U.S. patent application number 16/521789 was filed with the patent office on 2020-03-19 for factor viii compositions and methods of making and using same.
The applicant listed for this patent is BIOVERATIV THERAPEUTICS INC.. Invention is credited to Pei-Yun Chang, Sheng Ding, Nathan Geething, Haiyan Jiang, John Kulman, Tongyao Liu, Baisong Mei, Robert Peters, Volker Schellenberger, Joshua Silverman, Benjamin Spink, Willem P. Stemmer, Garabet G. Toby, Fatbardha Varfaj, Chia-wei Wang, Deping Wang.
Application Number | 20200087379 16/521789 |
Document ID | / |
Family ID | 48984579 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200087379 |
Kind Code |
A1 |
Schellenberger; Volker ; et
al. |
March 19, 2020 |
FACTOR VIII COMPOSITIONS AND METHODS OF MAKING AND USING SAME
Abstract
The present invention relates to compositions comprising factor
VIII coagulation factors linked to extended recombinant polypeptide
(XTEN), isolated nucleic acids encoding the compositions and
vectors and host cells containing the same, and methods of making
and using such compositions in treatment of factor VIII-related
diseases, disorders, and conditions.
Inventors: |
Schellenberger; Volker;
(Palo Alto, CA) ; Chang; Pei-Yun; (Menlo Park,
CA) ; Varfaj; Fatbardha; (Mountain View, CA) ;
Ding; Sheng; (Redwood City, CA) ; Silverman;
Joshua; (Sunnyvale, CA) ; Wang; Chia-wei;
(Milpitas, CA) ; Spink; Benjamin; (San Carlos,
CA) ; Stemmer; Willem P.; (Los Gatos, CA) ;
Geething; Nathan; (Santa Clara, CA) ; Kulman;
John; (Belmont, MA) ; Liu; Tongyao;
(Lexington, MA) ; Toby; Garabet G.; (North
Reading, MA) ; Jiang; Haiyan; (Belmont, MA) ;
Peters; Robert; (Needham, MA) ; Wang; Deping;
(Sharon, MA) ; Mei; Baisong; (Waban, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOVERATIV THERAPEUTICS INC. |
WALTHAM |
MA |
US |
|
|
Family ID: |
48984579 |
Appl. No.: |
16/521789 |
Filed: |
July 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14379192 |
Feb 20, 2015 |
10421798 |
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PCT/US2012/046326 |
Jul 11, 2012 |
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16521789 |
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61599400 |
Feb 15, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/04 20180101; A61P
7/02 20180101; A61K 38/00 20130101; C07K 2319/31 20130101; C07K
14/755 20130101; A61P 7/00 20180101 |
International
Class: |
C07K 14/755 20060101
C07K014/755 |
Claims
1. A recombinant factor VIII fusion protein comprising a factor
VIII polypeptide and an extended recombinant polypeptide (XTEN),
wherein the factor VIII polypeptide comprises an A1 domain
including an a1 acidic spacer region, an A2 domain including an a2
acidic spacer region, an A3 domain including an a3 acidic spacer
region, a C1 domain, a C2 domain and optionally all or a portion of
a B domain, and wherein the XTEN is linked to the factor VIII
polypeptide: (i) at the C-terminus of the factor VIII polypeptide;
(ii) within the B domain of the factor VIII polypeptide if all or a
portion of the B domain is present; (iii) within the A1 domain of
the factor VIII polypeptide; (iv) within the A2 domain of the
factor VIII polypeptide; (v) within the A3 domain of the factor
VIII polypeptide; (vi) within the C1 domain of the factor VIII
polypeptide; (vii) within the C2 domain of the factor VIII
polypeptide; (viii) at the N-terminus of the factor VIII
polypeptide, or (ix) between two domains of the factor VIII
polypeptide; and wherein when compared to a corresponding factor
VIII protein not linked to an XTEN, the fusion protein (a) retains
at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%,
200%, 300%, 400%, or 500% of the procoagulant activity in an in
vitro coagulation assay, or (b) exhibits reduced binding to an
anti-factor VIII antibody in an in vitro binding assay.
2. (canceled)
3. The recombinant factor VIII fusion protein of claim 1, wherein
the XTEN is linked to the factor VIII polypeptide at an insertion
site selected from Table 5, Table 6, Table 7, Table 8, or Table
9.
4. (canceled)
5. The recombinant factor VIII fusion protein of claim 1, wherein
the factor VIII polypeptide has 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 about 100% sequence identity to a
sequence selected from the group consisting of the sequences in
Table 1, the sequence depicted in FIG. 3, and the sequence depicted
in FIG. 4, when optimally aligned.
6. (canceled)
7. The recombinant factor VIII fusion protein of claim 1, wherein
the comprising at least a second XTEN is linked to the factor VIII
polypeptide within or optionally replacing the B domain of the
factor VIII polypeptide.
8. The recombinant factor VIII fusion protein of claim 1, wherein
the factor VIII polypeptide comprises a B-domain deleted variant of
human factor VIII, wherein the B-domain deletion starts from a
first position at about amino acid residue number 741 to about 750
and ending at a second position at about amino acid residue number
1635 to about 1648 with reference to full-length human factor VIII
sequence as set forth in FIG. 3.
9. (canceled)
10. The recombinant factor VIII fusion protein of claim 1, wherein
the fusion protein has 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 at least about 100% sequence identity compared
to a sequence of comparable length selected from the group
consisting of the sequences of Table 21, when optimally
aligned.
11-13. (canceled)
14. The recombinant factor VIII fusion protein of claim 1, wherein
the XTEN has 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 about 100% sequence identity compared to an XTEN of comparable
length selected from the group consisting of the sequences in Table
4, Table 13, Table 14, Table 15, Table 16, and Table 17, when
optimally aligned.
15-30. (canceled)
31. A recombinant factor VIII fusion protein comprising: a first
polypeptide comprising formula X: (A1)-a1-(A2)-a2-[B]; and a second
polypeptide comprising formula XI: a3-(A3)-(C1)-(C2); wherein the
first polypeptide and the second polypeptide are fused or exist as
a heterodimer; wherein, a) A1 is an A1 domain of factor VIII; b) A2
is an A2 domain of factor VIII; c) [B] is a B domain of factor
VIII, a fragment thereof, or is deleted or optionally not present;
d) A3 is an A3 domain of factor VIII; e) C1 is a C1 domain of
factor VIII; f) C2 is a C2 domain of factor VIII; g) a1, a2, and a3
are acidic spacer regions; wherein an XTEN is inserted into [B];
and wherein when compared to a corresponding factor VIII protein
not linked to an XTEN, the fusion protein retains at least about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400%,
or 500% of the exhibits procoagulant activity in an in vitro
coagulation assay.
32. The recombinant factor VIII fusion protein of claim 31, wherein
the first polypeptide and the second polypeptide form a single
polypeptide chain comprising the formula
(A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1)-(C2).
33-54. (canceled)
55. The recombinant factor VIII fusion protein of claim 31, wherein
the fusion protein exhibits a terminal half-life at least about 3
hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14
hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96
hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21
days when administered to a subject.
56-72. (canceled)
73. The recombinant factor VIII fusion protein of claim 31, wherein
the at least one XTEN has at least 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 about 100%
sequence identity compared to a sequence selected from AE42_1,
AE42_2, AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A, AE144_2A,
AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A,
AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F,
AG144_3, AG144_4, AE288_1, AE288_2, AG288_1, and AG288_2.
74-85. (canceled)
86. The recombinant factor VIII fusion protein of claim 1, wherein
the XTEN is linked to the factor VIII polypeptide at an insertion
site selected form the group consisting of residue numbers 18-32,
or 40, or 211-224, or 336-403, or 599, or 745-1640, or 1656-1728,
or 1796-1804, or 1900-1912, or 2171-2332.
87. A pharmaceutical composition comprising the recombinant factor
VIII fusion protein of claim 1 and a pharmaceutically acceptable
carrier.
88. A method of treating a coagulopathy in a subject, comprising
administering to the subject a composition comprising an effective
amount of the pharmaceutical composition of claim 87.
89-93. (canceled)
94. The method of claim 88, wherein said coagulopathy is hemophilia
A.
95-99. (canceled)
100. A nucleic acid encoding the recombinant factor VIII fusion
protein of claim 1, or the complement thereof.
101. The nucleic acid of claim 100, further comprising a nucleic
acid sequence encoding a signal peptide, wherein said sequence is
ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID
NO: 1613), or the complement thereof.
102-103. (canceled)
104. A method of producing the recombinant factor VIII fusion
protein of claim 1, comprising: (a) providing a host cell
comprising an expression vector comprising a nucleic acid encoding
the fusion protein of claim 1; (b) culturing the host cell under
conditions to effect production of the fusion protein; and (c)
recovering the fusion protein.
105-107. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/379,192, filed Feb. 20, 2015, which is a 35
U.S.C. .sctn. 371 filing of International Patent Application No.
PCT/US2012/046326, filed Jul. 11, 2012, which claims priority to
U.S. Provisional Patent Application Ser. No. 61/599,400, filed Feb.
15, 2012, the entire disclosures of which are hereby incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Factor VIII is an important component of the intrinsic
pathway of the blood coagulation cascade. In the circulation,
factor VIII is mainly complexed to von Willebrand factor. Upon
activation by thrombin, (Factor IIa), it dissociates from the
complex to interact with factor IXa in the intrinsic coagulation
cascade, which, in turn, activates factor X. Once removed from the
von Willebrand factor complex, activated factor VIII is
proteolytically inactivated by activated Protein C (APC), factor
Xa, and factor IXa, and is quickly cleared from the blood stream.
When complexed with normal von Willebrand factor protein, the
half-life of factor VIII is approximately 12 hours, whereas in the
absence of von Willebrand factor, the half-life of factor VIII is
reduced to 2 hours (Tuddenham E G, et al., Br J Haematol. (1982)
52(2):259-267).
[0003] In hemophilia, the clotting of blood is disturbed by a lack
of certain plasma blood clotting factors. Hemophilia A is a
deficiency of factor VIII, and is a recessive sex-linked, X
chromosome disorder that represents 80% of hemophilia cases. The
standard of care for the management of hemophilia A is replacement
therapy with recombinant factor VIII concentrates. Subjects with
severe hemophilia A have circulating procoagulant factor VIII
levels below 1-2% of normal, and are generally on prophylactic
therapy with the aim of keeping factor VIII above 1% between doses,
which can usually be achieved by giving factor VIII two to three
times a week. Persons with moderately severe hemophilia (factor
VIII levels of 2-5% of normal) constitute 25-30% hemophilia
incidents and manifest bleeding after minor trauma. Persons with
mild hemophilia A (factor VIII levels of 5-40% of normal) comprise
15-20% of all hemophilia incidents, and develop bleeding only after
significant trauma or surgery.
[0004] The in vivo activity of exogenously supplied factor VIII is
limited both by a short protein half-life and inhibitors that bind
to the factor VIII and diminish or destroy hemostatic function.
[0005] Up to 30% of hemophilia A patients receiving
exogenously-supplied factor VIII mount an IgG immune response
towards factor VIII (Towfighi, F., et al. Comparative measurement
of anti-factor VIII antibody by Bethesda assay and ELISA reveals
restricted isotype profile and epitope specificity. Acta Haematol
(2005) 114:84-90), which can result in the complete inhibition of
its procoagulant activity and/or promote more rapid clearance of
the factor VIII (Briet E et al. High titer inhibitors in severe
haemophilia A. A meta-analysis based on eight long-term follow-up
studies concerning inhibitors associated with crude or intermediate
purity factor VIII products. Throm. Haemost. (1994) 72: 162-164).
The IgG antibodies, called FVIII inhibitors, are primarily directed
towards the A2, A3 and C2 domains (Scandella D et al. Localization
of epitopes for human factor VIII inhibitor antibodies by
immunoblotting and antibody neutralization. Blood (1989)
74:1618-1626), but can arise against the A1, B and C1 domains, as
well. As such, treatment options for patients with FVIII inhibitors
are limited.
[0006] Large proteins such as factor VIII are normally given
intravenously so that the medicament is directly available in the
blood stream. It has been previously demonstrated that an
unmodified factor VIII injected intramuscularly yielded a maximum
circulating level of only 1.4% of the normal plasma level (Pool et
al, Ineffectiveness of Intramuscularly Injected Factor VIII
Concentrate in Two Hemophilic Patients. New England J. Medicine
(1966) 275(10):547-548). Formulations that could be administered
other than by the intravenous route would greatly simplify their
use, increase safety, and result in substantial cost savings.
[0007] Chemical modifications to a therapeutic protein can modify
its in vivo clearance rate and subsequent serum half-life. One
example of a common modification is the addition of a polyethylene
glycol (PEG) moiety, typically coupled to the protein via an
aldehyde or N-hydroxysuccinimide (NHS) group on the PEG reacting
with an amine group (e.g. lysine side chain or the N-terminus).
However, the conjugation step can result in the formation of
heterogeneous product mixtures that require extraction,
purification and/or other further processes, all of which
inevitably affect product yield and quality control. Also, the
pharmacologic function of coagulation factors may be hampered if
amino acid side chains in the vicinity of its binding site become
modified by the PEGylation process. Other approaches include the
genetic fusion of an Fc domain to the therapeutic protein, which
increases the size of the therapeutic protein, hence reducing the
rate of clearance through the kidney. In some cases, the Fc domain
confers the ability to bind to, and be recycled from lysosomes by
the FcRn receptor, resulting in increased pharmacokinetic
half-life. Unfortunately, the Fc domain does not fold efficiently
during recombinant expression, and tends to form insoluble
precipitates known as inclusion bodies. These inclusion bodies must
be solubilized and functional protein must be renatured from the
misfolded aggregate, which is a time-consuming, inefficient, and
expensive process.
SUMMARY OF THE INVENTION
[0008] The present invention relates to novel coagulation factor
VIII fusion protein compositions and the uses thereof.
Specifically, the compositions provided herein are particularly
used for the treatment or improvement of a condition associated
with hemophilia A, deficiencies of factor VIII, bleeding disorders
and coagulopathies. In one aspect, the present invention provides
compositions of isolated fusion proteins comprising a factor VIII
(FVIII) and one or more extended recombinant polypeptides (XTEN)
wherein the fusion protein exhibits procoagulant activity. A
subject XTEN useful for constructing such fusion proteins is
typically a polypeptide with a non-repetitive sequence and
unstructured conformation. In one embodiment, one or more XTEN is
linked to a coagulation factor FVIII ("CF") selected from native
human factor VIII, factor VIII B-domain deleted sequences ("FVIII
BDD"), and sequence variants thereof (all the foregoing
collectively "FVIII" or "CF"), resulting in a recombinant factor
VIII-XTEN fusion protein ("CFXTEN"). The factor VIII polypeptide
component of the CFXTEN comprises an A1 domain, an A2 domain, a C1
domain, a C2 domain, and optionally a B domain or a portion
thereof. In some embodiments, the FVIII is further characterized by
delineation of the aforementioned domains to comprise an acidic a1,
a2 and a3 spacer. In another embodiment, the present disclosure is
directed to pharmaceutical compositions comprising the fusion
proteins and the uses thereof in methods and regimens for treating
factor VIII-related conditions. The CFXTEN compositions have
enhanced pharmacokinetic and pharmacologic properties compared to
FVIII not linked to XTEN, which may permit more convenient dosing
and improved efficacy.
[0009] In a first aspect, the invention relates to recombinant
factor VIII fusion proteins comprising a factor VIII polypeptide
and one or more extended recombinant polypeptide (XTEN) linked to
the factor VIII. In some embodiments, the invention provides
recombinant factor VIII fusion proteins comprising a factor VIII
polypeptide and at least one extended recombinant polypeptide
(XTEN), wherein said factor VIII polypeptide comprises an A1 domain
including an a1 acidic spacer region, an A2 domain including an a2
acidic spacer region, an A3 domain including an a3 acidic spacer
region, C1 domain, C2 domain and optionally all or a portion of B
domain, and wherein said at least one XTEN is linked to said factor
VIII polypeptide at (i) the C-terminus of said factor VIII
polypeptide; (ii) within B domain of said factor VIII polypeptide
if all or a portion of B domain is present; (iii) within the A1
domain of said factor VIII polypeptide; (iv) within the A2 domain
of said factor VIII polypeptide; (v) within the A3 domain of said
factor VIII polypeptide; (vi) within the C1 domain of said factor
VIII polypeptide; (vii) within the C2 domain of said factor VIII
polypeptide; (viii) at the N-terminus of said factor VIII
polypeptide, or (ix) between two domains of said factor VIII
polypeptide, wherein the fusion protein retains at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or
500% of the procoagulant activity, when measured by an in vitro
coagulation assay, compared to a corresponding factor VIII not
linked to XTEN. In one embodiment, in the foregoing recombinant
factor VIII fusion protein the at least one XTEN is linked to said
factor VIII polypeptide at a site at or within 1 to 6 amino acids
of a site selected from Table 5, Table 6, Table 7, Table 8, and
Table 9. In other embodiments, the invention provides recombinant
factor VIII fusion proteins comprising a factor VIII polypeptide
and at least a first extended recombinant polypeptide (XTEN),
wherein said factor VIII polypeptide comprises an A1 domain
including an a1 acidic spacer region, an A2 domain including an a2
acidic spacer region, an A3 domain including an a3 acidic spacer
region, a C1 domain, a C2 domain and optionally all or a portion of
a B domain, and wherein said first XTEN is linked to said factor
VIII polypeptide at (i) the C-terminus of said factor VIII
polypeptide; (ii) within the B domain of said factor VIII
polypeptide if all or a portion of the B domain is present; (iii)
within the A1 domain of said factor VIII polypeptide; (iv) within
the A2 domain of said factor VIII polypeptide; (v) within the A3
domain of said factor VIII polypeptide; (vi) within the C1 domain
of said factor VIII polypeptide; or (vii) within the C2 domain of
said factor VIII polypeptide; and when compared to a corresponding
factor VIII protein not linked to XTEN, the fusion protein (a)
retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
100%, 200%, 300%, 400%, or 500% of the procoagulant activity in an
in vitro coagulation assay described herein or other such assays
known in the art, and/or (b) exhibits reduced binding to an
anti-factor VIII antibody in an in vitro binding assay described
herein or other such assays known in the art. I n one embodiment,
in the foregoing recombinant factor VIII fusion protein the at
least one XTEN is linked to said factor VIII polypeptide at a site
at or within 1 to 6 amino acids of a site selected from Table 5,
Table 6, Table 7, Table 8, and Table 9. In other embodiments, the
invention provides recombinant factor VIII fusion proteins
comprising a factor VIII polypeptide and at least a first extended
recombinant polypeptide (XTEN), wherein said factor VIII
polypeptide comprises an A1 domain including an a1 acidic spacer
region, an A2 domain including an a2 acidic spacer region, an A3
domain including an a3 acidic spacer region, a C1 domain, a C2
domain and optionally all or a portion of a B domain, and wherein
said first XTEN is linked to said factor VIII polypeptide at an
insertion site selected from Table 6 and Table 7 and wherein the
fusion protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% of the procoagulant
activity, when measured by an in vitro coagulation assay described
herein or other such assays known in the art, compared to a
corresponding factor VIII protein not linked to XTEN. Non-limiting
examples of the factor VIII protein not linked to XTEN includes
native FVIII, BDD FVIII, pBC100 and sequences from Table 1. In
another embodiment of the recombinant factor VIII fusion protein,
the factor VIII polypeptide has at least about 80% sequence
identity, or 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%, to about 100% sequence identity to a sequence
selected from the group consisting of the sequences of Table 1, the
sequence depicted in FIG. 3, and the sequence depicted in FIG. 4,
when optimally aligned. In yet another embodiment, the fusion
protein comprises at least another XTEN linked to said factor VIII
polypeptide at the C-terminus of said factor VIII polypeptideor
within or optionally replacing the B domain of said factor VIII
polypeptide. In a specific embodiment, the fusion protein comprises
at least one XTEN sequence located within or optionally replacing
the B domain of said factor VIII polypeptide. In another specific
embodiment, the fusion protein comprises at least one XTEN sequence
linked to said factor VIII polypeptide at the C-terminus of said
factor VIII polypeptide. In one embodiment, the recombinant factor
VIII fusion protein comprises a B-domain deleted variant of human
factor VIII, wherein the B-domain deletion starts from a first
position at about amino acid residue number 741 to about 750 and
ending at a second position at amino acid residue number 1635 to
about 1648 with reference to full-length human factor VIII sequence
as set forth in FIG. 3. In another embodiment, the recombinant
factor VIII fusion protein comprises a first XTEN sequence linked
to said factor VIII polypeptide at the C-terminus of said factor
VIII polypeptide, and at least a second XTEN within or replacing
the B domain of said factor VIII polypeptide, wherein the second
XTEN is linked to the C-terminal end of about amino acid residue
number 741 to about 750 and to the N-terminal end of amino acid
residue numbers 1635 to about 1648 with reference to full-length
human factor VIII sequence as set forth in FIG. 3, wherein the
cumulative length of the XTEN is at least about 100 amino acid
residues. In one embodiment, in the foregoing fusion protein, the
second XTEN links the factor VIII amino acids between N745 to P1640
or between S743 to Q1638 or between P747 to V1642 or between N745
and Q1656 or between N745 and 51657 or between N745 and T1667 or
between N745 and Q1686 or between R747 and V1642 or between T751
and T1667. In one embodiment, the recombinant factor VIII fusion
protein comprises a sequence having at least about 80% sequence
identity, 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%, to about 100% sequence
identity compared to a sequence of comparable length selected from
Table 21, when optimally aligned. In another embodiment, the
recombinant factor VIII fusion protein comprises at least a second
XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally
a fifth XTEN and optionally a sixth XTEN, wherein each of the
second, third, fourth, fifth, or sixth XTEN is linked to said
factor VIII polypeptide at a second, third, fourth, fifth, or sixth
site selected from the group consisting of an insertion site from
Table 5, Table 6, Table 7 Table 8, and Table 9; a location within 6
amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399,
416, 603, 1656, 1711, 1725, 1905 and 1910 of mature factor VIII; a
location between any two adjacent domains of said factor VIII
polypeptide, wherein said two adjacent domains are selected from
the group consisting of A1 and A2 domains, A2 and B domains, B and
A3 domains, A3 and C1 domains, and C1 and C2 domains; a location
within the B domain of said factor VIII polypeptide, wherein the
second XTEN is linked to the C-terminal end of about amino acid
residue number 741 to about 750 and to the N-terminal end of amino
acid residue numbers 1635 to about 1648 of a native factor VIII
sequence; and the C-terminus of said factor VIII polypeptide. In
one embodiment, the first XTEN is separated from the second XTEN by
at least 10 amino acids, at least 50 amino acids, at least 100
amino acids, at least 200 amino acids, at least 300 amino acids, or
at least 400 amino acids. In one embodiment of the recombinant
factor VIII fusion protein that comprises at least a second XTEN,
optionally a third XTEN, optionally a fourth XTEN, optionally a
fifth XTEN and optionally a sixth XTEN, each XTEN has at least
about 80% sequence identity, 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 about 100% sequence identity compared to an XTEN of comparable
length selected from the group consisting of the sequences in Table
4, Table 13, Table 14, Table 15, Table 16, and Table 17, when
optimally aligned. In yet another embodiment of the recombinant
factor VIII fusion protein that comprises at least a second XTEN,
optionally a third XTEN, optionally a fourth XTEN, optionally a
fifth XTEN and optionally a sixth XTEN, In preferred embodiments,
the recombinant factor VIII fusion protein exhibits a terminal
half-life at least about 3 hours, or 4 hours, or 6 hours, or 12
hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48
hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7
days, or 14 days, or 21 days when administered to a subject,
wherein said subject is selected from human and factor VIII/von
Willebrand factor double knock-out mouse. Further, in the
embodiments of this paragraph, the fusion protein exhibits reduced
binding to anti-factor VIII antibody or greater retained
procoagulant activity, or both as compared to a corresponding
factor VIII not linked to XTEN. In one embodiment, the procoagulant
activity of the recombinant factor VIII fusion protein is at least
30%, or 40%, 50%, 80%, 100%, 200%, 300%, 400%, or 500% greater
procoagulant activity in the presence of the anti-FVIII antibody
compared to a corresponding factor VIII not linked to XTEN when
each are assayed by an in vitro coagulation assay. In one
embodiment, the reduced binding of the fusion protein to
anti-factor VIII antibody is determined using a Bethesda assay
using anti-factor VIII antibody selected from the group consisting
of the antibodies of Table 10 and polyclonal antibody from a
hemophilia A patient with factor VIII inhibitors, wherein the
reduced binding and retained procoagulant activity of the fusion
protein is evidenced by a lower Bethesda titer of at least about 2,
4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200
Bethesda units for the fusion protein compared to that for the
factor VIII not linked to XTEN.
[0010] In one embodiment, the recombinant factor VIII fusion
protein can, for example, comprise one or more XTEN wherein the
XTEN has at least about 80% sequence identity, or 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%, to about
100% sequence identity compared to one or more XTEN of comparable
length selected from Table 4, Table 13, Table 14, Table 15, Table
16, and Table 17, when optimally aligned.
[0011] In another aspect, the invention relates to recombinant
factor VIII fusion proteins comprising FVIII and one or more XTEN
in specific N- to C-terminus configurations. In one embodiment of
the CFXTEN composition, the invention provides a recombinant factor
VIII fusion protein of formula I:
(XTEN).sub.x-CF-(XTEN).sub.y I
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity with sequenced from Table 1; x is either
0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an
extended recombinant polypeptide as described herein, including,
but not limited to sequences 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% or
100% sequence identity to sequences set forth in Table 4.
Accordingly, the CFXTEN fusion composition can have XTEN-CF,
XTEN-CF-XTEN, or CF-XTEN configurations.
[0012] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula II:
(XTEN).sub.x-(S).sub.x-(CF)-(XTEN).sub.y II
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity to sequences set forth in Table 1; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence or amino acids
compatible with restrictions sites; x is either 0 or 1 and y is
either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4.
[0013] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein,
wherein the fusion protein is of formula III:
(XTEN).sub.x-(S).sub.x-(CF)-(S).sub.y-(XTEN).sub.y III
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity to sequence set for in Table 1; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence or amino acids
compatible with restrictions sites; x is either 0 or 1 and y is
either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4.
[0014] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula IV:
(A1)-(XTEN).sub.u-(A2)-(XTEN).sub.vB)-(XTEN).sub.w-(A3)-(XTEN).sub.x-(C1-
)-(XTEN).sub.y-(C2)-(XTEN).sub.z IV
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B
is a B domain of FVIII which can be a fragment or a splice variant
of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of
FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1;
y is either 0 or 1; y is either 0 or 1 with the proviso that
u+v+x+y+z.gtoreq.1; and XTEN is an extended recombinant polypeptide
as described herein including, but not limited to sequences 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% or 100% sequence identity to sequences
set forth in Table 4.
[0015] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula V:
(XTEN).sub.t-(S).sub.a-(A1)-(S).sub.b-(XTEN).sub.u-(S).sub.b-(A2)-(S).su-
b.c-(XTEN).sub.v-(S).sub.c-(B)-(S).sub.d-(XTEN).sub.w-(S).sub.d-(A3)-(S).s-
ub.e-(XTEN).sub.x-(S).sub.e-(C1)-(S).sub.f--(XTEN).sub.y-(S).sub.f--(C2)-(-
S).sub.g-(XTEN).sub.z V
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B
is a B domain of FVIII which can be a fragment or a splice variant
of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of
FVIII; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence or
amino acids compatible with restrictions sites; a is either 0 or 1;
b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is
either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either
0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is
either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that t+u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In another
embodiment of formula V, the spacer sequence is glycine or a
sequence selected from Tables 11 and 12.
[0016] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VI:
(XTEN).sub.u-(S).sub.a-(A1)-(S).sub.b-(XTEN).sub.v-(S).sub.b-(A2)-(S).su-
b.c-(XTEN).sub.w-(S).sub.c-(A3)-(S).sub.d-(XTEN).sub.x-(S).sub.d-(C1)-(S).-
sub.e-(XTEN).sub.y-(S).sub.e-(C2)-(S).sub.f-(XTEN).sub.z VI
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1
is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer
sequence having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence or amino acids compatible
with restrictions sites; a is either 0 or 1; b is either 0 or 1; c
is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is
either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1,
x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In another
embodiment of formula V, the spacer sequence is glycine or a
sequence selected from Tables 11 and 12.
[0017] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VII:
(SP)-(XTEN).sub.x-(CS).sub.x-(S).sub.x-(FVIII_1-745)-(S).sub.y-(XTEN).su-
b.y-(S).sub.y-(FVIII_1640-2332)-(S).sub.z-(CS).sub.z-(XTEN).sub.z
VII
wherein independently for each occurrence, SP is a signal peptide,
preferably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS
is a cleavage sequence listed in Table 12, S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include amino acids compatible with restrictions sites,
"FVIII_1-745" is residues 1-745 of Factor FVIII and
"FVIII_1640-2332" is residues 1640-2332 of FVIII, x is either 0 or
1, y is either 0 or 1, and z is either 0 or 1, wherein
x+y+z.gtoreq.2; and XTEN is an extended recombinant polypeptide as
described herein including, but not limited to sequences 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% or 100% sequence identity sequences set forth
in Table 4. In one embodiment of formula VII, the spacer sequence
is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V,
the spacer sequence is glycine or a sequence selected from Tables
11 and 12.
[0018] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VIII:
(A1)-(S).sub.a-(XTEN).sub.v-(S).sub.a-(A2)-(B1)-(S).sub.b-(XTEN).sub.w-(-
S).sub.b-(B2)-(A3)-(S).sub.c-(XTEN).sub.x-(S).sub.c-(C1)-(S).sub.d-(XTEN).-
sub.y-(S).sub.d-(C2)-(S).sub.c-(XTEN).sub.z VIII
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B
domain that can have from residue 741 to 743-750 of FVIII or
alternatively from about residue 741 to about residues 745 of
FVIII; B2 is a fragment of the B domain that can have from residues
1635-1686 to 1689 of FVIII or alternatively from about residue 1640
to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is
a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer
sequence having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence or amino acids compatible
with restrictions sites; a is either 0 or 1; b is either 0 or 1; c
is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is
either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1,
x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In one
embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID
NO: 1612). In another embodiment of formula V, the spacer sequence
is glycine or a sequence selected from Tables 11 and 12.
[0019] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula IX:
(A1.sub.N)-(S).sub.a-(XTEN).sub.t-(S).sub.b-(A1.sub.C)-(A2.sub.N)-(S).su-
b.c-(XTEN).sub.u-(S).sub.d-(A2.sub.C)-(B.sub.N)--(S).sub.c-(XTEN).sub.v-(S-
).sub.f-(B.sub.C)-(A3.sub.N)-
(S).sub.g-(XTEN).sub.w-(S)h-(A3.sub.C)-(C1.sub.N)-(S).sub.i-(XTEN).sub.x--
(S).sub.j-(C1.sub.C)-(C2.sub.N)-(S).sub.k-(XTEN).sub.y(S).sub.l-(C2.sub.C)-
-(S).sub.m-(XTEN).sub.z IX
wherein independently for each occurrence, A1.sub.N is a fragment
of the A1 domain from at least residue number 1 (numbered relative
to native, mature FVIII) to no more than residue number 371,
A1.sub.c is a fragment of the A1 domain from at least residue
number 2 to no more than residue number 372, with the priviso that
no sequence of the A1.sub.N fragment is duplicated in the A1.sub.c
is a fragment; A2N is a fragment of the A2 domain from at least
residue number 373 to no more than residue number 739, A2.sub.c is
a fragment of the A2 domain from at least residue number 374 to no
more than residue number 740, with the priviso that no sequence of
the A2.sub.N fragment is duplicated in the A2.sub.c is a fragment;
B.sub.N is a fragment of the B domain from at least residue number
741 to no more than residue number 1647, B.sub.c is a fragment of
the B domain from at least residue number 742 to no more than
residue number 1648, with the priviso that no sequence of the
B.sub.N fragment is duplicated in the B.sub.c is a fragment;
A3.sub.N is a fragment of the A3 domain from at least residue
number 1649 to no more than residue number 2019, A3.sub.c is a
fragment of the A3 domain from at least residue number 1650 to no
more than residue number 2019, with the priviso that no sequence of
the A3.sub.N fragment is duplicated in the A3.sub.c is a fragment;
C1.sub.N is a fragment of the C1 domain from at least residue
number 2020 to no more than residue number 2171, C1, is a fragment
of the C1 domain from at least residue number 2021 to no more than
residue number 2172, with the priviso that no sequence of the
C1.sub.N fragment is duplicated in the C1, is a fragment; C2.sub.N
is a fragment of the C2 domain from at least residue number 2173 to
no more than residue number 2331, C2.sub.c is a fragment of the C2
domain from at least residue number 2174 to no more than residue
number 2332, with the priviso that no sequence of the C2.sub.N
fragment is duplicated in the C2.sub.c is a fragment; S is a spacer
sequence having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence or amino acids compatible
with restrictions sites; a is either 0 or 1; b is either 0 or 1; c
is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is
either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either
0 or 1; j is either 0 or 1; k is either 0 or 1; 1 is either 0 or 1;
m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is
either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1;
z is either 0 or 1 with the proviso that t+u+v+w+x+y+z.gtoreq.1;
and XTEN is an extended recombinant polypeptide as described herein
including, but not limited to sequences having at least about 80%
sequence identity, or 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%, to about 100% sequence identity
compared to one or more XTEN of comparable length selected from
Table 4. In one embodiment of formula IX, the spacer sequence is
GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the
spacer sequence is glycine or a sequence selected from Tables 11
and 12. In another embodiment of formula IX, Z is 1. In another
embodiment of the fusion protein of formula IX V is 1 and the XTEN
is linked to the C-terminal end of about amino acid residue number
741 to about 750 and to the N-terminal end of amino acid residue
numbers 1635 to about 1648 with reference to full-length human
factor VIII sequence as set forth in FIG. 3. In another embodiment
of the fusion protein of formula IX, the sum oft, u, v, w, x, y,
and z equals 2, 3, 4, 5, or 6. In another embodiment of formula IX,
the sum oft, u, v, w, x, y, and z equals 2, and v is 1 and z is 1.
In another embodiment of the fusion protein of formula IX, the sum
of t, u, v, w, x, y, and z equals 3, v and z each equal 1, and
either t, u, w, x or y is 1. In another embodiment of formula IX,
the sum of t, u, v, w, x, y, and z equals 4, v and w and z each
equal 1, and two of t, u, x or y is 1. In another embodiment of the
fusion protein of formula IX, the cumulative length of the XTENs is
between about 84 to about 3000 amino acid residues. In another
embodiment of formula IX, at least one XTEN is inserted immediately
downstream of an amino acid which corresponds to an amino acid in
mature native human factor VIII selected from the group consisting
of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603,
1656, 1711, 1725, 1905 and 1910. In another embodiment of the
fusion protein formula IX, each XTEN is linked to said fusion
protein at sites selected from Table 5, Table 6, Table 7, Table 8,
and Table 9. In another embodiment of the fusion protein formula
IX, each XTEN has at least about 80%, or 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 about 100% sequence identity compared to an XTEN of comparable
length selected from the group consisting of the sequences in Table
4, Table 13, Table 14, Table 15, Table 16, and Table 17, when
optimally aligned.
[0020] In another embodiment of the CFXTEN composition, the
invention provides a first recombinant factor VIII polypeptide of
formula X:
(A1)-a1-(A2)-a2-[B] X
and a second polypeptide comprising Formula XI:
a3-(A3)-(C1)-(C2) XI
wherein the first polypeptide and the second polypeptide are fused
or exist as a heterodimer; wherein, A1 is an A1 domain of factor
VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of
factor VIII, a fragment thereof, or is deleted; A3 is an A3 domain
of factor VIII; C1 is a C1 domain of factor VIII; C2 is a C2 domain
of factor VIII; a1, a2, and a3 are acidic spacer regions; wherein
the A1 domain comprises an XTEN permissive loop-1 (A1-1) region and
an XTEN permissive loop-2 (A1-2) region; wherein the A2 domain
comprises an XTEN permissive loop-1 (A2-1) region and an XTEN
permissive loop-2 (A2-2) region; wherein the A3 domain comprises an
XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2
(A3-2) region; wherein an XTEN sequence is inserted into at least
one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2; and
wherein the recombinant factor VIII protein exhibits procoagulant
activity. In one embodiment of the heterodimer, the first
polypeptide and the second polypeptide form a single polypeptide
chain comprising the formula
(A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1)-(C2). In one embodiment of the
foregoing, "fused" means a peptidic bond.
[0021] In another embodiment of the CFXTEN composition, the
invention provides a first recombinant factor VIII polypeptide of
formula X:
(A1)-a1-(A2)-a2-[B] X
and a second polypeptide comprising Formula XI:
a3-(A3)-(C1)-(C2) XI
wherein the first polypeptide and the second polypeptide are fused
or exist as a heterodimer; wherein, A1 is an A1 domain of factor
VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of
factor VIII, a fragment thereof, or is deleted; A3 is an A3 domain
of factor VIII; C1 is a C1 domain of factor VIII; C2 is a C2 domain
of factor VIII; a1, a2, and a3 are acidic spacer regions; wherein
an XTEN sequence is inserted into a3; and wherein the recombinant
factor VIII protein exhibits procoagulant activity. In one
embodiment of the heterodimer, the first polypeptide and the second
polypeptide form a single polypeptide chain comprising the formula
(A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1)-(C2). In one embodiment of the
foregoing, "fused" means a peptidic bond.
[0022] In embodiments of the foregoing formulae X and XI
polypeptides, the XTEN permissive loops are contained within
surface-exposed, flexible loop structures, and wherein A1-1 is
located between beta strand 1 and beta strand 2, A1-2 is located
between beta strand 11 and beta strand 12, A2-1 is located between
beta strand 22 and beta strand 23, A2-2 is located between beta
strand 32 and beta strand 33, A3-1 is located between beta strand
38 and beta strand 39 and A3-2 is located between beta strand 45
and beta strand 46, according to the secondary structure of mature
factor VIII stored as Accession Number 2R7E of the DSSP database.
In other embodiments of the foregoing formulae X and XI
polypeptides, the surface-exposed, flexible loop structure
comprising A1-1 corresponds to a region in native mature human
factor VIII from about amino acid 15 to about amino acid 45. In
other embodiments of the foregoing formulae X and XI polypeptides
the A1-1 corresponds to a region in native mature human factor VIII
from about amino acid 18 to about amino acid 41. In other
embodiments of the foregoing formulae X and XI poly peptides, the
surface-exposed, flexible loop structure comprising A1-2
corresponds to a region in native mature human factor VIII from
about amino acid 201 to about amino acid 232. In other embodiments
of the foregoing formulae X and XI polypeptides the A1-2
corresponds to a region in native mature human factor VIII from
about amino acid 218 to about amino acid 229. In other embodiments
of the foregoing formulae X and XI polypeptides, the
surface-exposed, flexible loop structure comprising A2-1
corresponds to a region in native mature human factor VIII from
about amino acid 395 to about amino acid 421. In other embodiments
of the foregoing formulae X and XI polypeptides, the A2-1
corresponds to a region in native mature human factor VIII from
about amino acid 397 to about amino acid 418. In other embodiments
of the foregoing formulae X and XI polypeptides, the
surface-exposed, flexible loop structure comprising A2-2
corresponds to a region in native mature human factor VIII from
about amino acid 577 to about amino acid 635. In other embodiments
of the foregoing formulae X and XI polypeptides, the A2-2
corresponds to a region in native mature human factor VIII from
about amino acid 595 to about amino acid 607. In other embodiments
of the foregoing formulae X and XI polypeptides, the
surface-exposed, flexible loop structure comprising A3-1
corresponds to a region in native mature human factor VIII from
about amino acid 1705 to about amino acid 1732. In other
embodiments of the foregoing formulae X and XL polypeptides, the
A3-1 corresponds to a region in native mature human factor VIII
from about amino acid 1711 to about amino acid 1725. In other
embodiments of the foregoing formulae X and XI polypeptides, the
surface-exposed, flexible loop structure comprising A3-2
corresponds to a region in native mature human factor VIII from
about amino acid 1884 to about amino acid 1917. In other
embodiments of the foregoing formulae X and XI polypeptides, the
A3-2 corresponds to a region in native mature human factor VIII
from about amino acid 1899 to about amino acid 1911. In other
embodiments of the foregoing formulae X and XI polypeptides, an
XTEN sequence is inserted into at least two of the regions A1-1,
A1-2, A2-1, A2-2, A3-1, or A3-2. In other embodiments of the
foregoing formulae X and XI polypeptides, an XTEN sequence is
inserted immediately downstream of an amino acid which corresponds
to an amino acid in mature native human factor VIII selected from
the group consisting of amino acid residue number 32, 220, 224,
336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In other
embodiments of the foregoing formulae X and XI polypeptides, an
additional XTEN sequence is inserted into the a3 acidic spacer
region. In other embodiments of the foregoing formulae X and XI
polypeptides, an additional XTEN sequence is inserted into the a3
acid spacer immediately downstream of an amino acid which
corresponds to amino acid 1656. In other embodiments of the
foregoing formulae X and XI polypeptides, the A1 domain comprises
an XTEN permissive loop-1 (A1-1) region and an XTEN permissive
loop-2 (A1-2) region wherein the A2 domain comprises an XTEN
permissive loop-1 (A2-1) region and an XTEN permissive loop-2
(A2-2) region, and wherein the A3 domain comprises an XTEN
permissive loop-1 (A3-1) region and an XTEN permissive loop-2
(A3-2) region, and wherein an additional XTEN sequence is inserted
into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or
A3-2. In other embodiments of the foregoing formulae X and XI
polypeptides, an additional XTEN sequence is inserted immediately
downstream of an amino acid which corresponds to an amino acid in
mature native human factor VIII selected from the group consisting
of amino acid residue number 32, 220, 224, 336, 339, 390, 399, 416,
603, 1656, 1711, 1725, 1905 and 1910. In the foregoing embodiments
of formulae X and XI polypeptides, the fusion protein exhibits at
least about 30%, 40%, 50%, 60%, 70%, or 80%, or 90% of the
procoagulant activity of the corresponding factor VIII not linked
to XTEN, wherein the procoagulant activity is assayed by an in
vitro coagulation assay.
[0023] In all embodiments, the polypeptide can, for example,
exhibit an in vitro procoagulant activity exceeding 0.5 IU/ml, or
1.0, or 1.5, or 2.0 IU/ml when expressed in cell-culture medium and
assayed by an in vitro coagulation assay. The procoagulant activity
can be measured by a chromogenic assay, a one stage clotting assay
(e.g., a aPTT) or both.
[0024] In some embodiments, wherein the recombinant factor VIII
fusion protein comprises a factor VIII and at least a first and a
second XTEN, the at least first XTEN is separated from the at least
second XTEN by at least 10 amino acids, at least 50 amino acids, at
least 100 amino acids, at least 200 amino acids, at least 300 amino
acids, or at least 400 amino acids.
[0025] In preferred embodiments, the recombinant factor VIII fusion
protein comprising a factor VIII and at least a first XTEN and,
optionally, at least a second, or optionally at least a third, or
optionally at least a fourth XTEN, the fusion protein exhibits
reduced binding to an anti-factor VIII antibody as compared to the
corresponding factor VIII not linked to XTEN. The reduced binding
can be assessed either in vivo or by an in vitro assay. In one
embodiment, the in vitro assay is an ELISA assay, wherein the
binding of an anti-FVIII antibody to the fusion protein is reduced
at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or at least about
40% or more compared to a FVIII not linked to XTEN. In another
embodiment, the in vitro assay is a Bethesda assay wherein the
reduced binding of the fusion protein is evidenced by a lower
Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30,
40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion
protein compared to that for a factor VIII not linked to XTEN. In
the in vitro assays, the anti-factor VIII antibody is selected from
an antibody of Table 10 and polyclonal antibody from a hemophilia A
patient with factor VIII inhibitors. In particular embodiments of a
recombinant factor VIII fusion protein comprising a factor VIII and
at least a first and a second XTEN exhibiting reduced binding to a
factor VIII inhibitor antibody, the first XTEN is linked to said
factor VIII polypeptide within a C2 domain of said factor VIII
polypeptide, and the second XTEN is linked to said factor VIII
polypeptide within an A1 or A2 domain of said factor VIII
polypeptide, wherein said fusion protein exhibits reduced binding
to a factor VIII inhibitor antibody as compared to the
corresponding factor VIII not linked to XTEN, wherein the factor
VIII inhibitor antibody is capable of binding to an epitope located
within the A1, A2 or C2 domain, and further wherein the fusion
protein exhibits procoagulant activity. In one embodiment of the
foregoing fusion protein, the second XTEN is linked to said factor
VIII polypeptide within the A2 domain of the factor VIII
polypeptide and the factor VIII inhibitor antibody binds to the A2
domain of the factor VII polypeptide. In another embodiment of the
foregoing fusion protein, the second XTEN is linked to said factor
VIII polypeptide within the C2 domain of the factor VIII
polypeptide and the factor VIII inhibitor antibody binds to the C2
domain of the factor VIII polypeptide. The binding of an
anti-factor VIII antibody to the fusion protein is reduced by at
least about 5%, 10%, 15%, 20%, 25%. 30%, 35% or 40% compared to the
corresponding factor VIII not linked to XTEN when assayed by an
ELISA assay, wherein the anti-factor VIII antibody is selected from
the group consisting of the antibodies in Table 10 and a polyclonal
antibody from a hemophilia A subject with factor VIII inhibitors.
The foregoing fusion proteins can further comprise at least three
XTENs, wherein the at least third XTEN is linked to the factor VIII
at a site selected from within or replacing the B domain, at the
C-terminus and at or within 1, 2, 3, 4, 5, or 6 amino acids of an
insertion site selected from Table 7 or Table 9. In the embodiments
with reduced binding to anti-factor VIII antibodies, the fusion
protein has greater procoagulant activity in the presence of the
anti-FVIII antibody of at least 10%, 20%, 30%, 40%, 50%, 80%, 100%,
200%, 300%, 400%, or 500% or more compared to a corresponding
factor VIII not linked to XTEN when assayed by an in vitro
coagulation assay (e.g., a chromogenic or one-stage clotting
assay).
[0026] In all embodiments, the XTEN of the fusion protein can, for
example, be characterized in that the XTEN comprise at least 36, or
at least 42, or at least 72, or at least 96, or at least 144, or at
least 288, or at least 400, or at least 500, or at least 576, or at
least 600, or at least 700, or at least 800, or at least 864, or at
least 900, or at least 1000, or at least 2000, to about 3000 amino
acid residues or even more residues; the sum of glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P) residues constitutes 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% of the total
amino acid residues of the XTEN; the XTEN is substantially
non-repetitive such that (i) the XTEN 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, or about 12 amino acid residues
consisting of four to six amino acids selected from glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P), wherein any two contiguous amino acid residues do not occur
more than twice in each of the non-overlapping sequence motifs; or
(iii) the XTEN sequence has a subsequence score of less than 10;
the XTEN has greater than 90%, or greater than 95%, or greater than
99% random coil formation as determined by GOR algorithm; the XTEN
has less than 2% alpha helices and 2% beta-sheets as determined by
Chou-Fasman algorithm; the XTEN lacks a predicted T-cell epitope
when analyzed by TEPITOPE algorithm, wherein the TEPITOPE threshold
score for said prediction by said algorithm has a threshold of -9,
and wherein said fusion protein exhibits a terminal half-life that
is longer than at least about 12 h, or at least about 24 h, or at
least about 48 h, or at least about 72 h, or at least about 96 h,
or at least about 120 h, or at least about 144 h, or at least about
21 days or greater. In one embodiment, the recombinant factor VIII
fusion protein comprises at least a second, or at least a third, or
at least a fourth XTEN, which can be identical or different to the
other XTEN. According to a different approach, the at least one, at
least a second, or at least a third, or at least a fourth XTEN of
the CFXTEN fusion protein each have at least about 80% sequence
identity, or 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%, to about 100% sequence identity compared to one
or more XTEN of comparable length selected from Table 4, Table 13,
Table 14, Table 15, Table 16, and Table 17, when optimally aligned.
In yet another different approach, the at least one, at least a
second, or at least a third, or at least a fourth XTEN of the
CFXTEN fusion protein each have at least 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%, to about 100% sequence
identity compared to a sequence selected from AE42_1, AE42_2,
AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A, AE144_2A,
AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A,
AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F,
AG144_3, AG144_4, AE288_1, AE288_2, AG288_1, and AG288_2.
[0027] In one embodiment, the factor VIII component of the CFXTEN
recombinant factor VIII fusion protein comprises one, two or three
amino acid substitutions selected from residues R1648, Y1680, and
R1689, numbered relative to mature human factor VIII, wherein the
substitutions are selected from alanine, glycine, and
phenylalanine. Non-limiting examples of said substitutions include
R1648A, Y1680F, and R1689A.
[0028] In another embodiment, the CFXTEN fusion protein exhibits an
apparent molecular weight factor of at least about 1.3, or at least
about two, or at least about three, or at least about four, or at
least about five, or at least about six, or at least about seven,
or at least about eight, or at least about nine, or at least about
10, when measured by size exclusion chromatography or comparable
method.
[0029] In some embodiments of the CFXTEN fusion proteins, one or
more of the XTEN is to the FVIII via one or two cleavage sequences
that each is cleavable by a mammalian protease selected from the
group consisting of factor XIa, factor XIIa, kallikrein, factor
VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2,
MMP-12, MMP13, MMP-17 and MMP-20, wherein cleavage at the cleavage
sequence by the mammalian protease releases the factor VIII
sequence from the XTEN sequence, and wherein the released factor
VIII sequence exhibits an increase in procoagulant activity
compared to the uncleaved fusion protein. In one embodiment, the
cleavage sequence(s) are cleavable by factor XIa.
[0030] According to a different approach, the CFXTEN fusion
proteins comprise at least three XTENs located at different
locations of the factor VIII polypeptide, wherein said different
locations are selected from: an insertion location at or within 1
to 6 amino acids from a site selected from Table 5, Table 6, Table
7 Table 8, and Table 9; a location at or within 1 to 6 amino acids
of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603,
1656, 1711, 1725, 1905 and 1910 of mature factor VIII; a location
between any two adjacent domains in the factor VIII sequence,
wherein said two adjacent domains are selected from the group
consisting of A1 and A2, A2 and B, B and A3, A3 and C1, and C1 and
C2; a location within an internal B domain deletion starting from a
first position at about amino acid residue number 741 to about 750
and ending at a second position at amino acid residue number 1635
to about 1648 with reference to full-length human factor VIII
sequence as set forth in FIG. 3 and the C-terminus of the factor
VIII sequence, wherein the cumulative length of the multiple XTENs
is at least about 100 to about 3000 amino acid residues and wherein
the fusion protein retains at least about 30%, or about 40%, or
about 50%, or about 60%, or about 70%, or about 80%, or about 90%
of the procoagulant activity compared to the corresponding factor
VIII not linked to XTEN, wherein the procoagulant activity is
assayed by an in vitro coagulation assay. In one embodiment of the
foregoing, the fusion protein exhibits a prolonged terminal
half-life when administered to a subject as compared to a
corresponding factor VIII polypeptide lacking said XTEN, wherein
said fusion protein exhibits a terminal half-life at least about 3
hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14
hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96
hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21
days when administered to a subject. In one embodiment, the subject
is selected from the group consisting of human and a factor
VIII/von Willebrand factor double knock-out mouse. In one
embodiment of the foregoing, the fusion protein does not comprise a
sequence selected from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP
(SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID
NO: 34), and
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG
SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing,
the fusion protein does not contain an XTEN sequence consisting
of
TABLE-US-00001 (SEQ ID NO: 59)
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP, (SEQ ID NO: 71)
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS, or (SEQ ID NO: 80)
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS.
[0031] In a further aspect, the invention concerns CFXTEN fusion
proteins with enhanced pharmacokinetic properties, including
enhanced parameters compared to FVIII not linked to XTEN, wherein
the enhanced properties include but are not limited to longer
terminal half-life, larger area under the curve, increased time in
which the blood concentration remains within the therapeutic
window, increased time between consecutive doses results in blood
concentrations within the therapeutic window, and decreased dose in
IU over time that can be administered compared to a FVIII not
linked to XTEN, yet still result in a blood concentration above a
threshold concentration needed for a procoagulant effect. In some
embodiments, a CFXTEN fusion proteins exhibit a prolonged terminal
half-life when administered to a subject as compared to a
corresponding factor VIII polypeptide lacking said XTEN. The
subject can be a human or a mouse, such as a factor VIII/von
Willebrand factor double knock-out mouse. In one embodiment of the
foregoing, the CFXTEN exhibits a terminal half-life that is at
least about two-fold, or about three fold, or about four-fold, or
about five-fold, or about 10-fold, or about 20-fold longer when
administered to a subject compared to the corresponding factor VIII
not linked to XTEN. In one embodiment, the CFXTEN fusion protein
exhibits a terminal half-life at least about 3 hours, or 4 hours,
or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or
24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or
144 hours, or 7 days, or 14 days, or 21 days when administered to
the subject. In other embodiments, the enhanced pharmacokinetic
property of the fusion proteins of the embodiments is the property
of maintaining a circulating blood concentration of procoagulant
fusion protein in a subject in need thereof above a threshold
concentration of 0.01 IU/ml, or 0.05 IU/ml, or 0.1 IU/ml, or 0.2
IU/ml, or 0.3 IU/ml, or 0.4 IU/ml or 0.5 IU/ml for a period that is
at least about two fold, or at least about three-fold, or at least
about four-fold, or at least about five-fold, or at least about
six-fold, or at least about eight-fold, or at least about ten-fold,
or at least about 20-fold, or at least about 40-fold, or at least
about 60-fold longer compared to the corresponding FVIII not linked
to XTEN and administered to a subject at a comparable dose. The
increase in half-life and time spent above the threshold
concentration permits less frequent dosing and decreased amounts of
the fusion protein (in moles equivalent) that are administered to a
subject, compared to the corresponding FVIII not linked to XTEN. In
one embodiment, administration of a subject fusion protein to a
subject using a therapeutically-effective dose regimen results in a
gain in time of at least two-fold, or at least three-fold, or at
least four-fold, or at least five-fold, or at least six-fold, or at
least eight-fold, or at least 10-fold, or at least about 20-fold,
or at least about 40-fold, or at least about 60-fold or higher
between at least two consecutive Cmax peaks and/or Cmin troughs for
blood levels of the fusion protein compared to the corresponding
FVIII not linked to the XTEN and administered using a comparable
dose regimen to a subject.
[0032] In preferred embodiments, the CFXTEN fusion proteins retain
at least about 30%, or about 40%, or about 50%, or about 60%, or
about 70%, or about 80%, or about 90% of the procoagulant activity
compared to the corresponding factor VIII not linked to XTEN,
wherein the procoagulant activity is assayed by an in vitro
coagulation assay such as, but not limited to a chromogenic assay
or a one- or two-stage clotting assay.
[0033] According to a different approach, the invention provides
recombinant factor VIII fusion proteins comprising a factor VIII
polypeptide and at least one extended recombinant polypeptide
(XTEN), wherein said factor VIII polypeptide comprises A1 domain,
A2 domain, A3 domain, C1 domain, C2 domain and optionally all or a
portion of B domain, and wherein said at least one XTEN is linked
to said factor VIII polypeptide at an insertion site selected form
residue numbers 18-32, or 40, or 211-224, or 336-403, or 599, or
745-1640, or 1656-1728, or 1796-1804, or 1900-1912, or 2171-2332;
and wherein the fusion protein retains at least about 30%, or about
40%, or about 50%, or about 60%, or about 70%, or about 80%, or
about 90% of the procoagulant activity compared to the
corresponding factor VIII not linked to XTEN. In one embodiment of
the foregoing, the fusion protein comprises at least a second XTEN,
or at least a third, or at least a fourth XTEN wherein the XTEN are
linked to the factor VIII at a site at or within 1 to 6 amino acids
of a site selected from Table 5, Table 6, Table 7, Table 8, and
Table 9. In another embodiment, the invention provides an
recombinant factor VIII fusion protein further comprising at least
a second XTEN, or at least a third, or at least a fourth XTEN
linked to said FVIII polypeptide at an insertion site selected from
Table 5, Table 6, Table 7, Table 8, Table 9, at or within 6 amino
acids to the N- or C-terminus side of an insertion location at one
or more insertion locations from FIG. 8 and within one or more
insertion ranges from FIG. 9 wherein at least two XTEN are
separated by an amino acid sequence of at least 100 to about 400
amino acids.
[0034] The invention provides CFXTEN wherein the XTEN have a Ratio
XTEN Radii of at least 2.3 or at least 2.5, and are separated by an
amino acid sequence of at least about 20 amino acid residues, or at
least about 50, or at least about 100, or at least about 200, or at
least about 300, or at least about 400 amino acid residues. In
other embodiments, the CFXTEN comprise at least four XTEN wherein
the XTEN have a Ratio XTEN Radii of at least 2.3, or at least 2.5,
or at least 2.8, and wherein at least three of the four of the XTEN
linked to the fusion protein are separated by an amino acid
sequence of at least about 20 amino acid residues, or at least
about 50, or at least about 100, or at least about 200, or at least
about 300, or at least about 400 amino acid residues, and the
fourth XTEN is linked within the B domain (or a fragment thereof)
or within the C domain (or the terminus thereof).
[0035] In some embodiments, the subject compositions are configured
to have reduced binding affinity for a clearance receptor in a
subject as compared to the corresponding FVIII not linked to the
XTEN. In one embodiment, the CFXTEN fusion protein exhibits binding
affinity for a clearance receptor of the FVIII in the range of
about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about
15%, or about 2% to about 10% of the binding affinity of the
corresponding FVIII not linked to the XTEN. In another embodiment,
a fusion protein with reduced affinity for a clearance receptor has
reduced active clearance and a corresponding increase in half-life
of at least about 2-fold, or 3-fold, or at least 4-fold, or at
least about 5-fold, or at least about 6-fold, or at least about
7-fold, or at least about 8-fold, or at least about 9-fold, or at
least about 10-fold, or at least about 12-fold, or at least about
15-fold, or at least about 17-fold, or at least about 20-fold
longer compared to the corresponding FVIII that is not linked to
the XTEN.
[0036] In an embodiment, the invention provides a recombinant
factor VIII fusion protein comprising FVIII and one or more XTEN
wherein the fusion protein exhibits increased solubility of at
least three-fold, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
seven-fold, or at least about eight-fold, or at least about
nine-fold, or at least about ten-fold, or at least about 15-fold,
or at least a 20-fold, or at least 40-fold, or at least 60-fold at
physiologic conditions compared to the FVIII not linked to
XTEN.
[0037] In a further aspect, the invention provides a pharmaceutical
composition comprising the fusion protein of any of the embodiments
described herein and a pharmaceutically acceptable carrier.
[0038] In another embodiment, the invention provides a method of
treating a coagulopathy in a subject, comprising administering to
said subject a composition comprising a clotting effective amount
of the pharmaceutical composition. In one embodiment of the method,
after said administration, a blood concentration of procoagulant
factor VIII is maintained at about 0.05, or 1, or 1.5 IU/ml or more
for at least 48 hours after said administration. In another
embodiment, the invention provides a method of clotting blood in a
subject, comprising contacting a clotting effective amount of the
pharmaceutical composition with the blood.
[0039] In another embodiment, the invention provides a method of
treating a coagulopathy in a subject with circulating inhibitors of
factor VIII, comprising administering to said subject a composition
comprising a therapeutically effective amount of the pharmaceutical
composition of CFXTEN, wherein the composition exhibits greater
procoagulant activity in said subject compared to a composition
comprising the corresponding factor VIII not linked to XTEN and
administered using a comparable amount. In one embodiment of the
method, the coagulopathy is hemophilia A. In another embodiment,
the coagulopathy is the result of trauma or surgery or
infection.
[0040] The invention provides a method of treating a bleeding
episode in a subject, comprising administering to said subject a
composition comprising a clotting effective amount of the CFXTEN
pharmaceutical composition, wherein the clotting effective amount
of the fusion protein arrests a bleeding episode for a period that
is at least three-fold, or at least four-fold, or at least
five-fold longer compared to a corresponding factor VIII not linked
to XTEN and administered using a comparable amount to said subject.
Non-limiting examples of a corresponding factor VIII not linked to
XTEN include native FVIII, the sequences of Table 1, BDD-FVIII, and
the pCB0114 FVIII.
[0041] In another embodiment, the invention provides a CFXTEN
recombinant factor VIII fusion protein for use in a pharmaceutical
regimen for treating a hemophilia A patient, said regimen
comprising a pharmaceutical composition comprising a CFXTEN fusion
protein. In one embodiment of the pharmaceutical regimen, the
regimen further comprises the step of determining the amount of
pharmaceutical composition comprising the CFXTEN needed to achieve
hemostasis in the hemophilia A patient. In another embodiment, the
pharmaceutical regimen for treating a hemophilia A 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 hemophilia A disease compared to the factor VIII not
linked to XTEN and administered using a comparable dose.
Non-limited examples of parameters improved include blood
concentration of procoagulant FVIII, a reduced activated partial
prothrombin (aPTT) assay time, a reduced one-stage or two-stage
clotting assay time, delayed onset of a bleeding episode, a reduced
chromogenic assay time, a reduced bleeding assay time, resolution
of a bleeding event, or a reduced Bethesda titer to native
FVIII.
[0042] In another aspect, the invention provides isolated nucleic
acid sequences encoding the fusion proteins of any one of the
embodiments of the CFXTEN fusion protein. In one embodiment, the
isolated nucleic acid is the complement of a sequence encoding a
CFXTEN fusion protein of the embodiments. In one embodiment, the
isolated nucleic acid further comprises a sequence encoding a
signal peptide, wherein said sequence is
ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID
NO: 1613), or the complement thereof. In another embodiment, the
invention provides an expression vector comprising the nucleic acid
encoding the fusion protein, or the complement thereof. In another
embodiment, the invention provides an isolated host cell comprising
the foregoing expression vector. In another embodiment, the
invention provides a method of producing the fusion protein of any
of the embodiments, comprising providing a host cell comprising the
expression vector; culturing the host cell to effect production of
the fusion protein; and recovering the fusion protein.
[0043] In one embodiment, the invention provides an isolated fusion
protein comprising a polypeptide having at least about 80% sequence
identity, or 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%, to about 100% sequence identity compared to a
sequence of comparable length selected from Table 21, when
optimally aligned.
[0044] In another embodiment, the invention provides an isolated
nucleic acid comprising a polynucleotide sequence selected from (a)
a sequence having at least about 80% sequence identity, or 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%,
to about 100% sequence identity compared to a sequence of
comparable length selected from Table 21, when optimally aligned,
or (b) the complement of the polynucleotide of (a). In another
embodiment, the isolated nucleic acid comprises the sequence
ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID
NO: 1613) linked to the 5' end of the nucleic acid of (a) or the
complement of the sequence linked to the 3' end of (b).
[0045] It is specifically contemplated that the recombinant factor
VIII fusion proteins can exhibit one or more or any combination of
the properties disclosed herein.
INCORPORATION BY REFERENCE
[0046] 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
[0047] 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.
[0048] FIGS. 1A-1C show a schematic representation of the FVIII
architecture and spatial arrangement of the domains during
processing and clotting, and is intended to represent both native
FVIII and B domain deleted variants. The A1 domain ranges from
residue 1 to 372 (numbering relative to the mature form of FVIII
sequence NCBI Protein RefSeq NP_000123 and encompassing a1
residues), A2 domain ranges from residue 373 to 740, B domain
ranges from residue 741 to 1648, A3 domain ranges from residue 1649
to 2019 (encompassing a3 acidic region), C1 domain ranges from 2020
to 2172, and the C2 domain ranges from residue 2173 to 2332. BDD
variants include deletions between the range 741 to 1648, leaving
some or no remnant residues, with a non-limiting BDD remnant
sequence being SFSQNPPVLKRHQR (SEQ ID NO: 1614). FIG. 1A shows the
domain architecture of a single chain FVIII prior to processing.
Arrows indicate the sites at residues R372, R740, R1648, and R1689
that are cleaved in the processing and conversion of FVIII to
FVIIIa. FIG. 1B shows the FVIII molecule that has been processed
into the heterodimer by the cleavage at the R1648 residue, with the
a3 acidic region of the A3 domain indicated on the N-terminus of
the A3. FIG. 1C shows the FVIII molecule processed into the FVIIIa
heterotrimer by the cleavage at the R372, R740, and R1689
residues.
[0049] FIG. 2 is a schematic of the coagulation cascade, showing
the intrinsic and extrinsic arms leading to the common pathway.
[0050] FIG. 3 depicts the amino acid sequence of mature human
factor VIII (SEQ ID NO: 1592).
[0051] FIG. 4 depicts a factor VIII sequence with a deletion of a
portion of the B domain (SEQ ID NO: 1593).
[0052] FIGS. 5A-5D illustrate several examples of CFXTEN
configurations of FVIII linked to XTEN (the latter shown as thick,
wavy lines). In all cases, the FVIII can be either native or a BDD
form of FVIII, or a single chain form in which the entire B domain,
including the native cleavage sites are removed. FIG. 5A shows,
left to right, three variations of single chain factor VIII with
XTEN linked to the N-terminus, the C-terminus, and two XTEN linked
to the N- and C-terminus. FIG. 5B shows six variations of mature
heterodimer FVIII with, left to right, an XTEN linked to the
N-terminus of the A1 domain; an XTEN linked to the C-terminus of
the C2 domain; an XTEN linked to the N-terminus of the A1 domain
and the C-terminus of the C2 domain; an XTEN linked to the
N-terminus of the A1 domain and to the N-terminus of the A3 domain;
an XTEN linked to the C-terminus of the C2 domain and to the
N-terminus of the A3 domain via residual B domain amino acids; and
an XTEN linked to the N-terminus of the A1 domain, the C-terminus
of the A2 domain via residual B domain amino acids, and to the
C-terminus of the C2 domain. FIG. 5C shows, left to right, three
variations of single chain factor VIII: an XTEN linked to the
N-terminus of the A1 domain, an XTEN linked within a surface loop
of the A1 domain and an XTEN linked within a surface loop of the A3
domain; an XTEN linked within a surface loop of the A2 domain, an
XTEN linked within a surface loop of the C2 domain and an XTEN
linked to the C terminus of the C2 domain; an XTEN linked to the
N-terminus of the A1 domain and within a surface loop of the C1
domain and to the C-terminus of the C domain. FIG. 5D shows six
variations of mature heterodimer FVIII with, left to right, an XTEN
linked to the N-terminus of the A1 domain, an XTEN linked within a
surface loop of the A1 domain, and an XTEN linked within a surface
loop of the A3 domain; an XTEN linked within a surface loop of the
A2 domain, and an XTEN linked within a surface loop of the C1
domain, and an XTEN linked to the C-terminus of the C2 domain; an
XTEN linked to the N-terminus of the A1 domain, an XTEN linked
within a surface loop of the A1 domain, an XTEN linked within a
surface loop of the A3 domain, and an XTEN linked to the C-terminus
of the C2 domain; an XTEN linked to the N-terminus of the A1
domain, an XTEN linked to the N-terminus of the A3 domain via
residual amino acids of the B domain, and an XTEN linked within a
surface loop of the C2 domain; an XTEN linked within a surface loop
of the A2 domain, an XTEN linked to the N-terminus of the A3 domain
via residual amino acids of the B domain, an XTEN linked within a
surface loop of the C1 domain, and an XTEN linked to the C-terminus
of the C2 domain; and an XTEN linked within the B domain or between
the residual B domain residues of the BDD variant (and the
invention also contemplates a variation in which the XTEN replaces
the entirety of the B domain, including all native cleavage sites,
linking the A2 and A3 domains, resulting in a single chain form of
factor VIII). This figure also embodies all variations in which one
or more XTEN sequences are inserted within the B domain and the
resulting fusions are cleaved at one or more sites (e.g., at R1648
site) during intracellular processing.
[0053] FIG. 6A is a graphic portrayal of a CFXTEN construct with an
XTEN inserted within the B domain and linked to the C-terminus of
the C2 domain illustrating the unstructured characteristic of the
XTEN leading to random coil formation that can cover portions of
the factor VIII proximal to the XTEN. In the lower panel (FIG. 6B),
the drawing depicts that when XTEN is in random coil, it can adopt
a conformation resulting in steric hindrance that blocks binding of
factor VIII inhibitor antibodies that would otherwise have affinity
for epitopes proximal to the XTEN site of insertion.
[0054] FIG. 7 is a graphic portrayal of the various analyses
performed on a FVIII B-domain deleted sequence to identify
insertion sites for XTEN within the FVIII sequence. Each of lines
A-H are on an arbitrary scale of Y axis values across the FVIII BDD
sequence such that low values represent areas with a high predicted
tolerance for XTEN insertion, with the residue numbers on the X
axis. Line A shows the domain boundaries; all discontinuities in
this line represent boundaries that are likely to accept XTEN. Line
B shows exon boundaries; i.e., each step in the line represents a
new exon. Line C shown regions that were not visible in the X-ray
structure due to a lack of order in the crystal. Lines labeled D
represents multiple predictions of order that were calculated using
the respective programs FoldIndex found on the World-Wide web site
bip.weizmann.ac.il/fldbin/findex (last accessed Feb. 23, 2011) (see
Jaime Prilusky, Clifford E. Felder, Tzviya Zeev-Ben-Mordehai, Edwin
Rydberg, Orna Man, Jacques S. Beckmann, Israel Silman, and Joel L.
Sussman, 2005, Bioinformatics based on the Kyte & Doolitlle
algorithm, as well as RONN found on the World-Wide web site
strubi.ox.ac.uk/RONN (last accessed Feb. 23, 2011) (see Yang, Z.
R., Thomson, R., McMeil, P. and Esnouf, R. M. (2005) RONN: the
bio-basis function neural network technique applied to the
detection of natively disordered regions in proteins Bioinformatics
21: 3369-3376. Lines E and F were calculated based on multiple
sequence alignments of FVIII genes from 11 mammals available in
GenBank. Line E represents the conservation of individual residues.
Line F represent the conservation of 3 amino acid segments of
FVIII. Lines G and H represent gaps and insertions observed in the
multiple sequence alignment of 11 mammalian FVIII genes. Line J
lists the XTEN insertion points by amino acid number that were
obtained based by combining the multiple measurements above.
[0055] FIG. 8 depicts the sites in a FVIII B-domain deleted
sequence (SEQ ID NO: 1594) identified as active insertion points
for XTEN using the information depicted in FIG. 8 and as confirmed
in the assays of Example 34.
[0056] FIG. 9 depicts the range of sites in a FVIII B-domain
deleted sequence (SEQ ID NO: 1595) identified for insertion of XTEN
using the information depicted in FIG. 8 and or Example 34 plus a
span of amino acids around each insertion point that are considered
suitable for insertion of XTEN.
[0057] FIG. 10 is a schematic of the assembly of a CFXTEN library
created by identifying insertion points as described for FIG. 7
followed by insertion of single XTEN (black bars) at the various
insertion points using molecular biology techniques. The constructs
are expressed and recovered, then evaluated for FVIII activity and
pharmacokinetic properties to identify those CFXTEN configurations
that result in enhanced properties.
[0058] FIG. 11 is a schematic of the assembly of a CFXTEN component
library in which segments of FVIII BDD domains, either singly or
linked to various lengths of XTEN (black bars) are assembled in a
combinatorial fashion into libraries of genes encoding the CFXTEN,
which can then be evaluated for FVIII activity and pharmacokinetic
properties to identify those CFXTEN configurations that result in
enhanced properties.
[0059] FIGS. 12A-12D illustrate several examples of CFXTEN
configurations with XTEN (shown as thick, wavy lines), with certain
XTEN releasable by inserting cleavage sequences (indicated by black
triangles) that are cleavable by procoagulant proteases. FIG. 12A
illustrates a scFVIII with two terminal releasable XTENS. FIG. 12B
illustrates the same configuration as FIG. 12A but with an
additional non-releasable XTEN linking the A3 and C1 domains. FIG.
12C illustrates a mature heterodimer FVIII with two terminal
releasable XTEN. FIG. 12D illustrates the same configuration as 10
C but with an additional non-releasable XTEN linking the A3 and C1
domains.
[0060] FIG. 13 is a schematic flowchart of representative steps in
the assembly, production and the evaluation of an XTEN.
[0061] FIG. 14 is a schematic flowchart of representative steps in
the assembly of a CFXTEN 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 containing a
gene encoding the FVIII, resulting in the gene 500 encoding an
FVIII-XTEN fusion protein.
[0062] FIG. 15 is a schematic flowchart of representative steps in
the assembly of a gene encoding fusion protein comprising a CF and
XTEN, its expression and recovery as a fusion protein, and its
evaluation as a candidate CFXTEN product.
[0063] FIGS. 16A-16F illustrate the use of donor XTEN sequences to
produce truncated XTENs. FIG. 16A provides the sequence of AG864
(SEQ ID NO: 1596), with the underlined sequence used to generate a
sequence length of 576 (SEQ ID NO: 1597). FIG. 16B provides the
sequence of AG864 (SEQ ID NO: 1598), with the underlined sequence
used to generate a sequence length of 288 (SEQ ID NO: 1599). FIG.
16C provides the sequence of AG864 (SEQ ID NO: 1600), with the
underlined sequence used to generate a sequence length of 144 (SEQ
ID NO: 1601). FIG. 16D provides the sequence of AE864 (SEQ ID NO:
1602), with the underlined sequence used to generate a sequence
length of 576 (SEQ ID NO: 1603). FIG. 16E provides the sequence of
AE864 (SEQ ID NO: 1604), with the underlined sequence used to
generate a sequence length of 288 (SEQ ID NO: 1605). FIG. 16F
provides the sequence of AE864 (SEQ ID NO: 1606) used to generate
four sequences of 144 length (SEQ ID NOS 1607-1610, respectively,
in order of appearance) (the double underline indicates the first
amino acid in the 144 sequence with the single underline
representing the balance of that sequence).
[0064] FIGS. 17A-17C are schematic representations of the design of
Factor VIII-XTEN expression vectors with different strategies
introducing XTEN elements into the FVIII coding sequence. FIG. 17A
shows an expression vector encoding XTEN fused to the 3' end of the
sequence encoding FVIII. FIG. 17B depicts an expression vector
encoding an XTEN element inserted into the middle of the coding
sequence encoding a single FVIII. FIG. 17C depicts an expression
vector encoding two XTEN elements: one inserted internal to the
FVIII coding sequence, and the other fused to the 3' end of the
FVIII coding sequence.
[0065] FIG. 18 illustrates the process of combinatorial gene
assembly of genes encoding XTEN. In this case, the genes are
assembled from 6 base fragments and each fragment is available in 4
different codon versions (A, B, C and D). This allows for a
theoretical diversity of 4096 in the assembly of a 12 amino acid
motif.
[0066] FIG. 19 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 41. 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.
[0067] FIGS. 20A-20C 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 (see Example 42). The GFP-XTEN was incubated in
(A) cynomolgus plasma and (C) rat kidney lysate for up to 7 days at
37.degree. C. In addition, GFP-XTEN administered to (B) 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 with antibodies against GFP.
[0068] FIG. 21 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 40.
The glucagon-XTEN constructs are 1) glucagon-Y288; 2)
glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results
indicate an increase in apparent molecular weight with increasing
length of XTEN moiety (see Example 40 for data).
[0069] FIG. 22 shows results of a Western blot of proteins
expressed by cell culture of cells transformed with constructs as
designated (Example 25). The samples in lanes 1-12 were: MW
Standards, FVIII (42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114,
pBC0135, pBC0136, pBC0137, pBC0145, pBC0149, and pBC0146,
respectively. Lanes 8, 9 and 12 show bands consistent with a FVIII
with a C-terminal XTEN288, with an estimated MW of 95 kDa. Lanes 7
and 11 show bands consistent with a FVIII with a C-terminal XTEN42,
with an estimated MW of 175 kDa. Lanes 2-6 show bands consistent
with FVIII and heavy chain. Lanes 10 and 23 show bands consistent
with heavy chain. Lane 7 shows a band consistent with heavy chain
and an attached XTEN42.
[0070] FIG. 23 shows the results of FVIII assay on samples obtained
from FVIII and von Willebrand factor double knock-out mice with
hydrodynamic plasmid DNA injection, as detailed in Example 36.
[0071] FIGS. 24A-24B are graphic and tabular portrayals of the
pharmacokinetic properties of rBDD-FVIII and the purified CFXTEN
fusion proteins pBC0145 and pBC0146 (with C-terminal XTEN)
administered to either HemA (FIG. 24A) or FVIII/VWF double
knock-out (FIG. 24B) mice as described in Example 30, showing the
enhanced half-life of the CFXTEN in both strains of mice.
[0072] FIGS. 25A-25B are graphic and tabular portrayals of the
pharmacokinetic properties of rBDD-FVIII and the CFXTEN fusion
proteins pSD0050 and pSD0062 (with internal inserted XTEN)
administered to either HemA (FIG. 25A) or FVIII/VWF double
knock-out mice (FIG. 25B) using a cell culture PK assay in HemA
mice. Dose, 5-minute recovery, and half-life (T1/2) are shown, as
described in Example 32, underscoring the enhanced recovery and
half-life of the CFXTEN compared to the positive control FVIII in
both strains of mice.
[0073] FIG. 26 is a graphic depiction of a titration of GMA8021
FVIII inhibitor using the pBC0114 BDD-FVIII AND CFXTEN construct
LSD0049.002 with three 144 amino acid XTEN insertions at residues
18, 745 and 2332. The data indicate a right-shift of approximately
0.7 order of magnitude in the amount of antibody in .mu.g/ml
required to inhibit the CFXTEN to the 50% level, compared to FVIII
positive control.
[0074] FIG. 27 is a schematic of the logic flow chart of the
algorithm SegScore. 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.
[0075] FIG. 28 depicts the application of the algorithm SegScore to
a hypothetical XTEN of 11 amino acids (SEQ ID NO: 1591) in order to
determine the repetitiveness. An XTEN sequence 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 the subsequence score of 1.89.
[0076] FIG. 29 is a graph of the individual construct values of the
ratio of FVIII activity in the assayed CFXTEN to that of the pBC114
FVIII positive control after exposure to the GMA8021 antibody to
FVIII, grouped according to the number of XTEN in the construct
fusion protein (see Example 28). The results show an essentially
linear relationship in the ability of the CFXTEN to retain FVIII
activity with increasing number of incorporated XTEN.
[0077] FIGS. 30A-30H depict the primary sequence and domain
structure of mature B-domain deleted (BDD) human FVIII construct
(Example 46). The location of the introduced NheI and ClaI
restriction sites is shown. Note that the amino acid numbering
corresponds to the amino acid positions in the primary sequence of
mature FVIII (FIGS. 30A-30H). Individual domains are bounded by
gray lines/boxes with domain identification in gray text. Acidic
regions (a1, a2, a3) are indicated with dashed boxes. Solid
wedges/triangles indicate sites of thrombin cleavage in the
activation of FVIII to FVIIIa. Unfilled wedges/triangle indicates
the site of intracellular proteolytic processing to the two-chained
form of FVIII. Hexagons indicate sites of N-linked glycosylation.
Circles indicate sites of Tyr sulfation. Unique non-native
restriction sites (NheI, GCTAG; ClaI, ATCGAT) introduced into cDNA
to facilitate XTEN insertion/recombination are highlighted in gray
with double underline.
[0078] FIG. 31 provides graphical representation of the FVIII
construct described in FIG. 30, indicating the domain organization
and the location of native and non-native restriction sites.
[0079] FIG. 32 shows the graphical ASAView outputs for structural
datasets 2R7E, 3CDZ, and PM0076106. Accessible Solvent Areas (ASA)
for the amino acids in domains A1, A2, A3, C1 and C2 are shown.
Analyses were performed on X-ray crystallographic coordinates 3CDZ
(Ngo et al., Structure 16: 597-606 (2008)) and 2R7E (Shen et al.,
Blood 111:1240-1247 (2008)) deposited in the Protein Data Bank
maintained by the Research Collaboratory for Structural
Bioinformatics (RCSB; http://www.rcsb.org/pdb), as well as on
atomic coordinates PM0076106 for the predicted refined FVIII
structure derived from a molecular dynamics simulation study
(Venkateswarlu, BMC Struct. Biol. 10:7 (2010)) deposited in the
Protein Model Database (http://mi.caspur.it/PMDB/main.php)
maintained by Consorzio Interuniversitario per le Applicazioni di
Supercalcolo per Universita e Riserca (CASPUR) and the Department
of Biochemical Sciences of the University of Rome.
[0080] FIG. 33 shows a structural representation of the location of
XTEN insertion sites. The central drawing corresponding to the
crystal structure of FVIII (PDB: 2R7E) is surrounded by detailed
view of domains A1, A2, A3, C1 and C2. Beta strands and alpha
helices are shown as ribbon representation. Loops are shown as
alpha carbon pipes. The amino acids at XTEN insertion sites are
shown as CPK sphere representation. The number in each graph
indicate the location of the XTEN insertion sites according to the
numbering in FIG. 30.
[0081] FIG. 34 shows a structural representation of the location of
XTEN insertion sites shown in FIG. 33 wherein the resulting
recombinant FVIII protein displays FVIII activity.
[0082] FIG. 35 shows a structural representation of the location of
XTEN insertion sites shown in FIG. 34 wherein the resulting
recombinant FVIII protein displays FVIII activity.
[0083] FIG. 36 shows a structural representation of the location of
XTEN insertion sites shown in FIG. 35 wherein the resulting
recombinant FVIII protein displays FVIII activity.
[0084] FIG. 37 shows a ClustalW multiple sequence alignment of
domains A1, A2, A3, C1 and C2 of FVIII showing the location of XTEN
insertions resulting in recombinant FVIII proteins displaying FVIII
activity (black box, white text) or displaying no FVIII activity
(grey box, bold text).
[0085] FIG. 38 shows a DSSP graphical representation of the
secondary structure of the two polypeptide chains in a native
active human FVIII crystal structure deposited under the identifier
2R7E at the Protein Data Bank (see Example 47). Amino acid sequence
numbering is the same as in the protein sequence in FIG. 30. The
beta sheet regions are shown as filled arrows and are designated R1
to P66. The location of the XTEN permissive loops is denoted by
crosshatched boxes. Domain A1 XTEN permissive loops are designated
Loop A1-1 and Loop A1-2. Domain A2 XTEN permissive loops are
designated Loop A2-1 and Loop A2-2. Domain A3 XTEN permissive loops
are designated Loop A3-1 and Loop A3-2.
[0086] FIG. 39 shows a DSSP graphical representation of the
secondary structure of the two polypeptide chains in a native
active human FVIII crystal structure deposited under the identifier
2R7E at the Protein Data Bank (see Example 47). Amino acid sequence
numbering is the same as in the protein sequence in FIG. 30. The
beta sheet regions are shown as filled arrows and are designated R1
to P66. The location of the XTEN permissive loops is denoted by
crosshatched boxes. Domain A1 XTEN permissive loops are designated
Loop A1-1 and Loop A1-2. Domain A2 XTEN permissive loops are
designated Loop A2-1 and Loop A2-2. Domain A3 XTEN permissive loops
are designated Loop A3-1 and Loop A3-2.
