U.S. patent application number 14/680865 was filed with the patent office on 2019-02-14 for fully human antibodies that bind to vascular endothelial growth factor receptor 2 (vegfr2).
This patent application is currently assigned to Sorrento Therapeutics, Inc.. The applicant listed for this patent is Sorrento Therapeutics, Inc.. Invention is credited to Randy Gastwirt, John Dixon Gray, Guodi Lu, Heyue Zhou.
Application Number | 20190048087 14/680865 |
Document ID | / |
Family ID | 51621080 |
Filed Date | 2019-02-14 |
View All Diagrams
United States Patent
Application |
20190048087 |
Kind Code |
A9 |
Gastwirt; Randy ; et
al. |
February 14, 2019 |
Fully Human Antibodies that Bind to Vascular Endothelial Growth
Factor Receptor 2 (VEGFR2)
Abstract
There is disclosed compositions and methods relating to
anti-VEGFR2 antibodies. More specifically, there is disclosed fully
human antibodies that bind VEGFR2, VEGFR2-binding fragments and
derivatives of such antibodies, and VEGFR2-binding polypeptides
comprising such fragments. Further still, there is disclosed
antibody fragments and derivatives and polypeptides, and methods of
using such antibodies, antibody fragments and derivatives and
polypeptides, including methods of treating or diagnosing subjects
having various cancers.
Inventors: |
Gastwirt; Randy; (San Diego,
CA) ; Zhou; Heyue; (San Diego, CA) ; Gray;
John Dixon; (San Diego, CA) ; Lu; Guodi; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorrento Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Sorrento Therapeutics, Inc.
San Diego
CA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170166644 A1 |
June 15, 2017 |
|
|
Family ID: |
51621080 |
Appl. No.: |
14/680865 |
Filed: |
April 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13854071 |
Mar 30, 2013 |
9029510 |
|
|
14680865 |
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61618658 |
Mar 30, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/94 20130101;
A61K 2039/505 20130101; C07K 2317/55 20130101; A61K 45/06 20130101;
A61K 39/3955 20130101; C07K 2317/56 20130101; C07K 2317/76
20130101; C07K 2317/92 20130101; C07K 2317/622 20130101; C07K
16/2863 20130101; C07K 2317/21 20130101; A61K 2039/507
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Claims
1. A fully human antibody of an IgG class that binds to a VEGFR2
epitope wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 13,
SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID
NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,
SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID
NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 53,
SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID
NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,
SEQ ID NO. 75, SEQ ID NO. 79, and SEQ ID NO. 81, and that has a
light chain variable domain sequence that is at least 95% identical
to the amino acid sequences selected from the group consisting of
SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ
ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.
32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ
ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO.
50, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ
ID NO. 62, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO.
72, SEQ ID NO. 76, and SEQ ID NO. 80.
2. The fully human antibody of claim 1, wherein the antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID
NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID
NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.
15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID
NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 25/SEQ ID NO. 26,
SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID
NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO.
35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID
NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44,
SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID
NO. 49/SEQ ID NO. 50, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO.
55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID
NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 65/SEQ ID NO. 66,
SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID
NO. 71/SEQ ID NO. 72, and SEQ ID NO. 75/SEQ ID NO. 76.
3. A fully human antibody Fab fragment, having a variable domain
region from a heavy chain and a variable domain region from a light
chain, wherein the heavy chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ
ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.
17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.
37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ
ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO.
57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ
ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO.
79, and SEQ ID NO. 81, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 26,
SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID
NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44,
SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 54, SEQ ID
NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 66,
SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 76, and SEQ
ID NO. 80.
4. The fully human antibody Fab fragment of claim 3, wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.
29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID
NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38,
SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID
NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.
47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 53/SEQ ID
NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58,
SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID
NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO.
69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, and SEQ ID NO.
75/SEQ ID NO. 76.
5. A single chain human antibody, having a variable domain region
from a heavy chain and a variable domain region from a light chain
and a peptide linker connecting the heavy chain and light chain
variable domain regions, wherein the heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 25,
SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID
NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43,
SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID
NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63,
SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID
NO. 75, SEQ ID NO. 79, and SEQ ID NO. 81, and that has a light
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32,
SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID
NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50,
SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID
NO. 62, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,
SEQ ID NO. 76, and SEQ ID NO. 80.
6. The fully human single chain antibody of claim 5, wherein the
single chain fully human antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 11/SEQ ID NO. 12,
SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID
NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.
21/SEQ ID NO. 22, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID
NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32,
SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID
NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.
