHRH 3xCarboplatin
3xCarboplatin 52 3xLHRH-XTEN- Seg 176 Seg 177 3xLHRH 9xCarboplatin
9xCarboplatin 53 XTEN-3xCarboplatin Seg 174 3xCarboplatin 54
XTEN-9xCarboplatin Seg 175 9xCarboplatin 55 1xLHRH-XTEN-Human
1xLHRH Human RNase RNase 56 3xLHRH-XTEN-Human 3xLHRH Human RNase
RNase 57 1xLHRH-XTEN-Bovine 1xLHRH Bovine RNase RNase 58
3xLHRH-XTEN-Bovine 3xLHRH Bovine RNase RNase 59
1xLHRH-XTEN-Ranpirnase 1xLHRH Ranpirnase 60 3xLHRH-XTEN-Ranpirnase
3xLHRH Ranpirnase 61 1xLHRH-XTEN-Pokeweed antiviral protein 1xLHRH
Pokeweed antiviral protein 62 3xLHRH-XTEN-Pokeweed antiviral
protein 3xLHRH Pokeweed antiviral protein 63 1xfolate-XTEN-3xDox
Seg 174 1xfolate 3xDoxorubicin 64 1xfolate-XTEN-9xDox Seg 175
1xfolate 9xDoxorubicin 65 3xfolate-XTEN-3xDox Seg 176 Seg 176
3xfolate 3xDoxorubicin 66 3xfolate-XTEN-9xDox Seg 176 Seg 177
3xfolate 9xDoxorubicin 67 XTEN-3xDox Seg 174 3xDoxorubicin 68
XTEN-9xDox Seg 175 9xDoxorubicin 69 1xfolate-XTEN-3xMMAE Seg 174
1xfolate 3xMMAE 70 1xfolate-XTEN-9xMMAE Seg 175 1xfolate 9xMMAE 71
3xfolate-XTEN-3xMMAE Seg 176 Seg 176 3xfolate 3xMMAE 72
3xfolate-XTEN-9xMMAE Seg 176 Seg 177 3xfolate 9xMMAE 73 XTEN-3xMMAE
Seg 174 3xMMAE 74 XTEN-9xMMAE Seg 175 9xMMAE 75
1xfolate-XTEN-3xMMAF Seg 174 1xfolate 3xMMAF 76
1xfolate-XTEN-9xMMAF Seg 175 1xfolate 9xMMAF 77
3xfolate-XTEN-3xMMAF Seg 176 Seg 176 3xfolate 3xMMAF 78
3xfolate-XTEN-9xMMAF Seg 176 Seg 177 3xfolate 9xMMAF 79 XTEN-3xMMAF
Seg 174 3xMMAF 80 XTEN-9xMMAF Seg 175 9xMMAF 81
1xfolate-XTEN-3xMertansine Seg 174 1xfolate 3xMertansine 82
1xfolate-XTEN-9xMertansine Seg 175 1xfolate 9xMertansine 83
3xfolate-XTEN-3xMertansine Seg 176 Seg 176 3xfolate 3xMertansine 84
3xfolate-XTEN-9xMertansine Seg 176 Seg 177 3xfolate 9xMertansine 85
XTEN-3xMertansine Seg 174 3xMertansine 86 XTEN-9xMertansine Seg 175
9xMertansine 87 1xfolate-XTEN- Seg 174 1xfolate 3xMaytansinoid
3xMaytansinoid DM4 DM4 88 1xfolate-XTEN- Seg 175 1xfolate
9xMaytansinoid 9xMaytansinoid DM4 DM4 89 3xfolate-XTEN- Seg 176 Seg
176 3xfolate 3xMaytansinoid 3xMaytansinoid DM4 DM4 90
3xfolate-XTEN- Seg 176 Seg 177 3xfolate 9xMaytansinoid
9xMaytansinoid DM4 DM4 91 XTEN-3xMaytansinoid DM4 Seg 174
3xMaytansinoid DM4 92 XTEN-9xMaytansinoid DM4 Seg 175
9xMaytansinoid DM4 93 1xfolate-XTEN-3xPaclitaxel Seg 174 1xfolate
3xPaclitaxel 94 1xfolate-XTEN-9xPaclitaxel Seg 175 1xfolate
9xPaclitaxel 95 3xfolate-XTEN-3xPaclitaxel Seg 176 Seg 176 3xfolate
3xPaclitaxel 96 3xfolate-XTEN-9xPaclitaxel Seg 176 Seg 177 3xfolate
9xPaclitaxel 97 XTEN-3xPaclitaxel Seg 174 3xPaclitaxel 98
XTEN-9xPaclitaxel Seg 175 9xPaclitaxel 99 1xfolate-XTEN-3xSN-38 Seg
174 1xfolate 3xSN-38 100 1xfolate-XTEN-9xSN-38 Seg 175 1xfolate
9xSN-38 101 3xfolate-XTEN-3xSN-38 Seg 176 Seg 176 3xfolate 3xSN-38
102 3xfolate-XTEN-9xSN-38 Seg 176 Seg 177 3xfolate 9xSN-38 103
XTEN-3xSN-38 Seg 174 3xSN-38 104 XTEN-9xSN-38 Seg 175 9xSN-38 105
1xfolate-XTEN- Seg 174 1xfolate 3xGemcitabine 3xGemcitabine 106
1xfolate-XTEN- Seg 175 1xfolate 9xGemcitabine 9xGemcitabine 107
3xfolate-XTEN- Seg 176 Seg 176 3xfolate 3xGemcitabine 3xGemcitabine
108 3xfolate-XTEN- Seg 176 Seg 177 3xfolate 9xGemcitabine
9xGemcitabine 109 XTEN-3xGemcitabine Seg 174 3xGemcitabine 110
XTEN-9xGemcitabine Seg 175 9xGemcitabine 111
1xfolate-XTEN-3xCarboplatin Seg 174 1xfolate 3xCarboplatin 112
1xfolate-XTEN-9xCarboplatin Seg 175 1xfolate 9xCarboplatin 113
3xfolate-XTEN-3xCarboplatin Seg 176 Seg 176 3xfolate 3xCarboplatin
114 3xfolate-XTEN-9xCarboplatin Seg 176 Seg 177 3xfolate
9xCarboplatin 115 XTEN-3xCarboplatin Seg 174 3xCarboplatin 116
XTEN-9xCarboplatin Seg 175 9xCarboplatin 117
1xoctreotide-XTEN-3xDox Seg 174 1xoctreotide 3xDoxorubicin 118
1xoctreotide-XTEN-9xDox Seg 175 1xoctreotide 9xDoxorubicin 119
3xoctreotide-XTEN-3xDox Seg 176 Seg 176 3xoctreotide 3xDoxorubicin
120 3xoctreotide-XTEN-9xDox Seg 176 Seg 177 3xoctreotide
9xDoxorubicin 121 XTEN-3xDox Seg 174 3xDoxorubicin 122 XTEN-9xDox
Seg 175 9xDoxorubicin 123 1xoctreotide-XTEN-3xMMAE Seg 174
1xoctreotide 3xMMAE 124 1xoctreotide-XTEN-9xMMAE Seg 175
1xoctreotide 9xMMAE 125 3xoctreotide-XTEN-3xMMAE Seg 176 Seg 176
3xoctreotide 3xMMAE 126 3xoctreotide-XTEN-9xMMAE Seg 176 Seg 177
3xoctreotide 9xMMAE 127 XTEN-3xMMAE Seg 174 3xMMAE 128 XTEN-9xMMAE
Seg 175 9xMMAE 129 1xoctreotide-XTEN-3xMMAF Seg 174 1xoctreotide
3xMMAF 130 1xoctreotide-XTEN-9xMMAF Seg 175 1xoctreotide 9xMMAF 131
3xoctreotide-XTEN-3xMMAF Seg 176 Seg 176 3xoctreotide 3xMMAF 132
3xoctreotide-XTEN-9xMMAF Seg 176 Seg 177 3xoctreotide 9xMMAF 133
XTEN-3xMMAF Seg 174 3xMMAF 134 XTEN-9xMMAF Seg 175 9xMMAF 135
1xoctreotide-XTEN- Seg 174 1xoctreotide 3xMertansine 3xMertansine
136 1xoctreotide-XTEN- Seg 175 1xoctreotide 9xMertansine
9xMertansine 137 3xoctreotide-XTEN- Seg 176 Seg 176 3xoctreotide
3xMertansine 3xMertansine 138 3xoctreotide-XTEN- Seg 176 Seg 177
3xoctreotide 9xMertansine 9xMertansine 139 XTEN-3xMertansine Seg
174 3xMertansine 140 XTEN-9xMertansine Seg 175 9xMertansine 141
1xoctreotide-XTEN- Seg 174 1xoctreotide 3xMaytansinoid
3xMaytansinoid DM4 DM4 142 1xoctreotide-XTEN- Seg 175 1xoctreotide
9xMaytansinoid 9xMaytansinoid DM4 DM4 143 3xoctreotide-XTEN- Seg
176 Seg 176 3xoctreotide 3xMaytansinoid 3xMaytansinoid DM4 DM4 144
3xoctreotide-XTEN- Seg 176 Seg 177 3xoctreotide 9xMaytansinoid
9xMaytansinoid DM4 DM4 145 XTEN-3xMaytansinoid DM4 Seg 174
3xMaytansinoid DM4 146 XTEN-9xMaytansinoid DM4 Seg 175
9xMaytansinoid DM4 147 1xoctreotide-XTEN- Seg 174 1xoctreotide
3xPaclitaxel 3xPaclitaxel 148 1xoctreotide-XTEN- Seg 175
1xoctreotide 9xPaclitaxel 9xPaclitaxel 149 3xoctreotide-XTEN- Seg
176 Seg 176 3xoctreotide 3xPaclitaxel 3xPaclitaxel 150
3xoctreotide-XTEN- Seg 176 Seg 177 3xoctreotide 9xPaclitaxel
9xPaclitaxel 151 XTEN-3xPaclitaxel Seg 174 3xPaclitaxel 152
XTEN-9xPaclitaxel Seg 175 9xPaclitaxel 153
1xoctreotide-XTEN-3xSN-38 Seg 174 1xoctreotide 3xSN-38 154
1xoctreotide-XTEN-9xSN-38 Seg 175 1xoctreotide 9xSN-38 155
3xoctreotide-XTEN-3xSN-38 Seg 176 Seg 176 3xoctreotide 3xSN-38 156
3xoctreotide-XTEN-9xSN-38 Seg 176 Seg 177 3xoctreotide 9xSN-38 157
XTEN-3xSN-38 Seg 174 3xSN-38 158 XTEN-9xSN-38 Seg 175 9xSN-38 159
1xoctreotide-XTEN- Seg 174 1xoctreotide 3xGemcitabine 3xGemcitabine
160 1xoctreotide-XTEN- Seg 175 1xoctreotide 9xGemcitabine
9xGemcitabine 161 3xoctreotide-XTEN- Seg 176 Seg 176 3xoctreotide
3xGemcitabine 3xGemcitabine 162 3xoctreotide-XTEN- Seg 176 Seg 177
3xoctreotide 9xGemcitabine 9xGemcitabine 163 XTEN-3xGemcitabine Seg
174 3xGemcitabine 164 XTEN-9xGemcitabine Seg 175 9xGemcitabine 165
1xoctreotide-XTEN- Seg 174 1xoctreotide 3xCarboplatin 3xCarboplatin
166 1xoctreotide-XTEN- Seg 175 1xoctreotide 9xCarboplatin
9xCarboplatin 167 3xoctreotide-XTEN- Seg 176 Seg 176 3xoctreotide
3xCarboplatin 3xCarboplatin 168 3xoctreotide-XTEN- Seg 176 Seg 177
3xoctreotide 9xCarboplatin 9xCarboplatin 169 XTEN-3xCarboplatin Seg
174 3xCarboplatin 170 XTEN-9xCarboplatin Seg 175 9xCarboplatin 171
1xoctreotide-XTEN- Seg 174 1xoctreotide 3xEverolimus 3xEverolimus
172 1xoctreotide-XTEN- Seg 175 1xoctreotide 9xEverolimus
9xEverolimus 173 3xoctreotide-XTEN- Seg 176 Seg 176 3xoctreotide
3xEverolimus 3xEverolimus 174 3xoctreotide-XTEN- Seg 176 Seg 177
3xoctreotide 9xEverolimus 9xEverolimus
175 XTEN-3xEverolimus Seg 174 3xEverolimus 176 XTEN-9xEverolimus
Seg 175 9xEverolimus 177 1xpasireotide-XTEN-3xDox Seg 174
1xpasireotide 3xDoxorubicin 178 1xpasireotide-XTEN-9xDox Seg 175
1xpasireotide 9xDoxorubicin 179 3xpasireotide-XTEN-3xDox Seg 176
Seg 176 3xpasireotide 3xDoxorubicin 180 3xpasireotide-XTEN-9xDox
Seg 176 Seg 177 3xpasireotide 9xDoxorubicin 181 XTEN-3xDox Seg 174
3xDoxorubicin 182 XTEN-9xDox Seg 175 9xDoxorubicin 183
1xpasireotide-XTEN- Seg 174 1xpasireotide 3xMMAE 3xMMAE 184
1xpasireotide-XTEN- Seg 175 1xpasireotide 9xMMAE 9xMMAE 185
3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide 3xMMAE 3xMMAE 186
3xpasireotide-XTEN- Seg 176 Seg 177 3xpasireotide 9xMMAE 9xMMAE 187
XTEN-3xMMAE Seg 174 3xMMAE 188 XTEN-9xMMAE Seg 175 9xMMAE 189
1xpasireotide-XTEN- Seg 174 1xpasireotide 3xMMAF 3xMMAF 190
1xpasireotide-XTEN- Seg 175 1xpasireotide 9xMMAF 9xMMAF 191
3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide 3xMMAF 3xMMAF 192
3xpasireotide-XTEN- Seg 176 Seg 177 3xpasireotide 9xMMAF 9xMMAF 193
XTEN-3xMMAF Seg 174 3xMMAF 194 XTEN-9xMMAF Seg 175 9xMMAF 195
1xpasireotide-XTEN- Seg 174 1xpasireotide 3xMertansine 3xMertansine
196 1xpasireotide-XTEN- Seg 175 1xpasireotide 9xMertansine
9xMertansine 197 3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide
3xMertansine 3xMertansine 198 3xpasireotide-XTEN- Seg 176 Seg 177
3xpasireotide 9xMertansine 9xMertansine 199 XTEN-3xMertansine Seg
174 3xMertansine 200 XTEN-9xMertansine Seg 175 9xMertansine 201
1xpasireotide-XTEN- Seg 174 1xpasireotide 3xMaytansinoid
3xMaytansinoid DM4 DM4 202 1xpasireotide-XTEN- Seg 175
1xpasireotide 9xMaytansinoid 9xMaytansinoid DM4 DM4 203
3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide 3xMaytansinoid
3xMaytansinoid DM4 DM4 204 3xpasireotide-XTEN- Seg 176 Seg 177
3xpasireotide 9xMaytansinoid 9xMaytansinoid DM4 DM4 205
-XTEN-3xMaytansinoid DM4 Seg 174 3xMaytansinoid DM4 206
-XTEN-9xMaytansinoid DM4 Seg 175 9xMaytansinoid DM4 207
1xpasireotide-XTEN- Seg 174 1xpasireotide 3xPaclitaxel 3xPaclitaxel
208 1xpasireotide-XTEN- Seg 175 1xpasireotide 9xPaclitaxel
9xPaclitaxel 209 3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide
3xPaclitaxel 3xPaclitaxel 210 3xpasireotide-XTEN- Seg 176 Seg 177
3xpasireotide 9xPaclitaxel 9xPaclitaxel 211 XTEN-3xPaclitaxel Seg
174 3xPaclitaxel 212 XTEN-9xPaclitaxel Seg 175 9xPaclitaxel 213
1xpasireotide-XTEN-3xSN-38 Seg 174 1xpasireotide 3xSN-38 214
1xpasireotide-XTEN-9xSN-38 Seg 175 1xpasireotide 9xSN-38 215
3xpasireotide-XTEN-3xSN-38 Seg 176 Seg 176 3xpasireotide 3xSN-38
216 3xpasireotide-XTEN-9xSN-38 Seg 176 Seg 177 3xpasireotide
9xSN-38 217 XTEN-3xSN-38 Seg 174 3xSN-38 218 XTEN-9xSN-38 Seg 175
9xSN-38 219 1xpasireotide-XTEN- Seg 174 1xpasireotide 3xGemcitabine
3xGemcitabine 220 1xpasireotide-XTEN- Seg 175 1xpasireotide
9xGemcitabine 9xGemcitabine 221 3xpasireotide-XTEN- Seg 176 Seg 176
3xpasireotide 3xGemcitabine 3xGemcitabine 222 3xpasireotide-XTEN-
Seg 176 Seg 177 3xpasireotide 9xGemcitabine 9xGemcitabine 223
-XTEN-3xGemcitabine Seg 174 3xGemcitabine 224 -XTEN-9xGemcitabine
Seg 175 9xGemcitabine 225 1xpasireotide-XTEN- Seg 174 1xpasireotide
3xCarboplatin 3xCarboplatin 226 1xpasireotide-XTEN- Seg 175
1xpasireotide 9xCarboplatin 9xCarboplatin 227 3xpasireotide-XTEN-
Seg 176 Seg 176 3xpasireotide 3xCarboplatin 3xCarboplatin 228
3xpasireotide-XTEN- Seg 176 Seg 177 3xpasireotide 9xCarboplatin
9xCarboplatin 229 -XTEN-3xCarboplatin Seg 174 3xCarboplatin 230
-XTEN-9xCarboplatin Seg 175 9xCarboplatin 231 1xpasireotide-XTEN-
Seg 174 1xpasireotide 3xEverolimus 3xEverolimus 232
1xpasireotide-XTEN- Seg 175 1xpasireotide 9xEverolimus 9xEverolimus
233 3xpasireotide-XTEN- Seg 176 Seg 176 3xpasireotide 3xEverolimus
3xEverolimus 234 3xpasireotide-XTEN- Seg 176 Seg 177 3xpasireotide
9xEverolimus 9xEverolimus 235 XTEN-3xEverolimus Seg 174
3xEverolimus 236 XTEN-9xEverolimus Seg 175 9xEverolimus 237
1xbombesin-XTEN-3xDox Seg 174 1xbombesin 3xDoxorubicin 238
1xbombesin-XTEN-9xDox Seg 175 1xbombesin 9xDoxorubicin 239
3xbombesin-XTEN-3xDox Seg 176 Seg 176 3xbombesin 3xDoxorubicin 240
3xbombesin-XTEN-9xDox Seg 176 Seg 177 3xbombesin 9xDoxorubicin 241
XTEN-3xDox Seg 174 3xDoxorubicin 242 XTEN-9xDox Seg 175
9xDoxorubicin 243 1xbombesin-XTEN-3xMMAE Seg 174 1xbombesin 3xMMAE
244 1xbombesin-XTEN-9xMMAE Seg 175 1xbombesin 9xMMAE 245
3xbombesin-XTEN-3xMMAE Seg 176 Seg 176 3xbombesin 3xMMAE 246
3xbombesin-XTEN-9xMMAE Seg 176 Seg 177 3xbombesin 9xMMAE 247
XTEN-3xMMAE Seg 174 3xMMAE 248 XTEN-9xMMAE Seg 175 9xMMAE 249
1xbombesin-XTEN-3xMMAF Seg 174 1xbombesin 3xMMAF 250
1xbombesin-XTEN-9xMMAF Seg 175 1xbombesin 9xMMAF 251
3xbombesin-XTEN-3xMMAF Seg 176 Seg 176 3xbombesin 3xMMAF 252
3xbombesin-XTEN-9xMMAF Seg 176 Seg 177 3xbombesin 9xMMAF 253
XTEN-3xMMAF Seg 174 3xMMAF 254 XTEN-9xMMAF Seg 175 9xMMAF 255
1xbombesin-XTEN- Seg 174 1xbombesin 3xMertansine 3xMertansine 256
1xbombesin-XTEN- Seg 175 1xbombesin 9xMertansine 9xMertansine 257
3xbombesin-XTEN- Seg 176 Seg 176 3xbombesin 3xMertansine
3xMertansine 258 3xbombesin-XTEN- Seg 176 Seg 177 3xbombesin
9xMertansine 9xMertansine 259 XTEN-3xMertansine Seg 174
3xMertansine 260 XTEN-9xMertansine Seg 175 9xMertansine 261
1xbombesin-XTEN- Seg 174 1xbombesin 3xMaytansinoid 3xMaytansinoid
DM4 DM4 262 1xbombesin-XTEN- Seg 175 1xbombesin 9xMaytansinoid
9xMaytansinoid DM4 DM4 263 3xbombesin-XTEN- Seg 176 Seg 176
3xbombesin 3xMaytansinoid 3xMaytansinoid DM4 DM4 264
3xbombesin-XTEN- Seg 176 Seg 177 3xbombesin 9xMaytansinoid
9xMaytansinoid DM4 DM4 265 XTEN-3xMaytansinoid DM4 Seg 174
3xMaytansinoid DM4 266 XTEN-9xMaytansinoid DM4 Seg 175
9xMaytansinoid DM4 267 1xbombesin-XTEN- Seg 174 1xbombesin
3xPaclitaxel 3xPaclitaxel 268 1xbombesin-XTEN- Seg 175 1xbombesin
9xPaclitaxel 9xPaclitaxel 269 3xbombesin-XTEN- Seg 176 Seg 176
3xbombesin 3xPaclitaxel 3xPaclitaxel 270 3xbombesin-XTEN- Seg 176
Seg 177 3xbombesin 9xPaclitaxel 9xPaclitaxel 271 XTEN-3xPaclitaxel
Seg 174 3xPaclitaxel 272 XTEN-9xPaclitaxel Seg 175 9xPaclitaxel 273
1xbombesin-XTEN-3xSN-38 Seg 174 1xbombesin 3xSN-38 274
1xbombesin-XTEN-9xSN-38 Seg 175 1xbombesin 9xSN-38 275
3xbombesin-XTEN-3xSN-38 Seg 176 Seg 176 3xbombesin 3xSN-38 276
3xbombesin-XTEN-9xSN-38 Seg 176 Seg 177 3xbombesin 9xSN-38 277
XTEN-3xSN-38 Seg 174 3xSN-38 278 XTEN-9xSN-38 Seg 175 9xSN-38 279
1xbombesin-XTEN- Seg 174 1xbombesin 3xGemcitabine 3xGemcitabine 280
1xbombesin-XTEN- Seg 175 1xbombesin 9xGemcitabine 9xGemcitabine 281
3xbombesin-XTEN- Seg 176 Seg 176 3xbombesin 3xGemcitabine
3xGemcitabine 282 3xbombesin-XTEN- Seg 176 Seg 177 3xbombesin
9xGemcitabine 9xGemcitabine 283 XTEN-3xGemcitabine Seg 174
3xGemcitabine 284 XTEN-9xGemcitabine Seg 175 9xGemcitabine 285
1xbombesin-XTEN- Seg 174 1xbombesin 3xCarboplatin 3xCarboplatin 286
1xbombesin-XTEN- Seg 175 1xbombesin 9xCarboplatin 9xCarboplatin 287
3xbombesin-XTEN- Seg 176 Seg 176 3xbombesin 3xCarboplatin
3xCarboplatin 288 3xbombesin-XTEN- Seg 176 Seg 177 3xbombesin
9xCarboplatin 9xCarboplatin 289 XTEN-3xCarboplatin Seg 174
3xCarboplatin 290 XTEN-9xCarboplatin Seg 175 9xCarboplatin *1x, 3x,
9x refers to the number of the indicated moiety conjugated to the
XTEN **refers to XTEN sequence from Table 3; e.g., Seg 174 ***
Monomethyl auristatin E **** Monomethyl auristatin F
V). Pharmaceutical Compositions
[0421] The present invention provides pharmaceutical compositions
comprising XTEN-payload conjugates of the disclosure. In one
embodiment, the pharmaceutical composition comprises a conjugate
selected from the group consisting of the conjugates set forth in
Table 21 and at least one pharmaceutically acceptable carrier. In
one embodiment, the pharmaceutical composition comprises a
conjugate comprising at least a first XTEN sequence having at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99%, or having 100%
sequence identity to a sequence selected from the group of
sequences set forth in Table 2 and Table 3 wherein the XTEN
sequences of the composition are substantially homogeneous in
length, and wherein the XTEN is conjugated to at least a first
payload selected from the group of payloads set forth in Tables 11,
12, 18, 19, and 21, and wherein the composition further comprises
at least one pharmaceutically acceptable carrier. In one
embodiment, the invention provides a pharmaceutical composition
comprising an XTEN-payload conjugate of any of the embodiments
described herein and at least one pharmaceutically acceptable
carrier.
[0422] The invention provides a method of preparing a
pharmaceutical composition, comprising the step of combining a
subject conjugate composition of the embodiments with at least one
pharmaceutically acceptable carrier into a pharmaceutically
acceptable formulation. The XTEN-payload conjugates of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the XTEN-payload is
combined in admixture with a pharmaceutically acceptable carrier
vehicle, such as aqueous solutions or buffers, pharmaceutically
acceptable suspensions and emulsions. Examples of non-aqueous
solvents include propyl ethylene glycol, polyethylene glycol and
vegetable oils. Therapeutic formulations are prepared for storage
by mixing the active ingredient having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers, as described in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980), in the form of lyophilized
formulations or aqueous solutions. The pharmaceutical compositions
can be administered by any suitable means or route, including
subcutaneously, subcutaneously by infusion pump, intramuscularly,
and intravenously. It will be appreciated that the preferred route
will vary with the disease and age of the recipient, and the
severity of the condition being treated. Osmotic pumps may be used
as slow release agents in the form of tablets, pills, capsules or
implantable devices. Syringe pumps may also be used as slow release
agents. Such devices are described in U.S. Pat. Nos. 4,976,696;
4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908;
6,572,585; 5,298,022; 5,176,502; 5,492,534; 5,318,540; and
4,988,337, the contents of which are incorporated herein by
reference. One skilled in the art, considering both the disclosure
of this invention and the disclosures of these other patents could
produce a syringe pump for the extended release of the compositions
of the present invention.
[0423] In another embodiment, the invention provides an
XTEN-payload conjugate of any of the embodiments described herein
for use in making a medicament useful for the treatment of a
condition including, but not limited to the conditions set forth in
Table 16.
VI). Methods of Treatment
[0424] The invention provides a method of treating a disease in a
subject, comprising administering to the subject an effective
amount of the XTEN-payload conjugate of any of the foregoing
embodiments to a subject in need thereof. In one embodiment, the
XTEN-payload comprises a single type of payload selected from
Tables 11, 12, 18, 19, and 21. In another embodiment, the
XTEN-payload comprises a two types of payloads selected from Tables
11, 12, 18, 19, and 21. In another embodiment, the XTEN-payload
comprises a two types of payloads in which one payload is selected
from Tables 11, 12, and 18 and the second payload is a targeting
moiety with binding affinity to a target of Table 20 or is a
targeting moiety of any one of Tables 18, 19, or 21. In another
embodiment, the XTEN-payload comprises more than three or more
types of payloads selected from Tables 11, 12, 18, 19, and 21. In
the method, the payload of the conjugate is one that is known in
the art to have a beneficial effect or has affinity to a disease
target when administered to a subject with a particular disease or
condition. In one embodiment, the payload(s) of the composition
mediate their therapeutic effect via a common biological pathway.
In the foregoing embodiments of the paragraph, the method is useful
in treating or ameliorating or preventing a disease selected from
cancer, cancer supportive care, cardiovascular, central nervous
system, endocrine disease, gastrointestinal, genitourinary,
hematological, HIV infection, hormonal system, inflammation,
autoimmune disease, infectious diseases, metabolic disease,
musculoskeletal disease, nephrology disorders, ophthalmologic
diseases, pain, and respiratory. With greater particularity, the
disease is selected from Table 16.
[0425] In some embodiments of the method of treatment, the
conjugate composition can be administered subcutaneously,
intramuscularly, or intravenously. In one embodiment, the
composition is administered using a therapeutically effective
amount. In one embodiment, administration of two or more
consecutive doses of the therapeutically effective amount results
in a gain in time spent within a therapeutic window for the
composition compared to the payload not linked to XTEN and
administered using comparable doses to a subject. The gain in time
spent within the therapeutic window can be at least three-fold
longer than unmodified payload, or alternatively, at least
four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold,
or nine-fold, or at least 10-fold, or at least 20-fold, or at least
about 30-fold, or at least about 50-fold, or at least about
100-fold longer than payload not linked to XTEN.
[0426] In one embodiment of the method of treatment, a smaller
moles/kg amount of about two-fold less, or about three-fold less,
or about four-fold less, or about five-fold less, or about six-fold
less, or about eight-fold less, or about 10-fold less or greater of
the conjugate or a pharmaceutical composition comprising the
conjugate is administered to a subject in need thereof in
comparison to the corresponding payload(s) not linked to the XTEN
under a dose regimen needed to maintain a therapeutic effect and
the conjugate achieves a comparable area under the curve as the
corresponding moles/kg amount of the payload(s) not linked to the
XTEN needed to maintain a therapeutic effect. In another
embodiment, the conjugate or a pharmaceutical composition
comprising the conjugate requires less frequent administration for
routine treatment of a subject, wherein the dose of conjugate or
pharmaceutical composition is administered about every four days,
about every seven days, about every 10 days, about every 14 days,
about every 21 days, or about monthly to the subject, and the
conjugate achieves a comparable area under the curve as the
corresponding payload(s) not linked to the XTEN and administered to
the subject. In yet other embodiments, an accumulatively smaller
amount of about 5%, or about 10%, or about 20%, or about 40%, or
about 50%, or about 60%, or about 70%, or about 80%, or about 90%
less of moles/kg of the conjugate is administered to a subject in
comparison to the corresponding amount of the payload(s) not linked
to the XTEN under a dose regimen needed to maintain an effective
blood concentration, yet the conjugate achieves at least a
comparable area under the curve as the corresponding payload(s) not
linked to the XTEN. The accumulatively smaller amount is measure
for a period of at least about one week, or about 14 days, or about
21 days, or about one month. In some embodiments of the method, the
therapeutic effect is a measured parameter, clinical symptom or
endpoint known in the art to be associated with the underlying
condition of the subject to be treated or prevented.
[0427] In one embodiment, the invention provides a method of
treating a cancer cell in vitro, comprising administering to a
culture of a cancer cell a composition comprising an effective
amount of an XTEN-payload composition, wherein a first payload is a
targeting moiety and the second payload is a toxin of Table 21. In
another embodiment, the invention provides a method of treating a
cancer in a subject, comprising administering to the subject a
pharmaceutical composition comprising an effective amount of an
XTEN-payload composition wherein a first payload is a targeting
moiety and the second payload is a toxin of Table 21. In one
embodiment of the method, the pharmaceutical composition comprises
the composition having the structure set forth in FIG. 117. In
another embodiment of the method, the cancer is selected from the
group consisting of non-small cell lung cancer or mesothelioma.
platinum-resistant ovarian cancer, endometrial cancer,
adenocarcinoma of the lung, and refractory advanced tumors. In
another embodiment of the method, the administration results in at
least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or
80%, or 90% greater improvement of at least one, two, or three
parameters associated with a cancer compared to an untreated
subject wherein the parameters are selected from the group
consisting of response rate as defined by the Response Evaluation
Criteria in Solid Tumors (RECIST), time-to-progression of the
cancer (relapse), discovery of local recurrence, discovery of
regional metastasis, discovery of distant metastasis, onset of
symptoms, hospitalization, increase in pain medication requirement,
requirement of salvage chemotherapy, requirement of salvage
surgery, requirement of salvage radiotherapy, time-to-treatment
failure, and increased time of survival.
[0428] In another aspect, the invention provides a regimen for
treating a subject with a disease, said regimen comprising a
composition comprising a conjugate of any of the embodiments
described herein. In one embodiment of the regimen, the regimen
further comprises the step of determining the amount of
pharmaceutical composition comprising the CFXTEN needed to achieve
a therapeutic effect in the patient.
[0429] The invention provides conjugates comprising a treatment
regimen for a diseased subject comprising administering a
pharmaceutical composition comprising a conjugate of any of the
embodiments described herein in two or more successive doses
administered at an effective amount, wherein the administration
results in the improvement of at least one parameter associated
with the disease.
VII). Conjugation Kits
[0430] In another aspect, the invention provides a kit to
facilitate the use of the XTEN-crosslinker conjugate compositions.
In one embodiment, the kit comprises an XTEN-crosslinker in a
formulation, a container and a label on or associated with the
container. In the foregoing embodiment, the XTEN-crosslinker can be
any one of the embodiments described herein. The container holds
the XTEN-crosslinker at a defined concentration in a buffer
suitable for use in a conjugation reaction to link to a
payload.
VIII). Pharmaceutical Kits
[0431] In another aspect, the invention provides a kit to
facilitate the use of the conjugate compositions. The kit comprises
a pharmaceutical composition provided herein, a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc., formed from a variety of materials such as glass or plastic.
The container holds a pharmaceutical composition as a formulation
that is effective for treating a subject and may have a sterile
access port (for example the container may be an intravenous
solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). The package insert can list the approved
indications for the drug, instructions for the reconstitution
and/or administration of the drug for the use for the approved
indication, appropriate dosage and safety information, and
information identifying the lot and expiration of the drug. In
another embodiment of the foregoing, the kit can comprise a second
container that can carry a suitable diluent for the pharmaceutical
composition, the use of which will provide the user with the
appropriate concentration to be delivered to the subject. In
another embodiment, the kit comprises, in at least a first
container: a first container: an amount of a conjugate composition
drug sufficient to administer in treatment of a subject with a
disease; an amount of a pharmaceutically acceptable carrier, a
second container that can carry a suitable diluent for the subject
composition, which will provide the user with the appropriate
concentration of the pharmaceutical composition to be delivered to
the subject; together with a label identifying the drug and storage
and handling conditions, and/or a sheet of the approved indications
for the drug and instructions for the reconstitution and/or
administration of the drug for the use for the treatment of a
approved indication, appropriate dosage and safety information, and
information identifying the lot and expiration of the drug.
IX). The Nucleic Acids Sequences of the Invention
[0432] The present invention provides isolated polynucleic acids
encoding the polypeptide components of the conjugates and sequences
complementary to polynucleic acid molecules encoding the
polypeptide components of the conjugates. In some embodiments, the
invention provides polynucleic acids encoding the XTEN of any of
the conjugate embodiments described herein, or the complement of
the polynucleic acid. In one embodiment, the polynucleic acids
encodes an XTEN selected from the group consisting of the XTEN set
forth in Tables 2 and 3, or the complement of the polynucleic
acid.
[0433] In other embodiments, the invention provides polynucleic
acids encoding the XTEN linked to cleavage sequences, affinity tags
and helper sequences protein of any of the embodiments described
herein, or the complement of the polynucleic acids. In one
embodiment, the polynucleic acids encodes a protein payload
selected from the group consisting of the protein payloads set
forth in Tables 7, 18, 19, and 21, or the complement of the
polynucleic acid.
[0434] In one embodiment, the invention encompasses methods to
produce polynucleic acids encoding the XTEN and the XTEN linked to
cleavage sequences, affinity tags and helper sequences protein
embodiments, or sequences complementary to the polynucleic acids,
including homologous variants thereof. In general, and as
illustrated in FIGS. 38 and 39, the methods of producing a
polynucleotide sequence coding for an XTEN and expressing the
resulting gene product include assembling nucleotides encoding the
XTEN, ligating the components in frame, incorporating the encoding
gene into an expression vector appropriate for a host cell,
transforming the appropriate host cell with the expression vector,
and culturing the host cell under conditions causing or permitting
the XTEN to be expressed in the transformed host cell, thereby
producing the XTEN polypeptide, which is recovered by methods
described herein or by standard protein purification methods known
in the art. Standard recombinant techniques in molecular biology
are used to make the polynucleotides and expression vectors of the
present invention.
[0435] In accordance with the invention, nucleic acid sequences
that encode XTEN or XTEN linked to cleavage sequences, affinity
tags and helper sequences protein (or its complement) are used to
generate recombinant DNA molecules that direct the expression in
appropriate host cells. Several cloning strategies are suitable for
performing the present invention, many of which are used to
generate a construct that comprises a gene coding for an XTEN or a
payload composition of the present invention, or its complement. In
one embodiment, the cloning strategy is used to create a gene that
encodes an XTEN that comprises nucleotides encoding the XTEN that
is used to transform a host cell for expression of the XTEN
composition. In the foregoing embodiments hereinabove described in
this paragraph, the genes can further comprise nucleotides encoding
cleavage sequences, affinity tags, and helper sequences. In another
embodiment, the cloning strategy is used to create a gene that
encodes a protein payload that comprises nucleotides encoding the
payload that is used to transform a host cell for expression of the
payload composition.
[0436] In designing a desired XTEN sequences, it was discovered
that the non-repetitive nature of the XTEN of the inventive
compositions is achieved despite use of a "building block"
molecular approach in the creation of the XTEN-encoding sequences.
This was achieved by the use of a library of polynucleotides
encoding peptide sequence motifs, described above, that are then
ligated and/or multimerized to create the genes encoding the XTEN
sequences (see FIGS. 38 and 39 and Examples). Thus, while the
XTEN(s) of the expressed polypeptide may consist of multiple units
of as few as four different sequence motifs, because the motifs
themselves consist of non-repetitive amino acid sequences, the
overall XTEN sequence is rendered non-repetitive. Accordingly, in
one embodiment, the XTEN-encoding polynucleotides comprise multiple
polynucleotides that encode non-repetitive sequences, or motifs,
operably linked in frame and in which the resulting expressed XTEN
amino acid sequences are non-repetitive.
[0437] In one approach, a construct is first prepared containing
the DNA sequence corresponding to XTEN. Exemplary methods for the
preparation of such constructs are described in the Examples. The
construct is then used to create an expression vector suitable for
transforming a host cell, such as a prokaryotic host cell (e.g., E.
coli) for the expression and recovery of the XTEN. Exemplary
methods for the creation of expression vectors, the transformation
of host cells and the expression and recovery of XTEN are described
in the Examples.
[0438] The gene encoding for the XTEN can be made in one or more
steps, either fully synthetically or by synthesis combined with
enzymatic processes, such as restriction enzyme-mediated cloning,
PCR and overlap extension, including methods more fully described
in the Examples. The methods disclosed herein can be used, for
example, to ligate short sequences of polynucleotides encoding XTEN
into longer XTEN genes of a desired length and sequence. In one
embodiment, the method ligates two, three, four or more
codon-optimized oligonucleotides encoding XTEN motif or segment
sequences of about 9 to 14 amino acids, or about 12 to 20 amino
acids, or about 18 to 36 amino acids, or about 48 to about 144
amino acids, or about 144 to about 288 or longer, or any
combination of the foregoing ranges of motif or segment lengths.
Alternatively, the disclosed method is used to multimerize
XTEN-encoding sequences into longer sequences of a desired length;
e.g., a gene encoding 36 amino acids of XTEN can be dimerized into
a gene encoding 72 amino acids, then 144, then 288, etc. Even with
multimerization, XTEN polypeptides can be constructed such that the
XTEN-encoding gene has low or virtually no repetitiveness through
design of the codons selected for the motifs of the shortest unit
being used, which can reduce recombination and increase stability
of the encoding gene in the transformed host. Genes encoding XTEN
with non-repetitive sequences are assembled from oligonucleotides
using standard techniques of gene synthesis. The gene design can be
performed using algorithms that optimize codon usage and amino acid
composition. In one method of the invention, a library of
relatively short XTEN-encoding polynucleotide constructs is created
and then assembled, as described above. The resulting genes are
then assembled with genes encoding payload peptide or polypeptide,
and the resulting genes used to transform a host cell and produce
and recover the XTEN-payload for evaluation of its properties, as
described herein.
[0439] The resulting polynucleotides encoding the XTEN and the
peptide sequences to which it is linked can then be individually
cloned into an expression vector. The nucleic acid sequence is
inserted into the vector by a variety of procedures. In general,
DNA is inserted into an appropriate restriction endonuclease
site(s) using techniques known in the art. Vector components
generally include, but are not limited to, one or more of a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable vectors containing one or more
of these components employs standard ligation techniques which are
known to the skilled artisan. Such techniques are well known in the
art and well described in the scientific and patent literature.
Various vectors are publicly available. The vector may, for
example, be in the form of a plasmid, cosmid, viral particle, or
phage that may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e., a vector, which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated. Representative
plasmids are illustrated in FIG. 17, with encoding regions for
different configurations of FVIII and XTEN components
portrayed.
[0440] The invention provides for the use of plasmid expression
vectors containing replication and control sequences that are
compatible with and recognized by the host cell, and are operably
linked to the gene encoding the polypeptide for controlled
expression of the polypeptide. The vector ordinarily carries a
replication site, as well as sequences that encode proteins that
are capable of providing phenotypic selection in transformed cells.
Such vector sequences are well known for a variety of bacteria,
yeast, and viruses. Useful expression vectors that can be used
include, for example, segments of chromosomal, non-chromosomal and
synthetic DNA sequences. "Expression vector" refers to a DNA
construct containing a DNA sequence that is operably linked to a
suitable control sequence capable of effecting the expression of
the DNA encoding the polypeptide in a suitable host. The
requirements are that the vectors are replicable and viable in the
host cell of choice. Low- or high-copy number vectors may be used
as desired.
[0441] Suitable vectors include, but are not limited to,
derivatives of SV40 and pcDNA and known bacterial plasmids such as
col EI, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith, et
al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids
such as RP4, phage DNAs such as the numerous derivatives of phage I
such as NM989, as well as other phage DNA such as M13 and
filamentous single stranded phage DNA; yeast plasmids such as the 2
micron plasmid or derivatives of the 2 m plasmid, as well as
centomeric and integrative yeast shuttle vectors; vectors useful in
eukaryotic cells such as vectors useful in insect or mammalian
cells; vectors derived from combinations of plasmids and phage
DNAs, such as plasmids that have been modified to employ phage DNA
or the expression control sequences; and the like. Yeast expression
systems that can also be used in the present invention include, but
are not limited to, the non-fusion pYES2 vector (Invitrogen), the
fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like. The
control sequences of the vector include a promoter to effect
transcription, an optional operator sequence to control such
transcription, a sequence encoding suitable mRNA ribosome binding
sites, and sequences that control termination of transcription and
translation. The promoter may be any DNA sequence, which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Promoters suitable for use in
expression vectors with prokaryotic hosts include the
.beta.-lactamase and lactose promoter systems [Chang et al.,
Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],
alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,
Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters
such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.
USA, 80:21-25 (1983)], all is operably linked to the DNA encoding
CFXTEN polypeptides. Promoters for use in bacterial systems can
also contain a Shine-Dalgarno (S.D.) sequence, operably linked to
the DNA encoding CFXTEN polypeptides.
EXAMPLES
Example 1: Construction of XTEN_AD36 Motif Segments
[0442] The following example describes the construction of a
collection of codon-optimized genes encoding motif sequences of 36
amino acids. As a first step, a stuffer vector pCW0359 was
constructed based on a pET vector and that includes a T7 promoter.
pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease
recognition site followed by a stuffer sequence that is flanked by
BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted
such that they generate compatible overhangs after digestion. The
stuffer sequence is followed by a truncated version of the GFP gene
and a His tag. The stuffer sequence contains stop codons and thus
E. coli cells carrying the stuffer plasmid pCW0359 form
non-fluorescent colonies. The stuffer vector pCW0359 was digested
with BsaI and KpnI to remove the stuffer segment and the resulting
vector fragment was isolated by agarose gel purification. The
sequences were designated XTEN_AD36, reflecting the AD family of
motifs. Its segments have the amino acid sequence [X].sub.3 where X
is a 12mer peptide with the sequences: GESPGGSSGSES (SEQ ID NO:
26), GSEGSSGPGESS (SEQ ID NO: 27), GSSESGSSEGGP (SEQ ID NO: 28), or
GSGGEPSESGSS (SEQ ID NO: 29). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00024 AD1for: (SEQ ID NO: 612)
AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1rev: (SEQ ID NO: 613)
ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for: (SEQ ID NO: 614)
AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev: (SEQ ID NO: 615)
ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT AD3for: (SEQ ID NO: 616)
AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev: (SEQ ID NO: 617)
ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA AD4for: (SEQ ID NO: 618)
AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC
[0443] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 619) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 620). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12mer repeats
ligated to one BbsI/KpnI segment. The products corresponding to the
length of 36 amino acids were isolated from the mixture by
preparative agarose gel electrophoresis and ligated into the
BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in
the resulting library designated LCW0401 showed green fluorescence
after induction, which shows that the sequence of XTEN_AD36 had
been ligated in frame with the GFP gene and that most sequences of
XTEN_AD36 had good expression levels.
[0444] We screened 96 isolates from library LCW0401 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 39 clones
were identified that contained correct XTEN_AD36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 22.
TABLE-US-00025 TABLE 22 DNA and Amino Acid Sequences for 36-mer
motifs SEQ SEQ ID ID File name Amino acid sequence NO: Nucleotide
sequence NO: LCW0401_001_ GSGGEPSESGSSGESP 621
GGTTCTGGTGGCGAACCGTCCGAGTCTGGTA 659 GFP-N_A01.ab1 GGSSGSESGESPGGSS
GCTCAGGTGAATCTCCGGGTGGCTCTAGCGG GSES
TTCCGAGTCAGGTGAATCTCCTGGTGGTTCC AGCGGTTCCGAGTCA LCW0401_002_
GSEGSSGPGESSGESP 622 GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGT 660
GFP-N_B01.ab1 GGSSGSESGSSESGSS CTTCAGGTGAATCTCCTGGTGGTTCCAGCGG EGGP
TTCTGAATCAGGTTCCTCCGAAAGCGGTTCT TCCGAGGGCGGTCCA LCW0401_003_
GSSESGSSEGGPGSSE 623 GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTG 661
GFP-N_C01.ab1 SGSSEGGPGESPGGSS GTCCAGGTTCCTCTGAAAGCGGTTCTTCTGA GSES
GGGTGGTCCAGGTGAATCTCCGGGTGGCTCC AGCGGTTCCGAGTCA LCW0401_004_
GSGGEPSESGSSGSSE 624 GGTTCCGGTGGCGAACCGTCTGAATCTGGTA 662
GFP-N_D01.ab1 SGSSEGGPGSGGEPSE GCTCAGGTTCTTCTGAAAGCGGTTCTTCCGA SGSS
GGGTGGTCCAGGTTCTGGTGGTGAACCTTCC GAGTCTGGTAGCTCA LCW0401_007_
GSSESGSSEGGPGSEG 625 GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTG 663
GFP-N_F01.ab1 SSGPGESSGSEGSSGP GTCCAGGTAGCGAAGGTTCTTCCGGTCCAGG GESS
TGAGTCTTCAGGTAGCGAAGGTTCTTCTGGT CCTGGTGAATCTTCA LCW0401_008_
GSSESGSSEGGPGESP 626 GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTG 664
GFP-N_G01.ab1 GGSSGSESGSEGSSGP GTCCAGGTGAATCTCCAGGTGGTTCCAGCGG GESS
TTCTGAGTCAGGTAGCGAAGGTTCTTCTGGT CCAGGTGAATCCTCA LCW0401_012_
GSGGEPSESGSSGSGG 627 GGTTCTGGTGGTGAACCGTCTGAGTCTGGTA 665
GFP-N_H01.ab1 EPSESGSSGSEGSSGP GCTCAGGTTCCGGTGGCGAACCATCCGAATC GESS
TGGTAGCTCAGGTAGCGAAGGTTCTTCCGGT CCAGGTGAGTCTTCA LCW0401_015_
GSSESGSSEGGPGSEG 628 GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCG 666
GFP-N_A02.ab1 SSGPGESSGESPGGSS GTCCAGGTAGCGAAGGTTCTTCTGGTCCAGG GSES
CGAATCTTCAGGTGAATCTCCTGGTGGCTCC AGCGGTTCTGAGTCA LCW0401_016_
GSSESGSSEGGPGSSE 629 GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCG 667
GFP-N_B02.ab1 SGSSEGGPGSSESGSS GTCCAGGTTCCTCCGAAAGCGGTTCTTCCGA EGGP
GGGCGGTCCAGGTTCTTCTGAAAGCGGTTCT TCCGAGGGCGGTCCA LCW0401_020_
GSGGEPSESGSSGSEG 630 GGTTCCGGTGGCGAACCGTCCGAATCTGGTA 668
GFP-N_E02.ab1 SSGPGESSGSSESGSS GCTCAGGTAGCGAAGGTTCTTCTGGTCCAGG EGGP
CGAATCTTCAGGTTCCTCTGAAAGCGGTTCT TCTGAGGGCGGTCCA LCW0401_022_
GSGGEPSESGSSGSSE 631 GGTTCTGGTGGTGAACCGTCCGAATCTGGTA 669
GFP-N_F02.ab1 SGSSEGGPGSGGEPSE GCTCAGGTTCTTCCGAAAGCGGTTCTTCTGA SGSS
AGGTGGTCCAGGTTCCGGTGGCGAACCTTCT GAATCTGGTAGCTCA LCW0401_024_
GSGGEPSESGSSGSSE 632 GGTTCTGGTGGCGAACCGTCCGAATCTGGTA 670
GFP-N_G02.ab1 SGSSEGGPGESPGGSS GCTCAGGTTCCTCCGAAAGCGGTTCTTCTGA GSES
AGGTGGTCCAGGTGAATCTCCAGGTGGTTCT AGCGGTTCTGAATCA LCW0401_026_
GSGGEPSESGSSGESP 633 GGTTCTGGTGGCGAACCGTCTGAGTCTGGTA 671
GFP-N_H02.ab1 GGSSGSESGSEGSSGP GCTCAGGTGAATCTCCTGGTGGCTCCAGCGG GESS
TTCTGAATCAGGTAGCGAAGGTTCTTCTGGT CCTGGTGAATCTTCA LCW0401_027_
GSGGEPSESGSSGESP 634 GGTTCCGGTGGCGAACCTTCCGAATCTGGTA 672
GFP-N_A03.ab1 GGSSGSESGSGGEPSE GCTCAGGTGAATCTCCGGGTGGTTCTAGCGG SGSS
TTCTGAGTCAGGTTCTGGTGGTGAACCTTCC GAGTCTGGTAGCTCA LCW0401_028_
GSSESGSSEGGPGSSE 635 GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCG 673
GFP-N_B03.ab1 SGSSEGGPGSSESGSS GTCCAGGTTCTTCCGAAAGCGGTTCTTCCGA EGGP
GGGCGGTCCAGGTTCTTCCGAAAGCGGTTCT TCTGAAGGCGGTCCA LCW0401_030_
GESPGGSSGSESGSEG 636 GGTGAATCTCCGGGTGGCTCCAGCGGTTCTG 674
GFP-N_C03.ab1 SSGPGESSGSEGSSGP AGTCAGGTAGCGAAGGTTCTTCCGGTCCGGG GESS
TGAGTCCTCAGGTAGCGAAGGTTCTTCCGGT CCTGGTGAGTCTTCA LCW0401_031_
GSGGEPSESGSSGSGG 637 GGTTCTGGTGGCGAACCTTCCGAATCTGGTA 675
GFP-N_D03.ab1 EPSESGSSGSSESGSS GCTCAGGTTCCGGTGGTGAACCTTCTGAATC EGGP
TGGTAGCTCAGGTTCTTCTGAAAGCGGTTCT TCCGAGGGCGGTCCA LCW0401_033_
GSGGEPSESGSSGSGG 638 GGTTCCGGTGGTGAACCTTCTGAATCTGGTA 676
GFP-N_E03.ab1 EPSESGSSGSGGEPSE GCTCAGGTTCCGGTGGCGAACCATCCGAGTC SGSS
TGGTAGCTCAGGTTCCGGTGGTGAACCATCC GAGTCTGGTAGCTCA LCW0401_037_
GSGGEPSESGSSGSSE 639 GGTTCCGGTGGCGAACCTTCTGAATCTGGTA 677
GFP-N_F03.ab1 SGSSEGGPGSEGSSGP GCTCAGGTTCCTCCGAAAGCGGTTCTTCTGA GESS
GGGCGGTCCAGGTAGCGAAGGTTCTTCTGGT CCGGGCGAGTCTTCA LCW0401_038_
GSGGEPSESGSSGSEG 640 GGTTCCGGTGGTGAACCGTCCGAGTCTGGTA 678
GFP-N_G03.ab1 SSGPGESSGSGGEPSE GCTCAGGTAGCGAAGGTTCTTCTGGTCCGGG SGSS
TGAGTCTTCAGGTTCTGGTGGCGAACCGTCC GAATCTGGTAGCTCA LCW0401_039_
GSGGEPSESGSSGESP 641 GGTTCTGGTGGCGAACCGTCCGAATCTGGTA 679
GFP-N_H03.ab1 GGSSGSESGSGGEPSE GCTCAGGTGAATCTCCTGGTGGTTCCAGCGG SGSS
TTCCGAGTCAGGTTCTGGTGGCGAACCTTCC GAATCTGGTAGCTCA LCW0401_040_
GSSESGSSEGGPGSGG 642 GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCG 680
GFP-N_A04.ab1 EPSESGSSGSSESGSS GTCCAGGTTCCGGTGGTGAACCATCTGAATC EGGP
TGGTAGCTCAGGTTCTTCTGAAAGCGGTTCT TCTGAAGGTGGTCCA LCW0401_042_
GSEGSSGPGESSGESP 643 GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGT 681
GFP-N_C04.ab1 GGSSGSESGSEGSSGP CTTCAGGTGAATCTCCAGGTGGCTCTAGCGG GESS
TTCCGAGTCAGGTAGCGAAGGTTCTTCTGGT CCTGGCGAGTCCTCA LCW0401_046_
GSSESGSSEGGPGSSE 644 GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCG 682
GFP-N_D04.ab1 SGSSEGGPGSSESGSS GTCCAGGTTCTTCCGAAAGCGGTTCTTCTGA EGGP
GGGCGGTCCAGGTTCCTCCGAAAGCGGTTCT TCTGAGGGTGGTCCA LCW0401_047_
GSGGEPSESGSSGESP 645 GGTTCTGGTGGCGAACCTTCCGAGTCTGGTA 683
GFP-N_E04.ab1 GGSSGSESGESPGGSS GCTCAGGTGAATCTCCGGGTGGTTCTAGCGG GSES
TTCCGAGTCAGGTGAATCTCCGGGTGGTTCC AGCGGTTCTGAGTCA LCW0401_051_
GSGGEPSESGSSGSEG 646 GGTTCTGGTGGCGAACCATCTGAGTCTGGTA 684
GFP-N_F04.ab1 SSGPGESSGESPGGSS GCTCAGGTAGCGAAGGTTCTTCCGGTCCAGG GSES
CGAGTCTTCAGGTGAATCTCCTGGTGGCTCC AGCGGTTCTGAGTCA LCW0401_053_
GESPGGSSGSESGESP 647 GGTGAATCTCCTGGTGGTTCCAGCGGTTCCG 685
GFP-N_H04.ab1 GGSSGSESGESPGGSS AGTCAGGTGAATCTCCAGGTGGCTCTAGCGG GSES
TTCCGAGTCAGGTGAATCTCCTGGTGGTTCT AGCGGTTCTGAATCA LCW0401_054_
GSEGSSGPGESSGSEG 648 GGTAGCGAAGGTTCTTCCGGTCCAGGTGAAT 686
GFP-N_A05.ab1 SSGPGESSGSGGEPSE CTTCAGGTAGCGAAGGTTCTTCTGGTCCTGG SGSS
TGAATCCTCAGGTTCCGGTGGCGAACCATCT GAATCTGGTAGCTCA LCW0401_059_
GSGGEPSESGSSGSEG 649 GGTTCTGGTGGCGAACCATCCGAATCTGGTA 687
GFP-N_D05.ab1 SSGPGESSGESPGGSS GCTCAGGTAGCGAAGGTTCTTCTGGTCCTGG GSES
CGAATCTTCAGGTGAATCTCCAGGTGGCTCT AGCGGTTCCGAATCA LCW0401_060_
GSGGEPSESGSSGSSE 650 GGTTCCGGTGGTGAACCGTCCGAATCTGGTA 688
GFP-N_E05.ab1 SGSSEGGPGSGGEPSE GCTCAGGTTCCTCTGAAAGCGGTTCTTCCGA SGSS
GGGTGGTCCAGGTTCCGGTGGTGAACCTTCT GAGTCTGGTAGCTCA LCW0401_061_
GSSESGSSEGGPGSGG 651 GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCG 689
GFP-N_F05.ab1 EPSESGSSGSEGSSGP GTCCAGGTTCTGGTGGCGAACCATCTGAATC GESS
TGGTAGCTCAGGTAGCGAAGGTTCTTCCGGT CCGGGTGAATCTTCA LCW0401_063_
GSGGEPSESGSSGSEG 652 GGTTCTGGTGGTGAACCGTCCGAATCTGGTA 690
GFP-N_H05.ab1 SSGPGESSGSEGSSGP GCTCAGGTAGCGAAGGTTCTTCTGGTCCTGG GESS
CGAGTCTTCAGGTAGCGAAGGTTCTTCTGGT CCTGGTGAATCTTCA LCW0401_066_
GSGGEPSESGSSGSSE 653 GGTTCTGGTGGCGAACCATCCGAGTCTGGTA 691
GFP-N_B06.ab1 SGSSEGGPGSGGEPSE GCTCAGGTTCTTCCGAAAGCGGTTCTTCCGA SGSS
AGGCGGTCCAGGTTCTGGTGGTGAACCGTCC GAATCTGGTAGCTCA LCW0401_067_
GSGGEPSESGSSGESP 654 GGTTCCGGTGGCGAACCTTCCGAATCTGGTA 692
GFP-N_C06.ab1 GGSSGSESGESPGGSS GCTCAGGTGAATCTCCGGGTGGTTCTAGCGG GSES
TTCCGAATCAGGTGAATCTCCAGGTGGTTCT AGCGGTTCCGAATCA LCW0401_069_
GSGGEPSESGSSGSGG 655 GGTTCCGGTGGTGAACCATCTGAGTCTGGTA 693
GFP-N_D06.ab1 EPSESGSSGESPGGSS GCTCAGGTTCCGGTGGCGAACCGTCCGAGTC GSES
TGGTAGCTCAGGTGAATCTCCGGGTGGTTCC AGCGGTTCCGAATCA LCW0401_070_
GSEGSSGPGESSGSSE 656 GGTAGCGAAGGTTCTTCTGGTCCGGGCGAAT 694
GFP-N_E06.ab1 SGSSEGGPGSEGSSGP CCTCAGGTTCCTCCGAAAGCGGTTCTTCCGA GESS
AGGTGGTCCAGGTAGCGAAGGTTCTTCCGGT CCTGGTGAATCTTCA LCW0401_078_
GSSESGSSEGGPGESP 657 GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCG 695
GFP-N_F06.ab1 GGSSGSESGESPGGSS GTCCAGGTGAATCTCCGGGTGGCTCCAGCGG GSES
TTCTGAATCAGGTGAATCTCCTGGTGGCTCC AGCGGTTCCGAGTCA LCW0401_079_
GSEGSSGPGESSGSEG 658 GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGT 696
GFP-N_G06.ab1 SSGPGESSGSGGEPSE CTTCAGGTAGCGAAGGTTCTTCCGGTCCTGG SGSS
CGAGTCTTCAGGTTCCGGTGGCGAACCGTCC GAATCTGGTAGCTCA
Example 2: Construction of XTEN_AE36 Segments
[0445] A codon library encoding XTEN sequences of 36 amino acid
length was constructed. The XTEN sequence was designated XTEN_AE36.