[0087] FIG. 40 shows a ClustalW multiple sequence alignment of
domains A1, A2, A3, C1 and C2 of FVIII showing the location of XTEN
insertions resulting in recombinant FVIII proteins displaying FVIII
activity (black box, white text) or displaying no FVIII activity
(grey box, bold text). The locations of the XTEN permissive loops
are indicated by dashed rectangles (see Example 47).
[0088] FIG. 41A presents a front view structural representation of
human FVIII (PDB:2R7E) showing the location of domains A1, A2, A3,
C1 and C2 (circled in dashed lined) and the locations of XTEN
permissive loops A1-1, A1-2, A2-1, A2-2, A3-1 and A3-2 highlighted
as CPK sphere representations. FIG. 41B presents a side view
structural representation of human FVIII (PDB:2R7E) showing the
location of domains A1, A2, A3, C1 and C2 (circled in dashed lined)
and the locations of XTEN permissive loops A1-1, A1-2, A2-1, A2-2,
A3-1 and A3-2 highlighted as CPK sphere representations.
[0089] FIGS. 42A, 42C, and 42E show the top view structural
representations of isolated human FVIII (PDB:2R7E) A domains
showing the location of XTEN permissive loops highlighted as CPK
sphere representations. FIGS. 42B, 42D and 42F show side view
structural representations of isolated human FVIII (PDB:2R7E) A
domains showing the location of XTEN permissive loops highlighted
as CPK sphere representations.
[0090] FIG. 43 shows sequences of various factor VIII B-domain
deletions and individual mutations. Lines 4-10 show various
B-domain deletions with indicated XTEN linking the flanking
B-domain residual or A3 domain residues. The R1648A mutation is
indicated by arrow in line 5 and 8, while the Y1680F mutation is
indicated by arrow in lines 8-10.
[0091] FIG. 44 is a bar graph of chromogenic and aPTT assay
activity of various CFXTEN with single XTEN insertions (Example
49).
[0092] FIG. 45 is a bar graph of chromogenic and aPTT assay
activity of various CFXTEN with 2 XTEN insertions (Example 49).
[0093] FIG. 46 is a bar graph of chromogenic and aPTT assay
activity of various CFXTEN with 3 XTEN insertions (Example 49).
[0094] FIG. 47 is a graph of plasma levels in DKO mice of various
administered CFXTEN with single XTEN insertions compared to a
BDD-FVIII control, demonstrating the 10- to 20-fold longer
half-life achieved by the XTEN insertions at various locations
(Example 50).
[0095] FIG. 48 is a graph of plasma levels in DKO mice of various
administered CFXTEN with one, two, and three XTEN insertions
compared to a BDD-FVIII control, demonstrating the increases in
half-life achieved by the inclusion of additional XTEN insertions
compared to single or two insertions (Example 51).
[0096] FIGS. 49A-49D are graphs of the plotted inhibition curves
for remaining factor VIII procoagulant activity in samples assayed
in the Bethesda assay with three hemophilia patient sera (FIGS.
49A-49C) or sheep anti-FVII (FIG. 49D) described in Example 52,
demonstrating a clear left-shift of the inhibition curve for the
two CFXTEN molecules compared to the FVIII not linked to XTEN.
DETAILED DESCRIPTION OF THE INVENTION
[0097] 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.
[0098] 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
[0099] In the context of the present application, the following
terms have the meanings ascribed to them unless specified
otherwise:
[0100] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0101] 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.
[0102] 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.
[0103] The term "domain," when used in reference to a factor VIII
polypeptide refers to either a full length domain or a functional
fragment thereof, for example, full length or functional fragments
of the A1 domain, A2 domain, A3 domain, B domain, C1 domain, and/or
C2 domain of factor VIII.
[0104] 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).
[0105] 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.
[0106] 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.
[0107] A "fragment" when applied to a protein, is a truncated form
of a native biologically active protein that retains at least a
portion of the therapeutic and/or biological activity. A "variant".
when applied to a 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 moiety" includes proteins modified deliberately, as
for example, by site directed mutagenesis, synthesis of the
encoding gene, insertions, or accidentally through mutations.
[0108] 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. 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.
[0109] As used herein, "internal XTEN" refers to XTEN sequences
that have been inserted into the sequence of the coagulation
factor. Internal XTENs can be constructed by insertion of an XTEN
sequence into the sequence of a coagulation factor such as FVIII,
either by insertion between two adjacent amino acids within a
domain ("intradomain") or between two domains ("interdomain") of
the coagulation factor or wherein XTEN replaces a partial, internal
sequence of the coagulation factor.
[0110] As used herein, "terminal XTEN" refers to XTEN sequences
that have been fused to or in the N- or C-terminus of the
coagulation factor or to a proteolytic cleavage sequence or linker
at the N- or C-terminus of the coagulation factor. Terminal XTENs
can be fused to the native termini of the coagulation factor.
Alternatively, terminal XTENs can replace a portion of a terminal
sequence of the coagulation factor.
[0111] The term "XTEN release site" refers to a cleavage sequence
in CFXTEN fusion proteins that can be recognized and cleaved by a
mammalian protease, effecting release of an XTEN or a portion of an
XTEN from the CFXTEN fusion protein. 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 FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa,
FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any
protease that is present during a clotting event. 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.
[0112] 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
domain of a factor VIII polypeptide, the XTEN may be linked to the
N-terminus, the C-terminus, or may be inserted in said domain.
[0113] As used herein, the term "site," when used to refer to an
insertion site of an XTEN within or to a biological polypeptide
such as a factor VIII, represents the amino acid position at which
the XTEN is linked. When numbered sites are described, such as a
first, second, third, fourth, fifth, or sixth site for the
insertion of an XTEN within or to the factor VIII, each site will
be understood to represent a distinct site in the factor VIII;
e.g., the second site is a different factor VIII location from the
first site, the third site is different from the second and the
first, etc.
[0114] "Activity" or "procoagulant activity" as applied to form(s)
of a CFXTEN polypeptide provided herein, refers to the ability to
bind to a target coagulation protein substrate or cofactor and
promote a clotting event, whether measured by an in vitro, ex vivo
or in vivo assay. Such assays include, but are not limited to,
one-stage clotting assays, two-stage clotting assays, chromogenic
assays, and ELISA assays. "Biological activity" refers to an in
vitro or in vivo biological function or effect, including but not
limited to either receptor or ligand binding, or an effect on
coagulation generally known in the art for the FVIII coagulation
factor, or a cellular, physiologic, or clinical response, including
arrest of a bleeding episode.
[0115] As used herein, the term "ELISA" refers to an enzyme-linked
immunosorbent assay as described herein or as otherwise known in
the art.
[0116] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for the subject vectors. 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.
[0117] "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."
[0118] An "isolated" polynucleotide or polypeptide-encoding nucleic
acid or other polypeptide-encoding 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. 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.
[0119] 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.
[0120] "Conjugated", "linked," "fused," and "fusion" are used
interchangeably herein. These terms refer to the joining together
of two or more chemical elements, sequences or components, by
whatever means including chemical conjugation or recombinant means.
For example, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence.
Generally, "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. 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).
[0121] 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.
[0122] "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.
[0123] 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.
[0124] 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.
[0125] "Recombinant" as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of in vitro
cloning, restriction and/or ligation steps, and other procedures
that result in a construct that can potentially be expressed as a
recombinant protein in a host cell.
[0126] 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.
[0127] "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%, and most preferably 100% identical.
[0128] "Ligation" 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.
[0129] 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.
[0130] 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.
[0131] "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.
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 maximal 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.
[0132] The term "non-repetitiveness" as used herein in the context
of a polypeptide refers to a lack or limited degree of internal
homology in a peptide or polypeptide sequence. The term
"substantially non-repetitive" can mean, for example, that there
are few or no instances of four contiguous amino acids in the
sequence that are identical amino acid types or that the
polypeptide has a subsequence score (defined infra) of 10 or less
or that there is no a pattern in the order, from N- to C-terminus,
of the sequence motifs that constitute the polypeptide sequence.
The term "repetitiveness" as used herein in the context of a
polypeptide refers to the degree of internal homology in a peptide
or polypeptide sequence. In contrast, a "repetitive" sequence may
contain multiple identical copies of short amino acid sequences.
For instance, a polypeptide sequence of interest may be divided
into n-mer sequences and the number of identical sequences can be
counted. Highly repetitive sequences contain a large fraction of
identical sequences while non-repetitive sequences contain few
identical sequences. In the context of a polypeptide, a sequence
can contain multiple copies of shorter sequences of defined or
variable length, or motifs, in which the motifs themselves have
non-repetitive sequences, rendering the full-length polypeptide
substantially non-repetitive. The length of polypeptide within
which the non-repetitiveness is measured can vary from 3 amino
acids to about 200 amino acids, about from 6 to about 50 amino
acids, or from about 9 to about 14 amino acids. "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.
[0133] 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.
[0134] "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.
[0135] The term "t.sub.1/2" as used herein means the terminal
half-life calculated as ln(2)/K.sub.el. K.sub.el 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. The terms "t.sub.1/2", "terminal half-life",
"elimination half-life" and "circulating half-life" are used
interchangeably herein.
[0136] "Active clearance" means the mechanisms by which a protein
is removed from the circulation other than by filtration or
coagulation, and which includes removal from the circulation
mediated by cells, receptors, metabolism, or degradation of the
protein.
[0137] "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 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.
[0138] 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 fusion
proteins 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.
[0139] "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.
[0140] A "reactive group" is a chemical structure that can be
coupled to a second reactive group. Examples for reactive groups
are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl
groups, aldehyde groups, azide groups. Some reactive groups can be
activated to facilitate coupling with a second reactive group.
Non-limiting examples for activation are the reaction of a carboxyl
group with carbodiimide, the conversion of a carboxyl group into an
activated ester, or the conversion of a carboxyl group into an
azide function.
[0141] "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.
[0142] 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.
[0143] The term "payload" as used herein refers to a protein or
peptide sequence that has biological or therapeutic activity; the
counterpart to the pharmacophore of small molecules. Examples of
payloads include, but are not limited to, coagulation factors,
cytokines, enzymes, hormones, and blood and growth factors.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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
fusion protein 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.
[0148] 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 fusion protein 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.
[0149] 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 fusion protein
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
[0150] 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 h
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.
II). Coagulation Factor VIII
[0151] The present invention relates, in part, to compositions
comprising factor VIII coagulation factor (CF) linked to one or
more extended recombinant proteins (XTEN), resulting in a CFXTEN
fusion protein composition. As used herein, "CF" refers to factor
VIII (FVIII) or mimetics, sequence variants and truncated versions
of FVIII, as described below.
[0152] "Factor VIII" or "FVIII" or "FVIII protein" means a blood
coagulation factor protein and species (including human, porcine,
canine, rat or murine FVIII proteins) and sequence variants thereof
that includes, but is not limited to the 2351 amino acid
single-chain precursor protein (with a 19-amino acid hydrophobic
signal peptide), the mature 2332 amino acid factor VIII cofactor
protein of approximately 270-330 kDa with the domain structure
A1-A2-B-A3-C1-C2, as well as the nonenzymatic "active" or cofactor
form of FVIII (FVIIIa) that is a circulating heterodimer of two
chains that form as a result of proteolytic cleavage after R1648 of
a heavy chain form composed of A1-A2-B (in the range of 90-220 kD)
of amino acids 1-1648 (numbered relative to the mature FVIII form)
and a light chain A3-C1-C2 of 80 kDa of amino acids 1649-2232, each
of which is depicted schematically in FIG. 1. Further, and as used
herein, each of A1, A2 and the A3 domain encompasses acidic spacer
regions; a1, a2, and a3 acidic regions, respectively. Thus, it will
be understood that CFXTEN constructs described as having A1, A2,
A3, B, C1 and C2 domains include the a1, a2 and a3 acidic regions.
As used herein, "Factor VIII" or "FVIII" or "FVIII polypeptide"
also includes variant forms, including proteins with substitutions,
additions and/or deletions so long as the variant retains a desired
biological activity such as procoagulant activity. Myriad
functional FVIII variants have been constructed and can be used as
recombinant FVIII proteins as described herein. See PCT Publication
Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635 A2, or WO
2012/006633 A2, all of which are incorporated herein by reference
in their entireties. A great many functional FVIII variants are
known. In addition, hundreds of nonfunctional mutations in FVIII
have been identified in hemophilia patients. See, e.g., Cutler et
al., Hum. Mutat. 19:274-8 (2002), incorporated herein by reference
in its entirety. In addition, comparisons between FVIII from humans
and other species have identified conserved residues that are
likely to be required for function. See, e.g., Cameron et al.,
Thromb. Haemost. 79:317-22 (1998) and U.S. Pat. No. 6,251,632,
incorporated herein by reference in their entireties.
[0153] In one embodiment, the human factor VIII domains are defined
by the following amino acid residues: A1, residues Ala1-Arg372; A2,
residues Ser373-Arg740; B, residues Ser741-Arg1648; A3, residues
Ser1649-Asn2019; C1, residues Lys2020-Asn2172; C2, residues
Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues
Ser1649-Tyr2332. In another embodiment, residues Arg336-Arg372 is
usually referred to as the a1 region, and the Arg372 is cleaved by
thrombin. In certain embodiments, the a2 region is part of the A1
domain. In another embodiment, residues Glu1649-Arg1689, is
referred to as the a3 acidic region. In certain embodiments, the a3
acidic region is a part of the A3 domain. In another embodiment, a
native FVIII protein has the following formula:
A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are the
structurally-related "A domains," B is the "B domain," C1 and C2
are the structurally-related "C domains," and a1, a2 and a3 are
acidic spacer regions. In the foregoing formula and referring to
the primary amino acid sequence position in FIG. 30, the A1 domain
of human FVIII extends from Ala1 to about Arg336, the a1 spacer
region extends from about Met337 to about Arg372, the A2 domain
extends from about Ser373 to about Tyr719, the a2 spacer region
extends from about Glu720 to about Arg740, the B domain extends
from about Ser741 to about Arg 1648, the a3 spacer region extends
from about Glu1649 to about Arg1689, the A3 domain extends from
about Ser1690 to about Asn2019, the C1 domain extends from about
Lys2020 to about Asn2172, and the C2 domain extends from about
Ser2173 to Tyr2332 (Saenko et al., 2005, J Thromb Hemostasis, 1,
922-930). Other than specific proteolytic cleavage sites,
designation of the locations of the boundaries between the domains
and regions of FVIII can vary in different literature references.
The boundaries noted herein are therefore designated as approximate
by use of the term "about."
[0154] Such factor VIII include truncated sequences such as
B-domain deleted "BDD" sequences in which a portion or the majority
of the B domain sequence is deleted (such as BDD sequences
disclosed or referenced in U.S. Pat. Nos. 6,818,439 and 7,632,921).
An example of a BDD FVIII is REFACTO.RTM. or XYNTHA.RTM.
(recombinant BDD FVIII), which comprises a first polypeptide
corresponding to amino acids 1 to 743 of FIG. 30, fused to a second
polypeptide corresponding to amino acids 1638 to 2332 of FIG. 30.
Exemplary BDD FVIII constructs which can be used to produce
recombinant proteins of the invention include, but are not limited
to FVIII with a deletion of amino acids corresponding to amino
acids 747-1638 of mature human FVIII (FIG. 30) (Hoeben R. C., et
al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein
by reference in its entirety), and FVIII with a deletion of amino
acids corresponding to amino acids 771-1666 or amino acids 868-1562
of mature human FVIII (FIG. 30) (Meulien P., et al. Protein Eng.
2(4): 301-6 (1988), incorporated herein by reference in its
entirety).
[0155] In addition, sequences that include heterologous amino acid
insertions or substitutions (such as aspartic acid substituted for
valine at position 75), or single chain FVIII (scFVIII) in which
the heavy and light chains are covalently connected by a linker. As
used herein, "FVIII" shall be any functional form of factor VIII
molecule with the typical characteristics of blood coagulation
factor VIII capable of correcting human factor VIII deficiencies
when administered to such a subject, e.g., a subject with
hemophilia A. FVIII or sequence variants have been isolated,
characterized, and cloned, as described in U.S. Pat. Nos.
4,757,006; 4,965,199; 5,004,804; 5,198,349, 5,250,421; 5,919,766;
6,228,620; 6,818,439; 7,138,505; 7,632,921; and 20100081615.
[0156] Human factor VIII is encoded by a single-copy gene residing
at the tip of the long arm of the X chromosome (q28). It comprises
nearly 186,000 base pairs (bp) and constitutes approximately 0.1%
of the X-chromosome (White, G. C. and Shoemaker, C. B., Blood
(1989) 73:1-12). The human FVIII amino acid sequence was deduced
from cDNA as shown in U.S. Pat. No. 4,965,199, which is
incorporated herein by reference in its entirety. Native mature
human FVIII derived from the cDNA sequence (i.e., without the
secretory signal peptide but prior to other post-translational
processing) is presented as FIG. 3.
[0157] The DNA encoding the mature factor VIII mRNA is found in 26
separate exons ranging in size from 69 to 3,106 bp. The 25
intervening intron regions that separate the exons range in size
from 207 to 32,400 bp. The complete gene consists of approximately
9 kb of exon and 177 kb of intron. The three repeat A domains have
approximately 30% sequence homology. The B domain contains 19 of
the approximately 25 predicted glycosylation sites, and the A3
domain is believed to contain a binding site for the von Willebrand
factor. The tandem C domains follow the A3 domain and have
approximately 37% homology to each other (White, G. C. and
Shoemaker, C. B., Blood (1989) 73:1-12).
[0158] The B domain separates the A2 and A3 domains of native
factor FVIII in the newly synthesized precursor single-chain
molecule. The precise boundaries of the B domain have been
variously reported as extending from amino acids 712 to 1648 of the
precursor sequence (Wood et al., Nature (1984) 312:330-337) or
amino acids 741-1648 (Pipe, S W, Haemophilia (2009) 15:1187-1196
and U.S. Pat. No. 7,560,107) or amino acids 740-1689 (Toole, J J.
Proc. Natl. Acad. Sci. USA (1986) 83:5939-5942). As used herein, "B
domain" means amino acids 741-1648 of mature factor VIII. As used
herein, "FVIII B domain deletion" or "FVIII BDD" means a FVIII
sequence with any, a fragment of, or all of amino acids 741 to 1648
deleted. In one embodiment, FVIII BDD variants retain remnant amino
acids of the B domain from the N-terminal end ("B1" as used herein)
and C-terminal end ("B2" as used herein). In one FVIII BDD variant,
the B domain remnant amino acids are SFSQNPPVLKRHQR (SEQ ID NO:
1614). In one FVIII BDD variant, the B1 remnant is SFS and the B2
remnant is QNPPVLKRHQR (SEQ ID NO: 1615). In another FVIII BDD
variant, the B1 remnant is SFSQN (SEQ ID NO: 1616) and the B2
remnant is PPVLKRHQR (SEQ ID NO: 1617). A "B-domain-deleted factor
VIII," "FVIII BDD," or "BDD FVIII" may have the full or partial
deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513,
7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885,
6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112,
and 6,458,563, each of which is incorporated herein by reference in
its entirety. In some embodiments, a B-domain-deleted factor VIII
sequence of the present invention comprises any one of the
deletions disclosed at col. 4, line 4 to col. 5, line 28 and
examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No.
6,346,513). In another embodiment, a B-domain deleted factor VIII
is the 5743/Q1638 B-domain deleted factor VIII (SQ version factor
VIII) (e.g., factor VIII having a deletion from amino acid 744 to
amino acid 1637, e.g., factor VIII having amino acids 1-743 and
amino acids 1638-2332 of full-length factor VIII). In some
embodiments, a B-domain-deleted factor VIII of the present
invention has a deletion disclosed at col. 2, lines 26-51 and
examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. Nos.
6,060,447, 5,595,886, and 6,228,620). In some embodiments, a
B-domain-deleted factor VIII has a deletion described in col. 1,
lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6,
lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines
17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line
36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and
example 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19,
line 21 and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to
col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line
43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of
U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No.
6,458,563. In some embodiments, a B-domain-deleted factor VIII has
a deletion of most of the B domain, but still contains
amino-terminal sequences of the B domain that are essential for in
vivo proteolytic processing of the primary translation product into
two polypeptide chain, as disclosed in WO 91/09122, which is
incorporated herein by reference in its entirety. In some
embodiments, a B-domain-deleted factor VIII is constructed with a
deletion of amino acids 747-1638, i.e., virtually a complete
deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265
(13): 7318-7323 (1990), incorporated herein by reference in its
entirety. A B-domain-deleted factor VIII may also contain a
deletion of amino acids 771-1666 or amino acids 868-1562 of factor
VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988),
incorporated herein by reference in its entirety. Additional B
domain deletions that are part of the invention include: deletion
of amino acids 982 through 1562 or 760 through 1639 (Toole et al.,
Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through
1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through
1646 (Kaufman (PCT published application No. WO 87/04187)),
747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648
(Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741
through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP
295597)), each of which is incorporated herein by reference in its
entirety. Each of the foregoing deletions may be made in any factor
VIII sequence utilized in the embodiments of the present
invention.
[0159] Proteins involved in clotting include factor I, factor II,
factor III, factor IV, factor V, factor VI, factor VII, factor
VIII, factor IX, factor X, factor XI, factor XII, factor XIII,
Protein C, and tissue factor (collectively or individually
"clotting protein(s)"). The interaction of the major clotting
proteins in the intrinsic and extrinsic clotting pathways is showed
in FIG. 2. The majority of the clotting proteins are present in
zymogen form, but when activated, exhibit a procoagulant protease
activity in which they activate another of the clotting proteins,
contributing to the intrinsic or extrinsic coagulation pathway and
clot formation. In the intrinsic pathway of the coagulation
cascade, FVIII associates with a complex of activated factor IX,
factor X, calcium, and phospholipid. The factor VIII heterodimer
has no enzymatic activity, but the heterodimer becomes active as a
cofactor of the enzyme factor IXa after proteolytic activation by
thrombin or factor Xa, with the activity of factor VIIIa
characterized by its ability to form a membrane binding site for
factors IXa and X in a conformation suitable for activation of the
factor X by factor IXa. Upon cleavage by thrombin, activated FVIII
(FVIIIa) dissociates from von Willebrand factor and binds to
negatively charged phospholipid PL, and the resulting complex
participates as a cofactor to factor IXa in the factor X activating
(tenase) complex. Within the C2 domain and amino acid residues 1649
through 1689 in the A3 domain are von Willebrand factor (vWF)
binding sites that act to complex with von Willebrand factor, the
resulting circulating complex protects FVIII from rapid degradation
in the blood (Weiss H J, et al. Stabilization of factor VIII in
plasma by the von Willebrand factor. Studies on posttransfusion and
dissociated factor VIII and in patients with von Willebrand's
disease. J Clin Invest (1977) 60:390).
[0160] Activated factor VIII is a heterotrimer comprised of the A1
domain and the A2 domain and the light chain including domains
A3-C1-C2. The activation of factor IX is achieved by a two-step
removal of the activation peptide (Ala 146-Arg 180) from the
molecule (Bajaj et al., Human factor IX and factor IXa, in METHODS
IN ENZYMOLOGY. 1993). The first cleavage is made at the Arg 145-Ala
146 site by either factor XIa or factor VIIa/tissue factor. The
second, and rate limiting cleavage is made at Arg 180-Val 181. The
activation removes 35 residues. Activated human factor IX exists as
a heterodimer of the C-terminal heavy chain (28 kDa) and an
N-terminal light chain (18 kDa), which are held together by one
disulfide bridge attaching the enzyme to the Gla domain. Factor IXa
in turn activates factor X in concert with activated factor VIII.
Alternatively, factors IX and X can both be activated by factor
VIIa complexed with lipidated tissue factor, generated via the
extrinsic pathway. Factor Xa then participates in the final common
pathway whereby prothrombin is converted to thrombin, and thrombin,
in turn converts fibrinogen to fibrin to form the clot.
[0161] Defects in the coagulation process can lead to bleeding
disorders (coagulopathies) in which the time taken for clot
formation is prolonged. Such defects can be congenital or acquired.
For example, hemophilia A and B are inherited diseases
characterized by deficiencies in FVIII and FIX, respectively.
Stated differently, biologically active factor VIII corrects the
coagulation defect in plasma derived from individuals afflicted
with hemophilia A. Recombinant FVIII has been shown to be effective
and has been approved for the treatment of hemophilia A in adult
and pediatric patients, and also is used to stop bleeding episodes
or prevent bleeding associated with trauma and/or surgery. Current
therapeutic uses of factor VIII can be problematic in the treatment
of individuals exhibiting a deficiency in factor VIII, as well as
those individuals with Von Willebrand's disease. In addition,
individuals receiving factor VIII in replacement therapy frequently
develop antibodies to these proteins that often reduce or eliminate
the procoagulant activity of the bound FVIII. Continuing treatment
is exceedingly difficult because of the presence of these
antibodies that reduce or negate the efficacy of the treatment.
[0162] In one aspect, the invention contemplates inclusion of FVIII
sequences in the CFXTEN fusion protein compositions that are
identical to human FVIII, sequences that have homology to FVIII
sequences, sequences that are natural, such as from humans,
non-human primates, mammals (including domestic animals), or
truncated version of FVIII; all of which retain at least a portion
of the procoagulant activity of native FVIII and that are useful
for preventing, treating, mediating, or ameliorating hemophilia A
or bleeding episodes related to trauma, surgery, or deficiency of
coagulation factor VIII. Sequences with homology to FVIII may be
found by standard homology searching techniques, such as NCBI
BLAST, or in public databases such as Chemical Abstracts Services
Databases (e.g., the CAS Registry), GenBank, The Universal Protein
Resource (UniProt) and subscription provided databases such as
GenSeq (e.g., Derwent).
[0163] In one embodiment, the FVIII incorporated into the subject
CFXTEN compositions is a recombinant polypeptide with a sequence
corresponding to a FVIII protein found in nature. In another
embodiment, the FVIII is a non-natural FVIII sequence variant,
fragment, homolog, or a mimetic of a natural sequence that retains
at least a portion of the procoagulant activity of the
corresponding native FVIII. In another embodiment, the FVIII is a
truncated variant with all or a portion of the B domain deleted
("FVIII BDD"), which can be in either heterodimeric form or can
remain as a single chain ("scFVIII"), the latter described in
Meulien et al., Protein Eng. (1988) 2(4):301-306. Non-limiting
examples of FVIII BDD are factor VIII sequences in which the amino
acids are deleted between residue number 741 and residue number
1640 (numbered relative to native, mature FVIII), or between
residue number 745 and residue number 1640, or between residue
number 745 and residue number 1640, or between residue number 741
and residue number 1690, or between residue number 745 and residue
number 1667, or between residue number 745 and residue number 1657,
or between residue number 747 and residue number 1642, or between
residue number 751 and residue number 1667.
[0164] In another embodiment, heterologous sequences are
incorporated into the FVIII, which may include XTEN, as described
more fully below. Table 1 provides a non-limiting list of amino
acid sequences of FVIII that are encompassed by the CFXTEN fusion
proteins of the invention. In some embodiments, FVIII incorporated
into CFXTEN fusion proteins include proteins that have at least
about 70% sequence identity, or alternatively 80%, 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 amino acid
sequence of comparable length selected from Table 1.
TABLE-US-00002 TABLE 1 FVIII amino acid sequences SEQ Name ID
(source) Amino Acid Sequence NO: FVIII
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 1 precursor
SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM polypeptide
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN (human)
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF
AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS
VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD
DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG
PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR
PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR
CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR
SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY
ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN
SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS
TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLS
DLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNE
KLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQL
DTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLF
KGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVW
QNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE
GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSV
EGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEK
KIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVL
QDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQ
QNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNE
KEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPA
ASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTY
KKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQ
GTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWK
SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCS
QNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQK
KTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPL
YRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR
KNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLL
VCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPT
FKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR
KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNK
CQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL
LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD
SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA
QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVT
GVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN
SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 2 mature
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS (human)
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD
PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID
SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST
SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL
SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK
VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR
MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL
PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF
TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP
QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRIKKHTA
HFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL
EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS
IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG
AKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV
ELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFL
RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN
ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD
QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS
SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH
MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF
ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL
VMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV
EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG
QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL
YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP
THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA
RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF
LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH
QIALRMEVLGCEAQDLY FVIII
MQVELYTCCFLCLLPFSLSATRKYYLGAVELSWDYMQSDLLSALHADTSFSSRVP 3 (Canine)
GSLPLTTSVTYRKTVFVEFTDDLFNIAKPRPPWMGLLGPTIQAEVYDTVVIVLKN
MASHPVSLHAVGVSYWKASEGAEYEDQTSQKEKEDDNVIPGESHTYVWQVLKE
NGPMASDPPCLTYSYFSHVDLVKDLNSGLIGALLVCKEGSLAKERTQTLQEFVLL
FAVFDEGKSWHSETNASLTQAEAQHELHTINGYVNRSLPGLTVCHKRSVYWHVI
GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTFLMDLGQFLLFCHIPSH
QHDGMEAYVKVDSCPEEPQLRMKNNEDKDYDDGLYDSDMDVVSFDDDSSSPFI
QIRSVAKKHPKTWVHYIAAEEEDWDYAPSGPTPNDRSHKNLYLNNGPQRIGKKY
KKVRFVAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI
NYVTPLHTGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS
FINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSWYLTEN
MQRFLPNADVVQPHDPEFQLSNIMHSINGYVFDNLQLSVCLHEVAYWYILSVGA
QTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFR
NRGMTALLKVSSCNRNIDDYYEDTYEDIPTPLLNENNVIKPRSFSQNSRHPSTKEK
QLKATTTPENDIEKIDLQSGERTQLIKAQSVSSSDLLMLLGQNPTPRGLFLSDLREA
TDRADDHSRGAIERNKGPPEVASLRPELRHSEDREFTPEPELQLRLNENLGTNTTV
ELKKLDLKISSSSDSLMTSPTIPSDKLAAATEKTGSLGPPNMSVHFNSHLGTIVFGN
NSSHLIQSGVPLELSEEDNDSKLLEAPLMNIQESSLRENVLSMESNRLFKEERIRGP
ASLIKDNALFKVNISSVKTNRAPVNLTTNRKTRVAIPTLLIENSTSVWQDIMLERN
TEFKEVTSLIHNETFMDRNTTALGLNHVSNKTTLSKNVEMAHQKKEDPVPLRAE
NPDLSSSKIPFLPDWIKTHGKNSLSSEQRPSPKQLTSLGSEKSVKDQNFLSEEKVVV
GEDEFTKDTELQEIFPNNKSIFFANLANVQENDTYNQEKKSPEEIERKEKLTQENV
ALPQAHTMIGTKNFLKNLFLLSTKQNVAGLEEQPYTPILQDTRSLNDSPHSEGIHM
ANFSKIREEANLEGLGNQTNQMVERFPSTTRMSSNASQHVITQRGKRSLKQPRLS
QGEIKFERKVIANDTSTQWSKNMNYLAQGTLTQIEYNEKEKRAITQSPLSDCSMR
NHVTIQMNDSALPVAKESASPSVRHTDLTKIPSQHNSSHLPASACNYTFRERTSGV
QEGSHFLQEAKRNNLSLAFVTLGITEGQGKFSSLGKSATNQPMYKKLENTVLLQP
GLSETSDKVELLSQVHVDQEDSFPTKTSNDSPGHLDLMGKIFLQKTQGPVKMNK
TNSPGKVPFLKWATESSEKIPSKLLGVLAWDNHYDTQIPSEEWKSQKKSQTNTAF
KRKDTILPLGPCENNDSTAAINEGQDKPQREAMWAKQGEPGRLCSQNPPVSKHH
QREITVTTLQPEEDKFEYDDTFSIEMKREDFDIYGDYENQGLRSFQKKTRHYFIAA
VERLWDYGMSRSPHILRNRAQSGDVQQFKKVVFQEFTDGSFTQPLYRGELNEHL
GLLGPYIRAEVEDNIVVTFKNQASRPYSFYSSLISYDEDEGQGAEPRRKFVNPNET
KIYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPA
HGRQVTVQEFALVFTIFDETKSWYFTENLERNCRAPCNVQKEDPTLKENFRFHAI
NGYVKDTLPGLVMAQDQKVRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA
VYNLYPGVFETVEMLPSQVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPLGMAS
GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDLLAPMIIHGI
MTQGARQKFSSLYVSQFIIMYSLDGNKWHSYRGNSTGTLMVFFGNVDSSGIKHNI
FNPPIIAQYIRLHPTHYSIRSTLRMELLGCDFNSCSMPLGMESKAISDAQITASSYLS
SMLATWSPSQARLHLQGRTNAWRPQANNPKEWLQVDFRKTMKVTGITTQGVKS
LLISMYVKEFLISSSQDGHNWTLFLQNGKVKVFQGNRDSSTPVRNRLEPPLVARY
VRLHPQSWAHHIALRLEVLGCDTQQPA FVIII (Pig)
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 4
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD
PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID
SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST
SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL
SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK
VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR
MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL
PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF
TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP
QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTA
HFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL
EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS
IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG
AKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV
ELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFL
RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN
ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD
QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS
SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH
MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF
ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL
VMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV
EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG
QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL
YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP
THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA
RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF
LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH
QIALRMEVLGCEAQDLY FVIII
AIRRYYLGAVELSWNYIQSDLLSVLHTDSRFLPRMSTSFPFNTSIMYKKTVFVEYK 5 (Mouse)
DQLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNMASHPVSLHAVGVSYWKAS
EGDEYEDQTSQMEKEDDKVFPGESHTYVWQVLKENGPMASDPPCLTYSYMSHV
DLVKDLNSGLIGALLVCKEGSLSKERTQMLYQFVLLFAVFDEGKSWHSETNDSY
TQSMDSASARDWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEIHSIF
LEGHTFFVRNHRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKV
DSCPEESQWQKKNNNEEMEDYDDDLYSEMDMFTLDYDSSPFIQIRSVAKKYPKT
WIHYISAEEEDWDYAPSVPTSDNGSYKSQYLSNGPHRIGRKYKKVRFIAYTDETF
KTRETIQHESGLLGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSPLHARRLPR
GIKHVKDLPIHPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDLASGLIGP
LLICYKESVDQRGNQMMSDKRNVILFSIFDENQSWYITENMQRFLPNAAKTQPQD
PGFQASNIMHSINGYVFDSLELTVCLHEVAYWHILSVGAQTDFLSIFFSGYTFKHK
MVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFRKRGMTALLKVSSCDKST
SDYYEEIYEDIPTQLVNENNVIDPRSFFQNTNHPNTRKKKFKDSTIPKNDMEKIEPQ
FEEIAEMLKVQSVSVSDMLMLLGQSHPTPHGLFLSDGQEAIYEAIHDDHSPNAIDS
NEGPSKVTQLRPESHHSEKIVFTPQPGLQLRSNKSLETTIEVKWKKLGLQVSSLPS
NLMTTTILSDNLKATFEKTDSSGFPDMPVHSSSKLSTTAFGKKAYSLVGSHVPLN
ASEENSDSNILDSTLMYSQESLPRDNILSIENDRLLREKRFHGIALLTKDNTLFKDN
VSLMKTNKTYNHSTTNEKLHTESPTSIENSTTDLQDAILKVNSEIQEVTALIHDGT
LLGKNSTYLRLNHMLNRTTSTKNKDIFHRKDEDPIPQDEENTIMPFSKMLFLSESS
NWFKKTNGNNSLNSEQEHSPKQLVYLMFKKYVKNQSFLSEKNKVTVEQDGFTK
NIGLKDMAFPHNMSIFLTTLSNVHENGRHNQEKNIQEEIEKEALIEEKVVLPQVHE
ATGSKNFLKDILILGTRQNISLYEVHVPVLQNITSINNSTNTVQIHMEHFFKRRKDK
ETNSEGLVNKTREMVKNYPSQKNITTQRSKRALGQFRLSTQWLKTINCSTQCIIKQ
IDHSKEMKKFITKSSLSDSSVIKSTTQTNSSDSHIVKTSAFPPIDLKRSPFQNKFSHV
QASSYIYDFKTKSSRIQESNNFLKETKINNPSLAILPWNMFIDQGKFTSPGKSNTNS
VTYKKRENIIFLKPTLPEESGKIELLPQVSIQEEEILPTETSHGSPGHLNLMKEVFLQ
KIQGPTKWNKAKRHGESIKGKTESSKNTRSKLLNHHAWDYHYAAQIPKDMWKS
KEKSPEIISIKQEDTILSLRPHGNSHSIGANEKQNWPQRETTWVKQGQTQRTCSQIP
PVLKRHQRELSAFQSEQEATDYDDAITIETIEDFDIYSEDIKQGPRSFQQKTRHYFI
AAVERLWDYGMSTSHVLRNRYQSDNVPQFKKVVFQEFTDGSFSQPLYRGELNEH
LGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKEDQRGEEPRRNFVKPNET
KIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLERDMHSGLIGPLLICHANTLNPA
HGRQVSVQEFALLFTIFDETKSWYFTENVKRNCKTPCNFQMEDPTLKENYRFHAI
NGYVMDTLPGLVMAQDQRIRWYLLSMGNNENIQSIHFSGHVFTVRKKEEYKMA
VYNLYPGVFETLEMIPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMAS
GSIRDFQITASGHYGQWAPNLARLHYSGSINAWSTKEPFSWIKVDLLAPMIVHGIK
TQGARQKFSSLYISQFIIMYSLDGKKWLSYQGNSTGTLMVFFGNVDSSGIKHNSF
NPPIIARYIRLHPTHSSIRSTLRMELMGCDLNSCSIPLGMESKVISDTQITASSYFTN
MFATWSPSQARLHLQGRTNAWRPQVNDPKQWLQVDLQKTMKVTGIITQGVKSL
FTSMFVKEFLISSSQDGHHWTQILYNGKVKVFQGNQDSSTPMMNSLDPPLLTRYL
RIHPQIWEHQIALRLEILGCEAQQQY FVIII BDD
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 6 variant
SFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM (U.S. Pat.
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN No.
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF 7,632,921,
AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS SEQ ID
VYWHVIGMGTTPEVHSIFLEGHIFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL NO: 3)
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD
DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG
PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR
PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR
CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR
SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY
ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN
SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF
IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE
HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN
ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL
NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF
HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK
MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG
MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII
HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK
HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS
YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ
GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL
LTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 7 BDD-2
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY
DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 8
BDD-3 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
(G1648) EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDY
DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 9
BDD-4 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQQSPRSFQKKTRHYFIAAVERLWDY
GMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR
AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV
QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVT
VQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD
TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG
VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ
ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ
KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI
RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWS
PSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMY
VKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS
WVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 10 BDD-5
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT
DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEED
QRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV
HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAP
CNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIH
FSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS
TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKE
PFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT
LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG
MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD
FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN
QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 11 BDD-6
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD
TISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR
AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFR
NQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE
TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR
IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAG
IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP
KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY
SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL
RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS
NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEV LGCEAQDLY
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 12
BDD-7 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQSPRSFQKKTRHYFIAAVERLWDYG
MSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA
EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ
HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTV
QEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL
PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVF
ETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQIT
ASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF
SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL
HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVK
EFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSW
VHQIALRMEVLGCEAQDLY FVIII
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 13 BDD-8
SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM precursor
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN (U.S. Pat.
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF No.
AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS 6,818,439
VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL SEQ ID
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD NO: 47)
DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG
PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR
PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR
CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR
SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY
ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN
SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF
IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE
HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN
ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL
NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF
HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK
MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG
MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII
HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK
HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS
YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ
GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL
LTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 14 BDD-9
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS mature
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV (U.S. Pat.
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM No.
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL 6,818,439)
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY
DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 15
BDD-10 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY
DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR
MEVLGCEAQDLY
FVIII ATRATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF 16
BDD-11 VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY
WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY
LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETK
NSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEV
HSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAY
VKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK
HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMA
YTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYS
RRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDL
ASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA
GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSG
YTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS
SCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQ
EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS
PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVED
NIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM
APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFA
LFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV
MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVE
MLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ
YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI
SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH
YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS
SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA
LRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 17 BDD-12
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY
DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 18
BDD-13 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV
DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE
TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP
KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK
HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY
DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK
AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW
APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR
STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ
GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR
MEVLGCEAQDLY
[0165] The present invention also contemplates CFXTEN comprising
FVIII with various amino acid deletions, insertions and
substitutions made in the FVIII sequences of Table 1 that retain
procoagulant activity. Examples of conservative substitutions for
amino acids in polypeptide sequences are shown in Table 2. In
embodiments of the CFXTEN in which the sequence identity of the
FVIII is less than 100% compared to a specific sequence disclosed
herein, the invention contemplates substitution of any of the other
19 natural L-amino acids for a given amino acid residue of the
given FVIII, which may be at any position within the sequence of
the FVIII, including adjacent amino acid residues. If any one
substitution results in an undesirable change in procoagulant
activity, then one of the alternative amino acids can be employed
and the construct protein evaluated by the methods described herein
(e.g., the assays of Table 49), or using any of the techniques and
guidelines for conservative and non-conservative mutations set
forth, for instance, in U.S. Pat. No. 5,364,934, the content of
which is incorporated by reference in its entirety, or using
methods generally known in the art. In a preferred substitution,
the FVIII component of the CFXTEN embodiments is modified by
replacing the R1648 residue (numbered relative to the native mature
form of FVIII) with glycine or alanine to prevent proteolytic
processing to the heterodimer form. In another substitution, the
FVIII component of the CFXTEN embodiments is modified by replacing
the Y1680 residue (numbered relative to the native mature form of
FVIII) with phenylalanine. In another embodiment, the FVIII
component of the CFXTEN embodiments is modified by replacing the
Y1680 residue (numbered relative to the native mature form of
FVIII) with phenylalanine and the R1648 residue (numbered relative
to the native mature form of FVIII) with glycine or alanine.
[0166] In one embodiment, the FVIII of the fusion protein
composition has one or more amino acid substitutions designed to
reduce the binding of FVIII inhibitors at epitopes recognized by
the antibodies of Table 9, including but not limited to
substitutions at Lys(377), Lys(466), Lys(380), Ser(488), Arg(489),
Arg(490), Leu(491), Lys(493), Lys(496), His(497), Lys(499),
Lys(512), Lys(523), Lys(556), Met (2199), Phe(2200), Leu(2252),
Val(2223), and Lys(2227). In addition, variants can include, for
instance, polypeptides wherein one or more amino acid residues are
added or deleted at or near the N- or C-terminus of the full-length
native amino acid sequence or of a domain of a FVIII so long as the
variant retains some if not all of the procoagulant activity of the
native peptide. The resulting FVIII sequences that retain at least
a portion (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or at least 95% or more) of the procoagulant activity in
comparison to native circulating FVIII are considered useful for
the fusion protein compositions of this invention. Examples of FVII
variants are known in the art, including those described in U.S.
Pat. Nos. 6,316,226; 6,818,439; 7,632,921; 20080227691, which are
incorporated herein by reference. In one embodiment, a FVIII
sequence variant has an aspartic acid substituted for valine at
amino acid position 75 (numbered relative to the native mature form
of FVIII).
TABLE-US-00003 TABLE 2 Exemplary conservative amino acid
substitutions Original Residue Exemplary Substitutions Ala (A) val;
leu; ile Arg (R) lys; gln; asn Asn (N) gin; his; lys; arg Asp (D)
Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Pro His (H) asn:
gin: lys: arg Ile (I) leu; val; met; ala; phe: norleucine Leu (L)
norleucine: ile: val; met; ala: phe Lys (K) arg: gin: asn Met (M)
leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) Gly Ser (S) Thr
Thr (T) Ser Trp (W) Tyr Tyr(Y) Trp: phe: thr: ser Val (V) Ile; leu;
met; phe; ala; norleucine
III). Extended Recombinant Polypeptides
[0167] In one aspect, the invention provides XTEN polypeptide
compositions that are useful as fusion protein partner(s) to link
to and/or incorporate within a FVIII polypeptide, resulting in a
CFXTEN fusion protein. XTEN are generally polypeptides with
non-naturally occurring, substantially non-repetitive sequences
having a low degree of 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
fusion protein partners in that they serve various roles,
conferring certain desirable pharmacokinetic, physicochemical,
pharmacologic, and pharmaceutical properties when linked to a FVIII
protein to a create a CFXTEN fusion protein. Such CFXTEN fusion
protein compositions have enhanced properties compared to the
corresponding FVIII not linked to XTEN, making them useful in the
treatment of certain conditions related to FVIII deficiencies or
bleeding disorders, as more fully described below.
[0168] The selection criteria for the XTEN to be fused to the FVIII
proteins used to create the inventive fusion proteins compositions
generally relate to attributes of physical/chemical properties and
conformational structure of the XTEN that is, in turn, used to
confer enhanced pharmaceutical, pharmacologic, and pharmacokinetic
properties to the FVIII fusion proteins compositions. The
unstructured characteristic and physical/chemical properties of the
XTEN result, in part, from the overall amino acid composition
disproportionately limited to 4-6 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 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 4 that, within varying ranges of length, possess similar
properties, many of which are documented in the Examples. The XTEN
of the present invention may exhibit one or more, or all of the
following advantageous properties: unstructured conformation,
conformational flexibility, enhanced 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 can make them
particularly useful as fusion protein partners. Non-limiting
examples of the enhanced properties that XTEN confer on the fusion
proteins comprising FVIII fused to XTEN, compared to FVIII not
linked to XTEN, include increases in the overall solubility and/or
metabolic stability, reduced susceptibility to proteolysis, reduced
immunogenicity, reduced rate of absorption when administered
subcutaneously or intramuscularly, reduced binding to FVIII
clearance receptors, reduced reactivity to anti-payload antibodies,
enhanced interactions with substrate, and/or enhanced
pharmacokinetic properties when administered to a subject. The
enhanced pharmacokinetic properties of the CFXTEN compositions
compared to FVIII 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, and enhanced absorption after
subcutaneous or intramuscular injection (an advantage compared to
commercially-available forms of FVIII that must be administered
intravenously). In addition, it is believed that the CFXTEN
compositions comprising cleavage sequences (described more fully,
below) permit sustained release of biologically active FVIII, such
that the administered CFXTEN acts as a depot. It is specifically
contemplated that the inventive CFXTEN fusion proteins can exhibit
one or more or any combination of the improved properties disclosed
herein. As a result of these enhanced properties, it is believed
that CFXTEN compositions permit less frequent dosing compared to
FVIII not linked to XTEN when administered at comparable dosages.
Such CFXTEN fusion protein compositions have utility to treat
certain factor VIII-related conditions, as described herein.
[0169] A variety of methods and assays are known in the art for
determining the physical/chemical properties of proteins such as
the CFXTEN compositions comprising 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. Methods to assay these properties include
analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size
exclusion, 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. Additional methods are disclosed in Arnau, et al.,
Prot Expr and Purif (2006) 48, 1-13.
[0170] The XTEN component(s) of the CFXTEN 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, are largely devoid of secondary
structure. In other cases, the XTEN sequences are substantially
devoid of secondary structure under physiologic conditions such
that the XTEN can adopt random coil conformation. "Largely devoid,"
as used in this context, means that at least 50% of the XTEN amino
acid residues of the XTEN sequence do not 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 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.
[0171] A variety of methods have been established in the art to
discern the presence or absence of secondary and tertiary
structures in a given polypeptide. 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 ("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).
[0172] In one embodiment, the XTEN sequences used in the subject
fusion protein compositions 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 of the fusion
protein compositions have a beta-sheet percentage ranging from 0%
to less than about 5% as determined by the Chou-Fasman algorithm.
In some embodiments, the XTEN sequences of the fusion protein
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. In some
embodiments, the XTEN sequences of the fusion protein compositions
have an alpha-helix percentage less than about 2% and a beta-sheet
percentage less than about 2%. The XTEN sequences of the fusion
protein compositions have a high degree of random coil percentage,
as determined by the GOR algorithm. In some embodiments, an XTEN
sequence have at least about 80%, at least about 90%, at least
about 91%, at least about 92%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, and most preferably at least about 99% random
coil, as determined by the GOR algorithm. In some embodiments, the
XTEN sequences of the fusion protein 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 other
embodiments, the XTEN sequences of the fusion protein 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.
1. Non-Repetitive Sequences
[0173] It is contemplated that the XTEN sequences of the CFXTEN
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 about 36, or at least 72, or at least 96, or at least 144, or
at least 288, or at least 400, or at least 500, or at least 600, or
at least 700, or at least 800, or at least 864, or at least 900, or
at least 1000, or at least 2000, to about 3000 or more amino acid
residues, or has a length ranging from about 36 to about 3000,
about 100 to about 500, about 500 to about 1000, about 1000 to
about 3000 amino acids and residues, in which no three contiguous
amino acids in the sequence 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 comprises motifs of 9 to 14 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 sequence of any two contiguous
amino acid residues in any one motif is not repeated more than
twice in the sequence motif.
[0174] 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 aspect, the repetitiveness of a polypeptide of a
predetermined length can be calculated (hereinafter "subsequence
score") according to the formula given by Equation 1:
Subsequence score i = 1 m Count i m I ##EQU00001## [0175] wherein:
m=(amino acid length of polypeptide)-(amino acid length of
subsequence)+1; and Count.sub.i=cumulative number of occurrences of
each unique subsequence within sequence,
[0176] 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. 27 depicts a logic flowchart of the
SegScore algorithm, while FIG. 28 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 198
3-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.
[0177] 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 CFXTEN 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 CFXTEN comprising at least two to about six XTEN in which
200 amino acids of 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 the embodiments of the CFXTEN fusion
protein compositions described herein, an XTEN component of a
fusion protein with a subsequence score of 10 or less (i.e., 9, 8,
7, etc.) is also substantially non-repetitive.
[0178] It is believed that 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 many of the enhanced physicochemical and
biological properties of the CFXTEN fusion proteins. These enhanced
properties include a higher degree of expression of the fusion
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 CFXTEN
compared to fusion proteins comprising polypeptides having
repetitive sequences. These enhanced properties permit more
efficient manufacturing, lower cost of goods, and facilitate the
formulation of XTEN-comprising pharmaceutical preparations
containing extremely high protein concentrations, in some cases
exceeding 100 mg/ml. Furthermore, the XTEN polypeptide sequences of
the embodiments 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
[0179] The present invention encompasses XTEN used as fusion
partners that comprise multiple units of shorter sequences, or
motifs, in which the amino acid sequences of the motifs are
non-repetitive. The non-repetitive property is met despite the use
of a "building block" approach using a library of sequence motifs
that are multimerized to create the XTEN sequences. Thus, 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.
[0180] 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, or even longer 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 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. 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 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
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. As a result of testing variables such as codon
optimization, assembly polynucleotides encoding sequence motifs,
expression of protein, charge distribution and solubility of
expressed protein, and secondary and tertiary structure, it was
discovered that XTEN compositions with the enhanced characteristics
disclosed herein mainly or exclusively include glycine (G), alanine
(A), serine (S), threonine (T), glutamate (E) and proline (P)
residues wherein the sequences are designed to be substantially
non-repetitive. 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 greater than 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 greater
than 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%. 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%. 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 96%, 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).
[0181] In still other embodiments, XTENs comprise substantially
non-repetitive sequences of greater than about 36 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 96%, or about 97%, or about 98%, or about 99%
of the sequence consists of non-overlapping sequence motifs of 9 to
14 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
sequence of any two contiguous amino acid residues in any one motif
is not repeated more than twice in the sequence motif. In other
embodiments, 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% of an XTEN sequence consists of
non-overlapping sequence motifs of 12 amino acid residues wherein
the motifs consist of four to six 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 sequence motif is not repeated more
than twice in the sequence motif. In other embodiments, 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% of an XTEN sequence consists of non-overlapping sequence motifs
of 12 amino acid residues wherein the motifs consist of 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 sequence motif is not repeated more than
twice in the sequence motif. In yet other embodiments, XTENs
consist of 12 amino acid sequence motifs wherein the amino acids
are 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 sequence motif is not
repeated more than twice in the sequence motif, and wherein the
content of any one amino acid type in the full-length XTEN does not
exceed 30%. The foregoing embodiments are examples of substantially
non-repetitive XTEN sequences. Additional examples are detailed
below.
[0182] In some embodiments, the invention provides CFXTEN
compositions comprising one, or two, or three, or four, five, six
or more 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 96%, 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 3, 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 3, resulting in a
family sequence. As used herein, "family" means that the XTEN has
motifs selected only from a single motif category from Table 3;
i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other
amino acids in the XTEN not from a family motif are selected to
achieve a needed property, such as to permit incorporation of a
restriction site by the encoding nucleotides, incorporation of a
cleavage sequence, or to achieve a better linkage to a FVIII
coagulation factor component of the CFXTEN. In some embodiments of
XTEN families, an XTEN sequence comprises multiple units of
non-overlapping sequence motifs of the AD motif family, or of the
AE motif family, or of the AF motif family, or of the AG motif
family, or of the AM motif family, or of the AQ motif family, or of
the BC family, or of the BD family, with the resulting XTEN
exhibiting the range of homology described above. In other
embodiments, the XTEN comprises multiple units of motif sequences
from two or more of the motif families of Table 3. These sequences
can be selected to achieve desired physical/chemical
characteristics, including such properties as net charge,
hydrophilicity, lack of secondary structure, or lack of
repetitiveness that are conferred by the amino acid composition of
the motifs, described more fully below. In the embodiments
hereinabove described in this paragraph, the motifs incorporated
into the XTEN can be selected and assembled using the methods
described herein to achieve an XTEN of about 36 to about 3000 amino
acid residues.
TABLE-US-00004 TABLE 3 XTEN Sequence Motifs of 12 Amino Acids and
Motif Families Motif Family MOTIF SEQUENCE SEQ ID NO: AD
GESPGGSSGSES 19 AD GSEGSSGPGESS 20 AD GSSESGSSEGGP 21 AD
GSGGEPSESGSS 22 AE, AM GSPAGSPTSTEE 23 AE, AM, AQ GSEPATSGSETP 24
AE, AM, AQ GTSESATPESGP 25 AE, AM, AQ GTSTEPSEGSAP 26 AF, AM
GSTSESPSGTAP 27 AF, AM GTSTPESGSASP 28 AF, AM GTSPSGESSTAP 29 AF,
AM GSTSSTAESPGP 30 AG, AM GTPGSGTASSSP 31 AG, AM GSSTPSGATGSP 32
AG, AM GSSPSASTGTGP 33 AG, AM GASPGTSSTGSP 34 AQ GEPAGSPTSTSE 35 AQ
GTGEPSSTPASE 36 AQ GSGPSTESAPTE 37 AQ GSETPSGPSETA 38 AQ
GPSETSTSEPGA 39 AQ GSPSEPTEGTSA 40 BC GSGASEPTSTEP 41 BC
GSEPATSGTEPS 42 BC GTSEPSTSEPGA 43 BC GTSTEPSEPGSA 44 BD
GSTAGSETSTEA 45 BD GSETATSGSETA 46 BD GTSESATSESGA 47 BD
GTSTEASEGSAS 48 *Denotes individual motif sequences that, when used
together in various permutations, results in a "family
sequence"
[0183] In some embodiments of XTEN families, an XTEN sequence
comprises multiple units of non-overlapping sequence motifs of the
AD motif family, the AE motif family, or the AF motif family, or
the AG motif family, or the AM motif family, or the AQ motif
family, or the BC family, or the BD family, with the resulting XTEN
exhibiting the range of homology described above. In other
embodiments, the XTEN comprises multiple units of motif sequences
from two or more of the motif families of Table 3, selected to
achieve desired physicochemical characteristics, including such
properties as net charge, 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 CFXTEN are presented in
Table 4. It is intended that a specified sequence mentioned
relative to Table 4 has that sequence set forth in Table 4, 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-00005 TABLE 4 XTEN Polypeptides SEQ XTEN ID Name Amino
Acid Sequence NO: AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS
49 AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 50 AE42_2
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 51 AE42_3
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 52 AG42_1
GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP 53 AG42_2
GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP 54 AG42_3
SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 55 AG42_4
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 56 AE48
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS 57 AM48
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS 58 AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS 59
EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAP AE144_1A
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS 60
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPG AE144_2A
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 61
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES
ATPESGPGTSESATPESGPG AE144_2B
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 62
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES
ATPESGPGTSESATPESGPG AE144_3A
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 63
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG
SPTSTEEGTSTEPSEGSAPG AE144_3B
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 64
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG
SPTSTEEGTSTEPSEGSAPG AE144_4A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 65
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPG AE144_4B
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 66
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPG AE144_5A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 67
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG
SPTSTEEGSPAGSPTSTEEG AE144_6B
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE 68
PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPG AF144
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGS 69
TSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPS
GESSTAPGTSPSGESSTAP AG144_1
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA 70
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASP AG144_2
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 71
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSS AG144_A
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 72
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSP AG144_B
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG 73
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSP AG144_C
GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPG 74
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSP AG144_F
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG 75
SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSP AG144_3
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG 76
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA
SPGTSSTGSPGASPGTSSTGSP AG144_4
GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 77
ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP
GSGTASSSPGSSTPSGATGSP AE288_1
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 78
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA
TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE288_2
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT 79
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG288_1
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP 80
GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS
PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS AG288_2
GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG 81
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP AF504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 82
SXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSP AF540
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 83
STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS
GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS
GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP
GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP
GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT
STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE
SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE
SSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG 84
SSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSS
ESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGG
EPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGG
SSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSS
GSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEG
GPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGG
PGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSS
GSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSG
SSESGSSEGGPGSEGSSGPGESS AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 85
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE
SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAP AF576
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 86
STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS
GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS
GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP
GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP
GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT
STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE
SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE
SSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGS
ASPGTSTPESGSASP AG576
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP 87
GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGS AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 88
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAP AD836
GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESG 89
ESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGES
PGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSES
GSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGG
SSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGP
GESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSES
GSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGS
ESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGP
GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSES
GSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEP
SESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGP
GESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSES
GSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGS
ESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 90
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE
SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AF864
GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT 91
STPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPS
GESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA
ESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGS
ASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPG
PGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPG
STSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGAS
ASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSES
PSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAE
SPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST
APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP
GSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGT
SPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSS
TAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG
ATGSP AG864_2
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 92
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS
GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS
TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP
AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 93
TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE
TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGA
SASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSE
SPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATS
GSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESS
TAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSAS
PGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTE
PSEGSAP AE912
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 94
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT
STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AM923
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAP 95
GSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGS
TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
EEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP
SGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSG
SETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGT
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGS
SPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 96
TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE
TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGP
EPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSST
AESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGES
STAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAP
GTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGS
SPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS
TGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGS
STPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTS
STAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAP BC 864
GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGS 97
EPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTST
EPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPAT
SGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSG
TEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTE
PSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPS
GSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGS
GASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTST
EPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEP
SEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSE
PGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPG
SAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPS
GSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGS
GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA BD864
GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETA 98
GSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETA
GTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETA
GTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSAS
GSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEA
GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA
GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSAS
GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEA
GSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSAS
GSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETA
GTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETA
GTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETA
GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETA
GTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGA
GTSESATSESGAGSETATSGSETA AE948
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS 99
PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSG
SETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEP
SEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGP AE1044
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT 100
STEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSE
SATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEP
SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS
APGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPES
GPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEP
ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGTSESATPESGPGTST AE1140
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGS 101
EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS
PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEP
SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSE
TPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEP
ATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEP
SEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA AE1236
GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 102
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE
TPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST
EPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP AE1332
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGT 103
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPAT
SGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS
EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST
EPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGS
EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATP
ESGPGTSESATPESGPGTSTEPSEGSAPGTST AE1428
GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGT 104
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE
GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP
GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS
EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSE
SATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP
GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSE
SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSPA AE1524
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS 105
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
SGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPES
GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGS
EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEP
ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGS
PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST
EEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGSPA
AE1620
GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGT 106
SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESA
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP
ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP
SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGP
GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS
EPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST AE1716
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS 107
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSE
SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS
APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGS
PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA
GSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP
SEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGSPAGSPTSTEEGTSESATPESGPGTSE AE1812
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 108
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETP
GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT
SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESA
TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP
ESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP AE1908
GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGS 109
PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTST
EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP
ESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSE
SATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEP
SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSE
GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETP
GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGS
PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP
SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSEP AE2004A
GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGS 110
PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEP
SEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES
GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGS
PAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP
ESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS
PAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSE AG948
GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG 111
TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASP
GTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPG
TSSTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSS
TGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG
PGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP
GSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPG
SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS
PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG
TASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGSP AG1044
GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG 112
TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSS
PSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPG
SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT
GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG
SPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPG
TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGT
GPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSST AG1140
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG 113
SSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPS
GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSG
ATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGA
TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSST
GSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATG
SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS
PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG
SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSAST
GTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSST AG1236
GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG 114
ASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTP
GSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSA
STGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGA
TGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG
SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP
GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGA
SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSP
SASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPG
TSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG
TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA
SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP AG1332
GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPG 115
SSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP
SGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT
GSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATG
SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP
GSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
TPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS
TPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPS
GATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST
GSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG
SPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG AG1428
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPG 116
TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSS
TPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTA
SSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGT
GPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG
PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP
GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPG
SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASP
GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS
GTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG
ATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASP AG1524
GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG 117
TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTP
GSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS
GTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA
SSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG
TPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPS
ASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG
TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA
TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT
GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSP
GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPG AG1620
GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPG 118
ASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSS
PSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTP
SGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGT
SSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT
GPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGS
PGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG
TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS
TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS
ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSA
STGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS
SSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST AG1716
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPG 119
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSS
PSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPG
SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPS
GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT
ASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS
SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP
GTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTP
GSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS
GTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSAST
GTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG
AG1812
GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG 120
SSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSS
PSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP
SGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSG
TASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGT
ASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG
SPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPG
SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG
TSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSG
TASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA
TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASP AG1908
GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPG 121
SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGA
SPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS
TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP
GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS
GATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS
TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSAST
GTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSP AG2004A
GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG 122
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGA
SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST
PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTS
STGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA
SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP
GASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSS
PSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGS
GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSG
ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS
TGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT
GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASP AE72B
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE 123
PATSGSETPG AE72C
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS 124
TEPSEGSAPG AE108A
TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA 125
PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS AE108B
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS 126
EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE 127
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGS AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP 128
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAPG AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS 129
TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET
PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE 130
SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG
PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG
TSTEPSEGSAPGSEPATSGSETPGTSESAT AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES 131
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSE
AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG 132
SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 133
SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE
PSEGSAPGSEPATS AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS 134
TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS
ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS 135
TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE 136
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE 137
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG
SAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS 138
TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS
ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG
PGTSTEPS AE540A
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 139
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS
PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS
PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE
TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP 140
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP
SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGTSESA AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS 141
TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE648A
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG 142
SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET
PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESAT AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG 143
SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA TS
AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 144
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE AE756A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 145
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG
SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE
PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA
TSGSETPGTSES AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE 146
SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE
PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE 147
SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTS
ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG
SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG
SAPGSEPATSGSETPGTSESAT AG72A
GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS 148
PGTPGSGTASS AG72B
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 149
TPGSGTASSSP AG72C
SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSST 150
PSGATGSPGA AG108A
SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG 151
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP AG108B
PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP 152
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS AG144A
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 153
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSS AG144B
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS 154
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASP AG180A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 155
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS AG216A
TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS 156
TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG AG252A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 157
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPG AG288A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 158
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS AG324A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 159
STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG
TPGSGTASSSPGSSTP AG360A
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 160
STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP
GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG396A
GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT 161
ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA
SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG
SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGASPGT AG432A
GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG 162
ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA
SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP
GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP S
AG468A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 163
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP
GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG AG504A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 164
STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP
GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS
GTASSSPGSSTP AG540A
TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS 165
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA
SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA
STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG576A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS 166
TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA
TGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT
GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTP
SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGASPG AG612A
STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT 167
GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP
GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASP
GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG
TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS
TGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS AG648A
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG 168
ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS
TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG684A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG 169
ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA
SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTG
TGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG
SPGASPG AG720A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG 170
ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS
TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG
TGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG756A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 171
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGASPG AG792A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 172
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG AG828A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 173
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST
GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG
SPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG288_DE
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG 1699
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
[0184] In other embodiments, the CFXTEN composition comprises one
or more non-repetitive XTEN sequences of lengths ranging from about
36 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 96%, or about 97%, or about 98%,
or about 99% to about 100% of the sequence consists of
non-overlapping 36 amino acid sequence motifs selected from one or
more of the polypeptide sequences of Tables 13-17, either as a
family sequence, or where motifs are selected from two or more
families of motifs.
[0185] In those embodiments wherein the XTEN component of the
CFXTEN fusion protein 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 3 or the XTEN sequences of Tables 4, and
13-17, 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. 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, e.g., to create a linker between the XTEN and
the FVIII components. In such cases where the XTEN component of the
CFXTEN comprises amino acids other than glycine (G), alanine (A),
serine (S), threonine (T), glutamate (E) and proline (P), it is
preferred that less than about 2% or less than about 1% of the
amino acids be hydrophobic residues such that the resulting
sequences generally lack secondary structure, e.g., not having more
than 2% alpha helices or 2% beta-sheets, as determined by the
methods disclosed herein. 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: cysteine (to avoid
disulfide formation and oxidation), methionine (to avoid
oxidation), asparagine and glutamine (to avoid desamidation). Thus,
in some embodiments, the XTEN component of the CFXTEN fusion
protein comprising other amino acids in addition to glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P) have a sequence with less than 5% of the residues contributing
to alpha-helices and beta-sheets as measured by the Chou-Fasman
algorithm and have at least 90%, or at least about 95% or more
random coil formation as measured by the GOR algorithm.
3. Length of Sequence
[0186] In another aspect, the invention provides XTEN of varying
lengths for incorporation into CFXTEN compositions wherein the
length of the XTEN sequence(s) are chosen based on the property or
function to be achieved in the fusion protein. Depending on the
intended property or function, the CFXTEN compositions comprise
short or intermediate length XTEN located internal to the FVIII
sequence or between FVIII domains and/or longer XTEN sequences that
can serve as carriers, located in the fusion proteins as described
herein. 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 for incorporation
into the subject CFXTEN encompass XTEN or fragments of XTEN with
lengths of about 6, or about 12, or about 36, or about 40, or about
42, or about 72 or about 96, or about 144, or about 288, or about
400, 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. Alternatively, 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 CFXTEN can vary without
adversely affecting the activity of a CFXTEN composition. In one
embodiment, one or more of the XTEN used in the CFXTEN disclosed
herein has 36 amino acids, 42 amino acids, 144 amino acids, 288
amino acids, 576 amino acids, or 864 amino acids in length and may
be selected from one of the XTEN family sequences; i.e., AD, AE,
AF, AG, AM, AQ, BC or BD. In another embodiment, two or more of the
XTEN used in the CFXTEN disclosed herein has 36 amino acids, 42
amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or
864 amino acids in length and may be selected from two of the XTEN
family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD, with
combinations of AE and AG family sequences preferred. In some
embodiments, CFXTEN comprising one or more of the XTEN used herein
contain XTEN selected from any one of the sequences in Table 4,
which may be linked to the FVIII component directly or via spacer
sequences disclosed herein.
[0187] In particular CFXTEN configuration designs, where the XTEN
serve as a flexible linker, or are inserted in external loops or
unordered regions of the FVIII sequence to increase the bulk,
flexibility, or hydrophilicity of the region, or are designed to
interfere with clearance receptors for FVIII to enhance
pharmacokinetic properties, or to interfere with binding of FVIII
inhibitors or other anti-FVIII antibodies, or where a short or
intermediate length of XTEN is used to facilitate tissue
penetration or to vary the strength of interactions of the CFXTEN
fusion protein with its target, or where it is desirable to
distribute the cumulative length of XTEN in segments of short or
intermediate length at multiple locations within the FVIII
sequence, the invention contemplates CFXTEN compositions with one,
two, three, four, five or more short or intermediate XTEN sequences
inserted between or within one or more FVIII domains or within
external loops, or at other sites in the FVIII sequence such as,
but not limited to, locations at or proximal to the insertion sites
identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or as
illustrated in FIGS. 8-9. In one embodiment of the foregoing, the
CFXTEN fusion protein contains multiple XTEN segments, e.g., at
least two, or at least three, or at least four, or at least five or
more XTEN segments in which the XTEN segments can be identical or
they can be different and wherein the CFXTEN retains at least 10%,
20%, 30%, 40%, 50%, 60%, 70% or more of the procoagulant activity
of native FVIII when assayed by one of the assays disclosed herein.
In other particular CFXTEN configuration designs, where the XTEN
serves as a carrier to increase the bulk of the fusion protein, or
to vary the strength of interactions of the CFXTEN fusion protein
with its target, or to enhance the pharmacokinetic properties of
the fusion protein, the invention contemplates CFXTEN compositions
with one or more intermediate or longer length XTEN sequences
inserted at the C-terminus, within the B domain (or the residual of
the BDD sequence) between or within one or more FVIII domains,
within external loops, or at other sites in the FVIII sequence such
as, but not limited to, insertion sites identified in Table 5,
Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS.
8-9. However, it is believed that the incorporation of multiple
XTEN of short to intermediate lengths into CFXTEN compositions
confers enhanced properties on the fusion proteins compared to
CFXTEN fusion proteins with the same number of amino acids in fewer
but longer length XTEN, yet still results in compositions with
procoagulant activity and extended half-life; the rationale of
which is detailed herein regarding the derived radii of multiple
XTEN.
[0188] In the embodiments wherein the CFXTEN fusion proteins
comprise multiple XTEN sequences, the cumulative length of the
total residues in the XTEN sequences is greater than about 100 to
about 3000, or about 200 to about 2000, or about 400 to about 1000
amino acid residues and the XTEN can be identical or they can be
different in sequence, net charge, or in length. In one embodiment
of CFXTEN comprising multiple XTEN, the individual XTEN sequences
each exhibit 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
a motif or an XTEN selected from Tables 3, 4, and 13-17 or a
fragment thereof, when optimally aligned with a sequence of
comparable length.
[0189] As described more fully below, methods are disclosed in
which the CFXTEN are designed by selecting the length of the XTEN
and its site of incorporation within the CFXTEN to confer a target
half-life, retention of procoagulant activity, reduced binding to
FVIII inhibitors or an enhanced physicochemical property (e.g.,
stability or solubility) of a CFXTEN fusion protein, encoding
constructs are created and expressed and the recombinant CFXTEN
fusion proteins are isolated and recovered. In general, XTEN
cumulative lengths longer that about 400 residues incorporated into
the CFXTEN compositions result in longer half-life compared to
shorter cumulative lengths, e.g., shorter than about 280 residues.
In one embodiment, CFXTEN fusion proteins designs are contemplated
that comprise at least a single XTEN as a carrier, with a long
sequence length of at least about 400, or at least about 600, or at
least about 800, or at least about 900, or at least about 1000 or
more amino acids. In another embodiment, multiple XTEN are
incorporated into the fusion protein to achieve cumulative lengths
of at least about 400, or at least about 600, or at least about
800, or at least about 900, or at least about 1000 or more amino
acids, wherein the XTEN can be identical or they can be different
in sequence or 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 CFXTEN fusion
protein. Both of the foregoing embodiments are designed to confer
increased bioavailability and/or increased terminal half-life after
administration to a subject compared to CFXTEN comprising shorter
cumulative XTEN lengths, yet still result in a procoagulant
activity and hemostasis effect. When administered subcutaneously or
intramuscularly, the Cmax is reduced but the area under the curve
(AUC) is increased in comparison to a comparable dose of a CFXTEN
with shorter cumulative length XTEN or FVIII not linked to XTEN,
thereby contributing to the ability to maintain effective levels of
the CFXTEN composition for a longer period of time and permitting
increased periods of 2, 4, 7, 10, 14 or 21 days between dosing, as
described more fully below. Thus, the XTEN confers the property of
a depot to the administered CFXTEN, in addition to the other
physicochemical properties described herein.
[0190] When XTEN are used as a carrier, the invention takes
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
physical/chemical and pharmacokinetic properties of fusion proteins
comprising the XTEN carrier. 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 3), result in a sequence with a higher
percentage (e.g., 90% or more) of random coil formation, as
determined by GOR algorithm, or reduced content of alpha-helices or
beta-sheets (e.g., less than 2%), 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 fusion protein
with a disproportionate increase in terminal half-life (e.g., as
much as 50, 100, 200 or more hours) compared to fusion proteins
with unstructured polypeptide partners with shorter sequence
lengths. The enhanced pharmacokinetic properties of the CFXTEN in
comparison to FVIII not linked to XTEN are described more fully,
below.
[0191] 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 a fragment is created from
the interior of a donor sequence, thereby resulting in a short or
intermediate length XTEN. In non-limiting examples, as
schematically depicted in FIG. 16A-C, an AG sequence of 864 amino
acid residues can be truncated to yield an AG sequence with 144
residues, an AG sequence with 288 residues, an AG sequence with 576
residues, or other intermediate lengths, while the AE sequence of
864 residues (as depicted in FIG. 16D, E) can be truncated to yield
multiple AE sequences of 144 residues, an AE sequence with 288 or
576 residues or other shorter or intermediate lengths. 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 4 or 13-17 to result in XTEN of a
desired length. In preferred embodiments, the CFXTEN comprising
multiple XTEN have XTEN exhibiting 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 100% sequence identity to sequences
selected from AE42_1, AE42_2, AE42_3, AG42_1, AG42_2, AG42_3,
AG42_4, AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A,
AE144_4B, AE144_5A, AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B,
AG144_C, AG144_F, AG144_3, AG144_4, AE288_1, AE288_2, AG288_1,
AG288_2, and AG288_DE.
4. Net Charge
[0192] In other embodiments, the unstructured characteristic of an
XTEN polypeptide can be enhanced by incorporation of amino acid
residues with a net charge and/or reduction of the overall
percentage (e.g. less than 5%, or 4%, or 3%, or 2%, or 1%) of
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 opposite charge. In some embodiments, the net charge
density of the XTEN of the compositions 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 or propeptide. 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 preferred embodiments, the XTEN will have an
isoelectric point between 1.5 and 4.5 and carry a net negative
charge under physiologic conditions.
[0193] 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% or 95% of the charged
residues separated by other 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 distribution of net negative charge in the extended sequence
lengths of XTEN can lead to an unstructured conformation 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 CFXTEN fusion protein
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 3
to provide the desired degree of net charge. Non-limiting examples
of AE XTEN include, but are not limited to the 36, 42, 144, 288,
576, 624, 864, and 912 AE family sequences of Tables 4 and 14 or
fragments thereof. In one embodiment, an XTEN sequence of Tables 4,
or 13-17 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 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.
[0194] 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 3, or those AM motifs of Table 3 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 4 and 16, 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.
[0195] Not to be bound by a particular theory, the XTEN of the
CFXTEN compositions with the higher net 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 CFXTEN compositions
with a low (or no) net charge would have a higher degree of
interaction with surfaces that can potentiate the activity of the
associated coagulation factor, given the known contribution of cell
(e.g., platelets) and vascular surfaces to the coagulation process
and the intensity of activation of coagulation factors (Zhou, R.,
et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al.
Biochemistry (2000) 39(32):9850-9858).
[0196] The XTEN of the compositions 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 limited 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 peptide, a linker bridge, or a reactive group
on a drug or small molecule to be conjugated to the XTEN backbone.
In one embodiment of the foregoing, the XTEN of the subject CFXTEN
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 alternatively only a single lysine
residue. Using the foregoing lysine-containing XTEN, fusion
proteins can be constructed that comprise XTEN, a FVIII coagulation
factor, plus a chemotherapeutic agent or other coagulation factor
or cofactor useful in the treatment of coagulopathy conditions,
wherein the maximum number of molecules of the agent incorporated
into the XTEN component is determined by the numbers of lysines or
other amino acids with reactive side chains (e.g., cysteine)
incorporated into the XTEN.
[0197] As hydrophobic amino acids impart structure to a
polypeptide, the invention provides that the content of hydrophobic
amino acids in the XTEN will typically be less than 5%, or less
than 2%, or less than 1% hydrophobic amino acid content. In one
embodiment, the amino acid content of methionine and tryptophan in
the XTEN component of a CFXTEN fusion protein is typically less
than 5%, or less than 2%, and most preferably less than 1%. In
another embodiment, the XTEN of the subject CFXTEN compositions
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.
5. Low Immunogenicity
[0198] In another aspect, the XTEN sequences provided herein have 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.
[0199] 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.
[0200] 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 CFXTEN fusion proteins 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
fusion proteins 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 FVIII fusion partner in the CFXTEN
compositions.
[0201] In one embodiment, the XTEN sequences utilized in the
subject fusion proteins 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 a CFXTEN 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.
[0202] In another embodiment, the inventive XTEN sequences,
including those incorporated into the subject CFXTEN fusion
proteins, are rendered substantially non-immunogenic by the
restriction of known proteolytic sites from the sequence of the
XTEN, reducing the processing of XTEN into small peptides that can
bind to MHC II receptors. In another embodiment, the XTEN sequence
is rendered substantially non-immunogenic by the use a sequence
that is substantially devoid of secondary structure, conferring
resistance to many proteases due to the high entropy of the
structure. Accordingly, the reduced TEPITOPE score and elimination
of known proteolytic sites from the XTEN render the XTEN
compositions, including the XTEN of the CFXTEN fusion protein
compositions, substantially unable to be bound by mammalian
receptors, including those of the immune system or active clearance
receptors that target FVIII. In one embodiment, an XTEN of a CFXTEN
fusion protein can have .gtoreq.100 nM K.sub.d binding to a
mammalian receptor, or greater than 500 nM K.sub.d, or greater than
1 .mu.M K.sub.d towards a mammalian cell surface receptor or
circulating polypeptide receptor.
[0203] Additionally, the non-repetitive sequence and corresponding
lack of epitopes of XTEN limit the ability of B cells to bind to or
be activated by XTEN. A repetitive sequence is recognized and can
form multivalent contacts with even a few B cells and, as a
consequence of the cross-linking of multiple T-cell independent
receptors, can stimulate B cell proliferation and antibody
production. In contrast, while an XTEN can make contacts with many
different B cells over its extended sequence, each individual B
cell may only make one or a small number of contacts with an
individual XTEN due to the lack of repetitiveness of the sequence.
Not being to be bound by any theory, XTENs typically have a much
lower tendency to stimulate proliferation of B cells and thus an
immune response. In one embodiment, the CFXTEN have reduced
immunogenicity as compared to the corresponding FVIII that is not
fused to an XTEN. In one embodiment, the administration of up to
three parenteral doses of a CFXTEN to a mammal result in detectable
anti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution
of 1:1000. In another embodiment, the administration of up to three
parenteral doses of a CFXTEN to a mammal result in detectable
anti-FVIII IgG at a serum dilution of 1:100 but not at a dilution
of 1:1000. In another embodiment, the administration of up to three
parenteral doses of a CFXTEN to a mammal result in detectable
anti-XTEN IgG at a serum dilution of 1:100 but not at a dilution of
1:1000. In the foregoing embodiments, the mammal can be a mouse, a
rat, a rabbit, or a cynomolgus monkey.
[0204] An additional feature of XTENs with non-repetitive sequences
relative to sequences with a high degree of repetitiveness is
non-repetitive XTENs form weaker contacts with antibodies.
Antibodies are multivalent molecules. For instance, IgGs have two
identical binding sites and IgMs contain 10 identical binding
sites. Thus antibodies against repetitive sequences can form
multivalent contacts with such repetitive sequences with high
avidity, which can affect the potency and/or elimination of such
repetitive sequences. In contrast, antibodies against
non-repetitive XTENs may yield monovalent interactions, resulting
in less likelihood of immune clearance such that the CFXTEN
compositions can remain in circulation for an increased period of
time. In addition, it is believed, as schematically portrayed in
FIG. 6, the flexible unstructured nature of XTEN provides steric
shielding of FVIII regions proximal to the XTEN site of insertion
and providing steric hindrance to binding by FVIII inhibitors.
[0205] In another aspect, a subject XTEN useful as a fusion partner
has a high hydrodynamic radius; a property that in some embodiments
confers a corresponding increased apparent molecular weight to the
CFXTEN fusion protein incorporating the XTEN, while in other
embodiments enhances steric hindrance to FVIII inhibitors and to
anti-FVIII antibodies, reducing their ability to bind to CFXTEN. As
detailed in Example 26, the linking of XTEN to therapeutic protein
sequences results in CFXTEN 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 an XTEN with a high hydrodynamic radius is
incorporated into a fusion protein comprising a therapeutic protein
can effectively enlarge the hydrodynamic radius of the composition
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
electrostatic repulsion between individual charges of the peptide
or 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 can have a greater proportional hydrodynamic radius
compared to polypeptides of a comparable sequence length and/or
molecular weight that have secondary and/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 CFXTEN to
desired cut-off values of apparent molecular weights or
hydrodynamic radii. Accordingly, in certain embodiments, the CFXTEN
fusion protein can be configured with an XTEN such that the fusion
protein 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 CFXTEN fusion protein can lead to reduced clearance
of the resulting fusion protein, an increase in terminal half-life,
and an increase in mean residence time.
[0206] Generally, the actual molecular weight of the mature form of
FVIII component is about 265 kDa, while in the case of a FVIII BDD,
it is about 165 kDa. The actual molecular weight of a CFXTEN fusion
protein for comprising a FVIII BDD plus one or more XTEN ranges
from about 200 to about 270 kDa, depending on the length of the
XTEN components. As described in the Examples, when the molecular
weights of the CFXTEN fusion proteins 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 fusion proteins
into which they are incorporated. In some embodiments, the CFXTEN
comprising a FVIII and at least 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 CFXTEN
fusion proteins comprising one or more XTEN exhibit 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 greater than the actual molecular weight of the
fusion protein. In one embodiment, the isolated CFXTEN fusion
protein 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 CFXTEN fusion
protein 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 fusion protein. It is
believed that the increased apparent molecular weight of the
subject CFXTEN compositions enhances the pharmacokinetic properties
of the fusion proteins by a combination of factors, which include
reduced active clearance, reduced binding by FVIII inhibitors, and
reduced loss in capillary and venous bleeding.
IV). CFXTEN Compositions
[0207] The present invention provides compositions comprising
fusion proteins having factor VIII linked to one or more XTEN
sequences, wherein the fusion protein acts to replace or augment
the amount of existing FVIII in the intrinsic or contact activated
coagulation pathway when administered into a subject. The invention
addresses a long-felt need in increasing the terminal half-life of
exogenously administered factor VIII to a subject in need thereof.
One way to increase the circulation half-life of a therapeutic
protein is to ensure that renal clearance or metabolism of the
protein is reduced. Another way to increase the terminal half-life
is to reduce the active clearance of the therapeutic protein,
whether mediated by receptors, active metabolism of the protein, or
other endogenous mechanisms. Both may be achieved by conjugating
the protein to a polymer, which, on one hand, is capable of
conferring an increased molecular size (or hydrodynamic radius) to
the protein and, hence, reduced renal clearance, and, on the other
hand, interferes with binding of the protein to clearance receptors
or other proteins that contribute to metabolism or clearance. Thus,
certain objects of the present invention include, but are not
limited to, providing improved FVIII molecules with a longer
circulation or terminal half-life, decreasing the number or
frequency of necessary administrations of FVIII compositions,
retaining at least a portion of the activity compared to native
coagulation factor VIII, and/or enhancing the ability to treat
coagulation deficiencies and uncontrolled bleedings more
efficiently, more effectively, more economically, and/or with
greater safety compared to presently available factor VIII
preparations.