41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID
NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID
NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72,
and SEQ ID NO. 75/SEQ ID NO. 76.
7. A method for treating a broad spectrum of mammalian cancers,
comprising administering an effective amount of an anti-VEGFR2
polypeptide, wherein the anti-VEGFR2 polypeptide is selected from
the group consisting of a fully human antibody of an IgG class that
binds to a VEGFR2 epitope with a binding affinity of at least
10.sup.-6M, a fully human antibody Fab fragment, having a variable
domain region from a heavy chain and a variable domain region from
a light chain, a single chain human antibody, having a variable
domain region from a heavy chain and a variable domain region from
a light chain and a peptide linker connecting the heavy chain and
light chain variable domain regions, and combinations thereof;
wherein the fully human antibody has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 25,
SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID
NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43,
SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID
NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63,
SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID
NO. 75, SEQ ID NO. 79, and SEQ ID NO. 81, and that has a light
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32,
SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID
NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50,
SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID
NO. 62, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72,
SEQ ID NO. 76, and SEQ ID NO. 80; wherein the Fab fully human
antibody fragment has the heavy chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5,
SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID
NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID
NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,
SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID
NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65,
SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID
NO. 79, and SEQ ID NO. 81, and that has the light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 14,
SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID
NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,
SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID
NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 54,
SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID
NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 76,
and SEQ ID NO. 80; and wherein the single chain human antibody has
the heavy chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ
ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO.
29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ
ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO.
47, SEQ ID NO. 49, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ
ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO.
67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 79, and
SEQ ID NO. 81, and that has the light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 26,
SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID
NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44,
SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 54, SEQ ID
NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 66,
SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 76, and SEQ
ID NO. 80.
8. The method for treating a broad spectrum of mammalian cancers of
claim 7, wherein the fully human antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 11/SEQ ID NO.
12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ
ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.
21/SEQ ID NO. 22, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID
NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32,
SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID
NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.
41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID
NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID
NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72,
and SEQ ID NO. 75/SEQ ID NO. 76.
9. The method for treating a broad spectrum of mammalian cancers of
claim 7, wherein the fully human antibody Fab fragment has both a
heavy chain variable domain region and a light chain variable
domain region wherein the antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 11/SEQ ID NO. 12,
SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID
NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.
21/SEQ ID NO. 22, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID
NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32,
SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID
NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.
41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID
NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID
NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72,
and SEQ ID NO. 75/SEQ ID NO. 76.
10. The method for treating a broad spectrum of mammalian cancers
of claim 7, wherein the fully human single chain antibody has both
a heavy chain variable domain region and a light chain variable
domain region, wherein the single chain fully human antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID
NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID
NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.
15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID
NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 25/SEQ ID NO. 26,
SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID
NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO.
35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID
NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44,
SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID
NO. 49/SEQ ID NO. 50, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO.
55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID
NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 65/SEQ ID NO. 66,
SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID
NO. 71/SEQ ID NO. 72, and SEQ ID NO. 75/SEQ ID NO. 76.
11. The method for treating a broad spectrum of mammalian cancers
of claim 7, wherein the mammalian cancer to be treated is selected
from the group consisting of solid tumors, blood borne tumors,
leukemias, hemangiomas, acoustic neuromas, neurofibromas,
trachomas, and pyogenic granulomas.
12. A method for treating a broad spectrum of mammalian cancers,
comprising administering an effective amount of an anti-VEGFR2
polypeptide and an antineoplastic agent: wherein the anti-VEGFR2
polypeptide is selected from the group consisting of a fully human
antibody of an IgG class that binds to a VEGFR2 epitope with a
binding affinity of at least 10.sup.-6M, a fully human antibody Fab
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connecting the heavy chain and light chain variable domain regions,
and combinations thereof; wherein the fully human antibody has a
heavy chain variable domain sequence that is at least 95% identical
to the amino acid sequences selected from the group consisting of
SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO.
11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ
ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ
ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.
49, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ
ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO.
69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 79, and SEQ ID NO. 81,
and that has a light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.
18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 28, SEQ
ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO.
38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ
ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO.
