U.S. patent application number 15/988944 was filed with the patent office on 2018-11-29 for modified antibodies.
The applicant listed for this patent is Janux Therapeutics, Inc.. Invention is credited to Ramesh Bhatt, David Campbell, Steven E. Cwirla, William J. Dower, Blake M. Williams.
Application Number | 20180339043 15/988944 |
Document ID | / |
Family ID | 64395985 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180339043 |
Kind Code |
A1 |
Campbell; David ; et
al. |
November 29, 2018 |
MODIFIED ANTIBODIES
Abstract
Provided herein are modified antibodies, pharmaceutical
compositions thereof, as well as nucleic acids, and methods for
making and discovering the same. The modified antibodies described
herein are modified with a peptide. The peptide binds at or near
the antigen binding site of the antibody at physiological pH, thus
reducing binding affinity of the antibody for a target antigen. At
acidic pH, the binding interaction of the peptide at or near the
antigen binding site is disrupted, thus enabling binding with a
target antigen.
Inventors: |
Campbell; David; (La Jolla,
CA) ; Bhatt; Ramesh; (La Jolla, CA) ; Dower;
William J.; (Menlo Park, CA) ; Cwirla; Steven E.;
(Menlo Park, CA) ; Williams; Blake M.; (Menlo
Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janux Therapeutics, Inc. |
La Jolla |
CA |
US |
|
|
Family ID: |
64395985 |
Appl. No.: |
15/988944 |
Filed: |
May 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62511771 |
May 26, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
A61K 2039/572 20130101; C07K 2319/00 20130101; A61K 47/6803
20170801; A61P 35/00 20180101; C07K 16/2878 20130101; C07K 16/2818
20130101; A61K 39/39558 20130101; A61K 38/02 20130101; A61K 2300/00
20130101; A61K 2039/577 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61K 38/02 20060101
A61K038/02; C07K 16/28 20060101 C07K016/28; A61K 47/68 20060101
A61K047/68 |
Claims
1. A modified antibody comprising a formula: A-L-P wherein A is an
antibody or antibody fragment that binds to a target antigen, P is
a peptide that reduces binding of A to the target antigen at
physiological pH and that does not reduce binding of A to the
target antigen at acidic pH, and L is a linking moiety that
connects A to P at physiological pH and in a tumor microenvironment
and L is bound to A outside an antigen binding site.
2. (canceled)
3. The modified antibody of claim 1, wherein at physiological pH P
is reversibly bound to A at or near the antigen binding site.
4. The modified antibody of claim 1, wherein P inhibits the binding
of A to the target antigen at physiological pH and P does not
inhibit the binding of A to the target antigen at acidic pH.
5. The modified antibody of claim 1, wherein in tissue other than
the tumor microenvironment, P sterically blocks A from binding to
the target antigen.
6. (canceled)
7. The modified antibody of claim 1, wherein the modified antibody
has an increased binding affinity for the target antigen in the
tumor microenvironment compared to the binding affinity of the
modified antibody for the target antigen in a non-tumor
microenvironment.
8. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least one histidine.
9. The modified antibody of claim 8, wherein the histidine forms a
binding interaction at or near the antigen binding site of A at
physiological pH.
10.-19. (canceled)
20. The modified antibody of claim 8, wherein at acidic pH P is
reversibly bound to L.
21. The modified antibody of claim 20, wherein L comprises a
peptide sequence with at least one aspartic acid or glutamic acid,
or a combination thereof.
22. The modified antibody of claim 21, wherein the histidine of P
forms an interaction with the aspartic acid or glutamic acid of
L.
23.-39. (canceled)
40. The modified antibody of claim 1, wherein A is a full length
antibody, a single-chain antibody, an Fab fragment, an Fab'
fragment, an (Fab')2 fragment, an Fv fragment, a divalent single
chain antibody, bispecific antibody, a trispecific antibody, a
tetraspecific antibody, or an antibody drug conjugate.
41.-49. (canceled)
50. The modified antibody of claim 1, wherein the target antigen is
CTLA4 and P comprises an amino acid sequence selected from the
group consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6,
Peptide 10, Peptide 13, Peptide 26, Peptide 14, and Peptide 15.
51.-60. (canceled)
61. The modified antibody of claim 1, wherein the target antigen is
PD-L1 and P comprises an amino acid sequence selected from the
group consisting of Peptide 20, Peptide 27, Peptide 21, Peptide 22,
Peptide 23, Peptide 28, Peptide 29, Peptide 30, Peptide 31, and
Peptide 24.
62.-76. (canceled)
77. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least two histidines.
78. (canceled)
79. The modified antibody of claim 77, wherein P comprises a
peptide sequence with at least two histidines and at least two
cysteines.
80. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least two charged amino acid residues wherein the
charged amino acid residues are selected from the group consisting
of aspartate, glutamate, and histidine.
81. (canceled)
82. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least three charged amino acid residues wherein
the charged amino acid residues are selected from the group
consisting of aspartate, glutamate, and histidine.
83. (canceled)
84. The modified antibody of claim 82, wherein P comprises a
peptide sequence with at least one histidine and at least two
aspartates.
85. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least one cysteine.
86. (canceled)
87. The modified antibody of claim 1, wherein P comprises a peptide
sequence with at least two cysteine amino acid residues.
88. (canceled)
89. The modified antibody of claim 87, wherein P comprises a
peptide sequence with at least two cysteines and at least three
charged amino acid residues wherein the charged amino acid residues
are selected from the group consisting of aspartate, glutamate, and
histidine.
90.-93. (canceled)
94. The modified antibody of claim 1, wherein P does not comprise a
lysine or arginine.
95. The modified antibody of claim 1, wherein P comprises at least
one histidine and at least one aspartate.
96. The modified antibody of claim 1, wherein P comprises at least
one histidine and at least one glutamate.
97. The modified antibody of claim 1, wherein P comprises at least
one histidine and at least two glutamates.
98. The modified antibody of claim 1, wherein P comprises at least
two histidines and at least one aspartate or at least one
glutamate.
99. The modified antibody of claim 1, wherein P comprises at least
one histidine, wherein at least one hydrogen bonding amino acid
residue is within two amino acid positions to the histidine,
wherein the hydrogen bonding amino acid residue is selected from
the group consisting of serine, threonine, tyrosine, asparagine,
and glutamine.
100.-105. (canceled)
106. The modified antibody of claim 1, wherein A-L-P does not
comprise a protease cleavage site that releases A from P in a tumor
microenvironment.
107. The modified antibody of claim 1, wherein L comprises a
peptide sequence with at least one histidine.
108. The modified antibody of claim 107, wherein at acidic pH L is
reversibly bound to P.
109. The modified antibody of claim 108, wherein P comprises a
peptide sequence with at least one aspartic acid or glutamic acid,
or a combination thereof.
110. The modified antibody of claim 109, wherein the histidine of L
forms an interaction with the aspartic acid or glutamic acid of
P.
111. A pharmaceutical composition, comprising: (a) a modified
antibody according to claim 1; and (b) a pharmaceutically
acceptable excipient.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/511,771 filed May 26, 2017 which is incorporated
by reference herein in its entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 24, 2018, is named 52426-701_201_SL.txt and is 73,393bytes
in size.
SUMMARY OF THE INVENTION
[0003] Disclosed herein, in some embodiments, are modified
antibodies comprising a formula: A-L-P wherein A is an antibody or
antibody fragment that binds to a target antigen, P is a peptide
that reduces binding of A to the target antigen at physiological pH
and that does not reduce binding of A to the target antigen at
acidic pH, and L is a linking moiety that connects A to P at
physiological pH and in a tumor microenvironment and L is bound to
A outside an antigen binding site. In some instances, at
physiological pH P is reversibly bound to A through ionic,
electrostatic, hydrophobic, Pi-stacking, and H-bonding
interactions, or a combination thereof. In some instances, at
physiological pH P is reversibly bound to A at or near the antigen
binding site. In some instances, P inhibits the binding of A to the
target antigen at physiological pH and P does not inhibit the
binding of A to the target antigen at acidic pH. In some instances,
in tissue other than the tumor microenvironment, P sterically
blocks A from binding to the target antigen. In some instances, at
the tumor microenvironment, P is removed from the antigen binding
site, and the antigen binding site of A is exposed. In some
instances, the modified antibody has an increased binding affinity
for the target antigen in the tumor microenvironment compared to
the binding affinity of the modified antibody for the target
antigen in a non-tumor microenvironment. In some instances, P
comprises a peptide sequence with at least one histidine. In some
instances, the histidine forms a binding interaction at or near the
antigen binding site of A at physiological pH. In some instances,
at acidic pH P is reversibly bound to A in a region of A that is
not the antigen binding site. In some instances, at acidic pH P is
reversibly bound to A in a region of A that is not the antigen
binding site through ionic, electrostatic, hydrophobic, Pi-stacking
and H-bonding interactions, or a combination thereof. In some
instances, P is resistant to cleavage by a protease. In some
instances, physiological pH is about pH 7.4. In some instances,
acidic pH is about pH 6.0 to about pH 7.0. In some instances, P
comprises a peptide sequence of at least 6 amino acids in length.
In some instances, P comprises a peptide sequence of at least 10
amino acids in length. In some instances, P comprises a peptide
sequence of at least 6 to 20 amino acids in length. In some
instances, P comprises a modified amino acid, a non-natural amino
acid, or a modified non-natural amino acids, or combination
thereof. In some instances, the modified amino acid or modified
non-natural amino acid comprises a post-translational modification.
In some instances, at acidic pH P is reversibly bound to L. In some
instances, L comprises a peptide sequence with at least one
aspartic acid or glutamic acid, or a combination thereof. In some
instances, the histidine of P forms an interaction with the
aspartic acid or glutamic acid of L. In some instances, L is a
peptide sequence having at least 5 to no more than 50 amino acids.
In some instances, L has a formula selected from the group
consisting of: (GS).sub.n, wherein n is an integer from 6 to 20
(SEQ ID NO: 1); (G.sub.2S).sub.n, wherein n is an integer from 4 to
13 (SEQ ID NO: 2); (G.sub.3S).sub.n, wherein n is an integer from 3
to 10 (SEQ ID NO: 3); and (G.sub.4S).sub.n, wherein n is an integer
from 2 to 8 (SEQ ID NO: 4); and (G).sub.n, wherein n is an integer
from 12 to 40 (SEQ ID NO: 5). In some instances, L has a formula
comprising (GGSGGD).sub.n, wherein n is an integer from 2 to 6 (SEQ
ID NO: 8). In some instances, L has a formula comprising
(GGSGGE).sub.n, wherein n is an integer from 2 to 6 (SEQ ID NO: 9).
In some instances, L has a formula comprising (GGGSGSGGGGS).sub.n,
wherein n is an integer from 1 to 3 (SEQ ID NO: 6). In some
instances, L has a formula comprising (GGGGGPGGGGP) .sub.n, wherein
n is an integer from 1 to 3 (SEQ ID NO: 7). In some instances, L
has a formula selected from (GX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 20 (SEQ ID NO: 24); (GGX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 13 (SEQ ID NO: 25); (GGGX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 10 (SEQ ID NO: 26); (GGGGX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 8 (SEQ ID NO: 27); (G.sub.zX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 15, and z is between 1 and 20 (SEQ ID NO: 28). In some
instances, L is resistant to cleavage by a protease. In some
instances, L comprises a modified amino acid. In some instances,
the modified amino acid comprises a post-translational
modification. In some instances, L comprises a non-natural amino
acid or a modified non-natural amino acid, or combination thereof.
In some instances, the modified non-natural amino acid comprises a
post-translational modification. In some instances, the target
antigen is selected from the group consisting of: 4-1BB, CTLA4,
PD-1, and PD-L1. In some instances, the target antigen is 4-1BB. In
some instances, the target antigen is CTLA4. In some instances, the
target antigen is PD-1. In some instances, the target antigen is
PD-L1. In some instances, A is a full length antibody, a
single-chain antibody, an Fab fragment, an Fab' fragment, an
(Fab')2 fragment, an Fv fragment, a divalent single chain antibody,
bispecific antibody, a trispecific antibody, a tetraspecific
antibody, or an antibody drug conjugate. In some instances, A is
selected from the group consisting of utomilumab, urelumab,
ipilimumab, tremelimumab, pembrolizumab, nivolumab, and
atezolizumab. In some instances, A is utomilumab. In some
instances, A is urelumab. In some instances, A is ipilimumab. In
some instances, A is tremelimumab. In some instances, A is
pembrolizumab. In some instances, A is nivolumab. In some
instances, A is atezolizumab. In some instances, P comprises an
amino acid sequence selected from the group consisting of Peptide
1, Peptide 2, Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide
26, Peptide 14, Peptide 15, Peptide 20, Peptide 27, Peptide 21,
Peptide 22, Peptide 23, Peptide 28, Peptide 29, Peptide 30, Peptide
31, and Peptide 24. In some instances, the target antigen is CTLA4
and P comprises an amino acid sequence selected from the group
consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6, Peptide
10, Peptide 13, Peptide 26, Peptide 14, and Peptide 15. In some
instances, the target antigen is CTLA4 and P comprises an amino
acid sequence according to Peptide 1. In some instances, the target
antigen is CTLA4 and P comprises an amino acid sequence according
to Peptide 2. In some instances, the target antigen is CTLA4 and P
comprises an amino acid sequence according to Peptide 5. In some
instances, the target antigen is CTLA4 and P comprises an amino
acid sequence according to Peptide 6. In some instances, the target
antigen is CTLA4 and P comprises an amino acid sequence according
to Peptide 10. In some instances, the target antigen is CTLA4 and P
comprises an amino acid sequence according to Peptide 13. In some
instances, the target antigen is CTLA4 and P comprises an amino
acid sequence according to Peptide 26. In some instances, the
target antigen is CTLA4 and P comprises an amino acid sequence
according to Peptide 14. In some instances, the target antigen is
CTLA4 and P comprises an amino acid sequence according to Peptide
15. In some instances, the target antigen is CTLA4 and P comprises
an amino acid sequence selected from the group consisting of
Peptide 10, Peptide 13, Peptide 26, Peptide 14, and Peptide 15. In
some instances, the target antigen is PD-L1 and P comprises an
amino acid sequence selected from the group consisting of Peptide
20, Peptide 27, Peptide 21, Peptide 22, Peptide 23, Peptide 28,
Peptide 29, Peptide 30, Peptide 31, and Peptide 24. In some
instances, the target antigen is PD-L1 and P comprises an amino
acid sequence according to Peptide 20. In some instances, the
target antigen is PD-L1 and P comprises an amino acid sequence
according to Peptide 27. In some instances, the target antigen is
PD-L1 and P comprises an amino acid sequence according to Peptide
21. In some instances, the target antigen is PD-L1 and P comprises
an amino acid sequence according to Peptide 22. In some instances,
the target antigen is PD-L1 and P comprises an amino acid sequence
according to Peptide 23. In some instances, the target antigen is
PD-L1 and P comprises an amino acid sequence according to Peptide
28. In some instances, the target antigen is PD-L1 and P comprises
an amino acid sequence according to Peptide 29. In some instances,
the target antigen is PD-L1 and P comprises an amino acid sequence
according to Peptide 30. In some instances, the target antigen is
PD-L1 and P comprises an amino acid sequence according to Peptide
31. In some instances, the target antigen is PD-L1 and P comprises
an amino acid sequence according to Peptide 24. In some instances,
the target antigen is PD-L1 and P comprises an amino acid sequence
selected from the group consisting of Peptide 27, Peptide 22,
Peptide 23, and Peptide 31. In some instances, A comprises a kappa
light chain amino acid sequence according to SEQ ID NO: 68. In some
instances, A comprises a heavy chain amino acid sequence according
to SEQ ID NO: 70. In some instances, the modified antibody
comprises an amino acid sequence according to SEQ ID NO: 69. In
some instances, P comprises an amino acid sequence selected from
the group consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6,
Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide
20, Peptide 21, Peptide 22, Peptide 23, Peptide 31, and Peptide 24.