Its segments have the amino acid sequence [X].sub.3 where X is a
12mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 30),
GSEPATSGSE TP (SEQ ID NO: 31), GTSESA TPESGP (SEQ ID NO: 32), or
GTSTEPSEGSAP (SEQ ID NO: 33). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00026 AE1for: (SEQ ID NO: 697)
AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AE1rev: (SEQ ID NO: 698)
ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AE2for: (SEQ ID NO: 699)
AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC AE2rev: (SEQ ID NO: 700)
ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3for: (SEQ ID NO: 701)
AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev: (SEQ ID NO: 702)
ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for: (SEQ ID NO: 703)
AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4rev: (SEQ ID NO: 704)
ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT
[0446] We also annealed the phosphoiylated oligonucleotide
3KpnIstopperFor: AGGTSCGTCTTCACTCGAGGGTAC (SEQ ID NO: 619) and the
non-phosphoylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 620). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12mer repeats
ligated to one BbsI/KpnI segment. The products corresponding to the
length of 36 amino acids were isolated from the mixture by
preparative agarose gel electrophoresis and ligated into the
BsaI/KpnI digested stuffier vector pCW0359. Most of the clones in
the resulting library designated LCW0402 showed green fluorescence
after induction which shows that the sequence of XTEN_AE36 had been
ligated in frame with the GFP gene and most sequences of XTEN_AE36
show good expression.
[0447] We screened isolates from library LCW0402 for high level of
fluorescence by stamping them onto agar plate containing IPTG. The
same isolates were evaluated by PCR and 48 isolates were identified
that contained segments with 36 amino acids as well as strong
fluorescence. These isolates were sequenced and 37 clones were
identified that contained correct XTEN_AE36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 23.
TABLE-US-00027 TABLE 23 DNA and Amino Acid Sequences for 36-mer
motifs SEQ SEQ Amino acid ID ID File name sequence NO: Nucleotide
sequence NO: LCW0402_002_ GSPAGSPTSTEEG 705
GGTAGCCCGGCAGGCTCTCCGACCTCTA 742 GFP-N_A07.ab1 TSESATPESGPGT
CTGAGGAAGGTACTTCTGAAAGCGCAAC STEPSEGSAP
CCCGGAGTCCGGCCCAGGTACCTCTACC GAACCGTCTGAGGGCAGCGCACCA LCW0402_003_
GTSTEPSEGSAPG 706 GGTACTTCTACCGAACCGTCCGAAGGCA 743 GFP-N_B07.ab1
TSTEPSEGSAPGT GCGCTCCAGGTACCTCTACTGAACCTTCC STEPSEGSAP
GAGGGCAGCGCTCCAGGTACCTCTACCG AACCTTCTGAAGGTAGCGCACCA LCW0402_004_
GTSTEPSEGSAPG 707 GGTACCTCTACCGAACCGTCTGAAGGTA 744 GFP-N_C07.ab1
TSESATPESGPGT GCGCACCAGGTACCTCTGAAAGCGCAAC SESATPESGP
TCCTGAGTCCGGTCCAGGTACTTCTGAAA GCGCAACCCCGGAGTCTGGCCCA LCW0402_005_
GTSTEPSEGSAPG 708 GGTACTTCTACTGAACCGTCTGAAGGTA 745 GFP-N_D07.ab1
TSESATPESGPGT GCGCACCAGGTACTTCTGAAAGCGCAAC SESATPESGP
CCCGGAATCCGGCCCAGGTACCTCTGAA AGCGCAACCCCGGAGTCCGGCCCA LCW0402_006_
GSEPATSGSETPG 709 GGTAGCGAACCGGCAACCTCCGGCTCTG 746 GFP-N_E07.ab1
TSESATPESGPGS AAACCCCAGGTACCTCTGAAAGCGCTAC PAGSPTSTEE
TCCTGAATCCGGCCCAGGTAGCCCGGCA GGTTCTCCGACTTCCACTGAGGAA LCW0402_008_
GTSESATPESGPG 710 GGTACTTCTGAAAGCGCAACCCCTGAAT 747 GFP-N_F07.ab1
SEPATSGSETPGT CCGGTCCAGGTAGCGAACCGGCTACTTC STEPSEGSAP
TGGCTCTGAGACTCCAGGTACTTCTACCG AACCGTCCGAAGGTAGCGCACCA LCW0402_009_
GSPAGSPTSTEEG 711 GGTAGCCCGGCTGGCTCTCCAACCTCCA 748 GFP-N_G07.ab1
SPAGSPTSTEEGS CTGAGGAAGGTAGCCCGGCTGGCTCTCC EPATSGSETP
AACCTCCACTGAAGAAGGTAGCGAACCG GCTACCTCCGGCTCTGAAACTCCA LCW0402_011_
GSPAGSPTSTEEG 712 GGTAGCCCGGCTGGCTCTCCTACCTCTAC 749 GFP-N_A08.ab1
TSESATPESGPGT TGAGGAAGGTACTTCTGAAAGCGCTACT STEPSEGSAP
CCTGAGTCTGGTCCAGGTACCTCTACTGA ACCGTCCGAAGGTAGCGCTCCA LCW0402_012_
GSPAGSPTSTEEG 713 GGTAGCCCTGCTGGCTCTCCGACTTCTAC 750 GFP-N_B08.ab1
SPAGSPTSTEEGT TGAGGAAGGTAGCCCGGCTGGTTCTCCG STEPSEGSAP
ACTTCTACTGAGGAAGGTACTTCTACCG AACCTTCCGAAGGTAGCGCTCCA LCW0402_013_
GTSESATPESGPG 714 GGTACTTCTGAAAGCGCTACTCCGGAGT 751 GFP-N_C08.ab1
TSTEPSEGSAPGT CCGGTCCAGGTACCTCTACCGAACCGTC STEPSEGSAP
CGAAGGCAGCGCTCCAGGTACTTCTACT GAACCTTCTGAGGGTAGCGCTCCA LCW0402_014_
GTSTEPSEGSAPG 715 GGTACCTCTACCGAACCTTCCGAAGGTA 752 GFP-N_D08.ab1
SPAGSPTSTEEGT GCGCTCCAGGTAGCCCGGCAGGTTCTCC STEPSEGSAP
TACTTCCACTGAGGAAGGTACTTCTACCG AACCTTCTGAGGGTAGCGCACCA LCW0402_015_
GSEPATSGSETPG 716 GGTAGCGAACCGGCTACTTCCGGCTCTG 753 GFP-N_E08.ab1
SPAGSPTSTEEGT AGACTCCAGGTAGCCCTGCTGGCTCTCC SESATPESGP
GACCTCTACCGAAGAAGGTACCTCTGAA AGCGCTACCCCTGAGTCTGGCCCA LCW0402_016_
GTSTEPSEGSAPG 717 GGTACTTCTACCGAACCTTCCGAGGGCA 754 GFP-N_F08.ab1
TSESATPESGPGT GCGCACCAGGTACTTCTGAAAGCGCTAC SESATPESGP
CCCTGAGTCCGGCCCAGGTACTTCTGAA AGCGCTACTCCTGAATCCGGTCCA LCW0402_020_
GTSTEPSEGSAPG 718 GGTACTTCTACTGAACCGTCTGAAGGCA 755 GFP-N_G08.ab1
SEPATSGSETPGS GCGCACCAGGTAGCGAACCGGCTACTTC PAGSPTSTEE
CGGTTCTGAAACCCCAGGTAGCCCAGCA GGTTCTCCAACTTCTACTGAAGAA LCW0402_023_
GSPAGSPTSTEEG 719 GGTAGCCCTGCTGGCTCTCCAACCTCCAC 756 GFP-N_A09.ab1
TSESATPESGPGS CGAAGAAGGTACCTCTGAAAGCGCAACC EPATSGSETP
CCTGAATCCGGCCCAGGTAGCGAACCGG CAACCTCCGGTTCTGAAACCCCA LCW0402_024_
GTSESATPESGPG 720 GGTACTTCTGAAAGCGCTACTCCTGAGTC 757 GFP-N_B09.ab1
SPAGSPTSTEEGS CGGCCCAGGTAGCCCGGCTGGCTCTCCG PAGSPTSTEE
ACTTCCACCGAGGAAGGTAGCCCGGCTG GCTCTCCAACTTCTACTGAAGAA LCW0402_025_
GTSTEPSEGSAPG 721 GGTACCTCTACTGAACCTTCTGAGGGCA 758 GFP-N_C09.ab1
TSESATPESGPGT GCGCTCCAGGTACTTCTGAAAGCGCTAC STEPSEGSAP
CCCGGAGTCCGGTCCAGGTACTTCTACTG AACCGTCCGAAGGTAGCGCACCA LCW0402_026_
GSPAGSPTSTEEG 722 GGTAGCCCGGCAGGCTCTCCGACTTCCA 759 GFP-N_D09.ab1
TSTEPSEGSAPGS CCGAGGAAGGTACCTCTACTGAACCTTC EPATSGSETP
TGAGGGTAGCGCTCCAGGTAGCGAACCG GCAACCTCTGGCTCTGAAACCCCA LCW0402_027_
GSPAGSPTSTEEG 723 GGTAGCCCAGCAGGCTCTCCGACTTCCA 760 GFP-N_E09.ab1
TSTEPSEGSAPGT CTGAGGAAGGTACTTCTACTGAACCTTCC STEPSEGSAP
GAAGGCAGCGCACCAGGTACCTCTACTG AACCTTCTGAGGGCAGCGCTCCA LCW0402_032_
GSEPATSGSETPG 724 GGTAGCGAACCTGCTACCTCCGGTTCTG 761 GFP-N_H09.ab1
TSESATPESGPGS AAACCCCAGGTACCTCTGAAAGCGCAAC PAGSPTSTEE
TCCGGAGTCTGGTCCAGGTAGCCCTGCA GGTTCTCCTACCTCCACTGAGGAA LCW0402_034_
GTSESATPESGPG 725 GGTACCTCTGAAAGCGCTACTCCGGAGT 762 GFP-N_A10.ab1
TSTEPSEGSAPGT CTGGCCCAGGTACCTCTACTGAACCGTCT STEPSEGSAP
GAGGGTAGCGCTCCAGGTACTTCTACTG AACCGTCCGAAGGTAGCGCACCA LCW0402_036_
GSPAGSPTSTEEG 726 GGTAGCCCGGCTGGTTCTCCGACTTCCAC 763 GFP-N_C10.ab1
TSTEPSEGSAPGT CGAGGAAGGTACCTCTACTGAACCTTCT STEPSEGSAP
GAGGGTAGCGCTCCAGGTACCTCTACTG AACCTTCCGAAGGCAGCGCTCCA LCW0402_039_
GTSTEPSEGSAPG 727 GGTACTTCTACCGAACCGTCCGAGGGCA 764 GFP-N_E10.ab1
TSTEPSEGSAPGT GCGCTCCAGGTACTTCTACTGAACCTTCT STEPSEGSAP
GAAGGCAGCGCTCCAGGTACTTCTACTG AACCTTCCGAAGGTAGCGCACCA LCW0402_040_
GSEPATSGSETPG 728 GGTAGCGAACCTGCAACCTCTGGCTCTG 765 GFP-N_F10.ab1
TSESATPESGPGT AAACCCCAGGTACCTCTGAAAGCGCTAC STEPSEGSAP
TCCTGAATCTGGCCCAGGTACTTCTACTG AACCGTCCGAGGGCAGCGCACCA LCW0402_041_
GTSTEPSEGSAPG 729 GGTACTTCTACCGAACCGTCCGAGGGTA 766 GFP-N_G10.ab1
SPAGSPTSTEEGT GCGCACCAGGTAGCCCAGCAGGTTCTCC STEPSEGSAP
TACCTCCACCGAGGAAGGTACTTCTACC GAACCGTCCGAGGGTAGCGCACCA LCW0402_050_
GSEPATSGSETPG 730 GGTAGCGAACCGGCAACCTCCGGCTCTG 767 GFP-N_A11.ab1
TSESATPESGPGS AAACTCCAGGTACTTCTGAAAGCGCTAC EPATSGSETP
TCCGGAATCCGGCCCAGGTAGCGAACCG GCTACTTCCGGCTCTGAAACCCCA LCW0402_051_
GSEPATSGSETPG 731 GGTAGCGAACCGGCAACTTCCGGCTCTG 768 GFP-N_B11.ab1
TSESATPESGPGS AAACCCCAGGTACTTCTGAAAGCGCTAC EPATSGSETP
TCCTGAGTCTGGCCCAGGTAGCGAACCT GCTACCTCTGGCTCTGAAACCCCA LCW0402_059_
GSEPATSGSETPG 732 GGTAGCGAACCGGCAACCTCTGGCTCTG 769 GFP-N_E11.ab1
SEPATSGSETPGT AAACTCCAGGTAGCGAACCTGCAACCTC STEPSEGSAP
CGGCTCTGAAACCCCAGGTACTTCTACTG AACCTTCTGAGGGCAGCGCACCA LCW0402_060_
GTSESATPESGPG 733 GGTACTTCTGAAAGCGCTACCCCGGAAT 770 GFP-N_F11.ab1
SEPATSGSETPGS CTGGCCCAGGTAGCGAACCGGCTACTTC EPATSGSETP
TGGTTCTGAAACCCCAGGTAGCGAACCG GCTACCTCCGGTTCTGAAACTCCA LCW0402_061_
GTSTEPSEGSAPG 734 GGTACCTCTACTGAACCTTCCGAAGGCA 771 GFP-N_G11.ab1
TSTEPSEGSAPGT GCGCTCCAGGTACCTCTACCGAACCGTC SESATPESGP
CGAGGGCAGCGCACCAGGTACTTCTGAA AGCGCAACCCCTGAATCCGGTCCA LCW0402_065_
GSEPATSGSETPG 735 GGTAGCGAACCGGCAACCTCTGGCTCTG 772 GFP-N_A12.ab1
TSESATPESGPGT AAACCCCAGGTACCTCTGAAAGCGCTAC SESATPESGP
TCCGGAATCTGGTCCAGGTACTTCTGAA AGCGCTACTCCGGAATCCGGTCCA LCW0402_066_
GSEPATSGSETPG 736 GGTAGCGAACCTGCTACCTCCGGCTCTG 773 GFP-N_B12.ab1
SEPATSGSETPGT AAACTCCAGGTAGCGAACCGGCTACTTC STEPSEGSAP
CGGTTCTGAAACTCCAGGTACCTCTACCG AACCTTCCGAAGGCAGCGCACCA LCW0402_067_
GSEPATSGSETPG 737 GGTAGCGAACCTGCTACTTCTGGTTCTGA 774 GFP-N_C12.ab1
TSTEPSEGSAPGS AACTCCAGGTACTTCTACCGAACCGTCC EPATSGSETP
GAGGGTAGCGCTCCAGGTAGCGAACCTG CTACTTCTGGTTCTGAAACTCCA LCW0402_069_
GTSTEPSEGSAPG 738 GGTACCTCTACCGAACCGTCCGAGGGTA 775 GFP-N_D12.ab1
TSTEPSEGSAPGS GCGCACCAGGTACCTCTACTGAACCGTC EPATSGSETP
TGAGGGTAGCGCTCCAGGTAGCGAACCG GCAACCTCCGGTTCTGAAACTCCA LCW0402_073_
GTSTEPSEGSAPG 739 GGTACTTCTACTGAACCTTCCGAAGGTA 776 GFP-N_F12.ab1
SEPATSGSETPGS GCGCTCCAGGTAGCGAACCTGCTACTTCT PAGSPTSTEE
GGTTCTGAAACCCCAGGTAGCCCGGCTG GCTCTCCGACCTCCACCGAGGAA LCW0402_074_
GSEPATSGSETPG 740 GGTAGCGAACCGGCTACTTCCGGCTCTG 777 GFP-N_G12.ab1
SPAGSPTSTEEGT AGACTCCAGGTAGCCCAGCTGGTTCTCC SESATPESGP
AACCTCTACTGAGGAAGGTACTTCTGAA AGCGCTACCCCTGAATCTGGTCCA LCW0402_075_
GTSESATPESGPG 741 GGTACCTCTGAAAGCGCAACTCCTGAGT 778 GFP-N_H12.ab1
SEPATSGSETPGT CTGGCCCAGGTAGCGAACCTGCTACCTC SESATPESGP
CGGCTCTGAGACTCCAGGTACCTCTGAA AGCGCAACCCCGGAATCTGGTCCA
Example 3: Construction of XTEN_AF36 Segments
[0448] A codon library encoding sequences of 36 amino acid length
was constructed. The sequences were designated XTEN_AF36. Its
segments have the amino acid sequence [X]3 where X is a 12mer
peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 34),
GTSTPESGSASP (SEQ ID NO: 35), GTSPSGESSTAP (SEQ ID NO: 36), or
GSTSSTAESPGP (SEQ ID NO: 37). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00028 AF1for: (SEQ ID NO: 779)
AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC AF1rev: (SEQ ID NO: 780)
ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA AF2for: (SEQ ID NO: 781)
AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC AF2rev: (SEQ ID NO: 782)
ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT AF3for: (SEQ ID NO: 783)
AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC AF3rev: (SEQ ID NO: 784)
ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT AF4for: (SEQ ID NO: 785)
AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC AF4rev: (SEQ ID NO: 786)
ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA
[0449] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 619) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 620). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12mer repeats
ligated to one BbsI/KpnI segment The products corresponding to the
length of 36 amino acids were isolated from the mixture by
preparative agarose gel electrophoresis and ligated into the
BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in
the resulting library designated LCW0403 showed green fluorescence
after induction which shows that the sequence of XTEN_AF36 had been
ligated in frame with the GFP gene and most sequences of XTEN_AF36
show good expression.
[0450] We screened 96 isolates from library LCW0403 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 44 clones
were identified that contained correct XTEN_AF36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 24.
TABLE-US-00029 TABLE 24 DNA and Amino Acid Sequences for 36-mer
motifs SEQ SEQ Amino acid ID ID File_name sequence NO: Nucleotide
sequence NO: LCW0403_004_GFP-N_A01.ab1 GTSTPESGSASPGT 787
GGTACTTCTACTCCGGAAAGCGGTTCC 831 SPSGESSTAPGTSP
GCATCTCCAGGTACTTCTCCTAGCGGT SGESSTAP GAATCTTCTACTGCTCCAGGTACCTCT
CCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_GFP-N_B01.ab1
GTSPSGESSTAPGS 788 GGTACTTCTCCGAGCGGTGAATCTTCT 832 TSSTAESPGPGTSP
ACCGCACCAGGTTCTACTAGCTCTACC SGESSTAP GCTGAATCTCCGGGCCCAGGTACTTCT
CCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_GFP-N_C01.ab1
GSTSSTAESPGPGT 789 GGTTCCACCAGCTCTACTGCTGAATCT 833 SPSGESSTAPGTST
CCTGGTCCAGGTACCTCTCCTAGCGGT PESGSASP GAATCTTCTACTGCTCCAGGTACTTCT
ACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_GFP-N_D01.ab1
GSTSSTAESPGPGS 790 GGTTCTACCAGCTCTACTGCAGAATCT 834 TSSTAESPGPGTSP
CCTGGCCCAGGTTCCACCAGCTCTACC SGESSTAP GCAGAATCTCCGGGTCCAGGTACTTCC
CCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_GFP-N_E01.ab1
GSTSSTAESPGPGT 791 GGTTCTACTAGCTCTACTGCTGAATCT 835 SPSGESSTAPGTST
CCTGGCCCAGGTACTTCTCCTAGCGGT PESGSASP GAATCTTCTACCGCTCCAGGTACCTCT
ACTCCGGAAAGCGGTTCTGCATCTCCA LCW0403_010_GFP-N_F01.ab1
GSTSSTAESPGPGT 792 GGTTCTACCAGCTCTACCGCAGAATCT 836 STPESGSASPGSTS
CCTGGTCCAGGTACCTCTACTCCGGAA ESPSGTAP AGCGGCTCTGCATCTCCAGGTTCTACT
AGCGAATCTCCTTCTGGCACTGCACCA LCW0403_O11_GFP-N_G01.ab1
GSTSSTAESPGPGT 793 GGTTCTACTAGCTCTACTGCAGAATCT 837 STPESGSASPGTST
CCTGGCCCAGGTACCTCTACTCCGGAA PESGSASP AGCGGCTCTGCATCTCCAGGTACTTCT
ACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_GFP-N_H01.ab1
GSTSESPSGTAPGT 794 GGTTCTACCAGCGAATCTCCTTCTGGC 838 SPSGESSTAPGSTS
ACCGCTCCAGGTACCTCTCCTAGCGGC ESPSGTAP GAATCTTCTACCGCTCCAGGTTCTACT
AGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_GFP-N_A02.ab1
GSTSSTAESPGPGS 795 GGTTCCACCAGCTCTACTGCAGAATCT 839 TSSTAESPGPGTSP
CCGGGCCCAGGTTCTACTAGCTCTACT SGESSTAP GCAGAATCTCCGGGTCCAGGTACTTCT
CCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_GFP-N_B02.ab1
GSTSSTAESPGPGT 796 GGTTCCACTAGCTCTACTGCAGAATCT 840 STPESGSASPGSTS
CCTGGCCCAGGTACCTCTACCCCTGAA ESPSGTAP AGCGGCTCTGCATCTCCAGGTTCTACC
AGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_GFP-N_C02.ab1
GSTSSTAESPGPGS 797 GGTTCTACTAGCTCTACTGCTGAATCT 841 TSSTAESPGPGTSP
CCGGGTCCAGGTTCTACCAGCTCTACT SGESSTAP GCTGAATCTCCTGGTCCAGGTACCTCC
CCGAGCGGTGAATCTTCTACTGCACCA LCW0403_017_GFP-N_D02.ab1
GSTSSTAESPGPGS 798 GGTTCTACCAGCTCTACCGCTGAATCT 842 TSESPSGTAPGSTS
CCTGGCCCAGGTTCTACCAGCGAATCC STAESPGP CCGTCTGGCACCGCACCAGGTTCTACT
AGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_GFP-N_E02.ab1
GSTSSTAESPGPGS 799 GGTTCTACCAGCTCTACCGCAGAATCT 843 TSSTAESPGPGSTS
CCTGGCCCAGGTTCCACTAGCTCTACC STAESPGP GCTGAATCTCCTGGTCCAGGTTCTACT
AGCTCTACCGCTGAATCTCCTGGTCCA LCW0403_019_GFP-N_F02.ab1
GSTSESPSGTAPGS 800 GGTTCTACTAGCGAATCCCCTTCTGGT 844 TSSTAESPGPGSTS
ACTGCTCCAGGTTCCACTAGCTCTACC STAESPGP GCTGAATCTCCTGGCCCAGGTTCCACT
AGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_GFP-N_H02.ab1
GSTSESPSGTAPGS 801 GGTTCTACTAGCGAATCTCCTTCTGGT 845 TSESPSGTAPGSTS
ACCGCTCCAGGTTCTACCAGCGAATCC ESPSGTAP CCGTCTGGTACTGCTCCAGGTTCTACC
AGCGAATCTCCTTCTGGTACTGCACCA LCW0403_024_GFP-N_A03.ab1
GSTSSTAESPGPGS 802 GGTTCCACCAGCTCTACTGCTGAATCT 846 TSSTAESPGPGSTS
CCTGGCCCAGGTTCTACCAGCTCTACT STAESPGP GCTGAATCTCCGGGCCCAGGTTCCACC
AGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_GFP-N_B03.ab1
GSTSSTAESPGPGS 803 GGTTCCACTAGCTCTACCGCAGAATCT 847 TSSTAESPGPGTSP
CCTGGTCCAGGTTCTACTAGCTCTACT SGESSTAP GCTGAATCTCCGGGTCCAGGTACCTCC
CCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_GFP-N_D03.ab1
GSSPSASTGTGPGS 804 GGTTCTAGCCCTTCTGCTTCCACCGGT 848 STPSGATGSPGSST
ACCGGCCCAGGTAGCTCTACTCCGTCT PSGATGSP GGTGCAACTGGCTCTCCAGGTAGCTCT
ACTCCGTCTGGTGCAACCGGCTCCCCA LCW0403_029_GFP-N_E03.ab1
GTSPSGESSTAPGT 805 GGTACTTCCCCTAGCGGTGAATCTTCT 849 STPESGSASPGSTS
ACTGCTCCAGGTACCTCTACTCCGGAA STAESPGP AGCGGCTCCGCATCTCCAGGTTCTACT
AGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_GFP-N_F03.ab1
GSTSSTAESPGPGS 806 GGTTCTACTAGCTCTACCGCTGAATCT 850 TSSTAESPGPGTST
CCGGGTCCAGGTTCTACCAGCTCTACT PESGSASP GCAGAATCTCCTGGCCCAGGTACTTCT
ACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_031_GFP-N_G03.ab1
GTSPSGESSTAPGS 807 GGTACTTCTCCTAGCGGTGAATCTTCT 851 TSSTAESPGPGTST
ACCGCTCCAGGTTCTACCAGCTCTACT PESGSASP GCTGAATCTCCTGGCCCAGGTACTTCT
ACCCCGGAAAGCGGCTCCGCTTCTCCA LCW0403_033_GFP-N_H03.ab1
GSTSESPSGTAPGS 808 GGTTCTACTAGCGAATCCCCTTCTGGT 852 TSSTAESPGPGSTS
ACTGCACCAGGTTCTACCAGCTCTACT STAESPGP GCTGAATCTCCGGGCCCAGGTTCCACC
AGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_GFP-N_A04.ab1
GSTSSTAESPGPGS 809 GGTTCCACCAGCTCTACCGCTGAATCT 853 TSESPSGTAPGSTS
CCGGGCCCAGGTTCTACCAGCGAATCC STAESPGP CCTTCTGGCACTGCACCAGGTTCTACT
AGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_GFP-N_B04.ab1
GSTSSTAESPGPGT 810 GGTTCTACCAGCTCTACTGCTGAATCT 854 SPSGESSTAPGTST
CCGGGTCCAGGTACTTCCCCGAGCGGT PESGSASP GAATCTTCTACTGCACCAGGTACTTCT
ACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_GFP-N_C04.ab1
GSTSESPSGTAPGS 811 GGTTCTACCAGCGAATCTCCTTCTGGC 855 TSESPSGTAPGTSP
ACCGCTCCAGGTTCTACTAGCGAATCC SGESSTAP CCGTCTGGTACCGCACCAGGTACTTCT
CCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_GFP-N_D04.ab1
GSTSESPSGTAPGS 812 GGTTCTACCAGCGAATCCCCTTCTGGT 856 TSESPSGTAPGTST
ACTGCTCCAGGTTCTACCAGCGAATCC PESGSASP CCTTCTGGCACCGCACCAGGTACTTCT
ACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_GFP-N_E04.ab1
GTSTPESGSASPGS 813 GGTACCTCTACTCCTGAAAGCGGTTCT 857 TSSTAESPGPGSTS
GCATCTCCAGGTTCCACTAGCTCTACC STAESPGP GCAGAATCTCCGGGCCCAGGTTCTACT
AGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_GFP-N_F04.ab1
GSTSESPSGTAPGS 814 GGTTCTACCAGCGAATCCCCTTCTGGC 858 TSESPSGTAPGTSP
ACTGCACCAGGTTCTACTAGCGAATCC SGESSTAP CCTTCTGGTACCGCACCAGGTACTTCT
CCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_GFP-N_G04.ab1
GSTSSTAESPGPGS 815 GGTTCTACTAGCTCTACCGCTGAATCT 859 TSSTAESPGPGSTS
CCTGGCCCAGGTTCCACTAGCTCTACC ESPSGTAP GCAGAATCTCCGGGCCCAGGTTCTACT
AGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_GFP-N_H04.ab1
GSTSSTAESPGPGS 816 GGTTCCACCAGCTCTACTGCAGAATCT 860 TSSTAESPGPGTST
CCTGGCCCAGGTTCTACTAGCTCTACC PESGSASP GCAGAATCTCCTGGTCCAGGTACCTCT
ACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_GFP-N_A05.ab1
GSTSSTAESPGPGS 817 GGTTCTACTAGCTCTACTGCTGAATCT 861 TSSTAESPGPGSTS
CCGGGCCCAGGTTCTACTAGCTCTACC ESPSGTAP GCTGAATCTCCGGGTCCAGGTTCTACT
AGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_GFP-N_B05.ab1
GTSPSGESSTAPGS 818 GGTACCTCCCCGAGCGGTGAATCTTCT 862 TSESPSGTAPGSTS
ACTGCACCAGGTTCTACTAGCGAATCC STAESPGP CCTTCTGGTACTGCTCCAGGTTCCACC
AGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_GFP-N_C05.ab1
GSTSESPSGTAPGT 819 GGTTCTACTAGCGAATCCCCGTCTGGT 863 SPSGESSTAPGSTS
ACTGCTCCAGGTACTTCCCCTAGCGGT STAESPGP GAATCTTCTACTGCTCCAGGTTCTACC
AGCTCTACCGCAGAATCTCCGGGTCCA LCW0403_057_GFP-N_D05.ab1
GSTSSTAESPGPGS 820 GGTTCTACCAGCTCTACCGCTGAATCT 864 TSESPSGTAPGTSP
CCTGGCCCAGGTTCTACTAGCGAATCT SGESSTAP CCGTCTGGCACCGCACCAGGTACTTCC
CCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_GFP-N_E05.ab1
GSTSESPSGTAPGS 821 GGTTCTACTAGCGAATCTCCTTCTGGC 865 TSESPSGTAPGTST
ACTGCACCAGGTTCTACCAGCGAATCT PESGSASP CCGTCTGGCACTGCACCAGGTACCTCT
ACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_GFP-N_F05.ab1
GTSTPESGSASPGS 822 GGTACCTCTACTCCGGAAAGCGGTTCC 866 TSESPSGTAPGSTS
GCATCTCCAGGTTCTACCAGCGAATCC STAESPGP CCGTCTGGCACCGCACCAGGTTCTACT
AGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_GFP-N_G05.ab1
GSTSSTAESPGPGT 823 GGTTCTACTAGCTCTACTGCAGAATCT 867 SPSGESSTAPGTSP
CCGGGCCCAGGTACCTCTCCTAGCGGT SGESSTAP GAATCTTCTACCGCTCCAGGTACTTCT
CCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_GFP-N_H05.ab1
GTSPSGESSTAPGT 824 GGTACCTCCCCTAGCGGCGAATCTTCT 868 SPSGESSTAPGTSP
ACTGCTCCAGGTACCTCTCCTAGCGGC SGESSTAP GAATCTTCTACCGCTCCAGGTACCTCC
CCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_GFP-N_A06.ab1
GSTSSTAESPGPGT 825 GGTTCCACTAGCTCTACTGCTGAATCT 869 STPESGSASPGSTS
CCTGGCCCAGGTACTTCTACTCCGGAA ESPSGTAP AGCGGTTCCGCTTCTCCAGGTTCTACT
AGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_GFP-N_B06.ab1
GSTSESPSGTAPGT 826 GGTTCTACTAGCGAATCTCCGTCTGGC 870 SPSGESSTAPGTSP
ACTGCTCCAGGTACTTCTCCTAGCGGT SGESSTAP GAATCTTCTACCGCTCCAGGTACTTCC
CCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_GFP-N_C06.ab1
GSTSESPSGTAPGT 827 GGTTCTACTAGCGAATCTCCTTCTGGT 871 STPESGSASPGSTS
ACCGCTCCAGGTACTTCTACCCCTGAA STAESPGP
AGCGGCTCCGCTTCTCCAGGTTCCACT
AGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_068_GFP-N_D06.ab1
GSTSSTAESPGPGS 828 GGTTCCACTAGCTCTACTGCTGAATCT 872 TSSTAESPGPGSTS
CCTGGCCCAGGTTCTACCAGCTCTACC ESPSGTAP GCTGAATCTCCTGGCCCAGGTTCTACC
AGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_GFP-N_E06.ab1
GSTSESPSGTAPGT 829 GGTTCTACTAGCGAATCCCCGTCTGGT 873 STPESGSASPGTST
ACCGCACCAGGTACTTCTACCCCGGAA PESGSASP AGCGGCTCTGCTTCTCCAGGTACTTCT
ACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_GFP-N_F06.ab1
GSTSESPSGTAPGT 830 GGTTCTACTAGCGAATCCCCGTCTGGT 874 STPESGSASPGTST
ACTGCTCCAGGTACTTCTACTCCTGAA PESGSASP AGCGGTTCCGCTTCTCCAGGTACCTCT
ACTCCGGAAAGCGGTTCTGCATCTCCA
Example 4: Construction of XTEN_AG36 Segments
[0451] A codon library encoding sequences of 36 amino acid length
was constructed. The sequences were designated XTEN_AG36. Its
segments have the amino acid sequence [X].sub.3 where X is a 12mer
peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 38),
GSSTPSGATGSP (SEQ ID NO: 39), GSSPSASTGTGP (SEQ ID NO: 40), or
GASPGTSSTGSP (SEQ ID NO: 41). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00030 AG1for: (SEQ ID NO: 875)
AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC AG1rev: (SEQ ID NO: 876)
ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT AG2for: (SEQ ID NO: 877)
AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC AG2rev: (SEQ ID NO: 878)
ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT AG3for: (SEQ ID NO: 879)
AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC AG3rev: (SEQ ID NO: 880)
ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA AG4for: (SEQ ID NO: 881)
AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC AG4rev: (SEQ ID NO: 882)
ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC
[0452] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTNCGTCTTCACTCGAGGGTAC (SEQ ID NO: 619) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 620). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12mer repeats
ligated to one BbsI/KpnI segment. The products corresponding to the
length of 36 amino acids were isolated from the mixture by
preparative agarose gel electrophoresis and ligated into the
BsaI/KpnI digested stuffier vector pCW0359. Most of the clones in
the resulting library designated LCW4A4 showed green fluorescence
after induction which shows that the sequence of XTEN_AG36 had been
ligated in frame with the GFP gene and most sequences of XTEN_AG36
show good expression.
[0453] We screened isolates from library LCW0404 for high level of
fluorescence by stamping them onto agar plate containing IPTG. The
same isolates were evaluated by PCR and 48 isolates were identified
that contained segments with 36 amino acids as well as strong
fluorescence. These isolates were sequenced and 44 clones were
identified that contained correct XTEN_AG36 segments. The file
names of the nucleotide and amino acid constructs and the sequences
for these segments are listed in Table 25.
TABLE-US-00031 TABLE 25 DNA and Amino Acid Sequences for 36-mer
motifs SEQ SEQ Amino acid ID ID File name sequence NO: Nucleotide
sequence NO: LCW0404_001_GFP-N_A07.ab1 GASPGTSSTGSPGT 883
GGTGCATCCCCGGGCACTAGCTCTACC 927 PGSGTASSSPGSST
GGTTCTCCAGGTACTCCTGGTAGCGGT PSGATGSP ACTGCTTCTTCTTCTCCAGGTAGCTCT
ACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_GFP-N_B07.ab1
GSSTPSGATGSPGS 884 GGTAGCTCTACCCCTTCTGGTGCTACC 928 SPSASTGTGPGSST
GGCTCTCCAGGTTCTAGCCCGTCTGCT PSGATGSP TCTACCGGTACCGGTCCAGGTAGCTCT
ACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_GFP-N_C07.ab1
GASPGTSSTGSPGS 885 GGTGCATCTCCGGGTACTAGCTCTACC 929 SPSASTGTGPGSST
GGTTCTCCAGGTTCTAGCCCTTCTGCT PSGATGSP TCCACTGGTACCGGCCCAGGTAGCTCT
ACCCCGTCTGGTGCTACTGGTTCCCCA LCW0404_007_GFP-N_D07.ab1
GTPGSGTASSSPGS 886 GGTACTCCGGGCAGCGGTACTGCTTCT 930 STPSGATGSPGASP
TCCTCTCCAGGTAGCTCTACCCCTTCT GTSSTGSP GGTGCAACTGGTTCCCCAGGTGCATCC
CCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_GFP-N_E07.ab1
GTPGSGTASSSPGA 887 GGTACCCCTGGCAGCGGTACTGCTTCT 931 SPGTSSTGSPGSRP
TCTTCTCCAGGTGCTTCCCCTGGTACC SASTGTGP AGCTCTACCGGTTCTCCAGGTTCTAGA
CCTTCTGCATCCACCGGTACTGGTCCA LCW0404_011_GFP-N_F07.ab1
GASPGTSSTGSPGS 888 GGTGCATCTCCTGGTACCAGCTCTACC 932 STPSGATGSPGASP
GGTTCTCCAGGTAGCTCTACTCCTTCT GTSSTGSP GGTGCTACTGGCTCTCCAGGTGCTTCC
CCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_012_GFP-N_G07.ab1
GTPGSGTASSSPGS 889 GGTACCCCGGGCAGCGGTACCGCATCT 933 STPSGATGSPGSST
TCCTCTCCAGGTAGCTCTACCCCGTCT PSGATGSP GGTGCTACCGGTTCCCCAGGTAGCTCT
ACCCCGTCTGGTGCAACCGGCTCCCCA LCW0404_014_GFP-N_H07.ab1
GASPGTSSTGSPGA 890 GGTGCATCTCCGGGCACTAGCTCTACT 934 SPGTSSTGSPGASP
GGTTCTCCAGGTGCATCCCCTGGCACT GTSSTGSP AGCTCTACTGGTTCTCCAGGTGCTTCT
CCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_GFP-N_A08.ab1
GSSTPSGATGSPGS 891 GGTAGCTCTACTCCGTCTGGTGCAACC 935 SPSASTGTGPGASP
GGCTCCCCAGGTTCTAGCCCGTCTGCT GTSSTGSP TCCACTGGTACTGGCCCAGGTGCTTCC
CCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_GFP-N_B08.ab1
GSSTPSGATGSPGS 892 GGTAGCTCTACTCCTTCTGGTGCTACC 936 STPSGATGSPGTPG
GGTTCCCCAGGTAGCTCTACTCCTTCT SGTASSSP GGTGCTACTGGTTCCCCAGGTACTCCG
GGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_GFP-N_C08.ab1
GSSTPSGATGSPGS 893 GGTAGCTCTACTCCGTCTGGTGCAACC 937 STPSGATGSPGASP
GGTTCCCCAGGTAGCTCTACTCCTTCT GTSSTGSP GGTGCTACTGGCTCCCCAGGTGCATCC
CCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_018_GFP-N_D08.ab1
GTPGSGTASSSPGS 894 GGTACTCCTGGTAGCGGTACCGCATCT 938 SPSASTGTGPGSST
TCCTCTCCAGGTTCTAGCCCTTCTGCA PSGATGSP TCTACCGGTACCGGTCCAGGTAGCTCT
ACTCCTTCTGGTGCTACTGGCTCTCCA LCW0404_023_GFP-N_F08.ab1
GASPGTSSTGSPGS 895 GGTGCTTCCCCGGGCACTAGCTCTACC 939 SPSASTGTGPGTPG
GGTTCTCCAGGTTCTAGCCCTTCTGCA SGTASSSP TCTACTGGTACTGGCCCAGGTACTCCG
GGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_GFP-N_G08.ab1
GSSTPSGATGSPGS 896 GGTAGCTCTACTCCGTCTGGTGCTACC 940 STPSGATGSPGASP
GGCTCTCCAGGTAGCTCTACCCCTTCT GTSSTGSP GGTGCAACCGGCTCCCCAGGTGCTTCT
CCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_GFP-N_A09.ab1
GTPGSGTASSSPGS 897 GGTACCCCTGGCAGCGGTACCGCTTCT 941 STPSGATGSPGSSP
TCCTCTCCAGGTAGCTCTACCCCGTCT SASTGTGP GGTGCTACTGGCTCTCCAGGTTCTAGC
CCGTCTGCATCTACCGGTACCGGCCCA LCW0404_030_GFP-N_B09.ab1
GSSTPSGATGSPGT 898 GGTAGCTCTACTCCTTCTGGTGCAACC 942 PGSGTASSSPGTPG
GGCTCCCCAGGTACCCCGGGCAGCGGT SGTASSSP ACCGCATCTTCCTCTCCAGGTACTCCG
GGTAGCGGTACTGCTTCTTCTTCTCCA LCW0404_031_GFP-N_C09.ab1
GTPGSGTASSSPGS 899 GGTACCCCGGGTAGCGGTACTGCTTCT 943 STPSGATGSPGASP
TCCTCTCCAGGTAGCTCTACCCCTTCT GTSSTGSP GGTGCAACCGGCTCTCCAGGTGCTTCT
CCGGGCACCAGCTCTACCGGTTCTCCA LCW0404_034_GFP-N_D09.ab1
GSSTPSGATGSPGS 900 GGTAGCTCTACCCCGTCTGGTGCTACC 944 STPSGATGSPGASP
GGCTCTCCAGGTAGCTCTACCCCGTCT GTSSTGSP GGTGCAACCGGCTCCCCAGGTGCATCC
CCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_GFP-N_E09.ab1
GASPGTSSTGSPGT 901 GGTGCTTCTCCGGGCACCAGCTCTACT 945 PGSGTASSSPGSST
GGTTCTCCAGGTACCCCGGGCAGCGGT PSGATGSP ACCGCATCTTCTTCTCCAGGTAGCTCT
ACTCCTTCTGGTGCAACTGGTTCTCCA LCW0404_036_GFP-N_F09.ab1
GSSPSASTGTGPGS 902 GGTTCTAGCCCGTCTGCTTCCACCGGT 946 STPSGATGSPGTPG
ACTGGCCCAGGTAGCTCTACCCCGTCT SGTASSSP GGTGCAACTGGTTCCCCAGGTACCCCT
GGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_GFP-N_G09.ab1
GASPGTSSTGSPGS 903 GGTGCTTCTCCGGGCACCAGCTCTACT 947 SPSASTGTGPGSST
GGTTCTCCAGGTTCTAGCCCTTCTGCA PSGATGSP TCCACCGGTACCGGTCCAGGTAGCTCT
ACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_GFP-N_H09.abi
GASPGTSSTGSPGS 904 GGTGCATCCCCGGGCACCAGCTCTACC 948 STPSGATGSPGSST
GGTTCTCCAGGTAGCTCTACCCCGTCT PSGATGSP GGTGCTACCGGCTCTCCAGGTAGCTCT
ACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_GFP-N_A10.ab1
GTPGSGTASSSPGS 905 GGTACCCCTGGTAGCGGTACTGCTTCT 949 STPSGATGSPGTPG
TCCTCTCCAGGTAGCTCTACTCCGTCT SGTASSSP GGTGCTACCGGTTCTCCAGGTACCCCG
GGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_GFP-N_C10.ab1
GSSPSASTGTGPGS 906 GGTTCTAGCCCTTCTGCTTCCACCGGT 950 STPSGATGSPGSST
ACTGGCCCAGGTAGCTCTACCCCTTCT PSGATGSP GGTGCTACCGGCTCCCCAGGTAGCTCT
ACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_GFP-N_D10.ab1
GASPGTSSTGSPGS 907 GGTGCTTCTCCTGGCACCAGCTCTACT 951 SPSASTGTGPGSSP
GGTTCTCCAGGTTCTAGCCCTTCTGCT SASTGTGP TCTACCGGTACTGGTCCAGGTTCTAGC
CCTTCTGCATCCACTGGTACTGGTCCA LCW0404_047_GFP-N_F10.ab1
GTPGSGTASSSPGA 908 GGTACTCCTGGCAGCGGTACCGCTTCT 952 SPGTSSTGSPGASP
TCTTCTCCAGGTGCTTCTCCTGGTACT GTSSTGSP AGCTCTACTGGTTCTCCAGGTGCTTCT
CCGGGCACTAGCTCTACTGGTTCTCCA LCW0404_048_GFP-N_G10.ab1
GSSTPSGATGSPGA 909 GGTAGCTCTACCCCGTCTGGTGCTACC 953 SPGTSSTGSPGSST
GGTTCCCCAGGTGCTTCTCCTGGTACT PSGATGSP AGCTCTACCGGTTCTCCAGGTAGCTCT
ACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_GFP-N_H10.ab1
GSSTPSGATGSPGT 910 GGTAGCTCTACCCCGTCTGGTGCTACT 954 PGSGTASSSPGSST
GGTTCTCCAGGTACTCCGGGCAGCGGT PSGATGSP ACTGCTTCTTCCTCTCCAGGTAGCTCT
ACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_GFP-N_A11.ab1
GASPGTSSTGSPGS 911 GGTGCATCTCCTGGTACCAGCTCTACT 955 SPSASTGTGPGSST
GGTTCTCCAGGTTCTAGCCCTTCTGCT PSGATGSP TCTACCGGTACCGGTCCAGGTAGCTCT
ACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_GFP-N_B11.ab1
GSSTPSGATGSPGS 912 GGTAGCTCTACCCCGTCTGGTGCTACT 956 STPSGATGSPGSST
GGCTCTCCAGGTAGCTCTACTCCTTCT PSGATGSP GGTGCTACTGGTTCCCCAGGTAGCTCT
ACCCCGTCTGGTGCAACTGGCTCTCCA LCW0404_052_GFP-N_C11.ab1
GASPGTSSTGSPGT 913 GGTGCATCCCCGGGTACCAGCTCTACC 957 PGSGTASSSPGASP
GGTTCTCCAGGTACTCCTGGCAGCGGT GTSSTGSP ACTGCATCTTCCTCTCCAGGTGCTTCT
CCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_GFP-N_D11.ab1
GSSTPSGATGSPGS 914 GGTAGCTCTACTCCTTCTGGTGCAACT 958 SPSASTGTGPGASP
GGTTCTCCAGGTTCTAGCCCGTCTGCA GTSSTGSP TCCACTGGTACCGGTCCAGGTGCTTCC
CCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_057_GFP-N_E11.ab1
GASPGTSSTGSPGS 915 GGTGCATCTCCTGGTACTAGCTCTACT 959 STPSGATGSPGSSP
GGTTCTCCAGGTAGCTCTACTCCGTCT SASTGTGP GGTGCAACCGGCTCTCCAGGTTCTAGC
CCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_GFP-N_F11.ab1
GTPGSGTASSSPGS 916 GGTACTCCTGGCAGCGGTACCGCATCT 960 STPSGATGSPGASP
TCCTCTCCAGGTAGCTCTACTCCGTCT GTSSTGSP GGTGCAACTGGTTCCCCAGGTGCTTCT
CCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_GFP-N_G11.ab1
GSSTPSGATGSPGT 917 GGTAGCTCTACCCCGTCTGGTGCAACC 961 PGSGTASSSPGSST
GGCTCCCCAGGTACTCCTGGTAGCGGT PSGATGSP ACCGCTTCTTCTTCTCCAGGTAGCTCT
ACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_GFP-N_H11.ab1
GSSPSASTGTGPGS 918 GGTTCTAGCCCTTCTGCATCCACCGGT 962 SPSASTGTGPGASP
ACCGGCCCAGGTTCTAGCCCGTCTGCT GTSSTGSP TCTACCGGTACTGGTCCAGGTGCTTCT
CCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_GFP-N_A12.ab1
GTPGSGTASSSPGS 919 GGTACCCCGGGTAGCGGTACCGCTTCT 963 STPSGATGSPGSNP
TCTTCTCCAGGTAGCTCTACTCCGTCT SASTGTGP GGTGCTACCGGCTCTCCAGGTTCTAAC
CCTTCTGCATCCACCGGTACCGGCCCA LCW0404_068_GFP-N_B12.ab1
GSSPSASTGTGPGS 920 GGTTCTAGCCCTTCTGCATCTACTGGT 964 STPSGATGSPGASP
ACTGGCCCAGGTAGCTCTACTCCTTCT GTSSTGSP GGTGCTACCGGCTCTCCAGGTGCTTCT
CCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_069_GFP-N_C12.ab1
GSSTPSGATGSPGA 921 GGTAGCTCTACCCCTTCTGGTGCAACC 965 SPGTSSTGSPGTPG
GGCTCTCCAGGTGCATCCCCGGGTACC SGTASSSP AGCTCTACCGGTTCTCCAGGTACTCCG
GGTAGCGGTACCGCTTCTTCCTCTCCA LCW0404_070_GFP-N_D12.ab1
GSSTPSGATGSPGS 922 GGTAGCTCTACTCCGTCTGGTGCAACC 966 STPSGATGSPGSST
GGTTCCCCAGGTAGCTCTACCCCTTCT PSGATGSP GGTGCAACCGGCTCCCCAGGTAGCTCT
ACCCCTTCTGGTGCAACTGGCTCTCCA LCW0404_073_GFP-N_E12.ab1
GASPGTSSTGSPGT 923 GGTGCTTCTCCTGGCACTAGCTCTACC 967 PGSGTASSSPGSST
GGTTCTCCAGGTACCCCTGGTAGCGGT
PSGATGSP ACCGCATCTTCCTCTCCAGGTAGCTCT ACTCCTTCTGGTGCTACTGGTTCCCCA
LCW0404_075_GFP-N_F12_abl GSSTPSGATGSPGS 924
GGTAGCTCTACCCCGTCTGGTGCTACT 968 SPSASTGTGPGSSP
GGCTCCCCAGGTTCTAGCCCTTCTGCA SASTGTGP TCCACCGGTACCGGTCCAGGTTCTAGC
CCGTCTGCATCTACTGGTACTGGTCCA LCW0404_080_GFP-N_G12.ab1
GASPGTSSTGSPGS 925 GGTGCTTCCCCGGGCACCAGCTCTACT 969 SPSASTGTGPGSSP
GGTTCTCCAGGTTCTAGCCCGTCTGCT SASTGTGP TCTACTGGTACTGGTCCAGGTTCTAGC
CCTTCTGCTTCCACTGGTACTGGTCCA LCW0404_081_GFP-N_H12.ab1
GASPGTSSTGSPGS 926 GGTGCTTCCCCGGGTACCAGCTCTACC 970 SPSASTGTGPGTPG
GGTTCTCCAGGTTCTAGCCCTTCTGCT SGTASSSP TCTACCGGTACCGGTCCAGGTACCCCT
GGCAGCGGTACCGCATCTTCCTCTCCA
Example 5S: Construction of XTEN_AE864
[0454] XTEN_AE864 was constructed from serial dimerization of
XTEN_AE36 to AE72, 144, 288, 576 and 864. A collection of XTEN_AE72
segments was constructed from 37 different segments of XTEN_AE36.