[0208] Accordingly, the present invention provides recombinant
factor VIII fusion protein compositions comprising an FVIII
covalently linked to one or more extended recombinant polypeptides
("XTEN"), resulting in a CFXTEN fusion protein composition. The
term "CFXTEN", as used herein, is meant to encompass fusion
polypeptides that comprise at least one payload region comprising a
FVIII or a portion of a FVIII that is capable of procoagulant
activity associated with a FVIII coagulation factor and at least
one other region comprising one or more XTEN polypeptides that may
be interspersed within the payload region and/or attached to the
terminus. In one embodiment, the FVIII is native FVIII. In another
embodiment, the FVIII is a sequence variant, fragment, homolog, or
mimetic of a natural sequence that retains at least a portion of
the procoagulant activity of native FVIII, as disclosed herein.
Non-limiting examples of FVIII suitable for inclusion in the
compositions include the sequences of Table 1 or sequences having
at least 80%, or 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 at least 98%, or at least 99% sequence identity to
a sequence of Table 1. In a preferred embodiment, the FVIII is a
B-domain deleted (BDD) FVIII sequence variant, such as those BDD
sequences from Table 1 or other such sequences known in the art. In
another preferred embodiment, the CFXTEN comprises a B-domain
deleted (BDD) FVIII sequence variant expressed with the native 19
amino acid signal sequence, which is cleaved during the maturation
of the protein.
[0209] The compositions of the invention include fusion proteins
that are useful, when administered to a subject in need thereof,
for mediating or preventing or ameliorating a condition associated
with factor VIII deficiencies or defects in endogenously produced
FVIII, or bleeding disorders associated with trauma, surgery,
factor VIII deficiencies or defects. Of particular interest are
CFXTEN fusion protein compositions for which an increase in a
pharmacokinetic parameter, increased solubility, increased
stability, or some other enhanced pharmaceutical property compared
to native FVIII is sought, or for which increasing the terminal
half-life would improve efficacy, safety, or result in reduced
dosing frequency and/or improve patient management. The CFXTEN
fusion proteins of the embodiments disclosed herein exhibit one or
more or any combination of the improved properties and/or the
embodiments as detailed herein. In some embodiments, the CFXTEN
fusion composition remains at a level above a threshold value of at
least 0.01-0.05, or 0.05 to 0.1, or 0.1 to 0.4 IU/ml when
administered to a subject, for a longer period of time when
compared to a FVIII not linked to XTEN and administered at a
comparable dose to a subject in need thereof (e.g., a subject such
as a human or mouse or monkey with hemophilia A).
[0210] The FVIII of the subject compositions, particularly those
disclosed in Table 1, together with their corresponding nucleic
acid and amino acid sequences, are available in public databases
such as Chemical Abstracts Services Databases (e.g., the CAS
Registry), GenBank, The Universal Protein Resource (UniProt),
subscription provided databases such as GenSeq (e.g., Derwent), as
well as in the patent and primary literature. Polynucleotide
sequences applicable for expressing the subject CFXTEN sequences
may be a wild type polynucleotide sequence encoding a given FVIII
(e.g., either full length or mature), or in some instances the
sequence may be a variant of the wild type polynucleotide sequence
(e.g., a polynucleotide which encodes the wild type biologically
active protein, wherein the DNA sequence of the polynucleotide has
been optimized, for example, for expression in a particular
species, or a polynucleotide encoding a variant of the wild type
protein, such as a site directed mutant or an allelic variant. It
is well within the ability of the skilled artisan to use a
wild-type or consensus cDNA sequence or a codon-optimized variant
of a FVIII to create CFXTEN constructs contemplated by the
invention using methods known in the art and/or in conjunction with
the guidance and methods provided herein, and described more fully
in the Examples.
[0211] In one embodiment, a CFXTEN fusion protein comprises a
single FVIII molecule exhibiting 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%, or at least about
99%, or 100% sequence identity to a sequence of Table 1 linked to a
single XTEN (e.g., an XTEN as described above) including, but not
limited to sequences of the AE or AG family with 42, 144, 288, 576,
or 864 amino acids, as set forth in Table 4. In another embodiment,
the CFXTEN comprises a single FVIII linked to two XTEN, wherein the
XTEN may be identical or they may be different. In another
embodiment, the CFXTEN fusion protein comprises a single FVIII
molecule linked to one, two, three, four, five, six or more XTEN
sequences, in which the FVIII is a sequence that has 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%, or
at least about 99%, or 100% sequence identity compared to a protein
sequence selected from Table 1, when optimally aligned, and the one
or more XTEN are each 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%, or at least about 99%, or
100% sequence identity compared to one or more sequences selected
from any one of Tables 3, 4, and 13-17, when optimally aligned. In
the foregoing embodiment, where the CFXTEN has two or more XTEN,
the XTEN may be identical or they may be different sequences. In
yet another embodiment, the CFXTEN fusion protein comprises a
single FVIII exhibiting 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%, or at least about 99%, or
100% sequence identity compared to sequences of comparable length
selected from Table 1, when optimally aligned, with the portions
interspersed with and linked by three, four, five, six or more XTEN
sequences that may be identical or may be different and wherein
each has 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%, or at least about 99%, or 100% sequence
identity compared to sequences selected from any one of Tables 3,
4, and 13-17, or fragments thereof, when optimally aligned. In yet
another embodiment, the invention provides a CFXTEN fusion protein
comprising a sequence with 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%, or at least about 99%, or
100% sequence identity to a sequence from Table 21, when optimally
aligned.
[0212] 1. CFXTEN Fusion Protein Configurations
[0213] The invention provides CFXTEN fusion protein compositions
with the CF and XTEN components linked in specific N- to C-terminus
configurations.
[0214] In one embodiment of the CFXTEN composition, the invention
provides a fusion protein of formula I:
(XTEN).sub.x-CF-(XTEN).sub.y I
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity with sequenced from Table 1; x is either
0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an
extended recombinant polypeptide as described herein, including,
but not limited to sequences 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% or
100% sequence identity to sequences set forth in Table 4.
Accordingly, the CFXTEN fusion composition can have XTEN-CF,
XTEN-CF-XTEN, or CF-XTEN configurations.
[0215] In another embodiment of the CFXTEN composition, the
invention provides a fusion protein of formula II:
(XTEN).sub.x-(S).sub.x-(CF)-(XTEN).sub.y II
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity to sequences set forth in Table 1; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence or amino acids
compatible with restrictions sites; x is either 0 or 1 and y is
either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4.
[0216] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein,
wherein the fusion protein is of formula III:
(XTEN).sub.x-(S).sub.x-(CF)-(S).sub.y-(XTEN).sub.y III
wherein independently for each occurrence, CF is a factor VIII as
defined herein, including sequences 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%
or 100% sequence identity to sequence set for in Table 1; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence or amino acids
compatible with restrictions sites; x is either 0 or 1 and y is
either 0 or 1 wherein x+y.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4.
[0217] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula IV:
(A1)-(XTEN).sub.u-(A2)-(XTEN).sub.v-(B)-(XTEN).sub.w-(A3)-(XTEN).sub.x-(-
C1)-(XTEN).sub.y-(C2)-(XTEN).sub.z IV
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B
is a B domain of FVIII which can be a fragment or a splice variant
of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of
FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1;
y is either 0 or 1; y is either 0 or 1 with the proviso that
u+v+x+y+z.gtoreq.1; and XTEN is an extended recombinant polypeptide
as described herein including, but not limited to sequences 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% or 100% sequence identity to sequences
set forth in Table 4.
[0218] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula V:
(XTEN).sub.t-(S).sub.a-(A1)-(S).sub.b-(XTEN).sub.u-(S).sub.b-(A2)-(S).su-
b.c-(XTEN).sub.v-(S).sub.c-(B)-(S).sub.d-(XTEN).sub.w-(S).sub.d-(A3)-(S).s-
ub.e-(XTEN).sub.x-(S).sub.e-(C1)-(S).sub.f-(XTEN).sub.y-(S).sub.f-(C2)-(S)-
.sub.g-(XTEN).sub.z V
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B
is a B domain of FVIII which can be a fragment or a splice variant
of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of
FVIII; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence or
amino acids compatible with restrictions sites; a is either 0 or 1;
b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is
either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either
0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is
either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that t+u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In another
embodiment of formula V, the spacer sequence is glycine or a
sequence selected from Tables 11 and 12.
[0219] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VI:
(XTEN).sub.u-(S).sub.a-(A1)-(S).sub.b-(XTEN).sub.v-(S).sub.b-(A2)-(S).su-
b.c-(XTEN).sub.w-(S).sub.c-(A3)-(S).sub.d-(XTEN).sub.x-(S).sub.d(C1)-(S).s-
ub.e-(XTEN).sub.y(S).sub.e-(C2)-(S).sub.f-(XTEN).sub.z VI
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1
is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer
sequence having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence or amino acids compatible
with restrictions sites; a is either 0 or 1; b is either 0 or 1; c
is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is
either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1,
x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In another
embodiment of formula V, the spacer sequence is glycine or a
sequence selected from Tables 11 and 12.
[0220] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VII:
(SP)-(XTEN).sub.x-(CS).sub.x-(S).sub.x-(FVIII_1-745)-(S).sub.y-(XTEN).su-
b.y-(S).sub.y-(FVIII_1635-2332)-(S).sub.z-(CS).sub.z-(XTEN).sub.z
VII
wherein independently for each occurrence, SP is a signal peptide,
preferably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS
is a cleavage sequence listed in Table 12, S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include amino acids compatible with restrictions sites,
"FVIII_1-745" is residues 1-745 of Factor FVIII and
"FVIII_1635-2332" is residues 1635-2332 of FVIII, x is either 0 or
1, y is either 0 or 1, and z is either 0 or 1, wherein
x+y+z.gtoreq.2; and XTEN is an extended recombinant polypeptide as
described herein including, but not limited to sequences 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% or 100% sequence identity sequences set forth
in Table 4. In one embodiment of formula VII, the spacer sequence
is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V,
the spacer sequence is glycine or a sequence selected from Tables
11 and 12.
[0221] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula VIII:
(A1)-(S).sub.a-(XTEN).sub.v-(S).sub.a-(A2)-(B1)-(S).sub.b-(XTEN).sub.w-(-
S).sub.b-(B2)-(A3)-(S).sub.c-(XTEN).sub.x-(S)-(C1)-(S).sub.d-(XTEN).sub.y--
(S).sub.d-(C2)-(S).sub.e-(XTEN).sub.z VIII
wherein independently for each occurrence, A1 is an A1 domain of
FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B
domain that can have from residue 741 to 743-750 of FVIII or
alternatively from about residue 741 to about residues 745 of
FVIII; B2 is a fragment of the B domain that can have from residues
1635-1686 to 1689 of FVIII or alternatively from about residue 1640
to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is
a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer
sequence having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence or amino acids compatible
with restrictions sites; a is either 0 or 1; b is either 0 or 1; c
is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is
either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1,
x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the
proviso that u+v+w+x+y+z.gtoreq.1; and XTEN is an extended
recombinant polypeptide as described herein including, but not
limited to sequences 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% or 100%
sequence identity to sequences set forth in Table 4. In one
embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID
NO: 1612). In another embodiment of formula V, the spacer sequence
is glycine or a sequence selected from Tables 11 and 12.
[0222] In another embodiment of the CFXTEN composition, the
invention provides a recombinant factor VIII fusion protein of
formula IX:
(A1.sub.N)-(S).sub.a-(XTEN).sub.t-(S).sub.b-(A1.sub.C)-(A2.sub.N)-(S).su-
b.c-(XTEN).sub.u-(S).sub.d-(A2.sub.C)-(B.sub.N)--(S).sub.e-(XTEN).sub.v-(S-
).sub.f-(B.sub.C)-(A3.sub.N)-
(S).sub.g-(XTEN).sub.w-(S).sub.h-(A3.sub.C)-(C1.sub.N)-(S).sub.i-(XTEN).s-
ub.x-(S).sub.j-(C1.sub.C)-(C2.sub.N)-(S).sub.k-(XTEN).sub.y-(S).sub.l-(C2.-
sub.C)-(S).sub.m-(XTEN).sub.z IX
wherein independently for each occurrence, A1.sub.N is a fragment
of the A1 domain from at least residue number 1 (numbered relative
to native, mature FVIII) to no more than residue number 371,
A1.sub.c is a fragment of the A1 domain from at least residue
number 2 to no more than residue number 372; A2.sub.N is a fragment
of the A2 domain from at least residue number 373 to no more than
residue number 739, A2.sub.c is a fragment of the A2 domain from at
least residue number 374 to no more than residue number 740;
B.sub.N is a fragment of the B domain from at least residue number
741 to no more than residue number 1647, B.sub.c is a fragment of
the B domain from at least residue number 742 to no more than
residue number 1648; A3.sub.N is a fragment of the A3 domain from
at least residue number 1649 to no more than residue number 2019,
A3.sub.c is a fragment of the A3 domain from at least residue
number 1650 to no more than residue number 2019; C1.sub.N is a
fragment of the C1 domain from at least residue number 2020 to no
more than residue number 2171, C1, is a fragment of the C1 domain
from at least residue number 2021 to no more than residue number
2172; C2.sub.N is a fragment of the C2 domain from at least residue
number 2173 to no more than residue number 2331, C2.sub.c is a
fragment of the C2 domain from at least residue number 2174 to no
more than residue number 2332; S is a spacer sequence having
between 1 to about 50 amino acid residues that can optionally
include a cleavage sequence or amino acids compatible with
restrictions sites; a is either 0 or 1; b is either 0 or 1; c is
either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either
0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1;
j is either 0 or 1; k is either 0 or 1; 1 is either 0 or 1; m is
either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either
0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is
either 0 or 1 with the proviso that t+u+v+w+x+y+z.gtoreq.1; and
XTEN is an extended recombinant polypeptide as described herein
including, but not limited to sequences having at least 90%
identity to sequences set forth in Table 4. In one embodiment of
formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In
another embodiment of formula IX, the spacer sequence is glycine or
a sequence selected from Tables 11 and 12.
[0223] The embodiments of formulae IV-VIII encompass CFXTEN
configurations wherein one or more XTEN of lengths ranging from
about 6 amino acids to .gtoreq.1000 amino acids (e.g., sequences
selected from any one of Tables 3, 4, and 13-17 or fragments
thereof, or sequences exhibiting at least about 90-99% or more
sequence identity thereto) are inserted and linked between
adjoining domains of the factor VIII or are linked to the N- or
C-terminus of the FVIII. In other embodiments of formulae V-VIII,
the invention further provides configurations wherein the XTEN are
linked to FVIII domains via spacer sequences which can optionally
comprise amino acids compatible with restrictions sites or can
include cleavage sequences (e.g., the sequences of Tables 11 and
12, described more fully below) such that the XTEN encoding
sequence can be, in the case of a restriction site, integrated into
a CFXTEN construct and, in the case of a cleavage sequence, the
XTEN can be released from the fusion protein by the action of a
protease appropriate for the cleavage sequence.
[0224] The embodiments of formulae VI-VIII differ from those of
formula V in that the FVIII component of formulae VI-VIII are only
the B-domain deleted forms ("FVIII BDD") of factor VIII that retain
short residual sequences of the B-domain, non-limiting examples of
sequences of which are provided in Table 1, wherein one or more
XTEN or fragments of XTEN of lengths ranging from about 6 amino
acids to .gtoreq.1000 amino acids (e.g., sequences selected from
any one of Tables 3, 4, and 13-17) are inserted and linked between
adjoining domains of the factor VIII and/or between the remnants of
the B domain residues, such as those of Table 8. The embodiment of
formula IX generally differs from those of the other formulae in
that the one or more XTEN are each inserted within domains of FVIII
rather than between domains, and/or has an XTEN linked to the
C-terminus of the FVIII (or is linked via a spacer sequence to the
C-terminus of the FVIII).
[0225] In some embodiments of a CFXTEN, the fusion protein
comprises a B-domain deleted form of FVIII wherein the B-domain
deletion starts from a first position at about amino acid residue
number 745 and ends at a second position at amino acid residue
number 1635 to about 1690 with reference to the full-length human
factor VIII sequence and an XTEN links the first position and the
second position of the B-domain deletion. In one embodiment of the
foregoing, the first position and the second position of the
B-domain deletion are selected from the positions of Table 8. In
another embodiment of the foregoing, at least one XTEN links the
first and second position wherein the at least one XTEN links
factor VIII amino acid residue 745 and amino acid residue 1640, or
amino acid residue 741 and amino acid residue 1640, or amino acid
residue 741 and amino acid residue 1690, or amino acid residue 745
and amino acid residue 1667, or amino acid residue 745 and amino
acid residue 1657, or amino acid residue 745 and amino acid residue
1657, or amino acid residue 747 and amino acid residue 1642, or
amino acid residue 751 and amino acid residue 1667. In one
embodiment of the CFXTEN, wherein the factor VIII comprises an XTEN
linking a first position and a second position of a B-domain
deletion described in the embodiments of this paragraph, the XTEN
is a sequence having at least 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% or 100% sequence identity
compared to a sequence of comparable length selected from any one
of Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17,
when optimally aligned, wherein the CFXTEN retains 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% of the procoagulant activity of native FVIII.
[0226] The invention contemplates all possible permutations of
insertions of XTEN between or within the domains of FVIII or at or
proximal to the insertion points of Table 5, Table 6, Table 7,
Table 8, and Table 9 or those illustrated in FIGS. 8-9, with
optional linking of an additional XTEN to the N- or C-terminus of
the FVIII, optionally linked via an additional cleavage sequence
selected from Table 12, resulting in a CFXTEN composition;
non-limiting examples of which are portrayed in FIGS. 5 and 12. In
one embodiment, the CFXTEN comprises a FVIII BDD sequence of Table
1 in which one or more XTEN that each has 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% or
more sequence identity compared to a sequence from any one of
Tables 3, 4, and 13-17 or fragments thereof are inserted between
any two of the residual B domain amino acids of the FVIII BDD
sequence, resulting in a single chain FVIII fusion protein, wherein
the CFXTEN retains 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% of the procoagulant
activity of native FVIII. In the foregoing embodiment, the CFXTEN
can have an additional XTEN sequence of any one of Tables 4, and
13-17 linked to the N- or C-terminus of the fusion protein. In
another embodiment, a CFXTEN comprises at least a first XTEN
inserted at a site set forth in Table 8, wherein the CFXTEN retains
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% of the procoagulant activity of native
FVIII. In one embodiment of a fusion protein of formula VII, the
CFXTEN comprises a FVIII BDD sequence of Table 1 in which two or
more XTEN that each has 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%, or 100% sequence
identity compared to a sequence from any one of Tables 3, 4, and
13-17 or fragments thereof are linked to a FVIII-BDD sequence in
which at least one XTEN is inserted from about 3 to about 20 amino
acid residues to the C-terminus side of the FVIII cleavage site
amino acid R740 and from about 3 to about 20 amino acid residues to
the N-terminus side of the FVIII cleavage site amino acid R1689 of
the residual B domain amino acids of the FVIII BDD sequence,
resulting in a single chain FVIII fusion protein, and one or two
XTEN are linked by a cleavage sequence to the N- and/or C-terminus
of the FVIII-BDD sequence, wherein the CFXTEN exhibits 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% of
the procoagulant activity of native FVIII after release of the XTEN
by cleavage of the cleavage sequences.
[0227] In one embodiment, the A3 domain comprises an a3 acidic
region or a portion thereof. In another embodiment, at least one
XTEN is inserted within the a3 acidic region or the portion
thereof, N-terminus of the a3 acidic region or the portion thereof,
C-terminus of the a3 acidic region or the portion thereof, or a
combination thereof. In certain embodiments, at least one XTEN is
inserted within the C2 domain, N-terminus of C2 domain, C-terminus
of C2 domain, or a combination thereof. In still other embodiments,
the Factor VIII comprises all or portion of B domain. In yet other
embodiments, at least one XTEN is inserted within all or a portion
of B domain, N-terminus of B domain, C-terminus of B domain, or a
combination thereof.
[0228] 2. CFXTEN Fusion Protein Configurations with Internal
XTEN
[0229] In another aspect, the invention provides CFXTEN configured
with one or more XTEN sequences located internal to the FVIII
sequence. In one embodiment, invention provides CFXTEN configured
with one or more XTEN sequences located internal to the FVIII
sequence to confer properties such as, but not limited to,
increased stability, increased resistance to proteases, increased
resistance to clearance mechanisms including but not limiting to
interaction with clearance receptors or FVIII inhibitors, and
increased hydrophilicity, compared to FVIII without the
incorporated XTEN.
[0230] The invention contemplates that different configurations or
sequence variants of FVIII can be utilized as the platform into
which one or more XTEN are inserted. These configurations include,
but are not limited to, native FVIII, FVIII BDD, and single chain
FVIII (scFVIII), and variants of those configurations. In the case
of scFVIII, the invention provides CFXTEN that can be constructed
by replacing one or multiple amino acids of the processing site of
FVIII. In one embodiment, the scFVIII utilized in the CFXTEN is
created by replacing the R1648 in the FVIII sequence RHQREITR (SEQ
ID NO: 1698) with glycine or alanine to prevent proteolytic
processing to the heterodimer form. It is specifically contemplated
that any of the CFXTEN embodiments disclosed herein with a 1648
FVIII residue can have a glycine or alanine substitution for the
arginine at position 1648. In some embodiments, the invention
provides CFXTEN comprising scFVIII wherein parts of the sequence
surrounding the R1648 processing site are replaced with XTEN, as
illustrated in FIGS. 10A and 10B. In one embodiment, at least about
60%, or about 70%, or about 80%, or about 90%, or about 95%, or
about 97% or more of the B-domain is replaced with an XTEN sequence
disclosed herein, including one or more of the R740, R1648, or
R1689 cleavage sites. In another embodiment, the CFXTEN has the
FVIII sequence of the B-domain between the FXIa cleavage sites at
R740 and R1689 (with at least 1-5 adjacent B-domain amino acids
also retained between the cut site and the start of the XTEN to
permit the protease to access the cut site) replaced with XTEN. In
another embodiment, the CFXTEN has the FVIII sequence of the
B-domain between the FXIa cleavage site at N745 and P1640 replaced
with XTEN. In other embodiments, the invention provides CFXTEN
FVIII BDD sequence variants in which portions of the B-domain are
deleted but only one of the FXI R740 or R1689 activation sites (and
1-5 adjacent amino acids of the B-domain) are left within the
construct, wherein the XTEN remains attached at one end to either
the light or heavy chain after cleavage by FXIa, as illustrated in
FIGS. 5B and 5D. In one embodiment of the foregoing, the CFXTEN
comprises a FVIII BDD sequence in which the amino acids between
N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or
between N745 and Q1656 or between N745 and S1657 or between N745
and T1667 or between N745 and Q1686 or between R747 and V1642 or
between T751 and T1667 are deleted and an XTEN sequence is linked
between these amino acids, connecting the heavy and light chains,
and can further comprise additional XTEN inserted either in
external surface loops, between FVIII domains, or at the N- or
C-termini of the FVIII BDD sequence, such as one or more insertion
sites from Table 5, Table 6, Table 7, Table 8, and Table 9 or those
illustrated in FIGS. 8-9. In another embodiment of the foregoing,
the CFXTEN comprises a FVIII BDD sequence in which the amino acids
between K713 to Q1686 or between residues 741 and 1648 are deleted
and an XTEN linked between the two amino acids, and additional XTEN
can be inserted either in surface loops, between FVIII domains, or
at the N- or C-termini of the FVIII BDD sequence, including but not
limited to one or more insertion sites from Table 5, Table 6, Table
7, Table 8, and Table 9 or those illustrated in FIGS. 8-9. In some
embodiments such CFXTEN sequences can have one or more XTEN
exhibiting 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%, or 100% sequence identity
to an XTEN sequence from any one of Tables 4 and 13-17.
[0231] The invention contemplates other CFXTEN with internal XTEN
in various configurations; schematics of exemplary configurations
are illustrated in FIGS. 5 and 10. The regions suitable for XTEN
insertion sites include the known domain boundaries of FVIII, exon
boundaries, known surface (external) loops and solvent accessible
surface area sites identified by X-ray crystallography analysis,
and structure models derived from molecular dynamic simulations of
FVIII, regions with a low degree of order (assessed by programs
described in FIG. 7 legend), regions of low homology/lack of
conservation across different species, and hydrophilic regions. In
another embodiment, XTEN insertion sites were selected based on
FVIII putative clearance receptor binding sites. In another
embodiment, CFXTEN comprises XTEN inserted at locations not within
close proximity to mutations implicated in hemophilia A listed in
the Haemophilia A Mutation, Search, Test and Resource Site
(HAMSTeRS) database were eliminated (Kemball-Cook G, et al. The
factor VIII Structure and Mutation Resource Site: HAMSTeRS version
4. Nucleic Acids Res. (1998) 26(1):216-219). In another embodiment,
potential sites for XTEN insertion include residues within FVIII
epitopes that are capable of being bound by anti-FVIII antibodies
occurring in sensitized hemophiliacs and that do not otherwise
serve as protein interactive sites. Regions and/or sites that are
considered for exclusion as XTEN insertion sites include
residues/regions of factor VIII that are important in various
interactions including other clotting proteins, residues
surrounding each arginine activating/inactivating cleavage site
acted on by the proteases thrombin, factor Xa, activated protein C,
residues surrounding the signal peptide processing site (residue 1)
if the construct contains the signal peptide, regions known to
interact with other proteins such as FIXa, FX/FXa, thrombin,
activated protein C, protein S cofactor to Protein C, von
Willebrand factor, sites known to interact with phospholipid
cofactors in coagulation, residues involved in domain interactions,
residues coordinating Ca.sup.++ or Cu.sup.++ ions, cysteine
residues involved in S-S intramolecular bonds, documented amino
acid insertion and point mutation sites in FVIII produced in
hemophilia A subjects affecting procoagulant activity, and mutation
sites in FVIII made in a research lab that affect procoagulant
activity. Sites considered for either insertion (to prolong
half-life) or for exclusion (needed to remove spent FVIIIa or FXa)
include regions known to interact with heparin sulfate proteoglycan
(HSPG) or low-density lipoprotein receptor-related protein
(LPR).
[0232] By analysis of the foregoing criteria, as described in
Example 34, different insertion sites or ranges of insertions sites
across the FVIII BDD sequence have been identified and/or confirmed
as candidates for insertion of XTEN, non-limiting examples of which
are listed in Table 5, Table 6, Table 7, Table 8, and Table 9 and
are shown schematically in FIGS. 8 and 9. In one embodiment, CFXTEN
comprise XTEN insertions between the individual domains of FVIII,
i.e., between the A1 and A2, or between the A2 and the B, or
between the B and the A3, or between the A3 and the C1, or between
the C1 and the C2 domains. In another embodiment, CFXTEN comprises
XTEN inserted within the B domain or between remnant residues of
the BDD sequence. In another embodiment, CFXTEN comprises XTEN
inserted at known exon boundaries of the encoding FVIII gene as
exons represent evolutionary conserved sequence modules that have a
high probability of functioning in the context of other protein
sequences. In another embodiment, CFXTEN comprise XTEN inserted
within surface loops identified by the x-ray structure of FVIII. In
another embodiment, CFXTEN comprise XTEN inserted within regions of
low order identified as having low or no detected electron density
by X-ray structure analysis. In another embodiment, CFXTEN comprise
XTEN inserted within regions of low order, predicted by structure
prediction algorithms such as, but not limited to FoldIndex, RONN,
and Kyte & Doolitlle algorithms. In another embodiment, CFXTEN
comprise XTEN inserted within sequence areas of high frequency of
hydrophilic amino acids. In another embodiment, CFXTEN comprise
XTEN inserted within epitopes capable of being bound by
naturally-occurring anti-FVIII antibodies in sensitized
hemophiliacs. In another embodiment, CFXTEN comprise XTEN inserted
within sequence areas of low sequence conservation and/or
differences in sequence segment length across FVIII sequences from
different species. In another embodiment, CFXTEN comprise XTEN
linked to the N-terminus and/or C-terminus. In another embodiment,
the invention provides CFXTEN configurations with inserted XTEN
selected from two or more of the criteria from the embodiments
listed above. In another embodiment, the invention provides CFXTEN
configurations with at least one, alternatively at least two,
alternatively at least three, alternatively at least four,
alternatively at least five or more XTEN inserted into a factor
VIII sequence wherein the points of insertion are at or proximal to
the N- or C-terminus side of the at least one, two, three, four, or
five, or six or more amino acids selected from the insertion
residue amino acids of Table 5, Table 6, Table 7, Table 8, and
Table 9 or those illustrated in FIGS. 8-9, or alternatively within
one, or within two, or within three, or within four, or within
five, or within six amino acids of the insertion residue amino
acids from Table 5, Table 6, Table 7, Table 8, and Table 9, or
within the various spans of the insertion residue amino acids
schematically portrayed for an exemplary FVIII BDD sequence in FIG.
9.
[0233] As described above, the one or more internally-located XTEN
or a fragment of XTEN can have a sequence length of 6 to 1000 or
more amino acid residues. In some embodiments, wherein the CFXTEN
have one or two or three or four or five or more XTEN sequences
internal to the FVIII, the XTEN sequences can be identical or can
be different. In one embodiment, each internally-located XTEN has
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
comparable lengths or fragments of XTEN or motifs selected from any
one of Tables 3, 4, and 13-17, when optimally aligned. In another
embodiment, the invention provides a CFXTEN configured with one or
more XTEN inserted internal to a FVIII BDD sequence with 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 a sequence of Table
1, wherein the insertions are located at the insertion points or
range of insertion points indicated in Table 5, Table 6, Table 7,
Table 8, and Table 9, FIG. 8 or within the range of insertions as
illustrated in FIG. 9. It will be understood by those of skill in
the art that an XTEN inserted within the FVIII sequence at an
insertion point of Table 5, Table 6, Table 7, Table 8, and Table 9
is linked by its N- and C-termini to flanking FVIII amino acids (or
via a linking spacer or cleavage sequences, as described above),
while an XTEN linked to the N- or C-terminus of FVIII would only be
linked to a single FVIII amino acid (or to a linking spacer or
cleavage sequence amino acid, as described above). By way of
example only, variations of CFXTEN with three internal XTEN could
have: XTEN (as described herein) incorporated between FVIII BDD
residues 741 and 1640, residues 18 and 19, and residues 1656 and
1657; or XTEN incorporated between FVIII BDD residues 741 and 1640,
residues 1900 and 1901, and at the C-terminus at residue 2332; or
XTEN incorporated between FVIII BDD residues 26 and 27, residues
1656 and 1657, and residues 1900 and 1901; or XTEN incorporated
between FVIII BDD residues 741 and 1640, residues 1900 and 1901,
and at the C-terminus at residue 2332.
[0234] In evaluating the CFXTEN fusion proteins with XTEN inserted
in the locations from Table 5, it was discovered that insertions in
certain regions of the FVIII sequence resulted in CFXTEN with good
expression and retention of procoagulant activity. Accordingly, in
preferred embodiments, the invention provides CFXTEN fusion
proteins configured with one, or two, or three, or four, or five,
or six or more XTEN, each 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 any one of
Tables 4, and 13-17 inserted internal or linked to a FVIII BDD
sequence with 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 a sequence of Table 1, wherein the insertions
are located at an insertion point within one, or two, or three, or
four, or five, or six or more ranges set forth in Table 7. In the
foregoing embodiments, the CFXTEN fusion proteins with the XTEN
insertions retain at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90% or more of the procoagulant activity compared to the
corresponding FVIII not linked to XTEN.
[0235] In evaluating the CFXTEN fusion proteins with XTEN inserted
in one or more locations from Table 5, it was surprisingly
discovered that a high percentage of fusion proteins with the XTEN
insertions retained procoagulant activity, as described in Example
25. Accordingly, the invention provides CFXTEN fusion proteins
configured with one, two, three, four, five, six or more XTEN
wherein the resulting fusion protein exhibits at least about 10%,
or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% or
more of the procoagulant activity compared to the corresponding
FVIII not linked to XTEN when assayed by a coagulation assay
described herein. In a preferred embodiment, the invention provides
CFXTEN fusion proteins comprising one, or two, or three, or four,
or five, or six or more XTEN, each 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 any one of
Tables 4, and 13-17 linked to a FVIII BDD sequence with 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 a sequence of Table
1, wherein the insertions are located at one or more insertion
points selected from Table 5, Table 6, Table 7, Table 8, and Table
9, and wherein the resulting fusion protein exhibits 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 more procoagulant activity
compared to the corresponding FVIII not linked to XTEN, when
assayed in vitro by an assay described herein (e.g., a chromogenic
assay). As the subject CFXTEN fusion proteins typically exhibit
increased terminal half-life compared to native FVIII, it will be
appreciated by one of skill in the art that a CFXTEN with lower
procoagulant activity relative to an equimolar amount of native
FVIII would nevertheless be acceptable when administered as a
therapeutic composition to a subject in need thereof. In another
embodiment, the CFXTEN fusion proteins comprising one, or two, or
three, or four, or five or more XTEN, each 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
any one of Tables 4, and 13-17 linked to a FVIII BDD sequence with
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 a
sequence of Table 1, wherein the insertions are located at one or
more insertion points or the range of insertion points selected
from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the
resulting fusion protein exhibits at least about 0.5 IU/ml, or at
least about 0.75 IU/ml, or at least about 1.0 IU/ml, or at least
about 1.5 IU/ml, or at least about 2.0 IU/ml, or at least about 2.5
IU/ml, or at least about 3 IU/ml, or at least about 4 IU/ml, or at
least about 5 IU/ml, or at least about 7 IU/ml, or at least about
10 IU/ml, or at least about 20 IU/ml, or at least about 30 IU/ml
FVIII activity when expressed in cell culture medium and assayed in
a chromogenic assay, wherein the culture and expression are
according to methods described herein; e.g., the methods of Example
25.
[0236] It is believed that the discovery of the insertions sites
wherein the FVIII retains at least a portion of its procoagulant
activity would also permit the insertion of other peptides and
polypeptides with either unstructured or structured characteristics
that are associated with the prolongation of half-life when fused
to a FVIII protein in one or more of those same sites. Non-limiting
examples include albumin, albumin fragments, Fc fragments of
immunoglobulins, the .beta. subunit of the C-terminal peptide (CTP)
of human chorionic gonadotropin, a HAP sequence, a transferrin, the
PAS polypeptides of U.S. Pat Application No. 20100292130,
polyglycine linkers, polyserine linkers, peptides and short
polypeptides of 6-40 amino acids of two types of amino acids
selected from glycine (G), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P) with varying degrees of secondary
structure from less than 50% to greater than 50%, amongst others,
would be suitable for insertion in the identified active insertions
sites of FVIII.
[0237] In the fusion protein embodiments described herein, the
CFXTEN fusion protein can further comprise one or more cleavage
sequence from Table 12 or other sequences known in the art, the
cleavage sequence being located between or within 6 amino acid
residues of the intersection of the FVIII and the XTEN sequences,
which may include two cleavage sequences in a given internal XTEN
sequence. In one embodiment, the CFXTEN comprising cleavage
sequences has two identical cleavage sequences, each located at or
near the respective ends of one or more internal XTEN such that the
XTEN is released from the fusion protein when cleaved by the
protease that binds to and cleaves that sequence. The sequences
that can be cleaved are described more fully below and exemplary
sequences are provided in Table 12.
TABLE-US-00006 TABLE 5 Insertion locations for XTEN linked to the
FVIII BDD sequence FVIII BDD XTEN Insertion Insertion Downstream
FVIII No. Point Residue Sequence Domain 1 0 (N-terminus) ATR A1 2 3
R RYY A1 3 17 M QSD A1 4 18 Q SDL A1 5 22 G ELP A1 6 24 L PVD A1 7
26 V DAR A1 8 28 A RFP A1 9 32 P RVP A1 10 38 F PFN A1 11 40 F NTS
A1 12 41 N TSV A1 13 60 N IAK A1 14 61 I AKP A1 15 65 R PPW A1 16
81 Y DTV A1 17 111 G AEY A1 18 116 D QTS A1 19 119 S QRE A1 20 120
Q REK A1 21 128 V FPG A1 22 129 F PGG A1 23 130 P GGS A1 24 182 G
SLA A1 25 185 A KEK A1 26 188 K TQT A1 27 205 G KSW A1 28 210 S ETK
A1 29 211 E TKN A1 30 216 L MQD A1 31 220 R DAA A1 32 222 A ASA A1
33 223 A SAR A1 34 224 S ARA A1 35 230 K MHT A1 36 243 P GLI A1 37
244 G LIG A1 38 250 R KSV A1 39 318 D GME A1 40 333 P QLR A1 42 334
Q LRM A1 43 336 R MKN a1 44 339 N NEE a1 45 345 D YDD a1 46 357 V
VRF a1 47 367 S FIQ a1 48 370 S RPY a1 49 375 A KKH A2 50 376 K KHP
A2 51 378 H PKT A2 52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2
55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59
441 A IQH A2 60 442 I QHE A2 61 463 I IFK A2 62 487 Y SRR A2 63 490
R LPK A2 64 492 P KGV A2 65 493 K GVK A2 66 494 G VKH A2 67 500 D
FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 K ESV A2 71 565 Q IMS
A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A GVQ A2 75 603 L EDP A2
76 616 S ING A2 77 686 G LWI A2 78 713 K NTG A2 79 719 Y EDS A2 80
730 L LSK A2 81 733 K NNA A2 82 745 N PPV B 83 1640 P PVL B 84 1652
R TTL B 85 1656 Q SDQ A3 86 1685 N QSP A3 87 1711 M SSS A3 88 1713
S SPH A3 89 1720 N RAQ A3 90 1724 S GSV A3 91 1725 G SVP A3 92 1726
S VPQ A3 93 1741 G SFT A3 94 1744 T QPL A3 95 1749 R GEL A3 96 1773
V TFR A3 97 1792 Y EED A3 98 1793 E EDQ A3 99 1796 Q RQG A3 100
1798 Q GAE A3 101 1799 G AEP A3 102 1802 P RKN A3 103 1803 R KNF A3
104 1807 V KPN A3 105 1808 K PNE A3 106 1827 K DEF A3 107 1844 E
KDV A3 108 1861 N TLN A3 109 1863 L NPA A3 110 1896 E RNC A3 111
1900 R APC A3 112 1904 N IQM A3 113 1905 I QME A3 114 1910 P TFK A3
115 1920 A ING A3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N
KCQ A3 119 2020 K CQT C1 120 2044 G QWA C1 121 2068 F SWI C1 122
2073 V DLL C1 123 2090 R QKF C1 124 2092 K FSS C1 125 2093 F SSL C1
126 2111 K WQT C1 127 2115 Y RGN C1 128 2120 T GTL C1 129 2125 V
FFG C1 130 2171 L NSC C1 131 2173 S CSM C2 132 2188 A QIT C2 133
2223 V NNP C2 134 2224 N NPK C2 135 2227 K EWL C2 136 2268 G HQW C2
137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y C
terminus of FVIII CT
Indicates an insertion point for XTEN based on the amino acid
number of mature full-length human FVIII, wherein the insertion
could be either on the N- or C-terminal side of the indicated amino
acid Downstream sequence in FVIII BDD with 746-1639 deletion
TABLE-US-00007 TABLE 6 Exemplary insertion locations for XTEN
linked to a FVIII polypeptide XTEN FVIII BDD Distance from
Insertion Insertion Downstream FVIII insertion No. Point Residue
Sequence Domain residue 9 32 P RVP A1 -3, +6 31 220 R DAA A1 -- 34
224 S ARA A1 +5 43 336 R MKN a1 -1, +6 44 339 N NEE a1 -4, +5 52
399 V LAP A2 -6, +3 56 416 P QRI A2 +6 75 603 L EDP A2 _6, +6 85
1656 Q SDQ B -3, +6 87 1711 M SSS A3 -6, +1 91 1725 G SVP A3 +6 113
1905 I QME A3 +6 114 1910 P TFK A3 -5, +6
Distance from insertion residue refers to the relative number of
amino acids away from the N-terminus (negative numbers) or
C-terminus (positive numbers) of the designated insertion residue
(residue "0") where an insertion may be made. The designation "-x"
refers to an insertion site which is x amino acids away on the
N-terminal side of the designated insertion residue. Similarly, the
designation "+x" refers to an insertion site which is x amino acids
away on the C-terminal side of the designated insertion residue.
For example, "-1, +2" indicates that the insertion is made at the
N-terminus or C-terminus of amino acid residues denoted -1, 0, +1
or +2.
TABLE-US-00008 TABLE 7 Further exemplary insertion locations for
XTEN linked to a FVIII polypeptide XTEN Insertion First Insertion
No. Point Range Residue FVIII Domain 3 18-32 Q A1 8 40 F A1 18
211-224 E A1 27 336-403 R A1, A2 43 599 A A2 47 745-1640 N B 50
1656-1728 Q B, A3 57 1796-1804 R A3 65 1900-1912 R A3 81 2171-2332
L C1, C2
indicates range of insertion sites numbered relative to the amino
acid number of mature human FVIII
TABLE-US-00009 TABLE 8 Exemplary XTEN insertion locations within
B-domain deleted variants of a FVIII polypeptide XTEN Insertion
First Insertion Second Insertion Point Range Residue Residue
740-1640 R P 740-1690 R S 741-1648 S R 743-1638 S Q 745-1638 N Q
745-1640 N P 745-1656 N Q 745-1657 N S 745-1667 N T 745-1686 N Q
747-1642 R V 751-1667 T T
indicates the amino acids linked within the B-domain deleted
variant and adjacent A3 domain, with the amino acids numbered
relative to the amino acid number of mature human FVIII indicates
the amino acids linked by an XTEN inserted in the BDD-FVIII
TABLE-US-00010 TABLE 9 Exemplary insertion locations for XTEN
linked to a FVIII polypeptide resulting in procoagulant activity
XTEN FVIII BDD Insertion Downstream FVIII No. Point Insertion
Residue Sequence Domain 2 3 R RYY A1 4 18 Q SDL A1 5 22 G ELP A1 7
26 V DAR A1 9 32 P RVP A1 11 40 F NTS A1 18 116 D QTS A1 19 119 S
QRE A1 26 188 K TQT A1 29 211 E TKN A1 30 216 L MQD A1 31 220 R DAA
A1 34 224 S ARA A1 35 230 K MHT A1 40 333 P QLR A1 43 336 R MKN a1
44 339 N NEE a1 52 399 V LAP A2 53 403 D DRS A2 55 409 S QYL A2 56
416 P QRI A2 60 442 I QHE A2 62 487 Y SRR A2 63 490 R LPK A2 66 494
G VKH A2 69 518 E DGP A2 74 599 A GVQ A2 75 603 L EDP A2 78 713 K
NTG A2 82 745 N PPV B 85 1656 Q SDQ A3 87 1711 M SSS A3 89 1720 N
RAQ A3 91 1725 G SVP A3 99 1796 Q RQG A3 102 1802 P RKN A3 110 1896
E RNC A3 111 1900 R APC A3 112 1904 N IQM A3 113 1905 I QME A3 114
1910 P TFK A3 121 2068 F SWI C1 130 2171 L NSC C1 135 2227 K EWL C2
137 2277 N GKV C2 140 2332 Y C terminus of FVIII C2 Downstream
sequence in FVIII BDD with 746-1639 deletion
[0238] In another aspect, the invention provides libraries of
components and methods to create the libraries derived from
nucleotides encoding FVIII segments, XTEN, and FVIII segments
linked to XTEN that are useful in the preparation of genes encoding
the subject CFXTEN. In a first step, a library of genes encoding
FVIII and XTEN inserted into the various single sites at or within
1-6 amino acids of an insertion site identified in Table 5 or
illustrated in FIGS. 8-9 are created, expressed, and the CFXTEN
recovered and evaluated for activity and pharmacokinetics as
illustrated in FIG. 15. Those CFXTEN showing enhanced properties
are then used to create genes encoding a FVIII segment and the
insertion site plus an XTEN, with components from each enhanced
insertion represented in the library, as illustrated in FIG. 11. In
one embodiment, the library components are assembled using standard
recombinant techniques in combinatorial fashion, as illustrated in
FIG. 11, resulting in permutations of CFXTEN with multiple internal
and N- and C-terminus XTEN, that can include the insertion sites of
or proximal to those Table 5, Table 6, Table 7, Table 8 and Table
9, or as illustrated in FIGS. 8-9. The resulting constructs would
then be evaluated for activity and enhanced pharmacokinetics, and
those candidates resulting in CFXTEN with enhanced properties,
e.g., reduced active clearance, resistance to proteases, reduced
immunogenicity, and enhance pharmacokinetics, compared to FVIII not
linked to XTEN, are evaluated further.
[0239] 3. XTEN Permissive Loops
[0240] As described in detail elsewhere herein and as illustrated
in FIGS. 33-36, the inventors have recognized that each FVIII "A"
domain comprise at least two "XTEN permissive loops" into which
XTEN sequences can be inserted without eliminating procoagulant
activity of the recombinant protein, or the ability of the
recombinant proteins to be expressed in vivo or in vitro in a host
cell. The inventors have identified the XTEN permissive loops as
regions with, among other attributes, high surface or solvent
exposure and high conformational flexibility. The A1 domain
comprises an XTEN permissive loop-1 (A1-1) region and an XTEN
permissive loop-2 (A1-2) region, the A2 domain comprises an XTEN
permissive loop-1 (A2-1) region and an XTEN permissive loop-2
(A2-2) region, the A3 domain comprises an XTEN permissive loop-1
(A3-1) region and an XTEN permissive loop-2 (A3-2) region.
[0241] In certain aspects a recombinant FVIII protein as described
above comprises at least one XTEN sequence inserted into at least
one of the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, or
A3-2, wherein the recombinant FVIII protein has procoagulant
activity and can be expressed in vivo or in vitro in a host cell.
In certain aspects a recombinant FVIII protein as described above
comprises at least two XTEN sequences inserted into FVIII, e.g.,
into two different XTEN permissive loops A1-1, A1-2, A2-1, A2-2,
A3-1, or A3-2, wherein the recombinant FVIII protein has
procoagulant activity and can be expressed in vivo or in vitro in a
host cell. Alternatively, a recombinant FVIII protein as described
above can comprise two or more XTEN sequences inserted into a
single XTEN permissive loop either with our without XTEN sequences
inserted into other XTEN permissive loops, wherein the recombinant
FVIII protein has procoagulant activity and can be expressed in
vivo or in vitro in a host cell. In certain aspects a recombinant
FVIII protein as described above can comprise at least one XTEN
sequence inserted into at least one of the XTEN permissive loops as
described above, and can further comprise one or more XTEN
sequences inserted into a3, wherein the recombinant FVIII protein
has procoagulant activity and can be expressed in vivo or in vitro
in a host cell. In certain aspects, a recombinant FVIII protein of
the invention can comprise three, four, five, six or more XTEN
sequences inserted into one or more XTEN permissive loops or into
a3, wherein the recombinant FVIII protein has procoagulant activity
and can be expressed in vivo or in vitro in a host cell.
[0242] In certain aspects a recombinant FVIII protein as described
above comprises at least one XTEN sequence inserted into a3,
wherein the recombinant FVIII protein has procoagulant activity and
can be expressed in vivo or in vitro in a host cell. In certain
aspects a recombinant FVIII protein of the invention comprises at
least one XTEN sequence inserted into a3, and further comprises one
or more XTEN sequences inserted into one or more XTEN permissive
loops as described above, wherein the recombinant FVIII protein has
procoagulant activity and can be expressed in vivo or in vitro in a
host cell.
[0243] The inventors have recognized that a recombinant FVIII
protein of the invention comprises at least two XTEN permissive
loops in each of the FVIII A domain regions which allows for
insertion of an XTEN sequence while having procoagulant activity
and still being able to be expressed in vivo or in vitro by a host
cell. Various crystal structures of FVIII have been determined, of
varying degrees of resolution. These structures of FVIII and
FVIIIa, determined by X-ray crystallography and molecular dynamic
simulation, were used to generate models of accessible surface area
and conformational flexibility for FVIII. For example, the crystal
structure of human FVIII has been determined by Shen et al. Blood
111: 1240-1247 (2008) and Ngo et al. Structure 16: 597-606 (2008).
The data for these structures is available from the Protein Data
Bank (pdb.org) under Accession Numbers 2R7E and 3CDZ,
respectively.
[0244] The predicted secondary structure of the heavy and light
chains of human FVIII according to the Shen et al. crystal
structure is reproduced in FIGS. 37A and 37B. The various beta
strands predicted from the Shen et al. crystal structure are
numbered consecutively in FIGS. 8A and 8B. In certain embodiments,
the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, and A3-2
are contained within surface-exposed, flexible loop structures in
the A domains of FVIII. A1-1 is located between beta strand 1 and
beta strand 2, A1-2 is located between beta strand 11 and beta
strand 12, A2-1 is located between beta strand 22 and beta strand
23, A2-2 is located between beta strand 32 and beta strand 33, A3-1
is located between beta strand 38 and beta strand 39 and A3-2 is
located between beta strand 45 and beta strand 46, according to the
secondary structure of mature FVIII stored as Accession Number 2R7E
of the PDB database (PDB:2R7E) and as shown in FIGS. 8A and 8B. The
secondary structure of PDB Accession Number 2R7E shown in FIGS. 8A
and 8B corresponds to the standardized secondary structure
assignment according to the DSSP program (Kabsch and Sander,
Biopolymers, 22:2577-2637 (1983)). The DSSP secondary structure of
the mature FVIII stored as PDB Accession Number 2R7E can be
accessed at the DSSP database, available at the world wide web site
swift.cmbi.ru.nl/gv/dssp/ (last accessed Feb. 9, 2012) (Joosten et
al., 39 (Suppl. 1): D411-D419 (2010)).
[0245] In certain aspects, a surface-exposed, flexible loop
structure comprising A1-1 corresponds to a region in native mature
human FVIII from about amino acid 15 to about amino acid 45 of FIG.
30. In certain aspects, A1-1 corresponds to a region in native
mature human FVIII from about amino acid 18 to about amino acid 41
of FIG. 30. In certain aspects, the surface-exposed, flexible loop
structure comprising A1-2 corresponds to a region in native mature
human FVIII from about amino acid 201 to about amino acid 232 of
FIG. 30. In certain aspects, A1-2 corresponds to a region in native
mature human FVIII from about amino acid 218 to about amino acid
229 of FIG. 30. In certain aspects, the surface-exposed, flexible
loop structure comprising A2-1 corresponds to a region in native
mature human FVIII from about amino acid 395 to about amino acid
421 of FIG. 30. In certain aspects, A2-1 corresponds to a region in
native mature human FVIII from about amino acid 397 to about amino
acid 418 of FIG. 30. In certain aspects, the surface-exposed,
flexible loop structure comprising A2-2 corresponds to a region in
native mature human FVIII from about amino acid 577 to about amino
acid 635 of FIG. 30. In certain aspects, A2-2 corresponds to a
region in native mature human FVIII from about amino acid 595 to
about amino acid 607 of FIG. 30. In certain aspects, the
surface-exposed, flexible loop structure comprising A3-1
corresponds to a region in native mature human FVIII from about
amino acid 1705 to about amino acid 1732 of FIG. 30. In certain
aspects, A3-1 corresponds to a region in native mature human FVIII
from about amino acid 1711 to about amino acid 1725 of FIG. 30. In
certain aspects, the surface-exposed, flexible loop structure
comprising A3-2 corresponds to a region in native mature human
FVIII from about amino acid 1884 to about amino acid 1917 of FIG.
3. In certain aspects, A3-2 corresponds to a region in native
mature human FVIII from about amino acid 1899 to about amino acid
1911 of FIG. 30.
[0246] In certain aspects a recombinant FVIII protein of the
invention comprises one or more XTEN sequences inserted into one or
more XTEN permissive loops of FVIII, or into the a3 region, wherein
the recombinant FVIII protein has procoagulant activity and can be
expressed in vivo or in vitro in a host cell. XTEN sequences to be
inserted include those that increase the in vivo half-life or the
in vivo or in vitro stability of FVIII.
[0247] In certain aspects, a recombinant FVIII protein of the
invention comprises an XTEN sequences inserted immediately
downstream of one or more amino acids corresponding to one or more
amino acids in mature native human FVIII including, but not limited
to: amino acid 18 of FIG. 30, amino acid 26 of FIG. 30, amino acid
40 of FIG. 30, amino acid 220 of FIG. 30, amino acid 224 of FIG.
30, amino acid 399 of FIG. 30, amino acid 403 of FIG. 30, amino
acid 599 of FIG. 30, amino acid 603 of FIG. 30, amino acid 1711 of
FIG. 30, amino acid 1720 of FIG. 30, amino acid 1725 of FIG. 30,
amino acid 1900 of FIG. 30, amino acid 1905 of FIG. 30, amino acid
1910 of FIG. 30, or any combination thereof, including
corresponding insertions in BDD-variants of FVIII described
herein.
[0248] In certain aspects, a recombinant FVIII protein of the
invention comprises at least one XTEN sequence inserted into the a3
region of FVIII, either alone or in combination with one or more
XTEN sequences being inserted into the XTEN permissive loops of the
A domains (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2 as described
above), wherein the recombinant FVIII protein has procoagulant
activity and can be expressed in vivo or in vitro in a host cell.
In certain aspects, at least one XTEN sequence is inserted into the
a3 region immediately downstream of an amino acid which corresponds
to amino acid 1656 of FIG. 30. In certain aspects, a recombinant
FVIII protein of the invention comprises an XTEN sequence inserted
into the a3 region as described, and further includes one or more
XTEN sequences inserted immediately downstream of one or more amino
acids corresponding to one or more amino acids in mature native
human FVIII including, but not limited to: amino acid 18 of FIG.
30, amino acid 26 of FIG. 30, amino acid 40 of FIG. 30, amino acid
220 of FIG. 30, amino acid 224 of FIG. 30, amino acid 399 of FIG.
30, amino acid 403 of FIG. 30, amino acid 599 of FIG. 30, amino
acid 603 of FIG. 30, amino acid 1711 of FIG. 30, amino acid 1720 of
FIG. 30, amino acid 1725 of FIG. 30, amino acid 1900 of FIG. 30,
amino acid 1905 of FIG. 30, amino acid 1910 of FIG. 30, or any
combination thereof.
[0249] It will be understood by one of skill in the art that the
foregoing aspects of permissive loops of a native FVIII protein
into which a heterologous protein can be inserted are also
applicable to the B-domain deleted FVIII variants described herein;
e.g., sequences set forth in Table 1. In practicing the present
invention, it will be understood that a BDD-FVIII sequence of Table
1 can be substituted for the recombinant FVIII protein of the
various embodiments described above, and it is believed that the
resulting constructs will similarly retain procoagulant
activity.
[0250] 4. Interference with FVIII Binding Agents
[0251] It is an object of the present invention to provide
procoagulant CFXTEN fusion protein compositions for use in human
patients suffering from coagulopathies, such as haemophilia A, who
have native or acquired antibodies, inhibitors, or other proteins
or molecules that bind to FVIII that affect the activity or
half-life of CFXTEN fusion proteins, wherein the CFXTEN retain a
greater amount of procoagulant activity compared to the
corresponding FVIII not linked to XTEN. As used herein, "FVIII
binding agent" means any molecule capable of binding to native
FVIII or to a recombinant factor VIII fusion protein of the
invention comprising factor VIII or a fragment thereof, whether
native, derived, or produced recombinantly. It is specifically
contemplated that FVIII binding agent includes anti-FVIII
antibodies and FVIII inhibitors, amongst other proteins capable of
specifically binding to FVIII. In one aspect, the invention
provides procoagulant CFXTEN fusion proteins that exhibit reduced
binding to an anti-FVIII antibody or FVIII inhibitor that
interferes with the procoagulant activity of FVIII. As used herein,
"anti-FVIII antibody" or "anti-factor VIII antibody" means an
antibody capable of binding FVIII or a FVIII component of a CFXTEN
of the invention, said antibody including but not limited to the
antibodies of Table 10 or polyclonal antibody from a hemophilia A
patient with FVIII inhibitors. The term antibody includes
monoclonal antibodies, polyclonal antibodies, antibody fragments
and antibody fragment clones. As used herein, "FVIII inhibitor" or
"anti-FVIII inhibitor antibody" means an antibody capable of
binding FVIII or a FVIII component of a CFXTEN of the invention and
that reduces by any means the procoagulant activity of FVIII or the
FVIII component of a CFXTEN. In another aspect, the invention
provides CFXTEN fusion proteins that retain procoagulant activity
in the presence of a FVIII inhibitor. In another aspect, the
invention provides CFXTEN fusion proteins comprising FVIII that
exhibit increased terminal half-life in the presence of a FVIII
binding agent compared to the FVIII not linked to XTEN.
[0252] The majority of inhibitory antibodies to human factor VIII
act by binding to epitopes located in the A2 domain or the C2
domain of factor VIII, disrupting specific functions associated
with these domains, (U.S. Pat. No. 6,770,744; Fulcher et al.
Localization of human factor FVIII inhibitor epitopes to two
polypeptide fragments. Proc. Natl. Acad. Sci. USA (1985)
82:7728-7732; Scandella et al. Epitope mapping of human factor VIII
inhibitor antibodies by deletion analysis of fVIII fragments
expressed in Escherichia coli. Proc. Natl. Acad. Sci. USA (1988)
85:6152-6156). While 68% percent of inhibitory antibodies are
reported to be directed against the A2 and/or C2 domain, 3% act
against the A1 domain and 46% against the a3 acidic region
(Lavigne-Lissalde, G., et al. Characteristics, mechanisms of
action, and epitope mapping of anti-factor VIII antibodies. Clin
Rev Allergy Immunol (2009) 37:67-79). For example, certain heavy
chain-specific inhibitors react with the 18.3-kD amino-terminal
segment of the A2 domain (Scandella D, et al. 1988); Lollar P et
al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies.
J Clin Invest 1994; 93:2497). FVIII contains a phospholipid binding
site in the C2 domain between amino acids 2302 and 2332, and there
is also a von Willebrand factor binding site in the C2 domain that
acts in conjunction with amino acids 1649-1689 in the A3 domain.
The C2 domain also has epitopes that, when bound by inhibitors,
block the activation of FVIII by thrombin or factor Xa. Inhibitors
binding specifically to the light chain recognize epitopes in the
A3 domain or a major antigenic region in the C2 domain and can
result in reduced procoagulant activity by preventing the binding
of FVIII to phospholipid or reducing the dissociation rate of FVIII
from von Willebrand factor (Gilles J G, et al. Anti-factor VIII
antibodies of hemophiliac patients are frequently directed towards
nonfunctional determinants and do not exhibit isotypic restriction.
Blood (1993) 82:2452; Shima M, et al. A factor VIII neutralizing
monoclonal antibody and a human inhibitor alloantibody recognizing
epitopes in the C2 domain inhibit factor VIII binding to von
Willebrand factor and to phosphatidylserine. Thromb Haemost (1993)
69:240). Non-limiting examples of monoclonal FVIII inhibitors are
listed in Table 9. In patients with high-titer inhibitors, there is
an increased risk of developing recurrent bleeding in particular
joints, which may ultimately result in decreased quality of life,
disability, or death from excessive blood loss (U.S. Pat.
Application No. 20120065077; Zhang et al., Clinic. Rev. Allerg.
Immunol., 37:114-124 (2009); Gouw and van den Berg, Semin. Thromb.
Hemost., 35:723-734 (2009))
[0253] While not intending to be bound by any particular theory, it
is believed that the unstructured characteristic of the XTEN
incorporated into the CFXTEN fusion proteins permits the XTEN to
adopt conformations that result in steric hindrance to inhibitors
that would otherwise bind to FVIII epitopes. As illustrated in FIG.
6, as the incorporated XTEN assumes various random coil
conformations, it spatially covers regions of the FVIII component
of the fusion protein and sterically interferes with the ability of
an inhibitor to bind to a FVIII epitope.
[0254] In one embodiment, the invention provides CFXTEN exhibiting
procoagulant activity and reduced binding in the presence of an
antibody binding to the C2 domain of factor VIII compared to the
corresponding factor VIII not linked to XTEN and/or to native
FVIII. In another embodiment, the invention provides CFXTEN
exhibiting procoagulant activity and reduced binding in the
presence of an antibody binding to the A2 domain of Factor VIII
compared to the corresponding factor VIII not linked to XTEN or to
native FVIII. In another embodiment, the invention provides CFXTEN
exhibiting procoagulant activity and reduced binding in the
presence of antibodies binding to the A2 and the C2 domain of
Factor VIII, compared to the corresponding factor VIII not linked
to XTEN or to native FVIII. In one embodiment, the invention
provides CFXTEN exhibiting procoagulant activity and reduced
binding, compared to the corresponding FVIII not linked to XTEN, in
the presence of an antibody selected from the group consisting of
the antibodies of Table 10. In one embodiment, the CFXTEN fusion
protein exhibits reduced binding to the antibody GMA8021. In
another embodiment, the CFXTEN fusion protein exhibits reduced
binding to the antibody GMA8008. In another embodiment, the CFXTEN
fusion protein exhibits reduced binding to the antibody ESH4. In
another embodiment, the CFXTEN fusion protein exhibits reduced
binding to the antibody ESH8. In another embodiment, the CFXTEN
fusion protein exhibits reduced binding to the antibody B02C11. In
another embodiment, the CFXTEN fusion protein exhibits reduced
binding and a greater degree of procoagulant activity, compared to
the corresponding FVIII not linked to XTEN, in the presence of
plasma from a hemophilia A subject with polyclonal antibody FVIII
inhibitors, wherein the greater degree of procoagulant activity is
determined by an in vitro assay such as a Bethesda assay or other
assay described herein.
[0255] The CFXTEN exhibiting reduced binding by FVIII inhibitors
can have one, or two, or three, or four, or five, or six or more
individual XTEN, embodiments of which are disclosed herein. In the
foregoing embodiments of this paragraph, a CFXTEN exhibits at least
5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%
or less binding to the antibody when assessed in vitro in an assay
capable of assaying the binding of an antibody to FVIII, such as
assays described herein below or those known in the art.
Alternatively, the reduced binding of the subject CFXTEN to the
FVIII-binding antibodies can be assessed by retention of a higher
degree of procoagulant activity in the presence of the antibody
compared to FVIII not linked to XTEN, as described in the Examples.
Thus, in the embodiments pertaining to reduced binding by FVIII
inhibitors described herein, a CFXTEN exhibits, when reacted with
the anti-FVIII antibody, at least 5%, or 10%, or 15%, or 20%, or
30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 100%, or 200%, or
300%, or 400%, or 500% or more activity in a coagulation assay
(such as described herein below) compared to the corresponding
FVIII not linked to XTEN and reacted with the antibody. In the
foregoing, the anti-FVIII antibody can be an antibody from Table 9
or a circulating anti-FVIII antibody from a hemophilia A subject.
In another embodiment, the invention provides CFXTEN in which the
assayed fusion protein, when assayed utilizing the Bethesda assay
and an anti-FVIII antibody selected from Table 10 or a polyclonal
anti-FVIII antibody preparation such as, but not limited to, plasma
from a hemophilia A subject with FVIII inhibitors, results in a
Bethesda titer with at least about 2, 4, 6, 8, 10, 12, 15, 20, 30,
40, 50, 60, 70, 80, 100, or 200 fewer Bethesda units compared to a
FVIII not linked to XTEN and assayed under comparable conditions.
In another embodiment, the invention provides CFXTEN in which the
assayed fusion protein results in less than 50%, or less than 40%,
or less than 30%, or less than 25%, or less than 20%, or less than
15%, or less than 14%, or less than 13%, or less than 12%, or less
than 11%, or less than 10% of the Bethesda Units compared to a
FVIII not linked to XTEN when assayed under comparable conditions
utilizing the Bethesda assay and a polyclonal anti-FVIII antibody
preparation such as, but not limited to, plasma from a hemophilia A
subject with FVIII inhibitors.
TABLE-US-00011 TABLE 10 Anti-factor VIII antibodies Antibody
Inhibitor Titer Designation Epitope BU/mg Reference BO2C11 C2
Domain 20000 U.S. Pat. No. 6,770,744 Met2199/Phe2200 Blood (2007)
110: 4234-4242 NMC VIII-5 C2 Domain U.S. Pat. No. 6,770,744
Glu2181-Val2243 ESH2 Light Chain ADI ESH4 Light Chain 39 U.S. Pat.
No. 6,770,744 2303-2332 Blood (2007) 110: 4234-4242 ESH8 C2 Domain
10000 U.S. Pat. No. 6,770,744 2248-2285 Blood (2007) 110: 4234-4242
RHD5 C1 Domain WO 2005/016455 (LMBP US Pat. Application 20090263380
6165CB) LE2E9 C1 Domain US Pat. Application 20090263380 Blood
(2000) 95: 156-163 I54 C2 Domain 1300 Blood (2007) 110: 4234-4242
F85 C2 Domain 6 Blood (2007) 110: 4234-4242 F100 C2 Domain 5 Blood
(2007) 110: 4234-4242 F137 C2 Domain 6 Blood (2007) 110: 4234-4242
I89 C2 Domain 1900 Blood (2007) 110: 4234-4242 I117 C2 Domain 1800
Blood (2007) 110: 4234-4242 I109 C2 Domain 1500 Blood (2007) 110:
4234-4242 Met2199/Phe2200 1B5 C2 Domain 930 Blood (2007) 110:
4234-4242 3C6 C2 Domain 71 Blood (2007) 110: 4234-4242 3D12 C2
Domain 2600 Blood (2007) 110: 4234-4242 Phe2196 D102 C2 Domain 3800
Blood (2007) 110: 4234-4242 3G6 C2 Domain 25000 Blood (2007) 110:
4234-4242 2-77 C2 Domain 25000 Blood (2007) 110: 4234-4242 B45 C2
Domain 21000 Blood (2007) 110: 4234-4242 B9 C2 Domain 31000 Blood
(2007) 110: 4234-4242 B11 C2 Domain 3300 Blood (2007) 110:
4234-4242 B75 C2 Domain Indeterminate Blood (2007) 110: 4234-4242
D105 C2 Domain 0.8 Blood (2007) 110: 4234-4242 Val2223/Lys2227 F77
C2 Domain 26000 Blood (2007) 110: 4234-4242 F178 C2 Domain 18000
Blood (2007) 110: 4234-4242 F67 C2 Domain 21000 Blood (2007) 110:
4234-4242 G99 C2 Domain 15000 Blood (2007) 110: 4234-4242
Val2223/Lys2227 G86 C2 Domain 4300 Blood (2007) 110: 4234-4242 I14
C2 Domain 44000 Blood (2007) 110: 4234-4242 I55 C2 Domain 10000
Blood (2007) 110: 4234-4242 2-117 C2 Domain >0.4 Blood (2007)
110: 4234-4242 GMA012 A2 domain GMA 497-510; 584-593 GMA8001 A3
Domain 156 GMA GMA8002 A1 Domain <1 GMA GMA8003 C2 Domain GMA
GMA8004 A1 Domain GMA GMA8005 A1A3/A1 Domain GMA GMA8006 C2 Domain
GMA GMA8008 C2 Domain 1047 GMA GMA8009 A2 Domain 7923 GMA GMA8010
LC Domain GMA GMA8011 C1 Domain 97 GMA GMA8012 A1A3 Domain 204 GMA
GMA8013 A3C2 Domain 30 GMA GMA8014 C2 Domain 7799 GMA GMA8015 A2
Domain 17079 GMA GMA8016 A2 Domain <1 GMA GMA8017 A2 Domain 334
GMA GMA8018 LC Domain 242 GMA GMA8019 CR-LC Domain GMA GMA8020 A1A3
Domain 196 GMA GMA8021 A2 Domain 33928 GMA 4A4 A2 Domain 40000 J
Thromb Haemost (2009) 7: 658-664 3E6 C2 Domain 41 Blood (2007) 110:
4234-4242
American Diagnostica Inc. internet site, URL located on the World
Wide Web at
americandiagnostica.com/html/Product_Detail.asp?idCategory=5&idSub-
Category=104&idpro=ESH-8 as it existed on Jan. 12, 2012 Green
Mountain Antibodies internet site, URL located on the World Wide
Web at greenmoab.com/product_details/16316/21582.html as it existed
on Jan. 12, 2012
[0256] Assays for Inhibitor and Antibody Binding
[0257] The fusion proteins of the invention may be assayed to
confirm reduced binding by FVIII inhibitors using methods known in
the art. The assays that can be used include, but are not limited
to, competitive and non-competitive assay systems using techniques
such as Western blots, radioimmunoassays, ELISA, "sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel
diffusion precipitin reactions, immunodiffusion assays,
agglutination assays, immunoradiometric assays, fluorescent
immunoassays, clotting assays, factor VIII inhibitor assays to name
but a few. Such assays are routine and well known in the art (see,
e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is
incorporated by reference herein in its entirety). Exemplary are
described briefly below but are not intended by way of
limitation.
[0258] The Bethesda assay and the Nijmegen modification of the
Bethesda assay are factor VIII inhibitor assays well-known as
methods to detect FVIII inhibitors (Kasper C K, et al. Proceedings:
A more uniform measurement of factor VIII inhibitors. Thromb Diath
Haemorrh. (1975) 34(2):612). However, the assays can be modified to
assay binding of inhibitors to FVIII compositions using inhibitors
such as polyclonal or monoclonal anti-FVIII antibodies, including
the antibodies of Table 10, and methods such as described in
Example 52. Briefly, the modified Bethesda assay involves mixing
titered volumes of the test sample with an equal volume of an
inhibitor at a set concentration. The mixtures are incubated for 2
hours at 37.degree. C. prior to analysis of the factor
concentration by a coagulation assay such as a chromogenic assay.
Similarly, a reference plasma with native factor VIII level is
incubated that then assayed as the positive control. The endpoint
is the titer resulting in 50% of the FVIII activity of the positive
control, reported as Bethesda units. In the Nijimegen modification
of the Bethesda assay, the assay samples are stabilized with
imidazole buffer and the control sample is mixed with deficient
plasma instead of buffer (Verbruggen B, et al. The Nijmegen
modification of the Bethesda assay for factor VIII:C inhibitors:
improved specificity and reliability. Thromb Haemost. (1995)
73(2):247-251).
[0259] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32 P or 125 I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0260] ELISA assays can detect antibodies to FVIII independent of
their ability to block the procoagulant activity of FVIII, and have
been utilized for the detection of anti-FVIII developing in
hemophilia A patients. In a population of 131 patients with
hemophilia A with inhibitors, the ELISA technique resulted in 97.7%
sensitivity and 78.8% specificity, and had a high negative
predictive value (98.6%) [Martin, P. G., et al. Evaluation of a
novel ELISA screening test for detection of factor VIII inhibitory
antibodies in haemophiliacs. Clin Lab Haematol (1999) 21:125-128].
Other investigators have found a highly significant correlation
between the Bethesda titer and the absorbance values in an ELISA
assay for detecting anti-FVIII Abs (Towfighi, F., et al.
Comparative measurement of anti-factor VIII antibody by Bethesda
assay and ELISA reveals restricted isotype profile and epitope
specificity. Acta Haematol (2005) 114:84-90), with the added
advantage of the ability to detect non-inhibitory anti-FVIII
antibodies. Assay protocols comprise preparing the binding ligand,
which may include a sample comprising either factor VIII
polypeptide or the CFXTEN fusion protein, coating the well of a 96
well microtiter plate with the antibody, adding the ligand test
sample and incubating, then adding a detection antibody and
incubating prior to washing and adding a alkaline phosphatase- or
peroxidase-conjugated secondary antibody and incubating for an
additional period before the addition of TMB substrate and
processing for reading by spectrophotometer at 450 nm. In ELISAs
the antibody or inhibitor of interest does not have to be
conjugated to a detectable compound; instead, a second antibody
(which recognizes the antibody or inhibitor of interest) conjugated
to a detectable compound may be added to the well. Further, instead
of coating the well with the antibody, the ligand may be coated to
the well. One of skill in the art would be knowledgeable as to the
parameters that can be modified to increase the signal detected as
well as other variations of ELISAs known in the art (see, e.g.,
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).
[0261] Standard or modified coagulation assays are used to measure
reduced binding of FVIII binding agents. In one exemplary method
(further described in Example 28), the optimal concentration of a
given FVIII inhibitor to utilize in the assay is first determined
by a titration experiment using varying amounts of the inhibitory
antibody incubated at 37.degree. C. for 2 hrs with the base vector
expressing wild-type FVIII containing a His/Myc double tag. The
FVIII activity is measured by the Coatest assay procedure described
herein. The lowest concentration that results in optimal inhibition
of FVIII activity is employed in the assay. In the assay, the FVIII
inhibitor antibody at the optimal concentration is mixed with
individual test samples and incubated at 37.degree. C. for 2 hrs.
The resulting test samples are then collected and utilized in the
Coatest activity assay, along with untreated aliquots of the CFXTEN
and positive control in order to assess the residual and baseline
FVIII activity for each test sample.
[0262] The invention provides methods of making CFXTEN that exhibit
reduced binding to FVIII binding agents, including FVIII
inhibitors, and retention of procoagulant activity. In one
embodiment, the method to make a CFXTEN with reduced binding to
FVIII inhibitors comprises the steps of selecting a FVIII sequence
with at least 90% sequence identity to a sequence of Table 1,
selecting one, two, three, four, five, or six or more XTEN each
with at least 70%, or at least 80%, or at least 90%, or at least
95-99% sequence identity to XTEN sequences of comparable length
from Table 4, creating expression constructs designed to locate
said XTEN at or proximal to locations selected from Table 5, Table
6, Table 7, Table 8, and Table 9, expressing and recovering the
resulting CFXTEN, and assaying the resulting fusion proteins in an
assay described herein in order to confirm the reduced binding of
the CFXTEN fusion protein. By the inventive method, a CFXTEN
exhibits at least 5% reduced, or at least 10% reduced, or at least
15% reduced, or at least 20% reduced, or at least 25% reduced, or
at least 40% reduced, or at least 50% reduced, or at least 60%
reduced, or at least 70% reduced, or at least 80% reduced binding
to a FVIII binding agent including, but not limited to the
antibodies of Table 10 or anti-FVIII antibodies from a hemophilia A
subject, and retains 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% procoagulant activity
compared to the corresponding FVIII not linked to XTEN.
[0263] Up to 8-10% of hemophilia A patients have antibodies that
bind FVIII without affecting its procoagulant properties; they are
not, therefore categorized as FVIII inhibitors. However, the
binding of antibodies to FVIII is believed to lead to immune
complexes that are cleared by the innate immune response or are
more susceptible to proteolytic degradation (Kazatchkine M D.
Circulating immune complexes containing anti-VIII antibodies in
multi-transfused patients with haemophilia A. Clin Exp Immunol.
(1980) 39(2):315-320). Accordingly, it is an object of the
invention to provide CFXTEN fusion proteins comprising one or more
XTEN that exhibit reduced binding of antibodies to FVIII that are
not inhibitors, wherein the degradation or clearance of the CFXTEN
is reduced at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or
50%, or 60%, or 70% or less compared to a corresponding FVIII not
linked to XTEN or to native FVIII bound by such antibodies. The
reduced binding of antibodies to CFXTEN compared to FVIII not
linked to XTEN or to native FVIII can be assayed by in vitro and in
vivo methods. In vitro methods include the aforementioned ELISA and
Western blot methods. The reduced degradation or clearance of
CFXTEN can be assessed in vivo by use of animal models or in human
clinical trials. In one type of trial, factor VIII or CFXTEN are
administered separately, preferably by intravenous infusion, to
cohorts of patients having factor VIII deficiency who have
antibodies that promote degradation or clearance of therapeutic
human factor VIII. The dosage of the administered test article is
in a range between 5 and 50 IU/kg body weight, preferably 10-45
IU/kg, and most preferably 40 IU/kg body weight. Approximately 1
hour after each administration, the recovery of factor VIII or
CFXTEN from blood samples is measured in a functional one-stage or
chromogenic coagulation assay to assess activity and by ELISA,
HPLC, or similar assay to qualify the amount of intact factor VIII
equivalent. Samples are taken again approximately 5-10 hours after
infusion, and recovery is measured. Total recovery and the rate of
disappearance of factor VIII from the samples is predictive of the
antibody titer, and the comparison of results from the factor VIII
and CFXTEN indicates the degree of reduced clearance and/or
degradation of the CFXTEN. In one embodiment, the CFXTEN fusion
protein exhibits at least 5% reduced, or at least 10% reduced, or
at least 15% reduced, or at least 20% reduced, or at least 25%
reduced, or at least 40% reduced, or at least 50% reduced, or at
least 60% reduced, or at least 70% reduced, or at least 80% reduced
binding to an anti-FVIII antibody that promotes clearance but does
not otherwise inhibit the procoagulant activity of intact native
FVIII. In another embodiment, the CFXTEN fusion protein exhibits at
least 5% reduced, or at least 10% reduced, or at least 15% reduced,
or at least 20% reduced, or at least 25% reduced, or at least 40%
reduced, or at least 50% reduced, or at least 60% reduced, or at
least 70% reduced, or at least 80% reduced binding to an anti-FVIII
antibody that promotes the degradation of FVIII. In the foregoing
embodiments of this paragraph, the reduced binding of the
anti-FVIII antibody is alternatively characterized by an increased
K.sub.D value of the FVIII antibody to the fusion protein compared
to the FVIII of at least two-fold, or three-fold, or four-fold, or
five-fold, or 10-fold, or 33-fold, or 100-fold, or 330-fold, or at
least 1000-fold compared to the binding to the corresponding FVIII
not linked to XTEN. In one embodiment, the CFXTEN fusion proteins
comprising one or more XTEN exhibiting reduced reactivity to an
anti-FVIII antibody exhibits an increased terminal half-life when
administered to a subject with anti-FVIII antibodies of at least 48
h, or at least 72 h, or at least 96 h, or at least 120 h, or at
least 144 h, or at least 14 days, or at least 21 days compared to
FVIII not linked to XTEN. In the foregoing embodiment, the subject
can be a human hemophilia A subject or it can be a mouse hemophilia
A subject with circulating anti-FVIII antibodies.
[0264] Another aspect of the present invention is the use of CFXTEN
fusion protein for a specific therapy of a coagulopathy in a
subject with a FVIII inhibitor. The invention provides a method of
treating a subject with circulating FVIII inhibitor(s) comprising
the step of administering a clotting-effective amount of a CFXTEN
fusion protein to the subject wherein the fusion protein exhibits
greater procoagulant activity and/or clotting-effective
concentrations of longer duration compared to either a
corresponding factor VIII not linked to XTEN or compared to native
factor VIII administered to the subject using a comparable amount
and route of administration. In one embodiment of the method, the
FVIII inhibitor in the subject is an anti-FVIII antibody. In
another embodiment, the FVIII inhibitor is a neutralizing
anti-FVIII antibody. In one embodiment, the FVIII inhibitor is an
anti-FVIII antibody that binds to the A1 domain of FVIII. In
another embodiment, the FVIII inhibitor is an anti-FVIII antibody
that binds to the A2 domain of FVIII. In another embodiment, the
FVIII inhibitor is an anti-FVIII antibody that binds to the A3
domain of FVIII. In another embodiment, the FVIII inhibitor is an
anti-FVIII antibody that binds to the C1 domain of FVIII. In
another embodiment, the FVIII inhibitor is an anti-FVIII antibody
that binds to the C2 domain of FVIII. In another embodiment, the
FVIII inhibitor is an anti-FVIII antibody that binds to both the C2
and A2 domain of FVIII. In another embodiment, the FVIII inhibitor
binds to a FVIII epitope capable of being bound by one or more
antibodies of Table 10. In another embodiment, the FVIII inhibitor
is a polyclonal antibody from a hemophilia A subject with FVIII
inhibitor antibodies.
[0265] An object of the present invention is the creation of CFXTEN
with XTEN inserted to maximize the steric interference of FVIII
binding agents that would otherwise bind to FVIII and neutralize
procoagulant activity or result in the clearance or degradation of
FVIII. Accordingly, in one approach the invention provides CFXTEN
comprising one or more XTEN wherein the XTEN are inserted proximal
to a binding site of a FVIII inhibitor or anti-FVIII antibody. In
one embodiment, an XTEN is linked to the FVIII at a location
selected from Table 5, Table 6, Table 7, Table 8, and Table 9 that
is within about 50, or about 100, or about 150, or about 200, or
about 250, or about 300 amino acids of a FVIII epitope that is
bound by an antibody of Table 10. In another embodiment, the XTEN
is linked to the FVIII within about 50, or about 100, or about 150,
or about 200, or about 250, or about 300 amino acids of a FVIII
epitope in the A2 or C2 domain that is bound by an antibody of
Table 10. Accordingly, the invention provides CFXTEN fusion
proteins comprising one or more XTEN wherein binding by FVIII
inhibitors to the FVIII component of the fusion protein is reduced
compared to the corresponding FVIII not linked to XTEN or to native
FVIII and the CFXTEN retains procoagulant activity. In the
foregoing embodiments hereinabove described in this paragraph, the
fusion proteins can be assayed by the assays described herein
below, the assays of the Examples, or other assays known in the
art, and the inhibitors can be an antibody of Table 10, can be
polyclonal anti-FVIII, or can be blood or plasma from a hemophilia
A subject with FVIII inhibitors.
[0266] In another aspect, CFXTEN are designed to maximize the
regions over which XTEN can adopt random coil conformations
covering the fusion protein, thereby resulting in steric hindrance
for anti-FVIII antibodies that would otherwise bind epitopes on the
FVIII component of the fusion protein. It is believed that the
incorporation of multiple XTEN into a CFXTEN provides a higher
total hydrodynamic radius of the XTEN component compared to CFXTEN
with fewer XTEN yet having approximately the same total of XTEN
amino acids. Empirically, the hydrodynamic radius for a protein can
be calculated based on size exclusion chromatography, and results
of several fusion proteins using such methods are described in the
Examples. Alternatively, the radius for XTEN polypeptides, such as
those incorporated in the embodiments disclosed herein, can be
approximated by mathematical formulae because the limited types of
amino acids utilized have known characteristics that can be
quantified. In one embodiment, the maximum radius of a single XTEN
polypeptide is calculated (hereinafter "XTEN Radius") according to
the formula given by Equation II:
XTEN Radius=( XTEN length 0.2037)+3.4627 II
[0267] In another embodiment, the sum of the maximum of the XTEN
Radii for all XTEN segments in a CFXTEN is calculated (hereinafter
"Sum XTEN Radii") according to the formula given by Equation
III:
Sum XTEN Radii = i = 1 n XTEN Radius i III ##EQU00002##
[0268] wherein: n=the number of XTEN segments
[0269] and i is an iterator
[0270] In another embodiment, the ratio of the SUM XTEN Radii of a
CFXTEN comprising multiple XTEN to that of an XTEN Radius for a
single XTEN of an equivalent length (in total amino acid residues
to that of the CFXTEN) is calculated (hereinafter "Ratio XTEN
Radii") according to the formula given by Equation IV:
Ratio XTEN Radii = i = 1 n XTEN Radius i ( i = 1 N XTEN Length i *
0.2037 ) + 3.4627 IV ##EQU00003##
[0271] wherein: n=the number of XTEN segments
[0272] and i is an iterator
[0273] In applying the Equations to the XTEN, it will be understood
by one of skill in the art that the calculated values represent
maximum values that could vary or be reduced depending on the host
cell utilized for expression of the XTEN polypeptide. It is
believed that while E. coli expression would result in XTEN that
achieves the calculated values, expression in eukaryotic host cells
in which XTEN may be glycosylated could result in a radius of the
polypeptide less than the maximum calculated value. Such
differences can be quantified by methods such as size exclusion
chromatography, the methods of which are detailed in the
Examples.