In some instances, P comprises a peptide sequence with at least two
histidines. In some instances, P comprises an amino acid sequence
selected from the group consisting of Peptide 5, Peptide 13,
Peptide 26, Peptide 14, Peptide 15, Peptide 31, and Peptide 24. In
some instances, P comprises a peptide sequence with at least two
histidines and at least two cysteines. In some instances, P
comprises a peptide sequence with at least two charged amino acid
residues wherein the charged amino acid residues are selected from
the group consisting of aspartate, glutamate, and histidine. In
some instances, P comprises an amino acid sequence selected from
the group consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6,
Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide
20, Peptide 27, Peptide 21, Peptide 22, Peptide 23, Peptide 28,
Peptide 29, Peptide 30, Peptide 31, and Peptide 24. In some
instances, P comprises a peptide sequence with at least three
charged amino acid residues wherein the charged amino acid residues
are selected from the group consisting of aspartate, glutamate, and
histidine. In some instances, P comprises an amino acid sequence
selected from the group consisting of Peptide 1, Peptide 2, Peptide
5, Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15,
Peptide 21, Peptide 23, Peptide 28, Peptide 31, and Peptide 24. In
some instances, P comprises a peptide sequence with at least one
histidine and at least two aspartates. In some instances, P
comprises a peptide sequence with at least one cysteine. In some
instances, P comprises an amino acid sequence selected from the
group consisting of Peptide 5, Peptide 6, Peptide 10, Peptide 13,
Peptide 26, Peptide 14, Peptide 15, Peptide 22, Peptide 23, Peptide
29, Peptide 30, Peptide 31, and Peptide 24. In some instances, P
comprises a peptide sequence with at least two cysteine amino acid
residues. In some instances, P comprises an amino acid sequence
selected from the group consisting of Peptide 6, Peptide 10,
Peptide 13, Peptide 26, Peptide 14, Peptide 22, Peptide 23, Peptide
29, Peptide 30, Peptide 31, and Peptide 24. In some instances, P
comprises a peptide sequence with at least two cysteines and at
least three charged amino acid residues wherein the charged amino
acid residues are selected from the group consisting of aspartate,
glutamate, and histidine. In some instances, P comprises an amino
acid sequence of formula GGX, wherein X is cysteine, alanine,
proline, methionine, histidine, or leucine. In some instances, P
comprises an amino acid sequence selected from the group consisting
of Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide 26,
Peptide 14, Peptide 15, Peptide 22, Peptide 23, Peptide 29, Peptide
31, and Peptide 24. In some instances, P comprises an amino acid
sequence of GGC. In some instances, P comprises an amino acid
sequence selected from the group consisting of Peptide 5, Peptide
6, Peptide 13, Peptide 26, Peptide 14, Peptide 31, and Peptide 24.
In some instances, P does not comprise a lysine or arginine. In
some instances, P comprises at least one histidine and at least one
aspartate. In some instances, P comprises at least one histidine
and at least one glutamate. In some instances, P comprises at least
one histidine and at least two glutamates. In some instances, P
comprises at least two histidines and at least one aspartate or at
least one glutamate. In some instances, P comprises at least one
histidine, wherein at least one hydrogen bonding amino acid residue
is within two amino acid positions to the histidine, wherein the
hydrogen bonding amino acid residue is selected from the group
consisting of serine, threonine, tyrosine, asparagine, and
glutamine. In some instances, the hydrogen bonding amino acid
residue is within one amino acid position to the histidine. In some
instances, the hydrogen bonding amino acid residue is serine. In
some instances, the hydrogen bonding amino acid residue is
threonine. In some instances, the hydrogen bonding amino acid
residue is tyrosine. In some instances, the hydrogen bonding amino
acid residue is asparagine. In some instances, the hydrogen bonding
amino acid residue is glutamine. In some instances, A-L-P does not
comprise a protease cleavage site that releases A from P in a tumor
microenvironment. In some instances, L comprises a peptide sequence
with at least one histidine. In some instances, at acidic pH L is
reversibly bound to P. In some instances, P comprises a peptide
sequence with at least one aspartic acid or glutamic acid, or a
combination thereof. In some instances, the histidine of L forms an
interaction with the aspartic acid or glutamic acid of P.
[0004] Disclosed herein, in some embodiments, are pharmaceutical
compositions, comprising: (a) a modified antibody according to any
of the disclosures herein; and (b) a pharmaceutically acceptable
excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0006] FIG. 1 exemplifies an antibody that does not comprise a
peptide modification. Such antibodies bind to unique antigens that
exist in abundance in tumor tissue. But, the unique antigens are
also found in some healthy tissues, which can trigger systemic
immune activation in a subject, and cause toxicity.
[0007] FIG. 2 shows an exemplary modified antibody. In this
example, the modified antibody is linked to a peptide which binds
at or near the antigen binding site of the modified antibody at pH
7.4. This reduces binding of the modified antibody to its target
antigen in healthy tissue. In tumor tissue, the acidic tumor
microenvironment disrupts the interaction of the peptide with the
modified antibody. The antigen binding site of the modified
antibody is exposed, and the modified antibody selectively binds to
its target antigen in tumor tissue.
[0008] FIG. 3 shows an exemplary modified antibody. The peptide is
linked to the antibody via a linking moiety. The linking moiety
creates a stable link between the antibody and peptide. The peptide
prevents the antibody from binding to its target antigen in
physiological pH, non-diseased tissue. A pH switch in tumor
microenvironments modulates the peptide/antibody affinity. The
peptide is released in tumor tissue, and enables the antibody to
bind to its target antigen.
[0009] FIG. 4 shows an exemplary modified antibody. In this
example, the peptide is engineered to contain a histidine. At
physiological pH, the histidine of the peptide interacts with the
antibody binding site. At acidic pH, such as in a tumor
microenvironment, the interaction between the peptide and the
antibody binding site is disfavored because the histidine is
protonated. The antibody binding site is available for binding to
its target antigen in a tumor microenvironment.
[0010] FIG. 5 is an exemplary schematic of phage panning screening
platform to identify pH responsive peptide candidates.
[0011] FIG. 6 illustrates a phagemid ELISA of a collection of
enriched clones resulting from three rounds of biopanning against
anti-mouse CTLA-4 (clone 9D9).
[0012] FIG. 7 is an exemplary phagemid competition ELISA from a
collection of enriched clones isolated after three rounds of
biopanning against anti-mouse CTLA-4 (clone 9D9).
[0013] FIG. 8 is an exemplary pH-dependent "binding" assay of a
collection of enriched clones isolated after three rounds of
biopanning against anti-mouse CTLA-4 (clone 9D9).
[0014] FIG. 9 illustrates a pH-dependent "dissociation" assay of a
collection of enriched clones isolated after three rounds of
biopanning against anti-mouse CTLA-4 (clone 9D9).
[0015] FIG. 10A-FIG. 10B illustrate pH responsive anti-CTLA4
peptide candidates identified with phage panning screening
platform. FIG. 10A illustrates peptide candidates identified that
exhibit pH dependent binding to anti-CTLA4. FIG. 10B illustrates
peptide candidates identified that exhibit pH dependent
dissociation to anti-CTLA4.
[0016] FIG. 11A-FIG. 11C illustrate significant pH responsiveness
of unoptimized peptide candidates Peptide 5 (FIG. 11A), Peptide 6
(FIG. 11B) and Peptide 10 (FIG. 11C).
[0017] FIG. 12 illustrates multiple pH response anti-CTLA4 peptide
candidates with pH dependent dissociation identified from biased
library.
[0018] FIG. 13A-FIG. 13B illustrate pH biased library generated
peptide candidates Peptide 14 (FIG. 13A) and Peptide 15 (FIG.
13B).
[0019] FIG. 14 illustrates a Peptide 10 ELISA to anti-mouse
antibody (clone 9D9).
[0020] FIG. 15 illustrates a Peptide 10 competition ELISA.
[0021] FIG. 16A-FIG. 16C illustrate octet binding curve of Peptide
15 (FIG. 16A), Peptide 17 (FIG. 16C) and Peptide 18 (FIG. 16C).
[0022] FIG. 17 illustrates pH-dependent binding of a-mCTLA-4 Fab to
Peptide 15 by ELISA.
[0023] FIG. 18 illustrates CTLA4 antibody/ligand competition
ELISA.
[0024] FIG. 19 illustrates a phagemid ELISA of a collection of
enriched clones resulting from biopanning against PD-L1
antibody.
[0025] FIG. 20 illustrates anti-PD-L1 phage competition ELISA.
[0026] FIG. 21 illustrates pH-dependent phagemid PD-L1 antibody
binding ELISA.
[0027] FIG. 22 illustrates pH-dependent phagemid PD-L1 antibody
dissociation ELISA.
[0028] FIG. 23 illustrates Peptide 23 PD-L1 antibody binding.
[0029] FIG. 24 illustrates Peptide 23 PD-L1 competition ELISA.
[0030] FIG. 25 illustrates PD-L1 antibody/ligand competition
ELISA.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Protein-based therapies, including antibody therapies, are
effective treatments for a variety of diseases. A strategy to
improve toxicity and side effects of such treatments is to engineer
a peptide that binds to the protein-based therapy at physiological
pH, but does not bind to the protein-based therapy at acidic pH.
While peptides have been shown to bind to antibodies with varying
affinities, peptides which bind to antibodies in a pH dependent
manner are not known. These pH-dependent peptides enable the
protein-based therapy to be activated in certain acidic
microenvironments while not affecting healthy tissues.
Certain Definitions
[0032] The terminology used herein is for the purpose of describing
particular cases only and is not intended to be limiting. As used
herein, the singular forms "a", "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise. Furthermore, to the extent that the terms
"including", "includes", "having", "has", "with", or variants
thereof are used in either the detailed description and/or the
claims, such terms are intended to be inclusive in a manner similar
to the term "comprising."
[0033] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, e.g., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the given value.
Where particular values are described in the application and
claims, unless otherwise stated the term "about" should be assumed
to mean an acceptable error range for the particular value.
[0034] As used herein, "physiological pH" is used to refer to the
pH of a non-diseased state cellular environment. In some
embodiments, physiological pH is greater than pH 6.9. In some
embodiments, physiological pH is about pH 7.0 to about pH 8.0. In
some embodiments, physiological pH is about 7.4.
[0035] Described herein are modified antibodies, pharmaceutical
compositions thereof, as well as nucleic acids, and methods for
discovering the same.
[0036] The modified antibodies described herein are connected by a
linking moiety to a peptide. The peptide is designed to reduce
binding of the modified antibody to its target antigen when at
physiological pH. At acidic pH, for example at a tumor
microenvironment, the peptide does not reduce binding of the
modified antibody to its target antigen. The peptide is designed to
activate the modified antibody at tumor microenvironments, thus
improving the safety profile of such therapies. While
antibody-based therapies have proven effective for some diseases in
some cases, there is a need for increased targeting of antibodies
to the disease site to reduce systemic-based toxicities.
[0037] Disclosed herein, in certain embodiments, are modified
antibodies comprising a formula:
A-L-P
wherein A is an antibody or antibody fragment that binds to a
target antigen, P is a peptide that reduces binding of A to the
target antigen at physiological pH, and that does not reduce
binding of A to the target antigen at acidic pH, and L is a linking
moiety that connects A to P at physiological pH and in a tumor
microenvironment and L is bound to A outside an antigen binding
site.
Peptide (P)
[0038] The peptide of the modified antibodies, in some embodiments,
reversibly binds to A in such a way that P sterically blocks,
inhibits, or reduces the binding of affinity of A for its target
antigen at physiological pH. In some embodiments, P reversibly
binds to A through ionic, electrostatic, hydrophobic, Pi-stacking,
or H-bonding interactions, or a combination thereof. In some
embodiments, P binds to the antigen binding site of A at
physiological pH. In some embodiments, P binds to A at amino acid
residues which are near the antigen binding site of A. In some
embodiments, P binds to amino acid residues within the antigen
binding site.
[0039] In some embodiments, at acidic pH, P is not reversibly bound
to the antigen binding site of A. In some embodiments, at acidic
pH, P is not reversibly bound to amino acid residues near the
antigen binding site of A. The peptide activates the modified
antibody at acidic pH by exposing the antigen binding site of A for
engagement with its respective target antigen. In some cases, P has
a different conformation at acidic pH, compared to the conformation
of P at physiological pH. In some embodiments, at acidic pH, P does
not form any interactions with A. In some embodiments, at acidic
pH, P does not form any interactions with the linking moiety (L).
In some embodiments, at acidic pH, P forms an interaction with L.
In some embodiments, P and L reversibly bind through ionic,
electrostatic, hydrophobic, Pi-stacking, or H-bonding interactions,
or a combination thereof.
[0040] In some cases, P comprises a peptide sequence. In some
cases, P comprises a peptide sequence disclosed in Table 1. In some
cases, P comprises a peptide sequence at least 80% identical to a
peptide sequence disclosed in Table 1. In some cases, P comprises a
peptide sequence at least 90% identical to a peptide sequence
disclosed in Table 1. In some cases, P comprises a peptide sequence
at least 95% identical to a peptide sequence disclosed in Table 1.
In some cases, the peptides disclosed herein are linear peptides.
In some cases, the peptides disclosed herein are cyclic
peptides.
TABLE-US-00001 TABLE 1 Peptide Sequences (P) SEQ Peptide ID
Sequence ID NO CTLA4 peptides Peptide 1 TLDDMSHVILYA 29 Peptide 2
VISDNHQIVWDL 30 Peptide 3 LTTQDHPLTILL 31 Peptide 4
GGWICHWLEPQEACTY 32 Peptide 5 GGCFEEHEQLVFQTHC 33 Peptide 6
GGCILPGQHESQAIAC 34 Peptide 7 GGCLSQMDFHDWLQYC 35 Peptide 8
GGTDCYLWDYKASCHQ 36 Peptide 9 GGKCDSLSYWQEIECS 37 Peptide 10
GGADCLLHDWDSACQI 38 Peptide 11 MQNVDEAPPLLL 39 Peptide 12
TNDWQGLLLNVF 40 Peptide 13 GGCQDSMFHHPNHC 41 Peptide 26
GGCGMHQHPLFVDC 42 Peptide 14 GGCSLSQHPNHSDC 43 Peptide 15
GGPCNQVECHHQFT 44 Peptide 16 GGCPSLHPQWIHVC 45 Peptide 17
GGCFDSANQHPNIVIC 46 Peptide 18 GGCHQDIEMPIYWC 47 Peptide 19
GGCQIHDPHTWHLC 48 PD-L1 peptides Peptide 20 QLFYPSTYHIID 49 Peptide
27 QVSPLYFYEELA 50 Peptide 21 HQALLDFYGDY 51 Peptide 22
GGMCHELFYSNLNWCQ 52 Peptide 23 GGHCVDMVDFYQQTCQ 53 Peptide 28
VDLLDGSLQDFY 54 Peptide 29 GGLCSTFYEPQVDICY 55 Peptide 30
SDFSGLLFYDYQ 56 Peptide 31 GGCVHFFHHQRPDC 57 Peptide 24
GGCHNKSGLFYHYC 58 Peptide 25 GGCFYPGHHHQLLC 59
[0041] In some embodiments, P comprises an amino acid sequence
selected from the group consisting of Peptide 1, Peptide 2, Peptide
5, Peptide 6, Peptide 10, Peptide 13, Peptide 26, Peptide 14,
Peptide 15, Peptide 20, Peptide 27, Peptide 21, Peptide 22, Peptide
23, Peptide 28, Peptide 29, Peptide 30, Peptide 31, and Peptide 24.