Cultures of E. coli harboring all 37 different 36-amino acid
segments were mixed and plasmid was isolated. This plasmid pool was
digested with BsaI/NcoI to generate the small fragment as the
insert. The same plasmid pool was digested with BbsI/NcoI to
generate the large fragment as the vector. The insert and vector
fragments were ligated resulting in a doubling of the length and
the ligation mixture was transformed into BL21Gold(DE3) cells to
obtain colonies of XTEN_AE72.
[0455] This library of XTEN_AE72 segments was designated LCW0406.
All clones from LCW0406 were combined and dimerized again using the
same process as described above yielding library LCW0410 of
XTEN_AE144. All clones from LCW0410 were combined and dimerized
again using the same process as described above yielding library
LCW0414 of XTEN_AE288. Two isolates LCW0414.001 and LCW0414.002
were randomly picked from the library and sequenced to verify the
identities. All clones from LCW0414 were combined and dimerized
again using the same process as described above yielding library
LCW0418 of XTEN_AE576. We screened 96 isolates from library LCW0418
for high level of GFP fluorescence. 8 isolates with right sizes of
inserts by PCR and strong fluorescence were sequenced and 2
isolates (LCW0418.018 and LCW0418.052) were chosen for future use
based on sequencing and expression data.
[0456] The specific clone pCW0432 of XTEN_AE864 was constructed by
combining LCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288
using the same dimerization process as described above.
Example 6: Construction of XTEN_AG864
[0457] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AG864 sequences starting from
segments of XTEN_AD36 listed in Example 1. These sequences were
assembled as described in Example 3. Several isolates from
XTEN_AG864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. A full length
clone of XTEN_AG864 had excellent solubility and showed half-life
exceeding 60 h in cynomolgus monkeys.
Example 7: Construction of CBD-XTEN-Cys, a Cysteine-Engineered
XTEN
[0458] A cysteine island (CysIsland) encoding the amino acid
sequence GGSPAGSCTSP (SEQ ID NO: 187) containing one cysteine was
introduced by annealed oligos in the CBD-stuffer-GFP vector to
obtain CBD-CysIsland-GFP, where CysIsland is flanked by the
restriction sites BsaI and BbsI. The CBD-stuffer-GFP vector is a
pET30 derivative from Novagen with TEV protease recognition site
between CBD and the stuffer. Constructs were previously generated
by replacing the stuffer in CBD-stuffer-GFP vector with genes
encoding XTEN_AE288 and XTEN_AE576. The plasmid of
CBD-XTEN_AE288-GFP was digested with BsaI/NcoI to generate the
small fragment as the insert. The plasmid of CBD-CysIsland-GFP was
digested with BbsI/NcoI to generate the large fragment as the
vector. The insert and vector fragments were ligated and the
ligation mixture was electroporated into BL21-Gold (DE3) cells to
obtain transformants of CBD-CysIsland-XTEN_AE288-GFP. Similarly,
the plasmid of CBD-CysIsland-XTEN_AE288-GFP was digested with
BsaI/NcoI to generate the small fragment as the insert. The plasmid
of CBD-XTEN_AE576-GFP was digested with BbsI/NcoI to generate the
large fragment as the vector. The insert and vector fragments were
ligated and the ligation mixture was electroporated into BL21-Gold
(DE3) cells to obtain transformants of
CBD-XTEN_AE576-CysIsland-XTEN_AE288-GFP. Finally, the plasmid of
CBD-XTEN_AE576-CysIsland-XTEN_AE288-GFP was digested with
BbsI/HindIII to remove GFP and ligate with annealed oligos for the
stop codon, and the ligation mixture was electroporated into
BL21-Gold (DE3) cells to obtain transformants of
CBD-XTEN_AE576-CysIsland-XTEN_AE288, which has the DNA and encoded
amino acid sequences that follow in Table 26. Additional constructs
can be created with cysteines inserted at different locations
within the XTEN sequence by the selection of restriction sites
appropriate for the given location, including multiple insertions.
The method could also be utilized to create lysine-engineered XTEN
by substitution of codons encoding lysine for those encoding
cysteine in the oligonucleotides.
TABLE-US-00032 TABLE 26 DNA and amino acid sequence of
Cys-engineered XTEN Clone Amino Acid Name DNA Sequence Sequence
CBD-TEV- ATGGCAAATACACCGGTATCAGGCAATTTGAAGGTTGA MANTPVSGNLKV AE576-
ATTCTACAACAGCAATCCTTCAGATACTACTAACTCAA EFYNSNPSDTTN CysIsland-
TCAATCCTCAGTTCAAGGTTACTAATACCGGAAGCAGT SINPQFKVTNTG AE288
GCAATTGATTTGTCCAAACTCACATTGAGATATTATTA SSAIDLSKLTLR
TACAGTAGACGGACAGAAAGATCAGACCTTCTGGGCTG YYYTVDGQKDQT
ACCATGCTGCAATAATCGGCAGTAACGGCAGCTACAAC FWADHAAIIGSN
GGAATTACTTCAAATGTAAAAGGAACATTTGTAAAAAT GSYNGITSNVKG
GAGTTCCTCAACAAATAACGCAGACACCTACCTTGAAA TFVKMSSSTNNA
TCAGCTTTACAGGCGGAACTCTTGAACCGGGTGCACAT DTYLEISFTGGT
GTTCAGATACAAGGTAGATTTGCAAAGAATGACTGGAG LEPGAHVQIQGR
TAACTATACACAGTCAAATGACTACTCATTCAAGTCTG FAKNDWSNYTQS
CTTCACAGTTTGTTGAATGGGATCAGGTAACAGCATAC NDYSFKSASQFV
TTGAACGGTGTTCTTGTATGGGGTAAAGAACCCGGTGG EWDQVTAYLNGV
CAGTGTAGTAGGTTCAGGTTCAGGATCCGAAAATCTGT LVWGKEPGGSVV
ATTTTCAGGGTGGGTCTCCAGGTAGCCCGGCTGGCTCT GSGSGSENLYFQ
CCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTAC GGSPGSPAGSPT
TCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCG STEEGTSESATP
AAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACT ESGPGTSTEPSE
TCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGG GSAPGSPAGSPT
CAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCA STEEGTSTEPSE
GCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCT GSAPGTSTEPSE
GGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAAC GSAPGTSESATP
CCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTC ESGPGSEPATSG
CAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA SETPGSEPATSG
GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGG SETPGSPAGSPT
TACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTA STEEGTSESATP
CTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGC ESGPGTSTEPSE
CCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTC GSAPGTSTEPSE
TACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTA GSAPGSPAGSPT
CTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAA STEEGTSTEPSE
AGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGA GSAPGTSTEPSE
ACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCG GSAPGTSESATP
CAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT ESGPGTSTEPSE
TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTC GSAPGTSESATP
CGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG ESGPGSEPATSG
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCG SETPGTSTEPSE
GAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGA GSAPGTSTEPSE
GTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCA GSAPGTSESATP
CCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCC ESGPGTSESATP
GGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAAC ESGPGSPAGSPT
CCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCC STEEGTSESATP
CAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA ESGPGSEPATSG
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGG SETPGTSESATP
TACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTA ESGPGTSTEPSE
CCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACC GSAPGTSTEPSE
TCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTC GSAPGTSTEPSE
TACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAG GSAPGTSTEPSE
CAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACC GSAPGTSTEPSE
GAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAG GSAPGTSTEPSE
CGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTA GSAPGSPAGSPT
CCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCA STEEGTSTEPSE
ACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC GSAPGTSESATP
TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTC ESGPGSEPATSG
CTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAG SETPGTSESATP
GGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGA ESGPGSEPATSG
GTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCA SETPGTSESATP
CCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT ESGPGTSTEPSE
GAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGA GSAPGTSESATP
GGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCC ESGPGSPAGSPT
CAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA STEEGSPAGSPT
GGTGGTAGCCCGGCTGGCTCTTGTACCTCTCCAGGTAC STEEGSPAGSPT
CTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCG STEEGTSESATP
AACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT ESGPGTSTEPSE
GAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACC GSAPGGSPAGSC
TGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAA TSPGTSESATPE
GCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAA SGPGSEPATSGS
CCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTC ETPGTSESATPE
TCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAA SGPGSEPATSGS
CCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCC ETPGTSESATPE
GGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC SGPGTSTEPSEG
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTT SAPGSPAGSPTS
CCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCT TEEGTSESATPE
ACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAG SGPGSEPATSGS
CGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCG ETPGTSESATPE
GCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGT SGPGSPAGSPTS
CCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCC TEEGSPAGSPTS
AGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAG TEEGTSTEPSEG
GTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGT SAPGTSESATPE
AGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTAC SGPGTSESATPE
TTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT SGPGTSESATPE
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAA SGPGSEPATSGS
CCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGA ETPGSEPATSGS
AAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTG ETPGSPAGSPTS
AACCGTCCGAGGGCAGCGCACCAGGTTAA (SEQ ID TEEGTSTEPSEG NO: 971)
SAPGTSTEPSEG SAPGSEPATSGS ETPGTSESATPE SGPGTSTEPSEG SAPG(SEQ ID NO:
972)
Example 8: Construction of Cys3-XTEN
[0459] A pair of primers was designed to introduce the restriction
site BamHI and cysteine island 1 of sequence GRATAEAAGCGTAEAA (SEQ
ID NO: 973) at the N-terminus of XTEN_AE432-1, and a partial
cysteine island 2 of sequence TAEAAG (SEQ ID NO: 974) and
restriction site BbsI at C-terminus of XTEN_AE432-1. A second pair
of primers was designed to introduce the restriction site BsaI and
a partial cysteine island 2 of sequence GCGTAEAA (SEQ ID NO: 975)
at the N-terminus of XTEN_AE432-2, and cysteine island 4 of
sequence TAEAAGCGTAEAAR (SEQ ID NO: 976) with an 8.times.His-tag
(H8) (SEQ ID NO: 20) and restriction site HindIII at the C-terminus
of XTEN_AE432-2. The XTEN_AE432-1 contains the 1-432 amino acid
sequence and XTEN_AE432-2 contains the 433-864 amino acid sequence
encoded by the XTEN_AE864 gene. These two pairs of primers were
used to amplify the XTEN_AE432-1 and XTEN_AE432-2 gene,
respectively, by polymerase chain reaction (PCR). The PCR products
of correct sizes were gel-purified and digested with the
restriction enzymes BamHI/BbsI and BsaI/HindIII, respectively, as
the inserts for ligation. A destination vector, a derivative of
pET30 (Novagen) includes a CBD (cellulose binding domain)-stuffer
with the flanking restriction sites BamHI and HindIII. The
destination vector was digested with the restriction enzymes
BamHI/HindIII to remove the stuffer and prepared as the vector. The
vector was ligated with the BamHI/BbsI digested PCR product of
XTEN_AE432-1 and BsaI/HindIII digested PCR of XTEN_AE432-2 above.
The ligation mixture was transformed into E. coli TOP10 competent
cells. Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. Thus, the final
plasmid yields the CBD-cysteine island 1-XTEN_AE432-cysteine island
2-XTEN_AE432-cysteine island 3-H8 ("H8" disclosed as SEQ ID NO: 20)
gene under the control of a T7 promoter. The DNA sequences and
protein sequences are provided in Table 27.
TABLE-US-00033 TABLE 27 Cys3-XTEN DNA and amino acid sequences
Clone Amino Acid Name DNA Sequence Sequence CBD-Cys1-
ATGGCTAATACCCCAGTGAGCGGCAACCTGAAAGTG MANTPVSGNLKVEFY AE432-
GAATTCTACAATAGCAACCCGAGCGACACCACCAAC NSNPSDTTNSINPQF Cys2-
AGCATTAATCCGCAGTTCAAAGTGACCAACACGGGT KVTNTGSSAIDLSKL AE432-
AGCTCCGCGATCGATCTGTCGAAGCTGACGCTGCGT TLRYYYTVDGQKDQT Cys3-H8
TACTATTACACGGTTGACGGTCAGAAAGATCAGACG FWADHAAIIGSNGSY (AC673)
TTCTGGGCTGACCATGCGGCCATTATTGGCAGCAAC NGITSNVKGTFVKMS ("H8"
GGTTCCTACAACGGTATCACGAGCAATGTCAAAGGC SSTNNADTYLEISFT disclosed
ACTTTTGTTAAGATGAGCTCTTCGACCAACAATGCC GGTLEPGAHVQIQGR as SEQ ID
GATACCTATCTGGAGATTAGCTTCACCGGTGGTACT FAKNDWSNYTQSNDY NO: 20)
CTGGAGCCGGGTGCACACGTTCAAATCCAAGGTCGC SFKSASQFVEWDQVT
TTCGCAAAGAATGACTGGAGCAACTATACCCAGTCC AYLNGVLVWGKEPGG
AATGACTACAGCTTCAAAAGCGCTAGCCAATTTGTT SVVGSGSGSGRATAE
GAATGGGATCAGGTCACCGCATACCTGAACGGCGTG AAGCGTAEAAGSPAG
CTGGTCTGGGGCAAGGAACCGGGTGGTAGCGTTGTC SPTSTEEGTSESATP
GGTTCTGGCAGCGGATCCggtcgtGCGACGGCAGAA ESGPGTSTEPSEGSA
GCCGCTGGCtgcGGTACTGCTGAAGCGGCAGGTAGC PGSPAGSPTSTEEGT
CCAGCTGGTAGCCCAACCTCTACCGAAGAAGGTACC STEPSEGSAPGTSTE
TCTGAATCCGCTACTCCAGAATCCGGTCCTGGTACT PSEGSAPGTSESATP
AGCACTGAGCCAAGCGAAGGTTCTGCTCCAGGCTCC ESGPGSEPATSGSET
CCGGCAGGTAGCCCTACCTCTACCGAAGAGGGCACT PGSEPATSGSETPGS
AGCACCGAACCATCTGAGGGTTCCGCTCCTGGCACC PAGSPTSTEEGTSES
TCCACTGAACCGTCCGAAGGCAGTGCTCCGGGTACT ATPESGPGTSTEPSE
TCCGAAAGCGCAACTCCGGAATCCGGCCCTGGTTCT GSAPGTSTEPSEGSA
GAGCCTGCTACTTCCGGCTCTGAAACTCCAGGTAGC PGSPAGSPTSTEEGT
GAGCCAGCGACTTCTGGTTCTGAAACTCCAGGTTCA STEPSEGSAPGTSTE
CCGGCGGGTAGCCCGACGAGCACGGAGGAAGGTACC PSEGSAPGTSESATP
TCTGAGTCGGCCACTCCTGAGTCCGGTCCGGGCACG ESGPGTSTEPSEGSA
AGCACCGAGCCGAGCGAGGGTTCAGCCCCGGGTACC PGTSESATPESGPGS
AGCACGGAGCCGTCCGAGGGTAGCGCACCGGGTTCT EPATSGSETPGTSTE
CCGGCGGGCTCCCCTACGTCTACGGAAGAGGGTACG PSEGSAPGTSTEPSE
TCCACTGAACCTAGCGAGGGCAGCGCGCCAGGCACC GSAPGTSESATPESG
AGCACTGAACCGAGCGAAGGCAGCGCACCTGGCACT PGTSESATPESGPGS
AGCGAGTCTGCGACTCCGGAGAGCGGTCCGGGTACG PAGSPTSTEEGTSES
AGCACGGAACCAAGCGAAGGCAGCGCCCCAGGTACC ATPESGPGSEPATSG
TCTGAATCTGCTACCCCAGAATCTGGCCCGGGTTCC SETPGTSESATPESG
GAGCCAGCTACCTCTGGTTCTGAAACCCCAGGTACT PGTSTEPSEGSAPGT
TCCACTGAACCAAGCGAAGGTAGCGCTCCTGGCACT STEPSEGSAPGTSTE
TCTACTGAACCATCCGAAGGTTCCGCTCCTGGTACG PSEGSAPGTSTEPSE
TCTGAAAGCGCTACCCCTGAAAGCGGCCCAGGCACC GSAPGTSTEPSEGSA
TCTGAAAGCGCTACTCCTGAGAGCGGTCCAGGCTCT PGTSTEPSEGSAPGS
CCAGCAGGTTCTCCAACCTCCACTGAAGAAGGCACC PAGSPTSTEEGTSTE
TCTGAGTCTGCTACCCCTGAATCTGGTCCTGGCTCC PSEGSAPTAEAAGCG
GAACCTGCTACCTCTGGTTCCGAAACTCCAGGTACC TAEAAGTSESATPES
TCGGAATCTGCGACTCCGGAATCTGGCCCGGGCACG GPGSEPATSGSETPG
AGCACGGAGCCGTCTGAGGGTAGCGCACCAGGTACC TSESATPESGPGSEP
AGCACTGAGCCTTCTGAGGGCTCTGCACCGGGTACC ATSGSETPGTSESAT
TCCACGGAACCTTCGGAAGGTTCTGCGCCGGGTACC PESGPGTSTEPSEGS
TCCACTGAGCCATCCGAGGGTTCAGCACCAGGTACT APGTSESATPESGPG
AGCACGGAACCGTCCGAGGGCTCTGCACCAGGTACG SPAGSPTSTEEGSPA
AGCACCGAACCGTCGGAGGGTAGCGCTCCAGGTAGC GSPTSTEEGSPAGSP
CCAGCGGGCTCTCCGACAAGCACCGAAGAAGGCACC TSTEEGTSESATPES
AGCACCGAGCCGTCCGAAGGTTCCGCACCAACCGCT GPGTSTEPSEGSAPG
GAAGCCGCAGGTtgtGGCACTGCGGAAGCTGCAGGT TSESATPESGPGSEP
ACAAGCGAGAGCGCGACTCCTGAATCTGGTCCGGGT ATSGSETPGTSESAT
AGCGAGCCTGCAACCAGCGGTTCTGAGACGCCGGGC PESGPGSEPATSGSE
ACTTCCGAATCTGCGACCCCGGAGTCCGGTCCAGGT TPGTSESATPESGPG
TCAGAGCCGGCGACGAGCGGTTCGGAAACGCCGGGT TSTEPSEGSAPGSPA
ACGTCTGAATCAGCCACGCCGGAGTCTGGTCCGGGT GSPTSTEEGTSESAT
ACCTCGACCGAACCAAGCGAAGGTTCGGCACCGGGT PESGPGSEPATSGSE
ACTAGCGAGAGCGCAACCCCTGAAAGCGGTCCGGGC TPGTSESATPESGPG
AGCCCGGCAGGTTCTCCAACCAGCACCGAAGAAGGT SPAGSPTSTEEGSPA
TCCCCTGCTGGTAGCCCGACCTCTACGGAGGAAGGT GSPTSTEEGTSTEPS
AGCCCTGCAGGTTCCCCAACTTCTACTGAGGAAGGT EGSAPGTSESATPES
ACTTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGT GPGTSESATPESGPG
ACCTCCACTGAACCGTCTGAAGGCTCTGCACCAGGC TSESATPESGPGSEP
ACTTCTGAGTCTGCTACTCCAGAAAGCGGCCCAGGT ATSGSETPGSEPATS
TCTGAACCAGCAACTTCTGGCTCTGAGACTCCAGGC GSETPGSPAGSPTST
ACTTCTGAGTCCGCAACGCCTGAATCCGGTCCTGGT EEGTSTEPSEGSAPG
TCTGAACCAGCTACTTCCGGCAGCGAAACCCCAGGT TSTEPSEGSAPGSEP
ACCTCTGAGTCTGCGACTCCAGAGTCTGGTCCTGGT ATSGSETPGTSESAT
ACTTCCACTGAGCCTAGCGAGGGTTCCGCACCAGGT PESGPGTSTEPSEGS
TCTCCGGCTGGTAGCCCGACCAGCACGGAGGAGGGT APTAEAAGCGTAEAA
ACGTCTGAATCTGCAACGCCGGAATCGGGCCCAGGT RHHHHHHHH (SEQ
TCGGAGCCTGCAACGTCTGGCAGCGAAACCCCGGGT ID NO: 978)
ACCTCCGAATCTGCTACACCGGAAAGCGGTCCTGGC
AGCCCTGCTGGTTCTCCAACCTCTACCGAGGAGGGT
TCACCGGCAGGTAGCCCGACTAGCACTGAAGAAGGT
ACTAGCACGGAGCCGAGCGAGGGTAGTGCTCCGGGT
ACGAGCGAGAGCGCAACGCCAGAGAGCGGTCCAGGC
ACCAGCGAATCGGCCACCCCTGAGAGCGGCCCAGGT
ACTTCTGAGAGCGCCACTCCTGAATCCGGCCCTGGT
AGCGAGCCGGCAACCTCCGGCTCAGAAACTCCTGGT
TCGGAACCAGCGACCAGCGGTTCTGAAACTCCGGGT
AGCCCGGCAGGCAGCCCAACGAGCACCGAAGAGGGT
ACCAGCACGGAACCGAGCGAGGGTTCTGCCCCGGGT
ACTTCCACCGAACCATCGGAGGGCTCTGCACCTGGT
AGCGAACCTGCGACGTCTGGTTCTGAAACGCCGGGT
ACCAGCGAAAGCGCTACCCCAGAATCCGGTCCGGGC
ACTAGCACCGAGCCATCGGAGGGCTCCGCACCAACT
GCAGAAGCGGCTGGTtgtGGCACCGCCGAAGCAGCT cgtCATCACCATCACCACCATCATCACTAA
(SEQ ID NO: 977)
Example 9: Construction of Lys2-XTEN
[0460] A pair of primers was designed to introduce the restriction
site BamHI and the amino acid sequence GRGSP (SEQ ID NO: 979) at
the N-terminus of XTEN_AE432-1, and the amino acid sequence TAEAAG
(SEQ ID NO: 974) and restriction site BbsI at the C-terminus of
XTEN_AE432-1. A second pair of primers was designed to introduce
the restriction site BsaI and an amino acid sequence with an
incorporated lysine of GKPGTAEAA (SEQ ID NO: 980) at the N-terminus
of XTEN_AE432-2, and an amino acid sequence with an incorporated
lysine of GKAT (SEQ ID NO: 981) with an 8.times.His-tag (H8) (SEQ
ID NO: 20) and restriction site HindIII at C-terminus of
XTEN_AE432-2. XTEN_AE432-1 contains the 1-432 amino acid sequence
and XTEN_AE432-2 contains the 433-864 amino acid sequence encoded
by the XTEN_AE864 gene. These two pairs of primers were used to
amplify the XTEN_AE432-1 and XTEN_AE432-2 gene, respectively, by
polymerase chain reaction (PCR). The PCR products of right sizes
were gel-purified and digested with the restriction enzymes
BamHI/BbsI and BsaI/HindIII, respectively, as the inserts for
ligation. A destination vector, derivative of pET30 (Novagen), of
CBD (cellulose binding domain)-stuffer with the flanking
restriction sites BamHI and HindIII was digested with the
restriction enzymes BamHI/HindIII to remove the stuffer and
prepared as the vector. Ligate the vector with the BamHI/BbsI
digested PCR product of XTEN_AE432-1 and BsaI/HindIII digested PCR
of XTEN_AE432-2 above. The ligation mixture was transformed into E.
coli TOP10 competent cells. Transformants were screened by DNA
miniprep and the desired constructs were confirmed by DNA
sequencing. Thus, the final plasmid yields the CBD-GRGSP (SEQ ID
NO: 979)-XTEN_AE432-TAEAAGKPGTAEAA (SEQ ID NO:
982)-XTEN_AE432-GKAT-H8 (SEQ ID NO: 20) gene under the control of a
T7 promoter. The DNA sequences and protein sequences are provided
in Table 28.
TABLE-US-00034 TABLE 28 Lvs2-XTEN DNA and amino acid sequences
Clone Amino Acid Name DNA Sequence Sequence CBD-R-
ATGGCTAATACCCCAGTGAGCGGCAACCTGAAAGT MANTPVSGNLKVEFY AE432-K-
GGAATTCTACAATAGCAACCCGAGCGACACCACCA NSNPSDTTNSINPQF AE432-K-
ACAGCATTAATCCGCAGTTCAAAGTGACCAACACG KVTNTGSSAIDLSKL H8
GGTAGCTCCGCGATCGATCTGTCGAAGCTGACGCT TLRYYYTVDGQKDQT (AC698)
GCGTTACTATTACACGGTTGACGGTCAGAAAGATC FWADHAAIIGSNGSY ("H8"
AGACGTTCTGGGCTGACCATGCGGCCATTATTGGC NGITSNVKGTFVKMS disclosed
AGCAACGGTTCCTACAACGGTATCACGAGCAATGT SSTNNADTYLEISFT as SEQ ID
CAAAGGCACTTTTGTTAAGATGAGCTCTTCGACCA GGTLEPGAHVQIQGR NO: 20)
ACAATGCCGATACCTATCTGGAGATTAGCTTCACC FAKNDWSNYTQSNDY
GGTGGTACTCTGGAGCCGGGTGCACACGTTCAAAT SFKSASQFVEWDQVT
CCAAGGTCGCTTCGCAAAGAATGACTGGAGCAACT AYLNGVLVWGKEPGG
ATACCCAGTCCAATGACTACAGCTTCAAAAGCGCT SVVGSGSGSGRGSPG
AGCCAATTTGTTGAATGGGATCAGGTCACCGCATA SPAGSPTSTEEGTSE
CCTGAACGGCGTGCTGGTCTGGGGCAAGGAACCGG SATPESGPGTSTEPS
GTGGTAGCGTTGTCGGTTCTGGCAGCGGATCCggt EGSAPGSPAGSPTST
cgtGGGTCTCCAGGTAGCCCAGCTGGTAGCCCAAC EEGTSTEPSEGSAPG
CTCTACCGAAGAAGGTACCTCTGAATCCGCTACTC TSTEPSEGSAPGTSE
CAGAATCCGGTCCTGGTACTAGCACTGAGCCAAGC SATPESGPGSEPATS
GAAGGTTCTGCTCCAGGCTCCCCGGCAGGTAGCCC GSETPGSEPATSGSE
TACCTCTACCGAAGAGGGCACTAGCACCGAACCAT TPGSPAGSPTSTEEG
CTGAGGGTTCCGCTCCTGGCACCTCCACTGAACCG TSESATPESGPGTST
TCCGAAGGCAGTGCTCCGGGTACTTCCGAAAGCGC EPSEGSAPGTSTEPS
AACTCCGGAATCCGGCCCTGGTTCTGAGCCTGCTA EGSAPGSPAGSPTST
CTTCCGGCTCTGAAACTCCAGGTAGCGAGCCAGCG EEGTSTEPSEGSAPG
ACTTCTGGTTCTGAAACTCCAGGTTCACCGGCGGG TSTEPSEGSAPGTSE
TAGCCCGACGAGCACGGAGGAAGGTACCTCTGAGT SATPESGPGTSTEPS
CGGCCACTCCTGAGTCCGGTCCGGGCACGAGCACC EGSAPGTSESATPES
GAGCCGAGCGAGGGTTCAGCCCCGGGTACCAGCAC GPGSEPATSGSETPG
GGAGCCGTCCGAGGGTAGCGCACCGGGTTCTCCGG TSTEPSEGSAPGTST
CGGGCTCCCCTACGTCTACGGAAGAGGGTACGTCC EPSEGSAPGTSESAT
ACTGAACCTAGCGAGGGCAGCGCGCCAGGCACCAG PESGPGTSESATPES
CACTGAACCGAGCGAAGGCAGCGCACCTGGCACTA GPGSPAGSPTSTEEG
GCGAGTCTGCGACTCCGGAGAGCGGTCCGGGTACG TSESATPESGPGSEP
AGCACGGAACCAAGCGAAGGCAGCGCCCCAGGTAC ATSGSETPGTSESAT
CTCTGAATCTGCTACCCCAGAATCTGGCCCGGGTT PESGPGTSTEPSEGS
CCGAGCCAGCTACCTCTGGTTCTGAAACCCCAGGT APGTSTEPSEGSAPG
ACTTCCACTGAACCAAGCGAAGGTAGCGCTCCTGG TSTEPSEGSAPGTST
CACTTCTACTGAACCATCCGAAGGTTCCGCTCCTG EPSEGSAPGTSTEPS
GTACGTCTGAAAGCGCTACCCCTGAAAGCGGCCCA EGSAPGTSTEPSEGS
GGCACCTCTGAAAGCGCTACTCCTGAGAGCGGTCC APGSPAGSPTSTEEG
AGGCTCTCCAGCAGGTTCTCCAACCTCCACTGAAG TSTEPSEGSAPTAEA
AAGGCACCTCTGAGTCTGCTACCCCTGAATCTGGT AGKPGTAEAAGTSES
CCTGGCTCCGAACCTGCTACCTCTGGTTCCGAAAC ATPESGPGSEPATSG
TCCAGGTACCTCGGAATCTGCGACTCCGGAATCTG SETPGTSESATPESG
GCCCGGGCACGAGCACGGAGCCGTCTGAGGGTAGC PGSEPATSGSETPGT
GCACCAGGTACCAGCACTGAGCCTTCTGAGGGCTC SESATPESGPGTSTE
TGCACCGGGTACCTCCACGGAACCTTCGGAAGGTT PSEGSAPGTSESATP
CTGCGCCGGGTACCTCCACTGAGCCATCCGAGGGT ESGPGSPAGSPTSTE
TCAGCACCAGGTACTAGCACGGAACCGTCCGAGGG EGSPAGSPTSTEEGS
CTCTGCACCAGGTACGAGCACCGAACCGTCGGAGG PAGSPTSTEEGTSES
GTAGCGCTCCAGGTAGCCCAGCGGGCTCTCCGACA ATPESGPGTSTEPSE
AGCACCGAAGAAGGCACCAGCACCGAGCCGTCCGA GSAPGTSESATPESG
AGGTTCCGCACCAACCGCTGAAGCCGCAGGTaaac PGSEPATSGSETPGT
cgGGCACTGCGGAAGCTGCAGGTACAAGCGAGAGC SESATPESGPGSEPA
GCGACTCCTGAATCTGGTCCGGGTAGCGAGCCTGC TSGSETPGTSESATP
AACCAGCGGTTCTGAGACGCCGGGCACTTCCGAAT ESGPGTSTEPSEGSA
CTGCGACCCCGGAGTCCGGTCCAGGTTCAGAGCCG PGSPAGSPTSTEEGT
GCGACGAGCGGTTCGGAAACGCCGGGTACGTCTGA SESATPESGPGSEPA
ATCAGCCACGCCGGAGTCTGGTCCGGGTACCTCGA TSGSETPGTSESATP
CCGAACCAAGCGAAGGTTCGGCACCGGGTACTAGC ESGPGSPAGSPTSTE
GAGAGCGCAACCCCTGAAAGCGGTCCGGGCAGCCC EGSPAGSPTSTEEGT
GGCAGGTTCTCCAACCAGCACCGAAGAAGGTTCCC STEPSEGSAPGTSES
CTGCTGGTAGCCCGACCTCTACGGAGGAAGGTAGC ATPESGPGTSESATP
CCTGCAGGTTCCCCAACTTCTACTGAGGAAGGTAC ESGPGTSESATPESG
TTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGTA PGSEPATSGSETPGS
CCTCCACTGAACCGTCTGAAGGCTCTGCACCAGGC EPATSGSETPGSPAG
ACTTCTGAGTCTGCTACTCCAGAAAGCGGCCCAGG SPTSTEEGTSTEPSE
TTCTGAACCAGCAACTTCTGGCTCTGAGACTCCAG GSAPGTSTEPSEGSA
GCACTTCTGAGTCCGCAACGCCTGAATCCGGTCCT PGSEPATSGSETPGT
GGTTCTGAACCAGCTACTTCCGGCAGCGAAACCCC SESATPESGPGTSTE
AGGTACCTCTGAGTCTGCGACTCCAGAGTCTGGTC PSEGSAPGKATHHHH
CTGGTACTTCCACTGAGCCTAGCGAGGGTTCCGCA HHHH (SEQ ID
CCAGGTTCTCCGGCTGGTAGCCCGACCAGCACGGA NO: 984)
GGAGGGTACGTCTGAATCTGCAACGCCGGAATCGG
GCCCAGGTTCGGAGCCTGCAACGTCTGGCAGCGAA
ACCCCGGGTACCTCCGAATCTGCTACACCGGAAAG
CGGTCCTGGCAGCCCTGCTGGTTCTCCAACCTCTA
CCGAGGAGGGTTCACCGGCAGGTAGCCCGACTAGC
ACTGAAGAAGGTACTAGCACGGAGCCGAGCGAGGG
TAGTGCTCCGGGTACGAGCGAGAGCGCAACGCCAG
AGAGCGGTCCAGGCACCAGCGAATCGGCCACCCCT
GAGAGCGGCCCAGGTACTTCTGAGAGCGCCACTCC
TGAATCCGGCCCTGGTAGCGAGCCGGCAACCTCCG
GCTCAGAAACTCCTGGTTCGGAACCAGCGACCAGC
GGTTCTGAAACTCCGGGTAGCCCGGCAGGCAGCCC
AACGAGCACCGAAGAGGGTACCAGCACGGAACCGA
GCGAGGGTTCTGCCCCGGGTACTTCCACCGAACCA
TCGGAGGGCTCTGCACCTGGTAGCGAACCTGCGAC
GTCTGGTTCTGAAACGCCGGGTACCAGCGAAAGCG
CTACCCCAGAATCCGGTCCGGGCACTAGCACCGAG
CCATCGGAGGGCTCCGCACCAggtAAAgcgaccCA TCACCATCACCACCATCATCACTAA (SEQ
ID NO: 983)
Example 10. Host Strain and Promoter for Expression of XTEN for
Conjugation
[0461] Plasmids pCW1054 expressing CBD-TEV site-XTEN_AE864 under
the control of the TM/ac promoter, pLCW0968.003 expressing CBD-TEV
site-XTEN_AE864-GFP under the control of the T7/lac promoter, and
pLCW0970.030 expressing CBD-TEV site-XTEN_AE864-GFP under the
control of the PhoA promoter, were transformed into both E. coli
BL21 DE3 and E. coli BL21 DE3 rne-131 (Lopez, P. J., et al. (1999)
Mol. Microbiol. 33, 188-199). The rne-131 mutation disrupts the 3'
endoribonucleolytic activity of RNase E (Kido, M., et al. (1996)
Journal of Bacteriology, 178: 3917-3925). Starter cultures of all
six tranformants were prepared by picking single colonies to
inoculate 2 mL of LB Broth media containing the appropriate
selective antibiotic. The starter cultures were grown overnight and
0.5 mL was used to inoculate, in quadruplicate, 25 mL of 2.times.YT
broth, for cells containing pCW1054 and pLCW0968.003, or 25 mL of
PhoA induction broth (Amunix recipe 136-1) for cells containing
pLCW0970.030. The cultures were shaken for 3 hours at 37.degree. C.
The temperature was then reduced to 26.degree. C. for all of the
cultures; and for cells containing pCW1054 and pLCW0968.003 protein
expression was induced with IPTG at 1.0 mM final concentration.
Induction of the PhoA promoter in pLCW070.030 is auto-induced upon
depletion of phosphate from the culture media. The cultures were
then shaken overnight at 26.degree. C. Samples of each culture were
lysed and 20 .mu.l of each lysate were subjected to non-reducing
SDS-PAGE using NuPAGE 4-12% Bis-Tris gel from Invitrogen, according
to manufacturer's specifications, with Coomassie staining. The
results (FIG. 43) showed that when either CBD-TEV
site-XTEN_AE864-GFP or CBD-TEV site-XTEN_AE864 is expressed under
the control of the T7/lac promoter the yield of the recombinant
protein is significantly higher in the E. coli BL21 DE3 rne-131
compared to the E. coli BL21 DE3 cells. Cells were also measured
for GFP fluorescence using a fluorescent plate reader. The results
(FIG. 44) showed that expression in E. coli BL21 DE3 cells of
CBD-TEV site-XTEN_AE864-GFP under the control of the PhoA promoter
is nearly three times greater than the expression of CBD-TEV
site-XTEN_AE864-GFP under the control of the T7/lac promoter. In
the E. coli BL21 DE3 rne-131 both the T7/lac and PhoA promoters
yielded similar levels of expression. Therefore, only upon
inhibition of Rnase E 3' riboendocleolytic activity does T7/lac
induction yield titers similar to that obtained using PhoA
induction. Likely the faster rate of T7 RNAP transcription
(Studier, W., et al. (1986) Journal of Molecular Biology 189:
113-130), relative to translation results in mRNA susceptible to
endonucleolytic attack, while in the case of PhoA induction where
transcription is mediated by the native E. coli RNA polymerase II,
the rate of transcription is closely coupled to the rate of
translation protecting the mRNA from endonucleolytic attack.
Example 11: Construction of 1.times.Amino-XTEN288
[0462] A pair of primers AE278BsaIfor-AACG and AE278-RH8HindIIIrev
("H8" disclosed as SEQ ID NO: 20) were used to PCR the plasmid
containing XTEN_AE864_003 in order to obtain the PCR product
XTEN_AE278. Gel-purification of the band of the right size was
performed, followed by digestion with BsaI/HindIII as the insert of
XTEN_AE278-R-H8 ("H8" disclosed as SEQ ID NO: 20). A digestion of
plasmid pCW1161, which encodes the gene of
N-term-RP11-R-AE432_3Cys-R-H8 ("H8" disclosed as SEQ ID NO: 20)
(construct AC763), with BsaI/HindIII was performed to remove the
fragment of AE4323Cys-R-H8 ("H8" disclosed as SEQ ID NO: 20) and
gel-purify the large fragment as the vector. Ligation of the vector
with the insert was performed and was used to transform BL21
competent cells to obtain the construct of N-term-RP11-R-AE288-R-H8
("H8" disclosed as SEQ ID NO: 20). The XTEN length between the two
trypsin digestion sites (R, Arginine) was calculated by XTEN_AE278
plus some flanking amino acids and equaled exactly 288 amino acids.
Thus, the construct is designed to produce the precursor
N-term-RP11-R-AE288-R-H8 ("8" disclosed as SEQ ID NO: 20) (sequence
in Table 29, below) and generate 1.times.Amino-XTEN288 (Seg 178 of
Table 3, with an N-terminal amino group for conjugation) after
removal of the N-term-RP11 tag and C-term 8.times.His-tag (SEQ ID
NO: 20) by trypsin digestion.
TABLE-US-00035 TABLE 29 DNA and amino acid sequence for
1xAmino-XTEN288 Clone Amino Acid Name DNA Sequence Sequence N-term-
ATGAAAAACCCAGAGCAAGCAGAAGAACAAGCTGAAGAACAG MKNPEQAEEQAEEQRE RP11-R-
CGCGAAGAAACACGTCCGCGTCCTCGCCCACGTCCACGTCCG ETRPRPRPRPRPRPRP
AE288-R- CGTCCACGCCCTCGTCCTCGTCCGCGCCCTCGTCCGagcgcg
RPRPRPRPSASRSAGS H8 "H8" tctcgttccgctGGGTCTCCAAcgGGCCCAGGTTCTGAACCA
PTGPGSEPATSGSETP disclosed
GCAACTTCTGGCTCTGAGACTCCAGGCACTTCTGAGTCCGCA GTSESATPESGPGSEP as SEQ
ID ACGCCTGAATCCGGTCCTGGTTCTGAACCAGCTACTTCCGGC ATSGSETPGTSESATP NO:
20) AGCGAAACCCCAGGTACCTCTGAGTCTGCGACTCCAGAGTCT ESGPGTSTEPSEGSAP
GGTCCTGGTACTTCCACTGAGCCTAGCGAGGGTTCCGCACCA GSPAGSPTSTEEGTSE
GGTTCTCCGGCTGGTAGCCCGACCAGCACGGAGGAGGGTACG SATPESGPGSEPATSG
TCTGAATCTGCAACGCCGGAATCGGGCCCAGGTTCGGAGCCT SETPGTSESATPESGP
GCAACGTCTGGCAGCGAAACCCCGGGTACCTCCGAATCTGCT GSPAGSPTSTEEGSPA
ACACCGGAAAGCGGTCCTGGCAGCCCTGCTGGTTCTCCAACC GSPTSTEEGTSTEPSE
TCTACCGAGGAGGGTTCACCGGCAGGTAGCCCGACTAGCACT GSAPGTSESATPESGP
GAAGAAGGTACTAGCACGGAGCCGAGCGAGGGTAGTGCTCCG GTSESATPESGPGTSE
GGTACGAGCGAGAGCGCAACGCCAGAGAGCGGTCCAGGCACC SATPESGPGSEPATSG
AGCGAATCGGCCACCCCTGAGAGCGGCCCAGGTACTTCTGAG SETPGSEPATSGSETP
AGCGCCACTCCTGAATCCGGCCCTGGTAGCGAGCCGGCAACC GSPAGSPTSTEEGTST
TCCGGCTCAGAAACTCCTGGTTCGGAACCAGCGACCAGCGGT EPSEGSAPGTSTEPSE
TCTGAAACTCCGGGTAGCCCGGCAGGCAGCCCAACGAGCACC GSAPGSEPATSGSETP
GAAGAGGGTACCAGCACGGAACCGAGCGAGGGTTCTGCCCCG GTSESATPESGPGTST
GGTACTTCCACCGAACCATCGGAGGGCTCTGCACCTGGTAGC EPSEGSAPSASRSAHH
GAACCTGCGACGTCTGGTTCTGAAACGCCGGGTACCAGCGAA HHHHHH (SEQ ID
AGCGCTACCCCAGAATCCGGTCCGGGCACTAGCACCGAGCCA NO: 986)
TCGGAGGGCTCCGCACCAagcgcctctcgctccgcaCATCAC CATCACCACCATCATCACTAA
(SEQ IDNO: 985)
Example 12: Optimization for Expression of XTEN Constructs with
RP11 Affinity Tag
[0463] 1. 1. Design and construction of constructs encoding
N-term-RP11-AE864-GFP and MalEss-AE48-RP11-AE864
[0464] A group of highly expressed native E. coli proteins
described by Ishihama Y. et al, BMC Genomics 2008, 9:102 were used
to generate a list of the first 20 N-terminal amino acids (column 3
of Table 30), from which the hydrophobic amino acids F, I, W, L, V,
M, C were converted to alanine or serine, or were deleted in order
to generate candidates to create helper sequences containing at
least 11 amino acids (column 4 of Table 30). For comparative
purposes, the first 20 amino acids of a known CBD sequence from a
well expressed construct built at Amunix (AC616) was also included
as a control.
TABLE-US-00036 TABLE 30 List of N-terminal helpers designed in the
study SEQ SEQ Protein ID N-term ID Plasmid description 1st 20aa NO:
Helpers NO: pSD0107 30S ribosomal MAENQYYGTG 987 MAENQYYGTG 999
protein S9 RRKSSAARVF RRKSSAAR pSD0108 50S ribosomal MAVQQNKPTR 988
MAAQQNKPTR 1000 protein L32 SKRGMRRSHD SKRGARRSHD pSD0109 30S
ribosomal MANIKSAKKR 989 MANAKSAKKR 1001 protein S20 AIQSEKARKH
AAQSEKARKH pSD0110 GrpE protein MSSKEQKTPE 990 MSSKEQKTPE 1002
HSP-70 cofactor GQAPEEHMD GQAPEE pSD0111 30S ribosomal MAKQSMKARE
991 MAKQSAKARE 1003 protein S14 VKRVALADKY AKRAASADKY pSD0112 30S
ribosomal MATVNQLVRK 992 MATANQAARK 1004 protein S12 PRARKVAKSN
PRARKAAKSN pSD0113 Elongation MSKEKFERTK 993 MSKEKAERTK 1005 factor
Tu PHVNVGTIGH PHANAGT (EF-Tu) (P-43) pSD0114 30S ribosomal
MSLSTEATAK 994 MSASTEATAK 1006 protein S15 IVSEFGRDAN AASEAGRDAN
pSD0115 Superoxide MSYTLPSLPY 995 MSYTAPSAPY 1007 dismutase
AYDALEPHFD AYDAAEPH [Mn] (EC 1.15.1.1) (MnSOD) pSD0116 rraB
MANPEQLEEQ 996 MANPEQAEEQ 1008 ribonuclcase REETRLIIEE REETR E
inhibitor protein B pSD0117 Cellulose MANTPVSGKL 997 MANTPASGNA
1009 Binding KVEFYNSNPS KAEAYNSNPS Protein (CBD) pSD0118 CBD (from
MANTPVSGKL 998 MANTPVSGNL 1010 previous KVEFYNSNPS KVEFYNSNPS
construct AC616)
[0465] DNA oligonucleotides for the 107N-F&R to 119N-F&R
series and RP11F&R sequences of Table 31 were synthesized at
Elim Biopharm (Hayward, Calif.). Solutions of each DNA pair (107N-F
and 107N-R, 108N-F and 108N-R, etc) was mixed at a 1:1 molar ratio,
were denatured at 95.degree. C. for 3 min, followed by cooling to
25.degree. C. at 0.1.degree. C./min to allow double strand DNA
annealing. The base vector LCW0970.030 (encoding CBD-AE864-GFP) was
digested with NdeI/BsaI and the larger fragment was gel-purified as
the vector. The vector was ligated with the annealed oligos
107-118N-F&R and PNK treated annealed RP11F&R oligos, and
the ligation products were transformed into E. coli BL21 competent
cells (New England Biolabs) to obtain the colonies designated
pSD0107 to pSD118. The clones pSD0107-109, pSD0111-112, and
pSD0114-118 were obtained and verified by DNA sequencing; clone
pSD0110.001 had one mutation of frame-shift and was used as the
stuffer vector.
[0466] The plasmid construct pCW1110 (encoding RP11-AE864) was
digested with BsaI/NotI and the smaller band of corresponding to
the nucleotides encoding AE864 was gel-purified as the insert.
pCW1139 (encoding MalEss-AE48-payload-AE864) was digested by
XhoI/BstXI/NotI and the larger fragment was gel-purified as the
vector. The annealed product of oligos of 119-AEN-F&R was
ligated with the insert and vector, and then transformed into E.
coli BL21 to obtain colonies with the plasmid, designated pSD0119.
The clones were sequence verified.
[0467] 2. Construction of N-term helper libraries based on
pSD0116
[0468] DNA oligonucleotide pairs of Stuffer-RP5for &
Stuffer-RP5revP, RP6-SASRSAforP & RP6-SASRSArev, L2for &
L2rev, L3for & L3rev, L4for & L4rev, L5for & L5rev,
L6for & L6rev, L7for & L7rev, L8for & L8rev, L9for
& L9rev, 10for & L10rev, L11for & L11rev, L12for &
L12rev, and L13for & L13rev (Table 31) were synthesized at Elim
Biopharm (Hayward, Calif.) and each pair was annealed as described
above (Section 1) to generate double strand DNA.
[0469] Plasmid pSD0110 was digested with NdeI/BsaI and the larger
fragment was gel-purified as the vector. The vector was ligated
with annealed oligos of Stuffer-RP5for&revP and
RP6-SASRSAforP&rev, and then transformed into E. coli BL21 to
obtain the colonies with the stuffer vector plasmid pCW1146
(Stuffer-RP11-XTEN_AE864_003-GFP). The clone was sequence
verified.
[0470] The NdeI/BsaI digested pSD0110 vector was ligated with
L5for&rev annealed oligos to obtain colonies of LCW1160
(L5).
[0471] The stuffer vector pCW1146 was digested with NdeI/BsaI and
the larger fragment was gel-purified as the vector. The vector was
ligated with annealed oligos of L2-4, and L6-13 for&rev as in
Table 31, and then transformed into E. coli BL21 to obtain the
colonies of constructs LCW1157 (L2), LCW1158 (L3), LCW1159 (L4),
LCW1163 (L6), LCW1171 (L7), LCW1172 (L8), LCW1203 (L9), LCW1204
(10), LCW1208 (L11), LCW1209 (L12), and LCW1210 (L13).