[0274] In order to design CFTEN that maximize the area over which
XTEN can adopt random coil conformations, it was discovered that
CFXTEN designs with Ratio XTEN Radii above 2 provide greater
coverage over the fusion protein than designs with values <2.
Accordingly, in one embodiment the invention provides CFXTEN in
which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3,
or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or
3.2, or 3.3, or 3.4, or 3.5 or greater. In some embodiments, the
invention provides CFXTEN in which the Ratio XTEN Radii is at least
2.0-3.5 or greater comprise at least three XTEN with each XTEN
having at least 42 to about 288 amino acids and wherein at least
two of the XTEN are linked to the fusion protein with no less than
about 100, or about 200, or about 300, or about 400, or about 500
amino acids of separation between the two XTEN. In other
embodiments, the invention provides CFXTEN in which the Ratio XTEN
Radii is at least 2.0-3.5 or greater comprise at least four XTEN
with each XTEN having at least 42 to about 288 amino acids and
wherein at least three of the XTEN are linked to the fusion protein
with no less than about 100, or about 200, or about 300, or about
400 amino acids of separation between any two of the three
XTEN.
[0275] In another embodiment, the invention provides a CFXTEN in
which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the
CFXTEN comprises at least three XTEN with each XTEN having at least
42 to about 288 amino acids and wherein at least two of the three
of the XTEN linked to the fusion protein are separated by an amino
acid sequence of at least 100, or about 200, or about 300 to about
400 amino acids, and the third XTEN is linked within the B domain
(or fragment thereof) or within the C domain (or the terminus
thereof). In another embodiment, the invention provides a CFXTEN in
which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the
CFXTEN comprises at least four XTEN with each XTEN having at least
42 to about 288 amino acids and wherein at least three of the four
of the XTEN linked to the fusion protein are separated by an amino
acid sequence of at least 300 to about 400 amino acids and the
fourth XTEN is linked within the B domain (or fragment thereof) or
within the C domain (or the terminus thereof).
[0276] In yet other embodiments, the invention provides CFXTEN in
which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the
CFXTEN comprises at least five XTEN with four XTEN having at least
42 to about 144 amino acids wherein at least four of the XTEN are
linked to the fusion protein with no less than about 100, 200, or
about 300, or about 400 amino acids of separation between any two
of the four XTEN and a fifth XTEN is linked within the B domain (or
fragment thereof) or within the C domain (or the terminus thereof).
In one embodiment, the invention provides a CFXTEN in which the
Ratio XTEN Radii is at least 2.0-3.5 or greater, the CFXTEN
comprises at least five XTEN with four XTEN having at least 42 to
about 144 amino acids wherein at least three of the XTEN linked to
the fusion protein are separated by an amino acid sequence of at
least 300 to about 400 amino acids, the fourth XTEN is linked
within the B domain (or fragment thereof) and a fifth XTEN is
linked within the C domain (or the terminus thereof).
[0277] In one aspect, the invention provides CFXTEN in which the
Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4,
or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or
3.3, or 3.4, or 3.5 or greater, and the composition does not
comprise certain sequences. In one embodiment of the foregoing, the
invention provides CFXTEN in which the Ratio XTEN Radii is at least
2.0-3.5 or greater with the proviso that the fusion protein does
not comprise a sequence from any one of Table 50 or Table 51. In
another embodiment of the foregoing, the invention provides CFXTEN
in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with
the proviso that the fusion protein does not comprise a sequence
having an AG family XTEN sequence. In another embodiment of the
foregoing, the invention provides CFXTEN in which the Ratio XTEN
Radii is at least 2.0-3.5 or greater with the proviso that the
fusion protein does not comprise a sequence selected from
GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32),
GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34). In
another embodiment of the foregoing, the invention provides CFXTEN
in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with
the proviso that the fusion protein does not comprise any one of
the sequences selected from GTPGSGTASSSP (SEQ ID NO: 31),
GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33),
GASPGTSSTGSP (SEQ ID NO: 34) and
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG
SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing,
the invention provides CFXTEN in which the Ratio XTEN Radii is at
least 2.0-3.5 or greater with the proviso that the fusion protein
does not comprise a sequence selected from
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG
SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST
EPSEGSAP (SEQ ID NO: 59),
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSS (SEQ ID NO: 71), or
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA
SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG
TASSSPGSSTPSGATGS (SEQ ID NO: 80). In another embodiment of the
foregoing, the invention provides CFXTEN in which the Ratio XTEN
Radii is at least 2.0-3.5 or greater with the proviso that the
fusion protein does not comprise an XTEN sequence consisting of
TABLE-US-00012 (SEQ ID NO: 59)
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP, (SEQ ID NO: 71)
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS
PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS, or (SEQ ID NO:
80) PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTG
PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS.
[0278] In one aspect, the present invention provides methods to
create CFXTEN with XTEN inserted to maximize the steric
interference of FVIII binding agents that would otherwise bind to
FVIII and neutralize procoagulant activity or result in the
clearance or degradation of FVIII. Accordingly, in one embodiment,
the invention provides a method comprising the steps of selecting a
FVIII sequence with at least 90% sequence identity to a sequence of
Table 1, selecting three or more XTEN from Table 4 in which the
Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4,
or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or
3.3, or 3.4, or 3.5 or greater, creating expression constructs
designed to locate said XTEN at or proximal to locations selected
from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the
three or more XTEN are at least 300 to 400 amino acids, expressing
and recovering the resulting CFXTEN, and assaying the resulting
fusion proteins in an assay described herein in order to confirm
the reduced binding of the CFXTEN fusion protein. By the inventive
method, a CFXTEN exhibits at least 5% reduced, or at least 10%
reduced, or at least 15% reduced, or at least 20% reduced, or at
least 25% reduced, or at least 40% reduced, or at least 50%
reduced, or at least 60% reduced, or at least 70% reduced, or at
least 80% reduced binding to a FVIII binding agent including, but
not limited to the antibodies of Table 10, and exhibits
procoagulant activity.
[0279] 5. CFXTEN Fusion Protein Configurations with Spacer and
Cleavage Sequences
[0280] In another aspect, the invention provides CFXTEN configured
with one or more spacer sequences 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 fusion protein compositions. Such
properties include, but are not limited to, inclusion of cleavage
sequence(s) to permit release of components, inclusion of amino
acids compatible with nucleotide restrictions sites to permit
linkage of XTEN-encoding nucleotides to FVIII-encoding nucleotides
or that facilitate construction of expression vectors, and linkers
designed to reduce steric hindrance in regions of CFXTEN fusion
proteins.
[0281] In an embodiment, a spacer sequence can be introduced
between an XTEN sequence and a FVIII component to decrease steric
hindrance such that the FVIII component may assume its desired
tertiary structure and/or interact appropriately with its target
substrate or processing enzyme. 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 such as, but not limited
to, glycine (G), alanine (A), serine (S), threonine (T), glutamate
(E), proline (P) and aspartate (D). The spacer can be a single
glycine residue, polyglycines or polyalanines, or is predominately
a mixture of combinations of glycine, serine and alanine residues.
In one embodiment, a spacer sequence, exclusive of cleavage site
amino acids, has about 1 to 10 amino acids that consist of amino
acids selected from glycine (G), alanine (A), serine (S), threonine
(T), glutamate (E), and proline (P) and are substantially devoid of
secondary structure; e.g., less than about 10%, or less than about
5% as determined by the Chou-Fasman and/or GOR algorithms. In one
embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In
another embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612)
linked to a cleavage sequence of Table 12. 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.
[0282] In a particular embodiment, the CFXTEN fusion protein
comprises one or more spacer sequences linked at the junction(s)
between the payload FVIII sequence and the one or more XTEN
incorporated into the fusion protein, wherein the spacer sequences
comprise amino acids that are compatible with nucleotides encoding
restriction sites. In another embodiment, the CFXTEN fusion protein
comprises one or more spacer sequences linked at the junction(s)
between the payload FVIII sequence and the one more XTEN
incorporated into the fusion protein wherein the spacer sequences
comprise amino acids that are compatible with nucleotides encoding
restriction sites and the amino acids and the one more spacer
sequence amino acids are chosen from glycine (G), alanine (A),
serine (S), threonine (T), glutamate (E), and proline (P). In
another embodiment, the CFXTEN fusion protein comprises one or more
spacer sequences linked at the junction(s) between the payload
FVIII sequence and one more XTEN incorporated into the fusion
protein wherein the spacer sequences comprise amino acids that are
compatible with nucleotides encoding restriction sites and the one
more spacer sequences are chosen from the sequences of Table 11.
The exact sequence of each spacer sequence is chosen to be
compatible with cloning sites in expression vectors that are used
for a particular CFXTEN construct. In one embodiment, the spacer
sequence has properties compatible with XTEN. In one embodiment,
the spacer sequence is GAGSPGAETA (SEQ ID NO: 178). For XTEN
sequences that are incorporated internal to the FVIII sequence,
each XTEN would generally be flanked by two spacer sequences
comprising amino acids compatible with restriction sites, while
XTEN attached to the N- or C-terminus would only require a single
spacer sequence at the junction of the two components and another
at the opposite end for incorporation into the vector. As would be
apparent to one of ordinary skill in the art, the spacer sequences
comprising amino acids compatible with restriction sites that are
internal to FVIII could be omitted from the construct when an
entire CFXTEN gene is synthetically generated.
TABLE-US-00013 TABLE 11 Spacer Sequences Compatible with
Restriction Sites Spacer Sequence Restriction Enzyme GSPG (SEQ ID
NO: 174) BsaI ETET (SEQ ID NO: 175) BsaI PGSSS (SEQ ID NO: 176)
BbsI GAP AscI GPA FseI GPSGP (SEQ ID NO: 177) SfiI AAA SacII TG
AgeI GT KpnI GAGSPGAETA (SEQ ID SfiI NO: 178) ASS XhoI
[0283] In another aspect, the present invention provides CFXTEN
configurations with cleavage sequences incorporated into the spacer
sequences. In some embodiments, spacer sequences in a CFXTEN fusion
protein composition comprise one or more cleavage sequences, which
are identical or different, wherein the cleavage sequence may be
acted on by a protease, as shown in FIG. 12, to release FVIII, a
FVIII component (e.g., the B domain) or XTEN sequence(s) from the
fusion protein. In one embodiment, the incorporation of the
cleavage sequence into the CFXTEN is designed to permit release of
the FVIII component that becomes active or more active (with
respect to its ability serve as a membrane binding site for factors
IXa and X) upon its release from the XTEN. In the foregoing
embodiment, the procoagulant activity of FVIII component of the
CFXTEN is increased after cleavage by at least 30%, or at least
40%, or at least 50%, or at least 60%, or at least 70%, or at least
80%, or at least 90% compared to the intact CFXTEN. The cleavage
sequences are located sufficiently close to the FVIII sequences,
generally within 18, or within 12, or within 6, or within 2 amino
acids of the FVIII sequence, such that any remaining residues
attached to the FVIII after cleavage do not appreciably interfere
with the activity (e.g., such as binding to a clotting protein) of
the FVIII, yet provide sufficient access to the protease to be able
to effect cleavage of the cleavage sequence. In some cases, the
CFXTEN comprising the cleavage sequences will also have one or more
spacer sequence amino acids between the FVIII and the cleavage
sequence or the XTEN and the cleavage sequence to facilitate access
of the protease; the spacer amino acids comprising any natural
amino acid, including glycine, serine and alanine as preferred
amino acids. In one embodiment, the cleavage site is a sequence
that can be cleaved by a protease endogenous to the mammalian
subject such that the CFXTEN can be cleaved after administration to
a subject. In such case, the CFXTEN can serve as a prodrug or a
circulating depot for the FVIII. In a particular construct of the
foregoing, the CFXTEN would have one or two XTEN linked to the N-
and/or the C-terminus of a FVIII-BDD via a cleavage sequence that
can be acted upon by an activated coagulation factor, and would
have an additional XTEN located between the processing amino acids
at position R740 and R1689 such that the XTEN could be released,
leaving a form of FVIII similar to native activated FVIII. In one
embodiment of the foregoing construct, the FVIII that is released
from the fusion protein by cleavage of the cleavage sequence
exhibits at least about a two-fold, or at least about a three-fold,
or at least about a four-fold, or at least about a five-fold, or at
least about a six-fold, or at least about a eight-fold, or at least
about a ten-fold, or at least about a 20-fold increase in activity
compared to the intact CFXTEN fusion protein.
[0284] Examples of cleavage sites contemplated by the invention
include, but are not limited to, a polypeptide sequence cleavable
by a mammalian endogenous protease selected from FXIa, FXIIa,
kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2,
granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian
proteases such as TEV, enterokinase, PreScission.TM. protease
(rhinovirus 3C protease), and sortase A. Sequences known to be
cleaved by the foregoing proteases and others are known in the art.
Exemplary cleavage sequences contemplated by the invention and the
respective cut sites within the sequences are presented in Table
12, as well as sequence variants thereof. For CFXTEN comprising
incorporated cleavage sequence(s), it is generally preferred that
the one or more cleavage sequences are substrates for activated
clotting proteins. For example, thrombin (activated clotting factor
II) acts on the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.
D., et al. (2008) Nucleic Acids Res., 36: D320], which is cut after
the arginine at position 4 in the sequence. Active FIIa is produced
by cleavage of FII by FXa in the presence of phospholipids and
calcium and is down stream from factor VIII in the coagulation
pathway. Once activated, its natural role in coagulation is to
cleave fibrinogen, which then in turn, begins clot formation. FIIa
activity is tightly controlled and only occurs when coagulation is
necessary for proper hemostasis. By incorporation of the LTPRSLLV
sequence (SEQ ID NO: 1618) into the CFXTEN between and linking the
FVIII and the XTEN components, the XTEN is removed from the
adjoining FVIII concurrent with activation of either the extrinsic
or intrinsic coagulation pathways when coagulation is required
physiologically, thereby selectively releasing FVIII. In another
embodiment, the invention provides CFXTEN with incorporated FXIa
cleavage sequences between the FVIII and XTEN component(s) that are
acted upon only by initiation of the intrinsic coagulation system,
wherein a procoagulant form of FVIII is released from XTEN by FXIa
to participate in the coagulation cascade. While not intending to
be bound by any particular theory, it is believed that the CFXTEN
of the foregoing embodiment would sequester the FVIII away from the
other coagulation factors except at the site of active clotting,
thus allowing for larger doses (and therefore longer dosing
intervals) with minimal safety concerns.
[0285] Thus, cleavage sequences, particularly those susceptible to
the procoagulant activated clotting proteins listed in Table 12,
would provide for sustained release of FVIII that, in certain
embodiments of the CFXTEN, can provide a higher degree of activity
for the FVIII component released from the intact form of the
CFXTEN, as well as additional safety margin for high doses of
CFXTEN administered to a subject. In one embodiment, the invention
provides CFXTEN comprising one or more cleavage sequences operably
positioned to release the FVIII from the fusion protein upon
cleavage, wherein the one or more cleavage sequences has at least
about 86%, or at least about 92%, or 100% sequence identity to a
sequence selected from Table 12.
[0286] In some embodiments, only the two or three amino acids
flanking both sides of the cut site (four to six amino acids total)
are incorporated into the cleavage sequence that, in turn, is
incorporated into the CFXTEN of the embodiments, providing, e.g.,
XTEN release sites. In other embodiments, the incorporated cleavage
sequence of Table 12 can have one or more deletions or insertions
or one or two or three amino acid substitutions for any one or two
or three amino acids in the known sequence, wherein the deletions,
insertions or substitutions result in reduced or enhanced
susceptibility but not an absence of susceptibility to the
protease, resulting in an ability to tailor the rate of release of
the FVIII from the XTEN. Exemplary substitutions within cleavage
sequences that are utilized in the CFXTEN of the invention are
shown in Table 12.
TABLE-US-00014 TABLE 12 Protease Cleavage Sequences Exemplary
Protease Acting Cleavage SEQ ID SEQ ID Upon Sequence Sequence NO:
Minimal Cut Site NO: FXIa KLTR.dwnarw.AET 179
KD/FL/T/R.dwnarw.VA/VE/GT/GV FXIa DFTR.dwnarw.VVG 180
KD/FL/T/R.dwnarw.VA/VE/GT/GV FXIIa TMTR.dwnarw.IVGG 181 NA
Kallikrein SPFR.dwnarw.STGG 182 --/--/FL/RY.dwnarw.SR/RT/--/--
FVIIa LQVR.dwnarw.IVGG 183 NA FIXa PLGR.dwnarw.IVGG 184
--/--/G/R.dwnarw.--/--/--/-- FXa IEGR.dwnarw.TVGG 185
IA/E/GFP/R.dwnarw.STI/VFS/--/G FIIa (thrombin) LTPR.dwnarw.SLLV 186
--/--/PLA/R.dwnarw.SAG/--/--/-- Elastase-2 LGPV.dwnarw.SGVP 187
--/--/--/VIAT.dwnarw.--/--/--/-- Granzyme-B VAGD.dwnarw.SLEE 188
V/--/--/D.dwnarw.--/--/--/-- MMP-12 GPAG.dwnarw.LGGA 189
G/PA/--/G.dwnarw.L/--/G/-- 190 MMP-13 GPAG.dwnarw.LRGA 191
G/P/--/G.dwnarw.L/--/GA/-- 192 MMP-17 APLG.dwnarw.LRLR 193
--/PS/--/--.dwnarw.LQ/--/LT/-- MMP-20 PALP.dwnarw.LVAQ 194 NA TEV
ENLYFQ.dwnarw.G 195 ENLYFQ.dwnarw.G/S 196 Enterokinase
DDDK.dwnarw.IVGG 197 DDDK.dwnarw.IVGG 198 Protease 3C
LEVLFQ.dwnarw.GP 199 LEVLFQ.dwnarw.GP 200 (PreScission .TM.)
Sortase A LPKT.dwnarw.GSES 201 L/P/KEAD/T.dwnarw.G/--/EKS/S 202
.dwnarw.indicates cleavage site NA: not applicable 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
[0287] 6. Exemplary CFXTEN Fusion Protein Sequences
[0288] Non-limiting examples of sequences of fusion proteins
containing a single FVIII linked to one or more XTEN are presented
in Table 21. The exemplary amino acid sequences of Table 21 (and
the DNA sequences that encode them) contain his tags for
purification purposes that, as would be apparent to one of skill in
the art, can be deleted from the sequence without having an effect
on the procoagulant activity of the CFXTEN fusion protein. In one
embodiment, the CFXTEN of Table 21 further comprise amino acids on
the N-terminus corresponding to that of native human FVIII (namely,
the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) to aid in the
expression and secretion of the CFXTEN fusion protein. In one
embodiment, a CFXTEN composition comprises a fusion protein having
at least about 80% sequence identity compared to a CFXTEN from
Table 21, alternatively at least about 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or about 100% sequence identity as compared to a CFXTEN from
Table 21, when optimally aligned. In another embodiment, a CFXTEN
composition comprises a fusion protein from Table 21 in which the
C-terminal his-his-his-his-his-his sequence (SEQ ID NO: 1700)
deleted. However, the invention also contemplates substitution of
any of the FVIII sequences of Table 1 for a FVIII component of the
CFXTEN of Table 21, and/or substitution of any sequence of any one
of Tables 3, 4, and 13-17 for an XTEN component of the CFXTEN of
Table 21. Generally, the resulting CFXTEN of the foregoing examples
retain at least a portion of the procoagulant activity of the
corresponding FVIII not linked to the XTEN. In the foregoing fusion
proteins hereinabove described in this paragraph, the CFXTEN fusion
protein can further comprise one or more cleavage sequences; e.g.,
a sequence from Table 12, the cleavage sequence being located
between the FVIII and the XTEN sequences or between adjacent FVIII
domains linked by XTEN. In some embodiments comprising cleavage
sequence(s), the intact CFXTEN composition has less activity but a
longer half-life in its intact form compared to a corresponding
FVIII not linked to the XTEN, but is designed such that upon
administration to a subject, the FVIII component is gradually
released from the fusion protein by cleavage at the cleavage
sequence(s) by endogenous proteases, whereupon the FVIII component
exhibits procoagulant activity.
[0289] The CFXTEN compositions of the embodiments can be evaluated
for activity using assays or in vivo parameters as described herein
(e.g., in vitro coagulation assays, assays of Table 49, or a
pharmacodynamic effect in a preclinical hemophilia model or in
clinical trials in humans, using methods as described in the
Examples or other methods known in the art for assessing FVIII
activity) to determine the suitability of the configuration or the
FVIII sequence variant, and those CFXTEN compositions (including
after cleavage of any incorporated XTEN-releasing cleavage sites)
that retain at least about 30%, or about 40%, or about 50%, or
about 55%, or about 60%, or about 70%, or about 80%, or about 90%,
or about 95% or more activity compared to native FVIII sequence are
considered suitable for use in the treatment of FVIII-related
conditions.
V). Properties of the CFXTEN Compositions of the Invention
[0290] (a) Pharmacokinetic Properties of CFXTEN
[0291] It is an object of the present invention to provide CFXTEN
fusion proteins and pharmaceutical compositions comprising CFXTEN
with enhanced pharmacokinetics compared to FVIII not linked to
XTEN. The pharmacokinetic properties of a FVIII enhanced by linking
a given XTEN to the FVIII include, but are not limited to, terminal
half-life, area under the curve (AUC), C.sub.max, volume of
distribution, maintaining the biologically active CFXTEN above a
minimum effective blood unit concentration for a longer period of
time compared to the FVIII not linked to XTEN. The enhanced
properties permit less frequent dosing and/or a longer-lived
procoagulant effect compared to a comparable dose of FVIII not
linked to XTEN. Enhancement of one or more of these properties can
resulting benefits in the treatment of factor VIII-related
conditions.
[0292] Exogenously administered factor VIII has been reported to
have a terminal half-life in humans of approximately 12-14 hours
when complexed with normal von Willebrand factor protein, whereas
in the absence of von Willebrand factor, the half-life of factor
VIII is reduced to 2 hours (Tuddenham E G, et al., Br J Haematol.
(1982) 52(2):259-267; Bjorkman, S., et al. Clin Pharmacokinet.
(2001) 40:815). As a result of the enhanced properties conferred by
XTEN, the CFXTEN, when used at the dose and dose regimen determined
to be appropriate for the subject and its underlying condition, can
achieve a circulating concentration resulting in a desired
procoagulant or clinical effect for an extended period of time
compared to a comparable dose of the corresponding FVIII not linked
to XTEN. As used herein, a "comparable dose" means a dose with an
equivalent moles/kg or International Units/kg (IU/kg) for the
composition that is administered to a subject. It will be
understood in the art that a "comparable dose" of FVIII not linked
to XTEN would represent a lesser weight of drug but would have
essentially the same IUs or mole-equivalents of CFXTEN in the
dose.
[0293] An international unit ("IU") of factor VIII is defined in
the art as the coagulant activity present in 1 ml of normal human
plasma. A normal, non-hemophilic individual human is expected to
have about 100 IU/dL factor VIII activity. In hemophilia A, the
doses required to treat are dependent on the condition. For minor
bleeding, doses of native or recombinant factor VIII of 20 to 40
IU/kg are typically administered, as necessary. For moderate
bleeding, doses of 30 to 60 IU/kg are administered as necessary,
and for major bleeding, doses of 80 to 100 IU/kg may be required,
with repeat doses of 20 to 25 IU/kg given every 8 to 12 hours until
the bleeding is resolved. For prophylaxis against bleeding in
patients with severe hemophilia A, the usual doses of native or
recombinant FVIII preparations are 20 to 40 IU/kg body weight at
intervals of about 2 to 3 days. A standard equation for estimating
an appropriate dose of a composition comprising FVIII is:
Required units=body weight (kg).times.desired factor VIII rise
(IU/dL or % of normal).times.0.5 (IU/kg per IU/dL).
[0294] In many cases, the therapeutic levels for FVIII in subjects
of different ages or degree of disease have been established and
are available in published literature or are stated on the drug
label for approved products containing the FVIII. For example, the
Subcommittee on Factor VIII and Factor IX of the Scientific and
Standardization Committee of the International Society on
Thrombosis and Haemostasis posted, on the ISTH Website 29 November,
2000, that the most widely used measure of hemophilia A is
established by determining the circulating concentrations of plasma
FVIII procoagulant levels, with persons with <1% (<0.01
IU/ml) factor VIII defined as severe; 1-5% (0.01-0.05 IU/ml) as
moderately severe; and .gtoreq.5-40% (0.05-<0.40 IU/ml) as mild,
where normal is 1 IU/ml of factor VIIIC (100%). The therapeutic
levels can be established for new compositions, including those
CFXTEN and pharmaceutical compositions comprising CFXTEN of the
disclosure, using standard methods. In practicing the present
invention, it will be understood that any dosage of CFXTEN that is
effective may be used for treating bleeding episodes or maintaining
hemostasis. The methods for establishing the therapeutic levels and
dosing schedules for a given composition are known to those of
skill in the art (see, e.g., Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 11.sup.th Edition,
McGraw-Hill (2005)). For example, by using dose-escalation studies
in subjects with the target condition to determine efficacy or a
desirable pharmacologic effect, appearance of adverse events, and
determination of circulating blood levels, the therapeutic blood
levels for a given subject or population of subjects can be
determined for a given drug or biologic. The dose escalation
studies would evaluate the activity of a CFXTEN through studies in
a subject or group of hemophilia A subjects. The studies would
monitor blood levels of procoagulant, as well as physiological or
clinical parameters as known in the art or as described herein for
one or more parameters associated with the factor VIII-related
condition, or clinical parameters associated with a beneficial
outcome, together with observations and/or measured parameters to
determine the no effect dose, adverse events, minimum effective
dose and the like, together with measurement of pharmacokinetic
parameters that establish the determined or derived circulating
blood levels. The results can then be correlated with the dose
administered and the blood concentrations of the therapeutic that
are coincident with the foregoing determined parameters or effect
levels. By these methods, a range of doses and blood concentrations
can be correlated to the minimum effective dose as well as the
maximum dose and blood concentration at which a desired effect
occurs or is maintained and the period for which it can be
maintained, thereby establishing the therapeutic blood levels and
dosing schedule for the composition. Thus, by the foregoing
methods, a C.sub.min blood level is established, below which the
CFXTEN fusion protein would not have the desired pharmacologic
effect and a C.sub.max blood level, above which side effects such
as thrombosis may occur (Brobrow, R S, JABFP (2005) 18(2):147-149),
establishing the therapeutic window for the composition.
[0295] One of skill in the art can, by the means disclosed herein
or by other methods known in the art, confirm that the administered
CFXTEN remains at therapeutic blood levels to maintain hemostasis
for the desired interval or requires adjustment in dose or length
or sequence of XTEN. Further, the determination of the appropriate
dose and dose frequency to keep the CFXTEN within the therapeutic
window establishes the therapeutically effective dose regimen; the
schedule for administration of multiple consecutive doses using a
therapeutically effective dose of the fusion protein to a subject
in need thereof resulting in consecutive C.sub.max peaks and/or
C.sub.min troughs that remain above therapeutically-effective
concentrations and result in an improvement in at least one
measured parameter relevant for the target condition. In one
embodiment, the CFXTEN or a pharmaceutical compositions comprising
CFXTEN administered at an appropriate dose to a subject results in
blood concentrations of the CFXTEN fusion protein that remains
above the minimum effective concentration to maintain hemostasis
for a period at least about two-fold longer compared to the
corresponding FVIII not linked to XTEN and administered at a
comparable dose; alternatively at least about three-fold longer;
alternatively at least about four-fold longer; alternatively at
least about five-fold longer; alternatively at least about six-fold
longer; alternatively at least about seven-fold longer;
alternatively at least about eight-fold longer; alternatively at
least about nine-fold longer, alternatively at least about ten-fold
longer, or at least about twenty-fold longer or greater compared to
the corresponding FVIII not linked to XTEN and administered at a
comparable dose. As used herein, an "appropriate dose" means a dose
of a drug or biologic that, when administered to a subject, would
result in a desirable therapeutic or pharmacologic effect (e.g.,
hemostasis) and/or a blood concentration within the therapeutic
window.
[0296] In practicing the invention, CFXTEN with longer terminal
half-life are generally preferred, so as to improve patient
convenience, to increase the interval between doses and to reduce
the amount of drug required to achieve a sustained effect. The
enhanced PK parameters allow for reduced dosing of the subject
compositions, compared to FVIII not linked to XTEN, particularly
for those hemophilia A subjects receiving routine prophylaxis.
[0297] As described more fully in the Examples pertaining to
pharmacokinetic characteristics of fusion proteins comprising XTEN,
it was observed that increasing the total length of the XTEN,
singly or in combination, confers a disproportionate increase in
the terminal half-life of a fusion protein comprising the XTEN.
Accordingly, the invention provides CFXTEN fusion proteins and
pharmaceutical compositions comprising CFXTEN wherein the CFXTEN
exhibits an enhanced half-life when administered to a subject. In
some embodiments, the invention provides monomeric CFXTEN fusion
proteins comprising one or more XTEN wherein the number and
location of the XTEN are selected to confer an increase in the
terminal half-life for the CFXTEN administered to a subject
compared to the corresponding FVIII not linked to the XTEN and
administered at a comparable dose, wherein the increase is at least
about two-fold longer, or at least about three-fold, or at least
about four-fold, or at least about five-fold, or at least about
six-fold, or at least about seven-fold, or at least about
eight-fold, or at least about nine-fold, or at least about
ten-fold, or at least about 15-fold, or at least a 20-fold, or at
least a 40-fold or greater increase in terminal half-life compared
to the FVIII not linked to the XTEN. In other embodiments, the
invention provides CXTEN compositions and pharmaceutical
compositions comprising CFXTEN wherein the administration of a
composition to a subject in need thereof results in a terminal
half-life that is at least 12 h greater, or at least about 24 h
greater, or at least about 48 h greater, or at least about 96 h
greater, or at least about 144 h greater, or at least about 7 days
greater, or at least about 14 days greater, or at least about 21
days greater compared to a comparable dose of FVIII not linked to
XTEN. In another embodiment, administration of a
coagulation-effective dose of a CFXTEN fusion protein to a subject
in need thereof can result in a gain in time between consecutive
doses necessary to maintain blood levels of about 0.1 IU/ml of at
least 48 h, or at least 72 h, or at least about 96 h, or at least
about 120 h, or at least about 7 days, or at least about 14 days,
or at least about 21 days between consecutive doses compared to a
FVIII not linked to XTEN and administered at a comparable dose.
[0298] In one embodiment, the present invention provides CFXTEN
fusion proteins and pharmaceutical compositions comprising CFXTEN
that exhibit, when administered to a subject in need thereof, an
increase in AUC of 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 a 100%, or at least about 150%, or at least about
200%, or at least about 300%, or at least about 500%, or at least
about 1000%, or at least about a 2000% compared to the
corresponding FVIII not linked to the XTEN and administered to a
subject at a comparable dose. The pharmacokinetic parameters of a
CFXTEN can be determined by standard methods involving dosing, the
taking of blood samples at timed intervals, and the assaying of the
protein using ELISA, HPLC, radioassay, clotting assays, the assays
of Table 49, or other methods known in the art or as described
herein, followed by standard calculations of the data to derive the
half-life and other PK parameters.
[0299] In one embodiment, a smaller IU 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 fusion protein is
administered in comparison to the corresponding FVIII not linked to
the XTEN under a dose regimen needed to maintain hemostasis and the
fusion protein achieves a comparable area under the curve as the
corresponding IU amount of the FVIII not linked to the XTEN needed
to maintain hemostasis. In another embodiment, the CFXTEN fusion
protein or a pharmaceutical compositions comprising CFXTEN requires
less frequent administration for routine prophylaxis of a
hemophilia A subject, wherein the dose of fusion protein 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 fusion protein achieves a
comparable area under the curve as the corresponding FVIII not
linked to the XTEN and administered to the subject. In yet other
embodiments, an accumulative smaller IU 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 the fusion protein
is administered to a subject in comparison to the corresponding IU
amount of the FVIII not linked to the XTEN under a dose regimen
needed to maintain a blood concentration of 0.1 IU/ml, yet the
fusion protein achieves at least a comparable area under the curve
as the corresponding FVIII not linked to the XTEN. The accumulative
smaller IU amount is measure for a period of at least about one
week, or about 14 days, or about 21 days, or about one month.
[0300] In one aspect, the invention provides CFXTEN compositions
designed to reduce binding by FVIII binding agents, thereby
increasing the terminal half-life of CFXTEN administered to a
subject, while still retaining procoagulant activity. It is
believed that the CFXTEN of the present invention have
comparatively higher and/or sustained activity achieved by reduced
active clearance of the molecule by the addition of unstructured
XTEN to the FVIII coagulation factor. The clearance mechanisms to
remove FVIII from the circulation have yet to be fully elucidated.
Uptake, elimination, and inactivation of coagulation proteins can
occur in the circulatory system as well as in the extravascular
space. Coagulation factors are complex proteins that interact with
a large number of other proteins, lipids, and receptors, and many
of these interactions can contribute to the elimination of CFs from
the circulation. The protein von Willebrand factor is an example of
a FVIII binding agent that binds to FVIII. Factor VIII and von
Willebrand factor (VWF) circulate in the blood as a tight,
non-covalently linked complex in which VWF serves as a carrier that
likely contributes to the protection of FVIII from active cleavage
mechanisms, yet nevertheless results in a limitation on the
terminal half-life of FVIII. For example: (i) VWF stabilizes the
heterodimeric structure of FVIII; (ii) VWF protects FVIII from
proteolytic degradation by phospholipid-binding proteases like
activated protein C and activated FX (FXa); (iii) VWF interferes
with binding of FVIII to negatively charged phospholipid surfaces
exposed within activated platelets; (iv) VWF inhibits binding of
FVIII to activated FIX (FIXa), thereby denying FVIII access to the
FX-activating complex; and (v) VWF prevents the cellular uptake of
FVIII (Lenting, P. J., et al., J Thrombosis and Haemostasis (2007)
5(7):1353-1360). In addition, LDL receptor-related protein (LRP1,
also known as .alpha.2-macrogobulin receptor or CD91) has been
identified as a candidate clearance receptor for FVIII, with LRP1
binding sites identified on both chains of the heterodimer form of
FVIII (Lenting P J, et al., J Biol Chem (1999) 274: 23734-23739;
Saenko E L, et al., J Biol Chem (1999) 274: 37685-37692). LRPs are
involved in the clearance of a diversity of ligands including
proteases, inhibitors of the Kunitz type, protease serpin
complexes, lipases and lipoproteins (Narita, et al., Blood (1998)
2:555-560). It has been shown that the light chain, but not the
heavy chain, of factor VIII binds to surface-exposed LRP1 receptor
protein (Lentig et al. (J Biol Chem (1999) 274(34):23734-23739; and
U.S. Pat. No. 6,919,311), which suggests that LRP1 may play an
essential role in the active clearance of proteins like FVIII.
While the VWF-FVIII interaction is of high affinity (<1 nM), the
complex is nevertheless in a dynamic equilibrium, such that a small
but significant portion of the FVIII molecules (5-8%) circulate as
a free protein (Leyte A, et al., Biochem J (1989) 257: 679-683; Noe
D A. Haemostasis (1996) 26: 289-303). As such, a portion of native
FVIII is unprotected by VWF, allowing active clearance mechanisms
to remove the unprotected FVIII from the circulation.
[0301] In one embodiment, the invention provides CFXTEN that
associate with VWF but have enhanced protection from active
clearance receptors conferred by the incorporation of two more XTEN
at one or more locations within the FVIII molecule (e.g., locations
selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or
FIGS. 8-9), wherein the XTEN interfere with the interaction of the
resulting CFXTEN with those clearance receptors with the result
that the pharmacokinetic properties of the CFXTEN is enhanced
compared to the corresponding FVIII not linked to XTEN. In another
embodiment, the invention provides CFXTEN that have reduced binding
affinity with VWF of at least 5% less, or about 10%, or about 20%,
or about 40%, or about 50%, or about 60%, or about 70% less, but
are nevertheless configured to have enhanced protection from active
clearance receptors conferred by the incorporation of XTEN at one
or more locations within the FVIII molecule, wherein the XTEN
interfere with the interaction of factor VIII with those receptors.
In the foregoing embodiments, the CFXTEN have an increased terminal
half-life of at least about 12 h, or 24 h, or 48 h, or 72 h, or 96
h, or 120 h, or 144 h, or 7 days, or 10 days, or 14 days, or 21
days compared to the FVIII not linked to XTEN. The invention
provides a method to create CFXTEN with reduced clearance wherein
the CFXTEN fusion proteins created with the multiple insertions are
evaluated for inhibition of binding to clearance receptors,
compared to FVIII not linked to XTEN, using in vitro binding assays
or in vivo pharmacokinetic models described herein or other assays
known in the art, and selecting those that demonstrate reduced
binding yet retain procoagulant FVIII activity. In addition, the
foregoing fusion proteins can be optimized to have increased Ratio
XTEN Radii of at least 2.0-3.5 in order to achieve pharmacokinetic
properties that are further enhanced. Table 5, Table 6, Table 7,
Table 8, and Table 9 and FIGS. 8-9 provide non-limiting examples of
XTEN insertion points within the factor VIII sequence. Using such
insertion points, the invention contemplates CFXTEN compositions
that have configurations with multiple XTEN inserted with about
100, or about 200, or about 300, or about 400, or about 500 amino
acids separating at least three XTEN to further increase the
protection against active clearance mechanisms and, hence, increase
the terminal half-life of the CFXTEN. Not to be bound by a
particular theory, the XTEN of the CFXTEN compositions with high
net charge (e.g., CFXTEN comprising AE family XTEN) are expected,
as described above, 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, the XTEN of the CFXTEN compositions
with a low (or no) net charge (e.g., CFXTEN comprising AG family
XTEN) are expected to have a higher degree of interaction with
surfaces that, while contributing to active clearance, can
potentiate the activity of the associated coagulation factor, given
the known contribution of cell (e.g., platelets) and vascular
surfaces to the coagulation process and the intensity of activation
of coagulation factors (Zhou, R., et al., Biomaterials (2005)
26(16):2965-2973; London, F., et al. Biochemistry (2000)
39(32):9850-9858). The invention, in part, takes advantage of the
fact that certain ligands wherein reduced binding to a clearance
receptor, either as a result of a decreased on-rate or an increased
off-rate, may be effected by the obstruction of a receptor site by
an inserted XTEN forming random coil, resulting in the reduced
binding. The choice of the particular configuration of the CFXTEN
fusion protein can be tested by methods disclosed herein to confirm
those configurations that reduce the degree of binding to a
clearance receptor such that a reduced rate of active clearance is
achieved. In one embodiment, the CFXTEN comprises a FVIII-XTEN
sequence that has one or more XTEN inserted at locations selected
from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9
wherein the terminal half-life of the CFXTEN is increased at least
about two-fold, or at least about three-fold, or at least about
four-fold, or at least about five-fold, or at least about six-fold,
or at least about eight-fold, or at least about ten-fold, or at
least about twenty-fold compared to a FVIII not linked to an XTEN.
In another embodiment, the CFXTEN comprises a FVIII-XTEN sequence
that has a first and at least a second XTEN inserted at a first and
second location selected from Table 5, Table 6, Table 7, Table 8,
and Table 9 or FIGS. 8-9 wherein the terminal half-life of the
CFXTEN is increased at least about two-fold, or at least about
three-fold, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
eight-fold, or at least about ten-fold, or at least about
twenty-fold compared to a FVIII not linked to an XTEN. In yet
another embodiment, the CFXTEN comprises a FVIII-XTEN sequence that
incorporates multiple XTEN sequences using three of more XTEN
insertion locations selected from Table 5, Table 6, Table 7, Table
8, and Table 9 or FIGS. 8-9 separated by about 100, or about 200,
or about 300, or about 400, or about 500 amino acids, wherein the
terminal half-life of the CFXTEN is increased at least about
two-fold, or at least about three-fold, or at least about
four-fold, or at least about five-fold, or at least about six-fold,
or at least about eight-fold, or at least about ten-fold, or at
least about twenty-fold compared to a FVIII not linked to an XTEN.
In the foregoing embodiments hereinabove described in this
paragraph, the XTEN incorporated into the CFXTEN configurations can
be identical or they can be different, and can have 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 to a sequence from any one
of Tables 3, 4, and 13-17, and can optionally include one or more
cleavage sequences from Table 12, facilitating release of one or
more of the XTEN from the CFXTEN fusion protein.
[0302] In one embodiment, the invention provides CFXTEN that
enhance the pharmacokinetics of the fusion protein by linking one
or more XTEN to the FVIII component of the fusion protein wherein
the fusion protein has an increase in apparent molecular weight
factor of at least about two-fold, or at least about three-fold, or
at least about four-fold, or at least about five-fold, or at least
about six-fold, or at least about seven-fold, or at least about
eight-fold, or at least about ten-fold, or at least about
twelve-fold, or at least about fifteen-fold, and wherein the
terminal half-life of the CFXTEN when administered to a subject is
increased at least about two-fold, or at least about four-fold, or
at least about eight-fold, or at least about 10-fold or more
compared to the corresponding FVIII not linked to XTEN. In the
foregoing embodiment, wherein at least two XTEN molecules are
incorporated into the CFXTEN, the XTEN can be identical or they can
be of a different sequence composition, net charge, or length. The
XTEN can have 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
to a sequence from any one of Tables 3, 4, and 13-17, and can
optionally include one or more cleavage sequences from Table 12,
facilitating release of one or more of the XTEN from the CFXTEN
fusion protein.
[0303] Thus, the invention provides CFXTEN compositions in which
the degree of activity, bioavailability, half-life or
physicochemical characteristic of the fusion protein can be
tailored by the selection and placement of the type and length of
the XTEN in the CFXTEN compositions. Accordingly, the invention
contemplates compositions in which a FVIII from Table 1 and XTEN or
XTEN fragment from any one of Tables 3, 4, or 13-17 are produced,
for example, in a configuration selected from any one of formulae
I-VIII or the XTEN are inserted at locations selected from Table 5,
Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 such that the
construct has the desired property.
[0304] The invention provides methods to produce the CFXTEN
compositions that can maintain the FVIII component at therapeutic
levels in a subject in need thereof for at least a two-fold, or at
least a three-fold, or at least a four-fold, or at least a
five-fold greater period of time compared to comparable dosages of
the corresponding FVIII not linked to XTEN. In one embodiment of
the method, the subject is receiving routine prophylaxis to prevent
bleeding episodes. In another embodiment of the method, the subject
is receiving treatment for a bleeding episode. In another
embodiment of the method, the subject is receiving treatment to
raise the circulating blood concentration of procoagulant FVIII
above 1%, or above 1-5%, or above 5-40% relative to FVIII
concentrations in normal plasma. "Procoagulant" as used herein has
its general meaning in the art and generally refers to an activity
that promotes clot formation, either in an in vitro assay or in
vivo. The method to produce the compositions that can maintain the
FVIII component at therapeutic levels includes the steps of
selecting one or more XTEN appropriate for conjugation to a FVIII
to provide the desired pharmacokinetic properties in view of a
given dose and dose regimen, creating a gene construct that encodes
the CFXTEN in one of the configurations disclosed herein,
transforming an appropriate host cell with an expression vector
comprising the encoding gene, expressing the fusion protein under
suitable culture conditions, recovering the CFXTEN, administration
of the CFXTEN to a mammal followed by assays to verify the
pharmacokinetic properties and the activity of the CFXTEN fusion
protein (e.g., the ability to maintain hemostasis or serve as a
procoagulant) and the safety of the administered composition. Those
compositions exhibiting the desired properties are selected for
further use. CFXTEN created by the methods provided herein can
result in increased efficacy of the administered composition by,
amongst other properties, maintaining the circulating
concentrations of the procoagulant FVIII component at therapeutic
levels for an enhanced period of time.
[0305] The invention provides methods to assay the CFXTEN fusion
proteins of differing composition or configuration in order to
provide CFXTEN with the desired degree of procoagulant and
therapeutic activity and pharmacokinetic properties, as well as a
sufficient safety profile. Specific in vitro and in vivo assays or
animal models are used to assess the activity and functional
characteristics of each configured CFXTEN and/or FVIII component to
be incorporated into CFXTEN, including but not limited to the
assays of the Examples, those assays of Table 49, as well as the
following assays or other such assays known in the art for assaying
the properties and effects of FVIII. Functional assays can be
conducted that allow determination of coagulation activity, such as
one-stage clotting assay and two-stage clotting assay (Barrowcliffe
T W, Semin Thromb Hemost. (2002) 28(3):247-256), activated partial
prothrombin (aPTT) assays (Belaaouaj A A et al., J. Biol. Chem.
(2000) 275:27123-8; Diaz-Collier J A. Haemost (1994) 71:339-46),
chromogenic FVIII assays (Lethagen, S., et al., Scandinavian J
Haematology (1986) 37:448-453), or animal model pharmacodynamic
assays including bleeding time or thrombelastography (TEG or
ROTEM), among others. Other assays include determining the binding
affinity of a CFXTEN for the target substrate using binding or
competitive binding assays, such as Biacore assays with chip-bound
receptors or binding proteins or ELISA assays, as described in U.S.
Pat. No. 5,534,617, assays described in the Examples herein,
radio-receptor assays, or other assays known in the art. Other
assays to determine the binding of FVIII inhibitors to CFXTEN
include the Bethesda assay or the Nijmegen modification of the
Bethesda assay. The foregoing assays can also be used to assess
FVIII sequence variants (assayed as single components or as CFXTEN
fusion proteins) and can be compared to the native FVIII to
determine whether they have the same degree of procoagulant
activity as the native CF, or some fraction thereof such that they
are suitable for inclusion in CFXTEN; e.g., at least about 10%, or
at least about 20$, or 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% of the activity compared
to the native FVIII.
[0306] Dose optimization is important for all drugs. A
therapeutically effective dose or amount of the CFXTEN varies
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the administered
fusion protein to elicit a desired response in the individual. For
example, a standardized single dose of FVIII for all patients
presenting with diverse bleeding conditions or abnormal clinical
parameters (e.g., neutralizing antibodies) may not always be
effective. Hemophilia A patients with trauma, who have undergone
surgery, or that have high titers of FVIII inhibitory antibodies
generally will require higher and more frequent dosing. Generally,
dosage level is adjusted in frequency, duration, and units in
keeping with the severity and duration of each patient's bleeding
episode. Accordingly, the CFXTEN is included in the
pharmaceutically acceptable carrier, delivery vehicle, or
stabilizer in an amount sufficient to deliver to a patient a
therapeutically effective amount of the fusion protein to stop
bleeding, as measured by standard clotting assays. A consideration
of these factors is well within the purview of the ordinarily
skilled clinician for the purpose of determining the
therapeutically or pharmacologically effective amount of the CFXTEN
and the appropriated dosing schedule, versus that amount that would
result in insufficient potency such that clinical improvement or
the arrest of bleeding is not achieved.
[0307] The invention provides methods to establish a dose regimen
for the CFXTEN pharmaceutical compositions of the invention. The
methods include administration of consecutive doses of a
therapeutically effective amount of the CFXTEN pharmaceutical
composition using variable periods of time between doses to
determine that interval of dosing sufficient to achieve and/or
maintain the desired parameter, blood level or clinical effect;
such consecutive doses of a therapeutically effective amount at the
effective interval establishes the therapeutically effective dose
regimen for the CFXTEN for a factor VIII-related disease state or
condition. A prophylactically effective amount refers to an amount
of CFXTEN required for the period of time necessary to prevent a
physiologic or clinical result or event; e.g., delayed onset of a
bleeding episode or maintaining blood concentrations of
procoagulant FVIII or equivalent above a threshold level (e.g.,
1-5% to 5-40% of normal). In the methods of treatment, the dosage
amount of the CFXTEN that is administered to a subject ranges from
about 5 to 300 IU/kg/dose, or from about 10 to 100 IU/kg/dose, or
from about 20 to about 65 IU/kg/dose, or from about 20 to about 40
IU/kg/dose for a subject. A suitable dosage may also depend on
other factors that may influence the response to the drug; e.g.,
bleeding episodes generally requiring higher doses at more frequent
intervals compared to prophylaxis.
[0308] In some embodiments, the method comprises administering a
therapeutically-effective amount of a pharmaceutical composition
comprising a CFXTEN fusion protein composition and at least one
pharmaceutically acceptable carrier to a subject in need thereof,
wherein the administration results in a greater improvement in at
least one parameter or physiologic condition associated with a
FVIII deficiency or coagulopathy, or results in a more favorable
clinical outcome mediated by the FVIII component of the CFXTEN
compared to the effect on the parameter, condition or clinical
outcome mediated by administration of a pharmaceutical composition
comprising a FVIII not linked to XTEN and administered at a
comparable dose. Non-limiting examples of parameters that are
improved include blood concentration of procoagulant FVIII, a
reduced activated partial prothrombin (aPTT) assay time, a reduced
one-stage or two-stage clotting assay time, delayed onset of a
bleeding episode, a reduced chromogenic FVIII assay time, a reduced
bleeding time, resolution of a bleeding event, or a reduced
Bethesda titer to the CFXTEN relative to native FVIII. In one
embodiment of the foregoing, the improvement is achieved by
administration of the CFXTEN pharmaceutical composition at a dose
that achieves a circulating concentration of procoagulant FVIII (or
equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal
FVIII levels), thereby establishing the therapeutically effective
dose. In another embodiment of the foregoing, the improvement is
achieved by administration of multiple consecutive doses of the
CFXTEN pharmaceutical composition using a therapeutically effective
dose regimen that maintains a circulating concentration of
procoagulant FVIII (or equivalent) above a threshold level (e.g.,
1-5% to 5-40% of normal FVIII levels) for the length of the dosing
period. In another embodiment of the method, the administration of
at least two consecutive doses of the CFXTEN pharmaceutical
composition using a therapeutically effective dose regimen
maintains a circulating concentration of procoagulant FVIII (or
equivalent) above about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, or
40% of normal FVIII levels for a period that is at least about
three-fold longer; alternatively at least about four-fold longer;
alternatively at least about five-fold longer; alternatively at
least about six-fold longer; alternatively at least about
seven-fold longer; alternatively at least about eight-fold longer;
alternatively at least about nine-fold longer or at least about
ten-fold longer compared to a FVIII not linked to XTEN and
administered using a therapeutically effective dose regimen
[0309] In one embodiment, the CFXTEN or a pharmaceutical
compositions comprising CFXTEN administered at a therapeutically
effective dose regimen results in a gain in time of at least about
three-fold longer; alternatively at least about four-fold longer;
alternatively at least about five-fold longer; alternatively at
least about six-fold longer; alternatively at least about
seven-fold longer; alternatively at least about eight-fold longer;
alternatively at least about nine-fold longer or at least about
ten-fold longer between at least two consecutive C.sub.max peaks
and/or C.sub.min troughs for blood levels of the fusion protein
compared to the corresponding biologically active protein of the
fusion protein not linked to the XTEN and administered at a
comparable dose regimen to a subject. In another embodiment, the
CFXTEN administered at a therapeutically effective dose regimen
results in a comparable improvement in one, or two, or three or
more measured parameters using less frequent dosing or a lower
total dosage in IUs of the fusion protein of the pharmaceutical
composition compared to the corresponding biologically active
protein component(s) not linked to the XTEN and administered to a
subject using a therapeutically effective dose regimen for the
FVIII. The measured parameters include any of the clinical,
biochemical, or physiological parameters disclosed herein, or
others known in the art for assessing subjects with factor
VIII-related conditions.
[0310] (b) Pharmacology and Pharmaceutical Properties of CFXTEN
[0311] The present invention provides CFXTEN compositions
comprising FVIII covalently linked to XTEN that have enhanced
pharmaceutical and pharmacology properties compared to FVIII not
linked to XTEN, as well as methods to enhance the therapeutic
and/or procoagulant effect of the FVIII components of the
compositions. In addition, the invention provides CFXTEN
compositions with enhanced properties compared to those art-known
fusion proteins of factor VIII containing albumin, immunoglobulin
polypeptide partners, polypeptides of shorter length and/or
polypeptide partners with repetitive sequences. In addition, CFXTEN
fusion proteins provide significant advantages over chemical
conjugates, such as pegylated constructs of FVIII, notably the fact
that recombinant CFXTEN fusion proteins can be made in host cell
expression systems, which can reduce time and cost at both the
research and development and manufacturing stages of a product, as
well as result in a more homogeneous, defined product with less
toxicity from both the product and metabolites of the CFXTEN
compared to pegylated conjugates.
[0312] As therapeutic agents, the CFXTEN possesses a number of
advantages over therapeutics not comprising XTEN, including one or
more of the following non-limiting properties: increased
solubility, increased thermal stability, reduced immunogenicity,
increased apparent molecular weight, reduced renal clearance,
reduced proteolysis, reduced metabolism, enhanced therapeutic
efficiency, less frequent dosage regimen with increased time
between doses capable of maintaining hemostasis in a subject with
hemophilia A, the ability to administer the CFXTEN composition
subcutaneously or intramuscularly, a "tailored" rate of absorption
when administered subcutaneously or intramuscularly, enhanced
lyophilization stability, enhanced serum/plasma stability,
increased terminal half-life, increased solubility in blood stream,
decreased binding by neutralizing antibodies, decreased active
clearance, tailored substrate binding affinity, stability to
degradation, stability to freeze-thaw, stability to proteases,
stability to ubiquitination, ease of administration, compatibility
with other pharmaceutical excipients or carriers, persistence in
the subject, increased stability in storage (e.g., increased
shelf-life), and the like. The net effect of the enhanced
properties is that the use of a CFXTEN composition can result in an
overall enhanced therapeutic effect compared to a FVIII not linked
to XTEN, result in economic benefits associated with less frequent
dosing, and/or result in improved patient compliance when
administered to a subject with a factor VIII-related condition.
[0313] The invention provides CFXTEN compositions and
pharmaceutical compositions comprising CFXTEN wherein the
administration of the composition results in an improvement in at
least one of the clinical or biochemical parameters disclosed
herein as being useful for assessing the subject diseases,
conditions or disorders. Non-limiting examples of parameters that
are improved include blood concentrations of procoagulant FVIII, a
reduced activated partial prothrombin (aPTT) assay time, a reduced
one-stage or two-stage clotting assay time, delayed onset of a
bleeding episode, a reduced chromogenic FVIII assay time, a reduced
bleeding time, resolution of a bleeding event, or a reduced
Bethesda titer to the CFXTEN relative to native FVIII. The enhanced
pharmacokinetic properties of the subject CFXTEN permits using an
accumulatively lower IU dose of fusion protein to maintain the
parameter compared to the corresponding FVIII component not linked
to the XTEN. In one embodiment, the total dose in IUs of an CFXTEN
of the embodiments needed to achieve and maintain the improvement
in the at least one parameter for about 2-7 days is at least about
three-fold lower, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
eight-fold, or at least about 10-fold lower compared to the
corresponding FVIII component not linked to the XTEN. In another
embodiment, the total dose in IUs of a subject CFXTEN needed to
achieve and maintain the improvement in the at least one parameter
over two, three or four consecutive doses is at least about
three-fold lower, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
eight-fold, or at least about 10-fold lower compared to the
corresponding FVIII component not linked to the XTEN.
Alternatively, the invention provides certain embodiments of CFXTEN
wherein the period between consecutive administrations that results
in achieving and maintaining the improvement in at least one
parameter is at least about three-fold, or at least about
four-fold, or at least about five-fold, or at least about six-fold,
or at least about eight-fold, or at least about 10-fold longer
compared to the corresponding FVIII component not linked to the
XTEN and administered at a comparable IU dose. Alternatively, the
invention provides certain embodiments of CFXTEN wherein
administration of 25 IU/kg results in a 30% improvement in a aPTT
assay (or similar coagulation assay) time in a hemophilia A subject
compared to 25 IU/kg of the corresponding FVIII not linked to XTEN
when assayed at about 2-7 days after administration. In yet another
embodiment, the invention provides CFXTEN wherein administration of
25 IU/kg results in a 30% improvement in a bleeding time assay time
in a hemophilia A subject compared to 25 IU/kg of the corresponding
FVIII not linked to XTEN when assayed at about 2-7 days after
administration.
[0314] In one embodiment, XTEN as a fusion partner increases the
solubility of the FVIII payload. Accordingly, where enhancement of
the pharmaceutical or physicochemical properties of the FVIII is
desirable, such as the degree of aqueous solubility or stability,
the length and/or the motif family composition of the XTEN
sequences incorporated into the fusion protein may each be selected
to confer a different degree of solubility and/or stability on the
respective fusion proteins such that the overall pharmaceutical
properties of the CFXTEN composition are enhanced. The CFXTEN
fusion proteins can be constructed and assayed, using methods
described herein, to confirm the physicochemical properties and the
choice of the XTEN length sequence or location adjusted, as needed,
to result in the desired properties. In one embodiment, the CFXTEN
has an aqueous solubility that is at least about 25% greater
compared to a FVIII not linked to the XTEN, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
75%, or at least about 100%, or at least about 200%, or at least
about 300%, or at least about 400%, or at least about 500%, or at
least about 1000% greater than the corresponding FVIII not linked
to XTEN.
[0315] The invention provides methods to produce and recover
expressed CFXTEN from a host cell with enhanced solubility and ease
of recovery compared to FVIII not linked to XTEN. In one
embodiment, the method includes the steps of transforming a
eukaryotic host cell with a polynucleotide encoding a CFXTEN with
one or more XTEN components of cumulative sequence length greater
than about 100, or greater than about 200, or greater than about
400, or greater than about 600, or greater than about 800, or
greater than about 1000, or greater than about 2000, or greater
than about 3000 amino acid residues, expressing the CFXTEN fusion
protein in the host cell under suitable culture and induction
conditions, and recovering the expressed fusion protein in soluble
form. In one embodiment, the one or more XTEN of the CFXTEN fusion
proteins each have at least about 80% sequence identity, or 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%,
to about 100% sequence identity compared to one or more XTEN
selected from any one of Tables 4, and 13-17, or fragments thereof,
and the FVIII have at least about 80% sequence identity, or 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 100% sequence identity compared to a FVIII selected from Table
1, and the CFXTEN components are in an N- to C-terminus
configuration selected from any one of the configuration
embodiments disclosed herein.
VI). Uses of the CFXTEN Compositions
[0316] The invention provides methods and regimens for achieving a
beneficial effect in a factor VIII-related condition by the
administration of compositions comprising CFXTEN. As used herein,
"factor VIII-related condition" is intended to include, but is not
limited to factor VIII deficiencies, bleeding disorders related to
factor VIII deficiency, hemophilia A, neutralization of factor VIII
by anti-FVIII antibodies or other factor VIII inhibitors, and
bleeding episodes resulting from trauma or surgery or vascular
injury and other such conditions that can be ameliorated or
corrected by administration of FVIII to a subject. The inventive
methods achieve a beneficial effect while addressing disadvantages
and/or limitations of other methods of treatment using factor VIII
preparations that have a relatively short terminal half-life,
require frequent administrations, are neutralized by inhibitors or
have unfavorable pharmacoeconomics.
[0317] Hemostasis is regulated by multiple protein factors, and
such proteins, as well as analogues thereof, have found utility in
the treatment of factor VIII-related conditions. However, the use
of commercially-available FVIII has met with less than optimal
success in the management of subjects afflicted with such
conditions. In particular, dose optimization and frequency of
dosing is important for FVIII used in maintaining circulating FVIII
concentrations above threshold levels needed for hemostasis, as
well as the treatment or prevention of bleeding episodes in
hemophilia A subjects. The fact that commercially-available FVIII
products have a short half-life necessitates frequent dosing in
order to achieve clinical benefit, which results in difficulties in
the management of such patients.
[0318] As established by the Subcommittee on Factor VIII and Factor
IX of the Scientific and Standardization Committee of the
International Society on Thrombosis and Haemostasis (posted on the
ISTH Website 29 November, 2000), the most widely used measure of
the severity of hemophilia A is established by determining the
circulating concentrations of plasma FVIII procoagulant levels,
with persons with <1% (<0.01 IU/ml) factor VIII defined as
severe; 1-5% (0.01-0.05 IU/ml) as moderately severe; and >5-40%
(0.05-<0.40 IU/ml) as mild, where normal is 1 IU/ml of factor
VIIIC (100%).
[0319] The invention provides methods of treating a subject
suffering from or at risk of developing a factor VIII-related
condition. More particularly, the invention provides methods for
treating or preventing controlling bleeding in subject. The subject
can be any animal but preferably is a human. In one embodiment, the
method comprises administering a coagulation-effective amount of a
CFXTEN composition to the subject in need thereof. In another
embodiment, the method comprises the step of administering to the
subject with a bleed a coagulation-effective amount of a
pharmaceutical composition that includes a CFXTEN, wherein the
administration results in an arrest or attenuation of the bleeding.
As used herein, "coagulation-effective amount" is an amount of a
FVIII composition that, when administered to a subject, is
sufficient to effect hemostasis or other beneficial or desired
therapeutic (including preventative) result. In practicing the
present invention, it will be understood that a
coagulation-effective amount can be administered in one or more
administrations. Precise coagulation-effective amounts of the
pharmaceutical composition to be administered will be guided by the
judgment of the practitioner, however, the unit dose will generally
depend on the severity or cause of the bleeding and the amount of
pre-existing FVIII in the subject. In a particular embodiment of
the method of treating a bleed, a coagulation-effective amount of a
pharmaceutical compositions comprising CFXTEN is administered to a
subject suffering from a bleeding episode, wherein the
administration results in the resolution of the bleeding for a
duration at least two-fold, or at least three-fold, or at least
four-fold longer compared to a FVIII not linked to XTEN and
administered to a comparable subject with a comparable bleed at a
comparable dose.
[0320] In another embodiment, the administration of a
coagulation-effective amount of a CFXTEN composition to a subject
with a factor VIII-related condition results in a 10%, or 20%, or
30%, or 40%, or 50%, or 60%, or 70% or greater improvement of one
or more biochemical, physiological or clinical parameters
associated with the FVIII condition, compared to the FVIII not
linked to XTEN, when measured at between 2 and 7 days after
administration. In another embodiment, the administration of a
coagulation-effective amount of a CFXTEN composition to the subject
in need thereof results in an improvement of one or more
biochemical, physiological or clinical parameters associated with
the FVIII condition for a period at least two-fold longer, or at
least four-fold longer, or at least five-fold longer, or at least
six-fold longer compared to period achieved by a FVIII not linked
to XTEN and administered at a comparable dose. Non-limiting
examples of parameters that are improved for a longer duration
include blood concentrations of procoagulant FVIII, a reduced
activated partial prothrombin (aPTT) assay time, a reduced
one-stage or two-stage clotting assay time, delayed onset of a
bleeding episode, a reduced chromogenic FVIII assay time, a reduced
bleeding time, among other FVIII-related parameters known in the
art. In the foregoing embodiments of the paragraph, the
administered CFXTEN comprises a FVIII with at least about 80%, or
at least about 90%, or at least about 95%, or at least about 97%,
or at least about 99% sequence identity to a factor VIII of Table 1
and one or more XTEN sequences with at least about 80%, or at least
about 90%, or at least about 95%, or at least about 97%, or at
least about 99% sequence identity to an XTEN of Table 4 inserted
into the FVIII at one or more locations selected from Table 5,
Table 6, Table 7, Table 8, and Table 9, or as depicted in FIGS.
8-9. In certain embodiments, at least one XTEN insertion site of
the CFXTEN is selected from amino acids 32, 220, 224, 336, 339,
390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered
relative to mature native human FVIII).
[0321] In a particular embodiment of the method of treatment, a
coagulation-effective amount of CFXTEN fusion protein administered
to a subject suffering from hemophilia A is sufficient to increase
the circulating FVIII procoagulant concentration to greater than
0.05 IU/ml and to maintain hemostasis for at least about 24 h, or
at least about 48 h, or at least about 72 h, or at least about 96
h, or at least about 120 h, or at least about 144 h, or at least
about 168 h, or greater. In another embodiment, the administration
of a coagulation-effective amount of a pharmaceutical composition
comprising CFXTEN to a subject in need thereof results in a greater
reduction in a one-stage clotting assay time of at least about 5%,
or about 10%, or about 20%, or about 30%, or about 40%, or about
50%, or about 60%, or about 70%, or more in a blood sample from the
subject at 2-7 days after the administration compared to the assay
time in a subject after administration of a comparable amount of
the corresponding FVIII not linked to XTEN. In another embodiment,
the administration of a therapeutically effective amount of a
CFXTEN or a pharmaceutical compositions comprising CFXTEN to a
subject in need thereof results in a greater reduction in the
activated partial prothrombin time of at least about 5%, or about
10%, or about 20%, or about 30%, or about 40%, or about 50%, or
about 60%, or about 70%, or more in a blood sample from the subject
2-7 days after administration compared to the activated partial
prothrombin time in a subject after administration of a comparable
amount of the corresponding FVIII not linked to XTEN. In another
embodiment, the administration of a CFXTEN or a pharmaceutical
compositions comprising CFXTEN to a subject in need thereof using a
therapeutically effective amount results in maintenance of
activated partial prothrombin times within 30% of normal in a blood
sample from the subject for a period of time that is at least
two-fold, or at least about three-fold, or at least about four-fold
longer compared to that of a FVIII not linked to XTEN and
administered to a subject using a comparable dose.
[0322] In one embodiment of the method of treatment, the CFXTEN
fusion protein is formulated and administered as a pharmaceutical
composition comprising the CFXTEN in admixture with a
pharmaceutically acceptable excipient. Methods for making
pharmaceutical formulations are well known in the art. Techniques
and formulations generally may be found in Remington's
Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton,
Pa. 1990 (See, also, Wang and Hanson, Parenteral Formulations of
Proteins and Peptides: Stability and Stabilizers, Journal of
Parenteral Science and Technology, Technical Report No. 10, Supp.
42-2S (1988)).
[0323] In another aspect, the invention provides a regimen for
treating a hemophilia A patient, said regimen comprising a
composition comprising a CFXTEN fusion protein. In one embodiment
of the regimen for treating a hemophilia A patient, the regimen
further comprises the step of determining the amount of
pharmaceutical composition comprising the CFXTEN needed to achieve
hemostasis in the patient. In some embodiments of the regimen, (i)
a smaller IU 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 of
the pharmaceutical composition comprising CFXTEN is administered to
a subject in need thereof in comparison to the corresponding
coagulation factor not linked to the XTEN under an otherwise same
dose regimen, and the fusion protein achieves a comparable area
under the curve (based on IU/ml) and/or a comparable therapeutic
effect as the corresponding FVIII not linked to the XTEN; (ii) the
pharmaceutical composition is administered less frequently (e.g.,
every three days, about every seven days, about every 10 days,
about every 14 days, about every 21 days, or about monthly) in
comparison to the corresponding FVIII not linked to the XTEN under
an otherwise same dose amount, and the fusion protein achieves a
comparable area under the curve and/or a comparable therapeutic
effect as the corresponding coagulation factor not linked to the
XTEN; or (iii) an accumulative smaller IU amount of at least about
20%, or about 30%, or about 40%, or about 50%, or about 60%, or
about 70%, or about 80%, or about 90% less of the pharmaceutical
composition is administered in comparison to the corresponding
FVIII not linked to the XTEN under an otherwise same dose schedule
and the CFXTEN fusion protein achieves a comparable therapeutic
effect as the corresponding FVIII not linked to the XTEN. The
accumulative smaller IU amount is measured for a period of at least
about one week, or about 14 days, or about 21 days, or about one
month. In the foregoing embodiments, the therapeutic effect can be
determined by any of the measured parameters described herein,
including but not limited to blood concentration of procoagulant
FVIII, a reduced activated partial prothrombin (aPTT) assay time, a
reduced one-stage or two-stage clotting assay time, delayed onset
of a bleeding episode, a reduced chromogenic FVIII assay time, a
reduced bleeding time, resolution of a bleeding event, or a reduced
Bethesda titer to the CFXTEN relative to native FVIII, fibrinogen
levels, or other assays known in the art for assessing
coagulopathies of FVIII. In another embodiment, the invention
provides CFXTEN for use in a regimen for a treating a hemophilia A
subject comprising administering an CFXTEN 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 disease compared to a FVIII not linked to XTEN and
administered using a comparable dose.
[0324] In one aspect, the present invention relates to a method of
preventing or treating the bleeding in a patient, optionally a
haemophilia A patient, having pre-existing inhibitor(s) against
FVIII. Inhibitory antibodies against FVIII commonly develop in
hemophiliacs, where the overall incidence of developing an
inhibitor is 15-30%, particularly in haemophiliacs who are heavily
exposed to FVIII concentrates (Algiman et al. Natural antibodies to
factor VIII (anti-hemophilic factor) in healthy individuals. PNAS
USA (1992) 89: 3795-3799). However, inhibitory antibodies also
occur in patients in auto-immune disorders, malignancies (such as
lymphoproliferative disorders, lymphomas and solid tumors), during
pregnancy and in the post-partum state. Inhibition can also occur
when antibodies interfere with the binding of FVIII to FIX and FX.
Simultaneously or alternatively, anti-FVIII antibodies can
interfere with the binding of von Willebrand factor and/or
phospholipids to FVIII, affecting coagulation and/or half-life of
FVIII. The presence of inhibitory antibodies is often first
detected with symptoms such as easy bruising and uncontrolled
bleeding, and is usually referred to as acquired hemophilia.
Anti-FVIII antibodies can be determined by different methods
including quantitation of anti-FVIII activity in coagulation
assays, ELISA for FVIII inhibitors and purification using
chromatography and immunoadsorption (Algiman et al., 1992).
Accordingly, the inventive methods are used in the treatment or
prevention of any condition associated with or characterized by the
presence of inhibitory antibodies to FVIII. In one embodiment, the
invention provides a method of treating a patient having a
pre-existing inhibitor against FVIII, the method comprising the
step of administering to the patient a coagulation-effective amount
of a CFXTEN fusion protein that must be administered to achieve
hemostasis, wherein the coagulation-effective amount of fusion
protein administered is reduced in comparison to the amount of
FVIII not linked to XTEN (or native FVIII) that must be
administered to achieve hemostasis. In the method, the reduced
amount of CFXTEN is about two-fold, or three-fold, or four-fold, or
five-fold less in IU/kg compared to the corresponding FVIII not
linked to XTEN. In another embodiment of the method, the amount of
CFXTEN that is administered as a dose to achieve hemostasis is at
least 20 to 40 IU/kg less, or 30 to 60 IU/kg less, or 40 to 80
IU/kg less, or 60 to 100 IU/kg less, or 100 to 140 IU/kg less, or
120 to 180 IU/kg less, or 140 to 200 IU/kg less compared to the
corresponding FVIII not linked to XTEN or to native FVIII required
to achieve hemostasis. In another embodiment, the invention
provides a method of treating a bleeding episode in a hemophilia A
subject having a titer of at least 10, or 20, or 30, or 40, or 50,
or 75, or 100, or 150, or 200 or more Bethesda units against a
FVIII not linked to XTEN, wherein the dose of CFXTEN fusion protein
required to arrest the bleeding epidose is at least two-fold, or
three-fold, or four-fold, or five-fold, or six-fold, or seven-fold,
or eight-fold, or nine-fold, or 10-fold less in comparison to the
amount of FVIII not linked to XTEN (or native FVIII) that must be
administered to achieve hemostasis in a comparable subject. It will
be understood by one of skill in the art that the amount of
procoagulant administered to maintain hemostasis will depend on the
severity of FVIII deficiency and/or the frequency or duration of
bleeding.
[0325] A particular object of the present invention relates to use
of CFXTEN with reduced binding by FVIII inhibitors that bind the A2
and/or C2 domains of Factor VIII as a drug. Such a drug is
advantageously used for maintaining hemostasis in a patient
suffering from haemophilia, wherein such patient has circulating
FVIII inhibitors directed against the A2 domain and/or C2 domain of
Factor VIII. In one embodiment, the invention provides a method of
treatment, the method comprising the step of administering to the
patient with a A2 domain-binding inhibitor a coagulation-effective
amount of a CFXTEN fusion protein, wherein the CFXTEN exhibits at
least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%
or less binding to an inhibitor that binds the A2 domain of FVIII,
compared to the FVIII not linked to XTEN or to native FVIII, and
wherein the administration results in hemostasis. In another
embodiment, the invention provides a method of treatment, the
method comprising the step of administering to the patient with a
C2 domain-binding inhibitor a coagulation-effective amount of a
CFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or
20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding
to an inhibitor that binds the C2 domain of FVIII, compared to the
FVIII not linked to XTEN or to native FVIII, and wherein the
administration results in hemostasis. The reduced binding of the
subject CFXTEN can be assayed directly by ELISA that detects FVIII
inhibitors, or measured indirectly by demonstration of reduced
inhibition of FVIII activity of the CFXTEN compared to native FVIII
in the presence of an inhibitor as measured by a factor VIII
chromogenic test or one-step assay as described herein, or other
suitable coagulation methods known in the art. Alternatively, the
subject CFXTEN can be measured for reduced (or absence of)
inhibition in the presence of known inhibitors by use of a modified
Bethesda assay. According to a particular aspect of the present
invention, a CFXTEN useful in the methods has reduced reactivity to
one or more antibodies from Table 10, as well as
naturally-occurring antibodies found in hemophilia patients. For
testing purposes, such and other inhibitory antibodies can be
obtained from humans (i.e. from the serum of patients which have
inhibitory antibodies) or can be obtained from mice, guinea pigs,
horses, goats, non-human primates and other mammals by immunization
with FVIII, or fragments thereof, more particularly with a fragment
comprising the all or part of the A2 or C2 domain, whether in
polyclonal or monoclonal form.
[0326] The invention further contemplates that the CFXTEN used in
accordance with the methods provided herein can be administered in
conjunction with other treatment methods and compositions (e.g.,
other coagulation proteins) useful for treating factor VIII-related
conditions, or conditions for which coagulation factor is
adjunctive therapy; e.g., bleeding episodes due to injury or
surgery.
[0327] In another aspect, the invention provides methods of
preparing a drug for a factor VIII-related condition, comprising
combining a factor VIII sequence selected from Table 1 with one or
more XTEN selected from Table 4 inserted in one or more insertion
sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9
to result in a drug that retains at least a portion of the activity
of the native FVIII. The invention provides a method of preparing a
pharmaceutical composition, comprising the step of combining the
drug of the foregoing embodiment with at least one pharmaceutically
acceptable carrier. In one embodiment of the method of preparing a
drug for a factor VIII-related condition, the factor VIII has a
sequence with at least about 80%, or at least about 90%, or at
least about 95%, or at least about 97%, or at least about 99%
sequence identity compared to a sequence selected from Table 1 and
the one or more XTEN has a sequence with at least about 80%, or at
least about 90%, or at least about 95%, or at least about 97%, or
at least about 99% sequence identity compared to a sequence
selected from any one of Tables 3, 4, and 13-17, or a fragment
thereof, wherein the one or more XTEN are inserted in one or more
locations selected from Table 5, Table 6, Table 7, Table 8, and
Table 9. In a particular embodiment of the foregoing, at least one
XTEN insertion site is selected from amino acids 32, 220, 224, 336,
339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered
relative to mature native human FVIII).In another embodiment of the
method, the CFXTEN comprises a sequence with at least about 80%, or
at least about 90%, or at least about 95%, or at least about 97%,
or at least about 99% sequence identity compared to a sequence
selected from any one of Table 21.
[0328] In another aspect, the invention provides a method of making
the CFXTEN compositions to achieve desired pharmacokinetic,
pharmacologic or pharmaceutical properties. In general, the steps
in the design and production of the inventive fusion protein
compositions, as illustrated in FIGS. 11-13, include: (1) the
selection of a FVIII (e.g., native proteins, sequences of Table 1,
analogs or derivatives with activity) to treat the particular
condition; (2) selecting one or more XTEN (e.g., sequences with at
least 80% identity to sequences set forth in Table 4) that will
confer the desired pharmacokinetic and physicochemical
characteristics on the resulting CFXTEN (e.g., the administration
of the CFXTEN composition to a subject results in the fusion
protein being maintained above 0.05-0.4 IU/ml for a greater period
compared to FVIII not linked to XTEN); (3) establishing a desired
N- to C-terminus configuration of the CFXTEN to achieve the desired
efficacy or PK parameters (e.g., selecting one or more insertion
sites from Table 5, Table 6, Table 7, Table 8, and Table 9); (4)
establishing the design of the expression vector encoding the
configured CFXTEN; (5) transforming a suitable host with the
expression vector; and (6) expressing and recovering the resultant
isolated CFXTEN fusion protein. In one embodiment of the method of
making CFXTEN, the XTEN for insertion are evaluated by the
application of Equation IV to maximize the Ratio XTEN Radii for the
fusion protein construct, with the XTEN resulting in values greater
than 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7,
or 2.8, or 2.9. or 3.0 being preferred. For those CFXTEN for which
an increase in half-life or an increased period of time spent above
the minimum coagulation-effective concentration is desired, the
XTEN chosen for incorporation generally have at least about 144, or
about 288, or about 432, or about 576, or about 864, or about 875,
or about 912, or about 923 amino acid residues where a single XTEN
is to be incorporated into the CFXTEN. In another embodiment, the
CFXTEN comprises a first XTEN of the foregoing lengths, and at
least a second XTEN of about 36, or about 42, or about 72, or about
144, or about 288, or about 576, or about 864, or about 875, or
about 912, or about 923, or about 1000 or more amino acid residues.
The location of the XTEN within the fusion protein can include one,
two, three, four, five or more locations selected from Table 5,
Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9. In one
embodiment, the method of design includes an insertion of XTEN into
the FVIII of at least one site selected from amino acids 32, 220,
224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910
(numbered relative to mature native human FVIII).
[0329] In another aspect, the invention provides methods of making
CFXTEN compositions to improve ease of manufacture, result in
increased stability, increased water solubility, and/or ease of
formulation, as compared to the native FVIII. In one embodiment,
the invention includes a method of increasing the water solubility
of a FVIII comprising the step of linking the FVIII with at least
about 80%, or about 90%, or about 95% identity to a sequence from
Table 1 to one or more XTEN at one, two, three, four, five or more
locations selected from Table 5, Table 6, Table 7, Table 8, and
Table 9 or FIGS. 8-9 wherein the XTEN is a sequence with at least
about 80%, or about 90%, or about 95% sequence identity compared to
a sequence from any one of Tables 3, 4, and 13-17 such that a
higher concentration in soluble form of the resulting CFXTEN can be
achieved, under physiologic conditions, compared to the FVIII in an
un-fused state. In a particular embodiment, the CFXTEN comprises a
FVIII linked to two, three, four, or five XTEN having at least
about 24, or about 36, or about 48, or about 60, or about 72, or
about 84, or about 96, or about 144, or about 288 amino acid
residues inserted at sites selected from Table 5, Table 6, Table 7,
Table 8, and Table 9 or FIGS. 8-9, in which the solubility of the
fusion protein under physiologic conditions is at least three-fold
greater than the corresponding FVIII not linked to XTEN, 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 30-fold, or at least 50-fold, or at
least 60-fold or greater than FVIII not linked to XTEN. Factors
that contribute to the property of XTEN to confer increased water
solubility of CFs when incorporated into a fusion protein include
the high solubility of the XTEN fusion partner and the low degree
of self-aggregation between molecules of XTEN in solution, as well
as expanding the hydrophilicity of FVIII external loops into which
the XTEN is inserted. In some embodiments, the method results in a
CFXTEN fusion protein wherein the water solubility is at least
about 20%, or at least about 30% greater, or at least about 50%
greater, or at least about 75% greater, or at least about 90%
greater, or at least about 100% greater, or at least about 150%
greater, or at least about 200% greater, or at least about 400%
greater, or at least about 600% greater, or at least about 800%
greater, or at least about 1000% greater, or at least about 2000%
greater under physiologic conditions, compared to the un-fused
FVIII. In one embodiment, the XTEN of the CFXTEN fusion protein is
a sequence with at least about 80%, or about 90%, or about 95%
sequence identity compared to a sequence from any one of Tables 3,
4, and 13-17. In another embodiment, the invention includes a
method of increasing the shelf-life of a FVIII comprising the step
of linking the FVIII with one or more XTEN at one or more sites
selected from Table 5, Table 6, Table 7, Table 8, and Table 9,
wherein the shelf-life of the resulting CFXTEN is extended compared
to the FVIII in an un-fused state. As used herein, shelf-life
refers to the period of time over which the procoagulant activity
of a FVIII or CFXTEN that is in solution, lyophilized or in some
other storage formulation remains stable without undue loss of
activity or that remains within release specifications established
for the pharmaceutical composition. A FVIII that degrades or
aggregates generally has reduced functional activity or reduced
bioavailability compared to one that remains in solution. Factors
that contribute to the ability of the method to extend the shelf
life of FVIII when incorporated into a fusion protein include
increased water solubility, reduced self-aggregation in solution,
and increased heat stability of the XTEN fusion partner. In
particular, the low tendency of XTEN to aggregate facilitates
methods of formulating pharmaceutical preparations containing
higher drug concentrations of CFs, and the heat-stability of XTEN
contributes to the property of CFXTEN fusion proteins to remain
soluble and functionally active for extended periods. The method
results in CFXTEN fusion proteins with prolonged or extended
shelf-life that exhibit greater activity relative to a FVIII
standard that has been subjected to the same storage and handling
conditions. The standard may be the un-fused full-length FVIII or a
commercially-available FVIII pharmaceutical composition. In one
embodiment, the method includes the step of formulating the
isolated CFXTEN with one or more pharmaceutically acceptable
excipients that enhance the ability of the XTEN to retain its
unstructured conformation and for the CFXTEN to remain soluble in
the formulation for a time that is greater than that of the
corresponding un-fused FVIII. In one embodiment, the method
comprises linking a FVIII selected from Table 1 to one or more XTEN
selected from any one of Tables 3, 4, and 13-17 inserted at one or
more sites selected from Table 5, Table 6, Table 7, Table 8, and
Table 9 and admixing with at least one pharmaceutically acceptable
excipient to create a pharmaceutical composition that retains
greater than about 100% of the procoagulant activity, or greater
than about 105%, 110%, 120%, 130%, 150% or 200% of the procoagulant
activity of a FVIII standard subjected to the same storage and
handling conditions when compared at a time point of at least 90
days, or at least 6 months, or at least 12 months. Shelf-life may
also be assessed in terms of functional activity remaining after
storage, normalized to functional activity when storage began. In
some embodiments, CFXTEN pharmaceutical compositions of the
invention retain about 50% more procoagulant activity, or about
60%, 70%, 80%, or 90% more of the procoagulant activity of a FVIII
standard when subjected to the same conditions for the same period
of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various
temperature conditions. In one embodiment, the CFXTEN
pharmaceutical composition retains at least about 50%, or about
60%, or at least about 70%, or at least about 80%, and most
preferably at least about 90% or more of its original activity in
solution when heated at 80.degree. C. for 10 min. In another
embodiment, the CFXTEN pharmaceutical composition retains at least
about 50%, preferably at least about 60%, or at least about 70%, or
at least about 80%, or alternatively at least about 90% or more of
its original activity in solution when heated or maintained at
37.degree. C. for about 7 days. In another embodiment, CFXTEN
pharmaceutical composition retains at least about 80% or more of
its functional activity after exposure to a temperature of about
30.degree. C. to about 70.degree. C. over a period of time of about
one hour to about 18 hours. In the foregoing embodiments
hereinabove described in this paragraph, the retained activity of
the CFXTEN pharmaceutical compositions is at least about two-fold,
or at least about three-fold, or at least about four-fold, or at
least about five-fold, or at least about six-fold greater at a
given time point than that of a corresponding pharmaceutical
composition comprising FVIII not linked to the XTEN.