In some embodiments, P comprises an amino acid sequence selected
from the group consisting of Peptide 10, Peptide 13, Peptide 26,
Peptide 14, Peptide 15, Peptide 27, Peptide 22, Peptide 23, and
Peptide 31. In some embodiments, P comprises an amino acid sequence
selected from the group consisting of Peptide 15 and Peptide 23. In
some embodiments, P comprises an amino acid sequence of Peptide 15.
In some embodiments, P comprises an amino acid sequence of Peptide
23.
[0042] In some embodiments, P is designed to incorporate amino acid
residues which cause a conformational change when triggered by an
environmental change. In some cases, the environmental change is
the difference in pH from normal, healthy tissue to an acidic pH
that is found at tumor microenvironments.
[0043] Cancer cells in a solid tumor are able to form a tumor
microenvironment in their surroundings to support the growth and
metastasis of the cancer cells. A tumor microenvironment is often
hypoxic. As the tumor mass increases, the interior of the tumor
grows farther away from existing blood supply, which leads to
difficulties in fully supplying oxygen to the tumor
microenvironment. As a result, the tumor cells tend to rely on
energy generated from lactic acid fermentation, which does not
require oxygen. As a consequence of using lactic acid fermentation
is that the tumor microenvironment is acidic (approximately pH
6.0-6.9) in contrast to other parts of the body which are typically
either neutral or slightly basic. For example, human blood plasma
has a pH of about 7.4.
[0044] In some embodiments, P contains at least one histidine
residue. In some embodiments, at physiological pH, at least one
histidine residue of P forms a binding interaction with at least
one amino acid residue of the antigen binding site of A. In some
embodiments, at physiological pH, at least one histidine residue of
P forms a binding interaction with at least one amino acid residue
that is near the antigen binding site of A. In some embodiments, at
acidic pH, at least one histidine residue of P forms a binding
interaction with a glutamic acid or aspartic acid located on L.
[0045] In some embodiments, P contains more than one histidine
residue. In some embodiments, P contains at least two histidine
residues. In some embodiments, at physiological pH, at least two
histidine residues of P form a binding interaction with an amino
acid residue of the antigen binding site of A. In some embodiments,
at physiological pH, at least two histidine residues of P form a
binding interaction with an amino acid residue that is near the
antigen binding site of A. In some embodiments, at physiological
pH, at least two histidine residues of P form binding interactions
with amino acid residues that are at or near the antigen binding
site of A. In some embodiments, at acidic pH, at least two
histidine residues of P form a binding interaction with a glutamic
acid or aspartic acid located on L.
[0046] In some embodiments, P contains at least two charged amino
acid residues. In some embodiments, P contains at least three
charged amino acid residues. In some embodiments, the charged amino
acid residues are selected from the group consisting of lysine,
arginine, histidine, aspartate, and glutamate. In some embodiments,
the charged amino acid residues are selected from the group
consisting of glutamate, histidine, and aspartate. In some
embodiments, P contains at least glutamate and histidine. In some
embodiments, P contains histidine and aspartate. In some
embodiments, P contains aspartate and glutamate. In some
embodiments, P contains at least one histidine and at least two
aspartate residues. In some embodiments, P contains at least two
histidine and at least one glutamate residues. In some embodiments,
P contains at least one histidine and at least two glutamate
residues. In some embodiments, P contains at least two histidine
and at least one aspartate residues.
[0047] In some embodiments, P contains at least two polar amino
acid residues. In some embodiments, P contains at least three polar
amino acids. In some embodiments, P contains at least four polar
amino acids. In some embodiments, the polar amino acid residues are
selected from the group consisting of serine, threonine, cysteine,
asparagine, glutamine, and tyrosine. In some embodiments, P
contains at least one glutamine. In some embodiments, P contains at
least two glutamines. In some embodiments, P contains at least one
glutamine and at least one serine. In some embodiments, P contains
at least one glutamine and at least one cysteine. In some
embodiments, P contains at least one glutamine and at least one
asparagine. In some embodiments, P contains at least two glutamines
and at least one threonine. In some embodiments, P contains at
least two glutamines, and at least one asparagine. In some
embodiments, P contains at least two glutamines, at least one
asparagine, and at least one cysteine. In some embodiments, P
contains at least two glutamines, at least one asparagine, at least
one cysteine, and at least one threonine.
[0048] In some embodiments, P contains at least one cysteine. In
some embodiments, P contains at least two cysteines. In some
embodiments, P contains at least three cysteines.
[0049] In some embodiments, P contains at least one glutamine and
at least one methionine. In some embodiments, P contains at least
two glutamines and at least one methionine. In some embodiments, P
contains at least two glutamines at least one methionine, and at
least one threonine.
[0050] In some embodiments, P contains at least one charged amino
acid residue and at least one polar amino acid residue. In some
embodiments, P contains at least one charged amino acid residue and
at least two polar amino acid residues. In some embodiments, P
contains at least one charged amino acid residue and at least three
polar amino acid residues. In some embodiments, P contains at least
two charged amino acid residues and at least one polar amino acid
residues. In some embodiments, P contains at least two charged
amino acid residues and at least two polar amino acid residues. In
some embodiments, P contains at least two charged amino acid
residues and at least three polar amino acid residues. In some
embodiments, P contains at least two charged amino acid residues
and at least four polar amino acid residues. In some embodiments, P
contains at least three charged amino acid residues and at least
one polar amino acid residues. In some embodiments, P contains at
least three charged amino acid residues and at least two polar
amino acid residues. In some embodiments, P contains at least three
charged amino acid residues and at least three polar amino acid
residues. In some embodiments, P contains at least two histidine
and at least two cysteine residues. In some embodiments, P contains
at least three charged amino acid residues and at least two
cysteines. In some embodiments, P contains at least one histidine,
at least one serine, and at least one glutamine. In some
embodiments, P contains at least one histidine, at least one
asparagine, and at least one glutamine.
[0051] In some embodiments, P comprises the sequence of GGX,
wherein X is cysteine, alanine, proline, methionine, histidine, or
leucine. In some embodiments, P comprises the sequence GGC. In some
embodiments, P comprises the sequence GGP. In some embodiments, P
comprises the sequence GGA. In some embodiments, P comprises the
sequence GGP. In some embodiments, P comprises the sequence GGM. In
some embodiments, P comprises the sequence GGH. In some
embodiments, P comprises the sequence GGL.
[0052] In some embodiments, P does not contain a tryptophan
residue. In some embodiments, P does not contain an arginine
residue. In some embodiments, P does not contain a lysine residue.
In some embodiments, P does not contain a lysine residue or an
arginine residue. In some embodiments, P does not contain a lysine
residue, an arginine residue, or a tryptophan residue.
[0053] In some embodiments, P comprises at least one histidine,
wherein at least one hydrogen bonding amino acid residue is within
two amino acid positions to the histidine. In some embodiments, the
hydrogen bonding amino acid residue is serine, threonine, tyrosine,
asparagine, glutamine or a combination thereof.
[0054] In some embodiments, the hydrogen bonding amino acid residue
is within one amino acid position to the histidine. In some
embodiments, serine is within one amino acid position to the
histidine. In some embodiments, threonine is within one amino acid
position to the histidine. In some embodiments, tyrosine is within
one amino acid position to the histidine. In some embodiments,
asparagine is within one amino acid position to the histidine. In
some embodiments, glutamine is within one amino acid position to
the histidine.
[0055] In some embodiments, the hydrogen bonding amino acid residue
is two amino acid positions away from the histidine. In some
embodiments, serine is two amino acid positions away from the
histidine. In some embodiments, threonine is two amino acid
positions away from the histidine. In some embodiments, tyrosine is
two amino acid positions away from the histidine. In some
embodiments, asparagine is two amino acid positions away from the
histidine. In some embodiments, glutamine is two amino acid
positions away from the histidine.
[0056] In some embodiments, P contains at least one aromatic amino
acid residue. In some embodiments, P contains at least two aromatic
amino acid residues. In some embodiments, P contains phenylalanine
and tyrosine. In some embodiments, the phenylalanine and the
tyrosine are next to each other in the amino acid sequence of P. In
some embodiments, P contains two phenylalanines. In some
embodiments, the phenylalanines are next to each other in the amino
acid sequence of P.
[0057] In some embodiments, P is a peptide sequence at least 5
amino acids in length. In some embodiments, P is a peptide sequence
at least 6 amino acids in length. In some embodiments, P is a
peptide sequence at least 10 amino acids in length. In some
embodiments, P is a peptide sequence at least 20 amino acids in
length. In some embodiments, P is resistant to cleavage by a
protease.
[0058] In some embodiments, P is not a natural binding partner of
A. In some embodiments, P does not comprise a mimotope. In some
embodiments, P contains a random amino acid sequence that does not
share any sequence homology to the natural binding partner of A. It
is advantageous to use sequences that do not share any sequence
homology to the natural binding partner of A to allow for greater
flexibility in the peptide design. This allows for building a
larger library of candidate peptide sequences for screening. In
some instances, P is a modified binding partner for A which
contains amino acid changes that at least slightly decrease
affinity and/or avidity of binding to A. In some embodiments, P
contains no or substantially no homology to A's natural binding
partner. In some embodiments, P is no more than 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%
similar to the natural binding partner of A.
[0059] In some embodiments, P comprises a modified amino acid or
non-natural amino acid, or a modified non-natural amino acid, or a
combination thereof. In some embodiments, the modified amino acid
or a modified non-natural amino acid comprises a post-translational
modification. In some embodiments P comprises a modification
including, but not limited to acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cystine, formation of pyroglutamate, formylation, gamma
carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic
processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
Modifications are made anywhere to P including the peptide
backbone, the amino acid side chains, and the terminus.
Linking Moiety (L)
[0060] In some embodiments, L is a peptide sequence of at least 5
amino acid residues. In some embodiments, L is a peptide sequence
of no more than 50 amino acids. Regarding the amino acid
composition of L, peptides are selected with properties that confer
flexibility and facilitate a conformational change of P during a
change in pH.
[0061] In some embodiments, L comprises a peptide sequence with at
least one histidine. In some embodiments, at acidic pH L is
reversibly bound to P. In some embodiments, P comprises a peptide
sequence with at least one aspartic acid or glutamic acid, or a
combination thereof. In some embodiments, the histidine of L forms
an interaction with the aspartic acid or glutamic acid of P.
[0062] In some embodiments, L is resistant to protease cleavage.
For example, glycine and serine residues generally provide protease
resistance. Examples of suitable linking moieties for connecting A
or C to P include, but are not limited to (GS).sub.n, wherein n is
an integer from 6 to 20 (SEQ ID NO: 1); (G.sub.2S).sub.n, wherein n
is an integer from 4 to 13 (SEQ ID NO: 2); (G.sub.3S).sub.n,
wherein n is an integer from 3 to 10 (SEQ ID NO: 3); and
(G.sub.4S).sub.n, wherein n is an integer from 2 to 8 (SEQ ID NO:
4); and (G).sub.n, wherein n is an integer from 12 to 40 (SEQ ID
NO: 5). Additional examples include, but are not limited to,
(GGGSGSGGGGS).sub.n, wherein n is an integer from 1 to 3 (SEQ ID
NO: 6), or (GGGGGPGGGGP).sub.n, wherein n is an integer from 1 to 3
(SEQ ID NO: 7).
[0063] In some embodiments, L binds to P at acidic pH, for example,
at a tumor microenvironment. In some embodiments, at acidic pH, L
is reversibly bound to P. In some embodiments, the interaction of L
and P at acidic pH is mediated through a histidine residue on P and
a glutamic acid or aspartic acid residue on L. Examples of such
linking moieties include, but are not limited to, (GGSGGD).sub.n,
wherein n is an integer from 2 to 6 (SEQ ID NO: 8); or
(GGSGGE).sub.n, wherein n is an integer from 2 to 6 (SEQ ID NO:
9).
[0064] In some embodiments, L has a formula comprising,
(GS).sub.x(GGSGGD).sub.y (GS).sub.z, wherein x is an integer from 0
to 20, y is an integer from 2 to 6, and z is an integer from 0 to
20 (SEQ ID NO: 10). In some embodiments, L has a formula
comprising, (G.sub.2S).sub.x(GGSGGD).sub.y (G.sub.2S).sub.z,
wherein x is an integer from 0 to 13, y is an integer from 2 to 6,
and z is an integer from 0 to 13 (SEQ ID NO: 11). In some
embodiments, L has a formula comprising,
(G.sub.3S).sub.x(GGSGGD).sub.y (G.sub.3S).sub.z, wherein x is an
integer from 0 to 10, y is an integer from 2 to 6, and z is an
integer from 0 to 10 (SEQ ID NO: 12). In some embodiments, L has a
formula comprising, (G.sub.4S).sub.x(GGSGGD).sub.y
(G.sub.4S).sub.z, wherein x is an integer from 0 to 8, y is an
integer from 2 to 6, and z is an integer from 0 to 8 (SEQ ID NO:
13). In some embodiments, L has a formula comprising,
(G).sub.x(GGSGGD).sub.y (G).sub.z, wherein x is an integer from 0
to 40, y is an integer from 2 to 6, and z is an integer from 0 to
40 (SEQ ID NO: 14). In some embodiments, L has a formula
comprising, (GGGSGSGGGGS).sub.x(GGSGGD).sub.y (GGGSGSGGGGS).sub.z,
wherein x is an integer from 0 to 3, y is an integer from 2 to 6,
and z is an integer from 0 to 3 (SEQ ID NO: 15). In some
embodiments, L has a formula comprising,
(GGGSGSGGGGP).sub.x(GGSGGD).sub.y (GGGSGSGGGGP).sub.z, wherein x is
an integer from 0 to 3, y is an integer from 2 to 6, and z is an
integer from 0 to 3 (SEQ ID NO: 16).
[0065] In some embodiments, L has a formula comprising,
(GS).sub.x(GGSGGE).sub.y(GS).sub.z, wherein x is an integer from 0
to 20, y is an integer from 2 to 6, and z is an integer from 0 to
20 (SEQ ID NO: 17). In some embodiments, L has a formula
comprising, (G.sub.2S).sub.x(GGSGGE).sub.y (G.sub.2S).sub.z,
wherein x is an integer from 0 to 13, y is an integer from 2 to 6,
and z is an integer from 0 to 13 (SEQ ID NO: 18). In some
embodiments, L has a formula comprising,
(G.sub.3S).sub.x(GGSGGE).sub.y (G.sub.3S).sub.z, wherein x is an
integer from 0 to 10, y is an integer from 2 to 6, and z is an
integer from 0 to 10 (SEQ ID NO: 19). In some embodiments, L has a
formula comprising, (G.sub.4S).sub.x(GGSGGE).sub.y
(G.sub.4S).sub.z, wherein x is an integer from 0 to 8, y is an
integer from 2 to 6, and z is an integer from 0 to 8 (SEQ ID NO:
20). In some embodiments, L has a formula comprising,
(G).sub.x(GGSGGE).sub.y(G).sub.z, wherein x is an integer from 0 to
40, y is an integer from 2 to 6, and z is an integer from 0 to 40
(SEQ ID NO: 21). In some embodiments, L has a formula comprising,
(GGGSGSGGGGS).sub.x(GGSGGE).sub.y (GGGSGSGGGGS).sub.z, wherein x is
an integer from 0 to 3, y is an integer from 2 to 6, and z is an
integer from 0 to 3 (SEQ ID NO: 22). In some embodiments, L has a
formula comprising, (GGGSGSGGGGP).sub.x(GGSGGE).sub.y
(GGGSGSGGGGP).sub.z, wherein x is an integer from 0 to 3, y is an
integer from 2 to 6, and z is an integer from 0 to 3 (SEQ ID NO:
23).