TABLE-US-00037 TABLE 31 List of DNA oligonucleotides SEQ ID Name
DNA Sequence NO: RP11F
CGTCCGCGTCCTCGCCCACGTCCACGTCCGCGTCCACGCCCTCGTC 1011
CTCGTCCGCGCCCTCGTCCGagcgcgtctcgttccgctGGGTCTCC RP11R
ACCTGGAGACCCAGCGGAACGAGACGCGCTCGGACGAGGGCGCGGA 1012
CGAGGACGAGGGCGTGGACGCGGACGTGGACGTGGGCGAGGACGCG 107N-F
TATGGCTGAAAATCAATATTATGGTACGGGGCGCCGGAAGAGTTCG 1013 GCCGCC 107N-R
GACGGGCGGCCGAACTCTTCCGGCGCCCCGTACCATAATATTGATT 1014 TTCAGCCA 108N-F
TATGGCAGCTCAGCAGAATAAGCCTACACGAAGTAAAAGAGGCGCG 1015 CGCCGGTCGCACGAT
108N-R GACGATCGTGCGACCGGCGCGCGCCTCTTTTACTTCGTGTAGGCTT 1016
ATTCTGCTGAGCTGCCA 109N-F
TATGGCAAATGCTAAGAGTGCAAAGAAACGGGCGGCACAAAGCGAA 1017 AAAGCTCGGAAACAT
109N-R GACGATGTTTCCGAGCTTTTTCGCTTTGTGCCGCCCGTTTCTTTGC 1018
ACTCTTAGCATTTGCCA 110N-F
TATGTCCAGCAAAGAACAGAAGACTCCGGAAGGTCAAGCGCCAGAG 1019 GAG 110N-R
GACGCTCCTCTGGCGCTTGACCTTCCGGAGTCTTCTGTTCTTTGCT 1020 GGACA 111N-F
TATGGCCAAACAAAGCGCTAAAGCCCGCGAGGCGAAACGTGCAGCC 1021 TCTGCGGACAAATAT
111N-R GACGATATTTGTCCGCAGAGGCTGCACGTTTCGCCTCGCGGGCTTT 1022
AGCGCTTTGTTTGGCCA 112N-F
TATGGCTACTGCAAATCAGGCCGCCCGTAAACCTCGAGCACGAAAG 1023 GCTGCTAAATCAAAT
112N-R GACGATTTGATTTAGCAGCCTTTCGTGCTCGAGGTTTACGGGCGGC 1024
CTGATTTGCAGTAGCCA 113N-F
TATGTCCAAAGAAAAAGCCGAACGGACCAAACCTCATGCTAACGCT 1025 GGCACG 113N-R
GACGCGTGCCAGCGTTAGCATGAGGTTTGGTCCGTTCGGCTTTTTC 1026 TTTGGACA 114N-F
TATGTCAGCGTCTACGGAGGCAACCGCAAAAGCTGCTAGTGAAGCG 1027 GGCCGTGATGCGAAT
114N-R GACGATTCGCATCACGGCCCGCTTCACTAGCAGCTTTTGCGGTTGC 1028
CTCCGTAGACGCTGACA 115N-F
TATGAGCTATACTGCACCGAGCGCACCGTATGCTTATGATGCAGCC 1029 GAACCTCAC
115N-R GACGGTGAGGTTCGGCTGCATCATAAGCATACGGTGCGCTCGGTGC 1030
AGTATAGCTCA 116N-F TATGGCAAACCCCGAACAGGCTGAGGAACAGAGAGAAGAAACA 1031
116N-R GACGTGTTTCTTCTCTCTGTTCCTCAGCCTGTTCGGGGTTTGCCA 1032 117N-F
TATGGCTAATACCCCTGCGAGCGGGAACGCCAAGGCGGAAGCTTAC 1033 AACAGTAATCCAAGC
117N-R GACGGCTTGGATTACTGTTGTAAGCTTCCGCCTTGGCGTTCCCGCT 1034
CGCAGGGGTATTAGCCA 118N-F
TATGGCAAATACACCGGTATCAGGCAATTTGAAGGTTGAATTCTAC 1035 AACAGCAATCCTTCA
118N-R GACGTGAAGGATTGCTGTTGTAGAATTCAACCTTCAAATTGCCTGA 1036
TACCGGTGTATTTGCCA 119- TCGAGCACGGGCAGCCCA 1037 AEN-F 119-
GACGTGGGCTGCCCGTGC 1038 AEN-R Stuffer-
TATGggctgaGGGTCTCaCGTCCGCGTCCTCGCCCACGTCCACGTC 1039 RP5for CGCGT
Stuffer- GGACGTGGACGTGGGCGAGGACGCGGACGtGAGACCCtcagccCA 1040 RP5revP
RP6- CCACGCCCTCGTCCTCGTCCGCGCCCTCGTCCGagcgcgtctcgtt 1041 SASRS ccgc
AforP RP6- ACCTgcggaacgagacgcgctCGGACGAGGGCGCGGACGAGGACGA 1042
SASRS GGGCGTGGACGC Arev L2for
tATGaaaAAYCCNGARCARGCNGARGARCARMGYGARGARACa 1043 L2rev
GACGTGTYTCYTCRCKYTGYTCYTCNGCYTGYTCNGGRTTTTTCA 1044 L3for
tATGGCNAAYCCNGARCARGCNGARGARCARMGYGARGARACa 1045 L3rev
GACGTGTYTCYTCRCKYTGYTCYTCNGCYTGYTCNGGRTTNGCCA 1046 L4for
tATGaaaAAcCCVGARCARGCDGARGAaCARGCDGARGAaCAgMGY 1047 GAaGARACa L4rev
GACGTGTYTCTTCRCKCTGTTCYTCHGCYTGTTCYTCHGCYTGYTC 1048 BGGGTTTTTCA
L5for tATGaRaCCNCGNCCNCGNCCNCGNCCNCGNCCNCGNCCNCGNCCN 1049
CGNCCNCGNCCNCGNCCNCGNCCNGGGTCTCC L5rev
ACCTGGAGACCCNGGNCGNGGNCGNGGNCGNGGNCGNGGNCGNGGN 1050
CGNGGNCGNGGNCGNGGNCGNGGNCGNGGTYTCA L6for
tATGaaaAAHMNIVGARCARGCWGARGAaCARGCDGARGAaCAgMG 1051 YGAaGARACa
L6rev GACGTGTYTCTTCRCKCTGTTCYTCHGCYTGTTCYTCWGCYTGYTC 1052
BKKDTTTTTCA L7for tATGAAAAAWCAMRARMARRMWRAARAAMAARMDRAARAACAGMGC
1053 GAAGARACA L7rev GACGTGTYTCTTCGCKCTGTTYTTYHKYTTKTTYTTYWKYYTKYTY
1054 KTGWTTTTTCA L8for
tATGAAAAAWCAMRARMARRMWRAARAAMAAGCDGAAGAACAGMGC 1055 GAAGARACA L8rev
GACGTGTYTCTTCGCKCTGTTCTTCHGCTIKTTYTTYWKYYTKYTY 1056 KTGWTTTTTCA
L9for tATGAAAAANCMNIGAACAAGAARAARAAMAAGCNGAAGAACARCG 1057
YGARGARACA L9rev GACGTGTYTCYTCRCGYTGTTCTTCNGCTIKTTYTTYTTCTTGTTC
1058 KKGNTTTTTCA L10for
tATGAAAAANCMMGAACAAGAARAARAAMAAGCNGAAGAAMARMRH 1059 RARRARAMA
L10rev GACGTKTYTYYTYDYKYTKTTCTTCNGCTTKTTYTTYTTCTTGTTC 1060
KKGNTTTTTCA L11for tATGAAAAAACAAGAACAAGAAAAAGAACAAGCGGAAGAACAAKCN
1061 VARKCNVARCGTGAGGAGACA L11rev
GACGTGTCTCCTCACGYTBNGMYTBNGMTTGTTCTTCCGCTTGTTC 1062
TTTTTCTTGTTCTTGTTTTTTCA L12for
tATGAAAAAACAAGAACAAGAAAAAGAACAAGCGGAAGAACAAKCV 1063
VAAKCVVAAKCVVAAKCVVAACGTGAGGAGACA L12rev
GACGTGTCTCCTCACGTTBBGMTTBBGMTTBBGMTTBBGMTTGTTC 1064
TTCCGCTTGTTCTTTTTCTTGTTCTTGTTTTTTCA L13for
tATGAAAAAACAAGAACAAGAAAAAGAACAAGCGGAAGAACAANNN 1065
NNNNNNNNNCGTGAGGAGACA L13rev
GACGTGTCTCCTCACGNNNNNNNNNNNNTTGTTCTTCCGCTTGTTC 1066
TTTTTCTTGTTCTTGTTTTTTCA
[0472] 3. Screening and Analysis of the N-Terminal Helper Libraries
LCW1157-1159
[0473] E. coli hosts transformed with the plasmids pSD0107 to
pSD0118 were expressed in shake flasks and the expression levels in
each were evaluated by measuring the fluorescence from the
C-terminal GFP reporter. Briefly, an overnight culture was grown
for each construct in SB media (with 12.5 .mu.g/ml tetracycline),
which was then used to inoculate a 200 ml culture of PhoA phosphate
depletion autoinduction media with 12.5 .mu.g/ml tetracycline (3
shake flasks were grown for each construct). After growing at
26.degree. C. with 225 rpm for 48 h, 100 .mu.l aliquot was taken
from each culture and the GFP expression level was measured with a
fluorescence plate reader with excitation wavelength of 395 nm and
emission wavelength of 510 nm. Two readings were taken for each
shake flask. Among the constructs tested, pSD0116 had the highest
fluorescence signal, followed by pSD0114 (FIG. 81), but the
expression levels of these two constructs were significantly lower
than the LCW0970.030 (pLCW970) positive control.
[0474] In order to further improve expression, three libraries
(LCW1157, 1158, and 1159) were built based on pSD0106, and were
screened in a high through-put format. Large numbers of colonies
from these libraries (Table 32) were picked to grow individually in
500 .mu.l SB media (with 12.5 .mu.g/ml tetracycline) in 96 deep
well plates overnight at 37.degree. C. shaking with 300 rpm. 20
.mu.l of the saturated culture was used to inoculate 500 .mu.l of
PhoA phosphate depletion autoinduction media (with 12.5 .mu.g/ml
tetracycline) in 96 deep well plates that were incubated at
26.degree. C. shaking with 300 rpm for 22-24 h. Expression was then
determined by placing 100 .mu.l of the culture into a 96 well plate
measuring the fluorescence from the C-terminal GFP reporter.
TABLE-US-00038 TABLE 32 Libraries LCW1157-1159 N-terminal helper
Number Library Description (sequence DNA) Diversity Screened LCW
Codon optimize helper ATGaaaAAY(C/T)CCN(A/G/ 98304 406 1157
sequence, Change 2.sup.nd C/T)GAR(A/G)CAR(A/G)GCN codon to Lys
(AAA) (A/C/T/G)GAR(A/G)GAR(A/ G)CAR(A/G)M(A/C)GY(C/T)
GAR(A/G)GAR(A/G)ACa (SEQ ID NO: 1067) LCW Codon optimize helper
ATGGCN(A/G/C/T)AAY(C/T) 393216 85 1158 sequence
CCN(A/G/C/T)GAR(A/G)CAR (A/G)GCN(A/G/C/T)GAR(A/
G)GAR(A/G)CAR(A/G)M(A/C) GY(C/T)GAR(A/G)GAR(A/G) ACa (SEQ ID NO:
1068) LCW Insert AEEQ (SEQ ID ATGaaaAAcCCV(A/G/C)GAR 20736 146 1159
NO: 1070) into helper (A/G)CAR(A/G)GCD(A/G/T) sequence, Use fewer
GAR(A/G)GAaCAR(A/G)GCD codons than LCW1157 (A/G/T)GAR(A/G)GAaCAgM
to keep diversity (A/C)GY(C/T)GAaGAR(A/G) manageable ACa (SEQ ID
NO: 1069)
[0475] After evaluation of the fluorescence signal, the six highest
expression clones and two low expression clones were chosen from
each 96 deep well plate tested and the expression of these clones
were tested again with 4 replicates. For all three libraries,
clones having higher expression than the pSD0116 construct were
identified (FIGS. 82A-C), and the correlation between the original
tests and retests was good (FIGS. 82D-F). Among the 3 libraries
tested, library LCW1159 gave higher expression level in general,
and the highest expression construct from this round of screening
(LCW1159.004) came from this library.
[0476] The 8 constructs with the highest expression levels from
these three libraries and controls were treated with Pop Culture
(EMD Millipore), were heat treated, and the resulting lysates were
analyzed with SDS-PAGE/Coomassie staining (FIG. 83). Expression
levels of the 8 constructs were confirmed to be higher than
controls (negative control, LSD0114 and LSD0116), and full-length
proteins were produced from these constructs. Lysates of 4 top
expression constructs (after Pop Culture and heat treatment) and a
negative control (AP32, a purified XTEN-GFP protein without RP11
tag) were each loaded onto a MacroCap SP column at pH of 8 and
conductivity of 6.5 mS/cm. The column was washed with 20 mM sodium
phosphate, pH 8, 100 mM NaCl, and the protein was eluted with 20 mM
sodium phosphate, pH 8, 500 mM NaCl. The samples of the load, flow
through, wash and elutate were analyzed by SDS-PAGE gel (FIG. 84).
The results demonstrated that the expression protein of the four,
which came from all three libraries, can be captured by MacroCap
SP; thus the binding was contributed to the presence of the RP11
tag in the protein since the negative control (not containing the
RP11 tag) didn't bind to the MacroCap SP column.
[0477] The plasmids of the clones chosen for retests were
minipreped and the DNA sequences of the N-terminal helpers were
analyzed. Codon bias was observed at several locations (Table 33).
For example, the 3rd amino acid of LCW1157, N, is encoded by AAC or
AAT. Most of the high expression clones (77%) in LCW1157 are
encoded by AAT at the 3rd amino acid, while most of the low
expression clones (88%) are encoded by AAC, indicating AAT is
preferred over AAC at this position for high expression. Similarly,
CCG is preferred at the 4th amino acid, while GCG at the 7th amino
acid is accumulated in low expression clones. These trends were
also observed in libraries LCW1158 and LCW1159, as well.
TABLE-US-00039 TABLE 33 Analysis of sequence results of high and
low expression clones in libraries LCW1157-1159* LCW1157 amino
acid# 3 4 5 6 7 8 9 10 11 12 13 2 N(AA+) P(CC+) E(GA+) Q(CA+)
A(GC+) E(GA+) E(GA+) Q(CA+) R or S(+G+) E(GA+) E(GA+) 14 codon
K(AAA) 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 1st 3rd 3rd 3rd T(AC+) high
A K(AAA) 0% 10% 57% 67% 33% 80% 70% 50% 30% 0% 63% 57% T(ACA)
total: 30 G 0% 43% 43% 33% 7% 20% 30% 50% 0% 0% 37% 43% C 23% 23%
0% 0% 13% 0% 0% 0% 70% 67% 0% 0% T 77% 23% 0% 0% 47% 0% 0% 0% 0%
33% 0% 0% low A 0% 13% 88% 50% 25% 38% 63% 25% 63% 0% 75% 63%
total: 8 G 0% 13% 13% 50% 63% 63% 38% 75% 0% 0% 25% 38% C 88% 0% 0%
0% 0% 0% 0% 0% 38% 38% 0% 0% T 13% 75% 0% 0% 13% 0% 0% 0% 0% 63% 0%
0% LCW1158 amino acid# 2 3 4 5 6 7 8 9 10 11 12 13 A(GC+) N(AA+)
P(CC+) E(GA+) Q(CA+) A(GC+) E(GA+) E(GA+) Q(CA+) R or S(+G+) E(GA+)
E(GA+) 14 codon 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 1st 3rd 3rd 3rd
T(AC+) high A 83% 0% 33% 17% 67% 50% 83% 50% 83% 83% 0% 67% 50%
T(ACA) total: 6 G 0% 0% 0% 83% 33% 0% 17% 50% 17% 0% 0% 33% 50% C
0% 33% 33% 0% 0% 33% 0% 0% 0% 17% 100% 0% 0% T 17% 67% 33% 0% 0%
17% 0% 0% 0% 0% 0% 0% 0% low A 0% 0% 0% 100% 0% 0% 0% 0% 0% 0% 0%
0% 100% total: 2 G 100% 0% 100% 0% 100% 100% 100% 100% 100% 0% 0%
100% 0% C 0% 0% 0% 0% 0% 0% 0% 0% 0% 100% 100% 0% 0% T 0% 100% 0%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0% LCW1159 amino acid# 3 4 5 6 7 8 9 10
2 N(AA+) P(CC+) E(GA+) Q(CA+) A(GC+) E(GA+) E(GA+) Q(CA+) codon
K(AAA) 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd high A K(AAA) N(AAC) 42% 67%
67% 50% 75% E(GAA) 67% total: 12 G 17% 33% 33% 0% 25% 33% C 42% 0%
0% 0% 0% 0% T 0% 0% 0% 50% 0% 0% low A 33% 100% 67% 0% 67% 67%
total: 3 G 33% 0% 33% 100% 33% 33% C 33% 0% 0% 0% 0% 0% T 0% 0% 0%
0% 0% 0% LCW1159 amino acid# 11 12 13 14 15 16 17 A(GC+) E(GA+)
E(GA+) Q(CA+) R or S(+G+) E(GA+) E(GA+) 18 codon 3rd 3rd 3rd 3rd
1st 3rd 3rd 3rd T(AC+) high A 50% 58% E(GAA) Q(CAG) 25% 0% E(GAA)
58% T(ACA) total: 12 G 17% 42% 0% 0% 42% C 0% 0% 75% 75% 0% T 33%
0% 0% 25% 0% low A 100% 100% 33% 0% 33% total: 3 G 0% 0% 0% 0% 67%
C 0% 0% 67% 67% 0% T 0% 0% 0% 33% 0% *The total numbers of high and
low expression clones analyzed are specified for each library, and
the percentages of A, G, C, or T in each of the location where the
codon was varied were analyzed.
[0478] 4. Screening and Analysis of the N-Terminal Helper Library
LCW1163
[0479] Since library LCW1159 had generally higher expression than
LCW1157 and LCW1158, the next library design was based on LCW1159,
with the introduction of more variants in the N-terminal helper
domain coding region. A total of 672 clones from this library
(theoretical diversity of 552%) was screened and retested in the
same way as libraries LCW1157-1159. Clones with higher expression
than LCW1159.004 (the highest from the previous round of screening)
were observed (FIG. 85). The top expression clone, LCW1163.029,
achieved a 40% improvement from LCW1159.004, while its expression
level was 27% lower than the CBD control. The sequence of the high
and low expression clones were analyzed, and the preferred
nucleotides for expression were identified and summarized (see
Table 34). Most of the locations have shown a preference of codon A
for high expression, while others have a preference for C.
TABLE-US-00040 TABLE 34 Analysis of sequence results of high and
low expression clones in the library LCW1163* Expression
(Percentage) nucleotide number #9 #10 #11 #12 #15 #18 #21 #24 #30
#33 #36 #43 #45 #51 high A 74 17 74 40 37 40 66 77 60 43 66 40 0 34
expression C 17 83 26 49 0 0 0 0 0 0 0 60 83 0 T 9 0 0 0 0 0 34 0 0
29 0 0 17 0 G 0 0 0 11 63 60 0 23 40 29 34 0 0 66 low A 20 70 3 20
40 20 60 50 30 10 20 50 0 60 expression C 60 30 70 30 0 0 0 0 0 0 0
50 50 0 T 20 0 0 0 0 0 40 0 0 40 0 0 50 0 G 0 0 0 50 60 80 0 50 70
50 80 0 0 40 preferred A C A A/C A A A C *The percentages of A, G,
C, or T in each of the location where the codon was varied were
analyzed, and the identified preferred nucleotides were summarized
in the bottom row
[0480] 5. Screening and Analysis of the RP11 Library LCW1160
[0481] At the same time, 168 colonies from library LCW1160 (the
library varying the coding region of RP11 tag without any
N-terminal helper domain (total theoretical diversity of
8.8.times.10.sup.12) were screened and analyzed. However, this
library had very low expression level in general (FIG. 86), and the
one outlier with high expression was found to encode a truncated
RP11 tag after performing plasmid miniprep and DNA sequencing
analysis. The screening results of the library, under these
experimental conditions, strongly suggest that an N-terminal helper
is required to achieve high expression levels.
[0482] 6. Screening of the N-Terminal Helper Libraries LCW1171,
LCW1172, LCW1203, and LCW1204.
[0483] More N-terminal libraries (LCW1171, LCW1172, LCW1203, and
LCW1204) were screened and analyzed in the same way as those
described above. LCW1171 and 1172 were designed similarly, while
LCW1171 allowed more amino acid changes in the helper sequence than
LCW1172. The screening results showed that LCW1171 in general had
much lower expression level than LCW1172 (FIGS. 94A and B). LCW1203
and 1204 were designed similarly, focusing on randomizing a
different region of the helper sequence compared with LCW1171 and
1172. LCW1204 allowed more amino acid changes than LCW1203, which
resulted in lower expression than LCW1203 in general (FIGS. 94C and
D). These results suggest that the expression level is sensitive to
amino acid changes in the helper domain sequences.
[0484] 7. Screening of the N-Terminal Helper Libraries LCW1208,
LCW1209, and LCW1210.
[0485] Three new N-terminal libraries (LCW1208, LCW1209, and
LCW1210) were designed to investigate the effect of further
elongation the N-terminal helper sequence (Table 35). LCW1208 and
LCW1210 introduced 4 more residues to the helper domain, while
LCW1209 introduced 8 more residues. The screening results showed a
general trend that LCW1209 had highest expression, followed by
LCW1208, and then LCW1210 (FIG. 95), which confirmed the beneficial
effect of adding more amino acid in the helper sequence.
TABLE-US-00041 TABLE 35 Libraries LCW1208-1210 Library N-terminal
Helper Sequence (amino acid) Diversity Number Screened LCW1208
MKKQEQEKEQAEEQBXBXREET (SEQ ID NO: 2304 336 1071) (B = A/S; X =
E/K/Q) LCW1209 MKKQEQEKEQAEEQBXBXBXBXREET (SEQ 104976 336 ID NO:
1072) (B = A/S; X = E/K/Q) LCW1210 MKKQEQEKEQAEEQZZZZREET (SEQ ID
NO: 262144 336 1073) (Z = any amino acid)
[0486] Additional residues in the helper sequences were
underlined.
[0487] Three new N-terminal libraries (LCW1208, LCW1209, and
LCW1210) were designed to investigate the effect of further
elongation the N-terminal helper sequence (Table 36). LCW1208 and
LCW1210 introduced 4 more residues to the helper domain, while
LCW1209 introduced 8 more residues. The screening results showed a
general trend that LCW1209 had highest expression, followed by
LCW1208, and then LCW1210 (FIGS. 95A-95C), which confirmed the
beneficial effect of adding more amino acids in the helper sequence
for improving expression.
[0488] The 4 top constructs with the highest expression levels from
the three libraries were chosen from the retest and the sequence of
their N-terminal helper sequences were analyzed (Table 36). The
current highest expressed construct (LCW 1209.029) achieved 90% of
the expression level of the CBD control by comparing the average
florescence after subtracting the negative control.
TABLE-US-00042 TABLE 36 Constructs with the highest expression
levels from Libraries LCW1208- 1210 and controls. SEQ Avg. ID
Sample name Fluorescence Helper sequence* NO: LCW 1208.009 9070
MKKQEQEKEQAEEQAESEREET 1074 LCW 1208.007 8870
MKKQEQEKEQAEEQAESEREET 1075 LCW 1208.008 8800
MKKQEQEKEQAEEQSQSQREET 1076 LCW 1208.010 8740
MKKQEQEKEQAEEQSESEREET 1077 LCW 1209.029 10010
MKKQEQEKEQAEEQAKAESEAEREET 1078 LCW 1209.015 9440
MKKQEQEKEQAEEQSKSQAEAEREET 1079 LCW 1209.023 8980
MKKQEQEKEQAEEQAQAQAEDEREET 1080 LCW 1209.010 8580
MKKQEQEKEQAEEQSKSKAEDEREET 1081 LCW 1210.030 8650
MKKQEQEKEQAEEQPEVQREET 1082 LCW 1210.032 8310
MKKQEQEKEQAEEQVENPREET 1083 LCW 1210.025 8100
MKKQEQEKEQAEEQELCEREET 1084 LCW 1210.009 8030
MKKQEQEKEQAEEQGIDTREET 1085 CBD control 10940 n/a Negative control
1630 n/a *Additional residues in the helper sequences were
underlined.
[0489] In summary, the screening results, under these experimental
conditions, strongly suggest that an N-terminal helper contributes
in achieving high expression levels.
Example 13: Fermentation of XTEN Using PhoA Induction--Evaluation
of Expression Yields
[0490] E. coli BL21 carrying the plasmids encoding
Helper_LCW1159.004-RP11-AE288-His8 ("His8" disclosed as SEQ ID NO:
20) (AC767), Helper_LCW1172.033-RP11-AE576-His8 ("His8" disclosed
as SEQ ID NO: 20) (AC780), and Helper_LCW1172.033-RP11-AE864-His8
("His8" disclosed as SEQ ID NO: 20) (AC786) were transformed into
the E. coli BL21 strain. Three 10 L fermentations were run for each
of the 3 strains. Glycerol stocks were used to inoculate 125 mL of
LB broth media containing 10 .mu.g/mL tetracyclin. The starter
cultures were then shaken overnight at 37.degree. C. The starter
culture was used to inoculate 4 L of fermentation batch media
containing -20 g ammonium sulfate, 10.4 g potassium phosphate
dibasic anhydrous, 5 g sodium citrate dihydrate; 4.6 g sodium
phosphate monobasic monohydrate; 106 g NZ BL4 soy peptone (Kerry
Bioscience #5X00043); 54 g yeast extract (Teknova #Y9020); 3.6 L
water, 0.05 mL polypropylene glycol; 5.2 mL trace elements solution
(Amunix recipe 144-1); 35 mL 1M magnesium sulfate; and 4 mL
Kanamycin (50 mg/mL)--in 10 L glass jacketed vessel with a B. Braun
Biostat B controller. The fermentation control settings were:
pH=6.9+/-0.1; dO.sub.2=10%; dissolved oxygen cascade in stirrer
only mode with a range of 125-1180 rpm; air flow of 5 liters per
minute of 90% oxygen; initial temperature 37.degree. C.; base
control 13% ammonium hydroxide; and no acid control. After 6 hours
of culture a 70% glycerol feed was initiated at a rate of 40 g/hr.
Upon the cultures reaching an OD600 of 50+/-10 OD the culture
temperature was lowered to 26.degree. C., 54 mL of 1M magnesium
sulfate was added, and a salt feed consisting of: 10 g/l ammonium
sulfate, 26 g/l potassium phosphate dibasic anhydrous, 2 g/l sodium
citrate dihydrate; 13 g/l sodium phosphate monobasic monohydrate;
15 g/l potassium phosphate monobasic anhydrous; 0.08% trace
elements solution, was started at a rate of 33 g/L and continued
for 6 hours. After a total fermentation run time of 64-70 hours the
culture was harvested by centrifugation yielding cell pellets
between 1.6-2.3 kilograms in wet weight. The pellets were stored
frozen at -80.degree. C. until further use. For titer analysis, end
of run fermentation whole broth samples were frozen in a 0.2 mL
volume, then later thawed, then 0.2 mL of water was added, then to
lyse and flocculate host proteins the samples were incubated at
85.degree. C. for 15 minutes, then transferred to 4.degree. C. for
15 minutes, followed centrifugation for 10 minutes at 20,000 g. The
resulting flocculated soluble lysates were assayed by C18 reversed
phased HPLC, and the A214 absorbance area corresponding of the
peaks representing the Helper-RP11-XTEN-His8 ("His8" disclosed as
SEQ ID NO: 20) was compared to that of purified reference standard.
Next, to determine the dry cell weight (DCW), aliquots of cells
were pelleted and the supernatant discarded. The cell pellet was
washed once with water, and was then dried in an oven at 80.degree.
C. for 3 days. The tubes containing cell pellets were weighed, the
weight of the tube was subtracted from the measured weights, and
the remaining weight was divided by the initial volume of each
sample (0.0002 L) to obtain the DCW. The results of the
fermentation growth, titer analysis, and dry cell weight are
summarized in Table 37 below. In 96-well plate screening assays,
when a library of RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO:
20) constructs without an N-terminal helper, LCW1160 were screened
(Example 12, FIG. 86) the expression level of the LCW 1160.006, the
highest expression construct, was only 50% of the expression of LCW
1159.004 or LCW 1172.033; constructs with helper sequences.
Therefore, it is expected that XTEN constructs with N-terminal
helper sequences will result in significantly higher expression
titers compared to XTEN constructs not having helper sequences.
TABLE-US-00043 TABLE 37 Measured expression parameters Total Run
Time Dry cell weight per Liter Titer by culture Average titer XTEN
Series Fermentation # Final O.D. (hours) of Ferm (g/L) volume (g/L)
(micromoles/L) Helper_LCW1159.004- EC846 140 67.5 95 0.5 0.7
RP11-AE288-His8 EC888 140 64 85 0.8 (AC767) ("His8" EC903 136 69
103 0.7 disclosed as SEQ ID NO: 20) Helper LCW1172.033- EC850 125
66 107 1.1 2.0 RP11-AE576-His8 EC869 140 70 90 2.1 (AC780) ("His8"
EC883 170 67 102 2.9 disclosed as SEQ ID NO: 20) Helper
LCW1172.033- EC873 135 68 91 2.0 2.1 RP11-AE864-His8 EC889 145 65.5
95 2.3 (AC786) ("His8" EC913 108 67 108 2.0 disclosed as SEQ ID NO:
20) Titer/Dry weight ferm Std error (mg of XTEN/g MW Titer Average
titer of the mean XTEN Series of E. coli) (g/mol) (micromoles/L)
(micromoles/L) (micromoles/L) Helper_LCW1159.004- 5.6 26399 212 271
76 RP11-AE288-His8 9.4 26399 357 (AC767) ("His8" 6.4 26399 243
disclosed as SEQ ID NO: 20) Helper LCW1172.033- 10.0 52929 189 388
182 RP11-AE576-His8 22.8 52929 430 (AC780) ("His8" 28.8 52929 545
disclosed as SEQ ID NO: 20) Helper LCW1172.033- 22.2 79284 280 272
41 RP11-AE864-His8 24.4 79284 308 (AC786) ("His8" 18.1 79284 228
disclosed as SEQ ID NO: 20)
Example 14: Purification of XTEN with RP11 and His8 Tags ("His8"
Disclosed as SEQ ID NO: 20)
[0491] 1. Expression, Lysis and Clarification
[0492] The fusion protein
MKNPEQAEEQAEEQREET-RP11-SASRSA-XTEN_AE432(C12,C217,C422)-SASRSA-His(8)]
("MKNPEQAEEQAEEQREET," "SASRSA" and "His8" disclosed as SEQ ID NOS
1086, 21 and 20, respectively), with the N-terminal helper sequence
from the library member LCW1159.004 described in Example 10, with
two affinity tags linked to XTEN at the N- and C-terminus,
respectively, was expressed in E. coli using a 4 L fermenter using
conditions described herein. After growth, the cells were harvested
by centrifugation and frozen at -80.degree. C. until use. The cell
pellet was resuspended in lysis buffer (20 mM sodium phosphate, 50
mM NaCl, 2 mM EDTA pH 8.0, 3 ml buffer per gram cell paste). The
cells were lysed by passing through an APV homogenizer three times
at a pressure of 830-900 bar. Lysis buffer (1 ml buffer per gram
cell paste) was used as a chase to retrieve hold up volume from the
homogenizer. The homogenized lysate was incubated in a water bath
at 85.degree. C. for 20 minutes, followed by quick cooling in ice
water bath for 20 minutes. After the heating and cooling treatment,
the lysate was centrifuged at 11000 RPM for 90 minutes in a SORVALL
centrifuge. After centrifugation, the supernatant was filtered
through two CUNO Bio cap 25 (BC0025L90SP08A) filters. The clarified
supernatant was stored at 4.degree. C. overnight.
[0493] 2. Capture Step: Toyopearl IMAC Chromatography
[0494] IMAC affinity chromatography was used as a capture step for
binding the XTEN with an intact C-terminal His-tag. Briefly, the
chromatography column BPG140/12 (GE Life Sciences) was packed with
2000 ml Toyopearl IMAC 650 M resin (TOSOH Biosciences). The column
was equilibrated with 2 column volumes (CVs) of equilibrium buffer
(20 mM sodium phosphate, 500 mM NaCl, pH 8.0). Clarified cell
lysate was adjusted to a final NaCl concentration of 500 mM using 5
M NaCl stock solution, and then was loaded onto the IMAC resin. The
column was washed with 2 column volumes of equilibrium buffer, and
then 2 column volumes of 20 mM sodium phosphate, 500 mM NaCl, 5 mM
Imidazole pH 8.0, followed by 2 column volumes of 20 mM sodium
phosphate, 5 mM imidazole pH 8.0 to remove salt. Elution was
performed with 2 column volumes of 20 mM sodium phosphate, 100 mM
imidazole, pH 8.0. The flow through, wash and elution fractions
were analyzed by non-reducing 4-12% Bis-Tris SDS-PAGE/Coomassie
staining and the fractions with the desired product were
pooled.
[0495] 3. Polishing/Capture Step: MacroCap SP Chromatography
[0496] Cation exchange chromatography was used as a polishing step
to ensure the N-terminal integrity of the product. MacroCap SP
resin (GE Life Sciences) was selected among several cation exchange
media due to its superior capacity and selectivity for the product.
1000 ml of MacroCap SP resin was packed in a BPG100/13 (GE Life
Sciences) chromatography column and equilibrated with 20 mM sodium
phosphate pH 8.0, 20 mM NaCl. The IMAC pool was loaded onto the
column and the resin was washed with 2 column volumes of 20 mM
sodium phosphate, 50 mM NaCl, pH 8.0 and 2 column volumes of 20 mM
sodium phosphate pH 8.0, 150 mM NaCl. The protein was eluted with 5
column volumes of linear gradient from 150 to 500 mM NaCl in 20 mM
sodium phosphate pH 8.0. Fractions were collected and analyzed by
4-12% Bis-Tris SDS/PAGE. Fractions the with desired product were
combined for the next step.
[0497] 4. Trypsin Digestion of Macrocap Sp Elution Pool
[0498] Trypsin (Sigma, Trypsin from Bovine Pancreas) digestion of
the SP elution pool was performed at 1:200 m/m enzyme/protein ratio
overnight at 37.degree. C.
[0499] 5. Polishing Step: Macrocap Q Chromatography
[0500] After trypsin digestion, the cleaved tags were separated
from the final product using Macrocap Q chromatography. The
BPG100/19 column (GE Life Sciences) was packed with 1500 ml column
volume of Macrocap Q resin (GE Life Sciences). The trypsin digested
Macrocap SP elution pool was incubated for 15 min at 80.degree. C.
with 20 mM DTT and 2 mM EDTA to reduce disulfide bonds and to
inactivate trypsin. The cooled protein solution was diluted to a
conductivity below 5 mS/cm with Milli-Q water and loaded onto the
Macrocap Q column equilibrated with 20 mM HEPES, 50 mM NaCl, pH
7.0. The column was washed with 2 column volumes of 20 mM HEPES, 50
mM NaCl, pH 7.0, then 2 column volumes of 20 mM HEPES, 2 mM TCEP,
150 mM NaCl pH7.0. The protein was eluted with a linear gradient
from 150 mM NaCl to 500 mM NaCl in 20 mM HEPES, pH 7.0 in 20 column
volumes. Fractions were analyzed by SDS-PAGE/silver staining.
[0501] 6. Concentration and Diafiltration (Final Formulation)
[0502] Selected MacroCap Q fractions were combined and concentrated
and using 10 KD Pellicon mini (Millipore) at a feed pressure <20
psi and retentate <8 psi, followed by 10.times. diafiltration
with 20 mM HEPES, 50 mM NaCl, pH 7.0 to achieve a final protein
concentration of >5 mg/ml.
[0503] 9. Purity Analysis of Proteins Purified with Different
Methods
[0504] One batch (Batch 1) was purified through three purification
steps as described above. Another batch (Batch 2) was purified from
the same fermented material but the MacroCap SP polishing step was
omitted. Truncated species of XTEN were detected by SDS-PAGE/silver
staining in MacroCap Q elution fractions for Batch 2 (FIG. 87A),
while the MacroCap Q elution fractions for Batch 1 were essentially
free from truncations (FIG. 87B). These results support that, under
the conditions employed, the MacroCap SP step based on the RP11 tag
is essential to ensure N-terminal integrity and overall product
quality.
Example 15: Construction of 1.times.Amino,
9.times.Thiol-XTEN432
[0505] The following sets of primers 5Afor&CI1BbsIrev-TGGC,
CI1BsaIfor-TGGC&CI2-AE38BbsIrev, and
C12-AE38BsaIfor&AatIICI3-2P were used to PCR plasmid pCW1164
containing XTEN_AE432 (C422) in order to obtain the PCR products of
AE-CI1, CI1-2 and CI2-3, respectively. CI1, 2&3 were designated
Cysteine Island1, 2&3, having the same amino acid sequence
TAEAAGCGTAEAA (SEQ ID NO: 189) but with different codon usages.
Gel-purification of the PCR products was performed to obtain bands
of the right sizes, which were digested with restriction enzymes
SbfI/BbsI, BsaI/BbsI and BsaI/AatII, respectively, as the inserts.
Digestion of the plasmid pCW1164, which encodes the gene of
N-term-RP11-R-XTEN_AE432 (C422)-R-H8 ("H8" disclosed as SEQ ID NO:
20), was performed with SbfI/AatII to remove the fragment of about
290 amino acids within XTEN_AE432 and gel-purification was
performed on the remaining large fragment as the vector. Ligation
of the vector with the three inserts of PCR products, above, was
performed and used to transform BL21 competent cells in order to
obtain the construct N-term-RP11-R-XTEN_AE432 (C319, C370,
C422)-R-H8 ("H8" disclosed as SEQ ID NO: 20). PCR was performed on
this construct with primers CI1BsaIfor-TGGC&AatIICI3-2P to
obtain a PCR product of around 360 bp in length. Gel-purification
of the band of the right size was performed, followed by digestion
with BsaI/AatII as the insert XTEN_AE120-3Cys, which contains three
Cysteine Islands.
[0506] Simultaneously, the codon-optimized DNA fragment of
XTEN_AE313-6Cys, containing six Cysteine Islands, was designed and
synthesized (Genscript). The fragment was digested by the flanking
restriction enzymes BsaI/BbsI and gel-purified as the insert
containing the first six Cysteine Islands of XTEN_AE432. Digestion
of the plasmid pCW1161, which encodes the gene of
N-term-RP11-R-XTEN_AE432_3Cys-R-H8 ("H8" disclosed as SEQ ID NO:
20), with BsaI/AatII was performed to remove the fragment of
XTEN_AE432_3Cys and gel-purification was performed to obtain the
large fragment as the vector. Ligation of the vector with the
BsaI/BbsI digested insert of XTEN_AE313-6Cys and BsaI/AatII
digested insert of XTEN_AE120-3Cys, above, was performed. The
ligated product was used to transform BL21 competent cells in order
to obtain the construct N-term-RP11-R-XTEN_AE432 (C12, C63, C114,
C165, C217, C268, C319, C370, C422)-R-H8 ("H8" disclosed as SEQ ID
NO: 20). The construct was designed to produce the precursor
N-term-RP11-R-AE432_9Cys-R-H8 ("H8" disclosed as SEQ ID NO: 20)
(sequence in Table 38, below), the product of which was used to
generate 1.times.Amino, 9-Thio-XTEN432 after removal of the
N-term-RP11 tag and C-term 8.times.His-tag (SEQ ID NO: 20) by
trypsin digestion. The final product contains nine cysteines in the
XTEN432 sequence (Seg 177).
TABLE-US-00044 TABLE 38 DNA and amino acid sequence for 1xAmino,
9-Thio-XTEN432 Clone Amino Acid Name DNA Sequence Sequence N-term-
ATGAAAAACCCAGAGCAAGCAGAAGAACAAGCTGAAGA MKNPEQAEEQAE RP11-R-
ACAGCGCGAAGAAACACGTCCGCGTCCTCGCCCACGTCC E AE432_
ACGTCCGCGTCCACGCCCTCGTCCTCGTCCGCGCCCTCGT QREETRPRPRPRP 9Cys-R-118
CCGagcgcgtctcgttccgctGGGTCTCCAACGGCAGAGGCAGCAG RPRPRPRPRPRPR
(''H8'' GTTGTGGTACAGCAGAAGCAGCTCCGGGTAGCGAGCCTG PSASRSAGSPTAE
disclosed CAACCAGCGGTTCTGAGACGCCGGGCACTTCCGAATCTG AAGCGTAEAAPG as
SEQ ID CGACCCCGGAGTCCGGTCCAGGTTCAGAGCCGGCGACGA S NO: 20)
GCGGTTCGGAAACGCCGGGTACTGCTGAAGCGGCTGGTT EPATSGSETPGTS
GTGGTACTGCTGAAGCTGCATCGACCGAACCAAGCGAAG ESATPESGPGSEP
GTTCGGCACCGGGTACTAGCGAGAGCGCAACCCCTGAAA ATSGSETPGTAEA
GCGGTCCGGGCAGCCCGGCAGGTTCTCCAACCAGCACCG AGCGTAEAASTE
AAGAAGGTTCCCCTGCTACTGCCGAAGCTGCAGGCTGCG P
GTACTGCGGAGGCGGCGTCCCCAACTTCTACTGAGGAAG SEGSAPGTSESAT
GTACTTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGTAC PESGPGSPAGSPT
CTCCACTGAACCGTCTGAAGGCTCTGCACCAGGCACTTCT STEEGSPATAEAA
GAGTCTGCTACTACCGCCGAAGCCGCTGGTTGTGGTACCG GCGTAEAASPTS
CAGAAGCTGCATCTGAGACTCCAGGCACTTCTGAGTCCG T
CAACGCCTGAATCCGGTCCTGGTTCTGAACCAGCTACTTC EEGTSESATPESG
CGGCAGCGAAACCCCAGGTACCTCTGAGTCTGCGACTCC PGTSTEPSEGSAP
AGAGTCTGGTACCGCGGAAGCGGCTGGTTGTGGTACTGC GTSESATTAEAA
AGAGGCAGCTGGTTCTCCGGCTGGTAGCCCGACCAGCAC G
GGAGGAGGGTACGTCTGAATCTGCAACGCCGGAATCGGG CGTAEAASETPG
CCCAGGTTCGGAGCCTGCAACGTCTGGCAGCGAAACCCC T
GGGTACCACGGCGGAAGCGGCAGGTTGTGGCACCGCGGA SESATPESGPGSE
GGCAGCAGCTGGTTCTCCAACCTCTACCGAGGAGGGTTC PATSGSETPGTSE
ACCGGCAGGTAGCCCGACTAGCACTGAAGAAGGTACTAG SATPESGTAEAA
CACGGAGCCGAGCGAGGGTAGTGCTCCGGGTACGAGCGA G
GAGCACGGCAGAAGCCGCTGGCtgcGGTACTGCTGAAGCG CGTAEAAGSPAG
GCAACCCCTGAGAGCGGCCCAGGTACTTCTGAGAGCGCC S
ACTCCTGAATCCGGCCCTGGTAGCGAGCCGGCAACCTCC PTSTEEGTSESAT
GGCTCAGAAACTCCTGGTTCGGAACCAGCGACCAGCGGT PESGPGSEPATSG
ACCGCTGAAGCCGCAGGTtgtGGCACTGCGGAAGCTGCAA SETPGTTAEAAG
CCGAAGAGGGTACCAGCACGGAACCGAGCGAGGGTTCTG C
CCCCGGGTACTTCCACCGAACCATCGGAGGGCTCTGCAC GTAEAAAGSPTS
CTGGTAGCGAACCTGCGACGTCTGGTTCTGAAACGCCGA T
CTGCAGAAGCGGCTGGTtgtGGCACCGCCGAAGCAGCTagcg EEGSPAGSPTSTE
cctctcgctccgcaCATCACCATCACCACCATCATCACTAA (SEQ EGTSTEPSEGSAP ID NO:
1087) GTSESTAEAAGC G TAEAATPESGPGT SESATPESGPGSE PATSGSETPGSEP
ATSGTAEAAGCG T AEAATEEGTSTEP SEGSAPGTSTEPS EGSAPGSEPATSG
SETPTAEAAGCG T AEAASASRSAHH H HHHHH (SEQ ID NO: 1088)
Example 16: Construction of 1.times.Amino,
9.times.Thiol-XTEN864
[0507] The primers PhoAfor&RP11-SASRSABsaIrevAGGT were used to
PCR the plasmid containing N-term-RP11 tag to obtain the PCR
product of N-term-RP11-R. Gel-purification of the band of the right
size was performed and was digested with NdeI/BsaI as the first
insert. Another PCR was performed with primers
AE432BsaIforAGGT&AE432_001BbsIrev-AACG on the plasmid
containing XTEN_AE864_003 in order to obtain the PCR product of
XTEN_AE432. Gel purification was performed on the band of the right
size, which was digested with BsaI/BbsI as the second insert.
Digestion of the construct N-term-RP11-R-AE432_9Cys-R-H8 ("H8"
disclosed as SEQ ID NO: 20) from Example 10 was performed with
NdeI/BsaI to remove the N-term-RP11-R fragment and gel purification
was performed to obtain the large fragment as the vector. Ligation
of the vector with the first and second inserts, above, was
performed and the product was used to transform BL21 competent
cells in order to obtain the construct N-term-RP11-R-XTEN_AE864
(C444, C495, C546, C597, C649, C700, C751, C802, C854)-R-H8 ("H8"
disclosed as SEQ ID NO: 20). The resulting construct was designed
to produce the precursor N-term-RP11-R-AE864_9Cys-R-H8 ("H8"
disclosed as SEQ ID NO: 20) (sequence in Table 39, below) the
product of which would generate 1.times.Amino, 9-Thio-XTEN864 after
removal of the N-term-RP11 tag and C-term 8.times.His-tag (SEQ ID
NO: 20) by trypsin digestion. The resulting product contains an
N-terminal amino group and nine cysteines in the XTEN864 sequence
for conjugation (Seg 175).
TABLE-US-00045 TABLE 39 DNA and amino acid sequence for 1xAmino,
9-Thio-XTEN864 Clone Amino Acid Name DNA Sequence Sequence N-term-
ATGAAAAACCCAGAGCAAGCAGAAGAACAAGCTGAAGA MKNPEQAEEQAE RP11-R-
ACAGCGCGAAGAAACACGTCCGCGTCCTCGCCCACGTCC E AE864_
ACGTCCGCGTCCACGCCCTCGTCCTCGTCCGCGCCCTCGT QREETRPRPRPRP 9Cys-R-118
CCGagcgcgtctcgttccgctGGGTCTCCAGGTAGCCCAGCTGGTA RPRPRPRPRPRPR
(''118'' GCCCAACCTCTACCGAAGAAGGTACCTCTGAATCCGCTA PSASRSAGSPGSP
disclosed CTCCAGAATCCGGTCCTGGTACTAGCACTGAGCCAAGCG AGSPTSTEEGTSE as
SEQ ID AAGGTTCTGCTCCAGGCTCCCCGGCAGGTAGCCCTACCTC SATPESGPGTSTE NO:
20) TACCGAAGAGGGCACTAGCACCGAACCATCTGAGGGTTC PSEGSAPGSPAGS
CGCTCCTGGCACCTCCACTGAACCGTCCGAAGGCAGTGCT PTSTEEGTSTEPS
CCGGGTACTTCCGAAAGCGCAACTCCGGAATCCGGCCCT EGSAPGTSTEPSE
GGTTCTGAGCCTGCTACTTCCGGCTCTGAAACTCCAGGTA GSAPGTSESATPE
GCGAGCCAGCGACTTCTGGTTCTGAAACTCCAGGTTCACC SGPGSEPATSGSE
GGCGGGTAGCCCGACGAGCACGGAGGAAGGTACCTCTGA TPGSEPATSGSET
GTCGGCCACTCCTGAGTCCGGTCCGGGCACGAGCACCGA PGSPAGSPTSTEE
GCCGAGCGAGGGTTCAGCCCCGGGTACCAGCACGGAGCC GTSESATPESGPG
GTCCGAGGGTAGCGCACCGGGTTCTCCGGCGGGCTCCCC TSTEPSEGSAPGT
TACGTCTACGGAAGAGGGTACGTCCACTGAACCTAGCGA STEPSEGSAPGSP
GGGCAGCGCGCCAGGCACCAGCACTGAACCGAGCGAAG AGSPTSTEEGTST
GCAGCGCACCTGGCACTAGCGAGTCTGCGACTCCGGAGA EPSEGSAPGTSTE
GCGGTCCGGGTACGAGCACGGAACCAAGCGAAGGCAGC PSEGSAPGTSESA
GCCCCAGGTACCTCTGAATCTGCTACCCCAGAATCTGGCC TPESGPGTSTEPS
CGGGTTCCGAGCCAGCTACCTCTGGTTCTGAAACCCCAGG EGSAPGTSESATP
TACTTCCACTGAACCAAGCGAAGGTAGCGCTCCTGGCAC ESGPGSEPATSGS
TTCTACTGAACCATCCGAAGGTTCCGCTCCTGGTACGTCT ETPGTSTEPSEGS
GAAAGCGCTACCCCTGAAAGCGGCCCAGGCACCTCTGAA APGTSTEPSEGSA
AGCGCTACTCCTGAGAGCGGTCCAGGCTCTCCAGCAGGT PGTSESATPESGP
TCTCCAACCTCCACTGAAGAAGGCACCTCTGAGTCTGCTA GTSESATPESGPG
CCCCTGAATCTGGTCCTGGCTCCGAACCTGCTACCTCTGG SPAGSPTSTEEGT
TTCCGAAACTCCAGGTACCTCGGAATCTGCGACTCCGGA SESATPESGPGSE
ATCTGGCCCGGGCACGAGCACGGAGCCGTCTGAGGGTAG PATSGSETPGTSE
CGCACCAGGTACCAGCACTGAGCCTTCTGAGGGCTCTGC SATPESGPGTSTE
ACCGGGTACCTCCACGGAACCTTCGGAAGGTTCTGCGCC PSEGSAPGTSTEP
GGGTACCTCCACTGAGCCATCCGAGGGTTCAGCACCAGG SEGSAPGTSTEPS
TACTAGCACGGAACCGTCCGAGGGCTCTGCACCAGGTAC EGSAPGTSTEPSE
GAGCACCGAACCGTCGGAGGGTAGCGCTCCAGGTAGCCC GSAPGTSTEPSEG
AGCGGGCTCTCCGACAAGCACCGAAGAAGGCACCAGCAC SAPGTSTEPSEGS
CGAGCCGTCCGAAGGTTCCGCACCAACGGCAGAGGCAGC APGSPAGSPTSTE
AGGTTGTGGTACAGCAGAAGCAGCTCCGGGTAGCGAGCC EGTSTEPSEGSAP
TGCAACCAGCGGTTCTGAGACGCCGGGCACTTCCGAATC TAEAAGCGTAEA
TGCGACCCCGGAGTCCGGTCCAGGTTCAGAGCCGGCGAC A
GAGCGGTTCGGAAACGCCGGGTACTGCTGAAGCGGCTGG PGSEPATSGSETP
TTGTGGTACTGCTGAAGCTGCATCGACCGAACCAAGCGA GTSESATPESGPG
AGGTTCGGCACCGGGTACTAGCGAGAGCGCAACCCCTGA SEPATSGSETPGT
AAGCGGTCCGGGCAGCCCGGCAGGTTCTCCAACCAGCAC AEAAGCGTAEAA
CGAAGAAGGTTCCCCTGCTACTGCCGAAGCTGCAGGCTG S
CGGTACTGCGGAGGCGGCGTCCCCAACTTCTACTGAGGA TEPSEGSAPGTSE
AGGTACTTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGT SATPESGPGSPAG
ACCTCCACTGAACCGTCTGAAGGCTCTGCACCAGGCACTT SPTSTEEGSPATA
CTGAGTCTGCTACTACCGCCGAAGCCGCTGGTTGTGGTAC EAAGCGTAEAAS
CGCAGAAGCTGCATCTGAGACTCCAGGCACTTCTGAGTC P
CGCAACGCCTGAATCCGGTCCTGGTTCTGAACCAGCTACT TSTEEGTSESATP
TCCGGCAGCGAAACCCCAGGTACCTCTGAGTCTGCGACT ESGPGTSTEPSEG
CCAGAGTCTGGTACCGCGGAAGCGGCTGGTTGTGGTACT SAPGTSESATTAE
GCAGAGGCAGCTGGTTCTCCGGCTGGTAGCCCGACCAGC AAGCGTAEAASE
ACGGAGGAGGGTACGTCTGAATCTGCAACGCCGGAATCG T
GGCCCAGGTTCGGAGCCTGCAACGTCTGGCAGCGAAACC PGTSESATPESGP
CCGGGTACCACGGCGGAAGCGGCAGGTTGTGGCACCGCG GSEPATSGSETPG
GAGGCAGCAGCTGGTTCTCCAACCTCTACCGAGGAGGGT TSESATPESGTAE
TCACCGGCAGGTAGCCCGACTAGCACTGAAGAAGGTACT AAGCGTAEAAGS
AGCACGGAGCCGAGCGAGGGTAGTGCTCCGGGTACGAGC P
GAGAGCACGGCAGAAGCCGCTGGCtgcGGTACTGCTGAAG AGSPTSTEEGTSE
CGGCAACCCCTGAGAGCGGCCCAGGTACTTCTGAGAGCG SATPESGPGSEPA
CCACTCCTGAATCCGGCCCTGGTAGCGAGCCGGCAACCT TSGSETPGTTAEA
CCGGCTCAGAAACTCCTGGTTCGGAACCAGCGACCAGCG AGCGTAEAAAGS
GTACCGCTGAAGCCGCAGGTtgtGGCACTGCGGAAGCTGC P
AACCGAAGAGGGTACCAGCACGGAACCGAGCGAGGGTT TSTEEGSPAGSPT
CTGCCCCGGGTACTTCCACCGAACCATCGGAGGGCTCTGC STEEGTSTEPSEG
ACCTGGTAGCGAACCTGCGACGTCTGGTTCTGAAACGCC SAPGTSESTAEAA
GACTGCAGAAGCGGCTGGTtgtGGCACCGCCGAAGCAGCTa GCGTAEAATPES
gcgcctctcgctccgcaCATCACCATCACCACCATCATCACTAA G (SEQ ID NO: 1089)
PGTSESATPESGP GSEPATSGSETPG SEPATSGTAEAA G CGTAEAATEEGT S
TEPSEGSAPGTST EPSEGSAPGSEPA TSGSETPTAEAAG CGTAEAASASRS A HHHHHHHH
(SEQ ID NO: 1090)
Example 17: Fermentation of XTEN for Conjugation
[0508] Starter cultures were prepared by inoculating glycerol
stocks of E. coli carrying the plasmid containing the appropriate
XTEN for conjugation protein sequences into 125 mL of LB Broth
media containing 50 .mu.g/mL kanamycin. The cultures were then
shaken overnight at 37.degree. C. The starter culture was used to
inoculate 2 L of fermentation batch media containing -12.5 g
ammonium sulfate, 15 g potassium phosphate dibasic anhydrous, 2.5 g
sodium citrate dihydrate; 8.5 g sodium phosphate monobasic
monohydrate; 50 g NZ BL4 soy peptone (Kerry Bioscience #5X00043);
25 g yeast extract (Teknova #Y9020); 1.8 L water, 0.5 mL
polypropylene glycol; 2.5 mL trace elements solution (Amunix recipe
144-1); 17.5 mL 1M magnesium sulfate; and 2 mL Kanamycin (50
mg/mL)--in 5 L glass jacketed vessel with a B. Braun Biostat B
controller. The fermentation control settings were: pH=6.9+/-0.1;
dO2=10%; dissolved oxygen cascade in stirrer only mode with a range
of 125-1180 rpm; air flow of 5 liters per minute of 90% oxygen;
initial temperature 37.degree. C.; base control 13% ammonium
hydroxide; and no acid control. After 6 hours of culture a 50%
glucose feed was initiated at a rate of 30 g/hr. After 20 hours of
culture, 25 mL of 1M magnesium sulfate and 3 mL of 1M IPTG were
added. After a total fermentation run time of 45 hours the culture
was harvested by centrifugation yielding cell pellets between
0.45-1.1 kilograms in wet weight for all constructs. The pellets
were stored frozen at -80.degree. C. until further use. Culture
samples at multiple time points in the fermentation were taken, the
cells were lysed, then cell debris was flocculated with heat and
rapid cooling, clarified soluble lysates were prepared by
centrifugation and analyzed by a regular non-reducing SDS-PAGE
using NuPAGE 4-12% Bis-Tris gel from Invitrogen according to
manufacturer's specifications with Coomassie staining. An example
of the accumulation of XTEN fusion protein as a function of
fermentation run time is shown in FIG. 45. The results showed that
the XTEN fusion protein constructs were expressed at fermentation
scale with titers >1 g/L, with an apparent MW of about 160 kDa
(note: the actual molecular weight are 100 kDa. The observed
migration in SDS-PAGE was comparable to that observed for other
XTEN-containing fusion proteins).