VII). The Nucleic Acids Sequences of the Invention
[0330] The present invention provides isolated polynucleic acids
encoding CFXTEN chimeric fusion proteins and sequences
complementary to polynucleic acid molecules encoding CFXTEN
chimeric fusion proteins, including homologous variants thereof. In
another aspect, the invention encompasses methods to produce
polynucleic acids encoding CFXTEN chimeric fusion proteins and
sequences complementary to polynucleic acid molecules encoding
CFXTEN chimeric fusion protein, including homologous variants
thereof. In general, and as illustrated in FIGS. 11-13, the methods
of producing a polynucleotide sequence coding for a CFXTEN fusion
protein and expressing the resulting gene product include
assembling nucleotides encoding FVIII and 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 fusion protein
to be expressed in the transformed host cell, thereby producing the
biologically-active CFXTEN polypeptide, which is recovered as an
isolated fusion protein by standard protein purification methods
known in the art. Standard recombinant techniques in molecular
biology is used to make the polynucleotides and expression vectors
of the present invention.
[0331] In accordance with the invention, nucleic acid sequences
that encode CFXTEN (or its complement) are used to generate
recombinant DNA molecules that direct the expression of CFXTEN
fusion proteins in appropriate host cells. For the purposes of the
invention, nucleic acid encoding a signal peptide corresponding to
that of native human FVIII (encoding MQIELSTCFFLCLLRFCFS (SEQ ID
NO: 1611)) can be added to any of the encoding constructs described
herein to aid in the expression and secretion of the CFXTEN fusion
protein. In one embodiment, the nucleic acid add is
ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID
NO: 1613), or the complement thereof.
[0332] Several cloning strategies are suitable for performing the
present invention, many of which is used to generate a construct
that comprises a gene coding for a fusion protein of the CFXTEN
composition of the present invention, or its complement. In some
embodiments, the cloning strategy is used to create a gene that
encodes a monomeric CFXTEN that comprises at least a first FVIII
and at least a first XTEN polypeptide, or their complement. In one
embodiment of the foregoing, the gene comprises a sequence encoding
a FVIII or sequence variant. In other embodiments, the cloning
strategy is used to create a gene that encodes a monomeric CFXTEN
that comprises nucleotides encoding at least a first molecule of
FVIII or its complement and a first and at least a second XTEN or
their complement that is used to transform a host cell for
expression of the fusion protein of the CFXTEN composition. In the
foregoing embodiments hereinabove described in this paragraph, the
genes can further comprise nucleotides encoding spacer sequences
that also encode cleavage sequence(s).
[0333] 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. 11 and 12 and Examples). Thus, while the
XTEN(s) of the expressed fusion protein 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.
[0334] In one approach, a construct is first prepared containing
the DNA sequence corresponding to CFXTEN fusion protein. DNA
encoding the FVIII of the compositions is obtained synthetically,
from a commercial source, or from a cDNA library prepared using
standard methods from tissue or isolated cells believed to possess
FVIII mRNA and to express it at a detectable level. If necessary,
the coding sequence can be obtained using conventional primer
extension procedures as described in Sambrook, et al., supra, to
detect precursors and processing intermediates of mRNA that may not
have been reverse-transcribed into cDNA. One can then use
polymerase chain reaction (PCR) methodology to amplify the target
DNA or RNA coding sequence to obtain sufficient material for the
preparation of the CFXTEN constructs containing the FVIII gene.
Assays can then be conducted to confirm that the hybridizing
full-length genes are the desired FVIII gene(s). By these
conventional methods, DNA can be conveniently obtained from a cDNA
library prepared from such sources. The FVIII encoding gene(s) can
also created by standard synthetic procedures known in the art
(e.g., automated nucleic acid synthesis using, for example one of
the methods described in Engels et al. (Agnew. Chem. Int. Ed.
Engl., 28:716-734 1989)), using DNA sequences obtained from
publicly available databases, patents, or literature references.
Such procedures are well known in the art and well described in the
scientific and patent literature. For example, sequences can be
obtained from Chemical Abstracts Services (CAS) Registry Numbers
(published by the American Chemical Society) and/or GenBank
Accession Numbers (e.g., Locus ID, NP_XXXXX, and XP_XXXXX) 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.
In one embodiment, the FVIII encoding gene encodes a protein
sequence from Table 1, or a fragment or variant thereof.
[0335] A gene or polynucleotide encoding the FVIII portion of the
subject CFXTEN protein, in the case of an expressed fusion protein
that comprises a single FVIII, is then cloned into a construct,
which is a plasmid or other vector under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system. In a later step, a second gene
or polynucleotide coding for the XTEN is genetically fused to the
nucleotides encoding the N- and/or C-terminus of the FVIII gene by
cloning it into the construct adjacent and in frame with the
gene(s) coding for the FVIII. This second step occurs through a
ligation or multimerization step. In the foregoing embodiments
hereinabove described in this paragraph, it is to be understood
that the gene constructs that are created can alternatively be the
complement of the respective genes that encode the respective
fusion proteins.
[0336] 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 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
42 amino acids, or about 42 to about 144 amino acids, or about 144
to about 288 amino acids, or 288 to about 864 amino acids or
longer, or any combination of the foregoing ranges of motif or
segment lengths.
[0337] 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.
[0338] 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 FVIII or regions of FVIII, as illustrated in FIGS. 11 and
12, and the resulting genes used to transform a host cell and
produce and recover the CFXTEN for evaluation of its properties, as
described herein.
[0339] In another aspect, the invention provides isolated nucleic
acids comprising a polynucleotide sequence encoding the CFXTEN
fusion protein embodiments described herein. In one embodiment, the
isolated nucleic acid comprises a polynucleotide sequence selected
from (a) a sequence having at least about 80% sequence identity, or
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%, to about 100% sequence identity compared to a sequence of
comparable length selected from Table 21, when optimally aligned,
or (b) the complement of the polynucleotide of (a). In another
embodiment, the isolated nucleic acid comprises the sequence
ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID
NO: 1613) linked to the 5' end of the nucleic acid of (a) or the
complement of the sequence linked to the 3' end of (b).
[0340] Polynucleotide Libraries
[0341] In another aspect, the invention provides libraries of
polynucleotides that encode XTEN sequences that are used to
assemble genes that encode XTEN of a desired length and
sequence.
[0342] In certain embodiments, the XTEN-encoding library constructs
comprise polynucleotides that encode polypeptide segments of a
fixed length. As an initial step, a library of oligonucleotides
that encode motifs of 9-14 amino acid residues can be assembled. In
a preferred embodiment, libraries of oligonucleotides that encode
motifs of 12 amino acids are assembled.
[0343] The XTEN-encoding sequence segments can be dimerized or
multimerized into longer encoding sequences, as depicted
schematically in FIG. 13. Dimerization or multimerization can be
performed by ligation, overlap extension, PCR assembly or similar
cloning techniques known in the art. This process of can be
repeated multiple times until the resulting XTEN-encoding sequences
have reached the organization of sequence and desired length,
providing the XTEN-encoding genes. As will be appreciated, a
library of polynucleotides that encodes, e.g., 12 amino acid motifs
can be dimerized and/or ligated into a library of polynucleotides
that encode 36 amino acids. Libraries encoding motifs of different
lengths; e.g., 9-14 amino acid motifs leading to libraries encoding
27 to 42 amino acids are contemplated by the invention. In turn,
the library of polynucleotides that encode 27 to 42 amino acids,
and preferably 36 amino acids (as described in the Examples) can be
serially dimerized into a library containing successively longer
lengths of polynucleotides that encode XTEN sequences of a desired
length for incorporation into the gene encoding the CFXTEN fusion
protein, as disclosed herein.
[0344] A more efficient way to optimize the DNA sequence encoding
XTEN is based on combinatorial libraries. The gene encoding XTEN
can be designed and synthesized in segment such that multiple codon
versions are obtained for each segment. These segments can be
randomly assembled into a library of genes such that each library
member encodes the same amino acid sequences but library members
comprise a large number of codon versions. Such libraries can be
screened for genes that result in high-level expression and/or a
low abundance of truncation products. The process of combinatorial
gene assembly is illustrated in FIG. 18. The genes in FIG. 18 are
assembled from 6 base fragments and each fragment is available in 4
different codon versions. This allows for a theoretical diversity
of 4096.
[0345] In some embodiments, libraries are assembled of
polynucleotides that encode amino acids that are limited to
specific sequence XTEN families; e.g., the AD, AE, AF, AG, AM, or
AQ sequences of Table 4. In other embodiments, libraries comprise
sequences that encode two or more of the motif family sequences
from Table 3. The names and sequences of representative,
non-limiting polynucleotide sequences of libraries that encode 36
mers are presented in Tables 13-17, and the methods used to create
them are described more fully in the respective Examples. In other
embodiments, libraries that encode XTEN are constructed from
segments of polynucleotide codons linked in a randomized sequence
that encode amino acids wherein 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 97%, or at least about 98%, or at least about 99% of
the codons are selected from the group consisting of condons for
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
and proline (P) amino acids. The libraries can be used, in turn,
for serial dimerization or ligation to achieve polynucleotide
sequence libraries that encode XTEN sequences, for example, of 42,
48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up
to a total length of about 3000 amino acids, as well as
intermediate lengths, in which the encoded XTEN can have one or
more of the properties disclosed herein, when expressed as a
component of a CFXTEN fusion protein. In some cases, the
polynucleotide library sequences may also include additional bases
used as "sequencing islands," described more fully below.
[0346] FIG. 14 is a schematic flowchart of representative,
non-limiting steps in the assembly of a XTEN polynucleotide
construct and a CFXTEN polynucleotide construct in the embodiments
of the invention. 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 than 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 containing a
gene encoding the FVIII, resulting in the gene 500 encoding an
FVIII-XTEN fusion protein.
[0347] One may clone the library of XTEN-encoding genes into one or
more expression vectors known in the art. To facilitate the
identification of well-expressing library members, one can
construct the library as fusion to a reporter protein. Non-limiting
examples of suitable reporter genes are green fluorescent protein,
luciferace, alkaline phosphatase, and beta-galactosidase. By
screening, one can identify short XTEN sequences that can be
expressed in high concentration in the host organism of choice.
Subsequently, one can generate a library of random XTEN dimers and
repeat the screen for high level of expression. Subsequently, one
can screen the resulting constructs for a number of properties such
as level of expression, protease stability, or binding to
antiserum.
[0348] One aspect of the invention is to provide polynucleotide
sequences encoding the components of the fusion protein wherein the
creation of the sequence has undergone codon optimization. Of
particular interest is codon optimization with the goal of
improving expression of the polypeptide compositions and to improve
the genetic stability of the encoding gene in the production hosts.
For example, codon optimization is of particular importance for
XTEN sequences that are rich in glycine or that have very
repetitive amino acid sequences. Codon optimization is performed
using computer programs (Gustafsson, C., et al. (2004) Trends
Biotechnol, 22: 346-53), some of which minimize ribosomal pausing
(Coda Genomics Inc.). In one embodiment, one can perform codon
optimization by constructing codon libraries where all members of
the library encode the same amino acid sequence but where codon
usage is varied. Such libraries can be screened for highly
expressing and genetically stable members that are particularly
suitable for the large-scale production of XTEN-containing
products. When designing XTEN sequences one can consider a number
of properties. One can minimize the repetitiveness in the encoding
DNA sequences. In addition, one can avoid or minimize the use of
codons that are rarely used by the production host (e.g. the AGG
and AGA arginine codons and one leucine codon in E. coli). In the
case of E. coli, two glycine codons, GGA and GGG, are rarely used
in highly expressed proteins. Thus codon optimization of the gene
encoding XTEN sequences can be very desirable. DNA sequences that
have a high level of glycine tend to have a high GC content that
can lead to instability or low expression levels. Thus, when
possible, it is preferred to choose codons such that the GC-content
of XTEN-encoding sequence is suitable for the production organism
that will be used to manufacture the XTEN.
[0349] In one embodiment, polynucleotide libraries are constructed
using the disclosed methods wherein all members of the library
encode the same amino acid sequence but where codon usage for the
respective amino acids in the sequence is varied or optimized for
the intended host cell. Such libraries can be screened for highly
expressing and genetically stable members that are particularly
suitable for the large-scale production of XTEN-containing
products. In one embodiment, the libraries are optimized for
expression in a eukaryotic host cell.
[0350] Optionally, one can sequence clones in the library to
eliminate isolates that contain undesirable sequences. The initial
library of short XTEN sequences allows some variation in amino acid
sequence. For instance one can randomize some codons such that a
number of hydrophilic amino acids can occur in a particular
position. During the process of iterative multimerization one can
screen the resulting library members for other characteristics like
solubility or protease resistance in addition to a screen for
high-level expression.
[0351] Once the gene that encodes the XTEN of desired length and
properties is selected, it is genetically fused at the desired
location to the nucleotides encoding the FVIII gene(s) by cloning
it into the construct adjacent and in frame with the gene coding
for FVIII, or alternatively between nucleotides encoding adjacent
domains of the FVIII, or alternatively within a sequence encoding a
given FVIII domain, or alternatively in frame with nucleotides
encoding a spacer/cleavage sequence linked to a terminal XTEN. The
invention provides various permutations of the foregoing, depending
on the CFXTEN to be encoded. For example, a gene encoding a CFXTEN
fusion protein comprising a FVIII and two XTEN, such as embodied by
formula VI, as depicted above, the gene would have polynucleotides
encoding FVIII, encoding two XTEN, which can be identical or
different in composition and sequence length. In one non-limiting
embodiment of the foregoing, the FVIII polynucleotides would encode
factor VIII and the polynucleotides encoding the C-terminus XTEN
would encode an XTEN of 288 amino acids and the polynucleotides
encoding an internal XTEN adjacent to the C-terminus of the A2
domain would encode an XTEN of 144 amino acids. The step of cloning
the FVIII genes into the XTEN construct can occur through a
ligation or multimerization step, as shown in FIG. 14. The
constructs encoding CFXTEN fusion proteins can be designed in
different configurations of the components XTEN, CF, and spacer
sequences, such as the configurations of formulae I-VIII. In one
embodiment, the construct comprises polynucleotide sequences
complementary to, or those that encode a monomeric polypeptide of
components in the following order (5' to 3') FVIII, an XTEN
internal to the B domain, and a C-terminal XTEN. In another
embodiment, the construct comprises polynucleotide sequences
complementary to, or those that encode a monomeric polypeptide of
components in the following order (5' to 3') FVIIII, spacer
sequence linked to the C-terminus, and XTEN. The spacer
polynucleotides can optionally comprise sequences encoding cleavage
sequences. As will be apparent to those of skill in the art,
multiple permutations of FVIII domains and inserted XTEN are
possible.
[0352] Homology, sequence similarity or sequence identity of
nucleotide or amino acid sequences may also be determined
conventionally by using known software or computer programs such as
the BestFit or Gap pairwise comparison programs (GCG Wisconsin
Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.
53711). BestFit uses the local homology algorithm of Smith and
Waterman (Advances in Applied Mathematics. 1981. 2: 482-489), to
find the best segment of identity or similarity between two
sequences. Gap performs global alignments: all of one sequence with
all of another similar sequence using the method of Needleman and
Wunsch, (Journal of Molecular Biology. 1970. 48:443-453). When
using a sequence alignment program such as BestFit, to determine
the degree of sequence homology, similarity or identity, the
default setting may be used, or an appropriate scoring matrix may
be selected to optimize identity, similarity or homology
scores.
[0353] Nucleic acid sequences that are "complementary" are those
that are capable of base-pairing according to the standard
Watson-Crick complementarity rules. As used herein, the term
"complementary sequences" means nucleic acid sequences that are
substantially complementary, as may be assessed by the same
nucleotide comparison set forth above, or as defined as being
capable of hybridizing to the polynucleotides that encode the
CFXTEN sequences under stringent conditions, such as those
described herein.
[0354] The resulting polynucleotides encoding the CFXTEN chimeric
fusion proteins 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.
[0355] 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.
[0356] The invention provides for the use of plasmid vectors
containing replication and control sequences that are compatible
with and recognized by the host cell, and are operably linked to
the CFXTEN gene for controlled expression of the CFXTEN fusion
proteins. 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 fusion protein 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.
[0357] Other 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 NM98 9, 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.
[0358] 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.
[0359] Examples of suitable promoters for directing the
transcription of the DNA encoding the FVIII polypeptide variant in
mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell.
Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., Science 222 (1983), 809-814), the CMV promoter
(Boshart et al., Cell 41:521-530, 1985) or the adenovirus 2 major
late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319,
1982). The vector may also carry sequences such as UCOE (ubiquitous
chromatin opening elements).
[0360] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter or the tpiA
promoter. Examples of other useful promoters are those derived from
the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, A. niger neutral a-amylase, A. niger acid
stable .alpha.-amylase, A. niger or A. awamoriglucoamylase (gluA),
Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae
triose phosphate isomerase or A. nidulans acetamidase. Preferred
are the TAKA-amylase and gluA promoters.
[0361] 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.
[0362] The invention contemplates use of other expression systems
including, for example, a baculovirus expression system with both
non-fusion transfer vectors, such as, but not limited to pVL941
Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen),
pVL1392 (Summers, et al., Virology 84:390-402 (1978) and
Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer
vectors such as, but not limited to, pAc7 00 (Summers, et al.,
Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700,
with different reading frames), pAc360 Invitrogen) and
pBlueBacHisA, B, C (; Invitrogen) can be used.
[0363] Examples of suitable promoters for directing the
transcription of the DNA encoding the FVIII polypeptide variant in
mammalian cells are the CMV promoter (Boshart et al., Cell
41:521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell.
Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2
major late promoter (Kaufman and Sharp, Mol. Cell. Biol,
2:1304-1319, 1982). The vector may also carry sequences such as
UCOE (ubiquitous chromatin opening elements).
[0364] The DNA sequences encoding the CFXTEN may also, if
necessary, be operably connected to a suitable terminator, such as
the hGH terminator (Palmiter et al., Science 222, 1983, pp.
809-814) or the TPI1 terminators (Alber and Kawasaki, J. Mol. Appl.
Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4,
1985, pp. 2093-2099). Expression vectors may also contain a set of
RNA splice sites located downstream from the promoter and upstream
from the insertion site for the CFXTEN sequence itself, including
splice sites obtained from adenovirus. Also contained in the
expression vectors is a polyadenylation signal located downstream
of the insertion site. Particularly preferred polyadenylation
signals include the early or late polyadenylation signal from SV40
(Kaufman and Sharp, ibid.), the polyadenylation signal from the
adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl.
Acids Res. 9:3719-3730, 1981). The expression vectors may also
include a noncoding viral leader sequence, such as the adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites; and enhancer sequences, such as the SV40 enhancer.
[0365] To direct the CFXTEN of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(a.k.a., a leader sequence, a prepro sequence, or a pre sequence)
may be included in the recombinant vector. The secretory signal
sequence is operably linked to the DNA sequences encoding the
CFXTEN, usually positioned 5' to the DNA sequence encoding the
CFXTEN fusion protein. The secretory signal sequence may be that,
normally associated with the native FVIII protein or may be from a
gene encoding another secreted protein. Non-limiting examples
include OmpA, PhoA, and DsbA for E. coli expression, ppL-alpha,
DEX4, invertase signal peptide, acid phosphatase signal peptide,
CPY, or INU1 for yeast expression, and IL2L, SV40, IgG kappa and
IgG lambda for mammalian expression. Signal sequences are typically
proteolytically removed from the protein during the translocation
and secretion process, generating a defined N-terminus. Methods are
disclosed in Arnau, et al., Protein Expression and Purification 48:
1-13 (2006).
[0366] The procedures used to ligate the DNA sequences coding for
the CFXTEN, the promoter and optionally the terminator and/or
secretory signal sequence, respectively, and to insert them into
suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook, J. et al., "Molecular Cloning: A Laboratory
Manual," 3.sup.rd edition, Cold Spring Harbor Laboratory Press,
2001). In this manner, a chimeric DNA molecule coding for a
monomeric CFXTEN fusion protein is generated within the construct.
Optionally, this chimeric DNA molecule may be transferred or cloned
into another construct that is a more appropriate expression
vector. At this point, a host cell capable of expressing the
chimeric DNA molecule can be transformed with the chimeric DNA
molecule.
[0367] Non-limiting examples of mammalian cell lines for use in the
present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL
1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et
al., J. Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL
10314), CHO-K1 (ATCC CCL 61), CHO-S (Invitrogen 11619-012), and
293-F (Invitrogen R790-7), and the parental and derivative cell
lines known in the art useful for expression of FVIII. A tk-ts13
BHK cell line is also available from the ATCC under accession
number CRL 1632. In addition, a number of other cell lines may be
used within the present invention, including Rat Hep I (Rat
hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548),
TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL
9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc.
Natl. Acad. Sci. USA 77:4216-4220, 1980).
[0368] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous polypeptides there from are described, e.g.
in U.S. Pat. Nos. 4,599,311, 4,931,373, 4,870,008, 5,037,743, and
4,845,075, all of which are hereby incorporated by reference.
Transformed cells are selected by a phenotype determined by a
selectable marker, commonly drug resistance or the ability to grow
in the absence of a particular nutrient, e.g. leucine. A preferred
vector for use in yeast is the POT1 vector disclosed in U.S. Pat.
No. 4,931,373. The DNA sequences encoding the CFXTEN may be
preceded by a signal sequence and optionally a leader sequence,
e.g. as described above. Further examples of suitable yeast cells
are strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H.
polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J.
Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).
Examples of other fungal cells are cells of filamentous fungi, e.g.
Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma
spp., in particular strains of A. oryzae, A. nidulans or A. niger.
The use of Aspergillus spp. for the expression of proteins is
described in, e.g., EP 272 277, EP 238 023, EP 184 438 The
transformation of F. oxysporum may, for instance, be carried out as
described by Malardier et al., 1989, Gene 78: 147-156. The
transformation of Trichoderma spp. may be performed for instance as
described in EP 244 234.
[0369] Other suitable cells that can be used in the present
invention include, but are not limited to, prokaryotic host cells
strains such as Escherichia coli, (e.g., strain DH5-a), Bacillus
subtilis, Salmonella typhimurium, or strains of the genera of
Pseudomonas, Streptomyces and Staphylococcus. Non-limiting examples
of suitable prokaryotes include those from the genera:
Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus;
Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus;
Paracoccus; Pseudomonas; Staphylococcus; Streptococcus;
Streptomyces; Thermoplasma; and Vibrio.
[0370] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in e.g., Kaufman
and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978),
725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603,
Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al.,
EMBO J. 1 (1982), 841-845.
[0371] Cloned DNA sequences are introduced into cultured mammalian
cells by, for example, calcium phosphate-mediated transfection
(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic
Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology
52d:456-467, 1973), transfection with many commercially available
reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) or
lipofectamine (Invitrogen) or by electroporation (Neumann et al.,
EMBO J. 1:841-845, 1982). To identify and select cells that express
the exogenous DNA, a gene that confers a selectable phenotype (a
selectable marker) is generally introduced into cells along with
the gene or cDNA of interest. Preferred selectable markers include
genes that confer resistance to drugs such as neomycin, hygromycin,
puromycin, zeocin, and methotrexate. The selectable marker may be
an amplifiable selectable marker. A preferred amplifiable
selectable marker is a dihydrofolate reductase (DHFR) sequence.
Further examples of selectable markers are well known to one of
skill in the art and include reporters such as enhanced green
fluorescent protein (EGFP), beta-galactosidase (.beta.-gal) or
chloramphenicol acetyltransferase (CAT). Selectable markers are
reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, Mass., incorporated herein by reference). The
person skilled in the art will easily be able to choose suitable
selectable markers. Any known selectable marker may be employed so
long as it is capable of being expressed simultaneously with the
nucleic acid encoding a gene product.
[0372] Selectable markers may be introduced into the cell on a
separate plasmid at the same time as the gene of interest, or they
may be introduced on the same plasmid. If, on the same plasmid, the
selectable marker and the gene of interest may be under the control
of different promoters or the same promoter, the latter arrangement
producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Pat. No.
4,713,339). It may also be advantageous to add additional DNA,
known as "carrier DNA," to the mixture that is introduced into the
cells.
[0373] After the cells have taken up the DNA, they are grown in an
appropriate growth medium, typically 1-2 days, to begin expressing
the gene of interest. As used herein the term "appropriate growth
medium" means a medium containing nutrients and other components
required for the growth of cells and the expression of the CFXTEN
of interest. Media generally include a carbon source, a nitrogen
source, essential amino acids, essential sugars, vitamins, salts,
phospholipids, protein and growth factors. For production of
gamma-carboxylated proteins, the medium will contain vitamin K,
preferably at a concentration of about 0.1 .mu.g/ml to about 5
.mu.g/ml. Drug selection is then applied to select for the growth
of cells that are expressing the selectable marker in a stable
fashion. For cells that have been transfected with an amplifiable
selectable marker the drug concentration may be increased to select
for an increased copy number of the cloned sequences, thereby
increasing expression levels. Clones of stably transfected cells
are then screened for expression of the FVIII polypeptide variant
of interest.
[0374] The transformed or transfected host cell is then cultured in
a suitable nutrient medium under conditions permitting expression
of the CFXTEN polypeptide after which the resulting peptide may be
recovered from the culture as an isolated fusion protein. The
medium used to culture the cells may be any conventional medium
suitable for growing the host cells, such as minimal or complex
media containing appropriate supplements. Suitable media are
available from commercial suppliers or may be prepared according to
published recipes (e.g. in catalogues of the American Type Culture
Collection). The culture conditions, such as temperature, pH and
the like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan.
[0375] Gene expression may be measured in a sample directly, for
example, by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad.
Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in
situ hybridization, using an appropriately labeled probe, based on
the sequences provided herein. Alternatively, antibodies may be
employed that can recognize specific duplexes, including DNA
duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein
duplexes. The antibodies in turn may be labeled and the assay may
be carried out where the duplex is bound to a surface, so that upon
the formation of duplex on the surface, the presence of antibody
bound to the duplex can be detected.
[0376] Gene expression, alternatively, may be measured by
immunological of fluorescent methods, such as immunohistochemical
staining of cells or tissue sections and assay of cell culture or
body fluids or the detection of selectable markers, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of sample fluids may be
either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be prepared against a native
sequence FVIII polypeptide or against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence
fused to FVIII and encoding a specific antibody epitope. Examples
of selectable markers are well known to one of skill in the art and
include reporters such as enhanced green fluorescent protein
(EGFP), beta-galactosidase (3-gal) or chloramphenicol
acetyltransferase (CAT).
[0377] Expressed CFXTEN polypeptide product(s) may be purified via
methods known in the art or by methods disclosed herein. Procedures
such as gel filtration, affinity purification (e.g., using an
anti-FVIII antibody column), salt fractionation, ion exchange
chromatography, size exclusion chromatography, hydroxyapatite
adsorption chromatography, hydrophobic interaction chromatography
and gel electrophoresis may be used; each tailored to recover and
purify the fusion protein produced by the respective host cells.
Additional purification may be achieved by conventional chemical
purification means, such as high performance liquid chromatography.
Some expressed CFXTEN may require refolding during isolation and
purification. Methods of purification are described in Robert K.
Scopes, Protein Purification: Principles and Practice, Charles R.
Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra.
Multi-step purification separations are also described in Baron, et
al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J.
Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it is
preferred that the CFXTEN fusion proteins of the invention are
substantially pure. Thus, in a preferred embodiment of the
invention the CFXTEN of the invention is purified to at least about
90 to 95% homogeneity, preferably to at least about 98%
homogeneity. Purity may be assessed by, e.g., gel electrophoresis,
HPLC, and amino-terminal amino acid sequencing.
VIII). Pharmaceutical Compositions
[0378] The present invention provides pharmaceutical compositions
comprising CFXTEN. In one embodiment, the pharmaceutical
composition comprises a CFXTEN fusion protein disclosed herein
admixed with at least one pharmaceutically acceptable carrier.
CFXTEN polypeptides of the present invention can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the polypeptide is combined in admixture with
a pharmaceutically acceptable carrier vehicle, such as aqueous
solutions, buffers, solvents and/or pharmaceutically acceptable
suspensions, emulsions, stabilizers or excipients. Examples of
non-aqueous solvents include propyl ethylene glycol, polyethylene
glycol and vegetable oils. Formulations of the pharmaceutical
compositions are prepared for storage by mixing the active CFXTEN
ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients (e.g., sodium
chloride, a calcium salt, sucrose, or polysorbate) or stabilizers
(e.g., sucrose, trehalose, raffinose, arginine, a calcium salt,
glycine or histidine), as described in Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980), in the form of
lyophilized formulations or aqueous solutions.
[0379] The pharmaceutical composition may be supplied as a
lyophilized powder to be reconstituted prior to administration. In
another embodiment, the pharmaceutical composition may be supplied
in a liquid form in a vial, the contents of which can be
administered directly to a patient. Alternatively, the composition
is supplied as a liquid in a pre-filled syringe for administration
of the composition. In another embodiment, the composition is
supplied as a liquid in a pre-filled vial that can be incorporated
into a pump.
[0380] 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.
[0381] In one embodiment, the CFXTEN pharmaceutical composition in
liquid form or after reconstitution (when supplied as a lyophilized
powder) comprises coagulation factor VIII with an activity of at
least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at
least 300 IU/ml, or at least 400 IU/ml, or an activity of at least
500 IU/ml, or an activity of at least 600 IU/ml, which composition
is capable of increasing factor VIII activity to at least 1.5% of
the normal plasma level in the blood for at least about 12 hours,
or at least about 24 hours, or at least about 48 hours, or at least
about 72 hours, or at least about 96 hours, or at least about 120
hours after administration of the factor VIII pharmaceutical
composition to a subject in need of routine prophylaxis. In another
embodiment, the CFXTEN pharmaceutical composition in liquid form or
after reconstitution (when supplied as a lyophilized powder)
comprises coagulation factor VIII with an activity of at least 50
IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least
300 IU/ml, or at least 400 IU/ml, or at least 500 IU/ml, or an
activity of at least 600 IU/ml, which composition is capable of
increasing factor VIII activity to at least 2.5% of the normal
plasma level in the blood for at least about 12 hours, or at least
about 24 hours, or at least about 48 hours, or at least about 72
hours, or at least about 96 hours, or at least about 120 hours
after administration to a subject in need of routine prophylaxis.
It is specifically contemplated that the pharmaceutical
compositions of the foregoing can be formulated to include one or
more excipients, buffers or other ingredients known in the art to
be compatible with administration by the intravenous route or the
subcutaneous route or the intramuscular route. Thus, in the
embodiments hereinabove described in this paragraph, the
pharmaceutical composition is administered subcutaneously,
intramuscularly or intravenously.
[0382] The compositions of the invention may be formulated using a
variety of excipients. Suitable excipients include microcrystalline
cellulose (e.g. Avicel PH102, Avicel PH101), polymethacrylate,
poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl
methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl
methylcellulose (Methocel K100M, Premium CR Methocel K100M,
Methocel E5, Opadry.RTM.), magnesium stearate, talc, triethyl
citrate, aqueous ethylcellulose dispersion (Surelease.RTM.), and
protamine sulfate. The slow release agent may also comprise a
carrier, which can comprise, for example, solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents. Pharmaceutically acceptable salts can
also be used in these slow release agents, for example, mineral
salts such as hydrochlorides, hydrobromides, phosphates, or
sulfates, as well as the salts of organic acids such as acetates,
proprionates, malonates, or benzoates. The composition may also
contain liquids, such as water, saline, glycerol, and ethanol, as
well as substances such as wetting agents, emulsifying agents, or
pH buffering agents. Liposomes may also be used as a carrier.
[0383] In another embodiment, the compositions of the present
invention are encapsulated in liposomes, which have demonstrated
utility in delivering beneficial active agents in a controlled
manner over prolonged periods of time. Liposomes are closed bilayer
membranes containing an entrapped aqueous volume. Liposomes may
also be unilamellar vesicles possessing a single membrane bilayer
or multilamellar vesicles with multiple membrane bilayers, each
separated from the next by an aqueous layer. The structure of the
resulting membrane bilayer is such that the hydrophobic (non-polar)
tails of the lipid are oriented toward the center of the bilayer
while the hydrophilic (polar) heads orient towards the aqueous
phase. In one embodiment, the liposome may be coated with a
flexible water soluble polymer that avoids uptake by the organs of
the mononuclear phagocyte system, primarily the liver and spleen.
Suitable hydrophilic polymers for surrounding the liposomes
include, without limitation, PEG, polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxethylacrylate,
hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and hydrophilic peptide sequences as described in
U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the
contents of which are incorporated by reference in their entirety.
Additional liposomal technologies are described in U.S. Pat. Nos.
6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966;
6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680;
and 4,684,479, the contents of which are incorporated herein by
reference. These describe liposomes and lipid-coated microbubbles,
and methods for their manufacture. Thus, one skilled in the art,
considering both the disclosure of this invention and the
disclosures of these other patents could produce a liposome for the
extended release of the polypeptides of the present invention.
[0384] For liquid formulations, a desired property is that the
formulation be supplied in a form that can pass through a 25, 28,
30, 31, 32 gauge needle for intravenous, intramuscular,
intraarticular, or subcutaneous administration.
[0385] 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.
IX). Pharmaceutical Kits
[0386] In another aspect, the invention provides a kit to
facilitate the use of the CFXTEN polypeptides. The kit comprises
the 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 the FVIII-related condition 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.
EXAMPLES
Example 1: Construction of XTEN_AD36 Motif Segments
[0387] 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 12 mer peptide with the sequences: GESPGGSSGSES (SEQ ID NO:
19), GSEGSSGPGESS (SEQ ID NO: 20), GSSESGSSEGGP (SEQ ID NO: 21), or
GSGGEPSESGSS (SEQ ID NO: 22). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00015 AD1for: (SEQ ID NO: 1619)
AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1rev: (SEQ ID NO: 1620)
ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for: (SEQ ID NO: 1621)
AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev: (SEQ ID NO: 1622)
ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT AD3for: (SEQ ID NO: 1623)
AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev: (SEQ ID NO: 1624)
ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA AD4for: (SEQ ID NO: 1625)
AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC
[0388] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12 mer 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.
[0389] 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 and the sequences
for these segments are listed in Table 13.
TABLE-US-00016 TABLE 13 DNA and Amino Acid Sequences for AD 36-mer
motifs (SEQ ID NOS 203-278, respectively, in order of appearance)
File name Amino acid sequence Nucleotide sequence LCW0401_001_GFP-
GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAGCTCA N_A01.ab1
SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCTAGCGGTTCCGAGTCA
GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA LCW0401_002_GFP-
GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA N_B01.ab1
SSGSESGSSESGSSEGGP GGTGAATCTCCTGGTGGTTCCAGCGGTTCTGAATCA
GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA LCW0401_003_GFP-
GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGTCCA N_C01.ab1
SSEGGPGESPGGSSGSES GGTTCCTCTGAAAGCGGTTCTTCTGAGGGTGGTCCA
GGTGAATCTCCGGGTGGCTCCAGCGGTTCCGAGTCA LCW0401_004_GFP-
GSGGEPSESGSSGSSESG GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGCTCA N_D01.ab1
SSEGGPGSGGEPSESGSS GGTTCTTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA
GGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCA LCW0401_007_GFP-
GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGTCCA N_F01.ab1
GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCA
GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCW0401_008_GFP-
GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA N_G01.ab1
SSGSESGSEGSSGPGESS GGTGAATCTCCAGGTGGTTCCAGCGGTTCTGAGTCA
GGTAGCGAAGGTTCTTCTGGTCCAGGTGAATCCTCA LCW0401_012_GFP-
GSGGEPSESGSSGSGGEP GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGCTCA N_H01.ab1
SESGSSGSEGSSGPGESS GGTTCCGGTGGCGAACCATCCGAATCTGGTAGCTCA
GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCA LCW0401_015_GFP-
GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA N_A02.ab1
GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA
GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA LCW0401_016_GFP-
GSSESGSSEGGPGSSESG GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA N_B02.ab1
SSEGGPGSSESGSSEGGP GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA
GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA LCW0401_020_GFP-
GSGGEPSESGSSGSEGSS GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA N_E02.ab1
GPGESSGSSESGSSEGGP GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA LCW0401_022_GFP-
GSGGEPSESGSSGSSESG GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA N_F02.ab1
SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA
GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA LCW0401_024_GFP-
GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA N_G02.ab1
SSEGGPGESPGGSSGSES GGTTCCTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA
GGTGAATCTCCAGGTGGTTCTAGCGGTTCTGAATCA LCW0401_026_GFP-
GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGCTCA N_H02.ab1
SSGSESGSEGSSGPGESS GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAATCA
GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCW0401_027_GFP-
GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA N_A03.ab1
SSGSESGSGGEPSESGSS GGTGAATCTCCGGGTGGTTCTAGCGGTTCTGAGTCA
GGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCA LCW0401_028_GFP-
GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA N_B03.ab1
SSEGGPGSSESGSSEGGP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA
GGTTCTTCCGAAAGCGGTTCTTCTGAAGGCGGTCCA LCW0401_030_GFP-
GESPGGSSGSESGSEGSS GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAGTCA N_C03.ab1
GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCCGGTCCGGGTGAGTCCTCA
GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCA LCW0401_031_GFP-
GSGGEPSESGSSGSGGEP GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA N_D03.ab1
SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA
GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA LCW0401_033_GFP-
GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA N_E03.ab1
SESGSSGSGGEPSESGSS GGTTCCGGTGGCGAACCATCCGAGTCTGGTAGCTCA
GGTTCCGGTGGTGAACCATCCGAGTCTGGTAGCTCA LCW0401_037_GFP-
GSGGEPSESGSSGSSESG GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA N_F03.ab1
SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA
GGTAGCGAAGGTTCTTCTGGTCCGGGCGAGTCTTCA LCW0401_038_GFP-
GSGGEPSESGSSGSEGSS GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAGCTCA N_G03.ab1
GPGESSGSGGEPSESGSS GGTAGCGAAGGTTCTTCTGGTCCGGGTGAGTCTTCA
GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA LCW0401_039_GFP-
GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA N_H03.ab1
SSGSESGSGGEPSESGSS GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA
GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA LCW0401_040_GFP-
GSSESGSSEGGPGSGGEP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA N_A04.ab1
SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCATCTGAATCTGGTAGCTCA
GGTTCTTCTGAAAGCGGTTCTTCTGAAGGTGGTCCA LCW0401_042_GFP-
GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCA N_C04.ab1
SSGSESGSEGSSGPGESS GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA
GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCCTCA LCW0401_046_GFP-
GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGTCCA N_D04.ab1
SSEGGPGSSESGSSEGGP GGTTCTTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA
GGTTCCTCCGAAAGCGGTTCTTCTGAGGGTGGTCCA LCW0401_047_GFP-
GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAGCTCA N_E04.ab1
SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAGTCA
GGTGAATCTCCGGGTGGTTCCAGCGGTTCTGAGTCA LCW0401_051_GFP-
GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCTGAGTCTGGTAGCTCA N_F04.ab1
GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCCGGTCCAGGCGAGTCTTCA
GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA LCW0401_053_GFP-
GESPGGSSGSESGESPGG GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA N_H04.ab1
SSGSESGESPGGSSGSES GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA
GGTGAATCTCCTGGTGGTTCTAGCGGTTCTGAATCA LCW0401_054_GFP-
GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCTTCA N_A05.ab1
GPGESSGSGGEPSESGSS GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCCTCA
GGTTCCGGTGGCGAACCATCTGAATCTGGTAGCTCA LCW0401_059_GFP-
GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCCGAATCTGGTAGCTCA N_D05.ab1
GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCTGGTCCTGGCGAATCTTCA
GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAATCA LCW0401_060_GFP-
GSGGEPSESGSSGSSESG GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGCTCA N_E05.ab1
SSEGGPGSGGEPSESGSS GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA
GGTTCCGGTGGTGAACCTTCTGAGTCTGGTAGCTCA LCW0401_061_GFP-
GSSESGSSEGGPGSGGEP GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA N_F05.ab1
SESGSSGSEGSSGPGESS GGTTCTGGTGGCGAACCATCTGAATCTGGTAGCTCA
GGTAGCGAAGGTTCTTCCGGTCCGGGTGAATCTTCA LCW0401_063_GFP-
GSGGEPSESGSSGSEGSS GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA N_H05.ab1
GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA
GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCW0401_066_GFP-
GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGCTCA N_B06.ab1
SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA
GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA LCW0401_067_GFP-
GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA N_C06.ab1
SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAATCA
GGTGAATCTCCAGGTGGTTCTAGCGGTTCCGAATCA LCW0401_069_GFP-
GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCATCTGAGTCTGGTAGCTCA N_D06.ab1
SESGSSGESPGGSSGSES GGTTCCGGTGGCGAACCGTCCGAGTCTGGTAGCTCA
GGTGAATCTCCGGGTGGTTCCAGCGGTTCCGAATCA LCW0401_070_GFP-
GSEGSSGPGESSGSSESG GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCCTCA N_E06.ab1
SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCCGAAGGTGGTCCA
GGTAGCGAAGGTTCTTCCGGTCCTGGTGAATCTTCA LCW0401_078_GFP-
GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGTCCA N_F06.ab1
SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAATCA
GGTGAATCTCCTGGTGGCTCCAGCGGTTCCGAGTCA LCW0401_079_GFP-
GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCTTCA N_G06.ab1
GPGESSGSGGEPSESGSS GGTAGCGAAGGTTCTTCCGGTCCTGGCGAGTCTTCA
GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA
Example 2: Construction of XTEN_AE36 Segments
[0390] 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 12
mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23),
GSEPATSGSETP (SEQ ID NO: 24), GTSESATPESGP (SEQ ID NO: 25), or
GTSTEPSEGSAP (SEQ ID NO: 26). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00017 AE1for: (SEQ ID NO: 1628)
AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AE1rev: (SEQ ID NO: 1629)
ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AE2for: (SEQ ID NO: 1630)
AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC AE2rev: (SEQ ID NO: 1631)
ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3for: (SEQ ID NO: 1632)
AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev: (SEQ ID NO: 1633)
ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for: (SEQ ID NO: 1634)
AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4rev: (SEQ ID NO: 1635)
ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT
[0391] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12 mer 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 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.
[0392] We screened 96 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 and the sequences
for these segments are listed in Table 14.
TABLE-US-00018 TABLE 14 DNA and Amino Acid Sequences for AE 36-mer
motifs (SEQ ID NOS 279-352, respectively, in order of appearance)
File name Amino acid sequence Nucleotide sequence LCW0402_002_GFP-
GSPAGSPTSTEEGTSE GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA N_A07.ab1
SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA GSAP
GGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA LCW0402_003_GFP-
GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCA N_B07.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCA GSAP
GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA LCW0402_004_GFP-
GTSTEPSEGSAPGTSE GGTACCTCTACCGAACCGTCTGAAGGTAGCGCACCA N_C07.ab1
SATPESGPGTSESATP GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCA ESGP
GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA LCW0402_005_GFP-
GTSTEPSEGSAPGTSE GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCA N_D07.ab1
SATPESGPGTSESATP GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA ESGP
GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCW0402_006_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCA N_E07.ab1
SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCA STEE
GGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA LCW0402_008_GFP-
GTSESATPESGPGSEP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA N_F07.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA GSAP
GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA LCW0402_009_GFP-
GSPAGSPTSTEEGSPA GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAA N_G07.ab1
GSPTSTEEGSEPATSG GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAA SETP
GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA LCW0402_011_GFP-
GSPAGSPTSTEEGTSE GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAA N_A08.ab1
SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCA GSAP
GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA LCW0402_012_GFP-
GSPAGSPTSTEEGSPA GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAA N_B08.ab1
GSPTSTEEGTSTEPSE GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAA GSAP
GGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA LCW0402_013_GFP-
GTSESATPESGPGTST GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCA N_C08.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCA GSAP
GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCW0402_014_GFP-
GTSTEPSEGSAPGSPA GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCA N_D08.ab1
GSPTSTEEGTSTEPSE GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAA GSAP
GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA LCW0402_015_GFP-
GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA N_E08.ab1
GSPTSTEEGTSESATP GGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAA ESGP
GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA LCW0402_016_GFP-
GTSTEPSEGSAPGTSE GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA N_F08.ab1
SATPESGPGTSESATP GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA ESGP
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA LCW0402_020_GFP-
GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCA N_G08.ab1
ATSGSETPGSPAGSPT GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA STEE
GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA LCW0402_023_GFP-
GSPAGSPTSTEEGTSE GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA N_A09.ab1
SATPESGPGSEPATSG GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA LCW0402_024_GFP-
GTSESATPESGPGSPA GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA N_B09.ab1
GSPTSTEEGSPAGSPT GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA STEE
GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA LCW0402_025_GFP-
GTSTEPSEGSAPGTSE GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA N_C09.ab1
SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_026_GFP-
GSPAGSPTSTEEGTST GGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAA N_D09.ab1
EPSEGSAPGSEPATSG GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA SETP
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA LCW0402_027_GFP-
GSPAGSPTSTEEGTST GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA N_E09.ab1
EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA LCW0402_032_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCA N_H09.ab1
SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCA STEE
GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA LCW0402_034_GFP-
GTSESATPESGPGTST GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCA N_A10.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_036_GFP-
GSPAGSPTSTEEGTST GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA N_C10.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA GSAP
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA LCW0402_039_GFP-
GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCA N_E10.ab1
EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCA GSAP
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW0402_040_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA N_F10.ab1
SATPESGPGTSTEPSE GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GSAP
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA LCW0402_041_GFP-
GTSTEPSEGSAPGSPA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA N_G10.ab1
GSPTSTEEGTSTEPSE GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA GSAP
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA LCW0402_050_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCA N_A11.ab1
SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCA SETP
GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA LCW0402_051_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCA N_B11.ab1
SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCA SETP
GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA LCW0402_059_GFP-
GSEPATSGSETPGSEP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCA N_E11.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCA GSAP
GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA LCW0402_060_GFP-
GTSESATPESGPGSEP GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA N_F11.ab1
ATSGSETPGSEPATSG GGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA SETP
GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA LCW0402_061_GFP-
GTSTEPSEGSAPGTST GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA N_G11.ab1
EPSEGSAPGTSESATP GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA ESGP
GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA LCW0402_065_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA N_A12.ab1
SATPESGPGTSESATP GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCA ESGP
GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA LCW0402_066_GFP-
GSEPATSGSETPGSEP GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCA N_B12.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCA GSAP
GGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA LCW0402_067_GFP-
GSEPATSGSETPGTST GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA N_C12.ab1
EPSEGSAPGSEPATSG GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA SETP
GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW0402_069_GFP-
GTSTEPSEGSAPGTST GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCA N_D12.ab1
EPSEGSAPGSEPATSG GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA LCW0402_073_GFP-
GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCA N_F12.ab1
ATSGSETPGSPAGSPT GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCA STEE
GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW0402_074_GFP-
GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA N_G12.ab1
GSPTSTEEGTSESATP GGTAGCCCAGCTGGTTCTCCAACCTCTACTGAGGAA ESGP
GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA LCW0402_075_GFP-
GTSESATPESGPGSEP GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA N_H12.ab1
ATSGSETPGTSESATP GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA ESGP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA
Example 3: Construction of XTEN_AF36 Segments
[0393] 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].sub.3 where X is a 12 mer
peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27),
GTSTPESGSASP (SEQ ID NO: 28), GTSPSGESSTAP (SEQ ID NO: 29), or
GSTSSTAESPGP (SEQ ID NO: 30). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00019 AF1for: (SEQ ID NO: 1636)
AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC AF1rev: (SEQ ID NO: 1637)
ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA AF2for: (SEQ ID NO: 1638)
AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC AF2rev: (SEQ ID NO: 1639)
ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT AF3for: (SEQ ID NO: 1640)
AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC AF3rev: (SEQ ID NO: 1641)
ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT AF4for: (SEQ ID NO: 1642)
AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC AF4rev: (SEQ ID NO: 1643)
ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA
[0394] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12 mer 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.
[0395] 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 and the sequences
for these segments are listed in Table 15.
TABLE-US-00020 TABLE 15 DNA and Amino Acid Sequences for AF 36-mer
motifs (SEQ ID NOS 353-440, respectively, in order of appearance)
File name Amino acid sequence Nucleotide sequence LCW0403_004_GFP-
GTSTPESGSASPGTSP GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA N_A01.ab1
SGESSTAPGTSPSGES GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAG STAP
GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_GFP-
GTSPSGESSTAPGSTS GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCA N_B01.ab1
STAESPGPGTSPSGES GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG STAP
GTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_GFP-
GSTSSTAESPGPGTSP GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAG N_C01.ab1
SGESSTAPGTSTPESG GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGG SASP
TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAG N_D01.ab1
STAESPGPGTSPSGES GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAG STAP
GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_GFP-
GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_E01.ab1
SGESSTAPGTSTPESG GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA LCW0403_010_GFP-
GSTSSTAESPGPGTST GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG N_F01.ab1
PESGSASPGSTSESPS GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG GTAP
GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_011_GFP-
GSTSSTAESPGPGTST GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG N_G01.ab1
PESGSASPGTSTPESG GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG SASP
GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_GFP-
GSTSESPSGTAPGTSP GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG N_H01.ab1
SGESSTAPGSTSESPS GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG GTAP
TTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCA N_A02.ab1
STAESPGPGTSPSGES GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_GFP-
GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAG N_B02.ab1
PESGSASPGSTSESPS GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAG GTAP
GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG N_C02.ab1
STAESPGPGTSPSGES GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG STAP
TACCTCCCCGAGCGGTGAATCTTCTACTGCACCA LCW0403_017_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG N_D02.ab1
ESPSGTAPGSTSSTAE GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAG SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCA N_E02.ab1
STAESPGPGSTSSTAE GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAG SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCA LCW0403_019_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG N_F02.ab1
STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGG SPGP
TTCCACTAGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG N_H02.ab1
ESPSGTAPGSTSESPS GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG GTAP
TTCTACCAGCGAATCTCCTTCTGGTACTGCACCA LCW0403_024_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAG N_A03.ab1
STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP
TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_GFP-
GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAG N_B03.ab1
STAESPGPGTSPSGES GTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGG STAP
TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_GFP-
GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAG N_D03.ab1
PSGATGSPGSSTPSGA GTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGG TGSP
TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA LCW0403_029_GFP-
GTSPSGESSTAPGTST GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAG N_E03.ab1
PESGSASPGSTSSTAE GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAG SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAG N_F03.ab1
STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGG SASP
TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_031_GFP-
GTSPSGESSTAPGSTS GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAG N_G03.ab1
STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGG SASP
TACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA LCW0403_033_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAG N_H03.ab1
STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP
TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACCGCTGAATCTCCGGGCCCA N_A04.ab1
ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA SPGP
GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_GFP-
GSTSSTAESPGPGTSP GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG N_B04.ab1
SGESSTAPGTSTPESG GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAG SASP
GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG N_C04.ab1
ESPSGTAPGTSPSGES GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAG N_D04.ab1
ESPSGTAPGTSTPESG GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAG SASP
GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_GFP-
GTSTPESGSASPGSTS GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG N_E04.ab1
STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA N_F04.ab1
ESPSGTAPGTSPSGES GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAG STAP
GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAG N_G04.ab1
STAESPGPGSTSESPS GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG GTAP
GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCTGGCCCA N_H04.ab1
STAESPGPGTSTPESG GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAG SASP
GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG N_A05.ab1
STAESPGPGSTSESPS GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGG GTAP
TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_GFP-
GTSPSGESSTAPGSTS GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA N_B05.ab1
ESPSGTAPGSTSSTAE GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG SPGP
GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_GFP-
GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG N_C05.ab1
SGESSTAPGSTSSTAE GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGG SPGP
TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA LCW0403_057_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG N_D05.ab1
ESPSGTAPGTSPSGES GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG STAP
GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG N_E05.ab1
ESPSGTAPGTSTPESG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG SASP
GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_GFP-
GTSTPESGSASPGSTS GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCA N_F05.ab1
ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA SPGP
GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_GFP-
GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCA N_G05.ab1
SGESSTAPGTSPSGES GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAG STAP
GTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_GFP-
GTSPSGESSTAPGTSP GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAG N_H05.ab1
SGESSTAPGTSPSGES GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG STAP
TACCTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_GFP-
GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_A06.ab1
PESGSASPGSTSESPS GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG GTAP
TTCTACTAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_GFP-
GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAG N_B06.ab1
SGESSTAPGTSPSGES GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG STAP
TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG N_C06.ab1
PESGSASPGSTSSTAE GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGG SPGP
TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_068_GFP-
GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_D06.ab1
STAESPGPGSTSESPS GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGG GTAP
TTCTACCAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA N_E06.ab1
PESGSASPGTSTPESG GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAG SASP
GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG N_F06.ab1
PESGSASPGTSTPESG GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA
Example 4: Construction of XTEN_AG36 Segments
[0396] 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 12 mer
peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 31),
GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), or
GASPGTSSTGSP (SEQ ID NO: 34). The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00021 AG1for: (SEQ ID NO: 1644)
AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC AG1rev: (SEQ ID NO: 1645)
ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT AG2for: (SEQ ID NO: 1646)
AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC AG2rev: (SEQ ID NO: 1647)
ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT AG3for: (SEQ ID NO: 1648)
AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC AG3rev: (SEQ ID NO: 1649)
ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA AG4for: (SEQ ID NO: 1650)
AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC AG4rev: (SEQ ID NO: 1651)
ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC
[0397] We also annealed the phosphorylated oligonucleotide
3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the
non-phosphorylated oligonucleotide pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide
pairs were ligated, which resulted in a mixture of products with
varying length that represents the varying number of 12 mer 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 LCW0404 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.
[0398] We screened 96 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 16.
TABLE-US-00022 TABLE 16 DNA and Amino Acid Sequences for AG 36-mer
motifs (SEQ ID NOS 441-528, respectively, in order of appearance)
File name Amino acid sequence Nucleotide sequence LCW0404_001_GFP-
GASPGTSSTGSPGTPGS GGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCA N_A07.ab1
GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAG SP
GTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_GFP-
GSSTPSGATGSPGSSPS GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG N_B07.ab1
ASTGTGPGSSTPSGATG GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG SP
TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_GFP-
GASPGTSSTGSPGSSPS GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG N_C07.ab1
ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGG SP
TAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCA LCW0404_007_GFP-
GTPGSGTASSSPGSSTPS GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAG N_D07.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGG
TGCATCCCCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_GFP-
GTPGSGTASSSPGASPG GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAG N_E07.ab1
TSSTGSPGSRPSASTGT GTGCTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGG GP
TTCTAGACCTTCTGCATCCACCGGTACTGGTCCA LCW0404_011_GFP-
GASPGTSSTGSPGSSTPS GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAG N_F07.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGG
TGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_012_GFP-
GTPGSGTASSSPGSSTPS GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCA N_G07.ab1
GATGSPGSSTPSGATGSP GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG
GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA LCW0404_014_GFP-
GASPGTSSTGSPGASPG GGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAG N_H07.ab1
TSSTGSPGASPGTSSTGSP GTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG
TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_GFP-
GSSTPSGATGSPGSSPS GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA N_A08.ab1
ASTGTGPGASPGTSSTG GGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAG SP
GTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAG N_B08.ab1
GATGSPGTPGSGTASSSP GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG N_C08.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGG
TGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_018_GFP-
GTPGSGTASSSPGSSPS GGTACTCCTGGTAGCGGTACCGCATCTTCCTCTCCAG N_D08.ab1
ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCATCTACCGGTACCGGTCCAGG SP
TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCA LCW0404_023_GFP-
GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAG N_F08.ab1
ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGG SP
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG N_G08.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG
TGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_GFP-
GTPGSGTASSSPGSSTPS GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAG N_A09.ab1
GATGSPGSSPSASTGTGP GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG
TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCA LCW0404_030_GFP-
GSSTPSGATGSPGTPGS GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAG N_B09.ab1
GTASSSPGTPGSGTASS GTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG SP
GTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCA LCW0404_031_GFP-
GTPGSGTASSSPGSSTPS GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG N_C09.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG
TGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA LCW0404_034_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG N_D09.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAG
GTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_GFP-
GASPGTSSTGSPGTPGS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG N_E09.ab1
GTASSSPGSSTPSGATG GTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAG SP
GTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCA LCW0404_036_GFP-
GSSPSASTGTGPGSSTPS GGTTCTAGCCCGTCTGCTTCCACCGGTACTGGCCCAG N_F09.ab1
GATGSPGTPGSGTASSSP GTAGCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGG
TACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_GFP-
GASPGTSSTGSPGSSPS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG N_G09.ab1
ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG SP
TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_GFP-
GASPGTSSTGSPGSSTPS GGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCA N_H09.ab1
GATGSPGSSTPSGATGSP GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG
GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_GFP-
GTPGSGTASSSPGSSTPS GGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAG N_A10.ab1
GATGSPGTPGSGTASSSP GTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGG
TACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_GFP-
GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAG N_C10.ab1
GATGSPGSSTPSGATGSP GTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGG
TAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_GFP-
GASPGTSSTGSPGSSPS GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAG N_D10.ab1
ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGG GP
TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA LCW0404_047_GFP-
GTPGSGTASSSPGASPG GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG N_F10.ab1
TSSTGSPGASPGTSSTGSP GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGG
TGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA LCW0404_048_GFP-
GSSTPSGATGSPGASPG GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG N_G10.ab1
TSSTGSPGSSTPSGATGSP GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG
TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_GFP-
GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG N_H10.ab1
GTASSSPGSSTPSGATG GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG SP
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_GFP-
GASPGTSSTGSPGSSPS GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAG N_A11.ab1
ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG SP
TAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAG N_B11.ab1
GATGSPGSSTPSGATGSP GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG
TAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCA LCW0404_052_GFP-
GASPGTSSTGSPGTPGS GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCA N_C11.ab1
GTASSSPGASPGTSSTG GGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAG SP
GTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_GFP-
GSSTPSGATGSPGSSPS GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAG N_D11.ab1
ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCATCCACTGGTACCGGTCCAGG SP
TGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_057_GFP-
GASPGTSSTGSPGSSTPS GGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAG N_E11.ab1
GATGSPGSSPSASTGTGP GTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGG
TTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_GFP-
GTPGSGTASSSPGSSTPS GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG N_F11.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGG
TGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_GFP-
GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA N_G11.ab1
GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAG SP
GTAGCTCTACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_GFP-
GSSPSASTGTGPGSSPS GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAG N_H11.ab1
ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGG SP
TGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_GFP-
GTPGSGTASSSPGSSTPS GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAG N_A12.ab1
GATGSPGSNPSASTGTGP GTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGG
TTCTAACCCTTCTGCATCCACCGGTACCGGCCCA LCW0404_068_GFP-
GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAG N_B12.ab1
GATGSPGASPGTSSTGSP GTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGG
TGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_069_GFP-
GSSTPSGATGSPGASPG GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAG N_C12.ab1
TSSTGSPGTPGSGTASSSP GTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAG
GTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCA LCW0404_070_GFP-
GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG N_D12.ab1
GATGSPGSSTPSGATGSP GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG
TAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCA LCW0404_073_GFP-
GASPGTSSTGSPGTPGS GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAG N_E12.ab1
GTASSSPGSSTPSGATG GTACCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGG SP
TAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCA LCW0404_075_GFP-
GSSTPSGATGSPGSSPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAG N_F12.ab1
ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG GP
TTCTAGCCCGTCTGCATCTACTGGTACTGGTCCA LCW0404_080_GFP-
GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAG N_G12.ab1
ASTGTGPGSSPSASTGT GTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGG GP
TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA LCW0404_081_GFP-
GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAG N_H12.ab1
ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG SP
TACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA
Example 5: Construction of XTEN_AE864
[0399] 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.
[0400] 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.
[0401] 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_AM144
[0402] A collection of XTEN_AM144 segments was constructed starting
from 37 different segments of XTEN_AE36, 44 segments of XTEN_AF36,
and 44 segments of XTEN_AG36.
[0403] Cultures of E. coli that harboring all 125 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_AM72.
[0404] This library of XTEN_AM72 segments was designated LCW0461.
All clones from LCW0461 were combined and dimerized again using the
same process as described above yielding library LCW0462. 1512
Isolates from library LCW0462 were screened for protein expression.
Individual colonies were transferred into 96 well plates and
cultured overnight as starter cultures. These starter cultures were
diluted into fresh autoinduction medium and cultured for 20-30 h.
Expression was measured using a fluorescence plate reader with
excitation at 395 nm and emission at 510 nm. 192 isolates showed
high level expression and were submitted for DNA sequencing. Most
clones in library LCW0462 showed good expression and similar
physicochemical properties suggesting that most combinations of
XTEN_AM36 segments yield useful XTEN sequences. Thirty isolates
from LCW0462 were chosen as a preferred collection of XTEN_AM144
segments for the construction of multifunctional proteins that
contain multiple XTEN segments. The file names of the nucleotide
and amino acid constructs and the sequences for these segments are
listed in Table 17.
TABLE-US-00023 TABLE 17 DNA and amino acid sequences for AM144
segments (SEQ ID NOS 529-594, respectively, in order of appearance)
Clone Sequence Trimmed Protein Sequence LCW462_r1
GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGC GTPGSGTASSSPGS
TCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCC STPSGATGSPGSSTP
CGTCTGGTGCAACCGGCTCCCCAGGTAGCCCGGCTGGCTCTC SGATGSPGSPAGSP
CTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG TSTEEGTSESATPES
AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAP
CTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGG GSSPSASTGTGPGS
TTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT SPSASTGTGPGASP
CCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTACCGAAC GTSSTGSPGTSTEPS
CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTG EGSAPGTSTEPSEG
AGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTG SAPGSEPATSGSETP AAACTCCA
LCW462_r5 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGGTTCTA
GSTSESPSGTAPGST CTAGCGAATCCCCTTCTGGTACCGCACCAGGTACTTCTCCGAG
SESPSGTAPGTSPSG CGGCGAATCTTCTACTGCTCCAGGTACCTCTACTGAACCTTCC
ESSTAPGTSTEPSEG GAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGC
SAPGTSTEPSEGSAP AGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT
GTSESATPESGPGA CCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTA
SPGTSSTGSPGSSTP GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC
SGATGSPGASPGTS GGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAGCGAATCT
STGSPGSTSESPSGT CCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTG
APGSTSESPSGTAP GCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTT
GTSTPESGSASP CTCCA LCW462_r9
GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACT GTSTEPSEGSAPGT
TCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA SESATPESGPGTSES
AGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTT ATPESGPGTSTEPSE
CTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG GSAPGTSESATPES
AGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAP
CACCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG GTSTEPSEGSAPGS
GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCC EPATSGSETPGSPA
CGGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCTCCTG GSPTSTEEGASPGT
GCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCTTC SSTGSPGSSPSASTG
TACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGT TGPGSSPSASTGTGP
ACTGGTCCA LCW462_r10 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC
GSEPATSGSETPGT TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA
SESATPESGPGTSES AGCGCTACTCCGGAATCCGGTCCAGGTTCTACCAGCGAATCT
ATPESGPGSTSESPS CCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG
GTAPGSTSESPSGT GTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGC
APGTSPSGESSTAP ACCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT
GASPGTSSTGSPGS TCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTA
SPSASTGTGPGSSTP CCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCGTC
SGATGSPGSSTPSG TGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCT
ATGSPGSSTPSGAT ACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTT
GSPGASPGTSSTGSP CTCCA LCW462_r15
GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTA GASPGTSSTGSPGS
GCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTP
TTCTGGTGCAACCGGCTCTCCAGGTACTTCTGAAAGCGCTACC SGATGSPGTSESAT
CCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT PESGPGSEPATSGSE
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT TPGSEPATSGSETP
CCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGT GTSESATPESGPGT
ACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCT STEPSEGSAPGTSTE
ACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACCGAA PSEGSAPGTSTEPSE
CCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCT GSAPGTSTEPSEGS
GAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCT APGSEPATSGSETP GAAACTCCA
LCW462_r16 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC
GTSTEPSEGSAPGSP CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP AACCTTCTGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAA
SEGSAPGTSESATP CTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT
ESGPGSEPATSGSE CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTG
TPGTSESATPESGP GTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG
GSPAGSPTSTEEGT GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC
SESATPESGPGTSTE TACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGC
PSEGSAPGSEPATS TACTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCC
GSETPGTSTEPSEGS GAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCT
APGSEPATSGSETP GAAACTCCA LCW462_r20
GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT
TCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTE
GAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCG PSEGSAPGTSTEPSE
TCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAG GSAPGTSTEPSEGS
GGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC APGTSTEPSEGSAP
GCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA GTSTEPSEGSAPGT
GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT SESATPESGPGTSES
TCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTACTG ATPESGPGTSTEPSE
AACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTT GSAPGSEPATSGSE
CTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCT TPGSPAGSPTSTEE
CCACCGAGGAA LCW462_r23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE AACCTTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCC
PSEGSAPGSTSESPS CTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGG
GTAPGSTSESPSGT CACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT
APGTSTPESGSASP CCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT
GSEPATSGSETPGT ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA
SESATPESGPGTSTE CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTACTGAAC
PSEGSAPGTSTEPSE CGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC
GSAPGTSESATPES CGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT
GPGTSESATPESGP CCGGCCCA LCW462_r24
GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTTCTA GSSTPSGATGSPGS
GCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTP
TTCTGGTGCTACTGGTTCTCCAGGTAGCCCTGCTGGCTCTCCG SGATGSPGSPAGSP
ACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA TSTEEGSPAGSPTST
CTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTC EEGTSTEPSEGSAP
CAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTC GASPGTSSTGSPGS
TAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGC SPSASTGTGPGTPG
AGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAGCTCTACTG SGTASSSPGSTSSTA
CTGAATCTCCTGGCCCAGGTACTTCTCCTAGCGGTGAATCTTC ESPGPGTSPSGESST
TACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCT APGTSTPESGSASP CCA
LCW462_r27 GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT TCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT
SESATPESGPGTSTE GAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCG
PSEGSAPGTSTEPSE TCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCG
GSAPGTSESATPES GAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCC
GPGTSESATPESGP GGCCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG
GTPGSGTASSSPGA GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTC
SPGTSSTGSPGASP TCCGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGCTGGC
GTSSTGSPGSPAGS TCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCG
PTSTEEGSPAGSPTS ACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGT
TEEGTSTEPSEGSAP AGCGCTCCA LCW462_r28
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACT GSPAGSPTSTEEGT
TCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT STEPSEGSAPGTSTE
GAACCTTCTGAGGGCAGCGCTCCAGGTACCTCTACCGAACCG PSEGSAPGTSTEPSE
TCTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT GSAPGTSESATPES
GAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT GPGTSESATPESGP
GGCCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG GTPGSGTASSSPGS
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTC STPSGATGSPGASP
TCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTACTGAA GTSSTGSPGTSTEPS
CCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC EGSAPGTSESATPE
CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGT SGPGTSTEPSEGSAP
AGCGCACCA LCW462_r38 GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACT
GSEPATSGSETPGT TCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCG
SESATPESGPGSEPA GCTACTTCCGGCTCTGAAACCCCAGGTAGCTCTACCCCGTCTG
TSGSETPGSSTPSGA GTGCAACCGGCTCCCCAGGTACTCCTGGTAGCGGTACCGCTT
TGSPGTPGSGTASS CTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTC
SPGSSTPSGATGSP CCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGT
GASPGTSSTGSPGS AGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC
STPSGATGSPGASP CGGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCTGCTA
GTSSTGSPGSEPATS CTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCCGA
GSETPGTSTEPSEGS GGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA
APGSEPATSGSETP AACTCCA LCW462_r39
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT
TCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAA STEPSEGSAPGTSES
AGCGCAACCCCTGAATCCGGTCCAGGTAGCCCTGCTGGCTCT ATPESGPGSPAGSP
CCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT TSTEEGSPAGSPTST
TCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC EEGTSTEPSEGSAP
GCTCCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GSPAGSPTSTEEGT
GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC STEPSEGSAPGTSTE
TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTCCCCG PSEGSAPGASPGTS
GGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTT STGSPGSSPSASTGT
CTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGG GPGSSPSASTGTGP
TACTGGTCCA LCW462_r41 GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTT
GSSTPSGATGSPGA CTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC
SPGTSSTGSPGSSTP GTCTGGTGCTACTGGCTCTCCAGGTAGCCCTGCTGGCTCTCCA
SGATGSPGSPAGSP ACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA
TSTEEGTSESATPES TCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC
GPGSEPATSGSETP CCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTA
GASPGTSSTGSPGS GCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCC
STPSGATGSPGSSPS TTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAGCGAATCC
ASTGTGPGSTSESPS CCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTG
GTAPGSTSESPSGT GCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT
APGTSTPESGSASP CTCCA LCW462_r42
GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTA GSTSESPSGTAPGST
CTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG SESPSGTAPGTSPSG
CGGCGAATCTTCTACCGCACCAGGTACCTCTGAAAGCGCTAC ESSTAPGTSESATPE
TCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGG SGPGTSTEPSEGSAP
TAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGC GTSTEPSEGSAPGT
ACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG STEPSEGSAPGTSES
TACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT ATPESGPGTSTEPSE
ACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCTACCCCG GSAPGSSTPSGATG
TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT SPGASPGTSSTGSP
CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSSTPSGATGSP CTCTCCA
LCW462_r43 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCT
GSTSSTAESPGPGTS CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAG
PSGESSTAPGTSPSG CGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACCGCT
ESSTAPGSTSSTAES GAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTC
PGPGSTSSTAESPGP CTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCC
GTSTPESGSASPGTS AGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCT
PSGESSTAPGSTSST ACCAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACCC
AESPGPGTSTPESGS CGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAGCTCTACCG
ASPGSTSSTAESPGP CTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGG
GSTSESPSGTAPGTS CACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCA
PSGESSTAP CCA LCW462_r45
GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA GTSTPESGSASPGST
CCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCT SESPSGTAPGSTSST
CTACTGCTGAATCTCCGGGCCCAGGTACCTCTACTGAACCTTC AESPGPGTSTEPSE
CGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGG GSAPGTSTEPSEGS
CAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG APGTSESATPESGP
TCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGG GTSESATPESGPGT
TACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCT STEPSEGSAPGTSTE
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGC PSEGSAPGTSESATP
GCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCC ESGPGTSTEPSEGS
GAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGT APGTSTEPSEGSAP AGCGCTCCC
LCW462_r47 GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC
GTSTEPSEGSAPGT TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCG
STEPSEGSAPGSEPA GCAACCTCCGGTTCTGAAACTCCAGGTACTTCTACTGAACCGT
TSGSETPGTSTEPSE CTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG
GSAPGTSESATPES AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG
GPGTSESATPESGP GCCCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG
GASPGTSSTGSPGS GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC
SPSASTGTGPGSSTP TACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCG
SGATGSPGSSTPSG TCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTG
ATGSPGSSTPSGAT CTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCG
GSPGASPGTSSTGSP GTTCTCCA LCW462_r54
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGC GSEPATSGSETPGS
GAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT EPATSGSETPGTSTE
GAACCTTCTGAGGGCAGCGCACCAGGTAGCGAACCTGCAACC PSEGSAPGSEPATS
TCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GSETPGTSESATPES
GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GPGTSTEPSEGSAP
GCACCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG GSSTPSGATGSPGS
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTC STPSGATGSPGASP
TCCGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACCCCG GTSSTGSPGSSTPSG
TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT ATGSPGASPGTSST
CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSPGSSTPSGATGSP CTCTCCA
LCW462_r55 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE AACCTTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCTA
PSEGSAPGTSESATP CTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCCGAAG
ESGPGTSTEPSEGS GCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCG
APGTSTEPSEGSAP CTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGG
GSTSESPSGTAPGTS TACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC
PSGESSTAPGTSPSG CCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGGCTGGC
ESSTAPGSPAGSPTS TCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC
TEEGTSESATPESGP CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA
GTSTEPSEGSAP GCGCTCCA LCW462_r57
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGS
GAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCT EPATSGSETPGSPA
GGCTCTCCGACCTCCACCGAGGAAGGTAGCCCGGCAGGCTCT GSPTSTEEGSPAGSP
CCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG TSTEEGTSESATPES
GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGC GPGTSTEPSEGSAP
GCACCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA GTSTEPSEGSAPGT
GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACT STEPSEGSAPGTSES
TCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCTACT ATPESGPGSSTPSG
CCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTT ATGSPGSSPSASTG
CCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA TGPGASPGTSSTGSP
CTGGTTCTCCA LCW462_r61 GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGGTAGC
GSEPATSGSETPGSP CCTGCTGGCTCTCCGACCTCTACCGAAGAAGGTACCTCTGAA
AGSPTSTEEGTSES AGCGCTACCCCTGAGTCTGGCCCAGGTACCTCTACTGAACCTT
ATPESGPGTSTEPSE CCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG
GSAPGTSTEPSEGS GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG
APGTSESATPESGP GTCCAGGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG
GTSTPESGSASPGST GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTA
SESPSGTAPGSTSST CTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTGAAA
AESPGPGTSESATP GCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGT
ESGPGTSTEPSEGS CCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG
APGTSTEPSEGSAP GTAGCGCTCCA LCW462_r64
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGT
TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTE
AACCTTCCGAAGGTAGCGCACCAGGTACCTCTACCGAACCGT PSEGSAPGTSTEPSE
CTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTG GSAPGTSESATPES
AGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCTG GPGTSESATPESGP
GCCCAGGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG GTPGSGTASSSPGS
GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTC STPSGATGSPGASP
TCCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCAGCTCT GTSSTGSPGSTSSTA
ACTGCTGAATCTCCTGGTCCAGGTACCTCTCCTAGCGGTGAAT ESPGPGTSPSGESST
CTTCTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGC APGTSTPESGSASP TTCTCCA
LCW462_r67 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT
GSPAGSPTSTEEGT TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACC
SESATPESGPGTSTE GAACCGTCTGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA
PSEGSAPGTSESATP ACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGC
ESGPGSEPATSGSE TCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC
TPGTSTEPSEGSAP GCACCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA
GSPAGSPTSTEEGT GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC
STEPSEGSAPGTSTE TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCTACC
PSEGSAPGTSTEPSE GAACCGTCCGAGGGCAGCGCTCCAGGTACTTCTACTGAACCT
GSAPGTSTEPSEGS TCTGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAA
APGTSTEPSEGSAP GGTAGCGCACCA LCW462_r69
GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTA GTSPSGESSTAPGST
CTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAG SSTAESPGPGTSPSG
CGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACT ESSTAPGTSESATPE
CCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGT SGPGTSTEPSEGSAP
AGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA GTSTEPSEGSAPGSS
CCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA PSASTGTGPGSSTPS
GCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCC GATGSPGASPGTSS
GGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAA TGSPGTSTPESGSAS
AGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT PGTSPSGESSTAPGT
CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGC SPSGESSTAP TCCA
LCW462_r70 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC
GTSESATPESGPGT TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE AACCGTCCGAAGGTAGCGCACCAGGTAGCCCTGCTGGCTCTC
PSEGSAPGSPAGSP CGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTT
TSTEEGSPAGSPTST CTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCG
EEGTSTEPSEGSAP CTCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGG
GSSPSASTGTGPGS TAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT
STPSGATGSPGSSTP ACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAACCGGCA
SGATGSPGSEPATS ACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACT
GSETPGTSESATPES CCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCT
GPGSEPATSGSETP GAAACCCCA LCW462_r72
GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT
TCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTE
GAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACCCCGTCT PSEGSAPGSSTPSG
GGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTA ATGSPGASPGTSST
CCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTC GSPGSSTPSGATGS
TCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG PGTSESATPESGPGS
TAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT EPATSGSETPGTSTE
ACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTAGCGAA PSEGSAPGSTSESPS
TCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGT GTAPGSTSESPSGT
CTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCG APGTSTPESGSASP CTTCTCCA
LCW462_r73 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCA
GTSTPESGSASPGST CTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTC
SSTAESPGPGSTSST TACTGCTGAATCTCCTGGCCCAGGTTCTAGCCCTTCTGCATCT
AESPGPGSSPSAST ACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCG
GTGPGSSTPSGATG GCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC
SPGASPGTSSTGSP AGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTAC
GSEPATSGSETPGT CTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGC
SESATPESGPGSPA AGGTTCTCCGACTTCCACTGAGGAAGGTTCTACTAGCGAATC
GSPTSTEEGSTSESP TCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCT
SGTAPGSTSESPSGT GGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCT
APGTSTPESGSASP TCTCCC LCW462_r78
GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACT GSPAGSPTSTEEGT
TCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SESATPESGPGTSTE
AACCGTCCGAAGGTAGCGCTCCAGGTTCTACCAGCGAATCTC PSEGSAPGSTSESPS
CTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGG GTAPGSTSESPSGT
TACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCA APGTSPSGESSTAP
CCAGGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GTSTEPSEGSAPGSP
AGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCT AGSPTSTEEGTSTEP
ACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGAACCTGCA SEGSAPGSEPATSG
ACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT SETPGTSESATPESG
CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC PGTSTEPSEGSAP AGCGCACCA
LCW462_r79 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC
GTSTEPSEGSAPGSP CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP AACCTTCTGAGGGTAGCGCACCAGGTACCTCCCCTAGCGGCG
SEGSAPGTSPSGESS AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTC
TAPGTSPSGESSTAP TACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA
GTSPSGESSTAPGST CCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTT
SESPSGTAPGSTSES CTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTAC
PSGTAPGTSTPESGS CCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAACCTGCAAC
ASPGSEPATSGSETP CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT
GTSESATPESGPGT GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
STEPSEGSAP GCACCA LCW462_r87
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC GSEPATSGSETPGT
TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA SESATPESGPGTSES
AGCGCTACTCCGGAATCCGGTCCAGGTACTTCTCCGAGCGGT ATPESGPGTSPSGES
GAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAAT STAPGSTSSTAESPG
CTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGC PGTSPSGESSTAPGS
TCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGT TSESPSGTAPGTSPS
ACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCA GESSTAPGSTSSTA
GCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACTCCGTC ESPGPGSSTPSGAT
TGGTGCAACCGGTTCCCCAGGTAGCTCTACCCCTTCTGGTGCA GSPGSSTPSGATGS
ACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACTGG PGSSTPSGANWLS CTCTCC
LCW462_r88 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGC
GSPAGSPTSTEEGSP CCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP AACCTTCCGAAGGTAGCGCTCCAGGTACCTCTACTGAACCTT
SEGSAPGTSTEPSE CCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG
GSAPGTSTEPSEGS GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG
APGTSESATPESGP GTCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGG
GASPGTSSTGSPGS TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCC
STPSGATGSPGASP CCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGT
GTSSTGSPGSSTPSG CTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG
ATGSPGTPGSGTAS CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGG
SSPGSSTPSGATGSP CTCTCCA LCW462_r89
GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTC GSSTPSGATGSPGT
CGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC PGSGTASSSPGSSTP
TTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCT SGATGSPGSPAGSP
ACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAG TSTEEGTSESATPES
TCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT GPGTSTEPSEGSAP
CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGT GTSESATPESGPGS
AGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT EPATSGSETPGTSES
GAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTACTGAA ATPESGPGTSTEPSE
CCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC GSAPGTSESATPES
CCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG GPGTSESATPESGP
TCCGGCCCA
Example 7: Construction of XTEN_AM288
[0405] The entire library LCW0462 was dimerized as described in
Example 6 resulting in a library of XTEN_AM288 clones designated
LCW0463. 1512 isolates from library LCW0463 were screened using the
protocol described in Example 6. 176 highly expressing clones were
sequenced and 40 preferred XTEN_AM288 segments were chosen for the
construction of multifunctional proteins that contain multiple XTEN
segments with 288 amino acid residues.
Example 8: Construction of XTEN_AM432
[0406] We generated a library of XTEN_AM432 segments by recombining
segments from library LCW0462 of XTEN_AM144 segments and segments
from library LCW0463 of XTEN_AM288 segments. This new library of
XTEN_AM432 segment was designated LCW0464. Plasmids were isolated
from cultures of E. coli harboring LCW0462 and LCW0463,
respectively. 1512 isolates from library LCW0464 were screened
using the protocol described in Example 6. 176 highly expressing
clones were sequenced and 39 preferred XTEN_AM432 segment were
chosen for the construction of longer XTENs and for the
construction of multifunctional proteins that contain multiple XTEN
segments with 432 amino acid residues.
[0407] In parallel we constructed library LMS0100 of XTEN_AM432
segments using preferred segments of XTEN_AM144 and XTEN_AM288.
Screening this library yielded 4 isolates that were selected for
further construction
Example 9: Construction of XTEN_AM875
[0408] 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.
[0409] We annealed the phosphorylated oligonucleotide
BsaI-AscI-KpnIforP:
AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID
NO: 1652) and the non-phosphorylated oligonucleotide
BsaI-AscI-KpnIrev:
TABLE-US-00024 (SEQ ID NO: 1653)
CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC
for introducing the sequencing island A (SI-A) which encodes amino
acids GASASGAPSTG (SEQ ID NO: 1654) and has the restriction enzyme
AscI recognition nucleotide sequence GGCGCGCC inside. The annealed
oligonucleotide pairs were ligated with BsaI and KpnI digested
stuffer vector pCW0359 prepared above to yield pCW0466 containing
SI-A. We then generated a library of XTEN_AM443 segments by
recombining 43 preferred XTEN_AM432 segments from Example 8 and
SI-A segments from pCW0466 at C-terminus using the same
dimerization process described in Example 5. This new library of
XTEN_AM443 segments was designated LCW0479.
[0410] We generated a library of XTEN_AM875 segments by recombining
segments from library LCW0479 of XTEN_AM443 segments and 43
preferred XTEN_AM432 segments from Example 8 using the same
dimerization process described in example 5. This new library of
XTEN_AM875 segment was designated LCW0481.
Example 10: Construction of XTEN_AM1318
[0411] We annealed the phosphorylated oligonucleotide
BsaI-FseI-KpnIforP:
AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID
NO: 1655) and the non-phosphorylated oligonucleotide
BsaI-FseI-KpnIrev:
CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO:
1656)
TABLE-US-00025 (SEQ ID NO: 1656)
CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG
for introducing the sequencing island B (SI-B) which encodes amino
acids GPEPTGPAPSG (SEQ ID NO: 1657) and has the restriction enzyme
FseI recognition nucleotide sequence GGCCGGCC inside. The annealed
oligonucleotide pairs were ligated with BsaI and KpnI digested
stuffer vector pCW0359 as used in Example 9 to yield pCW0467
containing SI-B. We then generated a library of XTEN_AM443 segments
by recombining 43 preferred XTEN_AM432 segments from Example 8 and
SI-B segments from pCW0467 at C-terminus using the same
dimerization process described in example 5. This new library of
XTEN_AM443 segments was designated LCW0480.