[0066] Additional examples of linking moieties include, but are not
limited to wherein L has a formula selected from (GX).sub.n,
wherein X is serine, aspartic acid, glutamic acid, threonine, or
proline and n is at least 20 (SEQ ID NO: 24); (GGX).sub.n, wherein
X is serine, aspartic acid, glutamic acid, threonine, or proline
and n is at least 13 (SEQ ID NO: 25); (GGGX).sub.n, wherein X is
serine, aspartic acid, glutamic acid, threonine, or proline and n
is at least 10 (SEQ ID NO: 26); (GGGGX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 8 (SEQ ID NO: 27); (G.sub.zX).sub.n, wherein X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 15, and z is between 1 and 20 (SEQ ID NO: 28).
[0067] In some embodiments, L comprises a modified amino acid or
non-natural amino acid, or a modified non-natural amino acid, or a
combination thereof. In some embodiments, the modified amino acid
or a modified non-natural amino acid comprises a post-translational
modification. In some embodiments L comprises a modification
including, but not limited, to acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cystine, formation of pyroglutamate, formylation, gamma
carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic
processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
Modifications are made anywhere to L including the peptide
backbone, or the amino acid side chains.
Antibody or Antibody Fragments (A)
[0068] In some embodiments, A is a full length antibody, a
single-chain antibody, a Fab Fragment, an Fab' fragment, an (Fab')2
fragment, an Fv fragment, a divalent single chain antibody, a
bispecific antibody, trispecific antibody, tetraspecific antibody,
or an antibody drug conjugate.
[0069] In some embodiments A is an antagonist, agonist,
conditionally active antibody, or a sweeping body.
[0070] In some embodiments, A is an antibody or antibody fragment
including, but not limited to, utomilumab, urelumab, ipilimumab,
tremelimumab, pembrolizumab, nivolumab, and atezolizumab. In some
embodiments, A is utomilumab. In some embodiments, A is urelumab.
In some embodiments, A is ipilimumab. In some embodiments, A is
tremelimumab. In some embodiments, A is pembrolizumab. In some
embodiments, A is nivolumab. In some embodiments, A is
atezolizumab.
[0071] In some embodiments, A binds to a target antigen. In some
embodiments, the target antigen includes, but is not limited to,
4-1BB, CTLA4, PD-1, and PD-L1. In some embodiments, the target
antigen is 4-1BB. In some embodiments, the target antigen is CTLA4.
In some embodiments, the target antigen is PD-1. In some
embodiments, the target antigen is PD-L1.
[0072] In some embodiments, A contains a modification so as to
increase the bioavailability, improve stability, or solubility of
the modified antibody. In some embodiments, A is conjugated to
polyethylene glycol, polysialic acid (PSA), HPMA copolymer,
dextran, albumin, a glycosyl group or a combination thereof.
[0073] In some embodiments, A comprises a modified amino acid or
non-natural amino acid, or a modified non-natural amino acid, or a
combination thereof. In some embodiments, the modified amino acid
or a modified non-natural amino acid comprises a post-translational
modification. In some embodiments A comprises a modification
including, but not limited to acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cystine, formation of pyroglutamate, formylation, gamma
carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic
processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
Modifications are made anywhere to A including the peptide
backbone, the amino acid side chains, or the termini or
terminus.
Modified Antibodies (A-L-P)
[0074] Disclosed herein, in certain embodiments, are modified
antibodies comprising a formula:
A-L-P
wherein A is an antibody or antibody fragment that binds to a
target antigen, P is a peptide that reduces binding of A to the
target antigen at physiological pH, and that does not reduce
binding of A to the target antigen at acidic pH, and L is a linking
moiety that connects A to P at physiological pH and in a tumor
microenvironment, and L is bound to A outside an antigen binding
site.
[0075] In some embodiments, A-L-P does not comprise a protease
cleavage site that releases A from P in a tumor microenvironment.
It is advantageous that L and P are not cleaved from the molecule
so that P can bind and re-bind to A depending upon the
microenvironment. For example, in a tumor microenvironment, P is
not bound to A thereby exposing the antigen binding site of A to
its target antigen. However, because P is not cleaved from the
molecule in the tumor microenvironment, if A-L-P were to then
diffuse into a non-tumor microenvironment, P can then resume
binding interactions with A, thereby preventing A from interacting
with its target antigen in the non-tumor microenvironment.
[0076] In some embodiments, the modified antibodies disclosed
herein comprise more than one P, one for each region of the
antibody that contains the antigen binding site, connected to A by
L. In some instances, L for each of the Ps is the same. In some
instances, L for each of the P is different. In some embodiments,
the Ps for each region of the antibody that contains the antigen
binding site is the same. In some embodiments, the Ps for each
region of the antibody that contains the antigen binding site is
different. In some embodiments, the modified antibodies disclosed
herein comprise one P.
[0077] In some embodiments, P is not a natural binding partner of
A. In some embodiments, P does not comprise a mimotope. In some
embodiments, P contains a random amino acid sequence that does not
share any sequence homology to the natural binding partner of A. It
is advantageous to use sequences that do not share any sequence
homology to the natural binding part of A to allow for greater
flexibility in the peptide design. This allows for building a
larger library of candidate peptide sequences for screening.
[0078] In some embodiments, the target antigen to which the
antibody or antibody fragment (A) binds is CTLA4, peptide (P) is
Peptide 15, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 10, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 13, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 26, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 14, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 1, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 2, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 5, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is CTLA4, peptide (P)
is Peptide 6, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60).
[0079] In some embodiments, the antibody or antibody fragment (A)
that binds to a target antigen is Atezolizumab, peptide (P) is
Peptide 23, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the antibody or antibody
fragment (A) that binds to a target antigen is Atezolizumab,
peptide (P) is Peptide 27, and linking moiety (L) is
GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60). In some embodiments,
the antibody or antibody fragment (A) that binds to a target
antigen is Atezolizumab, peptide (P) is Peptide 22, and linking
moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60). In some
embodiments, the antibody or antibody fragment (A) that binds to a
target antigen is Atezolizumab, peptide (P) is Peptide 31, and
linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60).
In some embodiments, the antibody or antibody fragment (A) that
binds to a target antigen is Atezolizumab, peptide (P) is Peptide
20, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID
NO: 60). In some embodiments, the antibody or antibody fragment (A)
that binds to a target antigen is Atezolizumab, peptide (P) is
Peptide 21, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the antibody or antibody
fragment (A) that binds to a target antigen is Atezolizumab,
peptide (P) is Peptide 28, and linking moiety (L) is
GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60). In some embodiments,
the antibody or antibody fragment (A) that binds to a target
antigen is Atezolizumab, peptide (P) is Peptide 29, and linking
moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60). In some
embodiments, the antibody or antibody fragment (A) that binds to a
target antigen is Atezolizumab, peptide (P) is Peptide 30, and
linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 60).
In some embodiments, the antibody or antibody fragment (A) that
binds to a target antigen is Atezolizumab, peptide (P) is Peptide
24, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID
NO: 60).
[0080] In some embodiments, the target antigen to which the
antibody or antibody fragment (A) binds is PD-L1, peptide (P) is
Peptide 23, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 27, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 22, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 31, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 20, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 21, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 28, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 29, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 30, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60). In some embodiments, the target antigen to which
the antibody or antibody fragment (A) binds is PD-L1, peptide (P)
is Peptide 24, and linking moiety (L) is GSSGGSGGSGGSGGGSGGGSGGSSGT
(SEQ ID NO: 60).
Polynucleotides Encoding Modified Antibodies
[0081] Also provided, in some embodiments, are polynucleotide
molecules encoding a modified antibody described herein. In some
embodiments, the polynucleotide molecules are provided as a DNA
construct. In other embodiments, the polynucleotide molecules are
provided as a messenger RNA transcript.
[0082] The polynucleotide molecules are constructed by known
methods such as by combining the genes encoding the domains either
separated by peptide linkers or, in other embodiments, directly
linked by a peptide bond, into a single genetic construct operably
linked to a suitable promoter, and optionally a suitable
transcription terminator, and expressing it in bacteria or other
appropriate expression system such as, for example CHO cells.
Depending on the vector system and host utilized, any number of
suitable transcription and translation elements, including
constitutive and inducible promoters, may be used. The promoter is
selected such that it drives the expression of the polynucleotide
in the respective host cell.
[0083] In some embodiments, the polynucleotide is inserted into a
vector, preferably an expression vector, which represents a further
embodiment. This recombinant vector can be constructed according to
known methods. Vectors of particular interest include plasmids,
phagemids, phage derivatives, virii (e.g., retroviruses,
adenoviruses, adeno-associated viruses, herpes viruses,
lentiviruses, and the like), and cosmids.
[0084] A variety of expression vector/host systems may be utilized
to contain and express the polynucleotide encoding the polypeptide
of the described antigen-binding protein. Examples of expression
vectors for expression in E. coli are pSKK (Le Gall et al., J
Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for
expression in mammalian cells.
[0085] Thus, the modified antibodies as described herein, in some
embodiments, are produced by introducing a vector encoding the
protein as described above into a host cell and culturing said host
cell under conditions whereby the protein domains are expressed,
may be isolated and, optionally, further purified.
Pharmaceutical Compositions
[0086] Also provided, in some embodiments, are pharmaceutical
compositions comprising a modified antibody described herein, a
vector comprising the polynucleotide encoding the polypeptide of
the modified antibody or a host cell transformed by this vector and
at least one pharmaceutically acceptable carrier. The term
"pharmaceutically acceptable carrier" includes, but is not limited
to, any carrier that does not interfere with the effectiveness of
the biological activity of the ingredients and that is not toxic to
the patient to whom it is administered. Examples of suitable
pharmaceutical carriers include phosphate buffered saline
solutions, water, emulsions, such as oil/water emulsions, various
types of wetting agents, sterile solutions etc. Such carriers can
be formulated by conventional methods and can be administered to
the subject at a suitable dose. Preferably, the compositions are
sterile. These compositions may also contain adjuvants such as
preservative, emulsifying agents and dispersing agents. Prevention
of the action of microorganisms may be ensured by the inclusion of
various antibacterial and antifungal agents.
[0087] In some embodiments of the pharmaceutical compositions, the
modified antibody described herein is encapsulated in
nanoparticles. In some embodiments, the nanoparticles are
fullerenes, liquid crystals, liposome, quantum dots,
superparamagnetic nanoparticles, dendrimers, or nanorods. In other
embodiments of the pharmaceutical compositions, the modified
antibody, is attached to liposomes. In some instances, the modified
antibody is conjugated to the surface of liposomes. In some
instances, the modified antibody is encapsulated within the shell
of a liposome. In some instances, the liposome is a cationic
liposome.
[0088] The modified antibodies described herein are contemplated
for use as a medicament. Administration is effected by different
ways, e.g. by intravenous, intraperitoneal, subcutaneous,
intramuscular, topical or intradermal administration. In some
embodiments, the route of administration depends on the kind of
therapy and the kind of compound contained in the pharmaceutical
composition. The dosage regimen will be determined by the attending
physician and other clinical factors. Dosages for any one patient
depends on many factors, including the patient's size, body surface
area, age, sex, the particular compound to be administered, time
and route of administration, the kind of therapy, general health
and other drugs being administered concurrently. An "effective
dose" refers to amounts of the active ingredient that are
sufficient to affect the course and the severity of the disease,
leading to the reduction or remission of such pathology and may be
determined using known methods.
Methods for Discovering Modified Antibodies that are Conditionally
Active at Tumor Microenvironments
[0089] Disclosed herein, in some embodiments, are methods for
identifying peptides which reduce binding of the modified antibody
to its target antigen when at physiological pH but do not affect
target antigen binding at acidic pH.
[0090] In some embodiments, a modified antibody is tested for
binding to a target antigen at a range of pH, from about pH 3 to
about pH 12. In a further aspect, the testing step further includes
testing for antigen binding within a pH range from about pH 5 to pH
10. In a further aspect, the testing step includes testing for
antigen binding within a pH range from about pH 6 to pH 8. In a
further aspect, the testing step further includes testing for
antigen binding within a pH range from about pH 6.7 to pH 7.5.
[0091] In additional embodiments, P is identified by directed
evolution techniques. In some embodiments, P is engineered to
introduce one, two, or more ionizable groups which interact at
protein-protein interfaces. Such ionizable groups, for example,
interact with A at the antigen binding site or near the antigen
binding site at physiological pH to block binding of A to its
target antigen. Such ionizable groups, for example, are engineered
into P using a histidine scanning library approach.
Methods for Generating Modified Antibodies
[0092] Methods for generating an antibody (or fragment thereof) for
a given target are known in the art. The structure of antibodies
and fragments thereof, variable regions of heavy and light chains
of an antibody (VH and VL) Fv, F(ab')2, Fab fragments, single chain
antibodies (scAb), single chain variable regions (scFv),
complementarity determining regions (CDR), and domain antibodies
(dAbs) are well understood. Methods for generating a polypeptide
having a desired antigen-binding domain of a target antigen are
known in the art.
[0093] Methods for modifying antibodies or antibody fragments to
couple additional polypeptides are known in the art. For example,
peptides and linking moieties are coupled to modify antibodies to
generate the modified antibodies of the disclosure.
[0094] In some embodiments, A, L, and P are expressed in a nucleic
acid construct. In some embodiments, A, L, and P are expressed in
the same nucleic acid construct. In some instances, the nucleic
acid constructs include, but are not limited to, constructs which
are capable of expression in a prokaryotic or eukaryotic cell.
[0095] In some embodiments, P is coupled to L and A through
covalent binding. For example, A and L are expressed as a single
transcript, and the terminal residue of L is a cysteine. P is then
coupled to L and A by a cysteine-cysteine disulfide bridge.
EXAMPLES
Example 1
Screening of Candidate Peptides
[0096] To identify peptides for conjugation to or expression with
an antibody of interest, a library of candidate peptides are
generated. The candidate peptides have variable amino acid
sequences and variable amino acid lengths. The candidate peptides
are then screened for their ability to bind to the antibody of
interest at pH 7.4 and at pH 6.0. Those candidate peptides that
bind to the antibody of interest at pH 6.0 are eliminated.
Candidate peptides that bind to the antibody of interest at pH 7.4,
but not at pH 6.0, are sequenced and motifs are analyzed.
Example 2
Screening of Modified Antibody Libraries
[0097] Libraries of candidate modified antibodies having different
amino acid sequences in the peptide and linker lengths and various
points of attachment to the antibody are generated.