Example 18: Purification of 1.times.Thiol-XTEN (Cysteine-Engineered
XTEN) Reagent with CBD and His8 Tags (SEQ ID NO: 20)
[0509] The example describes the purification of a
cysteine-engineered XTEN comprising a single cysteine residue.
[0510] Materials and Methods:
[0511] 1. Clarification
[0512] 20 gm of cell paste from the fermentation was resuspended in
100 ml of 20 mM sodium phosphate, pH 8.0 (Lysis Buffer). Cell
lysate was homogenized between 800-900 bars three times in a
homogenizer. 50 ml of lysis buffer was used as a chase to retrieve
hold-up volume from the homogenizer. The homogenized lysate was
incubated in a water bath at 85.degree. C. for 20 minutes, followed
by quick cooling in an ice water bath for 20 minutes. After the
heating & cooling treatment, the lysate was centrifuged at
11000 RPM for 90 minutes in SORVALL centrifuge. After
centrifugation, the supernatant was filtered through two CUNO Bio
cap 25 (BC0025L90SP08A) filters. Filters were chased with 40 ml of
lysis buffer. The final volume of clarified material was 230 ml.
The clarified supernatant was stored at 4.degree. C. overnight.
[0513] 2. Capture Step: Hydrophobic Interaction Chromatography
[0514] Hydrophobic interaction chromatography was used as a first
step using hydrophobic CBD tag of XTEN to ensure the capture of
N-terminal intact protein. Toyopearl Phenyl 650M (Part #0014783,
TOSOH Bioscience) was used to pack XK16 to 15 cm bed height (30 ml
column volume). The chromatography was performed using AKTA FPLC
(GE Biosciences). Toyopearl Phenyl resin was equilibrated with 2
column volume of 20 mM Sodium Phosphate, 1M Sodium Sulfate, pH 8.0
prior to loading. HIC load was prepared by adding Sodium Sulfate to
1 M concentration to above clarified lysate (final volume.about.250
ml). The sample was loaded on HIC resin (.about.4 mg/ml resin load)
at 2 ml/min. Load was completed by chasing with .about.9 column
volume of equilibrium buffer (20 mM Sodium Phosphate, 1M Sodium
Sulfate, pH 8.0) till UV215 was stable. Protein was step eluted
with 100% B (20 mM Sodium Phosphate, pH 8.0). A total of 7 column
volumes of elution buffer was applied to confirm complete elution
(FIG. 46).
[0515] Samples were analyzed by 4-12% Bis-Tris SDS-PAGE
(non-reducing) to determine elution pool (FIG. 47). Based on the
following gel elution, fractions E1, E2, E3 & E4 were pooled
for further processing. The total protein was estimated to be 85 mg
in HIC elution pool, with a 65% step yield determined by running
another quantitation gel.
[0516] 3. Polishing/Capture Step: Toyopearl IMAC Chromatography
[0517] IMAC affinity chromatography was used as a capture step for
binding to intact C-terminal His-tag of XTEN. A chromatography
column XK26 was packed with Toyopearl IMAC 650 M (Part #0014907,
TOSOH Biosciences) with 15 cm bed height (85 ml Column volume). The
column was equilibrated with 2 column volume of 20 mM sodium
phosphate, 10 mM imidazole, 0.25 M NaCl, pH 8.0 (equilibrium
buffer). IMAC load was prepared by adding SM NaCl to the HIC
elution pool to make 0.25 M NaCl in the final volume. The sample
was loaded on IMAC resin at 4 ml/min flow rate. The load was
completed by 2 column volumes of equilibrium buffer. Resin was
washed with 2 column volumes of 20 mM Sodium Phosphate, 10 mM
Imidazole, pH 8.0 to remove salt. A linear elution was performed
from 0 to 100% B in 7 column volume with buffer A (20 mM Sodium
Phosphate, 10 mM Imidazole, pH 8.0) and buffer B (20 mM Sodium
Phosphate, 200 mM imidazole, pH 8.0) followed by 2 CV of 100% B.
Presence of imidazole maintained UV 215 absorbance above 3500 mAu
with AKTA FPLC, so an elution peak was not observed. The flow
through, wash and elution fractions were analyzed by 4-12% Bis-Tris
non-reducing SDS-PAGE gel (FIGS. 48A and 48B). The gel shows the
successful removal of host cell protein and truncated XTEN species
(FIG. 48A). Based on the second gel (FIG. 48B), fractions C5, C6
& C7 were pooled. A total of 70 mg of protein was estimated in
the IMAC elution pool.
[0518] 4. Trypsin Digestion of IMAC Elution Pool
[0519] Trypsin digestion of IMAC elution pool was performed at a
1:200 m/m ratio. 0.35 mg of bovine trypsin from pancreas (Sigma,
cat #T1426) was incubated with 70 mg of Protein (IMAC elution pool)
overnight at 37.degree. C. Non-reducing 4-12% Bis-Tris SDS-PAGE
analysis was performed to confirm that cleavage of the CBD tag was
completed as shown in FIG. 49.
[0520] 5. Polishing Step: MacroCap Q Chromatography
[0521] After trypsin digestion, cleaved tags were separated using
MacroCap Q chromatography. An XK 16 column was packed with 18 cm
bed height with MacroCap Q (GE Life Sciences, Cat #17-5469-02).
Trypsin digested IMAC elution pool was incubated for 1 hour at
37.degree. C. with 2 mM TCEP to reduce dimers of XTEN prior to
loading on MacroCap Q column. Column was equilibrated with 20 mM
HEPES, pH 7.0. The sample was loaded at 4 ml/min (.about.2 mg/ml
resin load). Column loading was completed with additional 2 column
volume of 20 mM HEPES, 2 mM TCEP pH 7.0. The column was washed with
2 column volumes of (20 mM HEPES, 2 mM TCEP, 150 mM NaCl pH 7.0). A
linear gradient elution from 150 mM NaCl to 500 mM NaCl in 20 mM
HEPES, 2 mM TCEP, pH 7.0 buffer was performed over 20 column
volumes. UV 215 was observed at a high level during the entire
chromatography (FIG. 50) due to presence of TCEP, but the elution
peak was observed between 22 to 30 mS/cm. All samples were analyzed
by SDS-PAGE, silver staining and C18 RP-HPLC. Removal of cleaved
CBD was observed in the flow through (FIG. 51A), while very faint
bands of XTEN were observed in elution fractions (FIG. 51B) in
elution fractions E11, E12, F12, F11, F10 . . . F6). Separation of
truncation species of XTEN were observed by silver staining in
earlier elution fractions E11, E12, F12 & F11 as species
migrating faster than the main XTEN polypeptide (FIG. 51C). Based
on the above results, elution fractions were further analyzed by
C18 RP-HPLC. FIG. 52 shows the stacked chromatography profiles of
the RP-HPLC analysis. Earlier elution fractions had wide leading
shoulder indicating significant presence of truncated species.
Also, these earlier fractions (E12, F12 & F11) were enriched
with proteolysis tags. Based on the above analysis, elution
fractions F10, F9, F8, F7 and F6 were pooled. The elution pool was
analyzed again by C18 RP-HPLC (FIG. 53). The purified protein was
found to be 97.5% pure.
[0522] 6. Concentration and Diafiltration (Final Formulation)
[0523] The above MacroCap Q pool was concentrated using Amicon
Ultracel-15 (MWCO 10k) centrifugal device to 5 ml followed by
7.times. diafiltration with 20 mM HEPES pH 7.0 to a final protein
concentration of 8.13 mg/ml. Total of 43 mgs of protein was
purified from 20 gm of cell pellet. The overall recovery for three
step chromatography was estimated at .about.33%.
Example 19: Purification of 1.times.Amino-XTEN Reagent with CBD and
His8 Tags (SEQ ID NO: 20)
[0524] Purification of amino-XTEN was performed essentially as
described for the purification of the 1.times.Thiol-XTEN containing
one cysteine (see Example 15 for details). 20 gm of cell paste from
fermentation was homogenized in 100 ml of 20 mM sodium phosphate,
pH 8.0 (Lysis Buffer), heat treated and clarified by centrifugation
and filtration. Hydrophobic interaction chromatography was used as
a first step to capture N-terminal intact protein (FIG. 54).
Fractions E2, E3, & E4 were pooled for further processing. IMAC
affinity chromatography was used as a capture step to bind intact
C-terminal His-tag of XTEN (FIG. 55). Fractions E2 and E3 were
pooled. Trypsin digestion of IMAC elution pool was performed at
1:200 m/m ratio overnight at 37.degree. C. After trypsin digestion
cleaved tags were separated using MacroCap Q chromatography.
Fractions were analyzed by SDS-PAGE followed by silver staining
(FIG. 56) and C18 RP-HPLC (FIG. 57). Based on above analysis
elution fractions C10, C11 and C12 were pooled. Elution pool was
analyzed again by C18 RP-HPLC (FIG. 58). Protein was found to be
>98% pure. MacroCap Q pool was concentrated using Amicon
Ultracel-15 (MWCO 10k) centrifugal device at 3000 RPM to 5 ml
followed by 7.times. diafiltration with 20 mM HEPES pH 7.0 to final
protein concentration to 5.35 mg/ml.
Example 20: Purification and Assessment of RP11/His8-XTEN ("His8"
Disclosed as SEQ ID NO: 20) Two-Tag System
[0525] Using expression vectors as described above, two-tagged XTEN
proteins were constructed to encode fusion proteins with the
following amino acid sequences or components:
MKIKTGARILALSALTTMMFSASALAAPTTAGAG-Tag-XTEN_AE869(Am1)-RHHHHHHHH
("MKIKTGARILALSALTTMMFSASALAAPTTAGAG" and "RHHHHHHHH" disclosed as
SEQ ID NOS 1091 and 1092, respectively), where:
MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 1093) is a MalE recognition
sequence that is cleaved from the polypeptide expressed and
transported to the host cell periplasm, APTTAGAG (SEQ ID NO: 1094)
is a spacer, and Tag is from the following (tag name followed by
sequence in parentheses): RP5 (RPRPRPRPRPGR) (SEQ ID NO: 1095); RP7
(RPRPRPRPRPRPRPGR) (SEQ ID NO: 1096); KP5 (KPKPKPKPKPGR) (SEQ ID
NO: 1097); RP9 (RPRPRPRPRPRPRPRPRPGR) (SEQ ID NO: 1098); RP11
(RPRPRPRPRPRPRPRPRPRPRPGR) (SEQ ID NO: 1099); PSK4P9
(RPRPKPRPKPRPKPRPKPGR) (SEQ ID NO: 1100); and R6K5P11
(RPRPKPRPKPRPKPRPKPRPKPGR) (SEQ ID NO: 1101). All variations of the
constructs with tags were made and the proteins were expressed in
E. coli using the methods as described in Example 15. Soluble
extracts were prepared from the host cell for SDS-PAGE/Coomassie
staining analysis. By analysis, the N-terminal tag length and amino
acid composition did not affect protein expression noticeably (see
FIG. 59) for expression of constructs with RP7, RP9, RP11 and
R6K5P11 tags. Expressed proteins with the RP5, RP7, RP9 and RP11
tags proteins were further tested for binding to MacroCap SP resin.
Proteins with longer tags bound more efficiently to cation exchange
resin, and remained bound under more stringent wash conditions.
Thus RP11 tag was selected as N-terminal tag for purification of
XTEN using cation exchange chromatography. An additional experiment
showed that RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO: 20)
polypeptide was efficiently expressed in fermenter and most of the
expressed protein was found in the cell pellet fraction (FIG.
60).
Example 21: Purification of 1.times.Amino-XTEN reagent with RP11/H8
("H8" Disclosed as SEQ ID NO: 20) Two-Tag System
[0526] 1. Expression
[0527] The RP11-XTEN-His8 ("His8" disclosed as SEQ ID NO: 20)
precursor of 1.times.Amino-XTEN was produced by expression in
transformed E. coli using a 4 L fermention reaction as described.
Cells were harvested by centrifugation and frozen at -80.degree. C.
until use.
[0528] 2. Lysis and Clarification
[0529] 25 g of cell paste was resuspended in 75 mL of 20 mM sodium
phosphate pH 8.0, 50 mM NaCl, 2 mM EDTA. Lysis was performed by
passing the resuspended paste through a homogenizer at 800-900 bar
three times. Homogenate was held at 85.degree. C. in a water bath
for 15 min before it was quickly cooled down using an ice/water
bath until the temperature dropped to below 10.degree. C. The
treated homogenate was then centrifuged at 10, 000 rpm in SLA-3000
rotor for 60 minutes. Supernatant was collected and filtered using
a 0.22 .mu.m bottletop filter.
[0530] 3. Cation Exchange Capture Step
[0531] Cation exchange chromatography was used as a capture step to
ensure N-terminal integrity of the product. MacroCap SP resin (GE
Healthcare) was selected among several cation exchange media due to
its superior capacity and selectivity for the product. A 20 mL
MacroCap SP column was packed in a Redi-Sep housing and
equilibrated with 20 mM sodium phosphate (pH 8.0), 20 mM NaCl
buffer. The lysate was loaded onto the column by gravity. Three
column volumes (CV) of 20 mM sodium phosphate pH 8.0, 100 mM NaCl
was applied as the wash step before protein was step-eluted with 3
CVs of 20 mM sodium phosphate pH 8.0, 500 mM NaCl. Half CV
fractions (10 mL) were collected for elutions and analyzed by 4-12%
Bis-Tris SDS/PAGE (FIG. 61). Elution fractions 2-4 were combined
for the subsequent chromatography step.
[0532] 4. IMAC Polishing Step
[0533] A 20-mL ToyoPearl AF-Chelate column was packed in a Redi-Sep
column housing and charged with 100 mM nickel sulfate. The column
was equilibrated with 20 mM sodium phosphate pH 8.0, 500 mM NaCl
before the MacroCap SP pool was loaded onto the column by gravity.
Two wash steps were applied using 20 mM sodium phosphate (pH 8.0),
500 mM NaCl, 5 mM imidazole buffer followed by 20 mM sodium
phosphate (pH 8.0), 5 mM imidazole. Product was then eluted from
the column using 20 mM sodium phosphate, 100 mM imidazole and half
CV (10 mL) fractions were collected for 4 CVs of elution. Samples
from each step were examined by 4-12% Bis-Tris SDS/PAGE (FIG. 62).
Based on the gel, elutions 2 and 3 were pooled for further
processing. The overall yield was 30%.
[0534] 5. Trypsin Digestion of IMAC Elution Pool
[0535] Trypsin digestion of the IMAC pool was performed at 1:200
and 1:500 m/m ratio by adding 1 mg/mL bovine trypsin (Sigma, Cat
#T1426, Trypsin from Bovine Pancreas) to the IMAC pool. The
reaction mixtures were held at 37.degree. C. overnight and the
completion of digestion was confirmed by MALDI-TOF mass
spectrometry. Pre- and post-digest samples were analyzed by 4-12%
Bis-Tris SDS/PAGE stained by both Coomassie and silver stain (FIGS.
63A and 63B). Fainter staining on Coomassie-stained gel as well as
the shifting of the molecule weight for the post-digestion samples,
when compared to pre-digestion sample, indicates the successful
removal of both N- and C-terminal tags. A silver stained gel showed
homogeneous bands after digestion, suggesting the absence of
truncated species in the sample, supporting the conclusion that the
RP11/H8 ("H8" disclosed as SEQ ID NO: 20) two-tag system and
purification methods provides a homogeneous final XTEN product.
Example 22: Purification of XTEN with RP11 and His8 (SEQ ID NO: 20)
Affinity Tags
[0536] 1. Expression
[0537] The fusion protein RP11-XTEN-His8 ("His8" disclosed as SEQ
ID NO: 20), with two affinity tags linked to XTEN at the N- and
C-terminus, respectively, was expressed in E. coli using a 4 L
fermention reaction using conditions described.
[0538] 2. Lysis and Clarification
[0539] After growth, the cells were harvested by centrifugation and
frozen at -80.degree. C. until use. The cell pellet was resuspended
in lysis buffer (20 mM sodium phosphate, 50 mM NaCl, 2 mM EDTA pH
8.0, 3 ml buffer per gram cell paste). The cells were lysed by
passing through an APV homogenizer three times at a pressure of
830-900 bar. Lysis buffer (1 ml buffer per gram cell paste) was
used as a chase to retrieve hold up volume from the homogenizer.
The homogenized lysate was incubated in a water bath at 85.degree.
C. for 20 minutes, followed by quick cooling in ice water bath for
20 minutes. After the heating and cooling treatment, the lysate was
centrifuged at 11000 RPM for 90 minutes in a SORVALL centrifuge.
After centrifugation, the supernatant was filtered through two CUNO
Bio cap 25 (BC0025L90SP08A) filters. The clarified supernatant was
stored at 4.degree. C. overnight.
[0540] 3. Capture Step: Toyopearl IMAC Chromatography
[0541] IMAC affinity chromatography was used as a capture step for
binding the XTEN with an intact C-terminal His-tag. Briefly, the
chromatography column BPG140/12 (GE Life Sciences) was packed with
2000 ml Toyopearl IMAC 650 M resin (TOSOH Biosciences). The column
was equilibrated with 2 column volumes (CVs) of equilibrium buffer
(20 mM sodium phosphate, 500 mM NaCl, pH 8.0). Clarified cell
lysate was adjusted to a final NaCl concentration of 500 mM using 5
M NaCl stock solution, and then was loaded onto the IMAC resin. The
column was washed with 2 column volumes of equilibrium buffer, and
then 2 column volumes of 20 mM sodium phosphate, 500 mM NaCl, 5 mM
Imidazole pH 8.0, followed by 2 column volumes of 20 mM sodium
phosphate, 5 mM Imidazole pH 8.0 to remove salt. Elution was
performed with 2 column volumes of 20 mM sodium phosphate, 100 mM
Imidazole, pH 8.0. The flow through, wash and elution fractions
were analyzed by non-reducing 4-12% Bis-Tris SDS-PAGE/Coomassie
staining and the fractions with the desired product were
pooled.
[0542] 4. Polishing/Capture Step: MacroCap SP Chromatography
[0543] Cation exchange chromatography was used as a polishing step
to ensure the N-terminal integrity of the product. MacroCap SP
resin (GE Life Sciences) was selected among several cation exchange
media due to its superior capacity and selectivity for the product.
1000 ml of MacroCap SP resin was packed in a BPG100/13 (GE Life
Sciences) chromatography column and equilibrated with 20 mM sodium
phosphate pH 8.0, 20 mM NaCl. The IMAC pool was loaded onto the
column and the resin was washed with 2 column volumes of 20 mM
sodium phosphate, 50 mM NaCl, pH 8.0 and 2 column volumes of 20 mM
sodium phosphate pH 8.0, 150 mM NaCl. The protein was eluted with 5
column volumes of linear gradient from 150 to 500 mM NaCl in 20 mM
sodium phosphate pH 8.0. Fractions were collected and analyzed by
4-12% Bis-Tris SDS/PAGE. Fractions the with desired product were
combined for the next step.
[0544] 5. Trypsin Digestion of Macrocap SP Elution Pool
[0545] Trypsin (Sigma, Trypsin from Bovine Pancreas) digestion of
the SP elution pool was performed at 1:200 m/m enzyme/protein ratio
overnight at 37.degree. C.
[0546] 6. Polishing Step: Macrocap Q Chromatography
[0547] After trypsin digestion, the cleaved tags were separated
from the final product using Macrocap Q chromatography. The
BPG100/19 column (GE Life Sciences) was packed with 1500 ml column
volume of Macrocap Q resin (GE Life Sciences). The trypsin digested
Macrocap SP elution pool was incubated for 15 min at 80.degree. C.
with 20 mM DTT and 2 mM EDTA to reduce disulfide bonds and to
inactivate trypsin. The cooled protein solution was diluted to a
conductivity below 5 mS/cm with Milli-Q water and loaded onto the
Macrocap Q column equilibrated with 20 mM HEPES, 50 mM NaCl, pH
7.0. The column was washed with 2 column volumes of 20 mM HEPES, 50
mM NaCl, pH 7.0, then 2 column volumes of 20 mM HEPES, 2 mM TCEP,
150 mM NaCl pH7.0. The protein was eluted with a linear gradient
from 150 mM NaCl to 500 mM NaCl in 20 mM HEPES, pH 7.0 in 20 column
volumes. Fractions were analyzed by SDS-PAGE/silver staining.
[0548] 7. Concentration and Diafiltration (Final Formulation)
[0549] Selected MacroCap Q fractions were combined and concentrated
and using 10 KD Pellicon mini (Millipore) at a feed pressure <20
psi and retentate <8 psi, followed by 10.times. diafiltration
with 20 mM HEPES, 50 mM NaCl, pH 7.0 to achieve a final protein
concentration of >5 mg/ml.
[0550] 8. Purity Analysis of Proteins Purified with Different
Methods
[0551] One batch (Batch 1) was purified through three purification
steps as described above. Another batch (Batch 2) was purified from
the same fermented material but the MacroCap SP polishing step was
omitted. Truncated species of XTEN were detected by SDS-PAGE/silver
staining in MacroCap Q elution fractions for Batch 2 (FIG. 87A),
while the MacroCap Q elution fractions for Batch 1 were essentially
free from truncations (FIG. 87B). These results support that, under
the conditions employed, the MacroCap SP step based on the RP11 tag
is essential to ensure N-terminal integrity and overall product
quality and that the RP11/H8 ("H8" disclosed as SEQ ID NO: 20)
two-tag system and purification methods provides a homogeneous
final XTEN product.
Example 23: Conjugation of the DBCO-Mal Linker to
3.times.Thiol-XTEN to Make an XTEN Precursor
[0552] A 3.times.-Thiol-XTEN (XTEN_AE905(Am1,C8,C453,C898, Seg
174)) cysteine-engineered XTEN segment was prepared for reaction as
a 193 uM (16 mg/ml) solution in 20 mM HEPES, pH 7.0, 50 mM NaCl.
DBCO-Maleimide (Click Chemistry Tools, Inc., cat. #A108) was
dissolved in DMF to a final concentration of 50 mM. An aliquot of
the 3.times.Thiol-XTEN (5.1 mg, 320 .mu.l) was reduced with 10 mM
freshly reconstituted DTT at 70.degree. C. for 20 minutes. The
protein sample was diluted to 600 .mu.l total volume with water.
1200 .mu.l of 100% acetonitrile was added and the mixture was
centrifuged at 13,000 rpm for 5 minutes. The supernatant was
removed, 1000 .mu.l of 80% acetonitrile was added and the mixture
was centrifuged at 13,000 rpm for 1 minute. The wash step was
repeated once more. The pellet was dissolved in 300 .mu.l 100 mM
HEPES pH 7.0. A 7.7 .mu.l 50 mM solution of DBCO-Mal in DMF was
added (1:6 molar ratio of 3.times.Thiol-XTEN to DBCO-Maleimide) and
was incubated for 2 hours at 25.degree. C. Completion of
modification was monitored by C18 RP-HPLC analysis (FIG. 64A). The
protein mixture was purified by hydrophobic interaction
chromatography (HIC) using a 1.6 ml Toyopearl butyl column. Elution
was performed with a 30 column volume descending gradient of
ammonium sulfate from 1.05 M to 0.3M in 20 mM Phosphate, pH 7.0
buffer at 0.5 ml/min flow rate (FIG. 64B). Chromatographic
fractions were analyzed by C18 RP-HPLC (FIG. 64C).
Example 24: Trypsin Cleavage and Purification of Double Tagged
Precursor
[0553] Trypsin Digestion
[0554] A double-tagged (CBD/His8) ("His8" disclosed as SEQ ID NO:
20) precursor of XTEN_AE870_Am1,C1 stains well with Coomassie due
to the presence of CBD sequence, while a no-tagged version of
XTEN_AE870_Am1,C1 stains poorly with Coomassie, but can be detected
by silver staining. Therefore, trypsin digestion completeness was
monitored using both Coomassie staining (FIG. 65A) and silver
staining (FIG. 65B) techniques. The double tagged precursor of
XTEN_AE870_Am1,C1 was digested with different ratios of bovine
trypsin and proteomics grade porcine trypsin (positive control) in
20 mM phosphate buffer, pH 8. Overnight incubation at 37.degree. C.
allowed more complete digestion than overnight incubation at
25.degree. C. based on detection of remaining Coomassie stained 160
kDa band of double tagged precursor (FIG. 65A: all ratios refer to
mass/mass ratio of trypsin to substrate). FIG. 65B shows that
1:100, 1:200 and 1:500 ratio digests with bovine trypsin and 1:100
digests with porcine trypsin resulting in efficient digestion of
the XTEN precursor.
[0555] MacroCap Q Purification of Trypsin Digested Double Tagged
Precursor
[0556] 1:200 ratio of bovine trypsin to double tagged precursor
(mass/mass) was used for digestion at 37.degree. C. overnight. FIG.
66A shows >90% conversion of double tagged precursor to digested
product. This was demonstrated by disappearance of the Coomassie
stained band at .about.160 kDa after digestion and appearance of 20
kDa CBD fragment.
[0557] Trypsin digested material was subjected to step purification
using MacroCap Q anion exchange resin and following buffers: A: 20
mM HEPES, 50 mM NaCl, pH 7.5 and B: 20 mM HEPES, 500 mM NaCl, pH
7.5. Digested material was loaded by gravity and eluted in a
stepwise manner using 3 column volume washes of 0%, 20%, 40%, 60%,
80% and 100% Buffer B consecutively. XTEN_AE870 eluted in 60% B.
FIG. 66B shows that cleaved CBD fragment did not bind to MacroCap Q
under used conditions and was completely separated from XTEN.
Stepwise elution of XTEN from MacroCap Q only partially separated
truncated polypeptides. Better separation was achieved by gradient
elution of XTEN (FIG. 66C).
[0558] Test for Residual Trypsin Activity
[0559] To test the presence of residual trypsin activity in the
final formulated XTEN preparations, a protein sample was mixed with
synthetic [G2]GLP2 peptide at 10:1 mass/mass ratio. A positive
control for digestion contained [G2]GLP2 peptide and bovine
trypsin; A negative control contained [G2]GLP2 peptide only. All
samples were incubated overnight at 37.degree. C. After incubation
samples were quenched with 1% TFA and subjected to C18 RP-HPLC
analysis using Phenomenex Jupiter C185 um 300 A analytical column.
Buffer A contained 0.1% TFA, 99.9% HPLC grade H.sub.2O; Buffer B
contained 0.1% TFA, 99.9% HPLC grade Acetonitrile. Analysis was
performed using a gradient of 5% B to 50% B over 45 min elution
time. FIGS. 67A-67C show the results of RP-HPLC analyses of
residual trypsin activity in the XTEN_AE869(Am1,C2) final
preparation. FIG. 67A shows the intact GLP2 peptide (41 min
retention time). FIG. 67B shows tryptic digest of GLP2 peptide with
two characteristic tryptic fragments (33.5 min and 34 min retention
time). FIG. 67C shows that GLP2 peptide remained intact after
overnight incubation with XTEN and no tryptic fragments were
observed. This result indicates that final MacroCap Q purified
preparations do not contain any residual trypsin activity.
Example 25: Fermentation and Purification of Cysteine-Engineered
XTEN for Conjugation
[0560] E. coli containing AC292 on a plasmid was grown to
saturation overnight in 2.times.YT and then 200 ml of this culture
was used to inoculate a 25 L culture of 2.times.YT media in a
wavebag. Both cultures were in the presence of 50 .mu.g/ml
kanamycin. The second culture was grown to an OD600 of .about.1.0
at 37.degree. C., chilled to 26.degree. C., and induced with 12 ml
of 1M IPTG overnight. The cell pellet was harvested at 4000 rpm in
a SLA-3000 rotor spinning for 20 minutes. The cell pellet (184 g)
was resuspended in 736 ml of 20 mM Tris pH 6.8, 50 mM NaCl. The
resuspended cells were lysed with a microfluidizer at 20,000 psi
and then heated to 75.degree. C. for 15 minutes, followed by rapid
cooling on ice for 30 minutes. The lysate was then clarified by
centrifugation. The clarified lysate was then loaded on to a DE52
column, previously sanitized with NaOH and equilibrated with 20 mM
Tris pH 6.8, 50 mM NaCl. The column was washed with 5 column
volumes of 20 mM Tris pH 6.8, 50 mM NaCl, 5 column volumes of 20 mM
Tris pH 6.8, 150 mM NaCl and eluted with 5 column volumes of 20 mM
Tris pH 6.8, 250 mM NaCl. The pooled elution fractions. were then
loaded on to a macrocapQ column, previously sanitized with NaOH and
equilibrated with 20 mM Tris pH 6.8, 50 mM NaCl. The column was
washed with 9 column volumes of 20 mM Tris pH 6.8, 50 mM NaCl, 9
column volumes of 20 mM Tris pH 6.8, 100 mM NaCl and eluted with 9
column volumes of 20 mM Tris pH 6.8, 250 mM NaCl. The pooled
elution fractions were adjusted to a 15% w/v sodium sulfate and
then loaded on to a octyl sepharose FF column column, previously
sanitized with NaOH and equilibrated with Tris pH 7.5. The column
was washed with 4 column volumes of 20 mM Tris pH 7.515% w/v sodium
sulfate, and eluted with 4 column volumes of 20 mM Tris pH 7.5, 5%
w/v sodium sulfate. The sample was stored at 4.degree. C. and given
the lot #AP197. The purified cysteine-engineered XTEN could then
serve as a suitable reactant for conjugation with a payload, such
as a drug from Table 11, resulting in an XTEN-drug conjugate.
Example 26: Conjugation of GLP2-Cys to 1.times.Amino-XTEN to Result
in XTEN-Payload of GLP2-XTEN
[0561] A 1.times.Amino-XTEN (XTEN_AE869(Am1)) was prepared as 67 uM
(5.35 mg/ml) solution in 20 mM HEPES, pH 7.0, 50 mM NaCl.
Sulfo-SMCC (Thermo Scientific, cat. #22322) was prepared fresh as
100 mM solution in DMSO. 10 mg of amino-XTEN (1.87 ml) was mixed
with 15-molar excess of sulfo-SMCC (18.7 ul) and incubated for 1 hr
at 25.degree. C. Excess cross-linker was removed by centrifugal
filtration using Amicon Ultra-15, MWCO 5k centrifugal device. A
volume of 1.8 ml reaction mixture was mixed with 8 ml 20 mM HEPES
pH 7.0, 50 mM NaCl and centrifuged for 20 min in Sorvall RT6000
centrifuge at 3000 rpm, 4.degree. C. The procedure was repeated two
more times. Final volume of recovered retentate was 1.8 ml. The
GLP2-Cys peptide (CSBio, custom synthesis) was dissolved in 20 mM
HEPES pH 7.0, 50 mM NaCl to the final concentration 3 mg/ml.
N-Maleimide-XTEN was mixed with 2.3-fold molar excess of GLP2-Cys
peptide and was incubated for 1 hr at 25.degree. C. Completion of
the modification was monitored by C18 RP-HPLC. 20 .mu.g protein
samples were loaded on Phenomenex Jupiter C185 uM 300 A 4.6
mm.times.150 mm column. Proteins were eluted with 5-50% gradient of
acetonitrile in 0.1% trifluoroacetic acid and detected by
absorbance at 214 nm. Essentially all N-maleimide-XTEN was
converted into GLP2-Cys-XTEN conjugate, as demonstrated by HPLC and
electrospray mass spectrometry (ESI-MS analysis of samples
performed on 100 .mu.g protein samples desalted using NanoSep 3K
Omega centrifugal devices (Pall Corp.). Protein solutions in 50%
acetonitrile, 0.5% formic acid were infused into high-resolution
mass spectometer at flow rate 10 ul/min. Spectra were acquired in
800-1600 amu range and reconstructed into zero-charge spectra using
Bayesian Protein Reconstruction Software) (FIGS. 68A-68C).
Unreacted XTEN and GLP2 peptide were separated from the conjugate
by consecutive anion exchange (MacroCap Q) and hydrophobic
interaction (Toyopearl Phenyl) chromatographies. The results of
RP-HPLC and MS analyses demonstrated the high yield and purity of
the reactants and final product (FIGS. 68A-68C).
Example 27: Conjugation of GLP2-N-Mal to 1.times.Thiol-XTEN
(Cysteine-Engineered XTEN)
[0562] 1.times.Thiol-XTEN (XTEN_AE880(Am1,C8)(Seg 181) was prepared
as 122 uM (9.84 mg/ml) solution in 20 mM HEPES, pH 7.0, 50 mM NaCl.
GLP2-N-Maleimide peptide (CSBio, custom synthesis) was dissolved in
DMSO to the final concentration 3 mg/ml. 1.times.Thiol-XTEN (8.8 mg
in 900 .mu.l) was mixed with 3-fold molar excess of GLP2-N-Mal
peptide and incubated for 1 hr at 25.degree. C. Completion of the
modification and the resulting conjugate was monitored by C18
RP-HPLC (20 .mu.g protein samples were loaded on Phenomenex Jupiter
C185 uM 300 A 4.6 mm.times.150 mm column. Proteins were eluted with
5-50% gradient of acetonitrile in 0.1% trifluoroacetic acid and
detected by absorbance at 214 nm) and electrospray ionization mass
spectrometry (ESI-MS analysis of samples was performed on 100 .mu.g
protein samples desalted using NanoSep 3K Omega centrifugal devices
(Pall Corp.). Protein solutions in 50% acetonitrile, 0.5% formic
acid were infused into high-resolution mass spectrometer at flow
rate 10 .mu.l/min. Spectra were acquired in 800-1600 amu range and
reconstructed into zero-charge spectra using Bayesian Protein
Reconstruction Software.) The results of the analysis are shown in
FIGS. 69A-69B. The GLP2-XTEN conjugate was purified by preparative
C4 RP-HPLC (Vydac Protein C4 5.mu.300 A 10 mm.times.250 mm column)
using 5-50% gradient of acetonitrile in 0.1% TFA as mobile phase
(see FIG. 70A). Final HPLC-purified GLP2-XTEN conjugate was
analyzed using Phenomenex Jupiter C185 uM 300 A 4.6 mm.times.150 mm
column (see FIG. 70B). Theield of purified GLP2-XTEN conjugate was
6.2 mg (70%).
Example 28: Conjugation of DBCO-Mal to 1.times.Thiol-XTEN
[0563] 1.times.Thiol-XTEN (XTEN_AE880(Am1,C8) (Seg 181) was
prepared as a 150 uM (12 mg/ml) solution in 20 mM HEPES, pH 7.0, 50
mM NaCl. DBCO-Maleimide (Click Chemistry Tools, Inc., cat. #A108)
was dissolved in DMF to a final concentration of 50 mM. A volume of
200 ul (2.4 mg) 1.times.Thiol-XTEN was adjusted to 100 mM HEPES pH
7.0 using a 1M stock solution. A 1.2 .mu.l volume of 50 mM DBCO-Mal
in DMF was added to the protein solution (1:2 molar ratio of
1.times.Thiol-XTEN to DBCO-Maleimide reagent) and was incubated for
1 hr at 25.degree. C. Completion of the modification reaction was
monitored by C18 RP-HPLC (FIG. 71A). The protein mixture was
purified by hydrophobic interaction chromatography (HIC) using a
1.6 ml Toyopearl Butyl column. Elution was performed with a 30
column volume of a descending gradient of ammonium sulfate from
1.05 M to 0.3M in 20 mM Phosphate, pH 7.0 buffer at 0.5 ml/min flow
rate (FIG. 71B). Chromatographic fractions were analyzed by C18
RP-HPLC (FIG. 71C).
Example 29: Preparation of a Bispecific Conjugate from Monospecific
XTEN Precursors Linked by the N-Termini
[0564] The example describes the creation of an XTEN-payload
composition by linking two different XTEN-payload precursors in an
N- to N-terminus configuration; one with a payload A and one with a
payload B, resulting in a bispecific conjugate.
[0565] As a first step, XTEN molecules containing multiple
cysteines (cysteine-engineered XTEN) are prepared using a RP11-His8
("His8" disclosed as SEQ ID NO: 20) two-tag purification system,
described above, and are formulated in 20 mM HEPES, pH 7.0, 50 mM
NaCl. A Payload A-maleimide is dissolved in aqueous solution 20 mM
HEPES, pH 7.0, DMF or DMCO or any other appropriate solvent
depending on reagent solubility. The Payload A-maleimide is added
to the cysteine-engineered XTEN in a 2-6 molar excess over XTEN and
incubated for 1 hr at 25.degree. C. Completion of modification is
monitored by C18 RP-HPLC. The resulting Payload A-XTEN conjugate is
purified from contaminants and unreacted components using
preparative C4-C18 RP-HPLC. The Payload A-XTEN conjugate is
formulated in 20 mM HEPES, pH 7.0, 50 mM NaCl. Next, the Payload
A-XTEN conjugate is further modified by adding dibenzylcyclooctyne
(DBCO)-NHS ester or DBCO-sulfo-NHS ester in a 10-50 molar excess to
the XTEN and incubating 1-2 hrs at 25.degree. C. Completion of the
modification is monitored by analytical C18 RP-HPLC. If the
conjugation efficiency is low (for example, <90%) or multiple
unspecific products are formed, the DBCO-Payload A-XTEN conjugate
is purified using preparative C4-C18 RP-HPLC. If the efficiency of
DBCO-NHS ester conjugation is high (>90%) with no significant
side products, the DBCO-Payload A-XTEN conjugate is purified from
excess reagent by buffer exchange using a 10-30 kDa MWCO
centrifugal device, acetonitrile precipitation or anion exchange
chromatography.
[0566] To create the second XTEN-payload precursor, a Payload
B-maleimide is dissolved in aqueous solution 20 mM HEPES, pH 7.0,
DMF or DMCO or any other appropriate solvent depending on reagent
solubility. Payload B-maleimide is added to the second
cysteine-containing XTEN in 2-6 molar excess over XTEN
concentration and incubated for 1 hr at 25.degree. C. Completion of
modification is monitored by analytical C18 RP-HPLC. The resulting
Payload B-XTEN conjugate is purified from contaminants and
reactants using preparative C4-C18 RP-HPLC. The Payload B-XTEN
conjugate is formulated in 20 mM HEPES, pH 7.0, 50 mM NaCl.
Azide-PEG4-NHS ester is added in 10-50 molar excess to the Payload
B-XTEN and incubated 1-2 hrs at 25.degree. C. Completion of
modification is monitored by C18 RP-HPLC. If the conjugation
efficiency is low (for example <90%) or multiple unspecific
products are formed, the azide-Payload B-XTEN conjugate is purified
using preparative C4-C18 RP-HPLC. If the efficiency of DBCO-NHS
ester conjugation is high (>90%) with no significant side
products, the azide-Payload B-XTEN conjugate is purified from
excess reagent by buffer exchange using a 10-30 kDa MWCO
centrifugal device, acetonitrile precipitation or anion exchange
chromatography. The final product is created by mixing purified and
concentrated DBCO-Payload A-XTEN and azide-Payload B-XTEN proteins
in an equilmolar ratio in 20 mM HEPES pH 7.0 buffer, 50 mM NaCl and
incubated at 25.degree. C. for 1 hr or longer until the reaction is
complete. Completion of modification is monitored by C4 or C18
RP-HPLC. If necessary, the bispecific conjugate Payload
A-XTEN-Payload B is purified by preparative RP-HPLC, hydrophobic
interaction chromatography or anion exchange chromatography.
Example 30: Preparation of a Trimeric Conjugate from Monospecific
XTEN Precursors Linked by the N-Termini
[0567] Monospecific XTEN-payload precursors will be prepared as
N-terminal fusions of a Payload A linked to an XTEN molecule; e.g.
of lengths ranging from AE144 to AE890, containing a single
cysteine at the C-terminus (prepared and purified as described in
Example 25). Purified precursors are formulated in 20 mM HEPES, pH
7.0, 50 mM NaCl. Tris-(2-maleimidoethyl)amine (TMEA, Thermo
Scientific, cat. #33043) and dissolved in DMSO or DMF. Precursor
(4-6 molar excess over cross-linker) and TMEA reagent are mixed and
incubated for 1 hr at 25.degree. C. Completion of the modification
is monitored by C4 or C18 RP-HPLC or size exclusion chromatography.
The resulting trivalent Payload A-XTEN conjugate is purified from
protein reactants or partial product mixture by hydrophobic
interaction chromatography (HIC), anion exchange chromatography or
preparative C4-C18 RP-HPLC.
Example 31: Conjugation and Purification of FITC-X-XTEN
[0568] Purified protein derived from AC272, lot #AP197, was labeled
with FITC maleimide. The sample was reduced by incubating at room
temperature with 5 mM TCEP for 1 hour. The sample was then desalted
into PBS using DG-10 columns. The sample was labeled by adding a
25-fold molar excess of FITC-maleimide in DMSO and incubating at
room temperature for 2 hours. Note that the volume adjusted such
that the DMSO concentration was <5% of total solvent. The
reaction was quenched by adding 2 mM DTT and then the sample was
digested overnight with TEV protease. The sample was diluted two
fold with 20 mM Tris pH 7.5 and loaded onto a macrocapQ column,
previously sanitized with NaOH and equilibrated with 20 mM Tris pH
7.5. The column was washed with 5 column volumes of 20 mM Tris pH
7.5, 135 mM NaCl, 5 column volumes of 20 mM Tris pH 7.5, 175 mM
NaCl and eluted with 5 column volumes of 20 mM Tris pH 7.5, 250 mM
NaCl. The pooled elution fractions were then digested with TEV over
60 hours at 4 C to complete the digestion. The digested samples
were then twice passed over a 1 ml perloza column previously
sanitized with NaOH and equilibrated with 20 mM Tris pH 7.5, 135 mM
NaCl. To remove any free FITC the sample was then dialyzed against
20 mM Tris pH 7.5, 135 mM NaCl using a 10,000 MWCO membrane.
Co-migration of the OD214 protein signal and OD495 FITC signal in a
SEC column indicate successful conjugation of the XTEN with the
label, with minimal free dye contamination (FIG. 72B). The
successful conjugation is also indicated by apparent large MW of
the protein with FITC fluorescence in SDS PAGE (FIG. 72A).
Example 32: Purification of GFP-X-XTEN
[0569] GFP (AC219) was chemically cross-linked to XTEN by a
bifunctional cross-linker with an amine reactive group to couple to
the GFP lysines and a cysteine reactive group to couple to the free
cysteine engineered into the XTEN in AC292. GFP was labeled with
bi-functional cross-linker sulfo-SMCC by incubating at room
temperature for 2 hours. The protein was desalted into PBS using
DG-10 columns to remove free sulfo-SMCC. Purified protein derived
from AC272, lot #AP197 was reduced and desalted into PBS on DG-10
columns and mixed with the labeled GFP to allow for cross-linking.
The cross-linking reaction was quenched with 2 mM DTT and TEV added
to remove the CBD domain in a overnight incubation at 4.degree. C.
The following day additional TEV was added to complete the
digestion with an additional 60 hour 4.degree. C. incubation.
Following TEV digestion the sample was dilute to 100 ml in 20 mM
Tris pH 7.5 and loaded onto a macrocapQ column, previously
sanitized with NaOH and equilibrated with 20 mM Tris pH 7.5. The
column was washed with 5 column volumes of 20 mM Tris pH 7.5, 5
column volumes of 20 mM Tris pH 7.5, 50 mM NaCl, 5 column volumes
of 20 mM Tris pH 7.5, 100 mM NaCl, 5 column volumes of 20 mM Tris
pH 7.5, 150 mM NaCl, 5 column volumes of 20 mM Tris pH 7.5, 200 mM
NaCl, 5 column volumes of 20 mM Tris pH 7.5, 250 mM NaCl, 5 column
volumes of 20 mM Tris pH 7.5, 300 mM NaCl, and 5 column volumes of
20 mM Tris pH 7.5, 500 mM NaCl. The peak elution fractions were
pooled and stored at 4.degree. C. Cross-linking was confirm by
co-migration of the OD214 protein signal and OD395 GFP signal in a
SEC column, with the SEC output shown as overlays in FIG. 73.
Example 33: Pharmacokinetics of GFP-XTEN and FITC-XTEN
Conjugates
[0570] The pharmacokinetics of the GFP-XTEN and FITC-XTEN
cross-linked conjugates prepared as described in the Examples above
were tested in cynomolgus monkeys. GFP-XTEN and FITC-XTEN were
administered to male cynos IV at 2 mg/kg and dose volumes of 0.77
and 0.68 mL respectively. Blood samples (1.0 mL) were collected
into prechilled heparinized tubes at predose, 2, 4, 8, 24, 48, 72,
96, 120, 168, 216, 264, 336, 388, 432, 504 hour time points, and
processed into plasma. Quantitation was performed by ELISA assay
using the anti-XTEN antibody for both capture and detection in the
case of GFP-XTEN and anti-XTEN capture and anti-FITC detection in
the case of FITC-XTEN. A non-compartmental analysis was performed
in WinNonLin with all time points included in the fit to determine
the PK parameters. The pharmacokinetic results are summarized in
Table 40 and FIG. 74. The data show XTEN can extend the half-life
of molecules to which it is chemically conjugated in a manner
comparable to genetic fusions to payloads of similar size.
TABLE-US-00046 TABLE 40 PK parameters of conjugated XTEN-payload
compositions. Construct Cmax (ng/mL) AUC (hr*ng/mL) T 1/2 (hrs)
GFP-X-XTEN (AP197d) 52800 8220000 107 FITC-X-XTEN AP197e 18900
3930000 84.2
[0571] The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576,
GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys
to determine the effect of composition and length of the
unstructured polypeptides on PK parameters. Blood samples were
analyzed at various times after injection and the concentration of
GFP in plasma was measured by ELISA using a polyclonal antibody
against GFP for capture and a biotinylated preparation of the same
polyclonal antibody for detection. Results are summarized in FIG.
75. They show a surprising increase of half-life with increasing
length of the XTEN sequence. For example, a half-life of 10 h was
determined for GFP-XTEN_L288 (with 288 amino acid residues in the
XTEN). Doubling the length of the unstructured polypeptide fusion
partner to 576 amino acids increased the half-life to 20-22 h for
multiple fusion protein constructs; i.e., GFP-XTEN_L576,
GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the
unstructured polypeptide fusion partner length to 836 residues
resulted in a half-life of 72-75 h for XTEN_AD836-GFP. Thus,
increasing the polymer length by 288 residues from 288 to 576
residues increased in vivo half-life by about 10 h. However,
increasing the polypeptide length by 260 residues from 576 residues
to 836 residues increased half-life by more than 50 h. These
results show that there is a surprising threshold of unstructured
polypeptide length that results in a greater than proportional gain
in in vivo half-life. Thus, fusion proteins comprising extended,
unstructured polypeptides are expected to have the property of
enhanced pharmacokinetics compared to polypeptides of shorter
lengths.
Example 34: Preparation of 1.times.DBCO, 3.times.LHRH-XTEN by
Conjugation
[0572] An aliquot of 1.times.Amino, 3.times.Thiol-XTEN432
(XTEN_AE432 (Am1,C12,C217,C422)) was prepared as a 196 .mu.M (7.7
mg/ml) solution in 20 mM HEPES, pH 7.0, 50 mM NaCl. FIG. 88A shows
a C18 RP-HPLC and ESI-MS analysis of the protein. 2,2'-Dipyridyl
disulfide (DPD, Sigma, cat. #D5767) was dissolved in
dimethylformamide (DMF) to the final concentration 100 mM. 4 ml of
1.times.Amino, 3.times.Thiol-XTEN432 solution was mixed with 0.2 ml
1 M HEPES, pH 8.0 to adjust the pH to .about.7.5 and with 78 .mu.l
DPD solution (10.times. molar excess over protein). The reaction
mixture was incubated for 2 hours at 25.degree. C. and products of
the reaction were analyzed by C18 RP-HPLC (FIG. 88B).