[0412] We generated a library of XTEN_AM1318 segments by
recombining segments from library LCW0480 of XTEN_AM443 segments
and segments from library LCW0481 of XTEN_AM875 segments using the
same dimerization process as in Example 5. This new library of
XTEN_AM1318 segment was designated LCW0487.
Example 11: Construction of XTEN_AD864
[0413] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AD864 sequences starting from
segments of XTEN_AD36 listed in Example 1. These sequences were
assembled as described in Example 5. Several isolates from
XTEN_AD864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. One
intermediate construct of XTEN_AD576 was sequenced. This clone was
evaluated in a PK experiment in cynomolgus monkeys and a half-life
of about 20 h was measured.
Example 12: Construction of XTEN_AF864
[0414] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AF864 sequences starting from
segments of XTEN_AF36 listed in Example 3. These sequences were
assembled as described in Example 5. Several isolates from
XTEN_AF864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. One
intermediate construct of XTEN_AF540 was sequenced. This clone was
evaluated in a PK experiment in cynomolgus monkeys and a half-life
of about 20 h was measured. A full length clone of XTEN_AF864 had
excellent solubility and showed half-life exceeding 60 h in
cynomolgus monkeys. A second set of XTEN_AF sequences was assembled
including a sequencing island as described in Example 9.
Example 13: Construction of XTEN_AG864
[0415] 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 5. 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 14: Methods of Producing and Evaluating CFXTEN with
Internal and Terminal XTEN
[0416] The design, construction and evaluation of CFXTEN comprising
FVIII and one or more XTEN is accomplished using a systematic
approach. The regions suitable for XTEN insertion sites include,
but are to limited to regions at or proximal to the known domain
boundaries of FVIII, exon boundaries, known surface loops, regions
with a low degree of order, and hydrophilic regions. By analysis of
the foregoing, different regions across the sequence of the FVIII B
domain deleted (BDD) sequence have been identified as insertion
sites for XTEN, non-limiting examples of which are listed in Tables
5-8, and shown schematically in FIGS. 8 and 9. Initially,
individual constructs are created (using methods described, below)
in which DNA encoding a single XTEN or XTEN fragment of a length
ranging from 6 to 2004 amino acid residues is inserted into the
FVIII sequence corresponding to or near (e.g., within 6 amino
acids) each of the single insertion sites identified in Table 5,
Table 6, Table 7, Table 8, and Table 9, and the resulting
constructs are expressed and the recovered protein then evaluated
for their effects on retention of procoagulant activity using,
e.g., one of the in vitro assays of Table 49. For example, using
the methods described below, constructs are made in which an XTEN
sequence is inserted within the A1, A2, B, A3, C1 and C2 domain
sequences of FVIII, as well as linked to the C-terminus, and the
resulting expressed fusion proteins are evaluated in a chromogenic
assay of Table 49, compared to a FVIII not linked to XTEN. CFXTEN
fusion proteins can be further classified acting to high,
intermediate and low categories based on the activities they
exhibit. In those cases where the CFXTEN exhibits activity that is
comparable or modestly reduced compared to FVIII, the insertion
site is deemed favorable. In those cases where the activity is
intermediate, the insertion site can be adjusted from 1-6 amino
acids towards the N- or C-terminus of the insertion site and/or the
length or net charge of the XTEN may be altered and the resulting
construct(s) re-evaluated to determine whether the activity is
improved. Alternatively, the XTEN is inserted into the construct
with flanking cleavage sites; preferably sites that are susceptible
to cleavage by proteases found in clotting assays, such that the
XTEN is released during the activation of the FVIII component,
thereby providing additional information about the suitability of
the XTEN insertion site in the fusion protein.
[0417] Once all of the individual insertion sites are evaluated and
the favorable insertion sites are identified, libraries of
constructs are created with two, three, four, five or more XTEN
inserted in the permutations of favorable sites. The length and net
charge of the XTEN (e.g., XTEN of the AE versus AG family) are
varied in order to ascertain the effects of these variables on
FVIII activity and physicochemical properties of the fusion
protein. CFXTEN constructs that retain a desired degree of in vitro
procoagulant FVIII activity are then evaluated in vivo using mouse
and/or dog models of hemophilia A, as described in Examples below,
or other models known in the art. In addition, constructs are
assayed in the presence of FVIII inhibitors and other anti-FVIII
antibodies to determine constructs that retain activity. In
addition, CFXTEN constructs are made that incorporate cleavage
sequences at or near the junction(s) of FVIII and XTEN (e.g.,
sequences from Table 8) designed to release the XTEN and are
evaluated for enhancement of FVIII activity and effects on terminal
half-life. By the iterative process of making constructs combining
different insertion sites, varying the length and composition
qualities of the XTEN (e.g., different XTEN families), and
evaluation, the skilled artisan obtains, by the foregoing methods,
CFXTEN with desired properties, such as but not limited to of
procoagulant FVIII activity, reduced binding with FVIII inhibitors,
enhanced pharmacokinetic properties, ability to administer to a
subject by different routes, and/or enhanced pharmaceutical
properties.
Example 15: Methods of Producing and Evaluating CFXTEN Containing
FVIII and AE_XTEN
[0418] A general scheme for producing and evaluating CFXTEN
compositions is presented in FIG. 15, and forms the basis for the
general description of this Example. Using the disclosed methods
and those known to one of ordinary skill in the art, together with
guidance provided in the illustrative examples, a skilled artesian
can create and evaluate CFXTEN fusion proteins comprising XTEN and
FVIII or variants of FVIII known in the art. The Example is,
therefore, to be construed as merely illustrative, and not
limitative of the methods in any way whatsoever; numerous
variations will be apparent to the ordinarily skilled artisan. In
this Example, a CFXTEN of a factor VIII BDD linked to an XTEN of
the AE family of motifs is created.
[0419] The general scheme for producing polynucleotides encoding
XTEN is presented in FIGS. 11 and 12. FIG. 14 is a schematic
flowchart of representative steps in the assembly of an XTEN
polynucleotide construct in one of the embodiments of the
invention. 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 that can
multimerize 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 motif
libraries include specific sequence XTEN families; e.g., AD, AE,
AF, AG, AM, or AQ sequences of Table 3. As illustrated in FIG. 14,
the XTEN length, in this case, is 36 amino acid residues, but
longer lengths are also achieved by this general process. For
example, multimerization is performed by ligation, overlap
extension, PCR assembly or similar cloning techniques known in the
art that, in this case, result in a construct with 288 amino acid
residues. 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
can be 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 containing a gene encoding the FVIII,
resulting in the gene 500 encoding a CFXTEN fusion protein with a
288 amino acid XTEN linked to the C-terminus of the factor VIII. As
would be apparent to one of ordinary skill in the art, the methods
are applied to create constructs in alternative configurations and
with varying XTEN lengths or in multiple locations.
[0420] DNA sequences encoding FVIII are conveniently obtained by
standard procedures known in the art from a cDNA library prepared
from an appropriate cellular source, from a genomic library, or may
be created synthetically (e.g., automated nucleic acid synthesis)
using DNA sequences obtained from publicly available databases,
patents, or literature references. In the present example, a FVIII
B domain deleted (BDD) variant is prepared as described in Example
17. A gene or polynucleotide encoding the FVIII portion of the
protein or its complement is then cloned into a construct, such as
those described herein, which can be a plasmid or other vector
under control of appropriate transcription and translation
sequences for high level protein expression in a biological system.
A second gene or polynucleotide coding for the XTEN portion or its
complement is genetically fused to the nucleotides encoding the
terminus of the FVIII gene by cloning it into the construct
adjacent and in frame with the gene coding for the CF, through a
ligation or multimerization step. In this manner, a chimeric DNA
molecule coding for (or complementary to) the CFXTEN fusion protein
is generated within the construct. Optionally, a gene encoding for
a second XTEN is inserted and ligated in-frame internally to the
nucleotides encoding the FVIII-encoding region. The constructs are
designed in different configurations to encode various insertion
sites of the XTEN in the FVIII sequence, including those of Table
5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in
FIGS. 8-9. Optionally, this chimeric DNA molecule is transferred or
cloned into another construct that is a more appropriate expression
vector; e.g., a vector appropriate for a mammalian host cell such
as CHO, BHK and the like. At this point, a host cell capable of
expressing the chimeric DNA molecule is transformed with the
chimeric DNA molecule, described more completely, below, or by
well-known methods, depending on the type of cellular host, as
described supra.
[0421] Host cells containing the XTEN-FVIII expression vector are
cultured in conventional nutrient media modified as appropriate for
activating the promoter. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan. After expression of the fusion protein,
culture broth is harvested and separated from the cell mass and the
resulting crude extract retained for purification of the fusion
protein.
[0422] Gene expression is measured in a sample directly, for
example, by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad.
Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in
situ hybridization, using an appropriately labeled probe, based on
the sequences provided herein. Alternatively, gene expression is
measured by immunological of fluorescent methods, such as
immunohistochemical staining of cells to quantitate directly the
expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of sample fluids may be
either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be prepared against the FVIII
sequence polypeptide using a synthetic peptide based on the
sequences provided herein or against exogenous sequence fused to
FVIII and encoding a specific antibody epitope. Examples of
selectable markers are well known to one of skill in the art and
include reporters such as enhanced green fluorescent protein
(EGFP), beta-galactosidase ((3-gal) or chloramphenicol
acetyltransferase (CAT).
[0423] The CFXTEN polypeptide product is purified via methods known
in the art. Procedures such as gel filtration, affinity
purification, salt fractionation, ion exchange chromatography, size
exclusion chromatography, hydroxyapatite adsorption chromatography,
hydrophobic interaction chromatography or gel electrophoresis are
all techniques that may be used in the purification. Specific
methods of purification are described in Robert K. Scopes, Protein
Purification: Principles and Practice, Charles R. Castor, ed.,
Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step
purification separations are also described in Baron, et al., Crit.
Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr.
A. 679:67-83 (1994).
[0424] As illustrated in FIG. 15, the isolated CFXTEN fusion
proteins are characterized for their chemical and activity
properties. An isolated fusion protein is characterized, e.g., for
sequence, purity, apparent molecular weight, solubility and
stability using standard methods known in the art. The fusion
protein meeting expected standards is evaluated for activity, which
can be measured in vitro or in vivo by measuring one of the factor
VIII-associated parameters described herein, using one or more
assays disclosed herein, or using the assays of the Examples or
Table 49.
[0425] In addition, the CFXTEN FVIII fusion protein is administered
to one or more animal species to determine standard pharmacokinetic
parameters and pharmacodynamic properties, as described in Examples
25 and 26.
[0426] By the iterative process of producing, expressing, and
recovering CFXTEN constructs, followed by their characterization
using methods disclosed herein or others known in the art, the
CFXTEN compositions comprising CF and an XTEN are produced and
evaluated to confirm the expected properties such as enhanced
solubility, enhanced stability, improved pharmacokinetics and
reduced immunogenicity, leading to an overall enhanced therapeutic
activity compared to the corresponding unfused FVIII. For those
fusion proteins not possessing the desired properties, a different
sequence or configuration is constructed, expressed, isolated and
evaluated by these methods in order to obtain a composition with
such properties.
Example 16: Construction of Expression Plasmids for BDD FVIII
[0427] I. Construction of B Domain Deleted FVIII (BDD FVIII)
Expression Vectors
[0428] The expression vector encoding BDD FVIII was created by
cloning the BDD FVIII open reading frame into the pcDNA4 vector
(Invitrogen, CA) containing a polyA to allow for optimal mammalian
expression of the FVIII gene, resulting in a construct designated
pBC0100. Several natural sites were identified within this
construct for cloning use, including BsiWI 48, AflII 381, PshAI
1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI
4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527. To facilitate
assay development, nucleotides encoding Myc and His tag were
introduced into the FVIII open reading frame. pBC0100 was PCR
amplified using the following primers: 1) F8-BsiWI-F:
tattccCGTACGgccgccaccATGCAAATAGAGCTCTCCACCT (SEQ ID NO: 1658); 2)
F8-nostop-XhoI-R1: GGTGACCTCGAGcgtagaggtcctgtgcctcg (SEQ ID NO:
1659) to introduce BsiWI and XhoI in appropriate locations. The PCR
product was digested with BsiWI and XhoI. PcDNA4-Myc-His/C was
digested with Acc65I and XhoI, which generated two products of 5003
and 68 bps. The 5003 bps product was ligated with the digested
PCR'ed FVIII fragment and used for DHSalpha transformation. The
enzymes Acc65I and BsiWI create compatible ends but this ligation
destroys the site for future digestion. The resulting construct was
designated pBC0102 (pcDNA4-FVIII_3-Myc-His). To facilitate the
design and execution of future cloning strategies, especially ones
involving the creation of BDD FVIII expression constructs that
contain multiple XTEN insertions, we selected additional unique
restriction enzyme sites to incorporate, including BsiWI 908, NheI
1829 and ClaI 3281. The introduction of these sites was done via
the QuikChange method (Agilent, CA) individually. The resulting
construct was designated pBC0112 (pcDNA4-FVIII_4-Myc-His). To avoid
problems that may arise from the linker peptides that connects
between Myc/His and FVIII/Myc, and to remove restriction enzyme
sites that are preferred for future XTEN insertion, we mutated the
sequences encoding the peptide sequences from ARGHPF (SEQ ID NO:
1660) to GAGSPGAETA (SEQ ID NO: 178) (between FVIII and Myc), NMHTG
(SEQ ID NO: 1661) to SPATG (SEQ ID NO: 1662) (between Myc and His)
via the QuikChange method. The construct was designated pBC0114
(pcDNA4-FVIII_4-GAGSPGAETA-Myc-SPATG-His (`GAGSPGAETA` and `SPATG`
disclosed as SEQ ID NOS 178 and 1662, respectively)) (sequence in
Table 21), which was used as the base vector for the design and
creation of other expression vectors incorporating XTEN sequences.
Expression and FVIII activity data for this construct are presented
in
[0429] II. Construction of B Domain Deleted FVIII (BDD FVIII)
Expression Vectors
[0430] The gene encoding BDD FVIII is synthesized by GeneArts
(Regensburg, Germany) in the cloning vector pMK (pMK-BDD FVIII).
The BDD FVIII proteins contain 1457 amino acids at a total
molecular weight of 167539.66. There are 6 domains within the
wild-type FVIII protein, the A1, A2, B, A3, C1 and C2 domains. In
the BDD FVIII protein, most of the B domain has been deleted as it
was shown to be an unstructured domain and the removal of the
domain does not alter critical functions of this protein. The pMK
vector used by GeneArts contains no promoter, and can not be used
as an expression vector. Restriction enzyme sites NheI on the 5'
end and SfiI, SalI and XhoI on the 3' end are introduced to
facilitate subcloning of the DNA sequence encoding BDD FVIII into
expression vectors, such as CET1019-HS (Millipore). Several unique
restriction enzyme sites are also introduced into the FVIII
sequence to allow further manipulation (e.g., insertion,
mutagenesis) of the DNA sequences. Unique sites listed with their
cut site include, but are not limited to: SacI 391, AfiII 700, SpeI
966, PshAI 1417, Acc65I 2192, KpnI 2192, BamHI 2250, HindIII 2658,
PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893, SwaI 4265, OliI
4626, XbaI 4644, and BstBI 4673. The HindIII site resides at the
very end of the A2 domain and can potentially be used for
modification of the B domain. The synthesized pMK-BDD FVIII from
GeneArts does not contain a stop codon. The stop codon is
introduced by amplifying a 127 bp fragment of FVIII using the
following primers: 5'-GTGAACTCTCTAGACCCACCG-3' (SEQ ID NO: 1663);
5'-CTCCTCGAGGTCGACTCAGTAGAGGTCCTGTGCCTCG-3' (SEQ ID NO: 1664). The
fragment is digested with XbaI and SalI, and ligated to XbaI/SalI
digested pMK-BDD FVIII. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The construct named pBC0027 (pMK-BDD FVIII-STOP)
contains coding sequences that encode the BDD FVIII protein. The
pBC0027 construct is then digested with NheI/SalI, and ligated with
NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS
vector contains a human CMV promoter and a UCOE sequence to
facilitate gene expression. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The final construct is designated pBC0025
(CET1019-HS-BDD FVIII-STOP), which encodes the BDD FVIII protein
under the control of a human CMV promoter. Introduction of the
pBC0025 construct into mammalian cells is expected to allow
expression of the BDD FVIII protein with procoagulant activity.
Example 17: Construction of Expression Plasmids for BDD FVIII
Containing XTEN
[0431] 1. B Domain AE42_Insertion
[0432] Two PCR reactions were run in parallel to insert
XTEN_AE42_into the remaining B domain region of the BDD FVIII
constructs. The PCR reactions involved the following primers:
cgaaagcgctacgcctgagaGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCTGCCTC
GAGCccaccagtcttgaaacgcc (SEQ ID NO: 1665);
TGATATGGTATCATCATAATCGATTTCCTCTTGATCTGACTG (SEQ ID NO: 1666);
agcttgaggatccagagttc (SEQ ID NO: 1667);
tctcaggcgtagcgctttcgCTTGTCCCCTCTTCTGTTGAGGTGGGGGAGCCAGCAGGAGAACCTGGCG
CGCCgttttgagagaagcttcttggt (SEQ ID NO: 1668). The PCR products then
served as templates, and a second PCR was performed to introduce
the XTEN_AE42 into the FVIII encoding nucleotide sequences flanked
by BamHI and ClaI. This PCR product was digested with BamHI and
ClaI simultaneously with the digestion of PBC0114 with the same two
enzymes. The PCR product was ligated to the digested vector. This
construct was designated pBC0135
(pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA-Myc-SPATG-His) (`GAGSPGAETA`
and `SPATG` disclosed as SEQ ID NOS 178 and 1662, respectively),
and encodes the BDD FVIII with an AE42 XTEN incorporated within the
residual B-domain.
[0433] 2. AE42 Insertion and R1648A Mutation
[0434] The QuikChange method (Agilent, CA) was employed to
introduce an R1648A mutation into PBC0135. This construct was
designated pBC0149
(pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA-Myc-SPATG-His_R1648A)
(`GAGSPGAETA` and `SPATG` disclosed as SEQ ID NOS 178 and 1662,
respectively), eliminating that FVIII processing site.
[0435] 3. B Domain AE288 Insertion
[0436] XTEN_AE288 was PCR amplified using the following primers:
tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1669) and
tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1670). PBC0075 was used
as the template for this PCR reaction. The PCR product was digested
with AscI and XhoI, and PBC0135 was digested with the same enzymes.
The PCR product was ligated to the PBC0135 fragment. This construct
was designated pBC0136
(pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (`GAGSPGAETA`
and `SPATG` disclosed as SEQ ID NOS 178 and 1662, respectively),
and encodes the BDD FVIII with an AE288 XTEN incorporated within
the residual B-domain.
[0437] 4. AE288 Insertion and R1648A Mutation
[0438] XTEN_AE288 was PCR amplified using the following primers:
tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1671) and
tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1672). Construct pBC0075
was used as the template for this PCR reaction. The PCR product was
digested with AscI and XhoI, and pBC0149 was digested with the same
enzymes. The PCR product was ligated to the pBC0149 fragment. This
construct was designated pBC0137
(pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA-Myc-SPATG-His R1648A)
(`GAGSPGAETA` and `SPATG` disclosed as SEQ ID NOS 178 and 1662,
respectively) and contains an AE288 XTEN sequence internal to the B
domain, with the R1648A mutation eliminating that FVIII processing
site.
[0439] 3. B Domain AE144, AG144, AG288 Insertions with and without
R1648A Mutations
[0440] Select XTEN fragments were PCR amplified to introduce AscI
and XhoI sites to the 5' and 3' end respectively. The PCR product
was digested with AscI and XhoI, and pBC0135 (for R1648) or pBC0149
(for A1648) were digested with the same enzymes. The PCR product
was ligated to the pBC0135 or pBC0149 vector. These constructs were
designated pSD0005, 6, 7, 8, 17 and 18.
[0441] Construction of Expression Plasmids for BDD FVIII with XTEN
Insertion at the C Terminus
[0442] 1. C Terminal AE288 Insertion
[0443] XTEN_AE288 was PCR amplified using the following primers:
ggggccgaaacggccggtacctcagagtctgctacc (SEQ ID NO: 1673) and
tgttcggccgtttcggcccctggcgcactgccttc (SEQ ID NO: 1674). The
construct pBC0075 was used as the template for this PCR reaction.
The PCR product was digested with SfiI, and pBC0114 was digested
with the same enzyme. The PCR product was ligated to the digested
pBC0114 fragment. This construct was designated pBC0145
(pcDNA4-FVIII_4-XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (`GAGSPGAETA`
and `SPATG` disclosed as SEQ ID NOS 178 and 1662, respectively),
and encodes an AE288 sequence at the C-terminus of the BDD
FVIII.
[0444] 2. C Terminal AG288 Insertion
[0445] XTEN_AG288 was designed and synthesized by DNA2.0 (Menlo
Park, Calif.). The synthesized gene was PCR amplified using the
following primers: ggggccgaaacggccccgggagcgtcacc (SEQ ID NO: 1675)
and tgttcggccgtttcggcccctgacccggttgcccc (SEQ ID NO: 1676). The PCR
product was digested with SfiI, and PBC0114 based vector was
digested with the same enzyme. The PCR product was ligated to the
digested PBC0114 fragment. This construct was designated pBC0146
(pcDNA4-FVIII_4-XTEN_AG288-GAGSPGAETA-Myc-SPATG-His) (`GAGSPGAETA`
and `SPATG` disclosed as SEQ ID NOS 178 and 1662, respectively),
and encodes an AG288 sequence at the C-terminus of the BDD
FVIII.
[0446] 3. C Terminal AE/AG144, 288, 864 Insertions
AscI and XhoI sites were introduced into the PBC0114 based vector
via QuikChange methods using the primers: 5037-PBC0114-AscI-XhoI-F:
CAGGACCTCTACGGCGCgccagcctcgaGCGAACAAAAACTCATCTCAGAAGAGG (SEQ ID NO:
1677); 5038-PBC0114-AscI-XhoI-R:
CCTCTTCTGAGATGAGTTTTTGTTCGCtcgaggctggcGCGCCGTAGAGGTCCTG (SEQ ID NO:
1678). Various XTEN fragments were PCR amplified with AscI and XhoI
introduced into the 5' and 3' end respectively. The PCR product was
ligated to the digested PBC0114 vector. These constructs were
designated pSD0013, pSD0014, pSD0015, pSD0016, pSD0019 and
pSD0020.
[0447] Construction of Expression Plasmids for BDD FVIII with
Inter- and Intra-Domain XTEN Insertions
[0448] 1. AE7, AE42 and AE144 Insertions
[0449] Four distinct strategies are used for insertion of AE42 into
the designated sites (e.g., the natural or introduced restriction
sites BsiWI 48, AflI 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI
3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI
4500, PmeI 4527, BsiWI 908, NheI 1829 and ClaI 3281) within the BDD
FVIII encoding sequence, each contributing to the creation of
several constructs. By design, these insertions of AE42 create AscI
and XhoI sites flanked on either side of the insertion allowing for
introduction/substitution of longer XTENs, as well as XTEN with
different sequences or incorporated cleavage sequences, as needed.
Specifically, the constructs that contain XTEN_144 insertions are
listed in Table 21. These insertions were created by replacing
either AE7 or AE42 with a PCRed XTEN_144 fragment flanked by AscI
and XhoI sites.
[0450] 2. Double PCR-Mediated Method
[0451] Two PCR reactions are run in parallel to insert XTEN_AE42
into the designated site. The two PCR reactions introduce XTEN on
either the 3' or the 5' end via use of a long primer that contains
partial XTEN. The PCR products then serve as templates, and a
second PCR is performed to introduce the XTEN_AE42 into the FVIII
encoding nucleotide sequences flanked by select restriction enzyme
sites. This PCR product is digested with the appropriate enzymes
simultaneously with the digestion of PBC0114 using the same two
enzymes. The PCR product is ligated to the digested vector. Using
this method, constructs are created designated pBC0126, pBC0127,
pBC0128, and pBC0129, resulting in AE42 insertions at the R3, R3,
P130, L216 locations respectively. The sequences are listed in
Table 21. Select XTEN_144 sequences can then be PCRed to introduce
AscI and XhoI sites on either end of the fragment, and ligate to
digested FVIII-XTEN_AE42 construct. For instance, pSD0053 was
created by replacing the AE42 of pBC0129 with XTEN_AE144. Other
XTEN_144 constructs were created via the same strategy and are
listed in Table 21.
[0452] 3. QuikChange Mediated Two Step Cloning Method
[0453] The QuikChange method is employed to introduce XTEN_AE7
encoding sequences that are flanked by AscI and XhoI into
designated sites. The resulting intermediate construct is then
digested with AscI and XhoI. XTEN_AE42 or XTEN_AE144 is PCR
amplified to introduce the two sites and digested accordingly. The
vector and insert are then ligated to create the final constructs.
The sequences are listed in Table 21.
[0454] 4. Three PCR Type II Restriction Enzyme Mediated Ligation
Method
[0455] Three PCR reactions are performed to create two pieces of
FVIII encoding fragments flanked by one type I restriction enzyme
that correlates with a unique site within the FVIII_4 gene and one
type II enzyme (e.g. BsaI, BbsI, BfuAI), the third PCR reaction
created the XTEN_AE42 flanked by two type II restriction enzyme
sites. The three PCR fragments are digested with appropriate
enzymes and ligated into one linear piece that contains the
XTEN_AE42 insertion within a fragment of FVIII encoding sequences.
This product is then digested with appropriate unique enzymes
within the FVIII encoding sequences and ligated to the PBC0114
construct digested with the same enzymes, and result in constructs
designated pBC0130 (with XTEN insertion at residue P333), pBC0132
(with XTEN insertion at residue D403), pBC0133 (with XTEN insertion
at residue R490). The sequences are listed in Table 21. Select
XTEN_144 sequences can then be PCRed to introduce AscI and XhoI
sites on either end of the fragment, and ligate to digested
FVIII-XTEN_AE42 construct. For instance, pSD0001 and pSD0003 were
created by replacing the AE42 of pBC0132 with XTEN_AE144 and
XTEN_AG144 respectively. Other XTEN_144 constructs listed in Table
21 were created via the same strategy.
[0456] 5. Custom Gene Synthesis
[0457] Custom gene synthesis is performed by GeneArt (Regensburg,
Germany). The genes are designed so that they include nucleotides
encoding the XTEN_AE42 inserted in the designated site(s) and the
genes are flanked by two unique restriction enzyme sites selected
within the FVIII_4 gene. The synthesized genes and PBC0114 are
digested with appropriate enzymes and ligated to create the final
product with the BDD FVIII incorporating the XTEN_AE42 between the
restriction sites. Select XTEN_144 sequences can then be PCRed to
introduce AscI and XhoI sites on either end of the fragment, and
ligate to digested FVIII-XTEN_AE42 construct.
[0458] Construction of Expression Plasmids with Dual XTEN
Insertions in the B Domain and at the C Terminus
[0459] The construct pBC0136, which encodes the BDD FVIII with an
AE288 XTEN incorporated within the residual B-domain, is digested
with BamHI and ClaI, and the resulting 1372 bps fragment from this
digestion is the insert. The construct pBC0146 is digested with
BamHI and ClaI, and the 9791 bps piece from this digestion is the
vector. The vector and insert are ligated together to create
pBC0209, containing an AE288 insertion within the B domain and an
AG288 on the C terminus. The same strategy is utilized to create
constructs containing two AE288 insertions in the B domain and at
the C terminus, respectively, using PBC0145 as the vector.
[0460] Construction of Expression Plasmids with Multiple XTEN
Insertions
[0461] The construct pBC0127, which encodes an AE42 XTEN at the R3
position of FVIII, is digested with BsiWI and AflII, and the
resulting 468 bps fragment from this digestion is the insert. The
construct pBC0209 is digested with BsiWI and AflII, the 10830 bps
piece from this digestion is the vector. The vector and insert are
ligated together to create a construct designated pBC0210,
containing an AE42 insertion in the A1 domain, an extra three ATR
amino acid to restore the signal cleavage sequence, an AE288 XTEN
insertion within the B domain and an AG288 on the C terminus. The
same methodology is used to create constructs encoding multiple
XTEN at the natural and introduced restriction sites; e.g., BsiWI
48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI
3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI
4527, BsiWI 908, NheI 1829 and ClaI 3281.
[0462] Construction of BDD FVIII-Internal-XTEN_AE288 Expression
Vectors
[0463] Two BsaI restriction enzyme sites are introduced into the
PBC0027 pMK-BDD FVIII construct between the base pair 2673 and 2674
using the QuikChange method following manufacturer's protocol
(Agilent Technologies, CA). The inserted DNA sequences are
gggtctcccgcgccagggtctccc, and the resulting construct is designated
pBC0205 (sequence in Table 21). The DNA sequence encoding AE288 (or
other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288)
is then PCR'ed with primers that introduce BsaI sites on both the
5' and 3'. The pBC0205 vector and the insert (XTEN_288) are then
digested with BsaI and ligated to create pBC0206, which encodes the
FVIII gene with an XTEN_AE288 insertion within the B domain
(sequence in Table 21). The pBC0206 construct is then digested with
NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector
(Millipore). The CET1019-HS vector contains a human CMV promoter
and a UCOE sequence to facilitate gene expression. The ligated DNA
mixture is used to transform DH5a bacterial cells. Transformants
are screened by DNA miniprep and the desired constructs are
confirmed by DNA sequencing. The final construct is designated
pBC0207 (CET1019-HS-BDD FVIII-STOP), which encodes the BDD FVIII
protein under the control of a human CMV promoter (sequence in
Table 21). Introduction of the pBC0207 construct into mammalian
cells is expected to allow expression of the BDD FVIII protein with
an internal XTEN_AE288. The same protocol is used to introduce,
transform and express constructs containing other variants and
lengths of XTEN; e.g. AE42, AG42, AG288, AM288, AE864, AG864, or
other XTEN of Table 4.
[0464] Construction of BDD FVIII-/-XTEN_AE864 Expression
Vectors
[0465] The BDD FVIII fragment with NheI and SfiI flanking the 5'
and 3' end is generated by digesting the pBC0025 construct. This
digested fragment is then ligated to a NheI/SfiI digested pSecTag
vector (pBC0048 pSecTag-FVIII-/-XTEN_AE864) encoding the FVIII
followed by the XTEN_AE864 sequence. The ligated DNA mixture is
used to transform DH5a bacterial cells. Transformants are screened
by DNA miniprep and the desired constructs are confirmed by DNA
sequencing. The final construct is pBC0060, which encodes the BDD
FVIII-/-XTEN_AE864 protein under the control of a human CMV
promoter. Introduction of the pBC0060 construct into mammalian
cells is expected to express the FVIII protein with a C terminal
XTEN fusion (BDD FVIII-/-XTEN_AE864) with procoagulant
activity.
[0466] Construction of BDD FVIII-/FXI/-XTEN_AE864 Expression
Vectors
[0467] The BDD FVIII fragment with NheI and SfiI flanking the 5'
and 3' end is generated by digesting the pBC0025 construct. This
digested fragment is then ligated to a NheI/SfiI digested pSecTag
vector (pBC0047 pSecTag-FVIII-/FXI/-XTEN_AE864) encoding the FVIII
followed by the FXI cleavage sequence (/FXI/) and XTEN_AE864. The
ligated DNA mixture is used to transform DH5a bacterial cells.
Transformants are screened by DNA miniprep and the desired
constructs are confirmed by DNA sequencing. The final construct is
pBC0051, which encodes the BDD FVIII-/FXI/-XTEN_AE864 protein under
the control of a human CMV promoter. Introduction of the pBC0051
construct into mammalian cells is expected to express the FVIII
protein with a C terminal XTEN fusion (BDD FVIII-/FXI/-XTEN_AE864),
which could be subsequently cleaved by FXI, therefore liberating
the BDD FVIII protein with procoagulant activity.
[0468] Construction of BDD FVIII-/FXI/-XTEN Expression Vectors
Comprising AE288 or AG288
[0469] The fused AE864 XTEN sequence in pBC0060 is replaced by
digesting the XTEN sequences AE288 and AG288 with BsaI and HindIII.
A subsequent ligation step using the respective AE288 or AG288 XTEN
fragment and BsaI/HindIII digested pBC0051 allows the exchange of
the AE288 or AG288 sequences into the BDD FVIII expression vector.
The resulting final constructs are pBC0061 for BDD FVIII-AE288 and
pBC0062 for BDD FVIII-AG288. Introduction of the pBC0061 construct
into mammalian cells is expected to express the FVIII protein with
a C-terminal AE288 XTEN fusion (BDD FVIII-/-XTEN_AE288) with
procoagulant activity. Introduction of the pBC0062 construct into
mammalian cells is expected to express the FVIII protein with a
C-terminal AG288 XTEN fusion (BDD FVIII-/-XTEN_AG288) with
procoagulant activity.
[0470] Construction of BDD FVIII-/FXI/-XTEN Expression Vectors with
Alternate XTEN
[0471] The fused XTEN sequence in pBC0051 is replaced by digesting
DNA encoding other XTEN sequences (e.g. other variants and lengths
of XTEN; e.g. AE42, AG42, AG288, AM288) with BsaI and HindIII. A
ligation using the XTEN fragment and BsaI/HindIII digested pBC0051
allows the exchange of the various XTEN-encoding sequences into the
BDD FVIII expression vector, providing the alternate constructs.
Introduction of the alternate constructs into mammalian cells is
expected to express the FVIII protein with a C-terminal XTEN (BDD
FVIII-/FXI/-XTEN) that can be subsequently cleaved by FXI,
releasing the FVIII, resulting in procoagulant FVIII fusion with
procoagulant activity.
Example 18: Construction of Expression Plasmids for FVIII Signal
Peptide-XTEN-/FXI/-BDD FVIII
[0472] Construction of Expression Vectors for FVIII Signal
Peptide-XTEN_AE864
[0473] The coding sequences for the FVIII signal peptide is
generated by annealing the following two oligos:
5'-CTAGCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTG
GGTCTCC-3' (SEQ ID NO: 1679);
5'-ACCTGGAGACCCACTAAAGCAGAATCGCAAAAGGCACAGAAAGAAGCAGGTGGAGAGCTC
TATTTGCATG-3' (SEQ ID NO: 1680). The annealed oligos are flanked by
the NheI and BsaI restriction enzyme sites on either end, and is
ligated to NheI/BsaI digested pCW0645 vector which encodes the
FVII-XTEN_AE864. The ligated DNA mixture is used to transform DH5a
bacterial cells. Transformants is screened by DNA miniprep and the
desired constructs are confirmed by DNA sequencing. The final
construct is designated pBC0029, which encodes the signal
peptide-XTEN_AE864 protein under the control of a human CMV
promoter. This construct is used as an intermediate construct for
creating an expression construct with XTEN fused on the N-terminus
of the FVIII protein, and can also be used as a master plasmid for
creating expression constructs that allow XTEN fusion on the
N-terminus of a secreted protein.
[0474] Construction of Signal Peptide-XTEN_AE864-/FXI/-BDD FVIII
Expression Vectors
[0475] An 1800 bp fragment within the FVIII coding region is
amplified using primers that introduce NheI-BbsI-/FXI/-AgeI sites
on the 5' and endogenous KpnI restriction enzyme on the 3' end. The
NheI/KpnI digested FVIII fragment is ligated with NheI/KpnI
digested pBC0027 vector. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The resulting construct is designated pBC0052, which
contains sequences that encode the /FXI/-FVIII protein without the
FVIII signal peptide. This construct is used as an intermediate
construct for creating an expression construct with XTEN fused on
the N-terminus of the FVIII protein.
[0476] The pBC0052 vector is digested with BbsI/XhoI enzymes, and
is used to ligate with Bbsi/XhoI digested pBC0029. The ligated DNA
mixture is used to transform DH5a bacterial cells. Transformants
are screened by DNA miniprep and the desired constructs are
confirmed by DNA sequencing. The final construct is designated
pBC0053, which encodes the signal peptide-XTEN_AE864-/FXI/-BDD
FVIII protein under the control of a human CMV promoter.
Introduction of the pBC0053 construct into mammalian cells is
expected to express the FVIII protein with an N-terminal XTEN
fusion (signal peptide-XTEN_AE864-/FXI/-BDD FVIII), which could be
subsequently cleaved by FXI, therefore liberating the BDD FVIII
protein.
[0477] Construction of Signal Peptide-XTEN-/FXI/-BDD FVIII
Expression Vectors
[0478] The fused XTEN sequence in pBC0053 can be replaced by
digesting other XTEN fragments (e.g. AM, AF, AG) with BsaI and
BbsI. A ligation using the XTEN fragment and BsaI/BbsI digested
pBC0053 allows the exchange of various XTEN pieces (e.g. AM, AF,
AG) into the BDD FVIII expression vector. Various XTEN fusions can
increase the half lives of these proteins differently, allowing
modification of the properties (e.g. efficacy, potency) of these
proteins. Introduction of any of these fusion constructs into
mammalian cells is expected to express the FVIII protein with an
N-terminal XTEN fusion (signal peptide-XTEN-/FXI/-BDD FVIII), in
which the fused XTEN peptide can be subsequently cleaved by FXI,
generating the BDD FVIII protein.
Example 19: Construction of BDD FVIII with Interdomain XTEN
Insertion
[0479] Construction of BDD FVIII Expression Vectors with an XTEN
Insertion at the A2-B Domain Boundaries
[0480] The pBC0027 construct (pMK-BDD FVIII-STOP) is a cloning
vector designed to contain the BDD FVIII protein coding sequences,
but not a promoter positioned to initiate the expression of BDD
FVIII. This construct is used for manipulation of the coding
sequences of BDD FVIII as the vector backbone contains very few
restriction enzyme sites. therefore allowing easy cloning
strategies. The BDD FVIII proteins contain 1457 amino acids at a
total molecular weight of 167539.66. There are 6 domains within the
wild-type FVIII protein, the A1, A2, B, A3, C1 and C2 domains. In
the BDD FVIII protein, most of the B domain has been deleted as it
is believed to be an unstructured domain and the removal of the
domain does not alter critical functions of this protein. However,
the B domain boundaries seem to be excellent positions for creating
XTEN fusions to allow extension of the protein half lives.
[0481] Within the pBC0027 construct, there is a unique HindIII
restriction enzyme site at the boundary of A2-B junction. The XTEN
(e.g., sequences of Tables 4, or 13-17) are amplified using primers
that introduce a HindIII and FXI cleavage site on either end of the
XTEN coding sequence. The fused XTEN sequence can be altered by
amplifying various XTEN fragments. Various XTEN fusions can
increase the half lives of these proteins differently, allowing
modification of the properties (e.g. efficacy, potency) of these
proteins. The HindIII-/FXI/-XTEN-/FXI/-HindIII fragment is digested
with HindIII and ligated with HindIII digested pBC0027. The ligated
DNA mixture is used to transform DH5a bacterial cells.
Transformants are screened by DNA miniprep and the desired
constructs are confirmed by DNA sequencing. The final construct is
designated pBC0054, which encodes the BDD FVIII protein with an
interdomain XTEN fusion
(FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)) but not a promoter to
initiate gene expression.
[0482] The pBC0054 construct is digested with NheI/SalI, and
ligated with NheI/SalI digested CET1019-HS vector (Millipore). The
CET1019-HS vector contains a human CMV promoter and a UCOE sequence
to facilitate gene expression. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The final construct is designated pBC0055
(CET1019-HS-FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which
encodes the BDD FVIII protein with an interdomain (inter-A2/B
domain) XTEN fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2))
under the control of a human CMV promoter. Introduction of the
pBC0055 construct into mammalian cells is expected to express the
BDD FVIII protein with an interdomain XTEN fusion
(FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which could be
subsequently cleaved by FXI, therefore liberating the BDD FVIII
protein.
[0483] Construction of BDD FVIII Expression Vectors with an XTEN
Insertion at the A1-A2 Domain Boundaries
[0484] The pBC0027 construct is designed as a template for two PCR
reactions using the following four primers:
TABLE-US-00026 (Reaction I) (SEQ ID NO: 1681)
5'-ATGATGGCATGGAAGCCTAT-3'; (SEQ ID NO: 1682)
5'-ATCCCTCACCTTCGCCAGAACCTTCAGAACCCTCACCTTCAGAA
CCTTCACCAGAACCTTCACCATCTTCCGCTTCTTCATTATTTTT CAT-3'. (Reaction II)
(SEQ ID NO: 1683) 5'-TTCTGGCGAAGGTGAGGGATCTGAAGGCGGTTCTGAAGGTGAAG
GTGGCTCTGAGGGTTCCGAATATGATGATGATCTTACTGATTCTGA AAT-3'; (SEQ ID NO:
1684) 5'-TATTCTCTGTGAGGTACCAGC-3'.
[0485] The PCR products generated are 150 bps and 800 bps
respectively. The 800 bp product is used as the template for the
next round of PCR reaction with the 150 bp product as one primer
and 5'-TATTCTCTGTGAGGTACCAGC-3' (SEQ ID NO: 1685) as the other. The
product for the second round of PCR is 930 bps and is digested with
PshAI and ACC65I restriction enzymes. This PshAI/Acc65I flanked DNA
fragment is ligated with PshAI/Acc65I digested pBC0027. The ligated
DNA mixture is used to transform DH5a bacterial cells.
Transformants is screened by DNA miniprep and the desired
constructs are confirmed by DNA sequencing. The final construct is
designated pBC0058 (pMK-BDD FVIII-D345-XTEN_Y36), which encodes the
BDD FVIII protein with an interdomain (inter-A1/A2 domain) XTEN
fusion after the D345 residue.
[0486] The pBC0058 construct is digested with NheI/SalI, and
ligated with NheI/SalI digested CET1019-HS vector (Millipore). The
CET1019-HS vector contains a human CMV promoter and a UCOE sequence
to facilitate gene expression. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The final construct is designated pBC0059
(CET1019-HS-BDD FVIII D345-XTEN_Y36), which encodes the BDD FVIII
protein with an interdomain (inter-A1/A2 domain) XTEN fusion after
the D345 residue under the control of a human CMV promoter.
Introduction of the pBC0059 construct into mammalian cells is
expected to express the BDD FVIII protein with an interdomain XTEN
fusion (BDD FVIII D345-XTEN_Y36).
Example 20: Construction of FVIII with Intradomain XTEN
Insertion
[0487] Construction of BDD FVIII Expression Vectors with an XTEN
Insertion after P598 (within the A2 Domain)
[0488] The coding sequences for XTEN_Y36 is amplified using PCR
techniques with the following
TABLE-US-00027 primers: (SEQ ID NO: 1686)
5'-GAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCA
ATCCAGGTGAAGGTTCTGGTGAAGG-3' (SEQ ID NO: 1687)
5'-AACTCTGGATCCTCAAGCTGCACTCCAGCTTCGGAACCCTCA GAGCC-3'.
[0489] The 184 bp PCR product is flanked by the KpnI and BamHI
restriction enzyme sites on either end, and is ligated to
KpnII/BamHI digested pBC0027 vector which encodes the BDD FVIII
gene. The ligated DNA mixture is used to transform DH5a bacterial
cells. Transformants are screened by DNA miniprep and the desired
constructs are confirmed by DNA sequencing. The final construct is
designated pBC0056, which contains DNA sequences encoding the FVIII
protein with an XTEN_Y36 fusion after the P598 residue. This
cloning strategy is used to introduce various forms of XTEN into
the BDD FVIII protein by altering the template for the PCR reaction
and changing the primers accordingly.
[0490] The pBC0056 construct is digested with NheI/SalI, and
ligated with NheI/SalI digested CET1019-HS vector (Millipore). The
CET1019-HS vector contains a human CMV promoter and a UCOE sequence
to facilitate gene expression. The ligated DNA mixture is used to
transform DH5a bacterial cells. Transformants are screened by DNA
miniprep and the desired constructs are confirmed by DNA
sequencing. The final construct is designated pBC0057
(CET1019-HS-FVIII P598-XTEN_Y32), which encodes the BDD FVIII
protein with an intradomain (within A2 domain) XTEN fusion under
the control of a human CMV promoter. Introduction of the pBC0057
construct into mammalian cells is expected to express the BDD FVIII
protein with an intradomain XTEN fusion (FVIII P598-XTEN_Y32).
[0491] Construction of BDD FVIII Expression Vectors with Other
Intradomain XTEN Insertions
[0492] To introduce various XTEN segments into other intradomain
sites within BDD FVIII (e.g., the XTEN of Tables 4, or 13-17),
primers are designed that amplify XTEN with an overhang that can
anneal with BDD FVIII. The coding sequence of FVIII (pMK-BDD FVIII)
is designed with various unique restriction enzyme sites to allow
these specific insertions. The unique restriction enzymes are
listed below with their cut site: NheI 376, SacI 391, AfiII 700,
SpeI 966, PshAI 1417, Acc65I 2192, KpnI 2192, BamHI 2250, HindIII
2658, PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893, SwaI 4265,
OliI 4626, XbaI 4644, BstBI 4673, SaII4756, and XhoI 4762. The NheI
and SaII sites on either end of the coding sequence are used to
insert the DNA fragment into a human CMV promoter driven vector,
the CET1019-HS (Millipore) for expression in mammalian cells. These
constructs express the BDD FVIII protein with an XTEN fusion with
sequences listed in Table 21.
Example 21: Construction of FVIII with XTEN Insertions
[0493] CFXTEN with Two XTEN:
[0494] To obtain CFXTEN with two XTEN insertions in various regions
(from N-termini to C-termini: A1-R1, A1-R2, A2-R1, A2-R2, B domain,
a3, A3-R1, A3-R2, C-termini), constructs that expressed fusions
with single-XTEN insertions that retained FVIII activity were
utilized. The coding sequence of FVIII (pBC0114
pcDNA4-FVIII_4-X10-Myc-SPATG-His extra RE) (`SPATG` disclosed as
SEQ ID NO: 1662) was designed with various unique restriction
enzyme sites to allow these specific combinations. The unique
restriction enzymes are listed in Table 18 below with their
relative sites between different regions: BsiWI (between N-termini
and A1-R1), AfII (between A1-R1 and A1-R2), NheI (between A1-R2 and
A2-R1), KpnI (between A2-R1 and A2-R2), BamHI (between A2-R2 and B
domain), ClaI (between a3 and A3-R1), PflMI (between A3-R1 and
A3-R2), XbaI (between A3-R2 and C-termini), AgeI (between FVIII
C-termini and stop codon). Building blocks and restriction enzymes
for cloning the libraries were chosen, as listed in the table
below. The chosen components in each region were mixed at molar
ratio of 1:1, and two sets of DNA mixtures were digested with
unique restriction enzymes. DNA fragments were separated with 1%
agarose gel and purified by Qiagen gel extraction kit. DNA with
XTEN insertion in the first desired region was regarded as the
insert (the smaller DNA fragment in agarose gel), while DNA with
XTEN insertion in the second desired region was regarded as vector
(the bigger DNA fragment in agarose gel). The insert and vector
were ligated in order to reconstitute the plasmid. The ligated DNA
mixture was used to transform DH5a E. coli competent host cells.
Transformants were screened by rolling circle amplification (RCA)
and Sanger sequencing to cover approximately 3-4 times the
potential library size. Unique clones were identified and
minipreped. Two distinct restriction digestions were then used to
further confirm the integrity of XTEN in each region. The amino
acid and the encoding DNA sequences for the resulting CFXTEN fusion
proteins are listed in Table 21.
[0495] CFXTEN with One or Two XTEN Insertions within the B/a3
Domain and C Terminus:
[0496] The B/a3 domain and C-terminus of FVIII are unstructured
regions that tolerated XTEN insertions well. The B/a3 domain
further mediated interactions with other cofactors, including the
von Willibrand Factor. To investigate the optimal XTEN insertions
at the B/a3 domain, select deletions and mutations of the region
were made via PCR-based mutagenesis methods. Select PCR reactions
and the vectors were digested with unique restriction enzymes as
listed in Table 18. DNA fragments were separated with 1% agarose
gel and purified by Qiagen gel extraction kit. DNA with XTEN
insertion in the first desired region was regarded as the insert
(the smaller DNA fragment in agarose gel), while DNA with XTEN
insertion in the second desired region was regarded as vector (the
bigger DNA fragment in agarose gel). The insert and vector were
ligated in order to reconstitute the plasmid. The ligated DNA
mixture was used to transform DH5a E. coli competent host cells.
Transformants were screened by colony PCR and Sanger sequencing to
cover approximately 8.times. the potential library size. Unique
clones were identified and minipreped. One three-enzyme restriction
digestion was then used to further confirm the integrity of XTEN in
each region. The amino acid and the encoding DNA sequences for the
resulting CFXTEN fusion proteins are listed in Table 21.
TABLE-US-00028 TABLE 18 Cloning design for FVIII libraries with two
XTEN insertions Library Vector components Restriction ID Insert
components (XTEN region) (XTEN region) enzymes LSD0001 pSD0005,
pSD0006, pSD0007, pSD0008, pSD0013 (C-termini) NheI + ClaI pSD0017,
pSD0018, pBC0136, pBC0137 (B-domain) LSD0002 pSD0005, pSD0006,
pSD0007, pSD0008, pSD0014 (C-termini) NheI + ClaI pSD0017, pSD0018,
pBC0136, pBC0137 (B-domain) LSD0003 pSD0005, pSD0006, pSD0007,
pSD0008, pSD0019 (C-termini) NheI + ClaI pSD0017, pSD0018, pBC0136,
pBC0137 (B-domain) LSD0004 pSD0005, pSD0006, pSD0007, pSD0008,
pSD0020 (C-termini) NheI + ClaI pSD0017, pSD0018, pBC0136, pBC0137
(B-domain) LSD0005 pSD0045, pSD0046, pSD0048, pSD0049, pSD0001
(A2-R1) BsiWI + AflII pSD0050, pSD0051, pSD0052 (A1-R1) LSD0006
pSD0045, pSD0046, pSD0048, pSD0049, pSD0002 (A2-R1) BsiWI + AflII
pSD0050, pSD0051, pSD0052 (A1-R1) LSD0007 pSD0045, pSD0046,
pSD0048, pSD0049, pSD0003 (A2-R1) BsiWI + AflII pSD0050, pSD0051,
pSD0052 (A1-R1) LSD0008 pSD0045, pSD0046, pSD0048, pSD0049, pSD0004
(A2-R1) BsiWI + AflII pSD0050, pSD0051, pSD0052 (A1-R1) LSD0037
pSD0045, pSD0046, pSD0049, pSD0050, pSD0032 (A2-R1) BsiWI + AflII
pSD0051, pSD0052 (A1-R1) LSD0038 pSD0039 (a3) pSD0045, pSD0046,
pSD0049, BamHI + ClaI pSD0050, pSD0051, pSD0052 (A1-R1) LSD0039
pSD0039 (a3) pSD0032, pSD0001, pSD0003 (A2-R1) BamHI + ClaI LSD0040
pSD0040, pSD0010, pSD0041 (A3-R1) pSD0045, pSD0046, pSD0049, ClaI +
XbaI pSD0050, pSD0051, pSD0052 (A1-R1) LSD0041 pSD0040, pSD0010,
pSD0041 (A3-R1) pSD0032, pSD0001, pSD0003 (A2-R1) ClaI + XbaI
LSD0042 pSD0062, pSD0063, pSD0043, pSD0044 pSD0045, pSD0046,
pSD0049, ClaI + XbaI (A3-R2) pSD0050, pSD0051, pSD0052 (A1-R1)
LSD0043 pSD0062, pSD0063, pSD0043, pSD0044 pSD0032, pSD0001,
pSD0003 (A2-R1) ClaI + XbaI (A3-R2) LSD0044 pSD0062, pSD0063,
pSD0043, pSD0044 pSD0040, pSD0010, pSD0041 (A3-R1) PflMI + XbaI
(A3-R2) LSD0045 pSD0039 (a3) pSD0040, pSD0010, pSD0041 (A3-R1)
BamHI + ClaI LSD0046 pSD0039 (a3) pSD0062, pSD0063, pSD0043, BamHI
+ ClaI pSD0044 (A3-R2) LSD0047 pSD0046 (A1-R1) pSD0001, pSD0003
(A2-R1) BsiWI + AflII LSD0048 pSD0045, pSD0051 (A1-R1) pSD0003
(A2-R1) BsiWI + AflII pNL0006 PCR product LSD0003.006 (B Domain and
C BamHI + PflMI termini) pNL0007 PCR product LSD0003.006 (B Domain
and C ClaI + PflMI termini) pNL0008 PCR product LSD0003.009 (B
Domain and C ClaI + PflMI termini) pNL0009 PCR product pSD0039 (a3
Domain) BamHI + AscI pNL0010 LSD0003.006 (B Domain and C termini)
pNL0009 (a3 Domain) XbaI + AgeI
Example 22: Construction of BDD FVIII Expression Vectors with 3-5
XTEN Insertions at Sites 18/26, 403, 745/1656, 1720, 1900 or
2332
[0497] FVIII-fusion constructs with XTEN insertions at sites 18/26,
403, 745/1656, 1720, 1900 or 2332 were chosen to recombine and
generate constructs with 3, 4, 5 or 6 XTEN insertions.
[0498] Construction of BDD FVIII Expression Vectors with 3-5 XTEN
Insertions at Sites 26, 403, 1656, 1720, or 1900
[0499] The chosen constructs with single XTEN at the desired sites
were: pSD0050, pSD0001, pSD0039, pSD0010, and pSD0062. Constructs
with double XTENs at the desired sites included LSD0005.002,
LSD0038.001, LSD0040.002, LSD0042.013, LSD0039.010, LSD0041.008,
LSD0043.008, LSD0045.002, LSD0046.002, and LSD0044.002. Building
blocks and restriction enzymes for cloning the constructs were
chosen, as listed in Table 19 below. Chosen components were
digested with unique restriction enzymes. DNA of inserts and
vectors were separated with 1% agarose gel and purified by Qiagen
gel extraction kit. The insert and vector were ligated, and then
transformed into DH5.alpha. E. coli competent host cells. Four
colonies for each construct were analyzed by RCA and DNA
sequencing. Clones with desired XTEN insertions were minipreped.
Restriction digestions were then used to further confirm the
integrity of XTEN in each region. The amino acid and the encoding
DNA sequences for the resulting CFXTEN fusion proteins are listed
in Table 21. The resulting constructs were numbered pSD0077 to
pSD0092.
[0500] Construction of BDD FVIII Expression Vectors with 4-6 XTEN
Insertions at Sites 18, 403, 1656, 1720, 1900 or 2332
[0501] Constructs pSD0077 to pSD0092 served as building blocks to
generate 4- to 6-XTEN constructs with insertions at 18, 403, 1656,
1720, 1900 and 2332. Building block constructs and restriction
enzymes for cloning the constructs were chosen, as listed in Table
19 below. Chosen components were digested with unique restriction
enzymes. DNA of inserts and vectors were separated with 1% agarose
gel and purified by Qiagen gel extraction kit. The insert and
vector were ligated, and then transformed into DH5.alpha. E. coli
competent host cells. Eight colonies for each construct were
analyzed by colony PCR and DNA sequencing. Clones with desired XTEN
insertions were minipreped. Restriction digestions were then used
to further confirm the integrity of XTEN in each region. The amino
acid and the encoding DNA sequences for the resulting CFXTEN fusion
proteins are listed in Table 21. The resulting constructs were
numbered pBC0247 to pBC0257, pNL0022, 23, 24, 25, and 30
[0502] Construction of BDD FVIII Expression Vectors with 4-6 XTEN
Insertions at Sites 18, 403, 745, 1720, 1900 or 2332
[0503] Constructs pBC0247 to pBC0252, pBC0255, pNL0022 to pNL0025
served as building blocks to generate 4- to 6-XTEN constructs with
insertions at 18, 403, 745, 1720, 1900 and 2332. Building block
constructs and restriction enzymes for cloning the constructs were
chosen, as listed in Table 19 below. Chosen components were
digested with unique restriction enzymes. DNA of inserts and
vectors were separated with 1% agarose gel and purified by Qiagen
gel extraction kit. The insert and vector were ligated, and then
transformed into DH5.alpha. E. coli competent host cells. Eight
colonies for each construct were analyzed by colony PCR and DNA
sequencing. Clones with desired XTEN insertions were minipreped.
Restriction digestions were then used to further confirm the
integrity of XTEN in each region. The amino acid and the encoding
DNA sequences for the resulting CFXTEN fusion proteins are listed
in Table 21. The resulting constructs were numbered pBC0258 to
pBC0268.
TABLE-US-00029 TABLE 19 Cloning design for FVIII libraries with 3-5
XTEN insertions at sites 26, 403, 1656, 1720, or 1900 Construct
Vector components Restriction Name Insert components (XTEN region)
(XTEN region) enzymes pSD0077 pSD0050 (A1-R1) LSD0039.010 (A2-R1,
a3) BsiWI + AflII pSD0078 pSD0010 (A3-R1) LSD0005.002 (A1-R1,
A2-R1) ClaI + XbaI pSD0079 pSD0062 (A3-R2) LSD0005.002 (A1-R1,
A2-R1) ClaI + XbaI pSD0080 pSD0050 (A1-R1) LSD0045.002 (a3, A3-R1)
BsiWI + AflII pSD0081 pSD0050 (A1-R1) LSD0046.002 (a3, A3-R2) BsiWI
+ AflII pSD0082 pSD0050 (A1-R1) LSD0044.002 (A3-R1, A3-R2) BsiWI +
AflII pSD0083 pSD0010 (A3-R1) LSD0039.010 (A2-R1, a3) ClaI + XbaI
pSD0084 pSD0062 (A3-R2) LSD0039.010 (A2-R1, a3) ClaI + XbaI pSD0085
pSD0062 (A3-R2) LSD0041.008 (A2-R1, A3-R1) PflMI + XbaI pSD0086
pSD0062 (A3-R2) LSD0045.002 (a3, A3-R1) PflMI + XbaI pSD0087
LSD0039.010 (A2-R1, a3) LSD0040.002 (A1-R1, A3-R1) NheI + ClaI
pSD0088 LSD0039.010 (A2-R1, a3) LSD0042.013 (A1-R1, A3-R2) NheI +
ClaI pSD0089 LSD0044.002 (A3-R1, A3-R2) LSD0005.002 (A1-R1, A2-R1)
ClaI + XbaI pSD0090 LSD0044.002 (A3-R1, A3-R2) LSD0038.001 (A1-R1,
a3) ClaI + XbaI pSD0091 LSD0044.002 (A3-R1, A3-R2) LSD0039.010
(A2-R1, a3) ClaI + XbaI pSD0092 LSD0044.002 (A3-R1, A3-R2) pSD0077
(A1-R1, A2-R1, a3) ClaI + XbaI pBC0247 pSD0077 LSD0050.003 NheI +
BstBI pBC0248 pSD0078 LSD0050.003 NheI + BstBI pBC0249 pSD0079
LSD0050.003 NheI + BstBI pBC0250 pSD0080 LSD0050.003 NheI + BstBI
pBC0251 pSD0082 LSD0050.003 NheI + BstBI pBC0252 pSD0080
LSD0050.003 NheI + BstBI pBC0253 pSD0087 LSD0050.003 NheI + BstBI
pBC0254 pSD0088 LSD0050.003 NheI + BstBI pBC0255 pSD0089
LSD0050.003 NheI + BstBI pBC0256 pSD0090 LSD0050.003 NheI + BstBI
pBC0257 pSD0092 LSD0050.003 NheI + BstBI pNL0022 LSD0003.009
pSD0083 XbaI + AgeI pNL0023 LSD0003.009 pSD0084 XbaI + AgeI pNL0024
LSD0003.009 pSD0085 XbaI + AgeI pNL0025 LSD0003.009 pSD0086 XbaI +
AgeI pNL0030 LSD0003.009 pSD0091 XbaI + AgeI pBC0258 LSD0003.006
pBC0247 BamHI + ClaI pBC0259 LSD0003.006 pBC0248 BamHI + ClaI
pBC0260 LSD0003.006 pBC0249 BamHI + ClaI pBC0261 LSD0003.006
pBC0250 BamHI + ClaI pBC0262 LSD0003.006 pBC0251 BamHI + ClaI
pBC0263 LSD0003.006 pBC0252 BamHI + ClaI pBC0264 LSD0003.006
pBC0255 BamHI + ClaI pBC0265 LSD0003.006 pNL0022 BamHI + ClaI
pBC0266 LSD0003.006 pNL0023 BamHI + ClaI pBC0267 LSD0003.006
pNL0024 BamHI + ClaI pBC0268 LSD0003.006 pNL0025 BamHI + ClaI
Example 23: Construction of CFXTEN Expression Vectors with Three or
Four XTENs: The First XTEN in the B Domain, the Second XTEN at the
C-Terminus, and the Third or Fourth XTEN Insertion within the A1 or
A2 or A3 Domains
[0504] Libraries of CFXTEN fusion proteins were constructed with
three XTEN insertions by combining coagulation-active clones with
XTEN insertions in the A1, A2, or A3 domains and clones with XTEN
inserted within the B domain and at the C-terminus. Additional
libraries were constructed with a fourth XTEN added in the A1, A2,
or A3 domains to select members of the 3 XTEN libraries. The design
of the cloning scheme is summarized in the table below. DNA was
prepared for the inserts and vectors by restriction enzyme
digestion and agarose gel purification. After ligating the inserts
with the corresponding vectors, the ligated DNA mixture was used to
transform DH5.alpha. competent E. coli host cells. Transformants
were screened by RCA and sequencing to cover approximately 3-4
times the potential library size. Unique clones were identified and
mini-prepped. Three distinct restriction digestions were then used
to further confirm the integrity of each XTEN. The amino acid and
the encoding DNA sequences for the resulting CFXTEN fusion proteins
are listed in Table 21.
TABLE-US-00030 TABLE 20 Cloning design for FVIII libraries with 3
XTEN insertions at sites B domain, C-termini, and A1/A2/A3 domain
Library Insert components (XTEN Vector components Restriction ID
region) (XTEN region) enzymes LSD0049 LSD0003.006 (B domain and C-
pSD0045, pSD0046, pSD0049, pSD0050, BamHI + AgeI termini) pSD0051,
pSD0052 (A1-R1) LSD0050 LSD0003.009 (B domain and C- pSD0045,
pSD0046, pSD0049, pSD0050, BamHI + AgeI termini) pSD0051, pSD0052
(A1-R1) LSD0051 LSD0003.006 (B domain and C- pSD0032, pSD0001,
pSD0003 (A2-R1) BamHI + AgeI termini) LSD0052 LSD0003.009 (B domain
and C- pSD0032, pSD0001, pSD0003 (A2-R1) BamHI + AgeI termini)
LSD0053 pSD0040, pSD0010, pSD0041 LSD0003.006 (B domain and
C-termini) ClaI + XbaI (A3-R1) LSD0054 pSD0040, pSD0010, pSD0041
LSD0003.009 (B domain and C-termini) ClaI + XbaI (A3-R1) LSD0055
pSD0062, pSD0063, pSD0043, LSD0003.006 (B domain and C-termini)
ClaI + XbaI pSD0044 (A3-R2) LSD0056 pSD0062, pSD0063, pSD0043,
LSD0003.009 (B domain and C-termini) ClaI + XbaI pSD0044 (A3-R2)
LSD0057 pSD0001 (A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, NheI
+ BamHI LSD0049.012 (A1-R1, B domain and C-termini) LSD0058 pSD0003
(A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, NheI + BamHI
LSD0049.012 (A1-R1, B domain and C-termini) LSD0059 pNL0005 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011, NheI + BamHI LSD0049.012
(A1-R1, B domain and C-termini) LSD0060 pBC0246 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011, NheI + BamHI LSD0049.012
(A1-R1, B domain and C-termini) LSD0061 pSD0009 (A3-R1)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0062 pSD0010 (A3-R1)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0063 pNL0004 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0064 pSD0063 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0065 pNL0002 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0066 pSD0043 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0067 pNL0003 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0068 pSD0044 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011, ClaI + XbaI LSD0049.012
(A1-R1, B domain and C-termini) LSD0069 pSD0009 (A3-R1)
LSD0051.002, pBC0244, pBC0245 (A2-R1, B ClaI + XbaI domain and
C-termini) LSD0070 pSD0010 (A3-R1) LSD0051.002, pBC0244, pBC0245
(A2-R1, B ClaI + XbaI domain and C-termini) LSD0071 pNL0004 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2-R1, B ClaI + XbaI domain and
C-termini) LSD0072 pSD0063 (A3-R2) LSD0051.002, pBC0244, pBC0245
(A2-R1, B ClaI + XbaI domain and C-termini) LSD0073 pNL0002 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2-R1, B ClaI + XbaI domain and
C-termini) LSD0074 pSD0043 (A3-R2) LSD0051.002, pBC0244, pBC0245
(A2-R1, B ClaI + XbaI domain and C-termini) LSD0075 pNL0003 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2-R1, B ClaI + XbaI domain and
C-termini) LSD0076 pSD0044 (A3-R2) LSD0051.002, pBC0244, pBC0245
(A2-R1, B ClaI + XbaI domain and C-termini) pSD0093 pNL0004 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini) PflMI + XbaI pSD0094
pSD0063 (A3-R2) LSD0053.022 (A3-R1, B domain and C-termini) PflMI +
XbaI pSD0095 pNL0002 (A3-R2) LSD0053.022 (A3-R1, B domain and
C-termini) PflMI + XbaI pSD0096 pSD0043 (A3-R2) LSD0053.022 (A3-R1,
B domain and C-termini) PflMI + XbaI pSD0097 pNL0003 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini) PflMI + XbaI pSD0098
pSD0044 (A3-R2) LSD0053.022 (A3-R1, B domain and C-termini) PflMI +
XbaI pCS0001 pBC0168 (A1) LSD0055.021 (A3-R1, B domain and
C-termini) BsiWI + BamHI pCS0002 pBC0134 (A2_R2) LSD0055.021
(A3-R1, B domain and C-termini) BsiWI + BamHI pCS0003 pBC0179 (C1)
LSD0055.021 (A3-R1, B domain and C-termini) ApaI + XbaI pCS0004
pBC0143 (C1) LSD0055.021 (A3-R1, B domain and C-termini) ApaI +
XbaI pCS0005 pBC0182 (C2) LSD0055.021 (A3-R1, B domain and
C-termini) ApaI + XbaI pCS0006 pBC0144 (C2) LSD0055.021 (A3-R1, B
domain and C-termini) ApaI + XbaI pBC0269 pBC0165 (A1_R1)
LSD0003.006 (B domain and C-termini) BsiWI + BamHI pBC0270 pBC0132
(A2_R1) LSD0003.006 (B domain and C-termini) BsiWI + BamHI pBC0271
pBC0138 (A3_R1) LSD0003.006 (B domain and C-termini) ClaI + XbaI
pBC0272 pBC0176 (A3_R2) LSD0003.006 (B domain and C-termini) ClaI +
XbaI pBC0273 pSD0001 (A2_R1) LSD0003.006 (B domain and C-termini)
BsiWI + BamHI pBC0274 pSD0009 (A3_R1) LSD0003.006 (B domain and
C-termini) ClaI + XbaI pBC0275 pNL0004 (A3_R2) LSD0003.006 (B
domain and C-termini) ClaI + XbaI pBC0276 pBC0280 (A1_R1)
LSD0003.006 (B domain and C-termini) BsiWI + BamHI pBC0277 pBC0281
(A2_R1) LSD0003.006 (B domain and C-termini) BsiWI + BamHI pBC0278
pBC0282 (A3_R1) LSD0003.006 (B domain and C-termini) ClaI + XbaI
pBC0279 pBC0283 (A3_R2) LSD0003.006 (B domain and C-termini) ClaI +
XbaI pBC0285, 286, 287, 288, 289, 290, 291, 292, L09_01 pBC0284
(CT) 293 (B domain and A3 R2) XbaI + Aget L09_01 pSD0014 (CT)
pBC0285, 286, 287, 288, 289, 290, 291, 292, 293 (B domain and A3
R2) XbaI + Aget L09_01 pSD0020 (CT) pBC0285, 286, 287, 288, 289,
290, 291, 292, 293 (B domain and A3 R2) XbaI + Aget
TABLE-US-00031 TABLE 21 DNA and Amino Acid Sequences of FVIII-XTEN
Constructs Amino Construct acid sequence disclosed as DNA sequence
disclosed as Name SEQ ID NO: SEQ ID NO: pBC0114 595 596 pBC0126 597
598 pBC0127 599 600 pBC0165 601 602 pBC0183 603 604 pBC0184 605 606
pBC0166 607 608 pBC0185 609 610 pBC0167 611 612 pBC0128 613 614
pBC0168 615 616 pBC0129 617 618 pBC0169 619 620 pBC0130 621 622
pBC0131 623 624 pBC0132 625 626 pBC0170 627 628 pBC0133 629 630
pBC0171 631 632 pBC0134 633 634 pBC0172 635 636 pBC0135 637 638
pBC0149 639 640 pBC0136 641 642 pBC0137 643 644 pBC0138 645 646
pBC0139 647 648 pBC0140 649 650 pBC0173 651 652 pBC0174 653 654
pBC0175 655 656 pBC0176 657 658 pBC0177 659 660 pBC0178 661 662
pBC0141 663 664 pBC0179 665 666 pBC0180 667 668 pBC0142 669 670
pBC0143 671 672 pBC0181 673 674 pBC0182 675 676 pBC0144 677 678
pBC0145 679 680 pBC0146 681 682 pSD0001 683 684 pSD0002 685 686
pSD0003 687 688 pSD0004 689 690 pSD0005 691 692 pSD0006 693 694
pSD0007 695 696 pSD0008 697 698 pSD0009 699 700 pSD0010 701 702
pSD0011 703 704 pSD0012 705 706 pSD0013 707 708 pSD0014 709 710
pSD0017 711 712 pSD0018 713 714 pSD0019 715 716 pSD0020 717 718
pSD0015 719 720 pSD0016 721 722 pSD0021 723 724 pSD0022 725 726
pSD0023 727 728 pSD0024 729 730 pSD0025 731 732 pSD0026 733 734
pSD0027 735 736 pSD0028 737 738 pSD0029 739 740 pSD0030 741 742
pSD0031 743 744 pSD0032 745 746 pSD0033 747 748 pSD0034 749 750
pSD0035 751 752 pSD0036 753 754 pSD0037 755 756 pSD0038 757 758
pSD0039 759 760 pSD0040 761 762 pSD0041 763 764 pSD0042 765 766
pSD0043 767 768 pSD0044 769 770 pSD0062 771 772 pSD0063 773 774
pSD0045 775 776 pSD0046 777 778 pSD0047 779 780 pSD0048 781 782
pSD0049 783 784 pSD0050 785 786 pSD0051 787 788 pSD0052 789 790
pSD0053 791 792 pSD0054 793 794 pSD0055 795 796 pSD0056 797 798
pSD0057 799 800 pSD0058 801 802 pSD0059 803 804 pSD0060 805 806
pSD0061 807 808 LSD0001.002 809 810 LSD0001.005 811 812 LSD0001.006
813 814 LSD0001.011 815 816 LSD0001.012 817 818 LSD0001.013 819 820
LSD0001.016 821 822 LSD0001.021 823 824 LSD0002.001 825 826
LSD0002.002 827 828 LSD0002.014 829 830 LSD0003.004 831 832
LSD0003.006 833 834 LSD0003.009 835 836 LSD0003.014 837 838
LSD0004.010 839 840 LSD0004.011 841 842 LSD0004.014 843 844
LSD0004.016 845 846 LSD0004.022 847 848 LSD0003.016 849 850
LSD0005.002 851 852 LSD0005.004 853 854 LSD0005.005 855 856
LSD0005.011 857 858 LSD0005.018 859 860 LSD0006.002 861 862
LSD0006.005 863 864 LSD0006.007 865 866 LSD0006.011 867 868
LSD0007.002 869 870 LSD0007.004 871 872 LSD0007.013 873 874
LSD0008.001 875 876 LSD0008.002 877 878 LSD0008.006 879 880
LSD0008.009 881 882 LSD0008.017 883 884 LSD0002.025 885 886
LSD0002.013 887 888 LSD0003.025 889 890 LSD0004.025 891 892
LSD0003.005 893 894 LSD0007.008 895 896 LSD0044.002 897 898
LSD0044.005 899 900 LSD0044.039 901 902 LSD0044.022 903 904
LSD0044.003 905 906 LSD0044.001 907 908 LSD0038.001 909 910
LSD0038.003 911 912 LSD0038.008 913 914 LSD0038.012 915 916
LSD0038.013 917 918 LSD0038.015 919 920 LSD0039.001 921 922
LSD0039.003 923 924 LSD0039.010 925 926 LSD0045.001 927 928
LSD0045.002 929 930 LSD0042.014 931 932 LSD0042.023 933 934
LSD0042.006 935 936 LSD0042.013 937 938 LSD0042.001 939 940
LSD0042.039 941 942 LSD0042.047 943 944 LSD0042.003 945 946
LSD0042.004 947 948 LSD0042.008 949 950 LSD0042.038 951 952
LSD0042.082 953 954 LSD0042.040 955 956 LSD0037.002 957 958
LSD0037.009 959 960 LSD0037.011 961 962 LSD0047.002 963 964
LSD0047.005 965 966 LSD0048.007 967 968 LSD0046.001 969 970
LSD0046.002 971 972 LSD0046.003 973 974 LSD0040.011 975 976
LSD0040.042 977 978 LSD0040.002 979 980 LSD0040.008 981 982
LSD0040.021 983 984 LSD0040.037 985 986 LSD0040.046 987 988
LSD0040.003 989 990 LSD0040.006 991 992 LSD0040.007 993 994
LSD0040.010 995 996 LSD0040.039 997 998 LSD0040.052 999 1000
LSD0041.001 1001 1002 LSD0041.004 1003 1004 LSD0041.006 1005 1006
LSD0041.008 1007 1008 LSD0041.010 1009 1010 LSD0041.014 1011 1012
LSD0041.016 1013 1014 LSD0041.035 1015 1016 LSD0043.001 1017 1018
LSD0043.002 1019 1020 LSD0043.005 1021 1022 LSD0043.006 1023 1024
LSD0043.007 1025 1026 LSD0043.008 1027 1028 LSD0043.015 1029 1030
LSD0043.029 1031 1032 LSD0043.043 1033 1034 pSD0077 1035 1036
pSD0078 1037 1038 pSD0079 1039 1040 pSD0080 1041 1042 pSD0081 1043
1044 pSD0082 1045 1046 pSD0083 1047 1048 pSD0084 1049 1050 pSD0085
1051 1052 pSD0086 1053 1054 pSD0087 1055 1056 pSD0088 1057 1058
pSD0089 1059 1060 pSD0090 1061 1062 pSD0091 1063 1064 pSD0092 1065
1066 LSD0049.002 1067 1068 LSD0049.008 1069 1070 LSD0049.011 1071
1072 LSD0049.012 1073 1074 LSD0049.020 1075 1076 LSD0049.021 1077
1078 LSD0050.002 1079 1080
LSD0050.003 1081 1082 LSD0050.007 1083 1084 LSD0050.010 1085 1086
LSD0050.012 1087 1088 LSD0050.014 1089 1090 LSD0051.002 1091 1092
LSD0051.003 1093 1094 LSD0052.001 1095 1096 LSD0052.003 1097 1098
LSD0053.021 1099 1100 LSD0053.022 1101 1102 LSD0053.024 1103 1104
LSD0054.021 1105 1106 LSD0054.025 1107 1108 LSD0054.026 1109 1110
LSD0055.021 1111 1112 LSD0055.022 1113 1114 LSD0055.026 1115 1116
LSD0056.021 1117 1118 LSD0056.024 1119 1120 LSD0056.025 1121 1122
pNL0001 1123 1124 pNL0002 1125 1126 pNL0003 1127 1128 pNL0004 1129
1130 pNL0005 1131 1132 pNL0006 1133 1134 pNL0007 1135 1136 pNL0008
1137 1138 pNL0009 1139 1140 pNL0010 1141 1142 pBC0244 1143 1144
pBC0245 1145 1146 pBC0246 1147 1148 pBC0247 1149 1150 pBC0248 1151
1152 pBC0249 1153 1154 pBC0250 1155 1156 pBC0251 1157 1158 pBC0252
1159 1160 pBC0253 1161 1162 pBC0254 1163 1164 pBC0255 1165 1166
pBC0256 1167 1168 pBC0257 1169 1170 pBC0259 1171 1172 pBC0260 1173
1174 pBC0262 1175 1176 pBC0263 1177 1178 pBC0264 1179 1180 pBC0266
1181 1182 pBC0267 1183 1184 pBC0268 1185 1186 pNL0016 1187 1188
pNL0017 1189 1190 pNL0018 1191 1192 pNL0022 1193 1194 pNL0023 1195
1196 pNL0024 1197 1198 pNL0025 1199 1200 pNL0030 1201 1202
LSD0057.001 1203 1204 LSD0057.004 1205 1206 LSD0057.005 1207 1208
LSD0057.010 1209 1210 LSD0058.003 1211 1212 LSD0058.005 1213 1214
LSD0058.006 1215 1216 LSD0059.002 1217 1218 LSD0059.003 1219 1220
LSD0059.005 1221 1222 LSD0059.006 1223 1224 LSD0060.001 1225 1226
LSD0060.003 1227 1228 LSD0060.004 1229 1230 LSD0061.002 1231 1232
LSD0061.007 1233 1234 LSD0061.008 1235 1236 LSD0061.012 1237 1238
LSD0062.001 1239 1240 LSD0062.002 1241 1242 LSD0062.006 1243 1244
LSD0062.007 1245 1246 LSD0063.001 1247 1248 LSD0063.003 1249 1250
LSD0063.011 1251 1252 LSD0064.017 1253 1254 LSD0064.018 1255 1256
LSD0064.020 1257 1258 LSD0064.021 1259 1260 LSD0065.001 1261 1262
LSD0065.007 1263 1264 LSD0065.014 1265 1266 LSD0066.001 1267 1268
LSD0066.002 1269 1270 LSD0066.009 1271 1272 LSD0066.011 1273 1274
LSD0067.004 1275 1276 LSD0067.005 1277 1278 LSD0067.006 1279 1280
LSD0067.008 1281 1282 LSD0068.001 1283 1284 LSD0068.002 1285 1286
LSD0068.005 1287 1288 LSD0068.010 1289 1290 LSD0069.004 1291 1292
LSD0069.008 1293 1294 LSD0070.003 1295 1296 LSD0070.004 1297 1298
LSD0070.005 1299 1300 LSD0071.001 1301 1302 LSD0071.002 1303 1304
LSD0071.008 1305 1306 LSD0072.001 1307 1308 LSD0072.002 1309 1310
LSD0072.003 1311 1312 LSD0073.002 1313 1314 LSD0073.004 1315 1316
LSD0073.006 1317 1318 LSD0074.007 1319 1320 LSD0074.010 1321 1322
LSD0074.011 1323 1324 LSD0075.003 1325 1326 LSD0075.004 1327 1328
LSD0075.007 1329 1330 LSD0076.002 1331 1332 LSD0076.003 1333 1334
pSD0093 1335 1336 pSD0094 1337 1338 pSD0095 1339 1340 pSD0096 1341
1342 pSD0097 1343 1344 pSD0098 1345 1346 pSD0099 1347 1348 pSD0100
1349 1350 pSD0101 1351 1352 pSD0102 1353 1354 pSD0103 1355 1356
pSD0104 1357 1358 pCS0001 1359 1360 pCS0002 1361 1362 pCS0003 1363
1364 pCS0004 1365 1366 pCS0005 1367 1368 pCS0006 1369 1370 pBC0269
1371 1372 pBC0270 1373 1374 pBC0271 1375 1376 pBC0272 1377 1378
pBC0273 1379 1380 pBC0274 1381 1382 pBC0275 1383 1384 pBC0276 1385
1386 pBC0277 1387 1388 pBC0278 1389 1390 pBC0279 1391 1392 pBC0280
1393 1394 pBC0281 1395 1396 pBC0282 1397 1398 pBC0283 1399 1400
pBC0284 1401 1402 pBC0285 1403 1404 pBC0286 1405 1406 pBC0287 1407
1408 pBC0288 1409 1410 pBC0289 1411 1412 pBC0290 1413 1414 pBC0291
1415 1416 pBC0292 1417 1418 pBC0293 1419 1420 pBC0294 1421 1422
pBC0295 1423 1424 pBC0296 1425 1426 pBC0297 1427 1428 pBC0298 1429
1430 pBC0299 1431 1432 pBC0300 1433 1434 pBC0301 1435 1436 pBC0302
1437 1438 pBC0303 1439 1440 pBC0304 1441 1442 pBC0305 1443 1444
pBC0306 1445 1446 pBC0307 1447 1448 pBC0308 1449 1450 pBC0309 1451
1452 pBC0310 1453 1454 pBC0311 1455 1456 pBC0312 1457 1458 pBC0313
1459 1460 pBC0314 1461 1462 pBC0315 1463 1464 pBC0316 1465 1466
pBC0317 1467 1468 pBC0318 1469 1470 pBC0319 1471 1472 pBC0320 1473
1474 pBC0321 1475 1476 PBC0322 1477 1478 PBC0323 1479 1480 pNL0040
1481 1482 pNL0041 1483 1484 pNL0042 1485 1486 pNL0043 1487 1488
Example 24: Transfection of Mammalian Cells, Expression of
FVIII-XTEN and Assessment of FVIII Activity
[0505] Mammalian cells, including but not limited to CHO, BHK, COS,
and HEK293, are suitable for transformation with the vectors of the
Examples, above, in order to express and recover FVIII-XTEN fusion
protein. The following are details for methods used to express BDD
FVIII and FVIII-XTEN fusion protein constructs pBC0114, pBC0135,
pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149 by transient
transfection, which includes electroporation and chemical (PEI)
transfection methods.
[0506] Adherent HEK293 cells purchased from ATCC were revived in
medium of vendor's recommendation and passaged for a few
generations before multiple vials were frozen in the medium with 5%
DMSO. One vial was revived and passaged one more time before
transfection. The HEK293 cells were plated 1-2 days before
transfection at a density of approximately 7.times.10.sup.5 per ml
in one T175 per transfection, using 35 ml medium. On the day of
transfection the cells were trypsinized, detached and counted, then
rinsed in the medium until an even cell suspension was achieved.
The cells were counted and an appropriate volume of cells (based on
cell count above) were transferred to 50 mL centrifuge tube, such
that there were approximately 4.times.10.sup.6 cells per
transfection. Cells were centrifuged for 5 min at 500 RCF, the
supernatant discarded, and the cells resuspended in 10 ml of
D-PBS.
[0507] Electroporation:
[0508] For electroporation, an appropriate volume of resuspension
buffer was added using a micropipette (supplied in the Neon.TM.
Transfection System 100 .mu.L Kit), such that 110 .mu.l of buffer
was available per transfection. Separate volumes of 110 .mu.l of
cell suspension were added to each Eppendorf tube containing 11
.mu.l of plasmid DNA for each of the individual FVIII-XTEN
constructs for a total of 6 .mu.g (volume of DNA may be less, qs to
11 .mu.l with sterile H2O). A Neon.TM. Transfection Device was used
for transfection. The program was set to electroporate at 1100v for
a pulse width of 20 ms, for a total of two pulses. A Neon.TM. Tube
(supplied in the Neon.TM. Transfection System 100 .mu.L Kit) was
placed into Neon.TM. Pipette Station. A volume of 3 mL of
Electrolytic Buffer E2 (supplied in the Neon.TM. Transfection
System 100 .mu.L Kit) was added to the Neon.TM. Tube. Neon.TM.