[0098] The libraries are introduced via expression vectors
resulting in display of the modified antibodies on the surface of
bacterial cells. After expansion of the libraries by culture, cells
displaying the modified antibodies are tested for their abilities
to bind to target antigens at pH 7.4 and at pH 6.0. Cells are
contacted with fluorescently labeled target antigen and the cells
are sorted by FACs to isolate those cells which can bind to the
fluorescently labeled target antigen at pH 6.0, but not at pH 7.4.
The cells can be subjected to additional cycles by expansion by
growth in culture and again by subjecting the culture to all or
part of the screening steps.
Example 3
In Vitro Screening of a Modified Antibody for pH Dependent Binding
Affinity for its Target Antigen
[0099] An immunoabsorbant target displacement assay (TDA) is
described herein for discovery and validation of a modified
antibody for selective binding to its target antigen at an acidic
pH. In the TDA assay, the ability of a modified antibody to bind to
its target antigen is measured. The assay is conducted at pH 7.4,
and then repeated at pH 6.0.
[0100] Briefly, the target antigen is adsorbed to the wells of an
ELISA plate overnight at 4.degree. C. The plate is blocked by
addition of 2% non-fat dry milk in PBS, about 0.5% (v/v) Tween20
(PBST), and incubation at room temperature for about 1 hour. The
plate is then washed about three times with PBST. About 50 .mu.l of
superblock is added. About 50 .mu.l of the modified antibody is
dissolved in superblock and incubated at about 37.degree. C. for
different periods of time. The plate is washed about three times
with PBST. About 100 ml of anti-huIgG-HRP is added in about 2%
NEM/PBST and incubated at room temperature for about 1 hour. The
plate is washed about four times with PBST and about twice with
PBS. The assay is developed using TMB (Thermo Scientific) as per
manufacturer's instructions.
Example 4
Cytotoxicity Assay for Assessing pH Dependent Selectivity of
Modified Antibodies
[0101] Modified antibodies are selected which demonstrate binding
to its target antigen at pH 6.0, but with minimal or no binding to
its target antigen at pH 7.4 conditions. A candidate antibody, and
a peptide-modified candidate antibody validated in Example 3, is
tested in a cell based cytotoxicity assay.
[0102] Cell Culture: A549 (human epithelial cell line derived from
a lung carcinoma tissue), or an alternative cancer cell line
(DU145, LNCaP, or PC-3 cells) can be obtained from, for example,
the ATCC. Human umbilical vein endothelial cells (HUVEC) can be
isolated from human umbilical veins as described. (Grant et al.,
"Matrigel induces thymosin h 4 gene in differentiating endothelial
cells", J Cell Sci 1995; 108:3685-94). HUVEC cells can be used as a
positive control as a cell line that express ATP synthase on the
cell surface. Cells can be cultured in DMEM (Life Technologies,
Carlsbad, Calif.) with 1% penicillin streptomycin and 10% serum
replacement medium 3 (Sigma, St. Louis, Mo.) to minimize the
presence of plasminogen. Low-pH (6.7) medium can be prepared by
reducing bicarbonate to 10 mmol/L at 5% CO2 and supplementing with
34 mmol/L NaCl to maintain osmolality or incubation of 22 mmol/L
bicarbonate medium under 17% CO2 conditions. The method of lowering
pH used can be varied by experimental constraints and assay.
[0103] Flow cytometry: To assure ATP synthase is functional on the
cell surface of the tumor cell line, flow cytometry experiments can
be performed. For example, A549 Cell lines can be cultured in
varying pH medium (10, 22, and 44 mmol/L bicarbonate DMEM), under
hypoxia (0.5% 02, 5% CO2, N2 balanced) versus normoxia (21% O2, 5%
CO2) for 0, 12, 24, 48, and 72 hours. Live cells can be blocked,
incubated with anti-.beta.-subunit antibody, washed, blocked,
incubated with a secondary goat anti-rabbit antibody-FITC (Southern
Biotech, Birmingham, Ala.), and again washed, with all steps
performed at 4 degrees C. Propidium iodide (BD Biosciences, San
Jose, Calif.) can be included with all samples to discriminate
cells with compromised membranes. The mean fluorescent intensity of
FITC in 10,000 cells can be quantified by FACSCalibur flow
cytometer (Becton Dickinson, Franklin Lakes, N.J.) and cells with
propidium iodide uptake can be excluded to eliminate detection of
mitochondrial ATP synthase on CELLQuest software (BD
Biosciences).
[0104] Cell surface ATP generation assay: A549 or 1-LN cells
(60,000 per well) in 96-well plates can be refreshed with medium
and treated with a candidate antibody, a peptide-modified candidate
antibody, anti-beta-subunit antibody, rabbit IgG raised to bovine
serum albumin (Organon Teknika, West Chester, Pa.), piceatannol (a
known inhibitor of ATP synthase F1 used as a positive control,
Sigma), or medium alone for 30 minutes at 37 degrees C., 5% CO2.
Cells can be then incubated with 0.05 mmol/L ADP for 20 seconds.
Supernatants can be removed and assayed for ATP production by
CellTiterGlo luminescence assay (Promega, Madison, Wis.) as
described (23). Cell lysates can be similarly analyzed to confirm
that intracellular pools of ATP did not vary under any conditions.
Recordings can be made on the Luminoskan Ascent (Thermo Labsystems,
Helsinki, Finland). Data are expressed in moles of ATP per cell
based on standards determined for each independent experiment.
[0105] Cell proliferation assay: The effect of the candidate
modified antibody on cancer cell lines can be assessed with a
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium, inner salt (MTS) proliferation assay in serum-free
medium. Relative cell numbers in each well of a 96-well microplate
after incubation for 20 hours, 37 degrees C., and 5% CO.sub.2 in
the presence or absence of the candidate antibody can be determined
using the AQueous One Cell Proliferation Assay (Promega) per
protocol of the manufacturer. Medium pH can be regulated at 5%
CO.sub.2 through bicarbonate concentration.
[0106] Assessment of cellular cytotoxicity: To quantify cell death
and cell lysis, the activity of lactate dehydrogenase (LDH)
released from the cytosol into supernatant can be measured with the
Cytotoxicity Detection kit (Roche, Indianapolis, Ind.). Cancer
cells (e.g. A549 cells) (5,000 per well) treated with a candidate
antibody, a peptide-modified candidate antibody, anti-beta-subunit
antibody, rabbit IgG, cariporide, and Triton X (a detergent used to
permeabilize cells as a positive control) can be incubated at 37
degrees C. and 5% CO.sub.2 or 17% CO.sub.2 for 15 hours at neutral
and low pH conditions, respectively. An index of cytotoxicity can
be calculated by dividing the average absorbance from treated
samples in quadruplicate by the average absorbance from untreated
samples in quadruplicate corresponding to the same pH medium.
[0107] Assessment of cellular necrosis and apoptosis: To determine
the mode of candidate antibody induced cell death a histone-DNA
ELISA can be performed. The effects of a candidate antibody, a
peptide-modified candidate antibody, anti-beta-subunit antibody,
rabbit IgG, and cariporide on A549 cells (5,000 per well) can be
determined using an ELISA apoptosis and necrosis assay (Roche) that
is dependent on detection of extranuclear histone-DNA fragments.
Apoptosis or necrosis can be determined from, respectively, the
cell lysates or supernatants of quadruplicate samples after 15
hours of incubation at 37 degrees C., in the presence or absence of
agents. The apoptotic or necrotic indices can be calculated by
dividing the average absorbance from treated samples in
quadruplicate by the average absorbance from untreated samples in
quadruplicate corresponding to the same pH medium. Medium pH can be
regulated by incubation at 5% CO2 or 17% CO.sub.2.
Example 5
Biopanning
[0108] Biopanning with m13 phagemid p8 or p3 displayed peptide
libraries was either performed with Fc immobilized anti-mouse
CTLA-4 antibody (clone 9D9) on 96-well ELISA plates or with
biotin-conjugated antibody immobilized on streptavidin coated
paramagnetic beads. Following binding to target and washing steps,
specifically bound phage were recovered by elution at pH 2.2 and in
some instances recovery occurred through the use of acidic buffers
of higher pH levels. Enrichment of specific binding clones was
accomplished by 3-4 rounds of successive biopanning and
amplification. After 3 or 4 rounds of biopanning phage pools were
infected into TG1 cells and plated out on LB-ampicillin/agar plates
for clonal isolation and subsequent characterization.
Example 6
Phagemid Hit Identification ELISA
[0109] For hit identification, individual colonies were grown in
96-deep well plates for 2-4 hours and infected with helper phage to
produce peptide displayed phagemid following an overnight growth.
The next day the deep well plates were centrifuged to separate the
soluble phagemid from the E. coli cells. The phagemid containing
supernatants were then combined with PBS-Tween 20 (0.05%)+BSA (1%)
pH neutral blocking buffer and incubated in previously antibody
(anti-mouse CTLA-4, clone 9D9) coated and blocked wells. After
binding at 4 degrees the plates were washed and specifically bound
phage were detected by anti-m13 HRP conjugated antibodies using
standard TMB-based chromogenic ELISA procedures. Daughter plates or
individual wells were subjected to standard DNA sequencing for
peptide identification.
Example 7
Phagemid Competition ELISA Assay
[0110] Phagemid peptide clones were next tested to determine
whether they bound within the antigen binding space of the
antibody, by target-based competition assay. Antibody (anti-mouse
CTLA-4, clone 9D9) immobilized and blocked 96-well ELISA plates
similar to above were prepared. Mouse CTLA-4 was added to the well
to block the antigen binding site. After a brief incubation period
phagemid supernatants were added to the wells. Following an
incubation at 4 degrees the plates were washed and specifically
bound phage were detected by anti-m13 HRP conjugated antibodies
using standard TMB-based chromogenic ELISA procedures. Phagemid
clones binding within the antigenic binding pocket would be blocked
and be identified by a decreased ELISA signal, compared to a well
lacking previous antigen blockade.
Example 8
Phagemid pH "Binding" ELISA
[0111] To assess the pH dependent binding attributes of each of the
individual clones, an ELISA similar to that described above was
performed, but instead of combining the supernatants into pH
neutral buffers they were combined with acidic pH buffered blocking
buffer to achieve acidic pH levels such as pH 5.4 and then
transferred into antibody (anti-mouse CTLA-4, clone 9D9) coated and
blocked wells. Following incubation at 4 degrees the plates were
washed and then all subsequent steps buffers used were neutral pH.
As before, the specifically bound phage were similarly detected by
anti-m13 HRP conjugated antibodies using standard TMB-based
chromogenic ELISA procedures.
[0112] Table 2 below shows the ELISA results for pH-dependent
phagemid 9D9 Antibody binding.
TABLE-US-00002 TABLE 2 pH-dependent phagemid 9D9 Antibody binding
ELISA. ELISA Plate ELISA signal signal Ratio wells pH 7.2 pH 5.4
5.4:7.2 A07 2.173 1.546 0.712 B07 0.567 0.177 0.312 C07 1.700 0.799
0.470 D07 1.753 1.136 0.648 E07 0.501 0.219 0.436 F07 0.477 0.054
0.113 G07 0.738 0.062 0.084 H07 1.687 1.096 0.650 A08 1.616 0.215
0.133 B08 0.125 0.045 0.359 C08 1.956 1.222 0.625 D08 1.004 0.278
0.276 E08 1.153 0.119 0.103 F08 2.415 1.651 0.684 G08 2.291 2.037
0.889 H08 1.099 0.539 0.491 A09 0.837 0.329 0.392 B09 2.283 1.836
0.804 C09 1.352 0.220 0.162 D09 1.055 0.260 0.247 E09 0.538 0.059
0.110 F09 0.949 0.264 0.278 G09 1.266 0.516 0.408 H09 0.422 0.066
0.156 A10 1.088 0.304 0.279 B10 1.147 0.495 0.431 C10 1.530 0.234
0.153 D10 1.842 1.105 0.600 E10 0.844 0.276 0.327 F10 2.154 1.645
0.764 G10 1.034 0.375 0.363 H10 0.425 0.174 0.409 A11 0.626 0.217
0.347 B11 0.851 0.306 0.360 C11 0.861 0.181 0.211 D11 1.358 0.570
0.420 E11 0.990 0.430 0.434 F11 1.699 0.958 0.564 G11 0.053 0.049
0.915 H11 2.369 2.110 0.891 A12 0.518 0.257 0.496 B12 0.331 0.148
0.447 C12 0.295 0.049 0.168 D12 1.103 0.208 0.189 E12 1.263 0.246
0.195 F12 0.593 0.283 0.478 G12 1.064 0.432 0.406 H12 3.031 1.749
0.577
Example 9
Phagemid "Dissociation" Assay
[0113] To determine pH dependent dissociation attributes of the
individual phagemid displayed peptides, an ELISA similar to that
described above was performed. Phagemid supernatants were combined
with neutral pH PBS-Tween 20, BSA containing blocking buffer and
then transferred into antibody (anti-mouse CTLA-4, clone 9D9)
coated and blocked wells. After binding at 4 degrees the plates
were washed extensively. In the next step the phage were incubated
with either neutral or acidic pH buffers, such as pH 5.4 for
approximately 30 minutes and then washed again at neutral pH. As
described previously the specifically bound phage were similarly
detected by anti-m13 HRP conjugated antibodies using standard
TMB-based chromogenic ELISA procedures.
[0114] Table 3 below shows the ELISA results for pH-dependent
phagemid 9D9 Antibody dissociation.
TABLE-US-00003 TABLE 3 pH-dependent phagemid 9D9 Antibody
dissociation ELISA. ELISA signal ELISA signal Plate wells pH 7.2 pH
5.4 Ratio 5.4:7.2 A07 1.996 2.054 1.029 B07 0.578 0.377 0.652 C07
1.681 1.487 0.885 D07 1.851 1.775 0.959 E07 0.474 0.444 0.937 F07
0.400 0.063 0.156 G07 0.633 0.072 0.113 H07 1.611 1.541 0.956 A08
1.449 0.494 0.341 B08 0.113 0.043 0.386 C08 1.948 1.818 0.933 D08
0.947 0.575 0.607 E08 1.068 0.182 0.170 F08 2.421 1.986 0.820 G08
2.305 2.250 0.976 H08 1.007 0.980 0.974 A09 0.747 0.689 0.922 B09
2.372 2.129 0.897 C09 1.302 0.388 0.298 D09 0.987 0.617 0.625 E09
0.477 0.072 0.150 F09 0.866 0.556 0.642 G09 1.169 1.022 0.874 H09
0.302 0.054 0.179 A10 0.964 0.542 0.562 B10 1.250 0.833 0.666 C10
1.507 0.346 0.230 D10 1.896 1.815 0.957 E10 0.755 0.524 0.694 F10
2.184 2.180 0.998 G10 0.984 0.918 0.933 H10 0.371 0.354 0.955 A11
0.562 0.468 0.831 B11 0.928 0.714 0.770 C11 0.651 0.467 0.717 D11
1.383 0.978 0.707 E11 0.912 0.919 1.008 F11 1.581 1.595 1.009 G11
0.049 0.042 0.862 H11 2.382 2.320 0.974 A12 0.474 0.457 0.964 B12
0.380 0.278 0.732 C12 0.283 0.048 0.169 D12 1.055 0.266 0.252 E12
1.159 0.363 0.313 F12 0.505 0.490 0.969 G12 0.857 0.745 0.870 H12
2.366 0.184 0.078
Example 10
Peptide Antibody Dissociation
[0115] To determine pH dependent antibody dissociation attributes
of the individual peptides identified through biopanning described
above, the corresponding biotinylated peptides were chemically
synthesized an ELISA similar to the phagemid antibody binding
example described above was performed. To begin, peptides were
immobilized on neutravidin coated and blocked wells. After unbound
peptides were washed away, dilution series of anti-mouse CTLA-4
antibody (clone 9D9) were added to corresponding wells of peptides.