DBCO-sulfo-NHS (Click Chemistry Tools, Inc., cat. #A124) was
dissolved in anhydrous DMF to a final concentration of 10 mM. 0.865
ml of DBCO-sulfo-NHS solution was added to protein solution
(11.times. molar excess over protein). The reaction mixture was
incubated for 2 hours at 25.degree. C. and products of reaction
were analyzed by C18 RP-HPLC (FIG. 88C). A solution of 1M
ethanolamine pH 8.0 was added to a final concentration of 50 mM to
quench the unreacted DBCO-sulfo-NHS. The reaction mixture was
incubated for 2 hours at 25.degree. C. and then overnight at
4.degree. C. 500 mM Bond-Breaker.TM. TCEP Solution (Thermo
Scientific, cat. #77720) was added to a final concentration 20 mM.
The reaction mixture was incubated for 1 hour at 25.degree. C. and
the products of reaction were analyzed by C18 RP-HPLC (FIG. 88D).
The reaction mixture was diluted to 15 mL with 0.01% TFA and pH
adjusted to .about.3 using 10% TFA solution. The protein solution
was loaded on a preparative C4 RP-HPLC column Vydac C4250.times.22
mm (Grace Davison Discovery Sciences, cat. #214TP1022). The protein
was eluted with 1200 ml linear 5-50% gradient of acetonitrile in
0.01% TFA at 15 ml/min flow rate. Fractions containing
1.times.DBCO,3.times.Thiol-XTEN432 were adjusted to pH .about.7
with 1 M HEPES pH 8 and were concentrated by vacuum evaporation. A
Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 1102)
peptide modified with 3-maleimidopropionic acid at .epsilon.-amino
group of D-Lys (LHRH-Mal) was synthesized by CSBio Co. (Menlo Park,
Calif.). The peptide was dissolved to a final concentration 25
mg/ml in anhydrous DMF and was added to
1.times.DBCO,3.times.Thiol-XTEN432 solution in a 5.times. molar
excess over protein. The reaction mixture was incubated for 1 hr at
25.degree. C. and the products of the reaction were analyzed by C18
RP-HPLC (FIG. 88E). The reaction mixture was diluted to 15 mL with
0.01% TFA and the pH adjusted to .about.3 using a 10% TFA solution.
The protein solution was loaded on preparative C4 RP-HPLC column
Vydac C4 250.times.22 mm. The protein was eluted with 1200 ml
linear 5-50% gradient of acetonitrile in 0.01% TFA at 15 ml/min
flow rate. Fractions containing 1.times.DBCO,3.times.LHRH-XTEN432
were adjusted to .about.pH 7 with 1 M HEPES pH 8 and concentrated
to 2 ml by vacuum evaporation to yield the final product.
Example 35: Preparation of 1.times.Azide,3.times.MMAE-XTEN by
Conjugation
[0573] An aliquot of the fusion protein
1.times.Amino,3.times.Thiol-XTEN432 (XTEN_AE432(Am1,C12,C217,C422))
was prepared as 1% .mu.M (7.7 mg/ml) solution in 20 mM HEPES, pH
7.0, 50 mM NaCl. FIG. 89A shows a C18 RP-HPLC and ESI-MS analysis
of the protein. MA6-Val-Cit-PAB-MMAE (MMAE-Mal, custom synthesis by
Concortis Biosystems, Inc.) was dissolved in dimethylsulfoxide
(DMSO) to a final concentration 1 mg/ml. A 2.2 ml volume of
1.times.Amino,3.times.Thiol-XTEN432 solution was mixed with 2.5 ml
of MMAE-Mal (3.5.times. molar excess over protein). The reaction
mixture was incubated for 1 hr at 25.degree. C. and the products of
the reaction were analyzed by C18 RP-HPLC (FIG. 89B).
Azide-PEG4-NHS ester (Click Chemistry Tools, Inc., cat. #A103) was
dissolved in anhydrous DMF to a final concentration of 500 mM. The
reaction mixture (4.7 ml) was mixed with 0.235 ml 1 M HEPES, pH 8.0
and with 9.75 ul 500 mM azide-PEG4-NHS (10.times. molar excess over
protein). The reaction mixture was incubated for 2 hr at 25.degree.
C. and the products of reaction were analyzed by C18 RP-HPLC (FIG.
89C). The conjugation mixture was diluted to 15 ml with 0.01% TFA
and the pH was adjusted to .about.3 using 10% TFA. The protein
solution was loaded onto a preparative C4 RP-HPLC column Vydac
C4250.times.22 mm (Grace Davison Discovery Sciences, cat.
#214TP1022). The protein was eluted with 1200 ml linear 5-50%
gradient of acetonitrile in 0.01% TFA at 15 ml/min flow rate.
Fractions containing 1.times.Azide,3.times.MMAE-XTEN432 were
adjusted to .about.pH7 with 1 M HEPES pH 8 and concentrated by
vacuum evaporation to yield the final product.
Example 36: Preparation of 3.times.LHRH,3.times.MMAE-XTEN by
Conjugation
[0574] An aliquot of the fusion protein
1.times.DBCO,3.times.LHRH-XTEN432 was prepared as 143 .mu.M (6.26
mg/ml) solution in 20 mM HEPES, pH 7.0.
1.times.Azide,3.times.MMAE-XTEN432 was prepared as a 135 .mu.M
(5.90 mg/ml) solution in 20 mM HEPES, pH 7.0. The two protein
reactants were mixed in solution to yield a 1.1 molar excess of
1.times.DBCO,3.times.LHRH-XTEN432. The reaction mixture was
incubated overnight at 25.degree. C. Completion of the click
chemistry reaction was analyzed by SDS-PAGE (FIG. 90A) and RP-HPLC
(FIG. 90B). The conjugation mixture was diluted to 15 ml with 0.01%
TFA and pH adjusted to .about.3 using 10% TFA. The protein solution
was loaded onto a preparative C4 RP-HPLC column Vydac
C4250.times.10 mm (Grace Davison Discovery Sciences, cat.
#214TP510). The protein was eluted with 180 ml linear 5-50%
gradient of acetonitrile in 0.01% TFA at 2 ml/min flow rate.
Fractions containing 3.times.LHRH,3.times.MMAE-XTEN were adjusted
to .about.pH7 with 1 M HEPES pH 8 and concentrated by vacuum
evaporation to yield the final product.
Example 37: Preparation of 1.times.LHRH,3.times.MMAE-XTEN by
Conjugation
[0575] An aliquot of the fusion protein
1.times.Amino,3.times.Thiol-XTEN905 (XTEN_AE905(Am1,C8,C453,C898,
Seg 174)) was prepared as a 131 .mu.M (10.9 mg/ml) solution in 20
mM HEPES, pH 7.0, 50 mM NaCl. FIG. 91A shows a C18 RP-HPLC and
ESI-MS analysis of the protein. 2,2'-Dipyridyl disulfide (DPD,
Sigma, cat. #D5767) was dissolved in dimethylformamide (DMF) to the
final concentration 100 mM. The 1.times.Amino,3.times.Thiol-XTEN905
solution was adjusted pH to .about.7.5 using 1 M HEPES pH 8 and
mixed with DPD solution (10.times. molar excess over protein). The
reaction mixture was incubated for 2 hr at 25.degree. C. and the
products of the reaction were analyzed by C18 RP-HPLC (FIG. 91B).
DBCO-sulfo-NHS (Click Chemistry Tools, Inc., cat. #A124) was
dissolved in anhydrous DMF to a final concentration 10 mM.
DBCO-sulfo-NHS solution was added to the protein solution
(10.times. molar excess over protein). The reaction mixture was
incubated for 2 hr at 25.degree. C. and the products of the
reaction analyzed by C18 RP-HPLC (FIG. 91C). 1M ethanolamine pH 8.0
was added to a final concentration of 50 mM to quench unreacted
DBCO-sulfo-NHS. The reaction mixture was incubated for 2 hr at
25.degree. C. and then overnight at 4.degree. C. A 500 mM
Bond-Breaker.TM. TCEP Solution (Thermo Scientific, cat. #77720) was
added to a final concentration 20 mM. The reaction mixture was
incubated for 1 hour at 25.degree. C. and the products of the
reaction were analyzed by C18 RP-HPLC (FIG. 91D). The reaction
mixture was diluted to 15 mL with 0.01% TFA and the pH adjusted to
.about.3 using 10% TFA solution. The protein solution was loaded
onto preparative C4 RP-HPLC column Vydac C4 250.times.22 mm (Grace
Davison Discovery Sciences, cat. #214TP1022). The protein was
eluted with 1200 ml linear 5-50% gradient of acetonitrile in 0.01%
TFA at 15 ml/min flow rate. Fractions containing
1.times.DBCO,3.times.Thiol-XTEN905 were adjusted to .about.pH7 with
1 M HEPES pH 8 and concentrated by vacuum evaporation.
MA6-Val-Cit-PAB-MMAE (MMAE-Mal, custom synthesis by Concortis
Biosystems, Inc.) was dissolved in dimethylsulfoxide (DMSO) to a
final concentration 1 mg/ml. 1.times.DBCO,3.times.Thiol-XTEN905
solution was mixed with a 3.5.times. molar excess of MMAE-Mal. The
reaction mixture was incubated for 1 hr at 25.degree. C. and
products of the reaction were analyzed by C18 RP-HPLC. The
Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 1102)
peptide was modified with azido-hexanoic acid at the
.epsilon.-amino group of D-Lys (LHRH-Mal, custom synthesis by CS
Bio Co., Menlo Park, Calif.). The peptide was dissolved to a final
concentration 25 mg/ml in anhydrous DMF and added to
1.times.DBCO,3.times.MMAE-XTEN905 solution in 1.5-5.times. molar
excess over protein. The reaction mixture was incubated at
25.degree. C. and the products of the reaction were analyzed by C18
RP-HPLC. The final reaction mixture was diluted to 15 mL with 0.01%
TFA and pH adjusted to .about.3 using 10% TFA solution. The protein
solution was loaded on preparative C4 RP-HPLC column Vydac
C4250.times.22 mm. The protein was eluted with 1200 ml linear 5-50%
gradient of acetonitrile in 0.01% TFA at 15 ml/min flow rate.
Fractions containing 1.times.DBCO,3.times.LHRH-XTEN432 were
adjusted to .about.pH7 with 1 M HEPES pH 8 and concentrated by
vacuum evaporation to yield the final product.
Example 38: Preparation of
1.times.DBCO,3.times.Folate(.gamma.)-XTEN by Conjugation
[0576] An aliquot of the fusion protein
1.times.Amino,3.times.Thiol-XTEN432 (XTEN_AE432(Am1,C12,C217,C422))
was prepared as a 203 .mu.M (8.0 mg/ml) solution in 20 mM HEPES, pH
7.0, 50 mM NaCl. FIG. 107A shows a C18 RP-HPLC and ESI-MS analysis
of the protein. Folate-.gamma.-aminopentyl-maleimide
(FA(.gamma.)-Mal, custom synthesis by CPC Scientific) was dissolved
in dimethylformamide (DMF) to a final concentration 21 mg/ml (9.8
mM). A 1.1 ml volume of 1.times.Amino,3.times.Thiol-XTEN432
solution was mixed with 0.08 ml of FA(.gamma.)-Mal (3.5 molar
excess over protein). The reaction mixture was incubated for 2 hrs
at 25.degree. C. and the products of the reaction were analyzed by
C18 RP-HPLC (FIG. 107B). pH of the reaction mixture was adjusted
with 0.06 ml of 1 M HEPES pH 8.0 buffer. DBCO-sulfo-NHS (Click
Chemistry Tools, Inc., cat. #A124) was dissolved in anhydrous DMF
to a final concentration of 50 mM. 0. 53 ml of DBCO-sulfo-NHS
solution was added to protein solution (11.times. molar excess over
protein). The reaction mixture was incubated for 2 hours at
25.degree. C. and products of reaction were analyzed by C18 RP-HPLC
(FIG. 107C). A solution of 1M ethanolamine pH 8.0 was added to a
final concentration of 50 mM to quench the unreacted
DBCO-sulfo-NHS. The reaction mixture was incubated for 2 hours at
25.degree. C. and then overnight at 4.degree. C. The reaction
mixture was diluted to 15 mL with 0.01% TFA and pH adjusted to
.about.3 using 10% TFA solution. The protein solution was loaded on
a preparative C4 RP-HPLC column Vydac C4250.times.10 mm (Grace
Davison Discovery Sciences, cat. #214TP510). The protein was eluted
with 180 ml linear 5-50% gradient of acetonitrile in 0.01% TFA at 2
ml/min flow rate. Fractions containing
1.times.DBCO,3.times.FA(.gamma.)-XTEN432 were adjusted to
pH.about.7 with 1 M HEPES pH 8 and were concentrated by vacuum
evaporation to yield the final product.
[0577] The 1.times.DBCO,3.times.Folate(.alpha.)-XTEN conjugate was
prepared essentially as described above using
folate-alpha-Maleimide (FA(.alpha.)-Mal, custom synthesis by CPC
Scientific).
Example 39: Preparation of 3.times.FA(.gamma.),3.times.MMAE-XTEN by
Conjugation
[0578] An aliquot of the fusion protein
1.times.DBCO,3.times.FA(.gamma.)-XTEN432 was prepared as 191 .mu.M
(8.8 mg/ml) solution in 20 mM HEPES, pH 7.0.
1.times.Azide,3.times.MMAE-XTEN432 (prepared as described in
Example 32) was prepared as a 242 .mu.M (11.1 mg/ml) solution in 20
mM HEPES, pH 7.0. The two protein reactants were mixed in solution
to yield a 1.1 molar excess of
1.times.DBCO,3.times.FA(.gamma.)-XTEN432. The reaction mixture was
incubated overnight at 25.degree. C. Completion of the click
chemistry reaction was analyzed by RP-HPLC (FIG. 108). The
conjugation mixture was diluted to 15 ml with 0.01% TFA and pH
adjusted to .about.3 using 10% TFA. The protein solution was loaded
onto a preparative C4 RP-HPLC column Vydac C4250.times.10 mm (Grace
Davison Discovery Sciences, cat. #214TP510). The protein was eluted
with 180 ml linear 5-50% gradient of acetonitrile in 0.01% TFA at 2
ml/min flow rate. Fractions containing
3.times.FA(.gamma.),3.times.MMAE-XTEN were adjusted to .about.pH7
with 1 M HEPES pH 8 and concentrated by vacuum evaporation to yield
the final product. The final product was analyzed by size exclusion
chromatography (SEC-HPLC) (FIG. 109A), RP-HPLC (FIG. 109B) and
ESI-MS (FIG. 109C).
[0579] The 3.times.FA(.alpha.),3.times.MMAE-XTEN conjugate was
prepared essentially as described above using click reaction
between 1.times.DBCO,3.times.FA(.alpha.)-XTEN432 and
1.times.Azide,3.times.MMAE-XTEN432.
Example 40: Comparison of Viscosity in Solutions of Branched Versus
Linear XTEN
[0580] The viscosity of linear and branched XTENs (the latter as
trimeric or tetrameric configurations) can be measured using
various types of viscometers and rheometers. For example one can
measure the time required to draw 1 mL of liquid into a syringe
through a 30G needle as described by Miller, M. A., et al. (2010)
Langmuir, 26:1067. In order to compare monomeric linear versus
trimeric or tetrameric configurations of XTENs to be for viscosity,
constructs having equivalent molecular weights for the XTEN amino
acid component are prepared and then solutions are made at fixed,
equivalent concentrations of protein at 20, 50, 100 mg/ml each. The
solutions are then are evaluated using the method of Miller. Using
the method, data are obtained with known standards to prepare a
standard curve, and then the XTEN solutions are measured. It is
expected that results will show that the viscosity of equimolar
solutions of XTENs with similar molecular weight will be
significantly decrease with increasing branching; a conjugate of 3
arms of XTEN288 will have significantly lower viscosity compared to
an equal concentration of linear XTEN864, even though they have
equivalent numbers of amino acids. Similarly, it is expected that a
configuration with 4 arms of XTEN216 will have even lower viscosity
than a conjugate with 3 arms of XTEN288.
Example 41: Preparation of Her2-XTEN-PTX by Conjugation
[0581] An aliquot of the fusion protein
1.times.Amino,3.times.Thiol-XTEN432 (XTEN_AE432(Am1,C12,C217,C422))
is prepared as 196 .mu.M (7.7 mg/ml) solution in 20 mM HEPES, pH
7.0, 50 mM NaCl. Paclitaxel-Mal (PTX-Mal) is custom synthesized by
modification of paclitaxel with succinic anhydride followed by
conjugation to aminoethylmaleimide. PTX-Mal is dissolved in
dimethylformamide (DMF) and added to the protein in a 3.5-5.times.
molar excess. The reaction mixture is incubated for 1-2 hr at
25.degree. C. and the products of the reaction are analyzed by C18
RP-HPLC. Sulfo-SMCC (Thermo Scientific, cat. #22122) is dissolved
in anhydrous DMF to a final concentration 50 mM and added to the
protein solution in a 10.times. molar excess over protein. The
reaction mixture is incubated for 2 hr at 25.degree. C. and the
products of the reaction analyzed by C18 RP-HPLC (see FIG. 92). The
reaction mixture is diluted to 15 mL with 0.01% TFA and pH adjusted
to .about.3 using 10% TFA solution. The protein solution is loaded
on a preparative C4 RP-HPLC column Vydac C4250.times.22 mm (Grace
Davison Discovery Sciences, cat. #214TP1022). The protein is eluted
with a 1200 ml linear 5-50% gradient of acetonitrile in 0.01% TFA
at 15 ml/min flow rate. Fractions containing
1.times.Mal,3.times.PTX-XTEN432 are adjusted to .about.pH 7 with 1
M HEPES pH 8 and concentrated by vacuum evaporation. Herceptin
(Trastuzumab, Roche) antibody is reconstituted in water according
to the instructions and is buffer exchanged into PBS pH 7.2
containing 5 mM EDTA. DTT is added to the protein solution to a
1-10 mM final concentration and incubated for 5-30 min at
37.degree. C. Excess DTT is removed by gel filtration or cut-off
membrane filtration. 1.times.Mal,3.times.PTX-XTEN432 is added to
partially reduced antibody in a 3-4.times. molar excess. The
reaction mixture is incubated for 1 hr at 25.degree. C. and the
final products of the reaction are analyzed by SDS-PAGE and size
exclusion chromatography under non-reducing and reducing
conditions.
Example 42: Preparation of Iodoacetyl-XTEN by Conjugation
[0582] An aliquot of the fusion protein 1.times.Amino-XTEN869
(XTEN_AE869(Am1) was prepared as a 164 .mu.M (13.1 mg/ml) solution
in 20 mM HEPES, pH 7.0, 50 mM NaCl. A 1/20 volume of 1M HEPES pH 8
was added to the protein solution to adjust the pH of the protein
solution to .about.7.5. N-succinimidyl iodoacetate (SIA, Thermo
Scientific, cat. #22349) was dissolved in anhydrous
dimethylformamide (DMF) to a final concentration 100 mM and was
added in a 10.times. molar excess over the protein. The reaction
mixture was incubated for 1 hr at 25.degree. C. and the products of
the reaction are analyzed by C18 RP-HPLC (FIG. 93A). The excess SIA
was removed by buffer exchange using Vivaspin 500 ultracentrifugal
device (5,000 MWCO, VivaScience, cat. #VS0112). Modification of the
N-terminal amino group by iodoacetyl group did not change the
retention time of the modified XTEN (FIG. 93A), therefore the
covalent modification was confirmed by ESI-MS (FIG. 93B). Also, the
IA-XTEN conjugate efficiently reacted with the cysteine-containing
peptide HCKFWW (Bachem, cat. #H-3524) (FIG. 93C).
Example 43: In Vitro Cell-Based Screening of LHRH-XTEN-Drug
Conjugates for Activity and Specificity
[0583] LHRH-XTEN-drug conjugates are assessed for in vitro activity
and selectivity. Each LHRH-XTEN-drug conjugate, its corresponding
non-targeting XTEN-drug molecule and its respective free drug
control are tested in a CellTiter-Glo anti-proliferation assay
against a panel of LHRH receptor positive and negative cell lines
listed in Table 41. Appropriate assay conditions, including optimal
cell density and incubation time are determined using the
respective free drug as control. LHRH-XTEN-drug conjugates are
tested as follows: cells in log-phase are collected, counted and
plated at pre-determined cell density in a 96-well microtiter assay
plate. Adherent cells are allowed to attach to the plate by an
overnight incubation at 37.degree. C. with an atmosphere of 5%
CO.sub.2. The LHRH-XTEN-drug conjugates and corresponding controls
are introduced using appropriate dose range dilutions, in
duplicate, and the plates are incubated for an additional 2-5 days.
After the appropriate incubation period, CellTiter-Glo reagent is
added to each well, mixed for 2 minutes on an orbital shaker. The
plate is then centrifuged at 90.times.g and incubated at room
temperature for an additional 10 minutes to stabilize the
luminescent signal. Luminescence signals are then read on a
luminometer and the IC.sub.50 (half maximal inhibitory
concentration) values are calculated with GraphPad Prism or
equivalent software. Quantitative comparisons of the IC.sub.50
values will enable ranking of the constructs' activity for
inhibition of cell growth and selectivity against LHRH receptor
positive versus negative cell lines. It is expected that the
results would support the finding that the LHRH-XTEN-drug
conjugates will show highly selective potent killing on LHRH
receptor positive cells but not on LHRH receptor negative cells.
This will be in contrast to the free drug moiety whereby no
discrimination in cytotoxicity is expected between LHRH receptor
positive and negative cell lines. The XTEN-drug control is expected
to yield poor cytotoxic activity. LHRH-XTEN-drug conjugates with
favorable activity and cell line selectivity relative to controls
will be further verified for LHRH receptor association by the
addition of free competitive LHRH peptide in the assay, resulting
in impaired LHRH-XTEN-drug cytotoxicity, further verifying the
selective activity of the constructs.
TABLE-US-00047 TABLE 41 LHRH receptor positive and negative cell
lines Cell line Tissue LHRHR status MCF-7 Breast Positive
MDA-MB-231 Breast (HER2-/ER-/PR-) Positive HCC1806 Breast
(HER2-/ER-/PR-) Positive HCC1937 Breast (HER2-/ER-/PR-) Positive
OV-1063 Ovarian Positive EFO-21 Ovarian Positive EFO-27 Ovarian
Positive NIH: OVCAR-3 Ovarian Positive BG-1 Ovarian Positive HEC-1A
Endometrial Positive HEC-1B Endometrial Positive Ishikawa
Endometrial Positive KLE Endometrial Positive AN-3-CA Endometrial
Positive MiaPaCa Pancreatic Positive Panc-1 Pancreatic Positive rat
Dunning R-3327-H Prostate (androgen-dep) Positive PC-82 Prostate
(androgen-dep) Positive MDA-PCa-2b Prostate (androgen-indep)
Positive C4-2 Prostate (androgen-dep) Positive (derivative of
LNCaP) A549 Lung Positive UCI-107 Ovarian Negative SK-OV-3 Ovarian
Negative SW626 Ovarian Negative MFE-296 Endometrial Negative ER:
estrogen receptor; PR: progesterone receptor
Example 44: In Vitro Cell-Based Screening of Folate-XTEN-Drug
Conjugates for Activity and Specificity
[0584] Folate-XTEN-drug conjugates are first subjected to an in
vitro activity and selectivity screen. Each folate-XTEN-drug
conjugate, its corresponding non-targeting XTEN-drug molecule and
respective free drug control are tested in a CellTiter-Glo
anti-proliferation assay against a panel of folate receptor
positive and negative cell lines listed in Table 42. As culture
media contain high folic acid content, cells will be grown and the
assay performed in folic acid free-media containing 5-10%
heat-inactivated fetal calf serum (FCS) at 37.degree. C., in an
atmosphere of 5% CO.sub.2 (heat-inactivated FCS contains endogenous
level of folic acid sufficient for folate receptor expressing cells
to survive and proliferate). Appropriate assay conditions are
established, including optimal cell density and incubation times,
using folate-free media containing 5-10% FCS using the respective
free drug as control. Folate-XTEN-drug conjugates are then tested
as follows: cells in log-phase are collected, counted and plated at
pre-determined cell density in 96-well microtiter assay plates.
Adherent cells are allowed to attach to the plate by an overnight
incubation at 37.degree. C., 5% CO.sub.2. Folate-XTEN-drug
conjugates and corresponding controls are introduced using
appropriate dose range dilutions, in duplicate, and the plates are
incubated for an additional 2-5 days. After the appropriate
incubation period, CellTiter-Glo reagent is added to each well and
is mixed for 2 minutes on an orbital shaker. The plate is then
centrifuged at 90.times.g and incubated at room temperature for an
additional 10 minutes to stabilize the luminescent signal.
Luminescence signals are then read on a luminometer and the
IC.sub.50 (half maximal inhibitory concentration) values are
calculated with GraphPad Prism or equivalent software. Quantitative
comparisons of the IC.sub.50 values will enable ranking of the
constructs' activity for inhibition of cell growth and selectivity
against folate receptor positive versus negative cell lines. It is
expected that the results would support the finding that the
folate-XTEN-drug conjugates will show highly selective potent
killing on folate receptor positive cells but not on folate
receptor negative cells. This will be in contrast to the free drug
moiety where no discrimination in cytotoxicity is expected between
folate receptor positive and negative cell lines. The XTEN-drug
control is expected to yield poor cytotoxic activity.
Folate-XTEN-drug conjugates with favorable activity and cell line
selectivity relative to controls will be further verified for
folate receptor association by the addition of free competitive
folic acid in the assay, demonstrating impaired folate-XTEN-drug
cytotoxicity, further verifying the selective activity of the
constructs.
TABLE-US-00048 TABLE 42 Folate receptor positive and negative cell
lines Cell line Tissue Folate receptor status KB Nasopharyngeal
Positive IGROV Ovarian Positive SK-OV-3 Ovarian Positive HeLa
Cervical Positive LoVo Colorectal Positive SW620 Colorectal
Positive A549 Lung Negative A375 Multiple melanoma Negative LS-174T
Colorectal Negative SK-BR-3 Breast Negative
Example 45: In Vitro Serum Stability of LHRH-XTEN-Drug
Conjugates
[0585] As a measure of stability, LHRH-XTEN-drug conjugates are
incubated independently in normal human, cynomolgus monkey and
mouse plasma at 37.degree. C. for up to 2 weeks with aliquots
removed at periodic intervals and stored at -80.degree. C. till
analysis. The stability of LHRH-XTEN-drug conjugate can be assessed
either by the amount of free drug released or the integrity of the
LHRH-XTEN-drug conjugate over time. Free drug is quantitated with
RP-HPLC and/or LC-MS/MS whereas the amount of intact LHRH-XTEN-drug
conjugate is determined using an XTEN/drug and/or an LHRH/drug
ELISA.
[0586] For RP-HPLC analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated under vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelengths specific for the
particular drug, compared to known drug standards. For example,
doxorubicin is detected at 480 nm. For LC-MS/MS analysis, plasma
samples are treated with organic solvents such as acetonitrile or
acetone to precipitate proteins. Soluble fractions are evaporated
under vacuum, redissolved in loading solutions and analyzed by
RP-HPLC. Analytes are detected and quantitated by triple quadrupole
tandem mass spectrometry, compared to known drug standards.
Parental ion-daughter ion pairs will be determined experimentally
for each drug. For quantitative ELISA, optimal concentrations of
antibodies for LHRH-XTEN-drug conjugate in the ELISAs are
determined using criss-cross serial dilution analysis. An
appropriate capture antibody recognizing one component of the
conjugate is coated onto a 96-well microtiter plate by an overnight
incubation at 4.degree. C. The wells are blocked, washed and serum
stability samples added to the wells, each at varying dilutions to
allow optimal capture of the LHRH-XTEN-drug conjugate by the coated
antibody. After washing, detection antibody recognizing another
component of the conjugate is added and allowed to bind to the
conjugate captured on the plate. Wells are then washed again and
either streptavidin-horseradish peroxidase (complementary to
biotinylated version of detection antibody) or an appropriate
secondary antibody-horseradish peroxidase (complementary to
non-biotinylated version of detection antibody) is then added.
After appropriate incubation and a final wash step,
tetramethylbenzidine (TMB) substrate is added and the plate read at
450 nM. Concentrations of intact conjugate are then calculated for
each time point by comparing the colorimetric response to a
calibration curve prepared with LHRH-XTEN-drug in the relevant
plasma type. The t1/2 of the decay of the conjugate in human, cyno
and mouse serum is then defined using linear regression analysis of
the log concentrations vs. time.
Example 46: In Vitro Serum Stability of Folate-XTEN-Drug
Conjugates
[0587] As a measure of stability, folate-XTEN-drug conjugates are
incubated independently in normal human, cynomolgus monkey and
mouse plasma at 37.degree. C. for up to 2 weeks with aliquots
removed at periodic intervals and stored at -80.degree. C. until
analysis. The stability of folate-XTEN-drug conjugate is assessed
either by the amount of free drug or the integrity of the
folate-XTEN-drug conjugate over time. Free drug is quantitated with
HPLC and/or LC-MS/MS whereas the amount of intact folate-XTEN-drug
conjugate is determined using an XTEN/drug and/or folate/drug
ELISA.
[0588] For RP-HPLC analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated under vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelength specific for a
particular drug, compared to known drug standards. For example,
doxorubicin is detected at 480 nm. For LC-MS/MS analysis, plasma
samples will be treated with organic solvents such as acetonitrile
or acetone to precipitate proteins. Soluble fractions will be
evaporated in vacuum, redissolved in loading solutions and analyzed
by RP-HPLC. Analytes will be in-line detected and quantitated by
triple quadrupole tandem mass spectrometry. Parental ion-daughter
ion pairs will be determined experimentally for each drug.
Calibration standards will be prepared by adding known amounts of
free drug to corresponding plasma type and will be treated in
parallel with experimental samples. For quantitative ELISA, optimal
concentrations of antibodies for folate-XTEN-drug conjugate in the
ELISAs is determined using criss-cross serial dilution analysis. An
appropriate capture antibody recognizing one component of the
conjugate is coated onto a 96-well microtiter plate by an overnight
incubation at 4.degree. C. The wells are blocked, washed and serum
stability samples added to the wells, each at varying dilutions to
allow optimal capture of the folate-XTEN-drug conjugate by the
coated antibody. After washing, detection antibody recognizing
another component of the conjugate is added and allowed to bind to
the conjugate captured on the plate. Wells are then washed again
and either streptavidin-horseradish peroxidase (complementary to
biotinylated version of detection antibody) or an appropriate
secondary antibody-horseradish peroxidase (complementary to
non-biotinylated version of detection antibody) is then added.
After appropriate incubation and a final wash step,
tetramethylbenzidine (TMB) substrate is added and the plate is read
at 450 nM. Concentrations of intact conjugate are then calculated
for each time point by comparing the colorimetric response to a
calibration curve prepared with folate-XTEN-drug in the relevant
plasma type. The t1/2 of the decay of the conjugate in human, cyno
and mouse serum is then defined using linear regression analysis of
the log concentrations vs. time.
Example 47: In Vivo and Ex Vivo Imaging of LHRH-XTEN-Cy5.5
Conjugate
[0589] A Cy5.5 fluorescent tagged LHRH-XTEN molecule is used as a
surrogate to investigate the targeting and biodistribution
efficiency of LHRH-XTEN-drug conjugates. Experiments will be
carried out in nude mice bearing subcutaneous grown xenografts of
LHRH receptor positive tumor cells using in vivo, followed by ex
vivo, fluorescence imaging with IVIS 50 optical imaging system
(Caliper Life Sciences, Hopkinton, Mass.). In brief, female nu/nu
mice bearing LHRH receptor positive tumor cells are given a single
intravenous injection of high or low dose LHRH-XTEN-Cy5.5 and
corresponding doses of non-targeting Cy5.5 tagged XTEN control.
Whole body scans are acquired pre-injection and then at
approximately 8, 24, 48 and 72 hours post-injection on live
anesthetized animals using the IVIS 50 optical imaging system.
After measuring the distribution of fluorescence in the entire
animal at the last time point of 72 h, tumor and healthy organs
including liver, lung, heart, spleen and kidneys are excised and
their fluorescence registered and processed by the imaging system.
Cy5.5 excitation (615-665 nm) and emission (695-770 nm) filters are
selected to match the fluorescence agents' wavelengths. Small and
medium binning of the CCD chip is used and the exposure time
optimized to obtain at least several thousand counts from the
signals observable in each mouse in the image and to avoid
saturation of the CCD chip. To normalize images for quantification,
a background fluorescence image is acquired using background
excitation and emission filters for the Cy5.5 spectral region. The
intensity of fluorescence is expressed as different colors with
blue color reflecting the lowest intensity and red being indicative
of the highest intensity, and the resulting images are used to
assess the uptake of the conjugates and controls.
Example 48: In Vivo and Ex Vivo Imaging of Folate-XTEN-Cy5.5
Conjugates
[0590] A Cy5.5 fluorescent tagged folate-XTEN molecule is used as a
surrogate to investigate the targeting and biodistribution
efficiency of folate-XTEN-drug conjugates. Experiments will be
carried out in nude mice bearing subcutaneous grown xenografts of
folate receptor positive tumor cells using in vivo, followed by ex
vivo, fluorescence imaging with IVIS 50 optical imaging system
(Caliper Life Sciences, Hopkinton, Mass.). As culture media contain
high folate content, folate receptor positive tumor cells to be
transplanted onto these mice will be grown in folate-free cell
culture media containing 5-10% heat-inactivated FCS with no
antibiotics. Similarly, normal rodent chow contains a high
concentration of folic acid; nude mice used in this study will be
maintained on folate-free diet 2 weeks prior to tumor implantation
and for the duration of the imaging analysis to reduce serum folate
concentration.
[0591] In brief, female nu/nu mice bearing folate receptor positive
tumor cells are given a single intravenous injection of high or low
dose folate-XTEN-Cy5.5 and corresponding doses of non-targeting
Cy5.5 tagged XTEN control. Whole body scans are acquired
pre-injection and then at approximately 8, 24, 48 and 72 hours
post-injection on live anesthetized animals using the IVIS 50
optical imaging system. After measuring the distribution of
fluorescence in the entire animal at the last time point of 72 h,
tumor and healthy organs including liver, lung, heart, spleen and
kidneys are excised and their fluorescence registered and processed
by the imaging system. Cy5.5 excitation (615-665 nm) and emission
(695-770 nm) filters are selected to match the fluorescence agents'
wavelengths. Small and medium binning of the CCD chip is used and
the exposure time optimized to obtain at least several thousand
counts from the signals that were observable in each mouse in the
image and to avoid saturation of the CCD chip. To normalize images
for quantification, a background fluorescence image is acquired
using background excitation and emission filters for the Cy5.5
spectral region. The intensity of fluorescence is expressed as
different colors with blue color reflecting the lowest intensity
and red being indicative of the highest intensity, and the
resulting images are used to assess the uptake of the conjugates
and controls.
Example 49: Pharmacokinetic Analysis of LHRH-XTEN-Drug
Conjugates
[0592] The in vivo pharmacokinetics of LHRH-XTEN-drug constructs
are assessed using standard methods for protein compositions using
mice, rats, cynomolgus monkeys, and dogs. Compositions of
LHRH-XTEN-drug constructs are provided in an aqueous buffer
compatible with in vivo administration (for example:
phosphate-buffered saline, Tris-buffered saline or Hepes-buffered
saline). The compositions are administered at appropriate doses and
via multiple routes: most preferably via intravenous or
subcutaneous routes. Blood samples are collected at appropriate
time points ranging from 0.08 to 504 hours, and processed into
plasma. Plasma samples are analyzed for concentration of
LHRH-XTEN-drug conjugates by one of a variety of methods, including
ELISA, HPLC and/or LC-MS/MS. ELISA analysis are performed using a
sandwich ELISA format that can recognize 2 components of the
LHRH-XTEN-drug conjugate, for instance, XTEN/LHRH, XTEN/drug
moiety, LHRH/drug moiety and/or XTEN/XTEN combinations. Typically
antibody recognizing one component of the LHRH-XTEN-drug conjugate
is coated onto wells of a %-well microtiter plate. The wells are
blocked, washed and plasma samples are then added to the wells at
varying dilutions to allow capture of the conjugate by the coated
antibody. Wells are then washed extensively, and bound protein
detected using either a biotinylated antibody or an appropriate
secondary antibody against the second LHRH-XTEN-drug conjugate
component. Wells are then washed again and streptavidin-horseradish
peroxidase (complementary to the biotinylated detection antibody)
or a secondary antibody-horseradish peroxidase (complementary to a
non biotinylated detection antibody) is then added. After
appropriate incubation and a final wash step, tetramethylbenzidine
(TMB) substrate is added and the plate is read at 450 nM.
Concentrations of conjugate are then calculated for each time point
by comparing the colorimetric response to a LHRH-XTEN-drug
calibration curve. Pharmacokinetic parameters are calculated using
the WinNonLin software package.
[0593] For RP-HPLC analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated in vacuum, redissolved
in loading solutions and analyzed by RP-HPLC. Analytes are detected
by UV absorption at wavelength specific for a particular drug. For
example, doxorubicin is detected at 480 nm. Calibration standards
are prepared by adding known amounts of free drug to corresponding
plasma type and are assayed in parallel with experimental
samples.
[0594] For LC-MS/MS analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated under vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
are assayed in parallel with experimental samples.
[0595] It is expected that the results would support the finding
that addition of an XTEN to LHRH and drug moiety will greatly
increase the terminal half-life and enhance the pharmacokinetic
properties of targeting and drug moiety not linked to XTEN.
Example 50: Pharmacokinetic Analysis of Folate-XTEN-Drug
Conjugates
[0596] The in vivo pharmacokinetics of folate-XTEN-drug constructs
are assessed using standard methods for protein compositions using
mice, rats, cynomolgus monkeys, and dogs. As normal feed contains a
high concentration of folic acid (approx. 6 mg/kg mouse chow),
animals to be used in pharmacokinetic studies of folate conjugates
will be maintained on folate-free diet for 2 weeks prior to study
initiation and for the duration of the study. The compositions are
administered at appropriate doses and via multiple routes: most
preferably via intravenous or subcutaneous routes. Blood samples
are collected at appropriate time points ranging from 0.08 to 504
hours, and processed into plasma. Plasma samples are analyzed for
concentration of folate-XTEN-drug conjugates by a variety of
methods including ELISA, HPLC and/or LC-MS/MS.
[0597] ELISA analysis are performed using a sandwich ELISA format
that can recognize 2 components of the folate-XTEN-drug conjugate,
for instance, XTEN/folate, XTEN/drug moiety, folate/drug moiety
and/or XTEN/XTEN combinations. Typically antibody recognizing one
component of the folate-XTEN-drug conjugate is coated onto wells of
a 96-well microtiter plate. The wells are blocked, washed and
plasma samples are then added to the wells at varying dilutions to
allow capture of the conjugate by the coated antibody. Wells are
then washed extensively, and bound protein detected using either a
biotinylated antibody or an appropriate secondary antibody against
the second folate-XTEN-drug conjugate component. Wells are then
washed again and streptavidin-horseradish peroxidase (complementary
to the biotinylated detection antibody) or a secondary
antibody-horseradish peroxidase (complementary to a non
biotinylated detection antibody) is then added. After appropriate
incubation and a final wash step, tetramethylbenzidine (TMB)
substrate is added and the plate is read at 450 nM. Concentrations
of conjugate are then calculated for each time point by comparing
the colorimetric response to a folate-XTEN-drug calibration curve.
Pharmacokinetic parameters are calculated using the WinNonLin
software package.
[0598] For RP-HPLC analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated in vacuum, redissolved
in loading solutions and analyzed by RP-HPLC. Analytes are detected
by UV absorption at wavelength specific for a particular drug. For
example, doxorubicin is detected at 480 nm. Calibration standards
are prepared by adding known amounts of free drug to corresponding
plasma type and are assayed in parallel with experimental
samples.
[0599] For LC-MS/MS analysis, plasma samples are treated with
organic solvents such as acetonitrile or acetone to precipitate
proteins. Soluble fractions are evaporated under vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
are assayed in parallel with experimental samples.
[0600] It is expected that the results would support the finding
that addition of an XTEN to folate and drug moiety will greatly
increase the terminal half-life and enhance the pharmacokinetic
properties of targeting and drug moiety not linked to XTEN.
Example 51: In Vivo Efficacy and Toxicity Analysis of
LHRH-XTEN-Drug Conjugates
[0601] LHRH-XTEN-drug conjugate is intended for targeted delivery
of highly potent toxin to LHRH receptor positive tumor cells. As
such, the in vivo pharmacologic activity of LHRH-XTEN-drug
constructs can be assessed using human tumor cells expressing LHRH
receptor transplanted into nude mice.
[0602] Prior to beginning the efficacy study, an initial assessment
in nude mice is carried out to establish the maximum tolerated dose
(MTD) of the LHRH-XTEN-drug candidates. The MTD, the highest dose
that is tolerated by the animal for the study duration, will then
be used to calculate the dose range for the efficacy and toxicity
study in the standard xenograft model. Briefly, the MTD experiment
is carried out with 5 mice per group evaluating the intravenous
administration of LHRH-XTEN-drug conjugates at various dose level,
interval and duration. The starting MTD dose and number of dose
groups required is based on scientific literature, knowledge of the
targeting LHRH moiety, the nature of the drug moiety conjugated,
toxicological properties of closely related compounds, and data
from the initial pharmacokinetic studies (see, above). Standard MTD
parameters such as reduction in body weight, food and water
consumption and signs of piloerection, hunched, behavior patterns,
respiratory pattern, tremors, convulsions, prostration and
self-mutilation are monitored on a daily basis. The highest dose of
LHRH-XTEN-drug that does not cause unacceptable toxicity will be
assigned as the MTD. The tumor xenograft study will include 3 to 4
dosing levels of LHRH-XTEN-drug conjugate and will depend on the
results from the MTD study; with other parameters depending on the
tumor cell line chosen. Table 42 provides examples of tumor lines
that can be used in the xenograft study. Thus, an appropriate
number of LHRH receptor positive cells from the relevant human
tumor line are injected subcutaneously and allowed to form tumors,
the size of which will be measured with calipers and the volume
calculated as 0.5.times.L.times.W.sup.2, where L=measurement of
longest axis in millimeters and W=measurement of axis perpendicular
to L in millimeters. Following randomization of mice containing
tumor volume in the desired size range into groups of 8-10 animals,
vehicle control, free drug control and LHRH-XTEN-drug conjugate is
administered intravenously at the chosen doses and interval.
Cessation or regression of tumor growth is determined by measuring
the tumor size and volume at selected time points with calipers.
Body weights and food consumption are measured every 1 to 2 days to
assess gross toxicity. Survival of animals is monitored daily. At
the end of the study, all animals are sacrificed and clinical
pathology and histopathology on major organs is performed.
[0603] It is anticipated that the results would support the finding
that the LHRH-XTEN-drug conjugate will produce a positive
therapeutic index as exhibited by potent efficacy and low systemic
toxicity. In contrast, the non-LHRH targeted free drug dosed at
equimolar doses is expected to be less potent but highly more
toxic. It is expected that the vehicle control will display
uncontrolled tumor growth and severe toxicity.
Example 52: In Vivo Efficacy and Toxicity Analysis of
Folate-XTEN-Drug Conjugates
[0604] Folate-XTEN-drug conjugates are intended for targeted
delivery of the toxin component to folate receptor positive tumor
cells. The in vivo pharmacologic activity of folate-XTEN-drug
constructs is assessed using human tumor cell expressing folate
receptor xenograft onto nude mice. Prior to beginning the efficacy
study, an initial assessment in nude mice is carried out to
establish the maximum tolerated dose (MTD) of the folate-XTEN-drug
candidates. The MTD, the highest dose that will be tolerated by the
animal for the study duration, is then used to calculate the dose
range for the efficacy and toxicity study in the standard xenograft
model. As normal rodent chow contains a high concentration of folic
acid (6 mg/kg chow), mice to be used in these studies are
maintained on a folate-free diet for 2 weeks prior to study
initiation and for the duration of the study. The MTD experiment is
carried out with 5 mice per group evaluating the intravenous
administration of folate-XTEN-drug conjugates at various dose
level, interval and duration. The starting MTD dose and number of
dose groups required is based on scientific literature, knowledge
of the targeting folate moiety, the nature of the drug moiety
conjugated, toxicological properties of closely related compounds,
and data from the initial pharmacokinetic studies (see PK Example
above). Standard MTD parameters such as reduction in body weight,
food and water consumption and signs of piloerection, hunched,
behavior patterns, respiratory pattern, tremors, convulsions,
prostration and self-mutilation are carefully monitored on a daily
basis. The highest dose of folate-XTEN-drug that does not cause
unacceptable toxicity is assigned as the MTD.
[0605] The tumor xenograft study includes 3 to 4 dosing levels of
folate-XTEN-drug, depending on the results from the MTD study, with
other parameters depending on the tumor cell line chosen. Table 42
describes examples of tumor lines that can be used in the xenograft
study. To reduce folate content, folate receptor positive tumor
cells to be transplanted onto nude mice are grown in folate-free
cell culture media containing 5-10% heat-inactivated fetal calf
serum with no antibiotics. Similarly, to reduce serum folate
concentration, mice used in the xenograft studies are maintained on
folate-free diet 2 weeks prior to tumor implantation and for the
duration of the study. An appropriate number of folate receptor
positive cells from the relevant line are injected subcutaneously
and allowed to form tumors, the size of which are measured with
calipers and the volume calculated as 0.5.times.L.times.W.sup.2,
where L=measurement of longest axis in millimeters and
W=measurement of axis perpendicular to L in millimeters. Following
randomization of mice containing tumor volume in the desired size
range into groups of 8-10 animals, vehicle control, free drug
control and folate-XTEN-drug is administered intravenously at the
chosen doses and intervals. Cessation or regression of tumor growth
is determined through measuring the tumor size and volume at
selected time points with calipers. Body weight and food
consumption is measured every 1 to 2 days to assess gross toxicity.
Survival of animals is monitored daily. At the end of study, all
animals are sacrificed and major organs will be removed for
clinical pathology and histopathology examination.
[0606] It is anticipated that the targeted chemotherapeutic
folate-XTEN-drug conjugate will be more effective and less toxic
than free cytotoxic drug alone on folate receptor positive tumors
in the mouse model.
Example 53: Human Clinical Trial Designs for Evaluating
LHRH-XTEN-Drug Conjugates
[0607] Targeted chemotherapy is a modern approach aimed at
increasing the efficacy of systemic chemotherapy and reducing side
effects. LHRH is a peptide that functions in reproductive organs.
Because its receptors are particularly concentrated on certain
tumors but are not expressed in most normal tissue, the LHRH
receptor is an ideal target for selective destruction of malignant
tumors. Indeed, .about.52% of breast, .about.80% of ovarian and
endometrial, and .about.85% of prostate cancer specimens is
targetable via the LHRH receptor. Of note, LHRH-dependent therapies
would be especially useful for triple negative breast tumors, which
do not overexpress estrogen or progesterone receptors or HER2 and
are therefore unsuitable for treatment with many available targeted
drugs. Patients with advanced endometrial, ovarian, or prostate
cancer often have particularly poor outcomes, as these malignancies
can be prone to recurrence and/or resistant to current treatments.
Fusion of a XTEN carrying .gtoreq.1 copy of LHRH to a XTEN bearing
.gtoreq.3 drug molecules to create a targeted peptide-drug
conjugate is expected to have vastly improved therapeutic index and
half-life that will enable dosing at levels way below MTD, reduce
dosing frequency and cost (reduced drug required per dose).
[0608] Clinical evaluation of a LHRH-XTEN-drug composition is
conducted in patients suffering from advanced breast, endometrial,
ovarian, and prostate or bladder cancers, with trials designed to
confirm the efficacy and safety of the LHRH-XTEN-drug conjugate in
humans. Such studies in patients would comprise three phases.
First, a Phase I safety and pharmacokinetics study would be
conducted to determine the maximum tolerated dose (MTD) and to
characterize the dose-limiting toxicity, pharmacokinetics and
preliminary pharmacodynamics in humans. These initial studies could
be performed in patients with metastatic or unresectable cancers
and for which standard curative or palliative measures could not be
used or were no longer effective or tolerated, and LHRH receptor
positive status in the patients would be an enrollment criteria.
The scheme of the phase I study would be to use single escalating
doses of LHRH-XTEN-drug conjugate and to measure the biochemical,
PK, and clinical parameters, permitting the determination of the
MTD and the threshold and maximum concentrations in dosage and in
circulating drug that constitute the therapeutic window to be used
in subsequent Phase II and Phase III trials, as well as defining
potential toxicities and adverse events to be tracked in future
studies.
[0609] Phase II clinical studies of human patients would be
independently conducted in LHRH receptor positive advanced (stage 3
or 4) or recurrent breast, endometrial, ovarian, and prostate or
bladder cancer patients. The trial would evaluate the efficacy and
safety of LHRH-XTEN-drug conjugate alone and in combination with a
current chemotherapy employed in the specific indication. Patients
will receive intravenously administered LHRH-XTEN-drug clinical
candidate at a dose level and regimen pre-determined in Phase I
with or without the standard chemotherapeutic agent. A control arm
comprising of the chemotherapeutic agent plus placebo would be
included. The primary endpoint would be response rate as defined by
the Response Evaluation Criteria in Solid Tumors (RECIST).
Secondary endpoints will include safety and tolerability,
time-to-progression and overall survival.
[0610] A phase III efficacy and safety study is conducted in LHRH
receptor positive advanced (resistant, recurrent) breast,
endometrial, ovarian, and prostate or bladder cancer patients to
test ability to reach statistically significant clinical endpoints
such as progression-free-survival as measured by RECIST. The trial
will also be statistically powered for overall survival as a
secondary endpoint with projected enrollment in excess of 400
patients. Efficacy outcomes are determined using standard
statistical methods. Toxicity and adverse event markers are also
followed in the study to verify that the compound is safe when used
in the manner described.
Example 54: Human Clinical Trial Designs for Evaluating
Folate-XTEN-Drug Conjugates
[0611] Targeted chemotherapy is a modern approach aimed at
increasing the efficacy of systemic chemotherapy and reducing its
side effects. Folate, also known as folic acid, vitamin B.sub.9, is
a vital nutrient required by all living cells for nucleotide
biosynthesis and function as cofactor in certain biological
pathways. The folate receptor is a focus for the development of
therapies to treat fast dividing malignancies; in particular
ovarian cancer and non-small cell lung carcinoma. While folate
receptor expression is negligible in normal ovary, .about.90% of
epithelial ovarian cancers overexpress the folate receptor, as do
many lung adenocarinomas, thereby opening the possibility of
directed therapies. Fusion of a XTEN carrying .gtoreq.1 copy of
folate to a XTEN bearing .gtoreq.3 drug molecules to create a
targeted peptide-drug conjugate is expected to improve the
therapeutic index and the extended half-life will enable dosing at
levels way below maximum tolerated dose (MTD), reduce dosing
frequency and cost (reduced drug required per dose).
[0612] Clinical evaluation of folate-XTEN-drug composition is
conducted in patients with relapsed or refractory advanced tumors
or in patients suffering from platinum-resistant ovarian cancer and
non-small cell lung carcinoma who have failed using other
chemotherapies. Clinical trials are designed to determine the
efficacy and advantages of the folate-XTEN-drug conjugate over
standard therapies in humans. Such studies in patients would
comprise three phases. First, a Phase I safety and pharmacokinetics
study is conducted to determine the MTD and to characterize the
dose-limiting toxicity, pharmacokinetics and preliminary
pharmacodynamics in humans. These initial studies could be
performed in patients with folate receptor positive status that
have relapsed or have refractory advanced tumors and for which
standard curative or palliative measures could not be used or were
no longer effective or tolerated. The phase I study would use
single escalating doses of folate-XTEN-drug conjugate and would
measure biochemical, PK, and clinical parameters to permit the
determination of the MTD and establish the threshold and maximum
concentrations in dosage and in circulating drug that constitute
the therapeutic window to be used in subsequent Phase II and Phase
III trials as well as defining potential toxicities and adverse
events to be tracked in future studies.