Pipettes and 100 .mu.l Neon.TM. Tips were used to electroporate 100
.mu.l of cell-plasmid DNA mixture using the Neon.TM. Pipette
Station. The electroporation was executed and when complete, the
Neon.TM. Pipette was removed from the Station and the pipette with
the transfected cells was used to transfer the cells, with a
circular motion, into a 100 mm.times.20 mm petri plate containing
10 ml of Opti-MEM I Reduced-Serum Medium (1.times., Invitrogen),
such that transfected cells were evenly distributed on plate. The
cells for each transfection were incubated at 37.degree. C. for
expression. On day 3 post-transfection, a 10% volume of salt
solution of 10 mM Hepes, 5 mM CaCl.sub.2, and 4M NaCl was added to
each cell culture and gently mixed for 30 minutes. Each cell
culture was transferred to a 50 ml conical centrifuge tube and was
centrifuged at 3000 rpm for 10 minutes at 4.degree. C. The
supernatants for each culture were placed into a new 50 ml conical
tube and then split into aliquots of 5.times.1 ml in Eppendorf and
2.times.15 ml conical tubes for assay or were flash frozen before
testing for expression of FVIII-XTEN in ELISA and performance in an
FVIII activity assay, as described herein.
[0509] Chemical Transfection:
[0510] Chemical transfection can be accomplished using standard
methods known in the art. In the present Example, PEI is utilized,
as described.
[0511] Suspension 293 Cells are seeded the day before transfection
at 7.times.10.sup.5 cells/mL in sufficient Freestyle 293
(Invitrogen) medium to provide at least 30 ml working volume, and
incubated at 37.degree. C. On the day of transfection, an aliquot
of 1.5 ml of the transfection medium is held at room temperature,
to which 90 .mu.L of 1 mg/ml PEI is added and vortexed briefly. A
volume of 30 .mu.l of DNA encoding the FVIII-XTEN_AE288 construct
(concentration of 1 mg/ml) is added to the PEI solution, which is
vortexed for 30 sec. The mixture is held at room temperature for
5-15 min. The DNA/PEI mixture is added to the HEK293 cells and the
suspension is incubated at 37.degree. C. using pre-established
shake flask conditions. About four hours after the addition of the
DNA/PEI mix, a 1.times. volume of expansion media is added and the
cells incubated at 37.degree. C. for 5 days. On the day of harvest,
a 10% volume of salt solution of 10 mM Hepes, 5 mM CaCl.sub.2, and
4M NaCl is added to the cell culture and gently mixed for 30
minutes. The cell culture is transferred to a 50 ml conical
centrifuge tube and is centrifuged at 4000 rpm for 10 minutes at
4.degree. C. The supernatant is placed into a new 50 ml conical
tube and then split into aliquots of 5.times.1 ml in Eppendorf and
2.times.15 ml conical tubes for assay or are flash frozen before
testing for expression of FVIII-XTEN in ELISA and/or performance in
an FVIII activity assay, as described herein.
[0512] Generation of Stable Pools and Cell Lines that Produce
FVIII-XTEN
[0513] Stable pools are generated by culturing transfected cells
for 3-5 weeks in medium containing selection antibiotics such as
puromycin, with medium change every 2-3 days. Stable cells can be
used for either production or generation of stable clones. For
stable cell line selection during primary screening, cells from
stable pools either from on-going passaging or revived from frozen
vials are seeded in 96-well plates at a target density of 0.5
cell/well. About 1 week after seeding spent medium from wells with
single cell cluster as observed under microscope are tested for
expression of FVIII by activity assay or antigen measurement.
[0514] For additional rounds of screening, normalized numbers of
cells are seeded in multi-well plates. Spent medium is harvested
and tested for FVIII concentration by ELISA and FVIII activity
assay. Cells would also be harvested from the plates and counted
using Vi-Cell. Clones are ranked by (1) FVIII titers according to
ELISA and activity; (2) ratios of ELISA titer/cell count and
activity titer/cell count; and (3) integrity and homogeneity of
products produced by the clones as measured by Western blots. A
number of clones for each of the constructs are selected from the
primary screening for additional rounds of screening.
[0515] For the second round of screening, cells in 96-well plates
for the top clones selected from primary screening are first
expanded in T25 flasks and then seeded in duplicate 24-well plates.
Spent medium is collected from the plates for FVIII activity and
antigen quantification and cells harvested and counted by Vi-Cell.
Clones are ranked and then selected according to titers by ELISA
and activity assay, ELISA titer/cell and activity titer/cell count
ratios. Frozen vials are prepared for at least 5-10 clones and
again these clones were screened and ranked according to titers by
ELISA and activity, and ratios of ELISA titer/cell count and
activity titer/cell count, and product integrity and homogeneity by
Western blot, and 2-3 clones are selected for productivity
evaluation in shake flasks. Final clones are selected based on
specific productivity and product quality.
[0516] Production of FVIII-XTEN Secreted in Cell Culture Medium by
Suspension 293 Stable Clones
[0517] HEK293 stable cell clones selected by the foregoing methods
are seeded in shake flasks at 1-2.times.10.sup.5 cells/ml in
expression medium. Cell count, cell viability, FVIII activity and
antigen expression titers are monitored daily. On the day when
FVIII activity and antigen titers and product quality are optimal,
the culture is harvested by either centrifugation/sterile
filtration or depth filtration/sterile filtration. The filtrate is
either used immediately for tangential flow filtration (TFF)
processing and purification or stored in -80.degree. C. freezer for
TFF processing and purification later.
Example 25: Purification and Characterization of CFXTEN
Constructs
[0518] Exemplary methods for the purification and characterization
of CFXTEN constructs with one or more XTEN follow.
[0519] Purification of FVII-XTEN_AE864 by FVIII Affinity
Chromatography
[0520] CFXTEN containing supernatant is filtered using a Cuno
ZetaPlus Biocap filter and a Cuno BioAssure capsule and
subsequently concentrated by tangential flow filtration using a
Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using
the same tangential flow filtration cartridge the sample is
diafiltered into 10 mM histidine, 20 mM calcium chloride, 300 mM
sodium chloride, and 0.02% Tween 80 at pH 7.0. FVIIISelect resin
(GE 17-5450-01) selectively binds FVIII or B domain deleted FVIII
using a 13 kDa recombinant protein ligand coupled to a
chromatography resin. The resin is equilibrated with 10 mM
histidine, 20 mM calcium chloride, 300 mM sodium chloride, and
0.02% Tween 80 at pH 7.0 and the supernatant loaded. The column is
washed with 20 mM histidine, 20 mM calcium chloride, 300 mM sodium
chloride, and 0.02% Tween 80 at pH 7.0, then is washed with 20 mM
histidine, 20 mM calcium chloride, 1.0 M sodium chloride, and 0.02%
Tween 80 at pH 7.0, and eluted with 20 mM histidine, 20 mM calcium
chloride, 1.5 M sodium chloride, and 0.02% Tween 80 dissolved in
50% ethylene glycol at pH 7.0.
[0521] Concentration and Buffer Exchange by Tangential Flow
Filtration and Diafiltration
[0522] Supernatant batches totaling at least 10 L in volume, from
stable CHO cells lines expressing CFXTEN are filtered using a Cuno
ZetaPlus Biocap filter and a Cuno BioAssure capsule. They are
subsequently concentrated approximately 20-fold by tangential flow
filtration using a Millipore Pellicon 2 Mini cartridge with a
30,000 Da MWCO. Using the same tangential flow filtration cartridge
the sample is diafiltered with 10 mM histidine, 20 mM calcium
chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0 10
mM tris pH 7.5, 1 mM EDTA with 5 volumes worth of buffer exchange.
Samples are divided into 50 ml aliquots and frozen at -80.degree.
C.
[0523] Purification of CFXTEN by Anion Exchange Chromatography
[0524] Using an Akta FPLC system the sample is purified using a
SuperQ-650M column. The column is equilibrated into buffer A (0.02
mol/L imidazole, 0.02 mol/L glycine ethylester hydrochloride, 0.1 5
mol/L, NaCl, 2.5% glycerol, pH 6.9) and the sample loaded. The
sample is eluted using buffer B (5 mmol/L histidine HCl (His/HCl),
1.15 mol/L NaCl, pH 7.0). The 215 nm chromatogram is used to
monitor the elution profile. The eluted fractions are assayed for
FVIII by ELISA, SDS-PAGE or activity assay. Peak fractions are
pooled and stored or subjected to thrombin activation immediately
(O'Brien et al., Blood (1990) 75:1664-1672). Fractions are assayed
for FVIII activity using an aPTT based factor assay. A Bradford
assay is performed to determine the total amount of protein in the
load and elution fractions.
[0525] Purification of CFXTEN by Hydrophobic Interaction
Chromatography
[0526] CFXTEN samples in Buffer A (50 mmol/l histidine, 1 mmol/l
CaCl 2, 1 M NaCl, and 0.2 g/l Tween 80.RTM., pH 7.0) are loaded
onto a toyopearl ether 650M resin equilibrated in Buffer A. The
column is washed with 10 column volumes of Buffer A to remove DNA,
incorrectly folded forms and FVIII, and other contaminant proteins.
The CFXTEN is eluted with Buffer B (25 mmol/l histidine, 0.5 mmol/l
CaCl 2 and 0.4 mol/l NaCl, pH 7.0) as a single step elution (U.S.
Pat. No. 6,005,082). Fractions are assayed for FVIII activity using
an aPTT based factor assay. A Bradford assay is performed to
determine the total amount of protein in the load and elution
fractions.
[0527] Removal of Aggregated Protein from Monomeric CFXTEN with
Anion Exchange Chromatography
[0528] Using an Akta FPLC system the sample is purified using a
macrocap Q column. The column is equilibrated into buffer A (20 mM
MES, 1 mM CaCl2, pH 7.0) and the sample is loaded. The sample is
eluted using a linear gradient of 30% to 80% buffer B (20 mM MES, 1
mM CaCl2, pH 7.0+500 mM NaCl) over 20 column volumes. The 215 nm
chromatogram is used to monitor the elution profile. The fractions
corresponding to the early portion of the elution contain primarily
monomeric protein, while the late portion of the elution contains
primarily the aggregated species. Fractions from the macrocapQ
column is analyzed via size exclusion chromatography with 60 cm
BioSep G4000 column to determine which to pool to create an
aggregate free sample.
[0529] Activation of FVIII by Thrombin
[0530] Purified FVIII in 5 mmol/L histidine HCl (His/HCl), 1.15
mol/L NaCl, pH 7.0 is treated with thrombin at a 1:4 ratio of units
of human thrombin to units FVIII, and the sample is incubated at
37.degree. C. for up to 2 hours. To monitor the activation process,
aliquots of this sample are then withdrawn, and acetone
precipitated by the addition of 4.5 vol ice-cold acetone. The
sample is incubated on ice for 10 minutes, and the precipitate is
collected by centrifugation at 13,000 g in a microfuge for 3
minutes. The acetone is removed, and the precipitate is resuspended
in 30 .mu.L SDS-PAGE reducing sample buffer and boiled for 2
minutes. Samples are then assayed by SDS-PAGE or western blot. The
conversion of FVIII to FVIIIa is examined by looking for the
conversion of the heavy chain into 40 and 50 kDa fragments and the
conversion of the light chain into a 70 kDa fragment (O'Brien et
al., Blood (1990) 75:1664-1672).
[0531] SEC Analysis of CFXTEN
[0532] FVII-XTEN purified by affinity and anion exchange
chromatography is analyzed by size exclusion chromatography with 60
cm BioSep G4000 column. A monodispersed population with a
hydrodynamic radius of .about.10 nm/apparent MW of 1.7 MDa
(XTEN-288 fusion) or .about.12 nm/an apparent MW of 5.3 MDa
(XTEN-864 fusion) is indicative of an aggregation-free sample.
CFXTEN is expected to have an apparent molecular weight factor up
to or about 8 (for an XTEN-288 fusion with FVIII) or up to or about
.about.15 (for an XTEN-864 fusion with FVIII).
[0533] ELISA Based Concentration Determination of CFXTEN
[0534] The quantitative determination of factor VIII/CFXTEN antigen
concentrations using the double antibody enzyme linked
immuno-sorbent assay (ELISA) is performed using proven antibody
pairings (VisuLize.TM. FVIII Antigen kit, Affinity Biologicals,
Ontario Canada). Strip wells are pre-coated with sheep polyclonal
antibody to human FVIII. Plasma samples are diluted and applied to
the wells. The FVIII antigen that is present binds to the coated
antibody. After washing away unbound material, peroxidase-labeled
sheep detecting antibody is applied and allowed to bind to the
captured FVIII. The wells are again washed and a solution of TMB
(the peroxidase substrate tetramethylbenzidine) is applied and
allowed to react for a fixed period of time. A blue color develops
which changes to yellow upon quenching the reaction with acid. The
color formed is measured spectrophotometrically in a microplate
reader at 450 nm. The absorbance at 450 nm is directly proportional
to the quantity of FVIII antigen captured onto the well. The assay
is calibrated using either the calibrator plasma provided in the
kit or by substituting a CFXTEN standard in an appropriate
matrix.
[0535] Assessment of CFXTEN Activity Via a FXa Coupled Chromogenic
Substrate Assay
[0536] Using the Chromogenix Coamatic Factor VIII (Chromogenix, cat
#82258563) the activity of FVIII or CFXTEN comprising FVIII is
assessed as follows. In the presence of calcium ions and
phospholipids, factor X is activated to factor Xa by factor IXa.
This activation is greatly stimulated by factor VIII which acts as
a cofactor in this reaction. By using optimal amounts of Ca.sup.2+,
phospholipid and factor IXa, and an excess of factor X, the rate of
activation of factor X is linearly related to the amount of factor
VIII. Factor Xa hydrolyses the chromogenic substrate S-2765 thus
liberating the chromophoric group, pNA. The color is then read
spectrophotometrically at 405 nm. The generated factor Xa and thus
the intensity of color is proportional to the factor VIII activity
in the sample. Hydrolysis of S-2765 by thrombin formed is prevented
by the addition of the synthetic thrombin inhibitor 1-2581 together
with the substrate. The activity of an unknown sample is determined
by comparing final A405 of that sample to those from a standard
curve constructed from known FVIII amounts. By also determining the
amount of FVIII antigen present in the samples (via A280 or ELISA),
a specific activity of a sample is determine to understand the
relative potency of a particular preparation of FVIII. This enables
the relative efficiency of different isolation strategies or
construct designs for CFXTEN fusions to be assessed for activity
and ranked.
[0537] aPTT Based Assays for CFXTEN Activity Determination
[0538] CFXTEN acts to replace FVIII in the intrinsic or contact
activated coagulation pathway. The activity of this coagulation
pathway is assessed using an activated partial thromboplastin time
assay (aPTT). FVIII activity specifically is measured as follows: a
standard curve is prepared by diluting normal control plasma
(Pacific Hemostasis cat #100595) two-fold with FVIII deficient
plasma (cat #100800) and then conducting 6, 4-fold serial dilutions
again with factor VIII deficient plasma. This creates a standard
curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 IU/ml of
activity, where one unit of activity is defined as the amount of
FVIIIC activity in 1 ml of normal human plasma. A FVIII-deficient
plasma also is included to determine the background level of
activity in the null plasma. The sample is prepared by adding
CFXTEN to FVIII deficient plasma at a ratio of 1:10 by volume. The
samples is tested using an aPTT assay as follows. The samples are
incubated at 37 C in a molecular devices plate reader
spectrophotometer for 2 minutes at which point an equal volume of
aPTT reagent (Pacific Hemostasis cat #100402) is added and an
additional 3 minute 37 C incubation performed. After the incubation
the assay is activated by adding one volume of calcium chloride
(Pacific Hemostasis cat #100304). The turbidity is monitored at 450
nm for 5 minutes to create reaction profiles. The aPTT time, or
time to onset of clotting activity, is defined as the first time
where OD405 nm increased by 0.06 over baseline. A log-linear
standard curve is created with the log of activity relating
linearly to the aPTT time. From this the activity of the sample in
the plate well is determined and then the activity in the sample is
determined by multiplying by 11 to account for the dilution into
the FVIII deficient plasma. By also determining the amount of FVIII
antigen present in the samples (via A280 or ELISA), a specific
activity of a sample can be determine to understand the relative
potency of a particular preparation of FVIII. This enables the
relative efficiency of different isolation strategies or construct
designs for CFXTEN fusions to be ranked.
[0539] Western Blot Analysis of FVIII/FVIII-XTEN Expressed
Proteins
[0540] Samples were run on a 8% homogeneous SDS gel and
subsequently transferred to PVDF membrane. The samples in lanes
1-15 were: MW Standards, FVIII(42.5 ng), pBC0100B, pBC0114A,
pBC0100, pBC0114, pBC0126, pBC0127 (8/5/11; #9), pBC0128, pBC0135,
pBC0136, pBC0137, pBC0145, pBC0149, and pBC0146, respectively. The
membrane was initially blocked with 5% milk then probed with
anti-FVIII monoclonal antibody, GMA-012, specific to the A2 domain
of the heavy chain (Ansong C, Miles S M, Fay P J. J Thromb Haemost.
2006 April; 4(4):842-7). Insertion of XTEN288 in the B-domain was
observed for pBC0136 (lane 8, FIG. 22) and pBC0137 (lane 9, FIG.
22), whereas XTEN288 insertion at the C-terminus was observed for
pBC0146 (lane 12, FIG. 22). All of the assayed FVIII-XTEN proteins
revealed the presence of single chain protein with molecular weight
of at least 21 kDa higher than that of pBC0114 base construct or
FVIII standard. In addition, AE42 insertion was observed for
pBC0135 (lane 7, FIG. 22) and pBC0149 (lane 11, FIG. 22) with the
single chain running .about.5 kDa higher than that of pBC0114 base
protein and heavy chain running at .about.5 kDa higher than 90 kDa
band of the base protein.
[0541] Assay of Expressed FVIII by ELISA
[0542] To verify and quantitate the expression of FVIII-XTEN fusion
proteins of the constructs by cell culture, an ELISA assay was
established. Capture antibodies, either SAF8C-AP (Affinity
Biologicals), or GMA-8002 (Green Mountain Antibodies), or GMA011
antibodies (Green Mountain Antibodies) for FVIII-LC ELISA) or by
GMA016 antibodies were immobilized onto wells of an ELISA plate.
The wells were then incubated with blocking buffer (1.times.PBS/3%
BSA) to prevent non-specific binding of other proteins to the
anti-FVIII antibody. FVIII standard dilutions (.about.50 ng-0.024
ng range), quality controls, and cell culture media samples were
then incubated for 1.5 h in the wells to allow binding of the
expressed FVIII protein to the coated antibody. Wells were then
washed extensively, and bound protein is incubated with anti-FVIII
detection antibody, SAF8C-Biotinylated (Affinity Biologicals). Then
streptavidin-HRP, which binds the biotin conjugated to the FVIII
detection antibody, is added to the well and incubated for 1 h.
Finally, OPD substrate is added to the wells and its hydrolysis by
HRP enzyme is monitored with a plate reader at 490 nm wavelength.
Concentrations of FVIII-containing samples were then calculated by
comparing the colorimetric response at each culture dilution to a
standard curve. The results, in Table 22, below, show that
FVIII-XTEN of the various constructs are expressed at 0.4-1
.mu.g/ml in the cell culture media. The results obtained by ELISA
and the activity data indicate that FVIII-XTEN fusion proteins were
very well expressed using the described transfection methods.
Furthermore, under the experimental conditions, the results
demonstrate that the specific activity values of the FVIII-XTEN
proteins were similar or greater than that of pBC0114 base
construct (expressing BDD FVIII) and support that XTEN insertion
into the C-terminus or B-domain of FVIII results in preservation of
FVIII protein function.
[0543] Chromogenic Activity Assay for CFXTEN Fusion Protein
[0544] BDD FVIII and CFXTEN fusion protein constructs pBC0114,
pBC0135, pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149, in
various configurations, including XTEN AE288 and AG288 inserted at
the C-terminus of the FVIII BDD sequence and FVIII-XTEN fusion
proteins with AE42 and AE288 inserted after residue 745 (or residue
743) and before residue 1640 (or residue 1638) of the B-domain
(including constructs with the P1648 processing site mutated to
alanine), were expressed in transiently transfected Freestyle 293
cells, as described above, and tested for procoagulant activity.
The procoagulant activity of each of the FVIII-XTEN proteins
present in cell culture medium was assessed using a Chromogenix
Coamatic.RTM. Factor VIII assay, an assay in which the activation
of factor X was linearly related to the amount of factor VIII in
the sample. The assay was performed according to manufacturer's
instructions using the end-point method, which was measured
spectrophotometrically at OD405 nm. A standard curve was created
using purified FVIII protein at concentrations of 250, 200, 150,
100, 75, 50, 37.5, 25, 12.5, 6.25, 3.125 and 1.56 mU/ml. Dilutions
of factor VIII standard, quality controls, and samples were
prepared with assay buffer and PEI culture medium to account for
the effect of the medium in the assay performance. Positive
controls consist of purified factor VIII protein at 20, 40, and 80
mU/ml concentrations and cell culture medium of pBC0114 FVIII base
construct, lacking the XTEN insertions. Negative controls consisted
of assay buffer or PEI culture medium alone. The cell culture media
of the FVIII-XTEN constructs were obtained as described, above, and
were tested in replicates at 1:50, 1:150, and 1:450 dilutions and
the activity of each was calculated in U/ml. Each FVIII-XTEN
construct exhibited procoagulant activity that was at least
comparable, and in some cases greater than that of the base
construct positive control, and support that under the conditions
of the experiments, the linkage of XTEN, including AE288 or AG288,
at the C-terminus of FVIII or insertion of XTEN, including AE42 or
AE288 within the B-domain resulted in retention or even enhancement
of FVIII procoagulant activity.
TABLE-US-00032 TABLE 22 Results of ELISA and Chromogenic FVIII
activity assays FVIII- XTEN Activity Concentration Specific
Activity Construct (IU/ml) (.mu.g/ml) (IU/mg) Description of
Construct pBC0114 3.0 0.6 5000 BDD FVIII base construct used for
XTEN insertions pBC0146 7.4 0.6 12759 FVIII construct with XTEN
AG288 inserted at the C-terminus of FVIII pBC0145 3.1 0.6 4844
FVIII construct with XTEN AE288 inserted at the C-terminus of FVIII
pBC0135 4.0 1.0 4124 FVIII construct with XTEN AE42 inserted
between residue 745 and 1640 pBC0149 4.9 0.9 5581 FVIII construct
with XTEN AE42 inserted between residue 745 and 1640 and with
Arg1648 to Ala mutation pBC0136 2.7 0.4 7670 FVIII construct with
XTEN AE288 inserted between residue 745 and 1640 pBC0137 1.9 0.3
6013 FVIII construct with XTEN AE288 inserted between residue 745
and 1640 and with Arg1648 to Ala mutation
[0545] Coatest Assay for Cell Culture Sample Activity Assay
Containing CFXTEN Fusion Protein
[0546] Using the Coatest assay, the activity of FVIII or CFXTEN
comprising FVIII is assessed as follows.
[0547] Assay Matrix:
[0548] All wells in the same plate were adjusted to the same
percentage of media to control for matrix effects. The test samples
were diluted such that the OD405 reading would fall within the
linear range of the standard. The range of concentrations for the
FVIII standard was 100 mU/mL to 0.78 mU/mL, prepared by four-fold
serial dilutions of the FVIII standard in 1.times. Coatest buffer
(DiaPharma) plus the pre-determined percentage of culture
media.
[0549] The Coatest SP FVIII (DiaPharma) reagent package includes
the 10.times. Coatest buffer stock solution, factor IXa+factor X,
phospholipid, CaCl.sub.2 and substrate. The 1.times. Coatest
solution was prepared by adding 9.times. volume of cold ddH.sub.2O
to 1.times. volume of the stock. The cell culture media was then
added to the prepared 1.times. solution at a pre-determined ratio
to normalize the percentage of matrix in all test wells. Factor
IXa+factor X, phospholipid, and substrate were reconstituted
according to manufacturer's recommendations.
[0550] Coatest Assay Procedure:
[0551] Assay reagents were prepared and kept on ice until needed.
25 .mu.l of the diluted test samples and standards were added to a
96 well plate in duplicate. 50 .mu.l of phospholipid/factor
IXa/factor X was added to each well and mixed by gently tapping the
side of the plate. Plates were incubated at 37.degree. C. for 5 min
on a 37.degree. C. plate heater. 25 .mu.l of CaCl.sub.2 was added
to each well and mixed. The plates were incubated at 37.degree. C.
for 5 min on a plate heater. 50 .mu.l of substrate was then added
to each well, mixed, and the plates incubated at 37.degree. C. for
an additional 5-10 min until the top standard developed an OD405
reading of about 1.5. 25 .mu.l of 20% acetic acid was added to each
well with mixing to stop the reaction and wells were read at OD405
using a SpectraMAX.RTM. plus (Molecular Devices) spectrophotometer.
Data analysis was performed using the SoftMax program (version
5.2). The LLOQ varied per assay, but was generally 0.0039
IU/ml.
[0552] Results:
[0553] The data are presented in Tables 23-26. Table 23 presents
results from CFXTEN fusion proteins with XTEN inserted in single
sites chosed on the basis of criteria described herein, including
Example 34. The pBC00114 FVIII positive control showed good
expression and FVIII activity. Of the 106 single-XTEN fusion
proteins assayed, 68% retained measurable FVIII activity, with 30%
exhibiting 3+ to 4+ activity in the coagulation assay. Thirty-one
percent of the fusion proteins assayed had results below the limits
of quantitation (which may be due to poor expression, reflected in
the corresponding expression ELISA results). All four B-domain
insertion constructs exhibited good activity, as did the C-terminal
linked constructs, indicating that these are likely favorable
insertion sites
[0554] The results of the single insertion site data guided the
creation of XTEN constructs with 2 XTEN insertions, the results of
which are presented in Table 24. Overall, the positivity rate was
67%, with 31% of fusion proteins exhibiting 3+ to 4+ activity in
the coagulation assay.
[0555] The results of the foregoing data guided the creation of
XTEN constructs with 3 XTEN insertions, the results of which are
presented in Table 25. Overall, 92% of the samples had measurable
FVIII activity, with fully 79% exhibiting 3+ to 4+ activity in the
coagulation assay.
[0556] A limited number of constructs with 4 XTEN inserted in the
A1, A2 and A3 domains were created and assayed, with 4 of 5
exhibiting FVIII activity (Table 26), suggesting that insertion of
multiple XTEN does not compromise the ability of the resulting
fusion proteins to retain FVIII activity.
[0557] Conclusions:
[0558] Under the conditions of the experiments, the results support
that the criteria used to select XTEN insertion sites are valid,
that insertion of one or more XTEN into the selected sites of FVIII
is more likely than not to result in retention of procoagulant
activity of the resulting CFXTEN molecule, and that insertion of
three XTEN appears to result in a greater proportion of fusion
proteins retaining high levels of FVIII procoagulant activity
compared to single or double XTEN insertion constructs.
TABLE-US-00033 TABLE 23 Results of Coagulation Activity Assays for
CFXTEN comprising one XTEN Insertion Expression Site Domain
Construct Activity ELISA pBC0114 +++ +++ 3 A1 pBC0126 LLOQ* LLOQ 3
A1 pBC0127 + + 18 A1 pBC0165 ++ ++ 22 A1 pBC0183 +++ ++ 26 A1
pBC0184 ++ ++ 40 A1 pBC0166 ++ ++ 60 A1 pBC0185 LLOQ LLOQ 116 A1
pBC0167 LLOQ LLOQ 130 A1 pBC0128 LLOQ LLOQ 188 A1 pBC0168 ++ ++ 216
A1 pBC0129 ++ ++ 230 A1 pBC0169 LLOQ LLOQ 333 A1 pBC0130 ++ ++ 375
A2 pBC0131 LLOQ +++ 403 A2 pBC0132 ++ ++ 442 A2 pBC0170 ++ ++ 490
A2 pBC0133 + ++ 518 A2 pBC0171 LLOQ + 599 A2 pBC0134 ++ ++ 713 A2
pBC0172 + +++ 745 B pBC0135 +++ +++ 745 B pBC0149 +++ +++ 745 B
pBC0136 ++ ++ 745 B pBC0137 +++ +++ 1720 A3 pBC0138 +++ +++ 1796 A3
pBC0139 + ++ 1802 A3 pBC0140 + ++ 1827 A3 pBC0173 LLOQ LLOQ 1861 A3
pBC0174 LLOQ LLOQ 1896 A3 pBC0175 LLOQ LLOQ 1900 A3 pBC0176 +++ +++
1904 A3 pBC0177 + + 1937 A3 pBC0178 LLOQ LLOQ 2019 A3 pBC0141 LLOQ
+ 2068 C1 pBC0179 ++ ++ 2111 C1 pBC0180 LLOQ LLOQ 2120 C1 pBC0142
LLOQ + 2171 C2 pBC0143 ++ +++ 2188 C2 pBC0181 LLOQ LLOQ 2227 C2
pBC0182 ++ +++ 2277 C2 pBC0144 ++ ++ 2332 CT pBC0145 +++ +++ 2332
CT pBC0146 +++ +++ 403 A2 pSD0001 +++ +++ 599 A2 pSD0002 + + 403 A2
pSD0003 +++ +++ 599 A2 pSD0004 + + 745 B pSD0005 +++ ++ 745 B
pSD0006 +++ +++ 745 B pSD0007 +++ ++ 745 B pSD0008 +++ +++ 1720 A3
pSD0009 + + 1720 A3 pSD0010 ++ ++ 2171 C2 pSD0011 + ++ 2171 C2
pSD0012 + ++ 2332 CT pSD0013 +++ ++ 2332 CT pSD0014 +++ +++ 745 B
pSD0017 +++ +++ 745 B pSD0018 +++ +++ 2332 CT pSD0019 +++ +++ 2332
CT pSD0020 +++ +++ 2332 CT pSD0015 ++ ++ 2332 CT pSD0016 +++ +++ 0
N-term pSD0021 + + 32 A1 pSD0022 +++ +++ 65 A1 pSD0023 LLOQ LLOQ 81
A1 pSD0024 LLOQ LLOQ 119 A1 pSD0025 LLOQ LLOQ 211 A1 pSD0026 + +
220 A1 pSD0027 + + 224 A1 pSD0028 + + 336 A1 pSD0029 ++ +++ 339 A1
pSD0030 ++ +++ 378 A2 pSD0031 LLOQ ++ 399 A2 pSD0032 ++ ++ 409 A2
pSD0033 ++ ++ 416 A2 pSD0034 + + 487 A2 pSD0035 LLOQ + 494 A2
pSD0036 LLOQ + 500 A2 pSD0037 LLOQ + 603 A2 pSD0038 + + 1656 A3
pSD0039 +++ +++ 1656 A3 pNL009** ++++ ND 1711 A3 pSD0040 ++ + 1725
A3 pSD0041 LLOQ ++ 1749 A3 pSD0042 LLOQ LLOQ 1905 A3 pSD0043 ++ ++
1910 A3 pSD0044 + + 1900 A3 pSD0062 ++ ++ 1900 A3 pSD0063 +++ ++ 18
A1 pSD0045 +++ +++ 18 A1 pSD0046 +++ +++ 22 A1 pSD0047 LLOQ LLOQ 22
A1 pSD0048 LLOQ LLOQ 26 A1 pSD0049 +++ +++ 26 A1 pSD0050 +++ +++ 40
A1 pSD0051 +++ +++ 40 A1 pSD0052 +++ +++ 216 A1 pSD0053 LLOQ LLOQ
216 A1 pSD0054 LLOQ LLOQ 375 A2 pSD0055 LLOQ + 442 A2 pSD0056 LLOQ
LLOQ 442 A2 pSD0057 LLOQ LLOQ 1796 A3 pSD0058 LLOQ LLOQ 1796 A3
pSD0059 + + 1802 A3 pSD0060 + + 1802 A3 pSD0061 LLOQ LLOQ *LLOQ:
below the limits of quantitation **pNL009 includes a deletion of
745-1656
TABLE-US-00034 TABLE 24 Results of Coagulation Activity Assays for
CFXTEN comprising two XTEN Insertion 1 Insertion 2 Insertion
Insertion Site Domain Site Domain Construct Activity 745 B 2332 CT
LSD0001.002 +++ 745 B 2332 CT LSD0001.005 +++ 745 B 2332 CT
LSD0001.006 +++ 745 B 2332 CT LSD0001.011 +++ 745 B 2332 CT
LSD0001.012 +++ 745 B 2332 CT LSD0001.013 +++ 745 B 2332 CT
LSD0001.016 +++ 745 B 2332 CT LSD0001.021 +++ 745 B 2332 CT
LSD0002.001 +++ 745 B 2332 CT LSD0002.002 +++ 745 B 2332 CT
LSD0002.014 +++ 745 B 2332 CT LSD0003.004 +++ 745 B 2332 CT
LSD0003.006 +++ 745 B 2332 CT LSD0003.009 +++ 745 B 2332 CT
LSD0003.014 + 745 B 2332 CT LSD0004.010 +++ 745 B 2332 CT
LSD0004.011 LLOQ 745 B 2332 CT LSD0004.014 +++ 745 B 2332 CT
LSD0004.016 +++ 745 B 2332 CT LSD0004.022 +++ 745 B 2332 CT
LSD0003.016 +++ 0745 B 2332 CT pNL006 +++ 0745 B 2332 CT pNL007 +++
0745 B 2332 CT pNL008 ++ 1656 a3 2332 CT pNL010 +++ 26 A1 403 A2
LSD0005.002 ++ 26 A1 403 A2 LSD0005.004 ++ 40 A1 403 A2 LSD0005.005
++ 40 A1 403 A2 LSD0005.011 ++ 18 A1 403 A2 LSD0005.018 ++ 26 A1
599 A2 LSD0006.002 + 40 A1 599 A2 LSD0006.005 ++ 40 A1 599 A2
LSD0006.007 ++ 40 A1 599 A2 LSD0006.011 +++ 40 A1 403 A2
LSD0007.002 + 40 A1 403 A2 LSD0007.004 + 26 A1 403 A2 LSD0007.013
++ 26 A1 599 A2 LSD0008.001 ++ 40 A1 599 A2 LSD0008.002 ++ 26 A1
599 A2 LSD0008.006 + 18 A1 599 A2 LSD0008.009 ++ 40 A1 599 A2
LSD0008.017 + 745 B 2332 CT LSD0002.025 +++ 745 B 2332 CT
LSD0002.013 +++ 745 B 2332 CT LSD0003.025 +++ 745 B 2332 CT
LSD0004.025 +++ 745 B 2332 CT LSD0003.005 ++ 26 A1 403 A2
LSD0007.008 ++ 1720 A3 1900 A3 LSD0044.002 LLOQ 1725 A3 1900 A3
LSD0044.005 LLOQ 1720 A3 1900 A3 LSD0044.039 LLOQ 1711 A3 1905 A3
LSD0044.022 LLOQ 1720 A3 1905 A3 LSD0044.003 LLOQ 1725 A3 1905 A3
LSD0044.001 LLOQ 1656 A3 26 A1 LSD0038.001 ++ 1656 A3 18 A1
LSD0038.003 ++ 1656 A3 18 A1 LSD0038.008 +++ 1656 A3 40 A1
LSD0038.012 ++ 1656 A3 40 A1 LSD0038.013 ++ 1656 A3 26 A1
LSD0038.015 ++ 1656 A3 399 A2 LSD0039.001 + 1656 A3 403 A2
LSD0039.003 ++ 1656 A3 403 A2 LSD0039.010 ++ 1656 A3 1725 A3
LSD0045.001 + 1656 A3 1720 A3 LSD0045.002 ++ 1900 A3 18 A1
LSD0042.014 + 1900 A3 18 A1 LSD0042.023 + 1900 A3 26 A1 LSD0042.006
+ 1900 A3 26 A1 LSD0042.013 ++ 1900 A3 40 A1 LSD0042.001 + 1900 A3
40 A1 LSD0042.039 + 1900 A3 26 A1 LSD0042.047 + 1905 A3 18 A1
LSD0042.003 + 1905 A3 40 A1 LSD0042.004 LLOQ 1905 A3 26 A1
LSD0042.008 LLOQ 1905 A3 26 A1 LSD0042.038 LLOQ 1905 A3 40 A1
LSD0042.082 LLOQ 1910 A3 26 A1 LSD0042.040 LLOQ 18 A1 399 A2
LSD0037.002 ++ 26 A1 399 A2 LSD0037.009 + 40 A1 399 A2 LSD0037.011
++ 18 A1 403 A2 LSD0047.002 ++ 18 A1 403 A2 LSD0047.005 + 18 A1 403
A2 LSD0048.007 + 1656 A3 1900 A3 LSD0046.001 ++ 1656 A3 1900 A3
LSD0046.002 + 1656 A3 1905 A3 LSD0046.003 + 1711 A3 40 A1
LSD0040.011 LLOQ 1711 A3 26 A1 LSD0040.042 LLOQ 1720 A3 26 A1
LSD0040.002 + 1720 A3 40 A1 LSD0040.008 + 1720 A3 18 A1 LSD0040.021
+ 1720 A3 26 A1 LSD0040.037 LLOQ 1720 A3 18 A1 LSD0040.046 + 1725
A3 26 A1 LSD0040.003 LLOQ 1725 A3 40 A1 LSD0040.006 LLOQ 1725 A3 26
A1 LSD0040.007 LLOQ 1725 A3 18 A1 LSD0040.010 LLOQ 1725 A3 40 A1
LSD0040.039 LLOQ 1725 A3 18 A1 LSD0040.052 + 1720 A3 403 A2
LSD0041.001 + 1720 A3 399 A2 LSD0041.004 LLOQ 1711 A3 403 A2
LSD0041.006 LLOQ 1720 A3 403 A2 LSD0041.008 LLOQ 1725 A3 403 A2
LSD0041.010 LLOQ 1725 A3 403 A2 LSD0041.014 LLOQ 1725 A3 399 A2
LSD0041.016 LLOQ 1711 A3 403 A2 LSD0041.035 LLOQ 1900 A3 399 A2
LSD0043.001 LLOQ 1900 A3 403 A2 LSD0043.002 LLOQ 1905 A3 403 A2
LSD0043.005 LLOQ 1900 A3 399 A2 LSD0043.006 LLOQ 1900 A3 403 A2
LSD0043.007 LLOQ 1900 A3 403 A2 LSD0043.008 LLOQ 1905 A3 399 A2
LSD0043.015 LLOQ 1905 A3 403 A2 LSD0043.029 LLOQ 1910 A3 403 A2
LSD0043.043 LLOQ
TABLE-US-00035 TABLE 25 Results of Coagulation Activity Assays for
CFXTEN comprising three XTEN Insertion 1 Insertion 2 Insertion 3
Insertion Insertion Insertion Site Domain Site Domain Site Domain
Construct Activity 26 A1 403 A2 1656 A3 pSD0077 +++ 26 A1 403 A2
1720 A3 pSD0078 ++ 26 A1 403 A2 1900 A3 pSD0079 ++ 26 A1 1656 A3
1720 A3 pSD0080 +++ 26 A1 1656 A3 1900 A3 pSD0081 LLOQ 26 A1 1720
A3 1900 A3 pSD0082 + 403 A2 1656 A3 1720 A3 pSD0083 +++ 403 A2 1656
A3 1900 A3 pSD0084 +++ 403 A2 1720 A3 1900 A3 pSD0085 + 1656 A3
1720 A3 1900 A3 pSD0086 +++ 18 A1 745 B 2332 CT LSD0049.002 +++ 26
A1 745 B 2332 CT LSD0049.008 +++ 26 A1 745 B 2332 CT LSD0049.011
+++ 40 A1 745 B 2332 CT LSD0049.012 +++ 40 A1 745 B 2332 CT
LSD0049.020 +++ 18 A1 745 B 2332 CT LSD0049.021 +++ 40 A1 745 B
2332 CT LSD0050.002 +++ 18 A1 745 B 2332 CT LSD0050.003 +++ 26 A1
745 B 2332 CT LSD0050.007 LLOQ 18 A1 745 B 2332 CT LSD0050.010 +++
26 A1 745 B 2332 CT LSD0050.012 +++ 40 A1 745 B 2332 CT LSD0050.014
+++ 403 A2 745 B 2332 CT LSD0051.002 +++ 399 A2 745 B 2332 CT
LSD0051.003 +++ 403 A2 745 B 2332 CT LSD0052.001 +++ 399 A2 745 B
2332 CT LSD0052.003 +++ 1725 A3 745 B 2332 CT LSD0053.021 LLOQ 1720
A3 745 B 2332 CT LSD0053.022 +++ 1711 A3 745 B 2332 CT LSD0053.024
+++ 1720 A3 745 B 2332 CT LSD0054.021 +++ 1711 A3 745 B 2332 CT
LSD0054.025 ++ 1725 A3 745 B 2332 CT LSD0054.026 +++ 1900 A3 745 B
2332 CT LSD0055.021 +++ 1905 A3 745 B 2332 CT LSD0055.022 +++ 1900
A3 745 B 2332 CT LSD0055.026 +++ 1900 A3 745 B 2332 CT LSD0056.021
+++ 1900 A3 745 B 2332 CT LSD0056.024 +++ 1910 A3 745 B 2332 CT
LSD0056.025 +++ 0745 B 1900 A3 2332 CT pBC0294* 0745 B 1900 A3 2332
CT pBC0295* 0745 B 1900 A3 2332 CT pBC0296* 0745 B 1900 A3 2332 CT
pBC0297* 0745 B 1900 A3 2332 CT pBC0298* 0745 B 1900 A3 2332 CT
pBC0299* 0745 B 1900 A3 2332 CT pBC0300* 0745 B 1900 A3 2332 CT
pBC0301* 0745 B 1900 A3 2332 CT pBC0302* 0745 B 1900 A3 2332 CT
pBC0303* 0745 B 1900 A3 2332 CT pBC0304* 0745 B 1900 A3 2332 CT
pBC0305* 0745 B 1900 A3 2332 CT pBC0306* 0745 B 1900 A3 2332 CT
pBC0307* 0745 B 1900 A3 2332 CT pBC0308* 0745 B 1900 A3 2332 CT
pBC0309* 0745 B 1900 A3 2332 CT pBC0310* 0745 B 1900 A3 2332 CT
pBC0311* 0745 B 1900 A3 2332 CT pBC0312* 0745 B 1900 A3 2332 CT
pBC0313* 0745 B 1900 A3 2332 CT pBC0314* 0745 B 1900 A3 2332 CT
pBC0315* 0745 B 1900 A3 2332 CT pBC0316* 0745 B 1900 A3 2332 CT
pBC0317* 0745 B 1900 A3 2332 CT pBC0318* 0745 B 1900 A3 2332 CT
pBC0319* 0745 B 1900 A3 2332 CT pBC0320* 0018 A1 0745 B 2332 CT
pBC0269* 0403 A2 0745 B 2332 CT pBC0270* 1720 A3 0745 B 2332 CT
pBC0271* 1900 A3 0745 B 2332 CT pBC0272* 0403 A2 0745 B 2332 CT
pBC0273* 1720 A3 0745 B 2332 CT pBC0274* 1900 A3 0745 B 2332 CT
pBC0275* 0018 A1 0745 B 2332 CT pBC0276* 0403 A2 0745 B 2332 CT
pBC0277* 1720 A3 0745 B 2332 CT pBC0278* 1900 A3 0745 B 2332 CT
pBC0279* *Construct with R1648A mutation
TABLE-US-00036 TABLE 26 Results of Coagulation Activity Assays for
CFXTEN comprising four XTEN XTEN XTEN XTEN XTEN XTEN XTEN Insert 1
Insert 2 Insert 3 Insert 4 Insert 5 Insert 6 Construct ID Activity
26 403 1656 1720 -- -- pSD0087 +++ 26 403 1656 1900 -- -- pSD0088
+++ 26 403 1720 1900 -- -- pSD0089 LLOQ 26 1656 1720 1900 -- --
pSD0090 ++ 403 1656 1720 1900 -- -- pSD0091 ++ 0040 0403 745 2332
-- -- LSD0058.006* ++ 0018 0409 745 2332 -- -- LSD0059.002* + 0040
0409 745 2332 -- -- LSD0059.006* + 0040 0409 745 2332 -- --
LSD0060.001* + 0018 0409 745 2332 -- -- LSD0060.003* + 0040 1720
745 2332 -- -- LSD0061.002* + 0026 1720 745 2332 -- -- LSD0061.007*
++ 0018 1720 745 2332 -- -- LSD0061.008* ++ 0018 1720 745 2332 --
-- LSD0061.012* ++ 0018 1720 745 2332 -- -- LSD0062.001* ++ 0026
1720 745 2332 -- -- LSD0062.002* ++ 0018 1720 745 2332 -- --
LSD0062.006* ++ 0018 1900 745 2332 -- -- LSD0063.001* ++ 0018 1900
745 2332 -- -- LSD0064.017* ++ 0026 1900 745 2332 -- --
LSD0064.020* ++ 0040 1900 745 2332 -- -- LSD0064.021* ++ 0040 1905
745 2332 -- -- LSD0065.001* + 0018 1905 745 2332 -- -- LSD0065.014*
+ 0040 1905 745 2332 -- -- LSD0066.001* + 0026 1905 745 2332 -- --
LSD0066.002* + 0018 1905 745 2332 -- -- LSD0066.009* ++ 0018 1905
745 2332 -- -- LSD0066.011* ++ 0018 1910 745 2332 -- --
LSD0067.004* ++ 0018 1910 745 2332 -- -- LSD0067.005* + 0040 1910
745 2332 -- -- LSD0067.006* + 0026 1910 745 2332 -- -- LSD0067.008*
+ 0018 1910 745 2332 -- -- LSD0068.001* + 0026 1910 745 2332 -- --
LSD0068.002* + 0040 1910 745 2332 -- -- LSD0068.005* + 0018 1910
745 2332 -- -- LSD0068.010* ++ 0409 1720 745 2332 -- --
LSD0069.004* + 0403 1720 745 2332 -- -- LSD0069.008* + 0409 1720
745 2332 -- -- LSD0070.003* + 0403 1720 745 2332 -- -- LSD0070.004*
++ 0403 1720 745 2332 -- -- LSD0070.005* ++ 0403 1900 745 2332 --
-- LSD0071.001* ++ 0403 1900 745 2332 -- -- LSD0071.002* + 0409
1900 745 2332 -- -- LSD0071.008* ++ 0403 1900 745 2332 -- --
LSD0072.001* ++ 0403 1900 745 2332 -- -- LSD0072.002* + 0409 1900
745 2332 -- -- LSD0072.003* + 0409 1905 745 2332 -- -- LSD0073.002*
+ 0403 1905 745 2332 -- -- LSD0073.004* + 0403 1905 745 2332 -- --
LSD0073.006* + 0403 1905 745 2332 -- -- LSD0074.007* ++ 0409 1905
745 2332 -- -- LSD0074.010* + 0403 1905 745 2332 -- -- LSD0074.011*
+ 0409 1910 745 2332 -- -- LSD0075.004* + 0403 1910 745 2332 -- --
LSD0075.007* + 0403 1910 745 2332 -- -- LSD0076.002* + 0403 1910
745 2332 -- -- LSD0076.003* + 0403 1910 745 2332 -- -- pSD0093* +
1720 1900 745 2332 -- -- pSD0094* ++ 1720 1905 745 2332 -- --
pSD0095* + 1720 1910 745 2332 -- -- pSD0097* + 1720 1910 745 2332
-- -- pSD0098* + 0403 1656 1720 2332 -- -- pNL0022 + 0403 1656 1900
2332 -- -- pNL0023 + 0403 1720 1900 2332 -- -- pNL0024 LLOQ 1656
1720 1900 2332 -- -- pNL0025 + 0018 0403 1656 2332 -- -- pBC0247 ++
0018 0403 1720 2332 -- -- pBC0248 + 0018 0403 1900 2332 -- --
pBC0249 + 0018 1656 1720 2332 -- -- pBC0250 + 0018 1656 1900 2332
-- -- pBC0251 ++ 0018 1720 1900 2332 -- -- pBC0252 LLOQ 0018 0403
0745 2332 -- -- LSD57.005 ++ 0018 0745 1720 2332 -- -- LSD62.001 ++
0018 0745 1900 2332 -- -- pBC0262 ++ 0403 0745 1720 2332 -- --
LSD70.004 + 0403 0745 1900 2332 -- -- pBC0266 + 0745 1720 1900 2332
-- -- pBC0268 + 0188 1900 0745 2332 -- -- pCS0001* ND 0599 1900
0745 2332 -- -- pCS0002* ND 2068 1900 0745 2332 -- -- pCS0003* ND
2171 1900 0745 2332 -- -- pCS0004* ND 2227 1900 0745 2332 -- --
pCS0005* ND 2277 1900 0745 2332 -- -- pCS0006* ND 0403 1656 1720
1900 2332 -- pNL0030 LLOQ 0018 0403 1656 1720 2332 -- pBC0253 +
0018 0403 1656 1900 2332 -- pBC0254 + 0018 0403 1720 1900 2332 --
pBC0255 LLOQ 0018 1656 1720 1900 2332 -- pBC0256 + 0018 0403 0745
1720 2332 -- pBC0259* + 0018 0403 0745 1900 2332 -- pBC0260* + 0018
0745 1720 1900 2332 -- pBC0263 + 0403 0745 1720 1900 2332 --
pBC0267 LLOQ 0018 0403 1656 1720 1900 2332 pBC0257 LLOQ 0018 0403
0745 1720 1900 2332 pBC0264 LLOQ *Construct with R1648A
mutation
Example 26: Determination of XTEN Radii and Related Parameters
[0559] In order to quantify the hydrodynamic radii of the XTEN
components of CFXTEN fusion proteins and how the value of multiple
XTEN versus single XTEN varies, a series of formulae were created
based on empirically-derived data from size exclusion
chromatography assays of various fusion proteins comprising one or
more XTEN. It is believed that the incorporation of multiple XTEN
into a CFXTEN provides a higher total hydrodynamic radius of the
XTEN component compared to CFXTEN with fewer XTEN yet having
approximately the same total of XTEN amino acids. The maximum
radius of a single XTEN polypeptide is calculated (hereinafter
"XTEN Radius") according to the formula given by Equation II:
XTEN Radius=( XTEN length 0.2037)+3.4627 II
[0560] The sum of the maximum of the XTEN Radii for all XTEN
segments in a CFXTEN is calculated (hereinafter "Sum XTEN Radii")
according to the formula given by Equation III:
Sum XTEN Radii = i = 1 n XTEN Radius i III ##EQU00004##
[0561] wherein: n=the number of XTEN segments
[0562] and i is an iterator
[0563] The ratio of the SUM XTEN Radii of a CFXTEN comprising
multiple XTEN to that of an XTEN Radius for a single XTEN of an
equivalent length (in total amino acid residues to that of the
CFXTEN) is calculated (hereinafter "Ratio XTEN Radii") according to
the formula given by Equation IV:
Ratio XTEN Radii = i = 1 n XTEN Radius i ( i = 1 N XTEN Length i *
0.2037 ) + 3.4627 IV ##EQU00005##
[0564] wherein: n=the number of XTEN segments
[0565] and i is an iterator
[0566] Results:
[0567] Equation II was applied to XTEN of lengths 144, 288, 576 and
864. The results are presented in Table 27. Equation IV was applied
to various CFXTEN fusion proteins described herein with two, three,
or four XTEN. The Ratio of XTEN Radii has a value of 1 for all
CFXTEN that contain a single XTEN. The Ratio XTEN Radii are
presented in Table 28. The Ratio of XTEN Radii for pSD0092, which
contains 5 XTEN insertions, has a value of 3.31. Collectively, the
results indicate that the inclusion of multiple XTEN increases the
Ratio XTEN Radii to values greater than 2, with four insertions
resulting in higher values than three insertions.
TABLE-US-00037 TABLE 27 Results of Radii Calculations for CFXTEN
comprising XTEN XEN Length XTEN Radius 42 4.8 144 5.9 288 6.9 576
8.4 864 9.5
TABLE-US-00038 TABLE 28 Results of Radii Calculations for CFXTEN
comprising XTEN Insertion 1 Insertion 2 Insertion 3 Insertion 4
Ratio Insert Insert Insert Insert XTEN Site Domain Site Domain Site
Domain Site Domain Construct Radii 40 A1 pBC0166 1.00 745 B 2332 CT
LSD0001.002 1.67 745 B 2332 CT LSD0001.005 1.71 745 B 2332 CT
LSD0001.006 1.71 745 B 2332 CT LSD0001.011 1.71 745 B 2332 CT
LSD0001.012 1.71 745 B 2332 CT LSD0001.013 1.67 745 B 2332 CT
LSD0001.016 1.67 745 B 2332 CT LSD0001.021 1.67 745 B 2332 CT
LSD0002.001 1.67 745 B 2332 CT LSD0002.002 1.67 745 B 2332 CT
LSD0002.004 1.71 745 B 2332 CT LSD0002.008 1.67 745 B 2332 CT
LSD0002.014 1.67 745 B 2332 CT LSD0003.001 1.67 745 B 2332 CT
LSD0003.004 1.66 745 B 2332 CT LSD0003.006 1.67 745 B 2332 CT
LSD0003.009 1.67 745 B 2332 CT LSD0003.014 1.66 745 B 2332 CT
LSD0003.018 1.67 745 B 2332 CT LSD0004.010 1.66 745 B 2332 CT
LSD0004.011 1.67 745 B 2332 CT LSD0004.014 1.66 745 B 2332 CT
LSD0004.016 1.66 745 B 2332 CT LSD0004.022 1.66 745 B 2332 CT
LSD0003.016 1.67 26 A1 403 A2 LSD0005.002 1.71 26 A1 403 A2
LSD0005.004 1.71 40 A1 403 A2 LSD0005.005 1.71 40 A1 403 A2
LSD0005.011 1.71 18 A1 403 A2 LSD0005.018 1.71 26 A1 599 A2
LSD0006.002 1.71 40 A1 599 A2 LSD0006.005 1.71 40 A1 599 A2
LSD0006.007 1.71 40 A1 599 A2 LSD0006.011 1.71 40 A1 403 A2
LSD0007.002 1.71 40 A1 403 A2 LSD0007.004 1.71 26 A1 403 A2
LSD0007.013 1.71 26 A1 599 A2 LSD0008.001 1.71 40 A1 599 A2
LSD0008.002 1.71 26 A1 599 A2 LSD0008.006 1.71 18 A1 599 A2
LSD0008.009 1.71 40 A1 599 A2 LSD0008.017 1.71 745 B 2332 CT
LSD0002.025 1.71 745 B 2332 CT LSD0002.013 1.67 745 B 2332 CT
LSD0003.025 1.67 745 B 2332 CT LSD0004.025 1.67 745 B 2332 CT
LSD0003.005 1.66 26 A1 403 A2 LSD0007.008 1.71 1720 A3 1900 A3
LSD0044.002 1.71 1725 A3 1900 A3 LSD0044.005 1.71 1720 A3 1900 A3
LSD0044.039 1.71 1711 A3 1905 A3 LSD0044.022 1.71 1720 A3 1905 A3
LSD0044.003 1.71 1725 A3 1905 A3 LSD0044.001 1.71 1656 A3 26 A1
LSD0038.001 1.71 1656 A3 18 A1 LSD0038.003 1.71 1656 A3 18 A1
LSD0038.008 1.71 1656 A3 40 A1 LSD0038.012 1.71 1656 A3 40 A1
LSD0038.013 1.71 1656 A3 26 A1 LSD0038.015 1.71 1656 A3 399 A2
LSD0039.001 1.71 1656 A3 403 A2 LSD0039.003 1.71 1656 A3 403 A2
LSD0039.010 1.71 1656 A3 1725 A3 LSD0045.001 1.71 1656 A3 1720 A3
LSD0045.002 1.71 1900 A3 18 A1 LSD0042.014 1.71 1900 A3 18 A1
LSD0042.023 1.71 1900 A3 26 A1 LSD0042.006 1.71 1900 A3 26 A1
LSD0042.013 1.71 1900 A3 40 A1 LSD0042.001 1.71 1900 A3 40 A1
LSD0042.039 1.71 1900 A3 26 A1 LSD0042.047 1.71 1905 A3 18 A1
LSD0042.003 1.71 1905 A3 40 A1 LSD0042.004 1.71 1905 A3 26 A1
LSD0042.008 1.71 1905 A3 26 A1 LSD0042.038 1.71 1905 A3 40 A1
LSD0042.082 1.71 1910 A3 26 A1 LSD0042.040 1.71 18 A1 399 A2
LSD0037.002 1.71 26 A1 399 A2 LSD0037.009 1.71 40 A1 399 A2
LSD0037.011 1.71 18 A1 403 A2 LSD0047.002 1.71 18 A1 403 A2
LSD0047.005 1.71 18 A1 403 A2 LSD0048.007 1.71 1656 A3 1900 A3
LSD0046.001 1.71 1656 A3 1900 A3 LSD0046.002 1.71 1656 A3 1905 A3
LSD0046.003 1.71 1711 A3 40 A1 LSD0040.011 1.71 1711 A3 26 A1
LSD0040.042 1.71 1720 A3 26 A1 LSD0040.002 1.71 1720 A3 40 A1
LSD0040.008 1.71 1720 A3 18 A1 LSD0040.021 1.71 1720 A3 26 A1
LSD0040.037 1.71 1720 A3 18 A1 LSD0040.046 1.71 1725 A3 26 A1
LSD0040.003 1.71 1725 A3 40 A1 LSD0040.006 1.71 1725 A3 26 A1
LSD0040.007 1.71 1725 A3 18 A1 LSD0040.010 1.71 1725 A3 40 A1
LSD0040.039 1.71 1725 A3 18 A1 LSD0040.052 1.71 1720 A3 403 A2
LSD0041.001 1.71 1720 A3 399 A2 LSD0041.004 1.71 1711 A3 403 A2
LSD0041.006 1.71 1720 A3 403 A2 LSD0041.008 1.71 1725 A3 403 A2
LSD0041.010 1.71 1725 A3 403 A2 LSD0041.014 1.71 1725 A3 399 A2
LSD0041.016 1.71 1711 A3 403 A2 LSD0041.035 1.71 1900 A3 399 A2
LSD0043.001 1.71 1900 A3 403 A2 LSD0043.002 1.71 1905 A3 403 A2
LSD0043.005 1.71 1900 A3 399 A2 LSD0043.006 1.71 1900 A3 403 A2
LSD0043.007 1.71 1900 A3 403 A2 LSD0043.008 1.71 1905 A3 399 A2
LSD0043.015 1.71 1905 A3 403 A2 LSD0043.029 1.71 1910 A3 403 A2
LSD0043.043 1.71 26 A1 403 A2 1656 A3 pSD0077 2.30 26 A1 403 A2
1720 A3 pSD0078 2.30 26 A1 403 A2 1900 A3 pSD0079 2.30 26 A1 1656
A3 1720 A3 pSD0080 2.30 26 A1 1656 A3 1900 A3 pSD0081 2.30 26 A1
1720 A3 1900 A3 pSD0082 2.30 403 A2 1656 A3 1720 A3 pSD0083 2.30
403 A2 1656 A3 1900 A3 pSD0084 2.30 403 A2 1720 A3 1900 A3 pSD0085
2.30 1656 A3 1720 A3 1900 A3 pSD0086 2.30 26 A1 403 A2 1656 A3 1720
A3 pSD0087 2.83 26 A1 403 A2 1656 A3 1900 A3 pSD0088 2.83 26 A1 403
A2 1720 A3 1900 A3 pSD0089 2.83 26 A1 1656 A3 1720 A3 1900 A3
pSD0090 2.83 403 A2 1656 A3 1720 A3 1900 A3 pSD0091 2.83 26 A1 403
A2 1656 A3 1720 A3 pSD0092 2.83 18 A1 745 B 2332 CT LSD0049.002
2.24 26 A1 745 B 2332 CT LSD0049.008 2.24 26 A1 745 B 2332 CT
LSD0049.011 2.24 40 A1 745 B 2332 CT LSD0049.012 2.24 40 A1 745 B
2332 CT LSD0049.020 2.24 18 A1 745 B 2332 CT LSD0049.021 2.24 40 A1
745 B 2332 CT LSD0050.002 2.24 18 A1 745 B 2332 CT LSD0050.003 2.24
26 A1 745 B 2332 CT LSD0050.007 2.24 18 A1 745 B 2332 CT
LSD0050.010 2.24 26 A1 745 B 2332 CT LSD0050.012 2.24 40 A1 745 B
2332 CT LSD0050.014 2.24 403 A2 745 B 2332 CT LSD0051.002 2.24 399
A2 745 B 2332 CT LSD0051.003 2.24 403 A2 745 B 2332 CT LSD0052.001
2.24 399 A2 745 B 2332 CT LSD0052.003 2.24 1725 A3 745 B 2332 CT
LSD0053.021 2.24 1720 A3 745 B 2332 CT LSD0053.022 2.24 1711 A3 745
B 2332 CT LSD0053.024 2.24 1720 A3 745 B 2332 CT LSD0054.021 2.24
1711 A3 745 B 2332 CT LSD0054.025 2.24 1725 A3 745 B 2332 CT
LSD0054.026 2.24 1900 A3 745 B 2332 CT LSD0055.021 2.24 1905 A3 745
B 2332 CT LSD0055.022 2.24 1900 A3 745 B 2332 CT LSD0055.026 2.24
1900 A3 745 B 2332 CT LSD0056.021 2.24 1900 A3 745 B 2332 CT
LSD0056.024 2.24 1910 A3 745 B 2332 CT LSD0056.025 2.24
Example 27: Binding Interference of FVIII-XTEN to Anti-FVIII
Antibody
[0568] The ability of XTEN inserted into different locations of
CFXTEN fusion proteins to affect the binding of anti-FVIII
antibodies was determined by sandwich ELISA assays. Two anti-FVIII
antibodies; i.e. GMA-8021 (Green Mountain Antibodies, Burlington,
Vt.) and ESH8 (American Diagnostica Inc., Stamford, Conn.), that
bind to the A2 and C2 domains, respectively were utilized as
capture antibodies. A non-XTEN containing FVIII-His-Myc protein was
used as a calibration standard and positive control for all ELISAs.
Ten CFXTEN fusion proteins with single XTEN insertions in either
the A1, A2 or A3 domains were created that additionally contained
His and Myc affinity tags. The protein concentrations of each test
sample was normalized to 100% based on an anti-His capture-anti-Myc
detection ELISA run concurrently on the same plate as the
anti-FVIII antibody capture-anti-Myc detection ELISA.
[0569] Briefly, appropriate wells on a 96-well plate were coated
with GMA-8021, ESH8 or anti-His antibody overnight at 4.degree. C.,
then were washed and blocked with BSA. Equal volumes of the
respective control or fusion proteins were introduced into
duplicate wells and allowed to interact with coated GMA-8021, ESH8
or anti-His antibody for 2 h at room temperature. After incubation,
unbound material was washed away and a rabbit anti-Myc detection
antibody was added and incubated for an additional h at room
temperature. The plate was then washed and a peroxidase-conjugated
donkey anti-rabbit secondary antibody was introduced and incubated
for 1 h at room temperature. The plate was washed again, followed
by the addition of TMB substrate and the reaction was allowed to
proceed for 5-20 min. H2SO4 was introduced to stop the reaction and
absorbance was read by spectrophotometer at 450 nm.
[0570] Results:
[0571] The results are presented in Table 29. Collectively, the
results demonstrate that the two antibodies against the CFXTEN
fusion proteins with XTEN inserted into the A2 domain exhibited
reduced binding of FVIII compared to CFXTEN with XTEN inserted into
the A1 or A3 domain when the anti-FVIII capture antibody was
GMA-8021 (with binding affinity to the A2 domain). In contrast,
there was no discernible pattern of inhibition or enhancement of
binding by any of the CFXTEN when the anti-FVIII capture antibody
was ESH8, with binding affinity to the C2 domain.
TABLE-US-00039 TABLE 29 Binding Interference of FVIII-XTEN to
anti-FVIII Antibody Concentration on XTEN insertion aFVIII/Myc /
concentration on aHis/Myc (Domain, site, GMA-8021/Myc (A2 ESH8/Myc
Sample Tested XTEN) His/Myc domain) (C2 domain) FVIII-His-Myc None
100% 92% 104% FVIII-XTEN-His- A2, 403, AE144 100% 103% .+-. 1% 141%
.+-. 24% Myc A2, 403, AG144 100% 104% .+-. 6% 129% .+-. 12% A2,
399, AE144 100% 100% .+-. 8% 140% .+-. 18% A3, 1656, AG144 100%
153% 158% A1, 18, AE144 100% 129% 130% A1, 18, AG144 100% 150% 131%
A1, 26, AE144 100% 155% 87% A1, 26, AG144 100% 157% 147% A1, 40,
AE144 100% 137% 147% A1, 40, AG144 100% 164% .+-. 0% 153% .+-. 18%
aFVIII/Myc = GMA-8021/Myc or ESH8/Myc antibody condition; aHis/Myc
= anti-His/Myc antibody condition
Example 28: Activity Assay of CFXTEN Fusion Proteins in the
Presence of FVIII Inhibitors
[0572] Inhibitor Testing Titration Procedure:
[0573] Select antibodies inhibiting FVIII procoagulant activity
were purchased from commercial sources. The antibodies target
select domains of FVIII (e.g. A2, A3, C1, C2) and inhibit
FVIII-dependent procoagulant activity. In order to establish the
optimal concentration of FVIII inhibitors to utilize in the assay,
an initial titration experiment was performed using varying amounts
of each inhibitory antibody incubated at 37.degree. C. for 2 hrs
with the base vector expressing wild-type FVIII with a His/Myc
double tag, and a second sample with antibody and at least one
CFXTEN fusion protein. The samples were then utilized in a
coagulation assay to determine the FVIII activity. The activity was
measured by the Coatest assay procedure described herein. The
concentration that resulted in optimal inhibition of FVIII activity
was determined for each antibody individually.
[0574] Inhibitor Testing Procedure:
[0575] The FVIII inhibitor antibodies were then used at their
optimal concentration for assay of test samples. CFXTEN and
positive control samples were individually incubated with each
antibody at 37.degree. C. for 2 hrs and the samples were then
collected and utilized in the Coatest activity assay, along with
untreated aliquots of the CFXTEN and positive control. In some
cases, CFXTEN constructs with a R1648A mutation were tested to
determine the effect, if any, of this mutation on resistance to
inhibitors as measured by the retention of FVIII activity.
[0576] Results:
[0577] The results of the titration experiment are shown in FIG.
26. The data indicate a right-shift of approximately 0.7 order of
magnitude in the amount of antibody required to inhibit the
procoagulant activity of the CFXTEN LSD0049.002 to the 50% level,
compared to FVIII positive control, indicating that the CFXTEN with
three XTEN insertions (at insertion points corresponding to amino
acid residue 18, 745 and 2332 of the BDD-FVIII) had lower binding
with the antibody compared to FVIII, reflected in the retention of
coagulation activity.
[0578] The results of the Coatest assays are presented in Tables 30
and 31, for the FVIII inhibitor antibodies GMA8008 and GMA8021,
respectively. All of the untreated CFXTEN fusion protein constructs
tested exhibited procoagulant activity, as did the pBC00114 FVIII
positive control. The positive control sample pre-incubated with
FVIII inhibitor antibodies resulted in a sharp decrease in the
measured coagulation activity to 0.05-0.15 (5-15%) relative to the
untreated sample, as did the majority of the CFXTEN constructs
treated with the GMA8008 antibody to the C2 domain. However, three
CFXTEN fusion proteins retained at least twice the relative
remaining activity compared to the FVIII control; LSD0049.020,
LSD0053.024, and LSD0056.025, each with three XTEN inserts.
[0579] The CFXTEN samples showed a lower degree of inhibition with
the GMA8021 antibody to the A2 domain compared to untreated samples
that was further reduced by either the additional numbers of XTEN
inserts (tabular data shown in Table 30). FIG. 29 shows the graph
of median values of the ratio to control of retained activity
showing a linear relationship between numbers of XTEN inserted and
reduced inhibition to the GMA8021 antibody relative to the
inhibition of the FVIII control. Similarly, the means.+-.S.E. for
the ratio to control values were 2.26.+-.0.12 for 1 XTEN,
3.48.+-.0.26 for 2 XTEN and 5.70.+-.0.29 for 3 XTEN insertions.
CFTXEN with at least three XTEN inserts treated with the GMA8021
antibody had at least 4.5 to 9.2-fold greater retention of FVIII
activity compared to FVIII control. In addition, in those CFXTEN
with three XTEN insertions, constructs with a higher degree of
separation (in numbers of amino acid residues) between any two
insertions appeared to result in a higher degree of procoagulant
activity and, hence, less binding by the FVIII inhibitor antibody,
compared to insertions clustered more closely; e.g. on the
C-terminal side of the B-domain. The assay results of constructs
with the R1648A mutation appeared to be comparable to those without
the mutation.
[0580] Conclusions:
[0581] The results support that, under the conditions of the
experiments, insertion of XTEN into FVIII resulted in protection
against binding by FVIII inhibitors, with retention of procoagulant
activity, and that inclusion of multiple XTEN inserts increased
resistance to, in particular, the A2 domain inhibitor antibody.
Lastly, there appears to be an effect by having spatial separation
between the XTEN inserts.
TABLE-US-00040 TABLE 30 Results of Coagulation Assay with CFXTEN
treated with antibody GMA8008 to C2 Domain Relative Construct
Remaining Ratio to XTEN XTEN XTEN Name Activity Control Insertion 1
Insertion 2 Insertion 3 Mutations pBC0114 CT 0.05-0.15 1 pBC0149
0.1 0.8 0745_AE42_1 pSD0045 0.3 1.1 0018_AE144_5A pSD0046 0.3 1.0
0018_AG144_F pSD0050 0.2 0.9 0026_AG144_F pSD0051 0.3 1.3
0040_AE144_5A pSD0052 0.2 1.0 0040_AG144_F pSD0001 0.2 0.9
0403_AE144_2A pBC0136 0.2 1.2 0745_AE288_1 pBC0137 0.2 1.1
0745_AE288_1 R1648A pSD0013 0.1 0.9 2332_AE144_6B pSD0014 0.1 0.8
2332_AG144_1 pBC0145 0.1 0.6 2332_AE288_1 pSD0019 0.1 0.5
2332_AE288_1 pBC0146 0.1 0.7 2332_AG288_1 pSD0015 0.1 0.8
2332_AE864 LSD0038.008 0.1 0.9 0018_AG144_F 1656_AG144_C
LSD0038.013 0.1 0.6 0040_AG144_F 1656_AG144_C LSD003.09 0.1 0.9
0745_AE144_3B 2332_AE288_1 LSD003.06 0.0 0.8 0745_AE144_3B
2332_AE288_1 R1648A LSD0046.001 0.0 0.6 1656_AG144_C 1900_AG144_C
PSD077 0.1 1.0 0026_AG144_F 0403_AE144_2A 1656_AG144_C PSD080 0.1
1.0 0026_AG144_F 1656_AG144_C 1720_AG144_C PSD083 0.1 0.8
0403_AE144_2A 1656_AG144_C 1720_AG144_C PSD084 0.1 0.9
0403_AE144_2A 1656_AG144_C 1900_AE144_4A LSD0050.010 0.1 0.7
0018_AE144_5A 0745_AE144_3B 2332_AE288_1 LSD0049.021 0.0 0.6
0018_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.002 0.1 0.9
0018_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.008 0.1 0.9
0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.011 0.1 0.9
0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.020 0.2 2.6
0040_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0050.002 0.0 0.2
0040_AG144_F 0745_AE144_3B 2332_AE288_1 LSD0053.024 0.2 2.5
1711_AE144_4A 0745_AE144_3B 2332_AE288_1 LSD0054.021 0.2 1.5
1720_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0055.021 0.2 1.6
1900_AE144_4A 0745_AE144_3B 2332_AE288_1 R1648A LSD0056.021 0.2 1.6
1900_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0056.025 0.3 2.0
1910_AG144_C 0745_AE144_3B 2332_AE288_1 proportion of activity
remaining relative to corresponding untreated sample
The ratio of the relative remaining activity (relative to its own
control) compared to FVIII pBC0114 positive control
TABLE-US-00041 TABLE 31 Results of Coagulation Assay with CFXTEN
treated with antibody GMA8021 to A2 Domain Relative Construct
Remaining Ratio to XTEN XTEN XTEN Name Activity Control Insertion 1
Insertion 2 Insertion 3 Mutations pBC0114 0.05-0.15 1 pBC0149 0.2
1.3 0745_AE42_1 pSD0045 0.3 2.7 0018_AE144_5A pSD0046 0.2 2.1 0018
AG144 F pSD0050 0.2 2.4 0026_AG144_F pSD0051 0.3 3.1 0040_AE144_5A
pSD0052 0.3 2.7 0040_AG144_F pSD0001 0.2 1.6 0403_AE144_2A pBC0136
0.3 2.4 0745_AE288_1 PBC0137 0.3 2.4 0745_AE288_1 R1648A pSD0013
0.2 1.8 2332_AE144_6B pSD0014 0.2 2.1 2332_AG144_1 pBC0145 0.3 2.1
2332_AE288_1 pSD0019 0.3 2.3 2332_AE288_1 pBC0146 0.3 2.1
2332_AG288_1 pSD0015 0.3 2.8 2332_AE864 LSD0038.008 0.4 3.0
0018_AG144_F 1656_AG144_C LSD0038.013 0.4 3.0 0040_AG144_F
1656_AG144_C LSD003.09 0.3 3.6 0745_AE144_3B 2332_AE288_1 LSD003.06
0.3 3.4 0745_AE144_3B 2332_AE288_1 R1648A LSD0046.001 0.2 4.4
1656_AG144_C 1900_AG144_C PSD077 0.4 5.8 0026_AG144_F 0403_AE144_2A
1656_AG144_C PSD080 0.4 5.7 0026_AG144_F 1656_AG144_C 1720_AG144_C
PSD083 0.3 5.0 0403_AE144_2A 1656_AG144_C 1720_AG144_C PSD084 0.3
4.5 0403 AE144 2A 1656 AG144 C 1900 AE144 4A LSD0050.010 0.4 6.7
0018_AE144_5A 0745_AE144_3B 2332_AE288_1 LSD0049.021 0.4 6.7
0018_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.002 0.5 9.2
0018_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.008 0.4 5.9
0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.011 0.4 5.6
0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.020 0.3 5.0
0040_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0050.002 0.3 6.2
0040_AG144_F 0745_AE144_3B 2332_AE288_1 LSD0053.024 0.3 4.5
1711_AE144_4A 0745_AE144_3B 2332_AE288_1 LSD0054.021 0.5 5.2
1720_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0055.021 0.5 5.4
1900_AE144_4A 0745_AE144_3B 2332_AE288_1 R1648A LSD0056.021 0.5 5.1
1900_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0056.025 0.5 4.8
1910_AG144_C 0745_AE144_3B 2332_AE288_1 proportion of activity
remaining relative to corresponding untreated sample
The ratio of the relative remaining activity (relative to its own
control) compared to FVIII pBC0114 positive control
Example 29: Protein Purification of CFXTEN fusion proteins pBC0145
and pBC0146
[0582] Two CFXTEN constructs with C-terminal XTEN were utilized to
establish a purification method. For both pBC0145 with a C-terminal
XTEN of 288 amino acids of the AE family (see sequence in Table 21)
and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG
family (see sequence in Table 21), a tangential flow filtration
(TFF) step was used to buffer exchange the clarified conditioned
media from cell culture. Products were then captured using a strong
anion exchange chromatography resin, and then further purified
using VIIISelect affinity chromatography (GE Healthcare). An
additional size exclusion chromatography (GE Healthcare) was
applied to FVIII-pBC0146 as a third polish step to remove high
molecule weight species. The purity of both fusion proteins was
deemed acceptable by HPLC-SEC and was further confirmed by SDS-PAGE
analysis of the two CFXTEN constructs showing CFXTEN products at
expected sizes. The specific activity of both molecules was
comparable to B-domain deleted FVIII, as measured by aPTT
coagulation assay and ELISA determination of FVIII
concentration.
Example 30: Pharmacokinetics of CFXTEN Fusion Proteins pBC0145 and
pBC0146 in HemA and FVIII/VWF DKO Mice
[0583] Male FVIII knock-out (HemA) mice or FVIII/VWF double
knock-out (DKO) mice, 8-12 weeks old, were treated with a single
intravenous administration of either recombinant BDD-FVIII, the
CFXTEN pBC0145 or pBC0146 fusion purified proteins (from Example
23) at 200 IU/kg dose (n=4/time point). At select time points,
blood samples were collected via vena cava sampling. In HemA mice,
blood samples were collected at 5 min, 1 4, 8, 16, 20, 24, 32, and
48 hrs post-dosing for rBDD-FVIII, and at 5 min, 8, 16, 24, 32, 48,
55 and 72 hrs post-dosing for pBC0145 and pBC0146 fusion proteins.
In the FVIII/VWF DKO mice, blood samples were collected at 5 min,
30 min and 1 hr post-dosing for rBDD-FVIII, and at 5 min, 4, 8, 16
and 24 hr post-dosing for the pBC0145 and pBC0146 fusion proteins.
Plasma FVIII activity was measured by FVIII chromogenic assay and
the PK profile was analyzed by the WinNonlin program.
[0584] Results:
[0585] As show in Table 32 and FIG. 24, CFXTEN with the AE
C-terminus XTEN insertion (pBC0145) exhibited 1.6-fold and
14.1-fold FVIII half-life (T1/2) extension compared to rBDD FVIII
in HemA mice and FVIII/VWF DKO mice, respectively. The CFXTEN with
the AG C-terminus XTEN insertion (pBC0146) had 1.4-fold and
14.4-fold extended half-life compared to rBDD-FVIII in the HemA
mice and FVIII/VWF DKO mice, respectively. The magnitude of the
FVIII half-life extension conferred by XTEN insertion was much more
pronounced in the FVIII/VWF DKO mice compared to the HemA mice,
demonstrated by the 14-fold longer FVIII half-life from both
FVIII-AE-XTEN and FVIII-AG-XTEN compared to rBDD-FVIII. In
addition, in comparison to rBDD-FVIII, FVIII with C-terminal AE or
AG-XTEN insertion also had significantly improved FVIII recovery at
the 5 min interval, reduced clearance and volume of distribution,
and increased AUC in the DKO mice. Under the conditions of the
experiment, CFXTEN with C-terminus XTEN insertions demonstrated
great potential on FVIII half-life extension, and, when combined
with other FVIII intra-domain insertions could potentially further
extend FVIII half-life.
TABLE-US-00042 TABLE 32 Pharmacokinetic parameters of CFXTEN in
HemA and FVIII/VWF DKO mice 5 min Mouse Recovery T.sub.1/2 MRT Cl
Vss AUC_D T.sub.1/2 Fold Mouse Strain Treatment (%) (hr) (hr)
(mL/hr/kg) (mL/kg) (hrkgmIU/mL/mIU) Increase Strain HemA pBC0145 73
11.88 16.47 3.81 62.74 0.26 1.6 HemA pBC0146 64 10.54 13.31 5.66
75.34 0.18 1.4 rBDD- 89 7.58 11.02 4.33 47.68 0.23 FVIII FVIII/
pBC0145 74 3.38 3.76 13.06 63.68 0.0765 13.9 FVIII/ VWF VWF DKO DKO
pBC0146 61 3.45 3.61 17.40 86.63 0.0575 14.2 rBDD- 23 0.24 0.24
460.62 161.51 0.0022 FVIII Compared to rBDD-FVIII
Example 31: Cell Culture and Concentration of Cell Culture Media
for CFXTEN Fusion Proteins pSD0050 and pSD0062
[0586] CFXTEN construct variants pSD0050 with an intradomain AG
XTEN of 144 amino acids inserted after amino acid residue 26 of BDD
FVIII, pSD0062 with an intradomain AE XTEN of 144 amino acids
inserted after residue 1900 of BDD FVIII (Note: amino acid
numbering based full length FVIII), as well as a construct encoding
rBDD-FVIII, were transfected into HEK293F cells (Invitrogen,
Carlsbad, Calif.) using polyethyleneimine (PEI, Polysciences Inc.
Warrington, Pa.). The transiently transfected cells were grown in
293 Free Style medium media (Invitrogen, Carlsbad, Calif.) for 4
days and 50-100 ml cell culture media were then concentrated 10- to
20-fold by Centricon Spin Column (100 kDa MW cut-off) to reach
10-30 IU/ml FVIII activity. The concentrated materials were then
flash-frozen and stored at -80.degree. C. for future in vitro
analysis and in vivo pharmacokinetic studies.
Example 32: Pharmacokinetics of CFXTEN Fusion Proteins pSD0050 and
pSD0062 in HemA and FVIII/VWF DKO Mice
[0587] Male HemA or FVIII/VWF double knock-out (DKO) mice, 8-12
weeks old, were treated with a single intravenous administration of
cell culture concentrates from Example 31 containing either
recombinant BDD-FVIII, the CFXTEN pBD0050 or pBD062 at 100-300
IU/kg (n=3/group). At select time points, blood samples were
collected via retro orbital bleeds from the same set of mice. In
HemA mice, blood samples were collected at 5 min, 24 hr and 48 hr
post-dosing, while in FVIII/VWF DKO mice blood samples were
collected at 5 min, 8 hr and 16 hr. The FVIII activity of plasma
samples and cell culture concentrates were analyzed by a FVIII
chromogenic assay, and the PK profile of rBDD FVIII and FVIII-XTEN
variants were analyzed using the WinNonlin program.
[0588] Results:
[0589] The PK profiles of the two CFXTEN intradomain insertion
variants pSD0050 and pSD0062 and rBDD-FVIII in HemA mice and
FVIII/VWF DKO mice are shown in FIG. 25 and Table 33. In HemA mice,
a comparable initial recovery at the 5 min interval was observed
for the three test FVIII molecules. Both CFXTEN fusion proteins
demonstrated two-fold longer half-life compared to wild-type
BDD-FVIII. In FVIII-VWF DKO mice, because of the loss of VWF
protection, rBDD-FVIII had only a 15 min plasma half-life. In the
case of the two CFXTEN, however, half-life were extended to 3.15 hr
and 3.83 hr, respectively; values that are comparable to the CFXTEN
with 288 C-terminus XTEN insertions (Example 24), suggesting that
further extension of the XTEN length at a given insertion point may
not be necessary. Under the experimental conditions, the study
results clearly demonstrate that intradomain insertion of an XTEN
with 144 amino acid residues not only preserved FVIII activity, but
also provided similar FVIII half-life benefit as the C-terminus 288
amino acid XTEN insertion variants, suggesting that the combination
of the FVIII intradomain and C-terminus insertions may allow
further extension of FVIII half-life.