After binding at 4 degrees the plates were washed extensively as
before. Next the peptides were incubated with either neutral or
acidic pH buffers for approximately 30 minutes and then washed
again. The specifically peptide bound antibodies were detected by
either anti-mouse Fc or anti-mouse kappa chain HRP conjugated
antibodies using standard TMB-based chromogenic ELISA procedures.
The effects of pH upon affinity were determined by standard EC50
analysis of each of the distinct pH dilution series binding curves
and comparison of their respective EC50 values.
[0116] Similar approach was taken to determine pH-dependent
antibody dissociation attributes of the individual peptides to
PD-L1 antibody.
TABLE-US-00004 TABLE 4 pH-dependent CTLA-4 antibody-peptide
dissociation. SEQ ID EC50 [M] EC50 [M] pH 6.0: pH 7.2 Peptide
sequence NO pH 7.2 pH 6.0 EC 50 ratio 1 TLDDMSHVILYA 29 2.9E-09
1.9E-08 6.7 2 VISDNHQIVWDL 30 1.8E-09 9.5E-09 5.2 3 LTTQDHPLTILL 31
2.2E-09 7.9E-09 3.5 4 GGWICHWLEPQEACTY 32 1.9E-09 4.1E-09 2.1 5
GGCFEEHEQLVFQTHC 33 2.5E-10 2.5E-09 9.6 6 GGCILPGQHESQAIAC 34
1.2E-10 1.7E-09 15.0 7 GGCLSQMDFHDWLQYC 35 7.8E-09 2.5E-08 3.2 8
GGTDCYLWDYKASCHQ 36 2.4E-09 5.3E-09 2.2 9 GGKCDSLSYWQEIECS 37
5.9E-09 1.4E-08 2.4 10 GGADCLLHDWDSACQI 38 5.5E-11 8.7E-10 15.8 11
MQNVDEAPPLLL 39 7.0E-09 1.1E-08 1.5 12 TNDWQGLLLNVF 40 4.0E-09
5.1E-09 1.3 13 GGCQDSMFHHPNHC 41 7.4E-09 3.6E-08 4.9 26
GGCGMHQHPLFVDC 42 1.6E-10 3.3E-09 20.9 14 GGCSLSQHPNHSDC 43 4.6E-10
4.1E-09 8.9 15 PCNQVECHHQFT 61 4.3E-10 5.4E-09 12.4 23
GGHCVDMVDFYQQTCQ 53 Inactive -- -- (Control)
TABLE-US-00005 TABLE 5 pH-dependent PD-L1 antibody-peptide
dissociation. SEQ pH 6.0: pH pH 5.4: pH ID EC50 [M] EC50 [M] EC50
[M] 7.2 EC 50 7.2 EC 50 Peptide sequence NO pH 7.2 pH 6.0 pH 5.4
ratio ratio 20 QLFYPSTYHIID 49 1.3E-10 2.1E-10 8.4E-10 1.7 6.7 27
QVSPLYFYEELA 50 4.2E-10 9.0E-10 3.3E-09 2.2 7.8 21 HQALLDFYGDY 51
7.5E-10 1.8E-09 4.8E-09 2.5 6.4 22 GGMCHELFYSNLNWCQ 52 4.5E-11
7.0E-11 3.5E-10 1.5 7.8 23 GGHCVDMVDFYQQTCQ 53 3.0E-10 1.3E-09
3.9E-09 4.3 12.9 28 VDLLDGSLQDFY 54 1.8E-09 4.7E-09 9.1E-09 2.6 5.0
29 GGLCSTFYEPQVDICY 55 2.3E-10 4.9E-10 1.5E-09 2.1 6.5 30
SDFSGLLFYDYQ 56 9.5E-10 2.2E-09 5.6E-09 2.3 5.9 31 GGCVHFFEIHQRPDC
57 1.6E-10 3.6E-10 1.5E-09 2.3 9.4 24 GGCHNKSGLFYHYC 58 2.0E-10
5.6E-10 1.3E-09 2.8 6.6 10 GGADCLLHDWDSACQI 38 Inactive -- -- -- --
(Control)
Example 11
Peptide Antibody Competition
[0117] To determine whether the peptides could inhibit mouse CTLA-4
from binding the 9D9 antibody a competition binding assay was
utilized. Briefly, 9D9 antibody was preincubated against a dilution
series of peptides. Following this incubation the peptide-antibody
complexes were transferred to mouse CTLA-4 coated and blocked ELISA
plates and incubated further. Following a brief binding period the
plates were washed and target bound antibody was detected by either
anti-mouse Fc or anti-mouse kappa chain HRP conjugated antibodies
using standard TMB-based chromogenic ELISA procedures. The effects
of competition were determined by standard IC50 analysis.
Example 12
pH-dependent Binding Analysis by ForteBio Octet Bio-Layer
Interferometry (BLI)
[0118] To determine pH-dependent binding properties of peptides to
mouse anti-CTLA-4 antibody, clone 9D9, monovalent antibody Fab
fragment (mCTLA-4 Fab) was used on a ForteBio Octet Red96 System.
Peptides with C-terminal biotinylation were captured on ForteBio
High Precision Streptavidin (SAX) biosensors at 0.4 .mu.g/ml in 50
mM sodium phosphate buffer, pH 7.4 with 150 mM NaCl, 0.1% BSA and
0.02% Tween-20 (pH 7.4 Assay Buffer).
[0119] For analysis of neutral pH binding, after loading, sensors
were base-lined in pH 7.4 Assay Buffer followed by association with
mCTLA-4 Fab (2-fold dilution series starting at 2.5 .mu.M) in pH
7.4 Assay Buffer and dissociation in pH 7.4 Assay Buffer.
[0120] For analysis of acidic pH binding, after loading, sensors
were base-lined in 50 mM sodium phosphate buffer, pH 6.0 with 150
mM NaCl, 0.1% BSA and 0.02% Tween-20 (pH 6.0 Assay Buffer) followed
by association with mCTLA-4 Fab (2-fold dilution series starting at
2.5 .mu.M) in pH 6.0 Assay Buffer and dissociation in pH 6.0 Assay
Buffer.
[0121] Affinity measurements were determined by ForteBio Octet
analysis software using global fitting.
Example 13
Anti-mCTLA-4 (Clone 9D9) Fab Binding to Peptide 15 by ELISA
[0122] Binding of anti-mouse CTLA-4 (clone 9D9) (Bio X Cell, Cat
#BE0164) Fab fragment to Peptide 15 was determined by ELISA in
96-well plate. Briefly, 100 nM Peptide 15 with C-terminal biotin
was captured on Neutravidin coated plates for 1 hour in PBS-T (50
mM phosphate buffered saline pH 7.4+0.05% Tween-20)+0.5% BSA. After
washing in PBS-T, mCTLA-4 Fab diluted in either pH 7.4 Binding
Buffer (50 mM sodium phosphate+150 mM NaCl+0.5% BSA+0.05% Tween-20,
pH 7.4) or pH 6.0 Binding Buffer (50 mM sodium phosphate+150 mM
NaCl+0.5% BSA+0.05% Tween-20, pH 6.0) was captured for 1 hour.
After capture, wells were washed with either pH 7.4 Binding Buffer
or pH 6.0 Binding Buffer, followed by additional 5-minute
incubation with the appropriate pH Binding Buffer. Detection was
performed with HRP conjugated goat anti-mouse IgG H+L (Southern
Biotech.) for 1 hour at 1/2000 dilution in PBS-T+0.5% BSA followed
by TMB (3-minute development) with acid stop. Absorbance at 450 nm
was determined and data analysis was performed in GraphPad Prism by
nonlinear regression with 4-parameter logistic curve. Data is
expressed as mean+SD. All ELISA steps were performed at room
temperature.
Example 14
Mammalian Expression of Reformatted Antibody Peptide Fusions
[0123] This example outlines a way to reformat peptides found above
into recombinant antibody fusions. Antibodies are comprised of a
full length heavy chain framework complexed with an antibody light
chain. The heavy chain used is either a mouse Fc gamma 2a or mouse
Fc gamma 2b isotype. The full length heavy chain and light chain
sequences for 9D9 (anti-CTLA-4, clone 9D9, Curran et. al) are
synthesized for expression in mammalian cells. Additional light
chain constructs with 9D9 binding peptides fused by flexible
linkers are similarly synthesized. The individual antibody light
chain constructs are co-transfected along with the heavy chain
expression vector in mammalian cells. Specifically the proteins are
transiently produced in HEK293 suspension cell-based systems. The
resulting proteins are harvested from the media after 5-7 days and
FPLC purified to >95% purity via Protein A chromatography.
Proteins are dialyzed and exchanged into pH 7.4 PBS, sterile
filtered, quantitated by A280 absorbance, and stored either at 4
degrees or -80 degrees for longer term storage.
[0124] Listed below are exemplary antibody sequences comprising
light chain with the peptide and heavy chain sequences.
TABLE-US-00006 Antibody-peptide fusion Isotype Light Chain +
peptide Heavy Chain 9D9 + peptide 10 Mouse IgG2b Peptide 10-9D9
light chain 9D9 heavy chain (mouse fusion (mouse kappa) IgG2b) 9D9
+ peptide 13 Mouse IgG2b Peptide 13-9D9 light chain 9D9 heavy chain
(mouse fusion (mouse kappa) IgG2b) 9D9 + peptide 14 Mouse IgG2b
Peptide 14-9D9 light chain 9D9 heavy chain (mouse fusion (mouse
kappa) IgG2b) 9D9 + peptide 15 Mouse IgG2b Peptide 15-9D9 light
chain 9D9 heavy chain (mouse fusion (mouse kappa) IgG2b) 9D9 +
peptide 10 Mouse IgG2a Peptide 10-9D9 light chain 9D9 heavy chain
(mouse fusion (mouse kappa) IgG2a) 9D9 + peptide 13 Mouse IgG2a
Peptide 13-9D9 light chain 9D9 heavy chain (mouse fusion (mouse
kappa) IgG2a) 9D9 + peptide 14 Mouse IgG2a Peptide 14-9D9 light
chain 9D9 heavy chain (mouse fusion (mouse kappa) IgG2a) 9D9 +
peptide 15 Mouse IgG2a Peptide 15-9D9 light chain 9D9 heavy chain
(mouse fusion (mouse kappa) IgG2a) Atezolizumab + peptide 23 Human
IgG1 Peptide 23-Atezolizumab light Atezolizumab heavy chain chain
fusion (human kappa) (human IgG1)
TABLE-US-00007 9D9 light chain (mouse kappa) (SEQ ID NO: 62)
DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGV
PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPS
SEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT
KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Peptide 10-9D9 light chain fusion
(mouse kappa) (SEQ ID NO: 63)
GGADCLLHDWDSACQIGSSGGSGGSGGSGGGSGGGSGGSSGTDIVMTQTTLSLPVSLGDQ
ASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
VEAEDLGVYYCFQGSHVPYTEGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNE
YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK
TSTSPIVKSFNRNEC Peptide 13-9D9 light chain fusion (mouse kappa)
(SEQ ID NO: 64)
GGCQDSMFHHPNHCGSSGGSGGSGGSGGGSGGGSGGSSGTDIVMTQTTLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV
EAEDLGVYYCFQGSHVPYTEGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNEY
PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS
TSPIVKSFNRNEC Peptide 14-9D9 light chain fusion (mouse kappa) (SEQ
ID NO: 65)
GGCSLSQHPNHSDCGSSGGSGGSGGSGGGSGGGSGGSSGTDIVMTQTTLSLPVSLGDQASI
SCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDLGVYYCFQGSHVPYTEGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNEYP
KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTST
SPIVKSFNRNEC Peptide 15-9D9 light chain fusion (mouse kappa) (SEQ
ID NO: 66)
GGPCNQVECHHQFTGSSGGSGGSGGSGGGSGGGSGGSSGTDIVMTQTTLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV
EAEDLGVYYCFQGSHVPYTEGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNEY
PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS
TSPIVKSFNRNEC 9D9 heavy chain (mouse IgG2b) (SEQ ID NO: 67)
EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSY
NQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKT
TPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLY
TMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPN
LEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTH
REDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYIL
PPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKL
DIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK Atezolizumab light chain
(human kappa) (SEQ ID NO: 68)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKWYSASFLYSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATEGQGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSSPVTKSFNRGEC Peptide 23-Atezolizumab light chain
fusion (human kappa) (SEQ ID NO: 69)
GGHCVDMVDFYQQTCQGSSGGSGGSGGSGGGSGGGSGGSSGTDIQMTQSPSSLSASVGD
RVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC Atezolizumab heavy chain (human IgG1) (SEQ ID NO: 70)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Example 15
In Vitro Cell Based pH Dependent Binding of Masked 9D9
Antibodies
[0125] Masked 9D9 antibody binding is evaluated in isogenic 293
cells that stably express mouse CTLA-4 (mCTLA-4). Briefly, parent
Flp-In 293 cells that harbor a single FRT recombination site are
expanded in complete culture medium (DMEM GlutaMax 10% FBS) prior
to transfection. Once sufficient parent cells are grown, cells are
transfected with two vectors, one encoded the Flp recombinase and
the other encoded mCTLA-4 plus a hygromycin B resistance gene.
Cells successfully transfected are selected using hygromycin B.
After several days of growth in the presence of hygromycin B,
cellular foci are picked and expanded. Expanded foci are tested for
mCTLA-4 expression via flow cytometry using the unmasked 9D9
antibody. Cells that express mCTLA-4 (mCTLA-4 293) are expanded
further and cryopreserved.
[0126] mCTLA-4_293 cells are then used to evaluate pH dependent
binding of masked 9D9 antibodies. mCTLA-4_293 cells are grown to
50%-75% confluence, washed, incubated in buffered EDTA, and gently
scraped from the culture dish surface. Parent 293 cells are
expanded in parallel and used as controls. Cells are transferred to
a 15 mL falcon tube, spun down, and washed 3 times. Cells are
washed with complete culture medium in the absence of bicarb,
supplemented with MES and adjusted to pH 6.0, or supplemented with
HEPES and adjusted to pH 7.4. Cell are concentrated then incubated
with unmasked or masked 9D9 antibodies for 1 h at RT in complete
culture medium plus MES pH 6.0 or HEPES pH 7.4. Cells are washed 3
times in complete culture medium plus MES pH 6.0 or HEPES pH 7.4.
Cells are then fixed in buffered formalin for 15 min RT and washed
3 times in PBS pH 7.4. Cells are concentrated then incubated with
secondary AlexaFluor 555 anti-mouse antibody for 1 hr at RT in PBS
5% BSA 0.1% tween. Cells are washed 3 times with PBS 0.1% tween and
analyzed by flow cytometry.