[0613] Phase II clinical studies of human patients would be
independently conducted in folate receptor positive
platinum-resistant ovarian cancer patient population, non-small
cell lung carcinoma patients having failed numerous chemotherapies,
and patients suffering from relapsed or refractory advanced tumors.
The trials would evaluate the efficacy and safety of
folate-XTEN-drug conjugate alone and in combination with a current
chemotherapy employed in the specific indication. Patients will
receive intravenously administered folate-XTEN-drug conjugate at a
dose level and regimen determined in the Phase I study with or
without the standard chemotherapy agent. A control arm comprising
of the chemotherapy agent plus placebo would be included. The
primary endpoint would be response rate as defined by the Response
Evaluation Criteria in Solid Tumors (RECIST). Secondary endpoints
will include safety and tolerability, time-to-progression and
overall survival.
[0614] A phase III efficacy and safety study is conducted in
folate-receptor positive platinum-resistant ovarian cancer
patients, non-small cell lung carcinoma patients, or advanced tumor
relapsed or refractory patients cancer patients to test ability to
reach statistically significant clinical endpoints such as
progression-free-survival as measured by RECIST. The trial will
also be statistically powered for overall survival as a secondary
endpoint with projected enrollment in excess of 400 patients.
Efficacy outcomes are determined using standard statistical
methods. Toxicity and adverse event markers are also followed in
the study to verify that the compound is safe when used in the
manner described.
Example 55: Serum Stability of XTEN
[0615] A fusion protein containing XTEN_AE864 fused to the
N-terminus of GFP was incubated in monkey plasma and rat kidney
lysate for up to 7 days at 37.degree. C. Samples were withdrawn at
time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by
detection using Western analysis and detection with antibodies
against GFP as shown in FIG. 76. The sequence of XTEN_AE864 showed
negligible signs of degradation over 7 days in plasma. However,
XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days.
The in vivo stability of the fusion protein was tested in plasma
samples wherein the GFP_AE864 was immunoprecipitated and analyzed
by SDS PAGE as described above. Samples that were withdrawn up to 7
days after injection showed very few signs of degradation. The
results demonstrate the resistance of aaT-XTEN to degradation due
to serum proteases; a factor in the enhancement of pharmacokinetic
properties of the aaT-XTEN fusion proteins.
Example 56: Characterization of Secondary Structure of XTEN Linked
to Exendin-4
[0616] The XTEN_AE864-Ex4 was evaluated for degree of secondary
structure by circular dichroism spectroscopy. CD spectroscopy was
performed on a Jasco J-715 (Jasco Corporation, Tokyo, Japan)
spectropolarimeter equipped with Jasco Peltier temperature
controller (TPC-348WI). The concentration of protein was adjusted
to 0.2 mg/mL in 20 mM sodium phosphate pH 7.0, 50 mM NaCl. The
experiments were carried out using HELLMA quartz cells with an
optical path-length of 0.1 cm. The CD spectra were acquired at
5.degree., 25.degree., 45.degree., and 65.degree. C. and processed
using the J-700 version 1.08.01 (Build 1) Jasco software for
Windows. The samples were equilibrated at each temperature for 5
min before performing CD measurements. All spectra were recorded in
duplicate from 300 nm to 185 nm using a bandwidth of 1 nm and a
time constant of 2 sec, at a scan speed of 100 nm/min. The CD
spectrum shown in FIG. 77 shows no evidence of stable secondary
structure and is consistent with an unstructured polypeptide.
Example 57: Increasing Solubility and Stability of a Peptide
Payload by Linking to XTEN
[0617] In order to evaluate the ability of XTEN to enhance the
physicochemical properties of solubility and stability, fusion
proteins of glucagon plus shorter-length XTEN were prepared and
evaluated. The test articles were prepared in Tris-buffered saline
at neutral pH and characterization of the Gcg-XTEN solution was by
reverse-phase HPLC and size exclusion chromatography to affirm that
the protein was homogeneous and non-aggregated in solution. The
data are presented in Table 43. For comparative purposes, the
solubility limit of unmodified glucagon in the same buffer was
measured at 60 .mu.M (0.2 mg/mL), and the result demonstrate that
for all lengths of XTEN added, a substantial increase in solubility
was attained. Importantly, in most cases the glucagon-XTEN fusion
proteins were prepared to achieve target concentrations and were
not evaluated to determine the maximum solubility limits for the
given construct. However, in the case of glucagon linked to the
AF-144 XTEN, the limit of solubility was determined, with the
result that a 60-fold increase in solubility was achieved, compared
to glucagon not linked to XTEN. In addition, the glucagon-AF144 was
evaluated for stability, and was found to be stable in liquid
formulation for at least 6 months under refrigerated conditions and
for approximately one month at 37.degree. C. (data not shown).
[0618] The data support the conclusion that the linking of
short-length XTEN polypeptides to a biologically active protein
such as glucagon can markedly enhance the solubility properties of
the protein by the resulting fusion protein, as well as confer
stability at the higher protein concentrations.
TABLE-US-00049 TABLE 43 Solubility of Glucagon-XTEN constructs Test
Article Solubility Glucagon 60 .mu.M Glucagon-Y36 >370 .mu.M
Glucagon-Y72 >293 .mu.M Glucagon-AF108 >145 .mu.M
Glucagon-AF120 >160 .mu.M Glucagon-Y144 >497 .mu.M
Glucagon-AE144 >467 .mu.M Glucagon-AF144 >3600 .mu.M
Glucagon-Y288 >163 .mu.M
Example 58: Analytical Size Exclusion Chromatography of XTEN Linked
with Diverse Payloads
[0619] Size exclusion chromatography analyses were performed on
fusion proteins containing various therapeutic proteins and
unstructured recombinant proteins of increasing length. An
exemplary assay used a TSKGel-G4000 SWXL (7.8 mm.times.30 cm)
column in which 40 .mu.g of purified glucagon fusion protein at a
concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min
in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profiles were
monitored using OD214 nm and OD280 nm. Column calibration for all
assays were performed using a size exclusion calibration standard
from BioRad; the markers include thyroglobulin (670 kDa), bovine
gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine
myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative
chromatographic profiles of Glucagon-Y288, Glucagon-Y144,
Glucagon-Y72, Glucagon-Y36 are shown as an overlay in FIG. 78. The
data show that the molecular weight of each compound is
proportional to the length of the attached XTEN sequence. However,
the data also show that the apparent molecular weight of each
construct is significantly larger than that expected for a globular
protein (as shown by comparison to the standard proteins run in the
same assay). Based on the SEC analyses for all constructs
evaluated, the apparent molecular weights, the apparent molecular
weight factor (expressed as the ratio of apparent molecular weight
to the calculated molecular weight) and the hydrodynamic radius
(R.sub.H in nm) are shown in Table 44. The results indicate that
incorporation of different XTENs of 576 amino acids or greater
confers an apparent molecular weight for the fusion protein of
approximately 339 kDa to 760, and that XTEN of 864 amino acids or
greater confers an apparent molecular weight greater than
approximately 800 kDA. The results of proportional increases in
apparent molecular weight to actual molecular weight were
consistent for fusion proteins created with XTEN from several
different motif families; i.e., AD, AE, AF, AG, and AM, with
increases of at least four-fold and ratios as high as about
17-fold. Additionally, the incorporation of XTEN fusion partners
with 576 amino acids or more into fusion proteins with the various
payloads (and 288 residues in the case of glucagon fused to Y288)
resulted with a hydrodynamic radius of 7 nm or greater; well beyond
the glomerular pore size of approximately 3-5 nm. Accordingly, it
is expected that fusion proteins comprising growth and XTEN have
reduced renal clearance, contributing to increased terminal
half-life and improving the therapeutic or biologic effect relative
to a corresponding un-fused biologic payload protein.
TABLE-US-00050 TABLE 44 SEC analysis of various polypeptides
Apparent XTEN or Actual Apparent Molecular Construct fusion
Therapeutic MW MW Weight R.sub.H Name partner Protein (kDa) (kDa)
Factor (nm) AC14 Y288 Glucagon 28.7 370 12.9 7.0 AC28 Y144 Glucagon
16.1 117 7.3 5.0 AC34 Y72 Glucagon 9.9 58.6 5.9 3.8 AC33 Y36
Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 Glucagon 14.1 76.4 5.4 4.3
AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.2
5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1
7.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6
AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7
10.1 AC85 AE864 Exendin-4 83.6 938 11.2 8.9 AC114 AM875 Exendin-4
82.4 1344 16.3 9.4 AC143 AM875 hGH 100.6 846 8.4 8.7 AC302 AE912 +
AE144 hGH 119.1 2,287 19.2 11.0 AC227 AM875 IL-1ra 95.4 1103 11.6
9.2 AC228 AM1318 IL-1ra 134.8 2286 17.0 10.5 AC493 AE864 FIX 127.7*
3967 31.1 12.2 AC616 AE864 GLP2-2G 83.1 1427 17.2 10 AC647 AE864
Ghrelin 82.7 996 12 9.2 AC659 AE864 C-peptide 82.7 822 10 8.8 AC663
AE1296 C-peptide 122.2 2348 19.2 11.1 AC434 AE288 aaT 71.1 500 7.0
7.7 AC435 AE576 aaT 97.5 1,127 11.6 9.5 AC345 AM875 aaT 122.6 1,390
11.3 9.9 AC450 AE288 aHer2_scFv 56.2 312 5.5 6.7 AC451 AE576
aHer2_scFv 82.6 760 9.2 8.6 AC452 AE864 aHer2_scFv 109.1 1,390 12.7
9.9 *excluding glycosylation
Example 59: Analysis of Sequences for Secondary Structure by
Prediction Algorithms
[0620] Amino acid sequences can be assessed for secondary structure
via certain computer programs or algorithms, such as the well-known
Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:
222-45) and the Garnier-Osguthorpe-Robson, or "GOR" method (Gamier
J, Gibrat J F, Robson B. (1996). GOR method for predicting protein
secondary structure from amino acid sequence. Methods Enzymol
266:540-553). For a given sequence, the algorithms can predict
whether there exists some or no secondary structure at all,
expressed as total and/or percentage of residues of the sequence
that form, for example, alpha-helices or beta-sheets or the
percentage of residues of the sequence predicted to result in
random coil formation.
[0621] Several representative sequences from XTEN "families" have
been assessed using two algorithm tools for the Chou-Fasman and GOR
methods to assess the degree of secondary structure in these
sequences. The Chou-Fasman tool was provided by William R. Pearson
and the University of Virginia, at the "Biosupport" internet site,
URL located on the World Wide Web at
.fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it
existed on Jun. 19, 2009. The GOR tool was provided by Pole
Informatique Lyonnais at the Network Protein Sequence Analysis
internet site, URL located on the World Wide Web at
.npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl as it existed on Jun.
19, 2008.
[0622] As a first step in the analyses, a single XTEN sequence was
analyzed by the two algorithms. The AE864 composition is a XTEN
with 864 amino acid residues created from multiple copies of four
12 amino acid sequence motifs consisting of the amino acids G, S,
T, E, P, and A. The sequence motifs are characterized by the fact
that there is limited repetitiveness within the motifs and within
the overall sequence in that the sequence of any two consecutive
amino acids is not repeated more than twice in any one 12 amino
acid motif, and that no three contiguous amino acids of full-length
the XTEN are identical. Successively longer portions of the AF 864
sequence from the N-terminus were analyzed by the Chou-Fasman and
GOR algorithms (the latter requires a minimum length of 17 amino
acids). The sequences were analyzed by entering the FASTA format
sequences into the prediction tools and running the analysis. The
results from the analyses are presented in Table 45.
[0623] The results indicate that, by the Chou-Fasman calculations,
short XTEN of the AE and AG families, up to at least 288 amino acid
residues, have no alpha-helices or beta sheets, but amounts of
predicted percentage of random coil by the GOR algorithm vary from
78-99%. With increasing XTEN lengths of 504 residues to greater
than 1300, the XTEN analyzed by the Chou-Fasman algorithm had
predicted percentages of alpha-helices or beta sheets of 0 to about
2%, while the calculated percentages of random coil increased to
from 94-99%. Those XTEN with alpha-helices or beta sheets were
those sequences with one or more instances of three contiguous
serine residues, which resulted in predicted beta-sheet formation.
However, even these sequences still had approximately 99% random
coil formation.
[0624] The analysis supports the conclusion that: 1) XTEN created
from multiple sequence motifs of G, S, T, E, P, and A that have
limited repetitiveness as to contiguous amino acids are predicted
to have very low amounts of alpha-helices and beta-sheets; 2) that
increasing the length of the XTEN does not appreciably increase the
probability of alpha-helix or beta-sheet formation; and 3) that
progressively increasing the length of the XTEN sequence by
addition of non-repetitive 12-mers consisting of the amino acids G,
S, T, E, P, and A results in increased percentage of random coil
formation. Based on the numerous sequences evaluated by these
methods, it is concluded that XTEN created from sequence motifs of
G, S, T, E, P, and A that have limited repetitiveness (defined as
no more than two identical contiguous amino acids in any one motif)
are expected to have very limited secondary structure. With the
exception of motifs containing three contiguous serines, generally
any order or combination of sequence motifs from Table 1 can be
used to create an XTEN polypeptide that will result in an XTEN
sequence that is substantially devoid of secondary structure, and
that the effects of three contiguous serines is ameliorated by
increasing the length of the XTEN. Such sequences are expected to
have the characteristics described in the XTEN-containing
composition embodiments of the invention disclosed herein.
TABLE-US-00051 TABLE 45 CHOU-FASMAN and GOR prediction calculations
of polypeptide sequences SEQ SEQ ID No. Chou-Fasman GOR NAME
Sequence NO: Residues Calculation Calculation AE36:
GSPAGSPTSTEEGTSESATPESGPGT 1103 36 Residue totals: H: 0 94.44%
LCW0402_ STEPSEGSAP E: 0 002 percent: H: 0.0 E: 0.0 AE36:
GTSTEPSEGSAPGTSTEPSEGSAPGT 1104 36 Residue totals: H: 0 94.44%
LCW040_2 STEPSEGSAP E: 0 003 percent: H: 0.0 E: 0.0 AG36:
GASPGTSSTGSPGTPGSGTASSSPGS 1105 36 Residue totals: H: 0 77.78%
LCW0404_ STPSGATGSP E: 0 001 percent: H: 0.0 E: 0.0 AG36:
GSSTPSGATGSPGSSPSASTGTGPGS 1106 36 Residue totals: H: 0 83.33%
LCW0404_ STPSGATGSP E: 0 003 percent: H: 0.0 E: 0.0 AE42_1
TEPSEGSAPGSPAGSPTSTEEGTSES 1107 42 Residue totals: H: 0 90.48%
ATPESGPGSEPATSGS E: 0 percent: H: 0.0 E: 0.0 AE42_1
TEPSEGSAPGSPAGSPTSTEEGTSES 1108 42 Residue totals: H: 0 90.48%
ATPESGPGSEPATSGS E: 0 percent: H: 0.0 E: 0.0 AG42_1
GAPSPSASTGTGPGTPGSGTASSSPG 1109 42 Residue totals: H: 0 88.10%
SSTPSGATGSPGPSGP E: 0 percent: H: 0.0 E: 0.0 AG42_2
GPGTPGSGTASSSPGSSTPSGATGSP 1110 42 Residue totals: H: 0 88.10%
GSSPSASTGTGPGASP E: 0 percent: H: 0.0 E: 0.0 AE144_1
GSEPATSGSETPGTSESATPESGPGS 1111 144 Residue totals: H: 0 98.61%
EPATSGSETPGSPAGSPTSTEEGTST E: 0 EPSEGSAPGSEPATSGSETPGSEPAT percent:
H: 0.0 E: SGSETPGSEPATSGSETPGTSTEPSE 0.0 GSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAP AG144_1 PGSSPSASTGTGPGSSPSASTGTGPG 1112 144 Residue
totals: H: 0 91.67% TPGSGTASSSPGSSTPSGATGSPGSS E: 0
PSASTGTGPGASPGTSSTGSPGTPGS percent: H: 0.0 E:
GTASSSPGSSTPSGATGSPGTPGSGT 0.0 ASSSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSS AE288 GTSESATPESGPGSEPATSGSETPGT 1113 288 Residue
totals: H: 0 99.31% SESATPESGPGSEPATSGSETPGTSE E: 0
SATPESGPGTSTEPSEGSAPGSPAGS percent: H: 0.0 E:
PTSTEEGTSESATPESGPGSEPATSG 0.0 SETPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGT
SESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTSTEPSEGS AP AG288_2
GSSPSASTGTGPGSSPSASTGTGPGT 1114 288 Residue totals: H: 0 92.71
PGSGTASSSPGSSTPSGATGSPGSSP E: 0 SASTGTGPGASPGTSSTGSPGTPGSG percent:
H: 0.0 E: TASSSPGSSTPSGATGSPGTPGSGTA 0.0 SSSPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSP GASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASS SP AF504
GASPGTSSTGSPGSSPSASTGTGPGS 1115 504 Residue totals: H: 0 94.44%
SPSASTGTGPGTPGSGTASSSPGSST E: 0 PSGATGSPGSNPSASTGTGPGASPG percent:
H: 0.0 E: TSSTGSPGTPGSGTASSSPGSSTPSG 0.0 ATGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGSN PSASTGTGPGSSPSASTGTGPGSSTP
SGATGSPGSSTPSGATGSPGASPGTS STGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGASPGTSSTGS PGASPGTSSTGSPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSP
AD 576 GSSESGSSEGGPGSGGEPSESGSSGS 1116 576 Residue totals: H: 7
99.65% SESGSSEGGPGSSESGSSEGGPGSSE E: 0 percent: H: 1.2
SGSSEGGPGSSESGSSEGGPGSSESG E: 0.0 SSEGGPGESPGGSSGSESGSEGSSGP
GESSGSSESGSSEGGPGSSESGSSEG GPGSSESGSSEGGPGSGGEPSESGSS
GESPGGSSGSESGESPGGSSGSESGS GGEPSESGSSGSSESGSSEGGPGSGG
EPSESGSSGSGGEPSESGSSGSEGSS GPGESSGESPGGSSGSESGSGGEPSE
SGSSGSGGEPSESGSSGSGGEPSESG SSGSSESGSSEGGPGESPGGSSGSES
GESPGGSSGSESGESPGGSSGSESGE SPGGSSGSESGESPGGSSGSESGSSE
SGSSEGGPGSGGEPSESGSSGSEGSS GPGESSGSSESGSSEGGPGSGGEPSE
SGSSGSSESGSSEGGPGSGGEPSESG SSGESPGGSSGSESGESPGGSSGSES
GSSESGSSEGGPGSGGEPSESGSSGS SESGSSEGGPGSGGEPSESGSSGSGG
EPSESGSSGESPGGSSGSESGSEGSS GPGESSGSSESGSSEGGPGSEGSSGP GESS AE576
GSPAGSPTSTEEGTSESATPESGPGT 1117 576 Residue totals: H: 2 99.65%
STEPSEGSAPGSPAGSPTSTEEGTST E: 0 EPSEGSAPGTSTEPSEGSAPGTSESA percent:
H: 0.4 E: TPESGPGSEPATSGSETPGSEPATSG 0.0 SETPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGT
STEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGS
PTSTEEGTSESATPESGPGTSTEPSE GSAP AG576 PGTPGSGTASSSPGSSTPSGATGSPG
1118 576 Residue totals: H: 0 99.31% SSPSASTGTGPGSSPSASTGTGPGSS E:
3 TPSGATGSPGSSTPSGATGSPGASPG percent: H: 0.4 E:
TSSTGSPGASPGTSSTGSPGASPGTS 0.5 STGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGS PGSSPSASTGTGPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGSSTPSGAT
GSPGSSTPSGATGSPGSSPSASTGTG PGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTS STGS AF540 GSTSSTAESPGPGSTSSTAESPGPGS
1119 540 Residue totals: H: 2 99.65 TSESPSGTAPGSTSSTAESPGPGSTSS E:
0 percent: H: 0.4 TAESPGPGTSTPESGSASPGSTSESPS E: 0.0
GTAPGTSPSGESSTAPGSTSESPSGT APGSTSESPSGTAPGTSPSGESSTAP
GSTSESPSGTAPGSTSESPSGTAPGT SPSGESSTAPGSTSESPSGTAPGSTSE
SPSGTAPGSTSESPSGTAPGTSTPES GSASPGSTSESPSGTAPGTSTPESGS
ASPGSTSSTAESPGPGSTSSTAESPG PGTSTPESGSASPGTSTPESGSASPG
STSESPSGTAPGTSTPESGSASPGTST PESGSASPGSTSESPSGTAPGSTSESP
SGTAPGSTSESPSGTAPGSTSSTAES PGPGTSTPESGSASPGTSTPESGSAS
PGSTSESPSGTAPGSTSESPSGTAPG TSTPESGSASPGSTSESPSGTAPGSTS
ESPSGTAPGTSTPESGSASPGTSPSG ESSTAPGSTSSTAESPGPGTSPSGESS
TAPGSTSSTAESPGPGTSTPESGSAS PGSTSESPSGTAP AD836
GSSESGSSEGGPGSSESGSSEGGPGE 1120 836 Residue totals: H: 0 98.44%
SPGGSSGSESGSGGEPSESGSSGESP E: 0 GGSSGSESGESPGGSSGSESGSSESG percent:
H: 0.0 E: SSEGGPGSSESGSSEGGPGSSESGSS 0.0 EGGPGESPGGSSGSESGESPGGSSGS
ESGESPGGSSGSESGSSESGSSEGGP GSSESGSSEGGPGSSESGSSEGGPGS
SESGSSEGGPGSSESGSSEGGPGSSE SGSSEGGPGSGGEPSESGSSGESPGG
SSGSESGESPGGSSGSESGSGGEPSE SGSSGSEGSSGPGESSGSSESGSSEG
GPGSGGEPSESGSSGSEGSSGPGESS GSSESGSSEGGPGSGGEPSESGSSGE
SPGGSSGSESGSGGEPSESGSSGSGG EPSESGSSGSSESGSSEGGPGSGGEP
SESGSSGSGGEPSESGSSGSEGSSGP GESSGESPGGSSGSESGSEGSSGPGE
SSGSEGSSGPGESSGSGGEPSESGSS GSSESGSSEGGPGSSESGSSEGGPGE
SPGGSSGSESGSGGEPSESGSSGSEG SSGPGESSGESPGGSSGSESGSEGSS
GPGSSESGSSEGGPGSGGEPSESGSS GSEGSSGPGESSGSEGSSGPGESSGS
EGSSGPGESSGSGGEPSESGSSGSGG EPSESGSSGESPGGSSGSESGESPGG
SSGSESGSGGEPSESGSSGSEGSSGP GESSGESPGGSSGSESGSSESGSSEG
GPGSSESGSSEGGPGSSESGSSEGGP GSGGEPSESGSSGSSESGSSEGGPGE
SPGGSSGSESGSGGEPSESGSSGSSE SGSSEGGPGESPGGSSGSESGSGGEP
SESGSSGESPGGSSGSESGSGGEPSE SGSS AE864 GSPAGSPTSTEEGTSESATPESGPGT
1121 864 Residue totals: H: 2 99.77% STEPSEGSAPGSPAGSPTSTEEGTST E:
3 EPSEGSAPGTSTEPSEGSAPGTSESA percent: H: 0.2 E:
TPESGPGSEPATSGSETPGSEPATSG 0.4 SETPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGT
STEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPES GPGTSESATPESGPGSEPATSGSETP
GSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEP SEGSAP AF864 GSTSESPSGTAPGTSPSGESSTAPGS
1122 875 Residue totals: H: 2 95.20% TSESPSGTAPGSTSESPSGTAPGTSTP E:
0 percent: H: 0.2 ESGSASPGTSTPESGSASPGSTSESPS E: 0.0
GTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGTSPSGESSTAP
GTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGTSPSGESSTAPGSTSS
TAESPGPGTSTPESGSASPGTSTPES GSASPGSTSESPSGTAPGSTSESPSG
TAPGTSTPESGSASPGSTSSTAESPG PGTSTPESGSASPGSTSESPSGTAPG
TSPSGESSTAPGSTSSTAESPGPGTSP SGESSTAPGTSTPESGSASPGSTSST
AESPGPGSTSSTAESPGPGSTSSTAE SPGPGSTSSTAESPGPGTSPSGESST
APGSTSESPSGTAPGSTSESPSGTAP GTSTPESGPXXXGASASGAPSTXXX
XSESPSGTAPGSTSESPSGTAPGSTS ESPSGTAPGSTSESPSGTAPGSTSESP
SGTAPGSTSESPSGTAPGTSTPESGS ASPGTSPSGESSTAPGTSPSGESSTA
PGSTSSTAESPGPGTSPSGESSTAPG TSTPESGSASPGSTSESPSGTAPGSTS
ESPSGTAPGTSPSGESSTAPGSTSESP SGTAPGTSTPESGSASPGTSTPESGS
ASPGSTSESPSGTAPGTSTPESGSAS PGSTSSTAESPGPGSTSESPSGTAPG
STSESPSGTAPGTSPSGESSTAPGSTS STAESPGPGTSPSGESSTAPGTSTPES
GSASPGTSPSGESSTAPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPG
PGSTSSTAESPGPGTSPSGESSTAPG SSPSASTGTGPGSSTPSGATGSPGSS TPSGATGSP
AG864 GASPGTSSTGSPGSSPSASTGTGPGS 1123 864 Residue totals: H: 0
94.91% SPSASTGTGPGTPGSGTASSSPGSST E: 0 PSGATGSPGSSPSASTGTGPGASPGT
percent: H: 0.0 E: SSTGSPGTPGSGTASSSPGSSTPSGA 0.0
TGSPGTPGSGTASSSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSPGSSP
SASTGTGPGSSPSASTGTGPGSSTPS GATGSPGSSTPSGATGSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGS SPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSTPSGATGSPGSSTPSGA
TGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSP
GSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGTPG
SGTASSSPGASPGTSSTGSPGASPGT SSTGSPGASPGTSSTGSPGASPGTSS
TGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSP
GTPGSGTASSSPGSSTPSGATGSPGS STPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGS SPSASTGTGPGTPGSGTASSSPGSST
PSGATGSPGSSTPSGATGSPGASPGT SSTGSP AM875 GTSTEPSEGSAPGSEPATSGSETPGS
1124 875 Residue totals: H: 7 98.63% PAGSPTSTEEGSTSSTAESPGPGTST E:
3 PESGSASPGSTSESPSGTAPGSTSESP percent: H: 0.8 E:
SGTAPGTSTPESGSASPGTSTPESGS 0.3 ASPGSEPATSGSETPGTSESATPESG
PGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESG
PGTSTEPSEGSAPGSEPATSGSETPG SPAGSPTSTEEGSSTPSGATGSPGTP
GSGTASSSPGSSTPSGATGSPGTSTE PSEGSAPGTSTEPSEGSAPGSEPATS
GSETPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGASASGAPSTG
GTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSTSSTAESPGPGSTS
ESPSGTAPGTSPSGESSTAPGTPGSG TASSSPGSSTPSGATGSPGSSPSAST
GTGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGSTSSTAESPGP
GSTSSTAESPGPGTSPSGESSTAPGS EPATSGSETPGSEPATSGSETPGTST
EPSEGSAPGSTSSTAESPGPGTSTPES GSASPGSTSESPSGTAPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSA PGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGSEPATSGSETPGTS ESATPESGPGSPAGSPTSTEEGSSTPS
GATGSPGSSPSASTGTGPGASPGTSS TGSPGTSESATPESGPGTSTEPSEGS
APGTSTEPSEGSAP AM1318 GTSTEPSEGSAPGSEPATSGSETPGS 1125 1318 Residue
totals: H: 7 99.17% PAGSPTSTEEGSTSSTAESPGPGTST E: 0
PESGSASPGSTSESPSGTAPGSTSESP percent: H: 0.7 E:
SGTAPGTSTPESGSASPGTSTPESGS 0.0 ASPGSEPATSGSETPGTSESATPESG
PGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESAT
PESGPGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESG
PGTSTEPSEGSAPGSEPATSGSETPG SPAGSPTSTEEGSSTPSGATGSPGTP
GSGTASSSPGSSTPSGATGSPGTSTE PSEGSAPGTSTEPSEGSAPGSEPATS
GSETPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGPEPTGPAPSG
GSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGSPAGSPTSTEEGSPAGSPT
STEEGSTSSTAESPGPGSTSESPSGT APGTSPSGESSTAPGSTSESPSGTAP
GSTSESPSGTAPGTSPSGESSTAPGT STEPSEGSAPGTSESATPESGPGTSE
SATPESGPGSEPATSGSETPGTSESA TPESGPGTSESATPESGPGTSTEPSE
GSAPGTSESATPESGPGTSTEPSEGS APGTSPSGESSTAPGTSPSGESSTAP
GTSPSGESSTAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGSSPS
ASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGSSTPSGATGSPGSSTPSGAT
GSPGASPGTSSTGSPGASASGAPSTG GTSPSGESSTAPGSTSSTAESPGPGT
SPSGESSTAPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSSPSA
STGTGPGSSTPSGATGSPGASPGTSS TGSPGTSTPESGSASPGTSPSGESST
APGTSPSGESSTAPGTSESATPESGP GSEPATSGSETPGTSTEPSEGSAPGS
TSESPSGTAPGSTSESPSGTAPGTSTP ESGSASPGSPAGSPTSTEEGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTS TEEGTSESATPESGPGSEPATSGSET
PGSSTPSGATGSPGASPGTSSTGSPG SSTPSGATGSPGSTSESPSGTAPGTS
PSGESSTAPGSTSSTAESPGPGSSTPS GATGSPGASPGTSSTGSPGTPGSGT
ASSSPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAP AM923
MAEPAGSPTSTEEGASPGTSSTGSP 1126 924 Residue totals: H: 4 98.70%
GSSTPSGATGSPGSSTPSGATGSPGT E: 3 percent: H: 0.4
STEPSEGSAPGSEPATSGSETPGSPA E: 0.3 GSPTSTEEGSTSSTAESPGPGTSTPES
GSASPGSTSESPSGTAPGSTSESPSG TAPGTSTPESGSASPGTSTPESGSAS
PGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGSEPATSGSETPGSP
AGSPTSTEEGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGTSTEPS
EGSAPGTSTEPSEGSAPGSEPATSGS ETPGSPAGSPTSTEEGSPAGSPTSTE
EGTSTEPSEGSAPGASASGAPSTGG TSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSTSSTAESPGPGSTSE SPSGTAPGTSPSGESSTAPGTPGSGT
ASSSPGSSTPSGATGSPGSSPSASTG TGPGSEPATSGSETPGTSESATPESG
PGSEPATSGSETPGSTSSTAESPGPG STSSTAESPGPGTSPSGESSTAPGSEP
ATSGSETPGSEPATSGSETPGTSTEP SEGSAPGSTSSTAESPGPGTSTPESG
SASPGSTSESPSGTAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAP
GSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSEPATSGSETPGTSES
ATPESGPGSPAGSPTSTEEGSSTPSG ATGSPGSSPSASTGTGPGASPGTSST
GSPGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAP AE912
MAEPAGSPTSTEEGTPGSGTASSSP 1127 913 Residue totals: H: 8 99.45%
GSSTPSGATGSPGASPGTSSTGSPGS E: 3 percent: H:
PAGSPTSTEEGTSESATPESGPGTST 0.9 E: 0.3 EPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGS
PAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGP GSPAGSPTSTEEGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGSPAGSPT STEEGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGT
SESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGS
EPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSE GSAP
BC 864 GTSTEPSEPGSAGTSTEPSEPGSAGS 1128 Residue totals: H: 0 99.77%
EPATSGTEPSGSGASEPTSTEPGSEP E: 0 percent: H: 0
ATSGTEPSGSEPATSGTEPSGSEPAT E: 0 SGTEPSGSGASEPTSTEPGTSTEPSEP
GSAGSEPATSGTEPSGTSTEPSEPGS AGSEPATSGTEPSGSEPATSGTEPSG
TSTEPSEPGSAGTSTEPSEPGSAGSE PATSGTEPSGSEPATSGTEPSGTSEP
STSEPGAGSGASEPTSTEPGTSEPST SEPGAGSEPATSGTEPSGSEPATSGT
EPSGTSTEPSEPGSAGTSTEPSEPGS AGSGASEPTSTEPGSEPATSGTEPSG
SEPATSGTEPSGSEPATSGTEPSGSE PATSGTEPSGTSTEPSEPGSAGSEPA
TSGTEPSGSGASEPTSTEPGTSTEPSE PGSAGSEPATSGTEPSGSGASEPTST
EPGTSTEPSEPGSAGSGASEPTSTEP GSEPATSGTEPSGSGASEPTSTEPGS
EPATSGTEPSGSGASEPTSTEPGTST EPSEPGSAGSEPATSGTEPSGSGASE
PTSTEPGTSTEPSEPGSAGSEPATSG TEPSGTSTEPSEPGSAGSEPATSGTE
PSGTSTEPSEPGSAGTSTEPSEPGSA GTSTEPSEPGSAGTSTEPSEPGSAGT
STEPSEPGSAGTSTEPSEPGSAGTSE PSTSEPGAGSGASEPTSTEPGTSTEP
SEPGSAGTSTEPSEPGSAGTSTEPSE PGSAGSEPATSGTEPSGSGASEPTST
EPGSEPATSGTEPSGSEPATSGTEPS GSEPATSGTEPSGSEPATSGTEPSGT
SEPSTSEPGAGSEPATSGTEPSGSGA SEPTSTEPGTSTEPSEPGSAGSEPATS
GTEPSGSGASEPTSTEPGTSTEPSEP GSA *H: alpha-helix E: beta-sheet
Example 60: Analysis of Polypeptide Sequences for
Repetitiveness
[0625] Polypeptide amino acid sequences can be assessed for
repetitiveness by quantifying the number of times a shorter
subsequence appears within the overall polypeptide. For example, a
polypeptide of 200 amino acid residues has 192 overlapping 9-amino
acid subsequences (or 9-mer "frames"), but the number of unique
9-mer subsequences will depend on the amount of repetitiveness
within the sequence. In the present analysis, different sequences
were assessed for repetitiveness by summing the occurrence of all
unique 3-mer subsequences for each 3-amino acid frame across the
first 200 amino acids of the polymer portion divided by the
absolute number of unique 3-mer subsequences within the 200 amino
acid sequence. The resulting subsequence score is a reflection of
the degree of repetitiveness within the polypeptide.
[0626] The results, shown in Table 46, indicate that the
unstructured polypeptides consisting of 2 or 3 amino acid types
have high subsequence scores, while those of consisting of 12 amino
acids motifs of the six amino acids G, S, T, E, P, and A with a low
degree of internal repetitiveness, have subsequence scores of less
than 10, and in some cases, less than 5. For example, the L288
sequence has two amino acid types and has short, highly repetitive
sequences, resulting in a subsequence score of 50.0. The
polypeptide J288 has three amino acid types but also has short,
repetitive sequences, resulting in a subsequence score of 33.3.
Y576 also has three amino acid types, but is not made of internal
repeats, reflected in the subsequence score of 15.7 over the first
200 amino acids. W576 consists of four types of amino acids, but
has a higher degree of internal repetitiveness, e.g., "GGSG" (SEQ
ID NO: 1129), resulting in a subsequence score of 23.4. The AD576
consists of four types of 12 amino acid motifs, each consisting of
four types of amino acids. Because of the low degree of internal
repetitiveness of the individual motifs, the overall subsequence
score over the first 200 amino acids is 13.6. In contrast, XTEN's
consisting of four motifs contains six types of amino acids, each
with a low degree of internal repetitiveness have lower subsequence
scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5).
[0627] Conclusions: The results indicate that the combination of 12
amino acid subsequence motifs, each consisting of four to six amino
acid types that are essentially non-repetitive, into a longer XTEN
polypeptide results in an overall sequence that is non-repetitive.
This is despite the fact that each subsequence motif may be used
multiple times across the sequence. In contrast, polymers created
from smaller numbers of amino acid types resulted in higher
subsequence scores, although the actual sequence can be tailored to
reduce the degree of repetitiveness to result in lower subsequence
scores.
TABLE-US-00052 TABLE 46 Subsequence score calculations of
polypeptide sequences Seq SEQ ID Name Amino Acid Sequence NO: Score
J288 GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG 1130 33.3
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG K288
GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEG 1131 46.9
GGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGG
EGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGG
GEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGE
GEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGG
EGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEG
EGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE G L288
SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSS 1132 50.0
ESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSES
SESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSE
SSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESS
SSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSS
ESSSESSESSSSESSSESSESSSSESSSESSESSSSES Y288
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGS 1133 26.8
EGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGG
SEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGE
GSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGS
EGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEG
SGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEG
EGSEGGSEGEGSEGSGEGEGSEGSGE Q576
GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGGG 1134 18.5
EGGKPEGGKPEGEGKPGGGEGKPGGKPEGGGGKPEGEGKPGG
GGGKPGGKPEGEGKPGGGEGGKPEGKPGEGGEGKPGGKPEGG
GEGKPGGGKPGEGGKPGEGKPGGGEGGKPEGGKPEGEGKPGG
GEGKPGGKPGEGGKPEGGGEGKPGGKPGEGGEGKPGGGKPEG
EGKPGGGKPGGGEGGKPEGEGKPGGKPEGGGEGKPGGKPEGG
GKPEGGGEGKPGGGKPGEGGKPGEGEGKPGGKPEGEGKPGGE
GGGKPEGKPGGGEGGKPEGGKPGEGGKPEGGKPGEGGEGKPG
GGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGGKPEGGKPEGG
GEGKPGGGKPEGEGKPGGGEGKPGGKPEGGGGKPGEGGKPEG
GKPGGEGGGKPEGEGKPGGKPGEGGGGKPGGKPEGEGKPGEG
GEGKPGGKPEGGGEGKPGGKPEGGGEGKPGGGKPGEGGKPEG
GGKPGEGGKPGEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGG
GEGKPGGKPGGEGGGKPEGGKPGEGGKPEG U576
GEGKPGGKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKPG 1135 18.1
GKPGEGGKPEGGSGGKPGGGGKPGGKPGGEGSGKPGGKPEGG
GKPEGGSGGKPGGKPEGGSGGKPGGKPGSGEGGKPGGGKPGG
EGKPGSGKPGGEGSGKPGGKPEGGSGGKPGGKPEGGSGGKPG
GSGKPGGKPGEGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPG
GKPGEGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPG
GKPGSGGEGKPGGKPEGGSGGKPGGGKPGGEGKPGSGGKPGE
GGKPGSGGGKPGGKPGGEGEGKPGGKPGEGGKPGGEGSGKPG
GGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPEGGGGKPEG
SGKPGGGGKPEGSGKPGGGKPEGGSGGKPGGSGKPGGKPGEG
GGKPEGSGKPGGGSGKPGGKPEGGGKPEGGSGGKPGGKPEGG
SGGKPGGKPGGEGSGKPGGKPGSGEGGKPGGKPGEGSGGKPG
GKPEGGSGGKPGGSGKPGGKPEGGGSGKPGGKPGEGGKPGGE GSGKPGGSGKPG W576
GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGS 1136 23.4
GKPGGGSGKPGSGKPGGGGKPGSGSGKPGGGKPGGSGGKPGG
GSGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGGSGGKPG
KPGSGGGSGKPGKPGSGGSGGKPGKPGSGGSGGKPGKPGSGGS
GKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGKPGSGK
PGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGGKP
GSGSGKPGGGKPGSGSGKPGGGKPGGSGGKPGGSGGKPGKPG
SGGGSGKPGKPGSGGGSGKPGKPGGSGSGKPGSGKPGGGSGKP
GSGKPGSGGSGKPGKPGSGGSGGKPGKPGSGGGKPGSGSGKPG
GGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGSGKPGS
GKPGGGSGGKPGKPGSGGSGKPGSGKPGSGGSGKPGKPGGSGS
GKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSG
KPGGGGKPGSGSGKPGGSGGKPGKPGSGGSGGKPGKPGSGGS GKPGSGKPGGGSGGKPGKPGSGG
Y576 GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSG 1137 15.7
EGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEG
GSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSE
GSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEG
GSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGE
GGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSE
GEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSG
EGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGS
GEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSE
GSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGS
EGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEG
SEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGE
GSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEGSEGEGSEGSGE GEGSGEGSE AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEG 1138 13.6
GPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGS
SGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSES
GSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGS
GGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSS
GSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSES
GSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGG
SSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSE
SGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPG
SGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSE
SGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSE
GGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSS
GPGESSGSSESGSSEGGPGSEGSSGPGESS AE576
AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS 1139 6.1
TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG
SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE
EGTSESATPESGPGTSTEPSEGSAP AF540
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESP 1140 8.8
GPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGES
STAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSES
PSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGT
STPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASP
GTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSA
SPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAE
SPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESP
SGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTP
ESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGST
SSTAESPGPGTSTPESGSASPGSTSESPSGTAP Ap504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS 1141 7.0
SSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTG
PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGS
STPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST GSP AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST 1142 6.1
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSG
SETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS
EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP AF864
GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGT 1143 7.5
APGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPS
GTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSG
ESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTS
STAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGS
TSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASP
GSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESST
APGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAE
SPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESP
SGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTS
ESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGS
TSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAP
GSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGT
APGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESG
SASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSST
AESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTS
STAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGT
SPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGP
GTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATG SP AG868
GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS 1144 7.5
GTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSAS
TGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSP
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESP 1145 4.5
GPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
SASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
EEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGT
SPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESP
GPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSG
SETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESP
SGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTP
SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGT
SESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS AP AM1318
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESP 1146 4.5
GPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
SASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
EEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSES
ATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
STSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTA
PGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPE
SGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESAT
PESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPS
GESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSP
AGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGS
PGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESST
APGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSAST
GTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSG
ESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGS
PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTG
SPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAE
SPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
Example 61: Calculation of TEPITOPE Scores
[0628] TEPITOPE scores of 9mer peptide sequence can be calculated
by adding pocket potentials as described by Sturniolo [Sturniolo,
T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example,
separate Tepitope scores were calculated for individual HLA
alleles. Table 47 shows as an example the pocket potentials for
HLA*0101B, which occurs in high frequency in the Caucasian
population. To calculate the TEPITOPE score of a peptide with
sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual
pocket potentials in Table 47 were added. The HLA*0101B score of a
9mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 1147) is the
sum of 0, -1.3, 0, 0.9, 0, -1.8, 0.09, 0, 0.
[0629] To evaluate the TEPITOPE scores for long peptides one can
repeat the process for all 9mer subsequences of the sequences. This
process can be repeated for the proteins encoded by other HLA
alleles. Tables 48-51 give pocket potentials for the protein
products of HLA alleles that occur with high frequency in the
Caucasian population.
[0630] TEPITOPE scores calculated by this method range from
approximately -10 to +10. However, 9mer peptides that lack a
hydrophobic amino acid (FKLMVWY) (SEQ ID NO: 1148) in P1 position
have calculated TEPITOPE scores in the range of -1009 to -989. This
value is biologically meaningless and reflects the fact that a
hydrophobic amino acid serves as an anchor residue for HLA binding
and peptides lacking a hydrophobic residue in P1 are considered non
binders to HLA. Because most XTEN sequences lack hydrophobic
residues, all combinations of 9mer subsequences will have TEPITOPEs
in the range in the range of -1009 to -989. This method confirms
that XTEN polypeptides may have few or no predicted T-cell
epitopes.
TABLE-US-00053 TABLE 47 Pocket potential for HLA*0101B allele.
Amino Acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -2.4 -- -2.7 -2 -- -1.9 E
-999 0.1 -1.2 -0.4 -- -2.4 -0.6 -- -1.9 F 0 0.8 0.8 0.08 -- -2.1
0.3 -- -0.4 G -999 0.5 0.2 -0.7 -- -0.3 -1.1 -- -0.8 H -999 0.8 0.2
-0.7 -- -2.2 0.1 -- -1.1 I -1 1.1 1.5 0.5 -- -1.9 0.6 -- 0.7 K -999
1.1 0 -2.1 -- -2 -0.2 -- -1.7 L -1 1 1 0.9 -- -2 0.3 -- 0.5 M -1
1.1 1.4 0.8 -- -1.8 0.09 -- 0.08 N -999 0.8 0.5 0.04 -- -1.1 0.1 --
-1.2 P -999 -0.5 0.3 -1.9 -- -0.2 0.07 -- -1.1 Q -999 1.2 0 0.1 --
-1.8 0.2 -- -1.6 R -999 2.2 0.7 -2.1 -- -1.8 0.09 -- -1 S -999 -0.3
0.2 -0.7 -- -0.6 -0.2 -- -0.3 T -999 0 0 -1 -- -1.2 0.09 -- -0.2 V
-1 2.1 0.5 -0.1 -- -1.1 0.7 -- 0.3 W 0 -0.1 0 -1.8 -- -2.4 -0.1 --
-1.4 Y 0 0.9 0.8 -1.1 -- -2 0.5 -- -0.9
TABLE-US-00054 TABLE 48 Pocket potential for HLA*0301B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 2.3 -- -2.4 -0.6 -- -0.6 E
-999 0.1 -1.2 -1 -- -1.4 -0.2 -- -0.3 F -1 0.8 0.8 -1 -- -1.4 0.5
-- 0.9 G -999 0.5 0.2 0.5 -- -0.7 0.1 -- 0.4 H -999 0.8 0.2 0 --
-0.1 -0.8 -- -0.5 I 0 1.1 1.5 0.5 -- 0.7 0.4 -- 0.6 K -999 1.1 0 -1
-- 1.3 -0.9 -- -0.2 L 0 1 1 0 -- 0.2 0.2 -- -0 M 0 1.1 1.4 0 --
-0.9 1.1 -- 1.1 N -999 0.8 0.5 0.2 -- -0.6 -0.1 -- -0.6 P -999 -0.5
0.3 -1 -- 0.5 0.7 -- -0.3 Q -999 1.2 0 0 -- -0.3 -0.1 -- -0.2 R
-999 2.2 0.7 -1 -- 1 -0.9 -- 0.5 S -999 -0.3 0.2 0.7 -- -0.1 0.07
-- 1.1 T -999 0 0 -1 -- 0.8 -0.1 -- -0.5 V 0 2.1 0.5 0 -- 1.2 0.2
-- 0.3 W -1 -0.1 0 -1 -- -1.4 -0.6 -- -1 Y -1 0.9 0.8 -1 -- -1.4
-0.1 -- 0.3
TABLE-US-00055 TABLE 49 Pocket potential for HLA*0401B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 1.4 -- -1.1 -0.3 -- -1.7 E
-999 0.1 -1.2 1.5 -- -2.4 0.2 -- -1.7 F 0 0.8 0.8 -0.9 -- -1.1 -1
-- -1 G -999 0.5 0.2 -1.6 -- -1.5 -1.3 -- -1 H -999 0.8 0.2 1.1 --
-1.4 0 -- 0.08 I -1 1.1 1.5 0.8 -- -0.1 0.08 -- -0.3 K -999 1.1 0
-1.7 -- -2.4 -0.3 -- -0.3 L -1 1 1 0.8 -- -1.1 0.7 -- -1 M -1 1.1
1.4 0.9 -- -1.1 0.8 -- -0.4 N -999 0.8 0.5 0.9 -- 1.3 0.6 -- -1.4 P
-999 -0.5 0.3 -1.6 -- 0 -0.7 -- -1.3 Q -999 1.2 0 0.8 -- -1.5 0 --
0.5 R -999 2.2 0.7 -1.9 -- -2.4 -1.2 -- -1 S -999 -0.3 0.2 0.8 -- 1
-0.2 -- 0.7 T -999 0 0 0.7 -- 1.9 -0.1 -- -1.2 V -1 2.1 0.5 -0.9 --
0.9 0.08 -- -0.7 W 0 -0.1 0 -1.2 -- -1 -1.4 -- -1 Y 0 0.9 0.8 -1.6
-- -1.5 -1.2 -- -1
TABLE-US-00056 TABLE 50 Pocket potential for HLA*0701B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -1.6 -- -2.5 -1.3 -- -1.2 E
-999 0.1 -1.2 -1.4 -- -2.5 0.9 -- -0.3 F 0 0.8 0.8 0.2 -- -0.8 2.1
-- 2.1 G -999 0.5 0.2 -1.1 -- -0.6 0 -- -0.6 H -999 0.8 0.2 0.1 --
-0.8 0.9 -- -0.2 I -1 1.1 1.5 1.1 -- -0.5 2.4 -- 3.4 K -999 1.1 0
-1.3 -- -1.1 0.5 -- -1.1 L -1 1 1 -0.8 -- -0.9 2.2 -- 3.4 M -1 1.1
1.4 -0.4 -- -0.8 1.8 -- 2 N -999 0.8 0.5 -1.1 -- -0.6 1.4 -- -0.5 P
-999 -0.5 0.3 -1.2 -- -0.5 -0.2 -- -0.6 Q -999 1.2 0 -1.5 -- -1.1
1.1 -- -0.9 R -999 2.2 0.7 -1.1 -- -1.1 0.7 -- -0.8 S -999 -0.3 0.2
1.5 -- 0.6 0.4 -- -0.3 T -999 0 0 1.4 -- -0.1 0.9 -- 0.4 V -1 2.1
0.5 0.9 -- 0.1 1.6 -- 2 W 0 -0.1 0 -1.1 -- -0.9 1.4 -- 0.8 Y 0 0.9
0.8 -0.9 -- -1 1.7 -- 1.1
TABLE-US-00057 TABLE 51 Pocket potential for HLA*1501B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -0.4 -- -0.4 -0.7 -- -1.9 E
-999 0.1 -1.2 -0.6 -- -1 -0.7 -- -1.9 F -1 0.8 0.8 2.4 -- -0.3 1.4
-- -0.4 G -999 0.5 0.2 0 -- 0.5 0 -- -0.8 H -999 0.8 0.2 1.1 --
-0.5 0.6 -- -1.1 I 0 1.1 1.5 0.6 -- 0.05 1.5 -- 0.7 K -999 1.1 0
-0.7 -- -0.3 -0.3 -- -1.7 L 0 1 1 0.5 -- 0.2 1.9 -- 0.5 M 0 1.1 1.4
1 -- 0.1 1.7 -- 0.08 N -999 0.8 0.5 -0.2 -- 0.7 0.7 -- -1.2 P -999
-0.5 0.3 -0.3 -- -0.2 0.3 -- -1.1 Q -999 1.2 0 -0.8 -- -0.8 -0.3 --
-1.6 R -999 2.2 0.7 0.2 -- 1 -0.5 -- -1 S -999 -0.3 0.2 -0.3 -- 0.6
0.3 -- -0.3 T -999 0 0 -0.3 -- -0 0.2 -- -0.2 V 0 2.1 0.5 0.2 --
-0.3 0.3 -- 0.3 W -1 -0.1 0 0.4 -- -0.4 0.6 -- -1.4 Y -1 0.9 0.8
2.5 -- 0.4 0.7 -- -0.9
Example 62: GPCR Ca2+Mobilization Activity Assay
[0631] Recombinant GLP2-2G-XTEN was prepared as described in
Alters, S. et al. (2012) GLP2-2G-XTEN: a pharmaceutical protein
with improved serum half-life and efficacy in a rat Crohn's disease
model. PLoS One; 7(11): e50630. The conjugate GLP2-2G-XTEN was
prepared as described in Example 26 (FIGS. 68A-68B) and purified by
preparative RP-HPLC (FIGS. 70A-70B). A GPCR Ca2+ flux mobilization
activity assay was performed using an EMD Millipore ChemiSCREEN
human recombinant GLP-2 glucagon family receptor calcium-optimized
stable cell line, used according to manufacturer's instructions,
with results presented in FIG. 110. Both the recombinant and
conjugated GLP2-2G-XTEN were profiled as an eight-point, three-fold
serial dilution dose response curve. Dose response curves were
fitted using a 4PL-regression plot with respective Y-axis RFU, with
data expressed as a percentage of absolute maximum RFU against
concentration on the X-axis. Both recombinant GLP2-2G-XTEN and
conjugate GLP2-2G-XTEN exhibited dose-dependent agonist activity
with predicted EC50 potency values of 423 nM and 529 nM,
respectively.