TABLE-US-00043 TABLE 33 Pharmacokinetic parameters of CFXTEN in
HemA and FVIII/VWF DKO mice 5 min Mouse Recovery T.sub.1/2 MRT Cl
Vss AUC_D T.sub.1/2 Fold Strain Treatment (%) (hr) (hr) (mL/hr/kg)
(mL/kg) (hrkgmIU/mL/mIU) Increase HemA pSD0050 40 14.12 14.25 5.27
75.03 0.19 2.3 pSD0062 43 12.96 14.79 4.24 62.67 0.24 2.1 rBDD- 47
6.19 2.62 6.35 16.62 0.16 FVIII FVIII/ pSD0050 34 3.15 2.59 21.73
56.28 0.05 ~12 VWF pSD0062 35 3.83 3.71 18.51 68.69 0.05 ~15 DKO
rBDD- 23 ~0.25 FVIII Compared to rBDD-FVIII
Example 33: Pharmacokinetic Analysis of CFXTEN Fusion Polypeptides
in Rats
[0590] The pharmacokinetics of various CFXTEN fusion proteins,
compared to FVIII alone, are tested in Sprague-Dawley rats. CFXTEN
and FVIII are administered to female Sprague-Dawley rats (n=3) IV
through a jugular vein catheter at 3-10 .mu.g/rat. Blood samples
(0.2 mL) are collected into pre-chilled heparinized tubes at
predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and
processed into plasma. Quantitation of the test articles is
performed by ELISA assay using an anti-FVIII antibody for both
capture and detection. A non-compartmental analysis is performed in
WinNonLin with all time points included in the fit to determine the
PK parameters. Results are expected to show increased terminal
half-life and area under the curve, and a reduced volume of
distribution for the CFXEN compared to FVIII alone, and the results
are used in conjunction with results from coagulation and
pharmacodynamic assays to select those fusion protein
configurations with desired properties.
Example 34: Analysis of FVIII for XTEN Insertion Sites
[0591] The selection of XTEN insertion sites within the factor VIII
molecule was performed by predicting the locations of permissive
sites within loop structures or otherwise flexible surface exposed
structural elements. For these analyses, the atomic coordinates of
two independently determined X-ray crystallographic structures of
FVIII were use (Shen B W, et al. The tertiary structure and domain
organization of coagulation factor VIII. Blood. (2008) Feb. 1;
111(3):1240-1247; Ngo J C, et al. Crystal structure of human factor
VIII: implications for the formation of the factor IXa-factor VIIIa
complex. Structure (2008)16(4):597-606), as well as those of factor
VIII and factor VIIIa derived from molecular dynamic simulation
(MDS) (Venkateswarlu, D. Structural investigation of zymogenic and
activated forms of human blood coagulation factor VIII: a
computational molecular dynamics study. BMC Struct Biol. (2010)
10:7). Atomic coordinates in Protein Data Bank (PDB) format were
analyzed to identify regions of the FVIII/FVIIIa predicted to have
a high degree solvent accessible surface area using the algorithms
ASAView (Ahmad S, et al. ASAView: database and tool for solvent
accessibility representation in proteins. BMC Bioinformatics (2004)
5:51) and GetArea (Rychkov G, Petukhov M. Joint neighbors
approximation of macromolecular solvent accessible surface area. J
Comput Chem (2007) 28(12):1974-1989). The resulting set of sites
was then further prioritized on the basis of high predicted atomic
positional fluctuation based on the basis of the published results
of the MDS study. Sites within the acidic peptide regions flanking
the A1, A2, and A3 domains, as well as those that appeared by
visual inspection to be in areas other than surface exposed loops
were deprioritized. The resulting set of potential sites was
evaluated on the basis of interspecies sequence conservation, with
those sites in regions of high sequence conservation among 20
vertebrate species being ranked more favorably. Additionally,
putative clearance receptor binding sites, FVIII interaction sites
with other molecules (such as vWF, FIX), domain and exon boundaries
were also considered in fusion site selection. Finally, sites
within close proximity to mutations implicated in hemophilia A
listed in the Haemophilia A Mutation, Search, Test and Resource
Site (HAMSTeRS) database were eliminated (Kemball-Cook G, et al.
The factor VIII Structure and Mutation Resource Site: HAMSTeRS
version 4. Nucleic Acids Res. (1998) 26(1):216-219). Based on these
criteria, the construction of 42 FVIII-XTEN variants was proposed
for XTEN insertions. Of these, three represent XTEN insertions
within the residual B domain sequence, two represent extensions to
the C-terminus of the factor VIII molecule, and 37 represent XTEN
insertions within structurally defined inter- and intradomain
structural elements; i.e., residues 3, 18, 22, 26, 40, 60, 116,
130, 188, 216, 230, 333, 375, 403, 442, 490, 518, 599, 713, 745,
1720, 1796, 1802, 1827, 1861, 1896, 1900, 1904, 1937, 2019, 2068,
2111, 2120, 2171, 2188, 2227, 2277, and 2332.
Example 35: Functional Analysis of FVIII-XTEN Constructs
[0592] Two FVIII-XTEN fusion proteins, FVIII-AE288 (F8X-40) and
FVIII-AG288 (F8X-41), contain an AE288_1 XTEN or an AG288_1 XTEN,
respectively, fused at the C-terminus of FVIII C2 domain. To
determine if FVIII activity was retained after XTEN fusion, HEK293
cells were transfected separately with these two FVIII-XTEN fusion
constructs by using polyethylenimine (PEI) in serum-free medium. At
3 or 5 days post-transfection, the cell culture supernatant was
tested for FVIII activity by a two-stage chromogenic assay.
Purified recombinant FVIII, calibrated against WHO international
standard, was used to establish the standard curve in the
chromogeinic assay. The fusion protein products of both F8X-40 and
F8X-41 constructs were expressed at levels comparable to those of
wild-type BDD-FVIII constructs. (Table 34).
TABLE-US-00044 TABLE 34 FVIII Titer of FVIII-XTEN fusion proteins
in transient transfection cell culture FVIII Molecules FVIII
066.sup.a pBC 0114.sup.a F8X-40 F8X-41 FVIII activity Sample A 6.42
6.68 7.47 3.32.sup.b (IU/ml) Sample B 7.13 7.61 8.25 Not done
.sup.aBoth FVIII 066 and pBC 0114 contain B-domain deleted FVIII
without XTEN fusion. .sup.bThe F8X-41sample was from a 3-day
transfection while other samples were from a 5-day transient
transfection.
Example 36: Functional Analysis of FVIII-XTEN Constructs: FVIII
Activity and PK Properties
[0593] The half-life extension potential of the F8X-40 and F8X-41
constructs was evaluated in FVIII and von Willebrand factor double
knock-out mice by hydrodynamic plasmid DNA injection, with a
FVIIIFc DNA construct serving as a positive control. Mice were
randomly divided into 3 groups with 4 mice per group. Plasmid DNA
encoding BDD FVIIIFc fusion protein, F8X-40 or F8X-41, all sharing
the same DNA vector backbone, was administered to mice in the
respective groups. Approximately 100 micrograms of the appropriate
plasmid DNA was injected into each mouse via hydrodynamic
injection, and blood plasma samples were collected at 24 hours and
48 hours post-injection. The plasma FVIII activity was measured by
a two-stage chromogenic assay using calibrated recombinant FVIII as
a standard. As shown in FIG. 23, samples from the F8X-40 and F8X-41
groups showed higher plasma FVIII titers than did those from the
BDD FVIIIFc, suggesting FVIII fusion with XTEN prolongs the
half-life of FVIII in vivo. Taken together, these data support the
conclusion that FVIII-XTEN fusion proteins retained FVIII activity
in transient transfection and exhibited prolonged circulating
half-life in an animal model.
Example 37: Pharmacodynamic Evaluation of CFXTEN in Animal
Models
[0594] The in vivo pharmacologic activity of CFXTEN fusion proteins
are assessed using a variety of preclinical models of bleeding
including but not limited to those of hemophilia, surgery, trauma,
thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced
bleeding and hydrodynamic injection. These models are developed in
multiple species including mice, rat, rabbits, and dogs using
methods equivalent to those used and published for other FVIII
approaches. CFXTEN compositions are provided in an aqueous buffer
compatible with in vivo administration (for example:
phosphate-buffered saline or Tris-buffered saline). The
compositions are administered at appropriate doses, dosing
frequency, dosing schedule and route of administration as optimized
for the particular model. Efficacy determinations include
measurement of FVIII activity, one-stage clotting assay, FVIII
chromogenic assay, activated partial prothrombin time (aPTT),
bleeding time, whole blood clotting time (WBCT), thrombelastography
(TEG or ROTEM), among others.
[0595] In one example of a PD model, CFXTEN and FVIII are
administered to genetically-deficient or experimentally-induced
HemA mice. At various time points post-administration, levels of
FVIII and CFXTEN are measured by ELISA, activity of FVIII and
CFXTEN is measured by commercially-available FVIII activity kits
and clotting time is measured by aPTT assay. Overall, the results
can indicate that the CFXTEN constructs may be more efficacious at
inhibiting bleeding as compared to FVIII and/or equivalent in
potency to comparable dosage of FVIII with less frequent or more
convenient dosing intervals.
[0596] In a mouse bleeding challenge PD model CFXTEN and FVIII are
administered to genetically-deficient or experimentally-induced
HemA mice and effect on hemostatic challenge is measured.
Hemostatic challenge can include tail transaction challenge,
hemarthropthy challenge, joint bleeding or saphenous vein challenge
among others. At various time points post-administration levels of
FVIII and CFXTEN are measured by ELISA, activity of FVIII and
CFXTEN are measured by commercially available FVIII activity kit,
bleeding time is measured and clotting time is measured by aPTT
assay. Overall the results are expected to indicate that the CFXTEN
constructs are more efficacious at inhibiting bleeding as compared
to FVIII and/or equivalent in potency to comparable dosage of FVIII
with less frequent or more convenient dosing intervals, and the
results are used in conjunction with results from coagulation and
other assays to select those fusion protein configurations with
desired properties.
[0597] In a dog PD model, CFXTEN and FVIII are administered to
genetically-deficient hemophiliac dogs. At various time points post
administration, levels of FVIII and CFXTEN are measured by ELISA,
activity of FVIII and CFXTEN are measured by commercially available
FVIII activity kit and clotting time is measured by aPTT assay.
Overall the results indicates that the CFXTEN constructs may be
more efficacious at inhibiting bleeding as compared to FVIII and/or
equivalent in potency to comparable dosage of FVIII with less
frequent or more convenient dosing, and the results are used in
conjunction with results from coagulation and other assays to
select those fusion protein configurations with desired
properties.
[0598] In a dog bleeding challenge PD model CFXTEN and FVIII are
administered to genetically deficient hemophiliac dogs and effect
on hemostatic challenge is measured. Hemostatic challenge includes
cuticle bleeding time among others. At various time points
post-administration levels of FVIII and CFXTEN are measured by
ELISA, activity of FVIII and CFXTEN are measured by commercially
available FVIII activity kit, bleeding time is measured and
clotting time are measured by aPTT assay. Overall the results
indicate that the CFXTEN constructs may be more efficacious at
inhibiting bleeding as compared to FVIII and/or equivalent in
potency to comparable dosage of FVIII with less frequent or more
convenient dosing intervals, and the results are used in
conjunction with results from coagulation and other assays to
select those fusion protein configurations with desired
properties.
[0599] Additional preclinical models of bleeding include but are
not limited to those of hemophilia, surgery, trauma,
thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced
bleeding and hydrodynamic injection. These models can developed in
multiple species including mice, rat, rabbits, and dogs using
methods equivalent to those used and published for other FVIII
approaches. Overall the results indicate that the CFXTEN constructs
may be more efficacious at inhibiting bleeding as compared to FVIII
and/or equivalent in potency to comparable dosage of FVIII with
less frequent or more convenient dosing intervals, and the results
are used in conjunction with results from coagulation and other
assays to select those fusion protein configurations with desired
properties.
Example 38: CFXTEN with Cleavage Sequences
[0600] C-Terminal XTEN Releasable by FXIa
[0601] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site cleavage sequence is
incorporated into the CFXTEN that contains an amino acid sequence
that is recognized and cleaved by the FXIa protease (EC 3.4.21.27,
Uniprot P03951). Specifically the amino acid sequence KLTRAET (SEQ
ID NO: 1688) is cut after the arginine of the sequence by FXIa
protease. FXI is the procoagulant protease located immediately
before FVIII in the intrinsic or contact activated coagulation
pathway. Active FXIa is produced from FXI by proteolytic cleavage
of the zymogen by FXIIa. Production of FXIa is tightly controlled
and only occurs when coagulation is necessary for proper
hemostasis. Therefore, by incorporation of the KLTRAET cleavage
sequence (SEQ ID NO: 1688), the XTEN domain is only be removed from
FVIII concurrent with activation of the intrinsic coagulation
pathway and when coagulation is required physiologically. This
creates a situation where the CFXTEN fusion protein is processed in
one additional manner during the activation of the intrinsic
pathway.
[0602] C-Terminal XTEN Releasable by FIIa (Thrombin)
[0603] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the FIIa
protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequence
LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et al. (2008) Nucleic
Acids Res., 36: D320], is cut after the arginine at position 4 in
the sequence. Active FIIa is produced by cleavage of FII by FXa in
the presence of phospholipids and calcium and is down stream from
factor IX in the coagulation pathway. Once activated its natural
role in coagulation is to cleave fibrinogen (FIG. 2), which then in
turn, begins clot formation. FIIa activity is tightly controlled
and only occurs when coagulation is necessary for proper
hemostasis. Therefore, by incorporation of the LTPRSLLV sequence
(SEQ ID NO: 1618), the XTEN domain is only removed from FVIII
concurrent with activation of either the extrinsic or intrinsic
coagulation pathways, and when coagulation is required
physiologically. This creates a situation where CFXTEN fusion is
processed in one additional manner during the activation of
coagulation.
[0604] C-Terminal XTEN Releasable by Elastase-2
[0605] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site contains an amino acid sequence
that is recognized and cleaved by the elastase-2 protease (EC
3.4.21.37, Uniprot P08246). Specifically the sequence LGPVSGVP (SEQ
ID NO: 1689) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4 in the sequence. Elastase is
constitutively expressed by neutrophils and is present at all times
in the circulation. Its activity is tightly controlled by serpins
and is therefore minimally active most of the time. Therefore as
the long lived CFXTEN circulates, a fraction of it is cleaved,
creating a pool of shorter-lived FVIII to be used in coagulation.
In a desirable feature of the inventive composition, this creates a
circulating pro-drug depot that constantly releases a prophylactic
amount of FVIII.
[0606] C-Terminal XTEN Releasable by MMP-12
[0607] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site contains an amino acid sequence
that is recognized and cleaved by the MMP-12 protease (EC
3.4.24.65, Uniprot P39900). Specifically the sequence GPAGLGGA (SEQ
ID NO: 1690) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4 of the sequence. MMP-12 is
constitutively expressed in whole blood. Therefore as the long
lived CFXTEN circulates, a fraction of it is cleaved, creating a
pool of shorter-lived FVIII to be used in coagulation. In a
desirable feature of the inventive composition, this creates a
circulating pro-drug depot that constantly releases a prophylactic
amount of FVIII.
[0608] C-Terminal XTEN Releasable by MMP-13
[0609] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site contains an amino acid sequence
that is recognized and cleaved by the MMP-13 protease (EC 3.4.24.-,
Uniprot P45452). Specifically the sequence GPAGLRGA (SEQ ID NO:
1691) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320],
is cut after position 4. MMP-13 is constitutively expressed in
whole blood. Therefore as the long lived CFXTEN circulates, a
fraction of it is cleaved, creating a pool of shorter-lived FVIII
to be used in coagulation. In a desirable feature of the inventive
composition, this creates a circulating pro-drug depot that
constantly releases a prophylactic amount of FVIII.
[0610] C-Terminal XTEN Releasable by MMP-17
[0611] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site contains an amino acid sequence
that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-,
Uniprot Q9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO:
1692) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320],
is cut after position 4 in the sequence. MMP-17 is constitutively
expressed in whole blood. Therefore as the long lived CFXTEN
circulates, a fraction of it is cleaved, creating a pool of
shorter-lived FVIII to be used in coagulation. In a desirable
feature of the inventive composition, this creates a circulating
pro-drug depot that constantly releases a prophylactic amount of
FVIII.
[0612] C-Terminal XTEN Releasable by MMP-20
[0613] A CFXTEN fusion protein consisting of an XTEN protein fused
to the C-terminus of FVIII is created with an XTEN release site
cleavage sequence placed in between the FVIII and XTEN components,
as depicted in FIG. 12. Exemplary sequences are provided in Table
51. In this case, the release site contains an amino acid sequence
that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-,
Uniprot 060882). Specifically the sequence PALPLVAQ (SEQ ID NO:
1693) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320],
is cut after position 4 (depicted by the arrow). MMP-20 is
constitutively expressed in whole blood. Therefore as the long
lived CFXTEN circulates, a fraction of it is cleaved, creating a
pool of shorter-lived FVIII to be used in coagulation. In a
desirable feature of the inventive composition, this creates a
circulating pro-drug depot that constantly releases a prophylactic
amount of FVIII.
[0614] Optimization of the Release Rate of XTEN
[0615] Variants of the foregoing Examples can be created in which
the release rate of XTEN incorporated at the C-terminus, the
N-terminus, or internal XTEN is altered. As the rate of XTEN
release by an XTEN release protease is dependent on the sequence of
the XTEN release site, by varying the amino acid sequence in the
XTEN release site one can control the rate of XTEN release. The
sequence specificity of many proteases is well known in the art,
and is documented in several data bases. In this case, the amino
acid specificity of proteases is mapped using combinatorial
libraries of substrates [Harris, J. L., et al. (2000) Proc Natl
Acad Sci USA, 97: 7754] or by following the cleavage of substrate
mixtures as illustrated in [Schellenberger, V., et al. (1993)
Biochemistry, 32: 4344]. An alternative is the identification of
optimal protease cleavage sequences by phage display [Matthews, D.,
et al. (1993) Science, 260: 1113]. Constructs are made with variant
sequences and assayed for XTEN release using standard assays for
detection of the XTEN polypeptides.
Example 39: Human Clinical Trial Designs for Evaluating CFXTEN
Comprising FVIII
[0616] Kogenate.RTM. FS is recombinant human coagulation factor
VIII, intended for promoting hemostasis in hemophilia A subjects.
Due to its short half-life, Kogenate is dosed intravenously every
other day for prophylaxis and 8 to every 12 h in treatment of
bleeds until hemostasis is achieved. It is believed that fusion of
one or more XTEN to FVIII improves the half-life of the protein,
enabling a reduced dosing frequency using such CFXTEN-containing
fusion protein compositions.
[0617] Clinical trials are designed such that the efficacy and
advantages of CFXTEN, relative to Kogenate or other commercially
available FVIII preparations, can be verified in humans. Such
studies comprises three phases. First, a Phase I safety and
pharmacokinetics study in adult patients is conducted to determine
the maximum tolerated dose and pharmacokinetics and
pharmacodynamics in humans (either normal subjects or patients with
hemophilia), as well as to define potential toxicities and adverse
events to be tracked in future studies. The Phase I studies are
conducted in which single rising doses of CFXTEN compositions are
administered by the route (e.g., subcutaneous, intramuscular, or
intravenously) and biochemical, PK, and clinical parameters are
measured at defined intervals. This permits the determination of
the minimum effective dose and the maximum tolerated dose and
establishes the threshold and maximum concentrations in dosage and
circulating drug that constitute the therapeutic window for the
respective components, as well as bioavailability when administered
by the intramuscular or subcutaneous routes. From this information,
the dose and dose schedule that permits less frequent
administration of the CFXTEN compositions, yet retains the
pharmacologic response, is obtained. Thereafter, clinical trials
are conducted in patients with the condition, verifying the
effectiveness of the CFXTEN compositions under the dose conditions,
which can be conducted in comparison to a positive control such as
Kogenate to establish the enhanced properties of the CFXTEN
compositions.
[0618] Phase II and III clinical trials are conducted in patients
suffering from any disease in which factor VIII may be expected to
provide clinical benefit. For example, the CFXTEN is used in
clinical trials for treatment of indications approved for use of
factor VIII; such indications include bleeding episodes in
hemophilia A, patients with inhibitors to factor VIII, prevention
of bleeding in surgical interventions or invasive procedures in
hemophilia A patients with inhibitors to factor VIII, treatment of
bleeding episodes in patients with congenital factor VIII
deficiency, and prevention of bleeding in surgical interventions or
invasive procedures in patients with congenital factor VIII
deficiency. CFXTEN may also be indicated for use in additional
patient populations. A phase II dosing study is conducted in
hemophilia A patients where pharmacodynamic, coagulation, bleeding
and other physiologic, PK, safety and clinical parameters and
clinical endpoints appropriate for trials are measured as a
function of the dosing of the fusion proteins compositions,
yielding dose-ranging information on doses that is appropriate for
a subsequent Phase III trial, in addition to collecting safety data
related to adverse events. The PK parameters are correlated to the
physiologic, clinical and safety parameter data to establish the
therapeutic window and the therapeutic dose regimen for the CFXTEN
composition, permitting the clinician to establish the appropriate
dose ranges for the composition. In one trial, hemophilia A
patients with factor VIII inhibitors would be evaluated to
establish doses and dose regimen of CFXTEN pharmaceutical
compositions that result in achieving and maintaining hemostasis
and preventing or attenuating bleeding episodes. Finally, a phase
III efficacy study is conducted wherein patients are administered
the CFXTEN pharmaceutical composition and a positive control (such
as a commercially-available Kogenate) are administered using a
dosing schedule deemed appropriate given the pharmacokinetic and
pharmacodynamic properties of the respective compositions derived
from the Phase II findings, with all agents administered for an
appropriately extended period of time to achieve the study
endpoints. Parameters that are monitored include aPTT assay, one-
or two-stage clotting assays, control of bleeding episodes, or the
occurrence of spontaneous bleeding episodes; parameters that are
tracked relative to the placebo or positive control groups.
Efficacy outcomes are determined using standard statistical
methods. Toxicity and adverse event markers are also be followed in
this study to verify that the compound is safe when used in the
manner described. In another phase III trial, hemophilia A patients
with factor VIII inhibitors would be evaluated to establish the
effectiveness of CFXTEN pharmaceutical compositions in achieving
and maintaining hemostasis and preventing or attenuating bleeding
episodes.
Example 40: Analytical Size Exclusion Chromatography of XTEN Fusion
Proteins 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. 21. The
data show that the apparent 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, including a CFXTEN composition, 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 35. 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-00045 TABLE 35 SEC analysis of various polypeptides
Apparent XTEN or Molecular Construct fusion Therapeutic Actual
Apparent Weight Name partner Protein MW (kDa) MW (kDa) Factor
R.sub.H (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 AC227 AM875
IL-1ra 95.4 1103 11.6 9.2 AC228 AM1318 IL-1ra 134.8 2286 17.0
10.5
Example 41: Pharmacokinetics of Extended Polypeptides Fused to GFP
in Cynomolgus Monkeys
[0620] 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.
19. 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 42: Serum Stability of XTEN
[0621] 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. 20. 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 CFXTEN to degradation due to
serum proteases; a factor in the enhancement of pharmacokinetic
properties of the CFXTEN fusion proteins.
Example 43: Increasing Solubility and Stability of a Peptide
Payload by Linking to XTEN
[0622] 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 36. 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
CFXTEN 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).
[0623] 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-00046 TABLE 36 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 44: Analysis of Sequences for Secondary Structure by
Prediction Algorithms
[0624] 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 (Garnier
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.
[0625] 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.
[0626] As a first step in the analyses, a single XTEN sequence was
analyzed by the two algorithms. The AE864 composition is an 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 37.
[0627] 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.
[0628] The data provided herein suggests 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. Results further indicate that XTEN sequences defined
herein (including e.g., XTEN created from sequence motifs of G, S,
T, E, P, and A) have limited repetitiveness (including those with
no more than two identical contiguous amino acids in any one motif)
are expected to have very limited secondary structure. Any order or
combination of sequence motifs from Table 3 can be used to create
an XTEN polypeptide that will result in an XTEN sequence that is
substantially devoid of secondary structure, though three
contiguous serines are not preferred. The unfavorable property of
three contiguous series however, can be ameliorated by increasing
the length of the XTEN. Such sequences are expected to have the
characteristics described in the CFXTEN embodiments of the
invention disclosed herein.
TABLE-US-00047 TABLE 37 CHOU-FASMAN and GOR prediction calculations
of polypeptide sequences No. Chou-Fasman GOR SEQ NAME SEQ ID NO:
Residues Calculation Calculation AE36: 1489 36 Residue totals: H: 0
E: 0 94.44% LCW0402_002 percent: H: 0.0 E: 0.0 AE36: 1490 36
Residue totals: H: 0 E: 0 94.44% LCW0402_003 percent: H: 0.0 E: 0.0
AG36: 1491 36 Residue totals: H: 0 E: 0 77.78% LCW0404_001 percent:
H: 0.0 E: 0.0 AG36: 1492 36 Residue totals: H: 0 E: 0 83.33%
LCW0404_003 percent: H: 0.0 E: 0.0 AE42_1 1493 42 Residue totals:
H: 0 E: 0 90.48% percent: H: 0.0 E: 0.0 AE42_1 1494 42 Residue
totals: H: 0 E: 0 90.48% percent: H: 0.0 E: 0.0 AG42_1 1495 42
Residue totals: H: 0 E: 0 88.10% percent: H: 0.0 E: 0.0 AG42_2 1496
42 Residue totals: H: 0 E: 0 88.10% percent: H: 0.0 E: 0.0 AE144
1497 144 Residue totals: H: 0 E: 0 98.61% percent: H: 0.0 E: 0.0
AG144_1 1498 144 Residue totals: H: 0 E: 0 91.67% percent: H: 0.0
E: 0.0 AE288 1499 288 Residue totals: H: 0 E: 0 99.31% percent: H:
0.0 E: 0.0 AG288_2 1500 288 Residue totals: H: 0 E: 0 92.71
percent: H: 0.0 E: 0.0 AF504 1501 504 Residue totals: H: 0 E: 0
94.44% percent: H: 0.0 E: 0.0 AD 576 1502 576 Residue totals: H: 7
E: 0 99.65% percent: H: 1.2 E: 0.0 AE576 1503 576 Residue totals:
H: 2 E: 0 99.65% percent: H: 0.4 E: 0.0 AG576 1504 576 Residue
totals: H: 0 E: 3 99.31% percent: H: 0.4 E: 0.5 AF540 1505 540
Residue totals: H: 2 E: 0 99.65 percent: H: 0.4 E: 0.0 AD836 1506
836 Residue totals: H: 0 E: 0 98.44% percent: H: 0.0 E: 0.0 AE864
1507 864 Residue totals: H: 2 E: 3 99.77% percent: H: 0.2 E: 0.4
AF864 1508 875 Residue totals: H: 2 E: 0 95.20% percent: H: 0.2 E:
0.0 AG864 1509 864 Residue totals: H: 0 E: 0 94.91% percent: H: 0.0
E: 0.0 AM875 1510 875 Residue totals: H: 7 E: 3 98.63% percent: H:
0.8 E: 0.3 AM1318 1511 1318 Residue totals: H: 7 E: 0 99.17%
percent: H: 0.7 E: 0.0 AM923 1512 924 Residue totals: H: 4 E: 3
98.70% percent: H: 0.4 E: 0.3 AE912 1513 913 Residue totals: H: 8
E: 3 99.45% percent: H: 0.9 E: 0.3 BC 864 1514 Residue totals: H: 0
E: 0 99.77% percent: H: 0 E: 0 H: alpha-helix E: beta-sheet
Example 45: Analysis of Polypeptide Sequences for
Repetitiveness
[0629] In this Example, different polypeptides, including several
XTEN sequences, were assessed for repetitiveness in the amino acid
sequence. 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 length has a total of 165
overlapping 36-amino acid "blocks" (or "36-mers") and 198 3-mer
"subsequences", but the number of unique 3-mer subsequences will
depend on the amount of repetitiveness within the sequence. For the
analyses, different polypeptide sequences were assessed for
repetitiveness by determining the subsequence score obtained by
application of the following equation:
Subsequence score i = 1 m Count i m I ##EQU00006## [0630] wherein:
m=(amino acid length of polypeptide)-(amino acid length of
subsequence)+1; and Count.sub.i=cumulative number of occurrences of
each unique subsequence within sequence, In the analyses of the
present Example, the subsequence score for the polypeptides of
Table 38 were determined using the foregoing equation in a computer
program using the algorithm depicted in FIG. 27, wherein the
subsequence length was set at 3 amino acids. The resulting
subsequence score is a reflection of the degree of repetitiveness
within the polypeptide.
[0631] The results, shown in Table 38, indicate that the
unstructured polypeptides consisting of 2 or 3 amino acid types
have high subsequence scores, while those of consisting of the 12
amino acid 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: 1694),", 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), while XTEN
consisting of four motifs containing five types of amino acids were
intermediate; i.e., AE864, with a score of 7.2.
[0632] Conclusions:
[0633] The results indicate that the combination of 12 amino acid
subsequence motifs, each consisting of four to six amino acid types
that are non-repetitive, into a longer XTEN polypeptide results in
an overall sequence that is substantially non-repetitive, as
indicated by overall subsequence scores less than 10 and, in many
cases, less than 5. 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, with polypeptides consisting of two
amino acid type having higher scores that those consisting of three
amino acid types.
TABLE-US-00048 TABLE 38 Subsequence score calculations of
polypeptide sequences Seq Name SEQ ID NO: Score J288 1515 33.3 K288
1516 46.9 L288 1517 50.0 Y288 1518 26.8 Q576 1519 18.5 U576 1520
18.1 W576 1521 23.4 Y576 1522 15.7 AE288 1523 6.0 AG288_1 1524 6.9
AD576 1525 13.6 AE576 1526 6.1 AF540 1527 8.8 AF504 1528 7.0 AE864
1529 6.1 AF864 1530 7.5 AG864 1531 7.2 AG868 1532 7.5 AM875 1533
4.5 AE912 1534 4.5 AM923 1535 4.5 AM1296 1536 4.5
Example 46: Calculation of TEPITOPE Scores
[0634] TEPITOPE scores of 9 mer 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 39 shows as an example the pocket potentials for
HLA0101B, 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 39 were added. The HLA0101B score of a 9
mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 1695) is the
sum of 0, -1.3, 0, 0.9, 0, -1.8, 0.09, 0, 0.
[0635] To evaluate the TEPITOPE scores for long peptides one can
repeat the process for all 9 mer subsequences of the sequences.
This process can be repeated for the proteins encoded by other HLA
alleles. Tables 40-43 give pocket potentials for the protein
products of HLA alleles that occur with high frequency in the
Caucasian population.
[0636] TEPITOPE scores calculated by this method range from
approximately -10 to +10. However, 9 mer peptides that lack a
hydrophobic amino acid (FKLMVWY (SEQ ID NO: 1696)) 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 9 mer 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-00049 TABLE 39 Pocket potential for HLA0101B 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-00050 TABLE 40 Pocket potential for HLA0301B 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-00051 TABLE 41 Pocket potential for HLA0401B 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-00052 TABLE 42 Pocket potential for HLA0701B 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-00053 TABLE 43 Pocket potential for HLA1501B 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 46: Assessment of Insertion of XTEN into Permissive
Loops
[0637] XTEN AE42-4 Insertion
[0638] The construction and expression of FVIII with XTEN AE42
insertions were described in Example 17 and 24. Thus, where residue
X designates the site of insertion and residue Z designates the
next residue in the native FVIII polypeptide sequence, the
polypeptide resulting from insertion of XTEN AE42 would contain the
sequence:
TABLE-US-00054 (SEQ ID NO: 1697)
X-GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS-Z
[0639] 16 different sites in the FVIII sequence were selected for
XTEN AE42 insertion, and these were designed Batch 1. An additional
21 sites selected for XTEN AE42 insertion were designed Batch 2.
Collectively, the Batch 1 and Batch 2 sites represent 12 sites in
the A1 domain, 7 sites in the A2 domain, 10 sites in the A3 domain,
4 sites in the C1 domain, and 3 sites in the C2 domain. Locations
of Batch 1 and 2 sites in the 3-D structure of FVIII are depicted
in FIG. 32.
[0640] The location of these Batch 1 and Batch 2 insertion sites
results in 37 constructs designated pSD0001-pSD0004,
pSD0009-pSD0012, pSD0023-pSD0032, pSD0034-pSD0063 [the foregoing
ranges include all intermediate numbers, as well], the sequences of
which are set forth in Table 21 and the insertions sites of which
are set forth in Table 23.
[0641] In Vitro Assays
[0642] To assess FVIII tolerability to XTEN AE42-4 insertion, the
FVIII activity in culture media samples from FVIII-XTEN cell
cultures was analyzed using a FVIII chromogenic assay. Antigen
expression levels were analyzed by FVIII-HC (FVIII heavy chain) and
FVIII-LC (FVIII light chain) ELISA.
[0643] FVIII Activity Measurement by Chromogenic Assay
[0644] The FVIII activity was measured using the COATEST.RTM. SP
FVIII kit from DiaPharma (lot #N089019) and all incubations were
performed on a 37.degree. C. plate heater with shaking. Cell
culture harvests from transient transfection media of FVIII-XTEN
AE42-4 variants from 6 well plates were diluted to the desired
FVIII activity range using 1.times.FVIII COATEST.RTM. buffer. FVIII
standards were prepared in 1.times. FVIII COATEST.RTM. buffer
containing mock transfection media with matching culture media
concentration as the testing sample. The range of recombinant
Factor VIII (rFVIII) standard was from 100 mIU/mL to 0.78 mIU/mL.
The standards, diluted cell culture samples, and a pooled normal
human plasma assay control were added to Immulon.RTM. 2HB 96-well
plates in duplicates (25 .mu.L/well).
[0645] Freshly prepared IXa/FX/Phospholipid mix (50 .mu.L), 25
.mu.L of 25 mM CaCl2, and 50 .mu.L of FXa substrate were added
sequentially into each well, with 5 minutes incubation between each
addition. After incubating with the substrate, 25 .mu.L of 20%
acetic acid was added to terminate the color reaction, and the
absorbance at 405 nm was measured with a SpectraMAX.RTM. plus
(Molecular Devices) instrument.
[0646] Data analysis was performed using SoftMax Pro software
(version 5.2). The Lowest Level of Quantification (LLOQ) was 39
mIU/mL. Results are presented in Table 22.
[0647] Expression Measurement by FVIII-HC and FVIII-LC ELISA
[0648] Expression of variants was quantified using ELISA. The FVIII
antigen expression levels of DNA constructs corresponding to XTEN
insertions in the A1 and A2 domains of FVIII were analyzed by
FVIII-LC ELISA. The FVIII antigen expression levels of DNA
constructs corresponding to XTEN insertions in the A3, C1 and C2
domains of FVIII were analyzed by FVIII-HC ELISA. Results are
presented in Table 22.
[0649] FVIII-XTEN antigens in cell culture media after harvest were
captured by GMA011 antibodies (Green Mountain Antibodies) for
FVIII-LC ELISA) or by GMA016 antibodies (Green Mountain Antibodies)
for FVIII-HC ELISA. Immulon.RTM. 2HB 96-well plates were coated
with 100 .mu.l/well of anti-FVIII antibody (2 .mu.g/ml) by
overnight incubation at 4.degree. C. Plates were then washed four
times with Phosphate Buffer saline with Tween-20 (PBST) and blocked
with blocking buffer (PBST with 10% heat inactivated horse serum)
for 1 hour at room temperature.
[0650] Cell culture harvests from transient transfection media of
FVIII-XTEN variants from a 6-well plate were diluted to the desired
FVIII antigen range using 1.times. blocking buffer. FVIII standards
were prepared in 1.times.FVIII blocking buffer containing mock
transfection media with matching media concentration as the testing
samples. The range of rFVIII standard was from 50 ng/mL to 0.39
ng/mL.
[0651] Standards, diluted cell culture samples, and a pooled normal
human plasma assay control were added into Immulon.RTM. 2HB 96-well
plates in duplicates (100 .mu.L/well) and incubated at 37.degree.
C. for 2 hours. Following four times washing with PBST, 100 .mu.l
of HRP-sheep anti-hFVIII antibody (Affinity Biologicals, F8C-EIC-D)
were added into each well and plates were incubated for 1 hour at
37.degree. C. After another four washes with PBST, 100 .mu.l of TMB
Super Sensitive Substrate (BioFX) were added to each well, followed
by 5-10 min color development. To terminate the color reaction, 50
.mu.L of H2SO4 were added to each well, and the absorbance of at
450 nm was measured with a SpectraMAX plus (Molecular Devices)
instrument.
[0652] Data analysis was performed using SoftMax Pro software
(version 5.4). The Lowest Level of Quantification (LLOQ) was 0.0039
.mu.g/mL. Results are presented in Table 22.
[0653] Permissive sites into which XTEN sequences were inserted
without eliminating procoagulant activity of the recombinant
protein, or the ability of the recombinant proteins to be expressed
in the host cell were clustered within loops in each of the three A
domains of FVIII. FIG. 36 shows the location of insertion sites in
the recombinant FVIII proteins that showed FVIII activity on
domains A1, A2 and A3. FIG. 33 shows a structural representation
depicting the location of insertion sites in the recombinant FVIII
proteins that showed FVIII activity.
[0654] The permissive sites clustered in solvent exposed, highly
flexible surface loops (XTEN permissive loops). The A1 domain loops
were located in a region corresponding approximately to amino acid
positions 15 to 45, and 201 to 232, respectively, in the sequence
of mature human FVIII (FIG. 30). The A2 domain loops were located
in a region corresponding approximately to amino acid positions 395
to 421, and 577 to 635, respectively, in the sequence of mature
human FVIII (FIG. 30). The A3 domain loops were located in a region
corresponding approximately to amino acid positions 1705 to 1732,
and 1884 to 1917, respectively, in the sequence of mature human
FVIII (FIG. 30). FIGS. 37A and 37B show the location of the XTEN
permissive loops relative to secondary structure elements in the
tridimensional structure of FVIII.
Example 47: CFXTEN with Insertions of XTEN Having 144 Amino
Acids
[0655] Analysis of the preliminary data presented above (Example
46) suggested the existence of defined regions within the linear
polypeptide sequences and 3-D structures of the FVIII A domains
that can accommodate the insertion of XTEN sequences. To test this
hypothesis and further define the boundaries of putative regions
that can accommodate the insertion of XTEN sequences without loss
of FVIII activity, 23 additional insertion sites not present in
either Batch 1 or 2 were chosen and designated Batch 3.
[0656] Batch 3 constructs were generated by the insertion of a 144
residue XTEN AE polypeptide, comprising amino acid residues Gly
(G), Ala (A), Pro (P), Ser (S), Thr (T), and Glu (E), or a 144
residue XTEN AG polypeptide, comprising amino acid residues Gly
(G), Ala (A), Pro (P), Ser (S), and Thr (T). Five different version
of the 144 residue AE polypeptide were generated and designated
XTEN-AE144-2A, XTEN-AE144-3B, XTEN-AE144-4A, XTEN-AE144-5A,
XTEN-AE144-6B. The amino acid sequences are as set forth in Table
4. Five different versions of the 144 residue polypeptide were
generated and designated XTEN-AG144-1, XTEN-AG144-A, XTEN-AG144-B,
XTEN-AG144-C, and XTEN-AG144-F. The amino acid sequences are as set
forth in Table 4.
[0657] The 144 residue XTEN encoding DNA sequence was introduced by
the chemical synthesis of DNA segments (DNA 2.0, Redwood City,
Calif.) spanning the nearest unique restriction sites within the
base vector on either side of the site of insertion.
[0658] The DNA sequences corresponding to the XTEN 144 peptides
were inserted such that the resulting DNA construct would encode a
FVIII protein in which the XTEN 144 protein sequence is inserted
immediately after the residue indicated in the site selection, and
flanked by AscI and XhoI sites.
[0659] In addition to these sites, those sites from Batch 1 and 2
at which insertion of the XTEN AE42 polypeptide did not abolish
FVIII procoagulant activity were modified by excision of the AE42
polypeptide encoding DNA segment with restriction enzymes AscI and
XhoI, and introduction of XTEN AE144 and XTEN AG144 coding
sequences at the same sites. The location of these Batch 1, Batch 2
and Batch insertion sites is summarized in Table III. FIG. 34
presents a structural representation of FVIII showing the location
of the XTEN 144 insertion sites.
[0660] A total of 48 constructs with 144 XTEN inserts were created.
The constructs are pSD0001-pSD0004, pSD0009-pSD0012, pSD0023-63
[the foregoing ranges include all intermediate numbers, as well],
the sequences of which are set forth in Table 21 and the insertion
sites of which are detailed in Table 22.
[0661] Expression of FVIII-XTEN 144 Variants
[0662] FVIII variants with XTEN 144 insertions were tranfected into
HEK293F cells (Invitrogen, Carlsbad, Calif.) using
polyethyleneimine (PEI, Polysciences Inc. Warrington, Pa.) or
Lipofectamine transfection reagent (Invitrogen, Carlsbad, Calif.).
The transiently transfected cells were grown in 293 Free Style
medium or a mixture of 293 Free Style and CD Opti CHO media
(Invitrogen, Carlsbad, Calif.). The cell culture medium was
harvested 3-5 days after transfection and analyzed for FVIII
expression by chromogenic FVIII activity assay and FVIII ELISA
conducted as described herein.
[0663] Cell culture media from transient transfection were
concentrated 10-fold in Centricon.RTM. spin columns (100 kd
cut-off). Concentrated material was then flash frozen and stored at
-80.degree. C. for future in vitro analysis and in vivo PK
studies.
[0664] In Vitro Assays
[0665] To assess FVIII tolerability to insertions, the FVIII
activity in culture media samples from cell cultures was analyzed
using a FVIII chromogenic assay. Antigen expression levels were
analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light
chain) ELISA.
[0666] FVIII Activity Measurement by Chromogenic Assay and
Expression Measurement by FVIII-HC and FVIII-LC ELISA
[0667] Chromogenic and ELISA assay methods were conducted as
described. The results obtained are summarized in Table 23.
[0668] Permissive sites into which XTEN sequences were inserted
without eliminating procoagulant activity of the recombinant
protein, or the ability of the recombinant proteins to be expressed
in the host cell clustered within loops in each of the three A
domains of FVIII. The same XTEN permissive loop regions tolerating
the shorter XTEN sequences inserted were found to tolerate the
insertion of the longer XTEN sequences. FIG. 38 shows the location
of XTEN 144 insertion sites in the recombinant FVIII proteins that
showed FVIII activity on domains A1, A2 and A3. FIG. 35 shows a
structural representation depicting the location of insertion sites
in the recombinant FVIII proteins that showed FVIII activity.
[0669] These observation indicate that two regions within each of
the A domains of FVIII are able to accommodate insertion of XTEN
sequences without loss of FVIII cofactor activity. A structural
depiction of these so-called XTEN permissive loops (FIGS. 40 and
41) demonstrate that they occupy structurally analogous positions
in each of the A domains and project from one face of the FVIII
molecule. The identified XTEN permissive loops correspond to highly
flexible loops located between beta strands in the
three-dimensional structures of the A1, A2, and A3 domains, as
shown in FIGS. 37A and 37B.
[0670] The in vivo evaluation of XTEN 144 insertions on FVIII
Half-life Extension, as determined by pharmacokinetics, is
described in Example 32.
Example 48: Rescue or Enhancement of FVIII Expression by Insertion
of an XTEN Sequence within the a3 Acidic Peptide Region of
FVIII
[0671] Adherent HEK293 cells were transfected (as described in
Example 24) with FVIII-XTEN DNA constructs in which the coding
sequence of a B-domain deleted factor VIII contained 2 to 4 XTEN
insertions of 144 amino acid residues each, of composition and
insertion location as indicated in Table 44, below. At 5 days
post-transfection, cell culture supernatants were assayed for FVIII
activity by the chromogenic assay (as described in Example 25).
Results are shown in Table 44.
TABLE-US-00055 TABLE 44 Expression levels of FVIII Activity by
CFXTEN variants containing an XTEN at position 1720 and one, two,
or three additional XTEN insertions. Construct Domain, Position,
and Type of XTEN Insertion Activity Name A1 A2 a3 A3-1 A3-2
(mIU/mL) LSD0040.002 26 AG144 1720 AG144 175 LSD0041.008 403 AE144
1720 AG144 279 LSD0045.002 1656 AG144 1720 AG144 2598 PSD080.002 26
AG144 1656 AG144 1720 AG144 1081 PSD083.001 403 AE144 1656 AG144
1720 AG144 789 PSD082.001 26 AG144 1720 AG144 1900 AE144 <LLOQ
PSD090.003 26 AG144 1656 AG144 1720 AG144 1900 AE144 316
[0672] For the purpose of comparison, all FVIII-XTEN constructs had
an AG144 XTEN insertion at amino acid position 1720 (numbered
relative to full-length factor VIII) within the A3 domain.
Expression levels of FVIII-XTEN varians were determined by
chromogenic assay and expressed in units of mIU/mL. Constructs with
a single additional XTEN insertion at either position 26 in the A1
domain (LSD0040.002) or position 403 in the A2 domain (LSD0041.008)
yielded expression levels of 175 and 279 mIU/mL, respectively. In
contrast, a construct with a single additional XTEN insertion at
position 1656 within the a3 acidic peptide yielded an expression
level of 2598 mIU/mL, demonstrating enhancement of expression level
for the a3 XTEN insertion construct relative to the A1 and A2
insertion constructs. In addition, in comparison to the FVIII-XTEN
construct with XTEN insertions at positions 26 in the A1 domain and
1720 in the A3 domain (LSD0040.002), the construct with an
additional XTEN insertion at position 1656 within the a3 acidic
peptide region (PSD080.002) yielded significantly higher expression
(175 and 1081 mIU/mL, respectively). Consistent with these
findings, the construct with XTEN insertions at positions 403 in
the A2 domain and 1720 in the A3 domain (LSD0041.008) yielded an
expression level of 279 mIU/mL, whereas an additional XTEN
insertion at position 1656 within the a3 acidic peptide region
(PSD083.001) resulted in an increase in the expression level to 789
mIU/mL. Lastly, the FVIII-XTEN construct with an XTEN insertion at
position 26 within the A1 domain and two XTEN insertions at
positions 1720 and 1900 within the A3 domain (PSD082.001) did not
yield activity above the lower limit of quantitation. However, the
FVIII-XTEN construct with an additional XTEN insertion within the
a3 acidic peptide region (PSD090.003) resulted in detectable
activity, demonstrating that inclusion of an XTEN sequence within
the a3 domain can result in recovery of expression (as measured by
activity) in FVIII-XTEN constructs that are otherwise expressed at
levels below the lower limit of quantitation. Under the conditions
of the experiment, the results support the conclusion that
insertion of XTEN at the 1656 position and, by extension, within
the a3 region, results in enhanced expression of procoagulant
FVIII-XTEN compositions.
Example 49: Effect of XTEN Insertion on FVIII Activity Measured by
aPTT
[0673] A one stage activated partial prothrombin (aPTT) coagulation
assay was employed in addition to the chromogenic assay (as
described in Example 25) to determine FVIII activity of various
FVIII-XTEN fusion proteins.
[0674] Method:
[0675] The FVIII-XTEN aPTT activity was measured using the Sysmex
CA-1500 instrument (Siemens Healthcare Diagnostics Inc., Tarrytown,
N.Y.). To create a standard curve for the assay, WHO factor VIII
standard was diluted with 2% mock transfection media to 100 mU/mL
and a two-fold serial dilution series was then performed, with the
last standard being 0.78 mU/mL. FVIII-XTEN cell culture samples
were first diluted at 1:50 with aPTT assay buffer, further
dilutions were made with 2% mock transfection media when
needed.
After dilution, the aPTT assay was performed using Sysmex
instrument as follow: 50 .mu.l of diluted standards and samples
were mixed with 50 .mu.l human FVIII deficient plasma and then 50
.mu.l of aPTT reagent. The mixture was incubated at 37.degree. C.
for 4 min, and following incubation, 50 .mu.l of CaCl.sub.2 was
added to the mixture, and the clotting time was measured
immediately. To determine test samples FVIII activity, the clotting
time of the standards were plotted using semi-log scale (Clotting
time: Linear; Standard concentration: Log) to extrapolates the
equation between clotting time and FVIII activity, and FVIII-XTEN
activity was then calculated against the standard curve. The assay
sensitivity was 40 mU/mL factor VIII.
[0676] Results:
[0677] The results are summarized in FIGS. 44-46. When single XTEN
of 144 or 288 amino acids were inserted into the FVIII, all of the
FVIII-XTEN fusion proteins exhibiting activity in the chromogenic
assay were also active in aPTT assay. The aPTT activity followed
the trend of chromogenic assay, for example, those molecules that
showed low FVIII activity in the chromogenic assay also had low
aPTT values. Generally, the aPTT results for the fusion proteins
were lower than those obtained by the chromogenic assay, with a
chromogenic to aPTT ratio of 1.1 up to 2.2, as illustrated in FIG.
44, for the single XTEN insertions. The FVIII-XTEN fusion proteins
with multiple XTEN insertions, in general, showed further
reductions in aPTT activity in comparison to chromogenic assay.
Assays of FVIII-XTEN with two XTEN insertions showed activity with
all constructs, but with chromogenic/aPTT ratios approaching 4, in
some instances (FIG. 45). Assays of FVIII-XTEN with some three XTEN
insertions also showed activity in both assays, with
chromogenic/aPTT ratios approaching 5, in some instances (FIG. 46),
while the ratios for the BDD-FVIII control were more comparable
(right side of FIG. 46). Additionally, the site of XTEN insertion
appeared to contribute to the differences seen between aPTT and
chromogenic activities. For example, while some molecules with 2
XTEN insertions resulted in up to 4-fold lower activity than
chromogenic values, the aPTT activity of other FVIII molecules with
2 XTEN were fairly comparable to chromogenic activity (FIG. 45).
Some molecules with 3 XTEN insertions showed up to 5-fold lower
than chromogenic activities, other FVIII molecules with 3 XTEN have
aPTT activity less than 2-fold lower than chromogenic activity
(FIG. 45). Under the conditions of the experiment, the results
support the conclusion that FVIII-XTEN fusion protein constructs do
retain procoagulant activity, but that the chromogenic assay
generally provides higher activity levels than that in the aPTT
assay system employed in the study.
Example 50: Evaluations of the Effect of XTEN Insertion Site Onf
FVIII Half-Life Extension
[0678] Methods:
[0679] Six FVIII-XTEN fusion proteins with single XTEN AG-144
insertions at defined locations were tested in FVIII/VWF DKO mice
(as generally described in Example 32) to evaluate the effect of
XTEN insertion site on FVIII half-life. Six representative XTEN
variants (listed in table 1) with XTEN insertion in either within
A1, A2, a3, A3-region 1 (A3-R1), A3-region 2 (A3-R2) or at the
C-terminus were selected for this study, and BDD-FVIII generated
from the base vector was used as the control. FVIII/VWF DKO mice
were treated with a single intravenous administration of transient
transfaction cell culture media concentrate from the six FVIII-XTEN
constructs (or positive control media) at 100-200 IU/kg, and plasma
samples were subsequently collected at 5 min, 7 hours and 16 hours
post-dosing. Plasma FVIII activity was tested using the FVIII
chromogenic assay and FVIII-XTEN half-life was estimated using the
WinNonlin program. The study data are summarized in Table 45 and
FIG. 47.
[0680] Results:
[0681] A significantly longer half-life was observed for all
FVIII-XTEN variants tested compared to BDD-FVIII control, but the
degree of the half-life increase varied, with the variant with XTEN
at the 403 insertion site conferring the least half-life extension
at 10-fold (in comparison to control), while the 1900 insertion
variant conferred the most half-life extension at 18-fold. The
differences of XTEN insertion site on FVIII half-life extension may
reflect the roles of different FVIII domains in FVIII clearance in
vivo.
TABLE-US-00056 TABLE 45 FVIII-XTEN single AG-144 insertion variants
PK in FVIII/VWF DKO mice BDD- pSD- pSD- pSD- pSD- pSD- pSD-
Treatment FVIII 050 0003 0039 0010 063 014 Insertion site None 26
403 1656 1720 1900 CT Recovery 21.3 33.8 34.8 36.0 33.6 39.6 32.4
t1/2 0.25 3.15 2.4 3.3 4.28 4.54 3.91 (hr) t1/2 Increase 13 10 13
17 18 16 (fold)
Example 51: Evaluations of the Additive Effect of XTEN Ilsertions
on FVIII Half-Life Extension
[0682] Methods:
[0683] To evaluate the effects of multiple XTEN insertions on
FVIII-XTEN fusion protein half-life, the half-lives of FVIII-XTEN
variants with 1-3 XTEN insertions were determined in FVIII-XTEN DKO
mice using the cell culture concentrate from five constructs (as
generally described in Example 32). Five FVIII-XTEN variants were
tested in the study: pSD-062, with AE144 insertion at position 1900
(numbered relative to full-length factor VIII); pSD-0005 with AE144
in the FVIII B domain (B-domain amino acid position 745); pSD-0019
with AE288 at the FVIII C-terminus (CT); LSD0003.006 with AE144
inserted in the B-domain and AE288 inserted at the C-terminus, and
LSD0055.021 with three XTEN of AE144, AE144, and AE288 inserted at
position 1900, with the B domain and at the C-terminus. The
FVIII-XTEN half-life values were estimated using the WinNonlin
program.
[0684] Results:
[0685] The study results are summarized in Table 46, and the PK
curves are shown in FIG. 48. The study results clearly demonstrated
the additive effect of multiple XTEN insertions on FVIII half-life
extension. With single XTEN insertions, the half-life of FVIII was
extended from 0.25 hr to 3.2-4.0 hr, a 13 to 16-fold increase. When
the B and CT XTEN insertions were combined together, the FVIII
half-life was further extended to 10.6 hr, a 42-fold prolongation.
Finally, in the case of a third XTEN insertion added at position
1900 to the B/CT construct, the half-life reached 16 hr in the
FVIII-VWF DKO mice, a 64-fold increase.
TABLE-US-00057 TABLE 46 Additive effect of XTEN insertions on FVIII
t.sub.1/2 in FVIII/VWF DKO mice BDD- pSD- pSD- pSD- LSD- LSD-
Treatment FVIII 062 0005 0019 0003.006 0055.021 XTEN None 1900 B CT
B/CT 1900/B/CT Insertion site Recovery 21.3 35.3 44.9 33.3 39.0
37.2 t1/2 0.25 3.8 3.2 4.0 10.6 16.0 (hr) t1/2 Increase 15 13 16 42
64 (fold)
Example 52: Evaluation of FVIII-XTEN Interference with the Binding
of Anti-FVIII Antibodies Using the Bethesday Assay
[0686] The ability of XTEN insertions in the FVIII molecule to
interfere with binding by pre-existing anti-FVIII antibodies to the
FVIII-XTEN fusion protein was evaluated in order to determine their
utility in treating patients with anti-FVIII inhibitory
antibodies.
[0687] Methods:
[0688] To assess the binding of anti-FVIII antibodies, two
FVIII-XTEN variants (PSD088, with 144 XTEN inserted at the
locations of 26/403/1656/1900; and PSD-090, with 144 XTEN inserted
at the locations of 26/1656/1720/1900) were tested in comparison
with Refacto (a marketed rFVIII) against plasma samples from three
hemophilia A patients with factor VIII inhibitors (designated
04-483, 05-505, and GK1838-2079), as well as a sheep anti-FVIII
poly-clonal antibody from Affinity Biologicals Inc (F8C-EIA-C). The
Bethesda titer of the four anti-FVIII ab against the two FVIII-XTEN
variants (pSD-088 and pSD-090) and the Refacto control were
determined using modified Bethesda assay methods, detailed as
follows. Heat inactivated anti-FVIII antibody samples at various
dilutions were incubated with 1 IU/mL of each FVIII variant
(diluted in 1.times. in FVIII chromogenic assay buffer) at a 1:1
ratio. The FVIII/antibody mixtures were then incubated for 2 hours
in a 37.degree. C. incubator. After the incubation, the samples
were diluted for 10-fold with 1.times.FVIII chromogenic assay
buffer, and 25 .mu.L of diluted mixture were then used for a FVIII
chromogenic assay, The percentage of remaining FVIII activity was
calculated against the post-incubation activity of a known
non-neutralizing sample. Bethesda units were calculated using the
following formula: BU=dilution factor X (LN(percent of remaining
activity)+6.6438).
[0689] Results:
[0690] The results are listed in Table 47. Decreased Bethesda unit
(BU) titers were observed for all four antibodies when tested
against the two FVIII-XTEN variants, in comparison with Refacto. A
5 to 8-fold fold decrease against PSD-088 and a 3 to 5-fold
decrease against pSD-090, respectively, were obtained. The
inhibition curves against FVIII variants for each antibody were
plotted (FIG. 49) and compared to Refacto, and demonstrates a clear
left-shift of the inhibition curve for the two FVIII-XTEN
molecules, with the pSD-088 FVIII-XTEN variant resulting in a
further left-shift compared to pSD-090. These results clearly
demonstrate that: 1) both FVIII-XTEN variant fusion proteins are
more resistant to pre-existing anti-FVIII inhibitory antibodies
than Refacto; and 2) PSD-088 is more resistant to anti-FVIII
antibodies than PSD-090, which may provide information useful in
determining the differences on the XTEN insertion sites in
interfering with the binding of anti-FVIII antibodies. Under the
conditions of the experiment, the results provide some support for
the potential use of FVIII-XTEN compositions for treating
hemophilia A patients with factor VIII inhibitors.
TABLE-US-00058 TABLE 47 Anti-FVIII antibody Bethesda titer against
FVIII-XTEN variants Anti-FVIII ab. GK1838- F8C-EIA- FVIII 04-483
05-505 2079 C pSD-088 (16/403/1656/1900) 2.5 8 47 57 pSD-090
(26/1656/1720/1900) 3.4 12 55 96 Refacto 12 66 268 337
Example 53: Half-Life Evaluations of FVIII XTEN Fusion Molecules
Containing Four XTEN Insertions in Hemophilia A Mice
[0691] Methods: Eight FVIII-XTEN fusion proteins with four XTEN
insertions each at defined locations were tested in FVIII/VWF DKO
mice to evaluate the effect of the XTEN insertions on FVIII
half-life extension: LSD0071.001, contains 403-AG144, 1900-AE144,
745(B)-AE144, 2332(CT)-AE288 XTEN insertions (designated as the
FVIII amino acid number and the XTEN inserted); LSD0071.002,
containing 403-AE144, 1900-AE144, 745(B)-AE144, 2332(CT)-AE288 XTEN
insertions; LSD0072.001, containing 403-AG144, 1900-AG144,
745(B)-AE144, 2332(CT)-AE288 XTEN insertions; LSD0072.002,
containing 403-AE144, 1900-AG144, 745(B)-AE144, 2332(CT)-AE288 XTEN
insertions; pBC0247.004, containing 18-AG144, 403-AE144,
1656-AG144, 2332(CT)-AE288 XTEN insertions; pBC0251.002, containing
18-AG144, 1656-AG144, 1900-AE144, 2332(CT)-AE288 XTEN insertions;
pSD088, containing 26-AG144, 403-AE144, 1656)-AG144, 1900-AE144
XTEN insertions and pSD090, containing 26-AG144, 1656-Ag144,
1720-AG144, 1900-AE144 XTEN insertions. FVIII/VWF DKO mice were
treated, as generally described in Example 32, with a single
intravenous administration of FVIII-XTEN transfection cell media
concentrate of the eight constructs at 100-200 IU/kg, and plasma
samples were subsequently collected at 5 min, 8 hrs, 24 hrs, 48
hrs, 72 hrs and 96 hrs post-dosing. Plasma FVIII activity was
tested using the FVIII chromogenic assay and FVIII-XTEN half-life
was estimated using the WinNonlin program.
[0692] Results:
[0693] All of the eight FVIII XTEN fusion molecules containing four
XTEN insertions exhibited longer half-life than unmodified FVIII
(results in Table 48). Three molecules with XTEN insertions at
positions 403, 1900, B domain, and C-terminal achieved half-life up
to 16.3 hrs, which is a 65-fold improvement in comparison to
unmodified BDD FVIII. However, the molecules tested with XTEN
insertions at 26/403/1656/1900 (pSD088), or at 26/1656/1720/1900
(pSD090) showed half-life of 9.1 hrs and 9.5 hrs, respectively,
which, in comparison to BDD FVIII, represents an increase of
36-fold and 38-fold, respectively. pBC247.004 (XTEN insertions at
18/403/1656/CT) and pBC251.002 (XTEN insertions at 18/1900/1656/CT)
achieved half-life values of 14.1 hrs and 13 hrs, respectively. The
results demonstrate that multiple XTEN insertions (in this case,
four XTEN insertions for each FVIII molecule) can significantly
improve FVIII half-life. It further shows that the effect of XTEN
on FVIII half-life is insertion site dependent, even in the event
of multiple XTEN insertions.
TABLE-US-00059 TABLE 48 PK of FVIII-XTEN variants with four XTEN
insertions in FVIII/VWF DKO mice Treatment XTEN Insertions t1/2
(hr) t1/2 Increase (fold) BDD-FVIII None 0.25 NA LSD0071.001
403AG/1900AE/B/CT 16.2 64.8 LSD0071.002 403AE/1900AE/B/CT 16.3 65.2
LSD0072.001 403AG/1900AG/B/CT 11.8 47.2 LSD0072.002
403AE/1900AG/B/CT 16.1 64.4 pBC247.004 18/403/1656/CT 14.1 56.4
pBC251.002 18/1900/1656/CT 13.0 52 pSD088 26/403/1656/1900 9.1 36.4
pSD090 26/1656/1720/1900 9.5 38
TABLE-US-00060 TABLE 49 Exemplary Biological Activity, Exemplary
Assays and Preferred Indications Biologically Active Exemplary
Activity Protein Biological Activity Assays Preferred Indication:
Factor VIII Coagulation factor VIII is a Chromogenix assay
Hemophilia A; (Factor VIII; factor essential for (Rosen S, Scand J
bleeding; Octocog alfa; hemostasis. This gene Haematol (1984) 33
Factor VIII Moroctocog encodes coagulation (Suppl 40): 139-45);
deficiency; alfa; factor VIII, which participates Chromogenix
bleeding episodes Recombinant in the intrinsic pathway of Coamatic
.RTM. Factor VIII in patients with Antihemophilic blood
coagulation; factor VIII assay; one-stage factor VIII inhibitor;
factor; is a cofactor for factor IXa clotting assay Surgery-related
Nordiate; which, in the presence of Ca + (Lethagen, S., et al.,
hemorrhagic ReFacto; 2 and phospholipids, Scandinavian J episodes
Kogenate; converts factor X to the Haematology (1986) Kogenate
activated form Xa. This gene 37: 448-453. SF; Helixate; produces
two alternatively One-stage clotting Recombinate) spliced
transcripts. assay and two-stage Transcript variant I encodes
clotting assay a large glycoprotein, isoform (Barrowcliffe TW, a,
which circulates in plasma Semin Thromb and associates with von
Hemost. (2002) Willebrand 28(3): 247-256); factor in a noncovalent
Development of a complex. This protein simple undergoes multiple
chromogenic factor VIII cleavage events. Transcript assay for
variant 2 encodes a putative clinical use. small protein, isoform
b, (Wagenvoord RJ, which consists primarily of Hendrix HH, the
phospholipid binding Hemker HC. domain of factor VIIIc. This
Haemostasis binding domain is essential 1989; 19(4): 196-204) for
coagulant activity. Bethesda assay Defects in this gene results
(Verbruggen B, et al. in hemophilia A, a common Improvements in
factor recessive X-linked VIII inhibitor detection: coagulation
disorder. From Bethesda to Nijmegen. Semin Thromb Hemost. 2009 Nov;
35(8): 752-759)
TABLE-US-00061 TABLE 50 Exemplary CFXTEN comprising FVIII and
internal/external XTEN sequences (SEQ ID NOS 1537-1554,
respectively, in order of appearance) CFXTEN Name Amino Acid
Sequence FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-K127-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE144-
REKEDDKGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
V128-N745-
GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESG- PG
AE288-
SEPATSGSETPGTSTEPSEGSAPGVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL
P1640-
VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA
Y2332) RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS
LEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYD
DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY
KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA
SRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRY
YSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRF
LPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGY
TFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTG
DYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLK
RHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV
EDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTK
DEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS
WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMG
SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAG
MSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW
IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS
SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF
TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS
MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH
QIALRMEVLGCEAQDLY FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-A375-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE576-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
K376-N745-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN AE144-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
P1640- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
Y2332)
DNSPSFIQIRSVAGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP
SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRF
MAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL
PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI
CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIM
HSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE
TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNA
IEPRSFSQNGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS
EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDF
DIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF
TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA
EPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCH
TNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHA
INGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG
VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ
YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY
SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM
GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPK
EWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN
QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-Y1792-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AF144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR E1793-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AE864) LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR
SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG
ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYGGTSTPESGSASPGTSPSGESS
TAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGT
STPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEEDQRQ
GAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLV
CHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF
HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNL
YPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITA
SGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFI
IMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME
LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN
PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ
GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEE
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS
EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAP FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-Y2043-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR G2044-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Q2222-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AG864- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
V2223-
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
Y2332)
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR
SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG
ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNET
KTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV
TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV
MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKA
GIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGPGSSPSASTGT
GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG
TASSSGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS
QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR
MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQG
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG
TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA
SPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST
PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA
TGSPGSSTPSGATGSPGASPGTSSTGSPGVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS
MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH
QIALRMEVLGCEAQDLY FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFN
(A1-G1799-
IAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTS AE144-
QREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLV A1800-
CREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY F2093-
VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL
AE42- LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV
S2094- RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQR
V2223-
IGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT
AE42- DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERD
N2224-
LASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLE
AE42- DPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYE
N2225- DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYED
G2278-
ISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE
AE42- DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGS
K2279-
FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGGGSEP
Y2332)
ATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG
SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
GTSTEPSEGSAPGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK
DVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ
MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR
KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPL
GMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT
QGARQKFGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPGSSLYISQFIIMYS
LDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG
CDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVGPAGS
PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGGNNPKEWLQVDFQKTMKVTGVTT
QGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGGTEPSEGSAPGSPAGSPTSTEEGTSESAT
PESGPGSEPATSGSKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG
CEAQDLY FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARGPGSSPSASTGTGPGSSPSASTGTGPGTPGSG
(A1-R28-
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
AG144-F29-
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSGFPPRVPKSFPFNTS- V
G244- VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY
AG288- WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL
L245- VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA
R2090-
RAWPKMHTVNGYVNRSLPGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
AG576-
SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT
Q2091-
GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
Y2332-
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA
AG864)
STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGLIGCHRKSVY
WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH
DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK
HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK
DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR
GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS
LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP
GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP
PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA
VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI
RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM
APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD
ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAGINGYIMDTLPGLVMAQDQRIRWY
LLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGE
HLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWST
KEPFSWIKVDLLAPMIIHGIKTQGARGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS
PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS
GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFT
NMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM
YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQ
IALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG
SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS
APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
(A1-T1651-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG576-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR R1652-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN K1808-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AG144- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
P1809-
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
F2093-
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
AG288-
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
S2094-
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
Y2332)
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLR
QSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL
RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDT
TLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP
ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP
LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPE
SARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLK
EMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKN
LFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQI
VEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTP
STLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSS
HLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKK
VENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWN
EANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDT
ILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITGPGTPGSGT
ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASP
GTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT
ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSSGRTTLQSDQEEI
DYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRA
QSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS
FYSSLISYEEDQRQGAEPRKNFVKGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGPNETKTYFWKVQHHMAPTKD
EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSW
YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS
NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGM
STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI
KVDLLAPMIIHGIKTQGARQKFGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA
TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS
ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGSSLYISQFII
MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME
LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN
PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ
GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDAGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSG
(A1-A28-
ATGSPGPSGPGRFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV
AG42-F29-
YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEGGPGTPGSGTASSSPG E124-
SSTPSGATGSPGSSPSASTGTGPGASPGDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYS AG42-
YLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGGSPSASTGTGPGAS
D125-E124-
PGTSSTGSPGTPGSGTASSSPGSSTPSGAGKSWHSETKNSLMQDRDAASARAWPKMHTVN AG42-
GYVNSSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQT
D125-P333-
LLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPGSASTGTGPGASPGTSSTGSPGTPGSG
AG42- TASSSPGSSTPSGATGGQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK
Q334- HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF
Y2332)
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK
DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR
GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS
LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP
GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP
PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA
VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI
RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM
APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD
ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL
LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH
LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK
EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFF
GNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQIT
ASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK
SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ
SWVHQIALRMEVLGCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
(A1-D345-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR Y346-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN D403-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AE144-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDGGSEPATSGSETPGTSES R405-
ATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSE
R1797-
TPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGYD
AE288- DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDGGT
Q1798-
STPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAE
Y2322)
SPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPG
TSPSGESSTAPGRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLY
GEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTV
EDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF
DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS
IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGM
TALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEN
DIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDS
NNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTI
PSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESG
LMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRK
THIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKN
MEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGP
EKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKI
QEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN
DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALK
QFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI
PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNL
SLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKD
LFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLA
WDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWA
KQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR
SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG
ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRGGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG
PGTSTEPSEGSAPGQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL
EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI
QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR
KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG
MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQG
ARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL
HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHL
QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW
TLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII
(A1- ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
AE864- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN
Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG
PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTIS
VEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ
FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY
EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG
LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTF
KENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK
MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHI
RDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS
SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI
RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA
WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN
GKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-N745)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE288-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN
Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT
SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE
PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQ
EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN
RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP
YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD
LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC
NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT
VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT
QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI
RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLY
FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-S743)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE288-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
(Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN
Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQREITRTTLQSDQE
EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR
AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP
YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD
LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC
NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT
VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT
QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI
RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLY
FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-N745)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
AG288_2-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN
Y2332)-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AG288_2
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGPPVLKRHQREITRTTLQS
DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL
RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA
SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS
DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR
APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH
VFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC
QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH
GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII
ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK
ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC
EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP
ATG FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI
(A1-S743)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
AG288_2-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
(Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN
Y2332)-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
AG288_2
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGQNPPVLKRHQREITRTTLQS
DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL
RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA
SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS
DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR
APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH
VFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC
QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH
GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII
ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK
ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ
DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC
EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS
PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP
GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP
ATG FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
(A1-N745)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AE288-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (P1640-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AE288
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT
SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE
PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQAEITRTTLQSDQ
EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN
RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP
YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD
LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC
NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT
VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT
QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI
RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ
DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG
SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
(A1-S743)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AE288-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (Q1638-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AE288
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS
ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQAEITRTTLQSDQE
EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR
AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP
YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD
LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC
NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT
VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT
QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI
RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ
DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG
SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
(A1-N745)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
AG288_2-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (P1640-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
AG288_2
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSD
QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS
RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD
VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA
PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF
TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT
PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIK
TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY
IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ
DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG
FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
BDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ
(A1-S743)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR
AG288_2-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (Q1638-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD
Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
AG288_2
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP
FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS
KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP
GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQAEITRTTLQSD
QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS
RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD
VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA
PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF
TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT
PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIK
TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY
IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL
HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH
QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ
DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG
Sequence name reflects N- to C-terminus configuration of the FVIII
segments (amino acid spanning numbers relative to mature sequence)
and XTEN components
TABLE-US-00062 TABLE 51 Exemplary CFXTEN comprising FVIII, cleavage
sequences and XTEN sequences (SEQ ID NOS 1555-1590, respectively,
in order of appearance) CFXTEN Name Amino Acid Sequence SP-AE288-
MQIELSTCFFLCLLRFCFSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS-
ETP CS-L-
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
(FVIII_1-
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES-
GP 745)-
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
AE288-
EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVE
(FVIII_1686-
LSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLG
2332)-L-
PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSH
CS-AE288
TYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI
LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWH
VIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGM
EAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT
WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAI
QHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI
FKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK
RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV
AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF
RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD
GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK
NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP
AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD
TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML
PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA
RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY
RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG
MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV
TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL
TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGS
ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
AP SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS-
TEE CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS-
AP 745)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS
AE576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
2332)-L-
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG-
S CS-AE288
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT-
P
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS
PRSFQGPSGPATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFV
EFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEY
DDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGA
LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNG
YVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL
MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD
DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLY
SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP
LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI
MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE
TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE
PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP
SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY
FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG
PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH
MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD
ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS
MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA
GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW
IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG
IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM
FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE
FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME
VLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SP-
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV
(FVIII_1-
VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW
745)- KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK
AE576-
DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW
(FVIII_1686-
PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI
2332)-L-
TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD
CS-AE576
SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN
GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH
GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER
DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED
PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL
TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL
LSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS
TEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPR
SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGE
LNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKT
YFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQ
EFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE
CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA
WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM
VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ
ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK
SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS
WVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE
SGPGTSTEPSEGSAP SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS-
TEE CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS-
AP 745)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS
AE576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
2332)
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS
PRSFQGPEGPSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF
VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE
YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG
ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN
GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL
LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF
DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK
YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG
PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI
MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE
TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE
PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP
SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY
FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG
PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH
MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD
ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS
MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA
GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW
IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG
IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM
FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE
FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME
VLGCEAQDLY SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS-
TEE CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS-
AP 743)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS
AE288-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
2332)-L-
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG-
S CS-AE576
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT-
P
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPIEP
RSPSGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDD
QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL
VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY
VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM
DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK
KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR
RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI
CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS
INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF
MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS
FSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE
PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQS
GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS
SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV
HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDP
TFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK
MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD
FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI
SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR
MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVN
NPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQG
NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGIEPRSPSGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP SP-AG288-
MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA-
TG CS-L-
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP
(FVIII_1-
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST-
GT 743)-
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
AG576-
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSIEPRSPSGSPGATRRYYLGAVEL
(FVIII_1686-
SWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGP
2332)-L-
TIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT
CS-AG288
YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFIL
LFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHV
IGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME
AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTW
VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQ
HESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF
KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKR
NVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVA
YWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR
NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPS
GATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP
GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS
GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP
GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA
STGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR
NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP
YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL
EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ
MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKK
EEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMAS
GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQK
FSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI
RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAW
RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV
KVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQ
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP
SGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG
TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGSSTPSGATGS SP-AG576-
MQIELSTCFFLCLLRFCFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST-
GT CS-L-
GPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG
(FVIII_1-
SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA-
SS 745)-
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP
AG288-
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
(FVIII_1686-
GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP
2332)-L-
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT-
G CS-AE576
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS-
P
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SQSPRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF
VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE
YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG
ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN
GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL
LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF
DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK
YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG
PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI
MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE
TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE
PRSFSQNPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS
SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSP
SASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL
RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASR
PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD
LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI
QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK
KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA
SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ
KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHY
SIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA
WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNG
KVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPR
SFQGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP SP-
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV
(FVIII_1-
VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW
743)- KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK
AG576-
DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW
(FVIII_1686-
PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI
2332)-L-
TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD
CS-AG576
SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN
GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH
GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER
DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED
PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL
TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL
LSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSST
PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP
GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRS
FQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGEL
NEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY
FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE
FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD
QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE
CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA
WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM
VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ
ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK
SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS
WVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGS SP-AG288-
MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA-
TG CS-L-
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP
(FVIII_1-
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST-
GT 743)-
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP
AG288-
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQGPSGPATRRYYLGAV
(FVIII_1686-
ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLL
2332)-L-
GPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGS
CS-AE288
HTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHK
FILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYW
HVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDG
MEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPK
TWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA
IQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGE
IFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK
RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV
AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF
RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP
SGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDG
SFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKN
FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPA
HGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL
PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS
KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL
HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG
NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME
SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG
VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR
YLRIHPQSWVHQIALRMEVLGCEAQDLYGPSGPQSPRSFQGTSESATPESGPGSEPATSGSETP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS-
TEE CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS-
AP 743)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS
AG576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
2332)
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS
PRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVE
FTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYD
DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL
LVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY
VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM
DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK
KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR
RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI
CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS
INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF
MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS
FSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSS
TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST
GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIA
AVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI
RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAP
TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS
WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS
NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS
TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKV
DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH
NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFAT
WSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI
SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL
GCEAQDLY FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK S367-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL FXIa-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNSSLPGL AE42-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
F368- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSKLT
Y2332-
RAETGEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSGFIQIRSVAKKHPKTWVHY
FXIa-
IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESG
AE864
ILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYK
WTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVIL
FSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY
ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG
MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSD
QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR
AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS
FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK
DVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQME
DPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEY
KMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR
DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL
YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL
RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQV
NNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ
GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE
SGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK N745-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL FIXa-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
AG288-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
FIXa-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
P1640- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
Y2332-
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
FIXa-
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
AG864
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPLGR
IVGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG
SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG
ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGSSTPSGATGSGPLGRIVGGPPVLKRHQREITRTTLQSDQEEIDYDDT
ISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ
FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYE
EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG
PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN
YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYN
LYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS
GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIM
YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG
CDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEW
LQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSF
TPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYPLGRIVGGGASPGTSSTGSPG
SSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS
STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK V128-
EDDKVLQVRIVGGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGPGLQVRIVGG
FVIIa-
FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ
AG42- TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK
FVIIa-
SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH
G2044-
QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK
FVIIa- KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF
AG144-
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF
Y2332-
PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ
FVIIa-
IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV
AG576
CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC
HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE
ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS
SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF
RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA
YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC
RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV
FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGLQVRIVGGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGLQVRIVGGQWAPKLARLHYSGS
INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT
LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD
AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG
VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP
QSWVHQIALRMEVLGCEAQDLYLQVRIVGGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST
GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS
TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG
SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG
ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS
TGTGPGASPGTSSTGS AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
Thrombin-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES-
ATP AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE
KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS
LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG
LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ
IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP
STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY
ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF
KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE
NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT
SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN
KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ
LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ
EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS
LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL
KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI
PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS
LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP
TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN
HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT
ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR
HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL
LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH
HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL
LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH
AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS
WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS
SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN
MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV
KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR
MEVLGCEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA
BDD3- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
FXIIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYGTMTRIVGGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS
TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA
BDD3- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
Elastase-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP
GSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA
BDD3- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST
EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA
BDD3- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
Thrombin-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS
TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAP AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT
MMP-17-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG
AE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL
IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT
VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ
TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV
RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG
RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR
PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS
NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS
GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN
AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK
TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL
GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV
QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF
TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW
YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH
LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP
FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV
DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF
TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM
YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA
LRMEVLGCEAQDLYGAPLGLRLRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAP AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT
FXIIa-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG
AE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL
IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT
VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ
TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV
RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG
RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR
PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS
NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS
GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN
AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK
TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL
GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV
QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF
TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW
YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH
LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP
FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV
DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF
TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM
YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA
LRMEVLGCEAQDLYGTMTRIVGGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAP AG144-
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
FVIII
GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
BDD2-
SASTGTGPGASPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT
FXIa-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG
AG576 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL
IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT
VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ
TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV
RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG
RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR
PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS
NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS
GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN
AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK
TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL
GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV
QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF
TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW
YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH
LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP
FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV
DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF
TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM
YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA
LRMEVLGCEAQDLYGKLTRAETGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPG
SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG
SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT
GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGS AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
FXIa-FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS-
ETP BDD2-
GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT
AE864
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS
YWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL
VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR
AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI
SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL
TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL
NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY
PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM
ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED
TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA
YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ
SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY
RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE
TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV
TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM
AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW
RVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS
INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT
LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD
AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG
VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP
QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAP AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT
Y2332-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG
Thrombin-
AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL AE864
IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT
VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ
TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV
RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG
RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR
PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL
IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS
NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS
GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN
AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK
TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL
GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV
QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF
TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW
YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH
LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP
FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV
DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF
TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM
YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA
LRMEVLGCEAQDLYGLTPRSLLVGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAP AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
MMP-17-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT-
P AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE
KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS
LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG
LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ
IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP
STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY
ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF
KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE
NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT
SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN
KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ
LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ
EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS
LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL
KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI
PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS
LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP
TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN
HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT
ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR
HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL
LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH
HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL
LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH
AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS
WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS
SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN
MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV
KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR
MEVLGCEAQDLYGAPLGLRLRGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS
PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AF144-
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTA
FXIIa-
ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGT
FVIII-
SPSGESSTAPGTMTRIVGGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV
FXIIa-
VYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW
AF864 KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK
DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW
PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI
TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD
SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN
GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH
GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER
DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED
PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL
TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL
LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLL
RQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLR
LNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLF
GKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLT
KDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR
MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQ
RTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSR
NLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQN
VEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRI
SPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKE
KGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSG
VQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTS
GKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATE
SSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINE
GQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFD
IYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD
GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK
NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP
AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD
TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML
PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA
RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY
RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG
MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV
TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL
TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGSTSESPSGTAPGTSPSGESSTAP
GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESP
SGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT
SPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSG
TAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSP
SGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPG
PGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPE
SGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAP
GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA
ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGS
TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAES
PGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTST
PESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPG
PGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
FXIa-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE
KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS
LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG
LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ
IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP
STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY
ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF
KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE
NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT
SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN
KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ
LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ
EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS
LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL
KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI
PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS
LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP
TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN
HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT
ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR
HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL
LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH
HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF
DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL
LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH
AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS
WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS
SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN
MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV
KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR
MEVLGCEAQDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSP
TSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT
FXIa-FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS-
ETP BDD9-
GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT
AE864
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS
YWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL
VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR
AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI
SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL
TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL
NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY
PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM
ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL
EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED
TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA
YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ
SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY
RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE
TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV
TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM
AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW
RVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS
INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT
LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD
AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG
VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP
QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE
TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS
PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAP AE48-
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGKLTRAETGATRRYY
FXIa-FVIII
LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPW
BDD9- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVF
AE864 PGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ
TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK
SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH
QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK
KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF
PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ
IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV
CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC
HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE
ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS
SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF
RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA
YFSDVDLEKDVHSGLIGPLLVCITINTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC
RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV
FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP
LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ
GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLH
PTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGR
SNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ
NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS
EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST
EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA
BDD9- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AG288_2
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYKLTRAETGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP
SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG
SPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA
BDD9- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AG864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS
PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP
SGATGSPGASPGTSSTGSP
FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9
(1- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
745) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AG288_2-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
(1640-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
Y2332)-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
FXIa- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
AG864
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG
TPGSGTASSSPGSSTPSGATGSPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE
DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT
QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV
KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH
GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP
GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS
KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL
HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG
NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME
SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG
VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR
YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS
PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9
(1- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
743) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AG288_2-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
(1638-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
Y2332)-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
FXIa- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
AG864
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASP
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG
SGTASSSPGSSTPSGATGSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE
DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT
QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV
KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH
GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP
GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS
KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL
HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG
NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME
SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG
VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR
YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS
PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP BDD10 (1-
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA 745)
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK
AG288_2-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
(1640-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
Y2332)-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
FXIa-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
AG864 RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS
STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG
TPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE
DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT
QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV
KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH
GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP
GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS
KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL
HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG
NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME
SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG
VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR
YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS
PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS
SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP
GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG
TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP
GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA
BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL
AG288_2
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYKLTRAETGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
ATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG
ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG
FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA
BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG864
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH
SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV
YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP
MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP
PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS
KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA
QDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS
PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG
TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS
PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP
SGATGSPGASPGTSSTGSP FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA
BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE864
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD
SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG
LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL
KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL
WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF
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
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200087379A1).
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
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200087379A1).
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