Example 16
In Vivo Efficacy Evaluation of Masked 9D9 Antibodies
[0127] Efficacy of masked 9D9 antibodies is evaluated relative to
parent 9D9 in C57BL/6 or BALB/c mice bearing syngeneic MC38 tumor
xenografts. Briefly, mice are subcutaneously injected with 2
million MC38 tumor cells, respectively. Tumor volumes are recorded
3 times weekly. Mice bearing palpable tumors (50-75 mm.sup.3) are
randomized into treatment groups with comparable mean tumor
volumes. Antibodies formulated in PBS pH 7.4 are administered twice
weekly intraperitoneally (IP) 200 .mu.g per dose in a volume of 200
.mu.L (10 mg/kg IP b.i.w). Matched isotype antibodies are used as
negative controls. Percent tumor growth inhibition (TGI %) is
determined after two weeks of dosing for antibody treatment groups
relative to isotype controls.
Example 17
Tumor Actuated Binding of Masked 9D9 Antibodies Using In Vivo Near
Infrared Imaging
[0128] Selective tumor binding of masked near infrared (NIR)
labeled 9D9 antibodies is evaluated relative to parent NIR 9D9 in
mice bearing MC38 or CT26 tumor xenografts. Antibodies are labeled
with the NIR imaging probe, IRDye-800CW-NHS (IRDye
800CW-N-hydroxysuccinimide ester) according to manufacturer's
instructions. Briefly, they are incubated in the dark at room
temperature with IRDye800CW in 1.0 M potassium phosphate buffer (pH
9.0) for 2 hours. The unconjugated dye is removed by desalting spin
columns and purified according to manufacturer's instructions. NIR
masked 9D9, NIR 9D9, or NIR labeled isotype control are
administered intravenously to MC38 bearing C57Bl/6 mice via the
tail vein. Forty-eight hours after administration of the NIR
labeled antibodies, probe uptake and tissue distribution in the
tumor bearing mice is measured using a small-animal NIR scanner
(IVIS Spectrum In Vivo Imaging System). During imaging, the mice
are anesthetized with isoflurane gas and placed in the prone
position.
Example 18
In Vivo Efficacy Evaluation of Masked anti-CTLA-4 Antibodies
[0129] Efficacy of masked anti-CTLA-4 antibodies is evaluated
relative to parent anti-CTLA-4 antibody in C57BL/6 or BALB/c mice
bearing syngeneic MC38 or CT26 tumor xenografts, respectively. Mice
are subcutaneously injected with 1 million CT26 or 2 million MC38
tumor cells. Tumor volumes are recorded 3 times weekly for four
weeks. Mice bearing palpable tumors (50-100 mm.sup.3) are
randomized into treatment groups with comparable mean tumor
volumes. Antibodies formulated in PBS pH 7.4 are administered twice
weekly intraperitoneally (IP) 200 m per dose in a volume of 200
.mu.L (10 mg/kg IP b.i.w). Matched isotype antibodies are used as
negative controls. Percent tumor growth inhibition (TGI %) is
determined after three weeks from first dose for antibody treatment
groups relative to isotype controls.
Example 19
In Vivo Safety Evaluation of Masked anti-CTLA-4 Antibodies in
Combination with anti-PD-1 Antibodies
[0130] Five-week old female NOD mice are administered masked
anti-CTLA-4 antibodies, parent anti-CTLA-4 antibody, anti-PD-1
antibody, and/or isotype controls in combination at 10 mg/kg
intraperitoneally each article on days 0, 4 and 7. The following
six combinations are used: 1) isotype controls, 2) parent
anti-CTLA-4 antibody plus isotype control, 3) masked anti-CTLA-4
antibody plus isotype control, 4) anti-PD-1 antibody plus isotype
control, 5) anti-PD-1 antibody plus parent anti-CTLA-4, or 6)
anti-PD-1 antibody plus masked anti-CTLA-4 antibody. Mice are
monitored daily for the induction of diabetes by glucosuria plus
confirmation of two consecutive blood glucose levels .gtoreq.250
mg/dL. Monitoring is continued for a minimum of 7 days until 48
hours have passed with no new incidents of glucosuria.
Example 20
In Vivo Peripheral T-Cell Activation Using Masked anti-CTLA-4
Antibodies Alone or in Combination with anti-PD-1 Antibody
[0131] Four to six week old C57Bl/6 mice are administered masked
anti-CTLA-4 antibody, parent anti-CTLA-4 antibody, isotype control,
anti-PD-1 antibody plus isotype control, anti-PD-1 antibody plus
parent anti-CTLA-4 antibody, or anti-PD-1 antibody plus masked
anti-CTLA-4 antibody at 10 mg/kg intraperitoneally each article on
days 0, 4 and 7. On day 14 animals are euthanized. Blood and spleen
are harvested from animals. Blood is stored in EDTA blood
collection tubes. Spleens are digested using the mouse spleen
dissociation kit from Miltenyi Biotec. Lymphocytes from blood and
spleen derived cells are enriched using a Ficoll gradient. Enriched
lymphocytes are stained for FoxP3 followed by either Ki67 or ICOS
and measured via flow cytometry. Percent Ki67 or ICOS positive
cells are calculated from total number of FoxP3 positive cells.
[0132] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
70140PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence may
encompass 6-20 'GS' repeating units wherein some positions may be
absent 1Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 1 5 10 15 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser 20 25 30 Gly Ser Gly Ser Gly Ser Gly Ser 35 40
239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(39)This sequence may
encompass 4-13 'GGS' repeating units wherein some positions may be
absent 2Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly 20 25 30 Ser Gly Gly Ser Gly Gly Ser 35
340PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence may
encompass 3-10 'GGGS' repeating units wherein some positions may be
absent 3Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly Ser 35 40
440PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence may
encompass 2-8 'GGGGS' repeating units wherein some positions may be
absent 4Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40
540PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence may
encompass 12-40 'G' repeating units wherein some positions may be
absent 5Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly 35 40
633PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(33)This sequence may
encompass 1-3 'GGGSGSGGGGS' repeating units wherein some positions
may be absent 6Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Ser Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
Ser Gly Gly Gly Gly 20 25 30 Ser 733PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(33)This sequence may encompass 1-3
'GGGGGPGGGGP' repeating units wherein some positions may be absent
7Gly Gly Gly Gly Gly Pro Gly Gly Gly Gly Pro Gly Gly Gly Gly Gly 1
5 10 15 Pro Gly Gly Gly Gly Pro Gly Gly Gly Gly Gly Pro Gly Gly Gly
Gly 20 25 30 Pro 836PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURE(1)..(36)This sequence
may encompass 2-6 'GGSGGD' repeating units wherein some positions
may be absent 8Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly
Gly Ser Gly 1 5 10 15 Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser
Gly Gly Asp Gly Gly 20 25 30 Ser Gly Gly Asp 35 936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(36)This sequence may encompass 2-6
'GGSGGE' repeating units wherein some positions may be absent 9Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly 1 5 10
15 Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
20 25 30 Ser Gly Gly Glu 35 10116PRTArtificial SequenceDescription
of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(40)This region may encompass 0-20 'GS'
repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-20 'GS'
repeating units wherein some positions may be absent 10Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 20 25
30 Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Ser Gly Gly Asp Gly Gly
35 40 45 Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly
Gly Asp 50 55 60 Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Ser Gly Ser 65 70 75 80 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser 85 90 95 Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110 Gly Ser Gly Ser 115
11114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(39)This region may encompass
0-13 'GGS' repeating units wherein some positions may be
absentMISC_FEATURE(40)..(75)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(76)..(114)This region may encompass 0-13 'GGS'
repeating units wherein some positions may be absent 11Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Asp Gly Gly Ser
35 40 45 Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly
Asp Gly 50 55 60 Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly
Gly Ser Gly Gly 65 70 75 80 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser 85 90 95 Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Ser Gly 100 105 110 Gly Ser
12116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This region may encompass
0-10 'GGGS' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-10 'GGGS'
repeating units wherein some positions may be absent 12Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25
30 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Gly Gly Asp Gly Gly
35 40 45 Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly
Gly Asp 50 55 60 Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser 85 90 95 Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser 100 105 110 Gly Gly Gly Ser 115
13116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This region may encompass
0-8 'GGGGS' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-8 'GGGGS'
repeating units wherein some positions may be absent 13Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Asp Gly Gly
35 40 45 Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly
Gly Asp 50 55 60 Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 85 90 95 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser 115
14116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This region may encompass
0-40 'G' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-40 'G'
repeating units wherein some positions may be absent 14Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25
30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Asp Gly Gly
35 40 45 Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly
Gly Asp 50 55 60 Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Gly Gly Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly 115
15102PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(33)This region may encompass
0-3 'GGGSGSGGGGS' repeating units wherein some positions may be
absentMISC_FEATURE(34)..(69)This region may encompass 2-6 'GGSGGD'
repeating units wherein some positions may be
absentMISC_FEATURE(70)..(102)This region may encompass 0-3
'GGGSGSGGGGS' repeating units wherein some positions may be absent
15Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly 1
5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Gly Gly
Gly 20 25 30 Ser Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Gly Ser 35 40 45 Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly
Ser Gly Gly Asp Gly 50 55 60 Gly Ser Gly Gly Asp Gly Gly Gly Ser
Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Ser Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Ser Gly 85 90 95 Ser Gly Gly Gly Gly
Ser 100 16102PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURE(1)..(33)This region may
encompass 0-3 'GGGSGSGGGGP' repeating units wherein some positions
may be absentMISC_FEATURE(34)..(69)This region may encompass 2-6
'GGSGGD' repeating units wherein some positions may be
absentMISC_FEATURE(70)..(102)This region may encompass 0-3
'GGGSGSGGGGP' repeating units wherein some positions may be absent
16Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Pro Gly Gly Gly Ser Gly 1
5 10 15 Ser Gly Gly Gly Gly Pro Gly Gly Gly Ser Gly Ser Gly Gly Gly
Gly 20 25 30 Pro Gly Gly Ser Gly Gly Asp Gly Gly Ser Gly Gly Asp
Gly Gly Ser 35 40 45 Gly Gly Asp Gly Gly Ser Gly Gly Asp Gly Gly
Ser Gly Gly Asp Gly 50 55 60 Gly Ser Gly Gly Asp Gly Gly Gly Ser
Gly Ser Gly Gly Gly Gly Pro 65 70 75 80 Gly Gly Gly Ser Gly Ser Gly
Gly Gly Gly Pro Gly Gly Gly Ser Gly 85 90 95 Ser Gly Gly Gly Gly
Pro 100 17116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURE(1)..(40)This region may
encompass 0-20 'GS' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-20 'GS'
repeating units wherein some positions may be absent 17Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 20 25
30 Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Ser Gly Gly Glu Gly Gly
35 40 45 Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 50 55 60 Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Ser Gly Ser 65 70 75 80 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser 85 90 95 Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110 Gly Ser Gly Ser 115
18114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(39)This region may encompass
0-13 'GGS' repeating units wherein some positions may be
absentMISC_FEATURE(40)..(75)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(76)..(114)This region may encompass 0-13 'GGS'
repeating units wherein some positions may be absent 18Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Glu Gly Gly Ser
35 40 45 Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly 50 55 60 Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly 65 70 75 80 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser 85 90 95 Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Ser Gly 100 105 110 Gly Ser
19116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This region may encompass
0-10 'GGGS' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-10 'GGGS'
repeating units wherein some positions may be absent 19Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25
30 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Gly Gly Glu Gly Gly
35 40 45 Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 50 55 60 Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser 85 90 95 Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly
Gly Ser 100 105 110 Gly Gly Gly Ser 115 20116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(40)This region may encompass 0-8
'GGGGS' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-8 'GGGGS'
repeating units wherein some positions may be absent 20Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Glu Gly Gly
35 40 45 Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 50 55 60 Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 85 90 95 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser 115
21116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This region may encompass
0-40 'G' repeating units wherein some positions may be
absentMISC_FEATURE(41)..(76)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(77)..(116)This region may encompass 0-40 'G'
repeating units wherein some positions may be absent 21Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25
30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Glu Gly Gly
35 40 45 Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 50 55 60 Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Gly Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly 115
22102PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(33)This region may encompass
0-3 'GGGSGSGGGGS' repeating units wherein some positions may be
absentMISC_FEATURE(34)..(69)This region may encompass 2-6 'GGSGGE'
repeating units wherein some positions may be
absentMISC_FEATURE(70)..(102)This region may encompass 0-3
'GGGSGSGGGGS' repeating units wherein some positions may be absent
22Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly 1
5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Gly Gly
Gly 20 25 30 Ser Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Ser 35 40 45 Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly 50 55 60 Gly Ser Gly Gly Glu Gly Gly Gly Ser
Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Gly Gly Gly Ser Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Ser Gly 85 90 95 Ser Gly Gly Gly Gly
Ser 100 23102PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURE(1)..(33)This region may
encompass 0-3 'GGGSGSGGGGP' repeating units wherein some positions
may be absentMISC_FEATURE(34)..(69)This region may encompass 2-6
'GGSGGE' repeating units wherein some positions may be
absentMISC_FEATURE(70)..(102)This region may encompass 0-3
'GGGSGSGGGGP' repeating units wherein some positions may be absent
23Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Pro Gly Gly Gly Ser Gly 1
5 10 15 Ser Gly Gly Gly Gly Pro Gly Gly Gly Ser Gly Ser Gly Gly Gly
Gly 20 25 30 Pro Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Ser 35 40 45 Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly 50 55 60 Gly Ser Gly Gly Glu Gly Gly Gly Ser
Gly Ser Gly Gly Gly Gly Pro 65 70 75 80 Gly Gly Gly Ser Gly Ser Gly
Gly Gly Gly Pro Gly Gly Gly Ser Gly 85 90 95 Ser Gly Gly Gly Gly
Pro 100 2440PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence
may encompass at least 20 'GX' repeating units wherein some
positions may be absentMOD_RES(2)..(2)Ser, Asp, Glu, Thr or
ProMOD_RES(4)..(4)Ser, Asp, Glu, Thr or ProMOD_RES(6)..(6)Ser, Asp,
Glu, Thr or ProMOD_RES(8)..(8)Ser, Asp, Glu, Thr or
ProMOD_RES(10)..(10)Ser, Asp, Glu, Thr or ProMOD_RES(12)..(12)Ser,
Asp, Glu, Thr or ProMOD_RES(14)..(14)Ser, Asp, Glu, Thr or
ProMOD_RES(16)..(16)Ser, Asp, Glu, Thr or ProMOD_RES(18)..(18)Ser,
Asp, Glu, Thr or ProMOD_RES(20)..(20)Ser, Asp, Glu, Thr or
ProMOD_RES(22)..(22)Ser, Asp, Glu, Thr or ProMOD_RES(24)..(24)Ser,
Asp, Glu, Thr or ProMOD_RES(26)..(26)Ser, Asp, Glu, Thr or
ProMOD_RES(28)..(28)Ser, Asp, Glu, Thr or ProMOD_RES(30)..(30)Ser,
Asp, Glu, Thr or ProMOD_RES(32)..(32)Ser, Asp, Glu, Thr or
ProMOD_RES(34)..(34)Ser, Asp, Glu, Thr or ProMOD_RES(36)..(36)Ser,
Asp, Glu, Thr or ProMOD_RES(38)..(38)Ser, Asp, Glu, Thr or
ProMOD_RES(40)..(40)Ser, Asp, Glu, Thr or Pro 24Gly Xaa Gly Xaa Gly
Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 1 5 10 15 Gly Xaa Gly
Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 20 25 30 Gly
Xaa Gly Xaa Gly Xaa Gly Xaa 35 40 2539PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(39)This sequence may encompass at
least 13 'GGX' repeating units wherein some positions may be
absentMOD_RES(3)..(3)Ser, Asp, Glu, Thr or ProMOD_RES(6)..(6)Ser,
Asp, Glu, Thr or ProMOD_RES(9)..(9)Ser, Asp, Glu, Thr or
ProMOD_RES(12)..(12)Ser, Asp, Glu, Thr or ProMOD_RES(15)..(15)Ser,
Asp, Glu, Thr or ProMOD_RES(18)..(18)Ser, Asp, Glu, Thr or