Example 63: In Vitro Plasma Stability Assay of Conjugate
GLP2-2G-XTEN
[0632] Equal concentrations of recombinant GLP2-2G-XTEN and
conjugate GLP2-2G-XTEN were independently spiked into respective
rat, cynomolgus monkey and human plasma. Samples were incubated at
37.degree. C. for up to 10 days with an aliquot removed at
appropriate time interval and stored at -80.degree. C. until
analysis. The plasma stability of conjugated GLP2-2G-XTEN in the
various species was compared to that of recombinant GLP2-2G-XTEN on
an anti-XTEN/GLP2 ELISA performed in the respective plasma
matrices. The anti-XTEN/GLP2 ELISA comprised of the anti-XTEN mouse
antibody as a capture antibody and a biotinylated anti-human GLP2
antibody as a detection antibody. As shown in FIG. 111, the in
vitro stability in human plasma of conjugate GLP2-2G-XTEN is
comparable to that of recombinant GLP2-2G-XTEN having an calculated
stability half-life of >240 h. Similar in vitro stability was
also observed with the two GLP2-2G-XTEN proteins in rat and
cynomolgus monkey plasma (data not shown).
Example 64: Pharmacokinetics of Conjugate GLP2-2G-XTEN in Rats
[0633] Female SD strain rats (200-220 g) were randomly assigned
into groups of 3 animals each. Recombinant GLP-2G-XTEN and
conjugate GLP2-2G-XTEN were administered by subcutaneous injection
at 2 mg/kg into each animal. Blood samples (0.2 ml) were collected
in pre-chilled heparinized microtainer tubes at pre-dose, 0.08, 4,
8, 24, 48, 72, 96, 120 and 168 hours after test compound
administration. The blood was then processed to plasma and stored
immediately at -80.degree. C. until analysis. Plasma samples were
analyzed using an anti-XTEN/GLP2 ELISA that uses the mouse
anti-XTEN antibody as a capture antibody and a biotinylated
anti-human GLP2 antibody as a detection antibody. The ELISA was
performed using relevant recombinant GLP2-2G-XTEN or conjugate
GLP2-2G-XTEN as the respective ELISA calibration standards (FIG.
112). The calculated half-life of recombinant GLP2-2G-XTEN (36
(.+-.7) h) and conjugate GLP2-2G-XTEN (37 (.+-.7) h) was found to
be similar.
Example 65: Trimeric XTEN Conjugate Linked Via C-Terminal
Cysteines
[0634] A trimeric XTEN conjugate was prepared by the following
procedure. An aliquot of the XTEN protein
1.times.Amino,1.times.Thiol-XTEN432 (XTEN_AE432(Am1,C422)), with
one internal cysteine residue, was prepared as a 587 .mu.M (23.23
mg/ml) solution in 20 mM HEPES, pH 7.0, 50 mM NaCl.
Tris-[2-maleimidoethyl]amine (TMEA, Thermo Scientific, cat. #33043)
was dissolved in anhydrous DMF to a final concentration 10 mM. TMEA
was added to the protein solution to link to the thiol group of the
XTEN (5.times. molar excess of protein over linker). The reaction
mixture was incubated for 2 hrs at 25.degree. C. and the products
of the reaction were analyzed by SEC-HPLC (Phenomenex
BioSep-SEC-s4000600.times.7.80 mm, buffer: 50 mM Sodium Phosphate
pH 6.5, 300 mM NaCl, flow rate 0.5 ml/min, isocratic elution for 70
min). Linear XTEN_432, XTEN_864 and XTEN_1296 (having 432, 864, and
1296 amino acids, respectively) were analyzed under the same
conditions to identify reaction products (FIGS. 113A-113D). Peak 1
eluted at 28 min, the same time as XTEN_1296 and was identified as
a trimer of XTEN_432. Peak 2 eluted at 30.5 min, the same time as
XTEN_864 and was identified as a dimer of XTEN_432. Peak 3 eluted
at 35 min, the same time as XTEN_432 and was identified as XTEN_432
precursor. The yields of trimeric XTEN conjugate and dimeric XTEN
conjugate were 19% and 36%, respectively. Essentially identical
retention times for the trimeric conjugate 3.times.XTEN_432 and the
linear molecule XTEN_12% suggest that the apparent molecular weight
and hydrodynamic radius of an XTEN protein is not dependent on its
geometric configuration.
Example 66: Trimeric XTEN Conjugates Linked Via N-Terminal
.alpha.-Amino Groups
[0635] A trimeric XTEN conjugate was prepared by the following
procedure.
[0636] 1. Synthesis of 1.times.DBCO-XTEN288
[0637] An aliquot of the protein 1.times.Amino-XTEN288
(XTEN_AE288(Am1)) was prepared as a 758 .mu.M (20 mg/ml) solution
in 20 mM HEPES, pH 7.0, 50 mM NaCl. 2 ml of protein was mixed with
0.1 ml 1M HEPES pH 8.0 and 0.152 ml of 50 mM DBCO-Sulfo-NHS (Click
Chemistry Tools, cat. #A124) dissolved in anhydrous DMF to link the
DBCO group to the N-terminal amino group of the XTEN. The reaction
mixture was incubated for 2 hours at 25.degree. C. and analyzed by
analytical RP-HPLC (FIG. 114A). The reaction mixture was diluted to
15 mL with 0.01% TFA and pH adjusted to .about.3 using 10% TFA
solution. The protein solution was divided into two equal parts and
each fraction was loaded on a preparative C4 RP-HPLC column Vydac
C4250.times.10 mm (Grace Davison Discovery Sciences, cat.
#214TP510). The protein was eluted with a 180 ml linear 5-50%
gradient of acetonitrile in 0.01% TFA at 2 ml/min flow rate.
Fractions containing 1.times.DBCO-XTEN288 were adjusted to
pH.about.7 with 1 M HEPES pH 8 and were concentrated by vacuum
evaporation.
[0638] 2. Synthesis of 3.times.Azide-PEG4-TAEA
[0639] Tris(2-aminoethyl)amine (TAEA, Sigma Aldrich, cat. #225630)
was diluted in anhydrous DMF to the final concentration 200 mM.
Azido-PEG4-NHS ester (Click Chemistry Tools, cat. #AZ103) was
dissolved in anhydrous DMF to the final concentration 1 M.
Azido-PEG4-NHS was mixed in 5-fold molar excess with
Tris(2-aminoethyl)amine and incubated at 25.degree. C. for 1 hour.
3.times.Azide-PEG4-TAEA was purified using C18 RP-HPLC using
Phenomenex Jupiter C185u 300 .ANG. 150.times.4.60 mm column, buffer
A 0.1% TFA in water, buffer B 0.1% TFA in acetonitrile, flow rate 1
ml/min, gradient 5 to 50% B in 45 min. Chromatographic peaks were
collected and analyzed by MALDI-TOF MS and ESI-MS to detect the
product with MW of 966 Da. 3.times.Azide-PEG4-TAEA was identified
as a peak with retention time 33 min (FIG. 114B). The fraction was
neutralized using 1M HEPES pH 8.0 and concentrated by vacuum
evaporation.
[0640] 3. Synthesis of the Trimeric XTEN Conjugate
[0641] 1.times.DBCO-XTEN288 was prepared as a 7.85 mg/ml (293 uM)
solution in 20 mM HEPES, pH 7.0, 50 mM NaCl.
3.times.Azide-PEG4-TAEA was RP-HPLC purified and formulated in the
same buffer. A concentration of the synthesized linker was not
determined, and 1.times.DBCO-XTEN288 and 3.times.Azide-PEG4-TAEA
were mixed empirically in various ratios and incubated at
25.degree. C. for 4 hours. Conjugation products were analyzed using
SEC-HPLC (Phenomenex BioSep-SEC-s4000600.times.7.80 mm, buffer: 50
mM Sodium Phosphate pH 6.5, 300 mM NaCl, flow rate 0.5 ml/min,
isocratic elution for 70 min). Linear XTEN_288, XTEN_576 and
XTEN_864 were analyzed under the same conditions to identify
reaction products (FIG. 115). Peak 1 eluted at 30.5 min, the same
time as XTEN_864 and was identified as a trimer of XTEN288. Peak 2
eluted at 34 min, the same time as XTEN_576 and was identified as a
dimer of XTEN_288. Peak 3 eluted at 39 min, the same time as
XTEN_288 and was identified as XTEN_288 precursor. Peak 4
corresponded to low molecular weight compounds and was not included
into quantitation of XTEN species. The yield of the trimeric XTEN
conjugate under the optimized protein/linker ratio was 57%.
Essentially identical retention times for trimeric conjugate
3.times.XTEN_288 and linear molecule XTEN_864 confirms the earlier
observation that the apparent molecular weight and hydrodynamic
radius of an XTEN protein is not dependent on its geometric
configuration.
Example 67: Selective Cytotoxicity of
3.times.FA(.gamma.),3.times.MMAE-XTEN on KB Cells
[0642] The ability to selectively target and kill cells bearing
folate receptors was evaluated. Test articles of free MMAE, a
non-targeting 3.times.MMAE-XTEN conjugate (XTEN linked to toxin)
and the folate receptor-targeted
3.times.FA(.gamma.),3.times.MMAE-XTEN conjugate were evaluated in a
CellTiter-Glo anti-proliferation assay using the folate
receptor-positive KB cell line. As culture media contain high folic
acid content, KB cells were grown in folic acid-free media
containing 10% heat-inactivated fetal calf serum at 37.degree. C.,
5% CO2 for at least 7 days prior to the commencement of the cell
viability experiment, This medium was also utilized for the
execution of the experiment. In brief, KB cells were plated at
10,000 cells per well onto a 96-well microtiter assay plate. KB
cells were allowed to adhere to the plate by an overnight
incubation at 37.degree. C., 5% CO2. The spent media was then
removed and wells designated to contain folic acid competitor
received assay medium containing folic acid, while wells not
designated to have folic acid competitor received assay medium
only. The plate was incubated for 30 min at 37.degree. C., 5% CO2
before the assay media was aspirated and plate washed with assay
media. Free MMAE, 3.times.MMAE-XTEN and
3.times.FA(.gamma.),3.times.MMAE-XTEN in the presence or absence of
folic acid competitor was then added at an appropriate range of
doses. The plate was then further incubated for 2-4 h at 37.degree.
C., 5% CO2. Media was then removed, the plate washed and fresh
media introduced and the plate was allowed to incubate for an
additional 48-72 h. After the appropriate incubation period,
CellTiter-Glo reagent was added and the plate was read on a
luminometer. The IC50 of each test article was determined using a 4
parameter logistic curve fit using GraphPad Prism.
[0643] Results: As shown in FIG. 116, free MMAE drug moiety shows
highly potent killing of KB cells, with an IC50 of 0.8 nM, while 3
copies of MMAE conjugated to non-targeting XTEN resulted in at
least a 3 log reduction in cell killing (IC50 >1,000 nM).
Significantly, the addition of 3 copies of folate targeting domains
to the 3.times.MMAE-XTEN conjugate restored the cell killing, with
an IC 50 of 4.2 nM; a level of activity close to that observed for
free MMAE. Of equal importance, the introduction of folic acid as a
competitor to the targeted conjugate impaired the observed cell
killing activity of 3.times.FA(.gamma.),3.times.MMAE-XTEN on the KB
cell line. This reduction from potent cell killing of the
folate-XTEN drug conjugate (from 4.2 nM to >1,000 nM) supports
the conclusion that the detected cell toxicity was, under the
experimental conditions, facilitated by use of the folate as the
targeting mechanism for the drug conjugate against the KB cell
line.
Example 68: In Vitro Cell-Based Screening of Folate-XTEN-Drug
Conjugates for Activity and Specificity
[0644] The ability to selectively target and kill cells bearing
folate receptors using targeted folate-XTEN-drug conjugates is
evaluated using an in vitro-based screening and selectivity
assay.
[0645] Each folate-XTEN-drug conjugate, its corresponding
non-targeting XTEN-drug molecule and respective free drug control
will be tested in a CellTiter-Glo anti-proliferation assay against
a panel of folate receptor positive and negative cell lines. Choice
of cell lines is based on relevance to proposed clinical
application and includes KB, IGROV, SK-OV-3, HeLa, LoVo, SW620,
Madison 109, A549, A375, LS-174T, HT-29, 4T1, SK-BR-3. As culture
media contain high folic acid content, cells will be grown and
assay performed in folic acid free-media containing 5-10%
heat-inactivated fetal calf serum (FCS) at 37.degree. C., 5%
CO.sub.2. Heat-inactivated FCS contains endogenous level of folic
acid sufficient for folate receptor expressing cells to survive and
proliferate. Appropriate assay conditions including optimal cell
density and incubation time are pre-determined in folate-free media
containing 5-10% FCS using the respective free drug as control.
Folate-XTEN-drug conjugates are then tested as follows: cells in
log-phase are collected, counted and plated at pre-determined cell
density onto each well of a 96-well microtiter assay plate.
Adherent cells are allowed to attach to the plate by an overnight
incubation at 37.degree. C., 5% CO.sub.2. Folate-XTEN-drug
conjugates and corresponding controls are introduced in a dose
range in duplicates and plate incubated for an additional 2 to 5
days. Alternatively, cells can also be pulsed with folate-XTEN-drug
conjugates and corresponding controls for 2-6 h, washed, fresh
media introduced and allowed to incubate for an additional 48-72 h.
After the appropriate incubation period, CellTiter-Glo reagent is
added to each well, mixed for 2 minute on an orbital shaker. Plate
is then centrifuged at 90 g and incubated at room temperature for
an additional 10 minutes to stabilize the luminescent signal.
Luminescence signals are then read on a luminometer &
IC.sub.50s (half maximal inhibitory concentration) calculated with
GraphPad Prism or equivalent software. Quantitative comparisons of
IC.sub.50s will enable ranking of the compounds' activity for
inhibition of cell growth and selectivity against folate receptor
positive versus negative cell lines.
[0646] It is expected that the results would support the finding
that the folate-XTEN-drug conjugates will show highly selective
potent killing on folate receptor positive cells but not on folate
receptor negative cells. This will be in contrast to the free drug
moiety whereby no discrimination in the strong cytotoxicity is
expected between folate receptor positive and negative cell lines.
The XTEN-drug control is expected to yield poor cytotoxic activity.
The folate-XTEN-drug conjugate with the most favorable activity and
cell line selectivity relative to controls will be further verified
for folate receptor association by the addition of free competitive
folic acid in the assay and demonstrating impaired folate-XTEN-drug
cytotoxicity.
Example 69: In Vitro Cell-Based Screening of LHRH-XTEN-Drug
Conjugates for Activity and Specificity
[0647] The ability to selectively target and kill cells bearing
LHRH receptors using targeted LHRH-XTEN-drug conjugates is
evaluated using an in vitro-based screening and selectivity assay.
Each LHRH-XTEN-drug conjugate, its corresponding non-targeting
XTEN-drug molecule and respective free drug control are tested in a
CellTiter-Glo anti-proliferation assay against a panel of LHRH
receptor positive and negative cell lines. Choice of cell lines is
based on relevance to proposed clinical application and includes
MCF-7, MDA-MB-231, HCC1806, HCC1937, OV-1063, EFO-21, EFO-27,
NIH:OVCAR-3, BG-1, HEC-1A, HEC-1B, Ishikawa, KLE, AN-3-CA, MiaPaCa,
Panc-1, rat Dunning R-3327-H, PC-82, MDA-PCa-2b, C4-2 (derivative
of LNCaP), A549, A2780, UCI-107, SK-OV-3, SW 626, MFE-296.
Appropriate assay conditions, including optimal cell density and
incubation time, are pre-determined using the respective free drug
as control. LHRH-XTEN-drug conjugates are tested as follows: cells
in log-phase are collected, counted and plated at pre-determined
cell density onto each well of a 96-well microtiter assay plate.
Adherent cells are allowed to attach to the plate by an overnight
incubation at 37.degree. C., 5% CO2. LHRH-XTEN-drug conjugates and
corresponding controls are introduced in a dose range in duplicates
and plate incubated for an additional 2 to 5 days depending on cell
lines used. After the appropriate incubation period, CellTiter-Glo
reagent is added to each well, mixed for 2 minute on an orbital
shaker. Plate is then centrifuged at 90.times.g and incubated at
room temperature for an additional 10 minutes to stabilize the
luminescent signal. Luminescence signals are then read on a
luminometer & IC50s (half maximal inhibitory concentration) are
calculated with GraphPad Prism or equivalent software. Quantitative
comparisons of IC50s will enable ranking of the compounds' activity
for inhibition of cell growth and selectivity against LHRH receptor
positive versus negative cell lines.
[0648] It is expected that the results would support the finding
that the LHRH-XTEN-drug conjugates will show highly-selective and
potent killing of LHRH receptor positive cells but not on LHRH
receptor-negative cells. This will be in contrast to the free drug
moiety whereby no discrimination in the strong cytotoxicity is
expected between LHRH receptor positive and negative cell lines.
The XTEN-drug control without the LHRH targeting moiety is expected
to yield poor cytotoxic activity. The LHRH-XTEN-drug conjugate with
the most favorable activity and cell line selectivity relative to
controls are further verified for LHRH receptor association by the
addition of free competitive LHRH peptide in the assay and
demonstrating impaired LHRH-XTEN-drug cytotoxicity.
Example 69: In Vitro Serum Stability of LHRH-XTEN-Drug
Conjugates
[0649] As a measure of drug linkage stability, LHRH-XTEN-drug
conjugates are incubated independently in normal human, cynomolgus
monkey and rodent plasma at 37.degree. C. for up to 2 weeks with an
aliquot removed at periodic interval and stored at -80.degree. C.
till analysis. The stability of LHRH-XTEN-drug conjugate can be
assessed either by the amount of free drug released or the
integrity of the LHRH-XTEN-drug conjugate over time. Free drug is
quantitated with RP-HPLC and/or LC-MS/MS whereas the amount of
intact LHRH-XTEN-drug conjugate is determined by a XTEN/drug and/or
LHRH/drug ELISA. For RP-HPLC analysis, plasma samples are treated
with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelength specific for a
particular drug. For example, doxorubicin is detected at 480 nm.
Calibration standards are prepared by adding known amounts of free
drug to corresponding plasma type and are treated in parallel with
experimental samples. For LC-MS/MS analysis, plasma samples are
treated with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
is treated in parallel with experimental samples. For quantitative
ELISA, optimal concentrations of antibodies for LHRH-XTEN-drug
conjugate in the ELISAs are determined using criss-cross serial
dilution analysis. An appropriate capture antibody recognizing one
component of the conjugate is coated onto a 96-well microtiter
plate by an overnight incubation at 4.degree. C. The wells are
blocked, washed and serum stability samples added to the wells,
each at varying dilutions to allow optimal capture of the
LHRH-XTEN-drug conjugate by the coated antibody. After washing,
detection antibody recognizing another component of the conjugate
is added and allowed to bind to the conjugate captured on the
plate. Wells are then washed again and either
streptavidin-horseradish peroxidase (complementary to biotinylated
version of detection antibody) or an appropriate secondary
antibody-horseradish peroxidase (complementary to non-biotinylated
version of detection antibody) is then added. After appropriate
incubation and a final wash step, tetramethylbenzidine (TMB)
substrate is added and plate read at 450 nM. Concentrations of
intact conjugate are then calculated for each time point by
comparing the colorimetric response to a calibration curve prepared
with LHRH-XTEN-drug in the relevant plasma type. The t1/2 of the
conjugate in human, cyno and mouse serum is then defined using
linear regression analysis of the log concentrations vs. time.
Example 70: In Vitro Serum Stability of Folate-XTEN-Drug
Conjugates
[0650] As a measure of drug linkage stability, folate-XTEN-drug
conjugates are incubated independently in normal human, cynomolgus
monkey and rodent plasma at 37.degree. C. for up to 2 weeks with an
aliquot removed at periodic interval and stored at -80.degree. C.
till analysis. The stability of folate-XTEN-drug conjugate can be
assessed either by the amount of free drug or the integrity of the
folate-XTEN-drug conjugate over time. Presence of free drug is
quantitated with HPLC, LC-MS/MS and/or with the anti-proliferation
assay as described in the relevant section above. The amount of
intact folate-XTEN-drug conjugate is determined by a XTEN/drug
and/or folate/drug ELISA. For RP-HPLC analysis, plasma samples are
treated with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelength specific for a
particular drug. For example, doxorubicin is detected at 480 nm.
Calibration standards are prepared by adding known amounts of free
drug to corresponding plasma type and is treated in parallel with
experimental samples. For LC-MS/MS analysis, plasma samples are
treated with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
is treated in parallel with experimental samples. When using the
anti-proliferation assay as a detection method for the presence of
de-conjugated free drug, folate receptor positive or negative cell
line could be employed. Folic acid inhibitor is added when
assessment is performed in receptor positive cell line and not
required when done in receptor negative cell line. In either
receptor cell type, increasing concentration of de-conjugated free
drug contributes to increased cell toxicity. For quantitative
ELISA, optimal concentrations of antibodies for folate-XTEN-drug
conjugate in the ELISAs are determined using criss-cross serial
dilution analysis. An appropriate capture antibody recognizing one
component of the conjugate is coated onto a 96-well microtiter
plate by an overnight incubation at 4.degree. C. The wells are
blocked, washed and serum stability samples added to the wells,
each at varying dilutions to allow optimal capture of the
folate-XTEN-drug conjugate by the coated antibody. After washing,
detection antibody recognizing another component of the conjugate
is added and allowed to bind to the conjugate captured on the
plate. Wells are then washed again and either
streptavidin-horseradish peroxidase (complementary to biotinylated
version of detection antibody) or an appropriate secondary
antibody-horseradish peroxidase (complementary to non-biotinylated
version of detection antibody) is then added. After appropriate
incubation and a final wash step, tetramethylbenzidine (TMB)
substrate is added and plate read at 450 nM. Concentrations of
intact conjugate are then calculated for each time point by
comparing the colorimetric response to a calibration curve prepared
with folate-XTEN-drug in the relevant plasma type. The t1/2 of the
conjugate in human, cyno and mouse serum is then defined using
linear regression analysis of the log concentrations vs. time.
Example 71: In Vivo and Ex Vivo Imaging of LHRH-XTEN-Cy5.5
Conjugate
[0651] A Cy5.5 fluorescent tagged LHRH-XTEN conjugate molecule is
used as a surrogate to investigate the targeting and
biodistribution efficiency of LHRH-XTEN-drug conjugates.
Experiments are carried out in nude mice bearing subcutaneous grown
xenografts of LHRH receptor positive tumor cells using in vivo,
followed by ex vivo, fluorescence imaging with IVIS 50 optical
imaging system (Caliper Life Sciences, Hopkinton, Mass.). In brief,
female nu/nu mice bearing LHRH receptor positive tumor cells are
given a single intravenously injection of high or low dose
LHRH-XTEN-Cy5.5 and corresponding doses of non-targeting Cy5.5
tagged XTEN control. Whole body scans are acquired pre-injection
and then at approximately 8, 24, 48 and 72 hours post-injection on
live anesthetized animals using the IVIS 50 optical imaging system.
After measuring the distribution of fluorescence in the entire
animal at the last time point of 72 h, tumor and healthy organs
including liver, lung, heart, spleen and kidneys are excised and
their fluorescence registered and processed by the imaging system.
Cy5.5 excitation (615-665 nm) and emission (695-770 nm) filters are
selected to match the fluorescence agents' wavelengths. Small and
medium binning of the CCD chip is used and the exposure time
optimized to obtain at least several thousand counts from the
signals that were observable in each mouse in the image and to
avoid saturation of the CCD chip. To normalize images for
quantification, a background fluorescence image is acquired using
background excitation and emission filters for the Cy5.5 spectral
region. The intensity of fluorescence is expressed by different
colors with blue color reflecting the lowest intensity and red is
indicative of the highest intensity.
Example 72: In Vivo and Ex Vivo Imaging of Folate-XTEN-Cy5.5
Conjugates
[0652] A Cy5.5 fluorescent tagged folate-XTEN molecule is used as a
surrogate to investigate the targeting and biodistribution
efficiency of folate-XTEN-drug conjugates. Experiments are carried
out in nude mice bearing subcutaneous grown xenografts of folate
receptor positive tumor cells using in vivo, followed by ex vivo,
fluorescence imaging with IVIS 50 optical imaging system (Caliper
Life Sciences, Hopkinton, Mass.). As culture media contain high
folate content, folate receptor positive tumor cells to be
transplanted onto these mice are grown in folate-free cell culture
media containing 5-10% heat-inactivated FCS with no antibiotics.
Similarly, normal rodent chow contains a high concentration of
folic acid; nude mice used in this study are maintained on
folate-free diet 2 weeks prior to tumor implantation and for the
duration of the imaging analysis to reduce serum folate
concentration. Female nu/nu mice bearing folate receptor positive
tumor cells are given a single intravenously injection of high or
low dose folate-XTEN-Cy5.5 and corresponding doses of non-targeting
Cy5.5 tagged XTEN control. Whole body scans are acquired
pre-injection and then at approximately 8, 24, 48 and 72 hours
post-injection on live anesthetized animals using the IVIS 50
optical imaging system. After measuring the distribution of
fluorescence in the entire animal at the last time point of 72 h,
tumor and healthy organs including liver, lung, heart, spleen and
kidneys are excised and their fluorescence registered and processed
by the imaging system. Cy5.5 excitation (615-665 nm) and emission
(695-770 nm) filters are selected to match the fluorescence agents'
wavelengths. Small and medium binning of the CCD chip is used and
the exposure time optimized to obtain at least several thousand
counts from the signals that were observable in each mouse in the
image and to avoid saturation of the CCD chip. To normalize images
for quantification, a background fluorescence image is acquired
using background excitation and emission filters for the Cy5.5
spectral region. The intensity of fluorescence is expressed by
different colors with blue color reflecting the lowest intensity
and red is indicative of the highest intensity.
Example 73:Pharmacokinetic Analysis of LHRH-XTEN-Drug
Conjugates
[0653] The in vivo pharmacokinetics of LHRH-XTEN-drug constructs is
assessed using standard methods for protein compositions.
Pharmacokinetics are assessed in multiple species, however mice,
rats, cynomolgus monkeys, and dogs are preferred due to their
common usage in predicting human pharmacokinetics. Compositions of
LHRH-XTEN-drug constructs are provided in an aqueous buffer
compatible with in vivo administration (for example:
phosphate-buffered saline, Tris-buffered saline or Hepes-buffered
saline). The compositions are administered at appropriate doses and
via multiple routes: most preferably via intravenous or
subcutaneous routes. Blood samples are collected at appropriate
time points ranging from 0.08 to 504 hours, and processed into
plasma. Plasma samples will then be analyzed for concentration of
LHRH-XTEN-drug conjugates by one of a variety of methods including
ELISA, HPLC and/or LC-MS/MS. ELISA analysis will be performed using
a sandwich ELISA format that can recognize 2 components of the
LHRH-XTEN-drug conjugate, for instance, XTEN/LHRH, XTEN/drug
moiety, LHRH/drug moiety and/or XTEN/XTEN combinations. Typically
antibody recognizing one component of the LHRH-XTEN-drug conjugate
is coated onto wells of a 96-well microtiter plate. The wells are
blocked, washed and plasma samples that have been collected at
different time points are then added to the wells, each at varying
dilutions, to allow capture of the conjugate by the coated
antibody. Wells are then washed extensively, and bound protein
detected using either a biotinylated antibody or an appropriate
secondary antibody against the second LHRH-XTEN-drug conjugate
component. Wells are then washed again and streptavidin-horseradish
peroxidase (complementary to the biotinylated detection antibody)
or a secondary antibody-horseradish peroxidase (complementary to a
non biotinylated detection antibody) is then added. After
appropriate incubation and a final wash step, tetramethylbenzidine
(TMB) substrate is added and plate read at 450 nM. Concentrations
of conjugate are then calculated for each time point by comparing
the colorimetric response to a LHRH-XTEN-drug calibration curve.
Pharmacokinetic parameters are calculated using the WinNonLin
software package. For RP-HPLC analysis, plasma samples are treated
with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelength specific for a
particular drug. For example, doxorubicin is detected at 480 nm.
Calibration standards are prepared by adding known amounts of free
drug to corresponding plasma type and are treated in parallel with
experimental samples. For LC-MS/MS analysis, plasma samples are
treated with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
are treated in parallel with experimental samples. It is expected
that the results would support the finding that addition of an XTEN
to LHRH and drug moiety as a conjugate preparation will greatly
increase the terminal half-life and enhance pharmacokinetic
properties compared to targeting peptides and drug moiety not
linked to XTEN. This in turn will translate into a less frequent,
more convenient dosing regimen for such conjugates.
Example 74: Pharmacokinetic Analysis of Folate-XTEN-Drug
Conjugates
[0654] The in vivo pharmacokinetics of folate-XTEN-drug constructs
are assessed using standard methods for protein compositions.
Pharmacokinetics are assessed in multiple species, however mice,
rats, cynomolgus monkeys, and dogs are preferred due to their
common usage in predicting human pharmacokinetics. As normal feed
contains a high concentration of folic acid (example 6 mg/kg mouse
chow), animals to be used in pharmacokinetic studies of folate
conjugates are maintained on folate-free diet for 2 weeks prior to
study initiation and for the duration of the study. Compositions of
folate-XTEN-drug constructs are typically provided in an aqueous
buffer compatible with in vivo administration (for example:
phosphate-buffered saline, Tris-buffered saline or Hepes-buffered
saline). The compositions would be administered at appropriate
doses and via multiple routes: most preferably via intravenous or
subcutaneous routes. Blood samples would be collected at
appropriate time points ranging from 0.08 to 504 hours, and
processed into plasma. Plasma samples will then be analyzed for
concentration of folate-XTEN-drug conjugates by a variety of
methods including ELISA, HPLC and/or LC-MS/MS. ELISA analysis are
performed using a sandwich ELISA format that can recognize 2
components of the folate-XTEN-drug conjugate, for instance,
XTEN/folate, XTEN/drug moiety, folate/drug moiety and/or XTEN/XTEN
combinations. Typically antibody recognizing one component of the
folate-XTEN-drug conjugate is coated onto wells of a 96-well
microtiter plate. The wells are blocked, washed and plasma samples
that have been collected at different time points are then added to
the wells, each at varying dilutions, to allow capture of the
conjugate by the coated antibody. Wells are then washed
extensively, and bound protein detected using either a biotinylated
antibody or an appropriate secondary antibody against the second
LHRH-XTEN-drug conjugate component. Wells are then washed again and
streptavidin-horseradish peroxidase (complementary to the
biotinylated detection antibody) or a secondary
antibody-horseradish peroxidase (complementary to a non
biotinylated detection antibody) is then added. After appropriate
incubation and a final wash step, tetramethylbenzidine (TMB)
substrate is added and plate read at 450 nM. Concentrations of
conjugate are then calculated for each time point by comparing the
colorimetric response to a folate-XTEN-drug calibration curve.
Pharmacokinetic parameters are calculated using the WinNonLin
software package. For RP-HPLC analysis, plasma samples are treated
with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are detected by UV absorption at wavelength specific for a
particular drug. For example, doxorubicin is detected at 480 nm.
Calibration standards are prepared by adding known amounts of free
drug to corresponding plasma type and are treated in parallel with
experimental samples. For LC-MS/MS analysis, plasma samples are
treated with organic solvents such as acetonitrile or acetone to
precipitate proteins. Soluble fractions are evaporated in vacuum,
redissolved in loading solutions and analyzed by RP-HPLC. Analytes
are in-line detected and quantitated by triple quadrupole tandem
mass spectrometry. Parental ion-daughter ion pairs are determined
experimentally for each drug. Calibration standards are prepared by
adding known amounts of free drug to corresponding plasma type and
are treated in parallel with experimental samples. It is expected
that the results would support the finding that addition of an XTEN
to folate and drug moiety as a conjugate preparation will greatly
increase the terminal half-life and enhance pharmacokinetic
properties compared to targeting peptides and drug moiety not
linked to XTEN. This in turn will translate into a less frequent,
more convenient dosing regimen for such conjugates.
Example 75: In Vivo Efficacy and Toxicity Analysis of
LHRH-XTEN-Drug Conjugates
[0655] LHRH-XTEN-drug conjugate is intended for targeted delivery
of highly potent toxin to LHRH receptor positive tumor cells. As
such, the in vivo pharmacologic activity of LHRH-XTEN-drug
constructs can be assessed using human tumor cell expressing LHRH
receptor transplanted onto nude mice. Prior to beginning the
efficacy study, an initial assessment in nude mice is carried out
to establish the maximum tolerated dose (MTD) of the LHRH-XTEN-drug
candidates. The MTD, the highest dose that is tolerated by the
animal for the study duration, will then be used to calculate the
dose range for the efficacy and toxicity study in the standard
xenograft model. Briefly, the MTD experiment are carried out with 5
mice per group evaluating the intravenous administration of
LHRH-XTEN-drug conjugates at various dose level, interval and
duration. The starting MTD dose and number of dose groups required
are based on scientific literature, knowledge of the targeting LHRH
moiety, the nature of the drug moiety conjugated, toxicological
properties of closely related compounds, and data from the initial
pharmacokinetic studies (see section above). Standard MTD
parameters such as reduction in body weight, food and water
consumption and signs of piloerection, hunched, behavior patterns,
respiratory pattern, tremors, convulsions, prostration and
self-mutilation is carefully monitored on a daily basis. The
highest dose of LHRH-XTEN-drug that does not cause unacceptable
toxicity is assigned as the MTD. The tumor xenograft study will
include 3 to 4 dosing levels of LHRH-XTEN-drug conjugate and will
depend on the results from the MTD study; with other parameters
depending on the tumor cell line chosen. Example 69 describes
examples of tumor lines that can be used in the xenograft study.
Thus an appropriate number of LHRH receptor positive cells from the
relevant human tumor line are injected subcutaneously and allowed
to form tumors, the size of which is measured with calipers and the
volume calculated as 0.5.times.L.times.W.sup.2, where L=measurement
of longest axis in millimeters and W=measurement of axis
perpendicular to L in millimeters. Following randomization of mice
containing tumor volume in the desired size range into groups of
8-10 animals, vehicle control, free drug control and LHRH-XTEN-drug
conjugate are administered intravenously at the chosen doses and
interval. Cessation or regression of tumor growth is determined
through measuring the tumor size and hence volume at selected time
points with calipers. Body weights and food consumption are
measured every 1 to 2 days to assess gross toxicity. Survival of
animals is monitored daily. At the end of the study, all animals
are sacrificed and clinical pathology and histopathology on major
organs are performed. Targeted cytotoxins are among the most
promising strategies for the selective elimination of malignant
cells. It is anticipated that the results would support the finding
that the LHRH-XTEN-drug conjugate will produce an excellent
therapeutic index as exhibited by potent efficacy and low systemic
toxicity. In contrast, the non-LHRH targeted free drug dosed at
equimolar doses will not only be less potent but more highly toxic.
The vehicle control is expected to display uncontrolled tumor
growth and severe toxicity.
Example 76: In Vivo Efficacy and Toxicity Analysis of
Folate-XTEN-Drug Conjugates
[0656] Folate-XTEN-drug conjugate is intended for targeted delivery
of highly potent toxin to folate receptor positive tumor cells. As
such, the in vivo pharmacologic activity of folate-XTEN-drug
constructs can be assessed using human tumor cell expressing folate
receptor xenograft onto nude mice. Prior to beginning the efficacy
study, an initial assessment in nude mice are carried out to
establish the maximum tolerated dose (MTD) of the folate-XTEN-drug
candidates. The MTD, the highest dose that is tolerated by the
animal for the study duration, will then be used to calculate the
dose range for the efficacy and toxicity study in the standard
xenograft model. As normal rodent chow contains a high
concentration of folic acid (6 mg/kg chow), mice to be used in
these studies are maintained on folate-free diet for 2 weeks prior
to study initiation and for the duration of the study. The MTD
experiment is carried out with 5 mice per group evaluating the
intravenous administration of folate-XTEN-drug conjugates at
various dose level, interval and duration. The starting MTD dose
and number of dose groups required is based on scientific
literature, knowledge of the targeting folate moiety, the nature of
the drug moiety conjugated, toxicological properties of closely
related compounds, and data from the initial pharmacokinetic
studies (see section above). Standard MTD parameters such as
reduction in body weight, food and water consumption and signs of
piloerection, hunched, behavior patterns, respiratory pattern,
tremors, convulsions, prostration and self-mutilation is carefully
monitored on a daily basis. The highest dose of folate-XTEN-drug
that does not cause unacceptable toxicity is assigned as the MTD.
The tumor xenograft study will include 3 to 4 dosing levels of
folate-XTEN-drug and will depend on the results from the MTD; with
other parameters depending on the tumor cell line chosen. Example
69 describes examples of tumor lines that can be used in the
xenograft study. To reduce folate content, folate receptor positive
tumor cells to be transplanted onto nude mice are grown in
folate-free cell culture media containing 5-10% heat-inactivated
fetal calf serum with no antibiotics. Similarly, to reduce serum
folate concentration, mice used in the xenograft studies are
maintained on folate-free diet 2 weeks prior to tumor implantation
and for the duration of the study. Thus an appropriate number of
folate receptor positive cells from the relevant line are injected
subcutaneously and allowed to form tumors, the size of which is
measured with calipers and the volume calculated as
0.5.times.L.times.W.sup.2, where L=measurement of longest axis in
millimeters and W=measurement of axis perpendicular to L in
millimeters. Following randomization of mice containing tumor
volume in the desired size range into groups of 8-10 animals,
vehicle control, free drug control and folate-XTEN-drug are
administered intravenously at the chosen doses and interval.
Cessation or regression of tumor growth is determined through
measuring the tumor size and hence volume at selected time points
with calipers. Body weights and food consumption are measured every
1 to 2 days to assess gross toxicity. Survival of animals is
monitored daily. At the end of study, all animals are sacrificed
and clinical pathology and histopathology on major organs are
performed. Targeted cytotoxins are among the most promising
strategies for the selective elimination of malignant cells. It is
anticipated that targeted chemotherapeutic folate-XTEN-drug
conjugate will be more effective and less toxic than free cytotoxic
drug alone on folate receptor positive tumors.
Example 76: Clinical Applications of LHRH-XTEN-Drug Conjugates
[0657] Targeted chemotherapy is a modern approach aimed at
increasing the efficacy of systemic chemotherapy and reducing its
side effects. Brentuximab vedotin (Adcentris), approved for Hodgkin
lymphoma and systemic anaplastic large cell lymphoma is a leading
example of effective toxin-targeted therapy. LHRH is a peptide that
functions in reproductive organs. Because its receptors are
particularly concentrated on certain tumors but are not expressed
in most normal tissue, LHRH receptor is an ideal target for
selective destruction of malignant tumors. Indeed, .about.52% of
breast, .about.80% of ovarian and endometrial, and .about.85% of
prostate cancer specimens is targetable via the LHRH receptor. Of
note, LHRH-dependent therapies would be especially useful for
triple negative breast tumors, which do not overexpress estrogen or
progesterone receptors or HER2 and are therefore unsuitable for
treatment with many available targeted drugs. Patients with
advanced endometrial, ovarian, or prostate cancer often have
particularly poor outcomes, as these malignancies can be prone to
recurrence and/or resistant to current treatments. In support of
this, clinical studies with AEZS-108, a targeted doxorubicin analog
of LHRH, indicate that each of these cancer types is susceptible to
LHRH-based therapies. Fusion of a XTEN carrying .gtoreq.1 copy of
LHRH to a XTEN bearing: 3 drug molecules to create a targeted
peptide-drug conjugate is expected to have vastly improved
therapeutic index and half-life that will enable dosing at levels
way below MTD, reduce dosing frequency and cost (reduced drug
required per dose).
[0658] Clinical evaluation of LHRH-XTEN-drug composition are
conducted in patients suffering from advanced breast, endometrial,
ovarian, and prostate or bladder cancers. Clinical trials are
designed such that the efficacy and advantages of the
LHRH-XTEN-drug conjugate can be verified in humans. Such studies in
patients would comprise three phases. First, a Phase I safety and
pharmacokinetics study is conducted to determine the maximum
tolerated dose (MTD) and to characterize the dose-limiting
toxicity, pharmacokinetics and preliminary pharmacodynamics in
humans. These initial studies are performed in patients with
metastatic or unresectable cancers and for which standard curative
or palliative measures could not be used or were no longer
effective or tolerated. To enhance treatment efficacy, LHRH
receptor positive status would be an enrollment criteria;
determined by immunohistochemistry of primary tumors or metastatic
specimens and/or by LHRH-targeted molecular imaging agent. The
scheme of the phase I study is to use single escalating doses of
LHRH-XTEN-drug conjugate and measure the biochemical, PK, and
clinical parameters. This would permit the determination of the MTD
and establish the threshold and maximum concentrations in dosage
and in circulating drug that constitute the therapeutic window to
be used in subsequent Phase II and Phase III trials. It also
defines potential toxicities and adverse events to be tracked in
future studies.
[0659] Phase II clinical studies of human patients are
independently conducted in LHRH receptor positive advanced (stage 3
or 4) or recurrent breast, endometrial, ovarian, and prostate or
bladder cancer patients. The trial evaluates the efficacy and
safety of LHRH-XTEN-drug conjugate alone and in combination with a
current chemotherapy employed in that specific indication. Patients
receive intravenously administered LHRH-XTEN-drug clinical
candidate at a dose level and regimen pre-determined in Phase I
with or without the standard chemo-agent A control arm comprising
of the chemo-agent plus placebo is included. The primary endpoint
is response rate as defined by the Response Evaluation Criteria in
Solid Tumors (RECIST). Secondary endpoints include safety and
tolerability, time-to-progression and overall survival.
[0660] A phase III efficacy and safety study is structured to
replicate or modify the phase II trial design in LHRH receptor
positive advanced (resistant, recurrent) breast, endometrial,
ovarian, and prostate or bladder cancer patients depending on the
phase II clinical observations. Refinement of patient enrollment
criteria, further patient stratification (example LHRH receptor
expression level), dosage, regimen, status of standard chemo-agent
etc. could be further adjusted. The primary endpoint is
progression-free-survival, as measured by RECIST, in patients
defined as LHRH receptor positive. The trial is statistically
powered for overall survival as a secondary endpoint with projected
enrollment in excess of 400 patients. Incidence of adverse events,
serious adverse event and deaths is assessed.
[0661] It is anticipated that LHRH-XTEN-drug candidate will
demonstrate anticancer activity without cardiotoxicity even in
these highly-pretreated patient populations.
Example 76: Clinical Applications of Folate-XTEN-Drug
Conjugates
[0662] Targeted chemotherapy is a modern approach aimed at
increasing the efficacy of systemic chemotherapy and reducing its
side effects. Brentuximab vedotin (Adcentris), approved for Hodgkin
lymphoma and systemic anaplastic large cell lymphoma is a leading
example of effective toxin targeted therapy. Folate, also known as
folic acid, vitamin B.sub.9, is a vital nutrient required by all
living cells for nucleotide biosynthesis and function as cofactor
in certain biological pathways. It is especially important in
aiding rapid cell division and growth. As such, the folate receptor
is a focus for the development of therapies to treat fast dividing
malignancy in particular ovarian cancer and non-small cell lung
carcinoma. Some ovarian tumor type is likely to recur after initial
successes with surgery and platinum-based chemotherapy, to which
the regrowth can become resistant to available therapies. While
folate receptor expression is negligible in normal ovary,
.about.90% of epithelial ovarian cancers overexpress the folate
receptor, as do many lung adenocarinomas, thereby opening the
possibility of directed therapies. In support of this, clinical
studies with EC-145, a targeted vinca alkaloid analog of folate,
indicated that platinum-resistant ovarian cancer and non-small cell
lung carcinoma are susceptible to folate-based therapies. Fusion of
a XTEN carrying 2:1 copy of folate to a XTEN bearing .gtoreq.3 drug
molecules to create a targeted peptide-drug conjugate is expected
to have vastly improved therapeutic index and half-life that will
enable dosing at levels way below maximum tolerated dose (MTD),
reduce dosing frequency and cost (reduced drug required per
dose).
[0663] Clinical evaluation of folate-XTEN-drug composition is
conducted in patients with relapsed or refractory advanced tumors
or specifically in patients suffering from platinum-resistant
ovarian cancer and non-small cell lung carcinoma who have failed
numerous chemotherapies. Clinical trials are designed such that the
efficacy and advantages of the folate-XTEN-drug conjugate can be
verified in humans. Such studies in patients would comprise three
phases. First, a Phase I safety and pharmacokinetics study is
conducted to determine the MTD and to characterize the
dose-limiting toxicity, pharmacokinetics and preliminary
pharmacodynamics in humans. These initial studies are performed in
patients with relapsed or refractory advanced tumors and for which
standard curative or palliative measures could not be used or were
no longer effective or tolerated. To enhance treatment efficacy,
folate receptor positive status is an enrollment criteria,
determined by immunohistochemistry of primary tumors or metastatic
specimens and/or by folate-targeted molecular imaging agent. The
scheme of the phase I study is to use single escalating doses of
folate-XTEN-drug conjugate and measure the biochemical, PK, and
clinical parameters. This would permit the determination of the MTD
and establish the threshold and maximum concentrations in dosage
and in circulating drug that constitute the therapeutic window to
be used in subsequent Phase II and Phase III trials. It also
defines potential toxicities and adverse events to be tracked in
future studies.
[0664] Phase II clinical studies of human patients are
independently conducted in folate receptor positive
platinum-resistant ovarian cancer patient population; non-small
cell lung carcinoma patients having failed numerous chemotherapies;
and patients suffering from relapsed or refractory advanced tumors.
The trial evaluates the efficacy and safety of folate-XTEN-drug
conjugate alone and in combination with a current chemotherapy
employed in the specific indication. Patients receive intravenously
administered folate-XTEN-drug conjugate at a dose level and regimen
pre-determined in Phase I with or without the standard chemo-agent.
A control arm comprising of the chemo-agent plus placebo is
included. The primary endpoint is response rate as defined by the
Response Evaluation Criteria in Solid Tumors (RECIST). Secondary
endpoints would include safety and tolerability,
time-to-progression and overall survival.
[0665] A phase III efficacy and safety study is structured to
replicate or modify the phase II trial design in folate receptor
positive platinum-resistant ovarian cancer patients; non-small cell
lung carcinoma patients; and advanced tumor relapsed or refractory
patients depending on the phase II clinical observations.
Refinement of patient enrollment criteria, further patient
stratification (example folate receptor expression level), dosage,
regimen, status of standard chemo-agent etc., is further adjusted.
The primary endpoint is progression-free-survival, as measured by
RECIST, in patients defined as folate receptor positive. The trial
will also be statistically powered for overall survival as a
secondary endpoint with projected enrollment in excess of 400
patients. Incidence of adverse events, serious adverse event and
deaths is also assessed.
[0666] It is anticipated that folate-XTEN-drug candidate will
demonstrate anticancer activity without severe toxicity even in
these highly pretreated patient populations.
TABLE-US-00058 TABLE 52 XTEN-cleavage sequence-affinity tag
Polypeptide Sequences SEQ Construct Amino Acid Sequence ID NO:
AC710 APTTAGAGRPRPRPRPRPRPRPRPRPRPRPGRGSPGSPAGSPTSTE 1149
EGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT
PESGPGTSTEPSEGSAPGRHHHHHHHH AC698
ANTPVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLT 1150
LRYYYTVDGQKDQTFWADHAAIIGSNGSYNGITSNVKGTFVKM
SSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSN
DYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSVVGSGSGSG
RGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPTAEAAGKPGTAEAAGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP
SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT
STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG KATHHHHHHHH AC815
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1151
GSPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSAS RSAHHHHHHHH AC816
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1152
GSPTAEAAGCGTAEAAGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPTAEAAGCGTAEAAG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPTAEAAGCGTAEAASASRSAHHHHHHHH AC763
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1153
GSPTAEAAGCGTAEAAGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAE
AAGCGTAEAASASRSAHHHHHHHH AC767
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1154
GSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG
SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APSASRSAHHHHHHHH
AC769 KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1155
GSPTAEAAGCGTAEAAPGSEPATSGSETPGTSESATPESGPGSEP
ATSGSETPGTAEAAGCGTAEAASTEPSEGSAPGTSESATPESGPG
SPAGSPTSTEEGSPATAEAAGCGTAEAASPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATTAEAAGCGTAEAASETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGTAEAAGCGTAEAAGSPA
GSPTSTEEGTSESATPESGPGSEPATSGSETPGTTAEAAGCGTAE
AAAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESTAEAAG
CGTAEAATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGT
AEAAGCGTAEAATEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPTAEAAGCGTAEAASASRSAHHHHHHHH AC771
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1156
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPTAEAAGCGTAEAAPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTAEAAGCGTAEAASTEPSEGSAP
GTSESATPESGPGSPAGSPTSTEEGSPATAEAAGCGTAEAASPTS
TEEGTSESATPESGPGTSTEPSEGSAPGTSESATTAEAAGCGTAE
AASETPGTSESATPESGPGSEPATSGSETPGTSESATPESGTAEAA
GCGTAEAAGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
TTAEAAGCGTAEAAAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESTAEAAGCGTAEAATPESGPGTSESATPESGPGSEPATS
GSETPGSEPATSGTAEAAGCGTAEAATEEGTSTEPSEGSAPGTST
EPSEGSAPGSEPATSGSETPTAEAAGCGTAEAASASRSAHHHHH HHH AC817
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1157
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTES ASRSAHHHHHHHH AC818
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1158
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTESASRSAHHHHHHHH AC780
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1159
GSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE
GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPSASRSAHHHHHHHH AC785
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1160
GSPTEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATPESGPGTSE
SATPESGPGSEPATSGSETPGSEPATSGTAEAAGCGTAEAATEEG
TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAEAAGCGTAE AASASRSAHHHHHHHH
AC765 KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1161
GSPTPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAEAAG
CGTAEAASASRSAHHHHHHHH AC766
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1162
GSPTGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPTAEAAGCGTAEAAGSPAGSPTS
TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESTAEAAGCGTAE
AATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPT
AEAAGCGTAEAASASRSAHHHHHHHH AC768
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1163
GSPTPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESTAEAAGCGTAEAATP
ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGTAEAAGCGT
AEAATEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPTAE
AAGCGTAEAASASRSAHHHHHHHH AC786
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1164
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS
GSETPGTSESATPESGPGTESASRSAHHHHHHHH AC792
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1165
GSPGSTSSTAESPGPGSTSSTAESPGPGCTSESPSGTAPGSTSSTAE
SPGPGSTSSTAESPGPGTSTPESGSASPGSTSCSPSGEAPGTSPSGE
SSTAPGSTSESPSGTAPGSTSESPSGTAPETSPSGESCTAPGSTSAS RSAHHHHHHHH AC793
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1166
GSPGTPGSGTASSSPGSSTPSGATGSPGCAGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTCSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTACSSPGSS SASRSAHHHHHHHH AC798
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1167
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG
SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS
GSETPGTSESATPESGPGTESASKSAHHHHHHHH AC809
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1168
GSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG
SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTACSEGS APSASRSAHHHHHHHH
AC810 KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1169
GSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT
STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG
SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSC ASASRSAHHHHHHHH
AC831 KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1170
GSPGSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAE
SPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGE
SSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSES
PSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSE
SPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST
PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTS
TPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGS
TSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPG
TSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAP
GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSAS
PGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESP
GPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGS
ASPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSTPESG
SASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGE
SSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGSTSST
AESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTS
ESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTS
PSGESSTAPGTSSASRSAHHHHHHHH AC832
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1171
GSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT
ASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSST
PSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGS
STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS
SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS
SPGSSTPSGATGSPGSSTPSGATGSPGASSASRSAHHHHHHHH AC819
KKQEQEEKKAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1172
GSPGSCAGSPTSTEEGTSESACPESGPGTSTEPSEGSCPGSPAGSP
TSTEEGTCTEPSEGSAPGTSTEPCSGSAPGTSESATPESCPGSEPA
TSGSETPGSCPATSGSETPGSPAGSCTSTEEGTSESATPESCPGTE SASRSAHHHHHHHH AC820
KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1173
GSPTGCGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
TSCTPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSC GSAPSASRSAHHHHHHHH
AC821 KNPEQAEEQAEEQREETRPRPRPRPRPRPRPRPRPRPRPSASRSA 1174
GSPTGCGSEPATSGSETPGTSESATPESGPGSEPATSGSCTPGTSE
SATPESGPGTSTEPSEGSAPGSPAGSPCSTEEGTSESATPESGPGS
EPATSGSETPGTSESCTPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
TSCTPSEGSAPGTSESATPESGPGTSESATPESGPGCSESATPESGP
GSEPATSGSETPGSEPATSGSETCGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGCAPGSEPATSGSETPGTSESATPESGPGTSTEPSC GSAPSASRSAHHHHHHHH
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210322518A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210322518A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References