ProMOD_RES(21)..(21)Ser, Asp, Glu, Thr or ProMOD_RES(24)..(24)Ser,
Asp, Glu, Thr or ProMOD_RES(27)..(27)Ser, Asp, Glu, Thr or
ProMOD_RES(30)..(30)Ser, Asp, Glu, Thr or ProMOD_RES(33)..(33)Ser,
Asp, Glu, Thr or ProMOD_RES(36)..(36)Ser, Asp, Glu, Thr or
ProMOD_RES(39)..(39)Ser, Asp, Glu, Thr or Pro 25Gly Gly Xaa Gly Gly
Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly 1 5 10 15 Gly Xaa Gly
Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly 20 25 30 Xaa
Gly Gly Xaa Gly Gly Xaa 35 2640PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATURE(1)..(40)This
sequence may encompass at least 10 'GGGX' repeating units wherein
some positions may be absentMOD_RES(4)..(4)Ser, Asp, Glu, Thr or
ProMOD_RES(8)..(8)Ser, Asp, Glu, Thr or ProMOD_RES(12)..(12)Ser,
Asp, Glu, Thr or ProMOD_RES(16)..(16)Ser, Asp, Glu, Thr or
ProMOD_RES(20)..(20)Ser, Asp, Glu, Thr or ProMOD_RES(24)..(24)Ser,
Asp, Glu, Thr or ProMOD_RES(28)..(28)Ser, Asp, Glu, Thr or
ProMOD_RES(32)..(32)Ser, Asp, Glu, Thr or ProMOD_RES(36)..(36)Ser,
Asp, Glu, Thr or ProMOD_RES(40)..(40)Ser, Asp, Glu, Thr or Pro
26Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly Gly Xaa 1
5 10 15 Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly Gly
Xaa 20 25 30 Gly Gly Gly Xaa Gly Gly Gly Xaa 35 40
2740PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(40)This sequence may
encompass at least 8 'GGGGX' repeating units wherein some positions
may be absentMOD_RES(5)..(5)Ser, Asp, Glu, Thr or
ProMOD_RES(10)..(10)Ser, Asp, Glu, Thr or ProMOD_RES(15)..(15)Ser,
Asp, Glu, Thr or ProMOD_RES(20)..(20)Ser, Asp, Glu, Thr or
ProMOD_RES(25)..(25)Ser, Asp, Glu, Thr or ProMOD_RES(30)..(30)Ser,
Asp, Glu, Thr or ProMOD_RES(35)..(35)Ser, Asp, Glu, Thr or
ProMOD_RES(40)..(40)Ser, Asp, Glu, Thr or Pro 27Gly Gly Gly Gly Xaa
Gly Gly Gly Gly Xaa Gly Gly Gly Gly Xaa Gly 1 5 10 15 Gly Gly Gly
Xaa Gly Gly Gly Gly Xaa Gly Gly Gly Gly Xaa Gly Gly 20 25 30 Gly
Gly Xaa Gly Gly Gly Gly Xaa 35 40 28315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURE(1)..(315)This sequence may encompass at
least 15 'GZX' repeating units wherein 'Z' is an integer between
1-20 and some positions may be absentMISC_FEATURE(1)..(20)This
region may encompass 1-20 'G' repeating units wherein some
positions may be absentMOD_RES(21)..(21)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(22)..(41)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(42)..(42)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(43)..(62)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(63)..(63)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(64)..(83)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(84)..(84)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(85)..(104)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(105)..(105)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(106)..(125)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(126)..(126)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(127)..(146)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(147)..(147)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(148)..(167)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(168)..(168)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(169)..(188)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(189)..(189)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(190)..(209)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(210)..(210)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(211)..(230)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(231)..(231)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(232)..(251)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(252)..(252)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(253)..(272)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(273)..(273)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(274)..(293)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(294)..(294)Ser, Asp, Glu, Thr or
ProMISC_FEATURE(295)..(314)This region may encompass 1-20 'G'
repeating units wherein some positions may be
absentMOD_RES(315)..(315)Ser, Asp, Glu, Thr or Pro 28Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly
Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25
30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly
35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Xaa Gly 50 55 60 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 65 70 75 80 Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly
Xaa Gly Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly 115 120 125 Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140 Gly Gly
Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155
160 Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly
165 170 175 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa Gly
Gly Gly 180 185 190 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 195 200 205 Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 210 215 220 Gly Gly Gly Gly Gly Gly Xaa Gly
Gly Gly Gly Gly Gly Gly Gly Gly 225 230 235 240 Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly 245 250 255 Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 260 265 270 Xaa
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 275 280
285 Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
290 295 300 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa 305 310 315
2912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Thr Leu Asp Asp Met Ser His Val Ile Leu Tyr Ala
1 5 10 3012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Val Ile Ser Asp Asn His Gln Ile Val Trp Asp Leu
1 5 10 3112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Leu Thr Thr Gln Asp His Pro Leu Thr Ile Leu Leu
1 5 10 3216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Gly Gly Trp Ile Cys His Trp Leu Glu Pro Gln Glu
Ala Cys Thr Tyr 1 5 10 15 3316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Gly Gly Cys Phe Glu Glu His
Glu Gln Leu Val Phe Gln Thr His Cys 1 5 10 15 3416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Gly
Gly Cys Ile Leu Pro Gly Gln His Glu Ser Gln Ala Ile Ala Cys 1 5 10
15 3516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Gly Gly Cys Leu Ser Gln Met Asp Phe His Asp Trp
Leu Gln Tyr Cys 1 5 10 15 3616PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Gly Gly Thr Asp Cys Tyr Leu
Trp Asp Tyr Lys Ala Ser Cys His Gln 1 5 10 15 3716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gly
Gly Lys Cys Asp Ser Leu Ser Tyr Trp Gln Glu Ile Glu Cys Ser 1 5 10
15 3816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Gly Gly Ala Asp Cys Leu Leu His Asp Trp Asp Ser
Ala Cys Gln Ile 1 5 10 15 3912PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Met Gln Asn Val Asp Glu Ala
Pro Pro Leu Leu Leu 1 5 10 4012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Thr Asn Asp Trp Gln Gly Leu
Leu Leu Asn Val Phe 1 5 10 4114PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Gly Gly Cys Gln Asp Ser Met
Phe His His Pro Asn His Cys 1 5 10
4214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Gly Gly Cys Gly Met His Gln His Pro Leu Phe Val
Asp Cys 1 5 10 4314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 43Gly Gly Cys Ser Leu Ser Gln His Pro
Asn His Ser Asp Cys 1 5 10 4414PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 44Gly Gly Pro Cys Asn Gln Val
Glu Cys His His Gln Phe Thr 1 5 10 4514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Gly
Gly Cys Pro Ser Leu His Pro Gln Trp Ile His Val Cys 1 5 10
4614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gly Gly Cys Phe Asp Ser Ala Asn Gln His Pro Asn
Met Cys 1 5 10 4714PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 47Gly Gly Cys His Gln Asp Ile His His
Pro Ile Tyr Trp Cys 1 5 10 4814PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Gly Gly Cys Gln Ile His Asp
Pro His Thr Trp His Leu Cys 1 5 10 4912PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Gln
Leu Phe Tyr Pro Ser Thr Tyr His Ile Ile Asp 1 5 10
5012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gln Val Ser Pro Leu Tyr Phe Tyr Glu Glu Leu Ala
1 5 10 5111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51His Gln Ala Leu Leu Asp Phe Tyr Gly Asp Tyr 1 5
10 5216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gly Gly Met Cys His Glu Leu Phe Tyr Ser Asn Leu
Asn Trp Cys Gln 1 5 10 15 5316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 53Gly Gly His Cys Val Asp Met
Val Asp Phe Tyr Gln Gln Thr Cys Gln 1 5 10 15 5412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Val
Asp Leu Leu Asp Gly Ser Leu Gln Asp Phe Tyr 1 5 10
5516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Gly Gly Leu Cys Ser Thr Phe Tyr Glu Pro Gln Val
Asp Ile Cys Tyr 1 5 10 15 5612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 56Ser Asp Phe Ser Gly Leu Leu
Phe Tyr Asp Tyr Gln 1 5 10 5714PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 57Gly Gly Cys Val His Phe Phe
His His Gln Arg Pro Asp Cys 1 5 10 5814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Gly
Gly Cys His Asn Lys Ser Gly Leu Phe Tyr His Tyr Cys 1 5 10
5914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Gly Gly Cys Phe Tyr Pro Gly His His His Gln Leu
Leu Cys 1 5 10 6026PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 60Gly Ser Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Ser
Ser Gly Thr 20 25 6112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Pro Cys Asn Gln Val Glu Cys
His His Gln Phe Thr 1 5 10 62219PRTMus sp. 62Asp Ile Val Met Thr
Gln Thr Thr Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn
Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val
Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly
Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp
Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser
Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210
215 63261PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 63Gly Gly Ala Asp Cys Leu Leu His Asp Trp Asp
Ser Ala Cys Gln Ile 1 5 10 15 Gly Ser Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Ser
Ser Gly Thr Asp Ile Val Met Thr Gln 35 40 45 Thr Thr Leu Ser Leu
Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser 50 55 60 Cys Arg Ser
Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu 65 70 75 80 Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 85 90
95 Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
100 105 110 Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu
Ala Glu 115 120 125 Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His
Val Pro Tyr Thr 130 135 140 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Ala Asp Ala Ala Pro 145 150 155 160 Thr Val Ser Ile Phe Pro Pro
Ser Ser Glu Gln Leu Thr Ser Gly Gly 165 170 175 Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 180 185 190 Val Lys Trp
Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 195 200 205 Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 210 215
220 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
225 230 235 240 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val
Lys Ser Phe 245 250 255 Asn Arg Asn Glu Cys 260 64259PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
64Gly Gly Cys Gln Asp Ser Met Phe His His Pro Asn His Cys Gly Ser 1
5 10 15 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser Gly
Gly 20 25 30 Gly Ser Gly Gly Ser Ser Gly Thr Asp Ile Val Met Thr
Gln Thr Thr 35 40 45 Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
Ser Ile Ser Cys Arg 50 55 60 Ser Ser Gln Ser Ile Val His Ser Asn
Gly Asn Thr Tyr Leu Glu Trp 65 70 75 80 Tyr Leu Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile Tyr Lys Val 85 90 95 Ser Asn Arg Phe Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 100 105 110 Gly Thr Asp
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu 115 120 125 Gly
Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly 130 135
140 Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val
145 150 155 160 Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
Gly Ala Ser 165 170 175 Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys
Asp Ile Asn Val Lys 180 185 190 Trp Lys Ile Asp Gly Ser Glu Arg Gln
Asn Gly Val Leu Asn Ser Trp 195 200 205 Thr Asp Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Met Ser Ser Thr Leu 210 215 220 Thr Leu Thr Lys Asp
Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu 225 230 235 240 Ala Thr
His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg 245 250 255
Asn Glu Cys 65259PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 65Gly Gly Cys Ser Leu Ser Gln His
Pro Asn His Ser Asp Cys Gly Ser 1 5 10 15 Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Gly Ser Gly Gly 20 25 30 Gly Ser Gly Gly
Ser Ser Gly Thr Asp Ile Val Met Thr Gln Thr Thr 35 40 45 Leu Ser
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg 50 55 60
Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp 65
70 75 80 Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
Lys Val 85 90 95 Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser 100 105 110 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu 115 120 125 Gly Val Tyr Tyr Cys Phe Gln Gly
Ser His Val Pro Tyr Thr Phe Gly 130 135 140 Gly Gly Thr Lys Leu Glu
Ile Lys Arg Ala Asp Ala Ala Pro Thr Val 145 150 155 160 Ser Ile Phe
Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser 165 170 175 Val
Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys 180 185
190 Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp
195 200 205 Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
Thr Leu 210 215 220 Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser
Tyr Thr Cys Glu 225 230 235 240 Ala Thr His Lys Thr Ser Thr Ser Pro
Ile Val Lys Ser Phe Asn Arg 245 250 255 Asn Glu Cys
66259PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Gly Gly Pro Cys Asn Gln Val Glu Cys His His
Gln Phe Thr Gly Ser 1 5 10 15 Ser Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Gly Ser Gly Gly 20 25 30 Gly Ser Gly Gly Ser Ser Gly
Thr Asp Ile Val Met Thr Gln Thr Thr 35 40 45 Leu Ser Leu Pro Val
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg 50 55 60 Ser Ser Gln
Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp 65 70 75 80 Tyr
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val 85 90
95 Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
100 105 110 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp Leu 115 120 125 Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro
Tyr Thr Phe Gly 130 135 140 Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
Asp Ala Ala Pro Thr Val 145 150 155 160 Ser Ile Phe Pro Pro Ser Ser
Glu Gln Leu Thr Ser Gly Gly Ala Ser 165 170 175 Val Val Cys Phe Leu
Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys 180 185 190 Trp Lys Ile
Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp 195 200 205 Thr
Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu 210 215
220 Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu
225 230 235 240 Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
Phe Asn Arg 245 250 255 Asn Glu Cys 67453PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Glu Ala Lys Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser
Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly
Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Ile Thr Val
Ser Thr Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala
Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 130 135
140 Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser
145 150 155 160 Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu
Leu Gln Ser 165 170 175 Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val
Pro Ser Ser Thr Trp 180 185 190 Pro Ser Gln Thr Val Thr Cys Ser Val
Ala His Pro Ala Ser Ser Thr 195 200 205 Thr Val Asp Lys Lys Leu Glu
Pro Ser Gly Pro Ile Ser Thr Ile Asn 210 215 220 Pro Cys Pro Pro Cys
Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu 225 230 235 240 Glu Gly
Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val 245 250 255
Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val 260
265 270 Ser Glu Asp Asp Pro Asp Val Arg Ile Ser Trp Phe Val Asn Asn
Val 275 280 285 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
Tyr Asn Ser 290 295 300 Thr Ile Arg Val Val Ser Ala Leu Pro Ile Gln
His Gln Asp Trp Met 305 310 315 320 Ser Gly Lys Glu Phe Lys Cys Lys
Val Asn Asn Lys Asp Leu Pro Ser 325 330 335 Pro Ile Glu Arg Thr Ile
Ser Lys Ile Lys Gly Leu Val Arg Ala Pro 340 345 350 Gln Val Tyr Ile
Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp 355 360 365 Val Ser
Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser 370 375 380
Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr 385
390 395 400 Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser
Lys Leu 405 410 415 Asp Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser
Phe Ser Cys Asn 420 425 430 Val Arg His Glu Gly Leu Lys Asn Tyr Tyr
Leu Lys Lys Thr Ile Ser 435 440 445 Arg Ser Pro Gly Lys 450
68214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
69256PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Gly Gly His Cys Val Asp Met Val Asp Phe Tyr
Gln Gln Thr Cys Gln 1 5 10 15 Gly Ser Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Ser
Ser Gly Thr Asp Ile Gln Met Thr Gln 35 40 45 Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 50 55 60 Cys Arg Ala
Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln 65 70 75 80 Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu 85 90
95 Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
100 105 110 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr 115 120 125 Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe
Gly Gln Gly Thr 130 135 140 Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe 145 150 155 160 Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys 165 170 175 Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 180 185 190 Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 195 200 205 Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 210 215
220 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
225 230 235 240 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 245 250 255 70448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 70Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305
310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
* * * * *