58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 66, SEQ ID NO. 68, SEQ
ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 76, and SEQ ID NO. 80; wherein
the antineoplastic agent is selected from the group consisting of
anti-metabolites/anti-cancer agents, pyrimidine analogs, purine
analogs, folate antagonists; antiproliferative/antimitotic agents,
vinca alkaloids, microtubule disruptors, taxane, vincristin,
vinblastin, nocodazole, epothilones and navelbine,
epidipodophyllotoxins, actinomycin, amsacrine, anthracyclines,
bleomycin, busulfan, camptothecin, carboplatin, chlorambucil,
cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin,
doxorubicin, epirubicin, hexamethylmelamineoxaliplatin,
iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone,
nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide, etoposide; antibiotics, dactinomycin
(actinomycin D), daunorubicin, doxorubicin, idarubicin,
anthracyclines, mitoxantrone, bleomycins, plicamycin, mitomycin;
L-asparaginase; antiplatelet agents; antiproliferative/antimitotic
alkylating agents, mechlorethamine, cyclophosphamide, melphalan,
chlorambucil, ethylenimines, methylmelamines, alkyl
sulfonates-busulfan, nitrosoureas, trazenes-dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites; platinum
coordination complexes, procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs, aromatase inhibitors;
anticoagulants; fibrinolytic agents; aspirin, dipyridamole,
ticlopidine, clopidogrel, abciximab; antimigratory agents;
antisecretory agents; immunosuppressives; anti-angiogenic
compounds; angiotensin receptor blocker; nitric oxide donors;
anti-sense oligonucleotides; antibodies; cell cycle inhibitors,
differentiation inducers; mTOR inhibitors, topoisomerase
inhibitors, corticosteroids; growth factor signal transduction
kinase inhibitors; mitochondrial dysfunction inducers, caspase
activators; and chromatin disruptors
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 13/854,071, filed Mar. 30, 2013. U.S. patent
application Ser. No. 13/854,071 claims the benefit of the filing
date under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application
No. 61/618,658, filed Mar. 30, 2012.
TECHNICAL FIELD
[0002] The present disclosure provides compositions and methods for
fully human anti-VEGFR2 antibodies. More specifically, the present
disclosure provides human antibodies that bind VEGFR2,
VEGFR2-binding fragments and derivatives of such antibodies, and
VEGFR2-binding polypeptides comprising such fragments. Further
still, the present disclosure provides antibody fragments and
derivatives and polypeptides, and methods of using such antibodies,
antibody fragments and derivatives and polypeptides, including
methods of treating or diagnosing subjects having VEGFR2-related
disorders or conditions.
BACKGROUND
[0003] Angiogenesis is an important cellular event in which
vascular endothelial cells proliferate, prune and reorganize to
form new vessels from preexisting vascular networks. There are
compelling evidences that the development of a vascular supply is
essential for normal and pathological proliferative processes
(Folkman and Klagsbrun (1987) Science 235:442-447). Delivery of
oxygen and nutrients, as well as the removal of catabolic products,
represent rate-limiting steps in the majority of growth processes
occurring in multicellular organisms. Thus, it has been generally
assumed that the vascular compartment is necessary, not only for
organ development and differentiation during embryogenesis, but
also for wound healing and reproductive functions in the adult.
[0004] Angiogenesis is also implicated in the pathogenesis of a
variety of disorders, including but not limited to, cancer,
proliferative retinopathies, age-related macular degeneration,
rheumatoid arthritis (RA), and psoriasis. Angiogenesis is essential
for the growth of most primary solid tumors and their subsequent
metastasis. Tumors can absorb sufficient nutrients and oxygen by
simple diffusion up to a size of 1-2 mm, at which point their
further growth requires the elaboration of vascular supply. This
process is thought to involve recruitment of the neighboring host
mature vasculature to begin sprouting new blood vessel capillaries,
which grow towards, and subsequently infiltrate, the tumor mass. In
addition, tumor angiogenesis involves the recruitment of
circulating endothelial precursor cells from the bone marrow to
promote neovascularization Kerbel, Carcinogenesis 21:505-515, 2000;
and Lynden et al., Nat. Med. 7:1194-1201, 2001.
[0005] While induction of new blood vessels is considered to be the
predominant mode of tumor angiogenesis, some tumors may grow by
co-opting existing host blood vessels. The co-opted vasculature
then regresses, leading to tumor regression that is eventually
reversed by hypoxia-induced angiogenesis at the tumor margin.
Holash et al., Science 284:1994-1998, 1999.
[0006] In many instances, the process begins with the activation of
existing vascular endothelial cells in response to a variety of
cytokines and growth factors. In cancer, tumor released cytokines
or angiogenic factors stimulate vascular endothelial cells by
interacting with specific cell surface receptors. The activated
endothelial cells secrete enzymes that degrade the basement
membrane of the vessels, allowing invasion of the endothelial cells
into the tumor tissue. Once situated, the endothelial cells
differentiate to form new vessel offshoots of pre-existing vessels.
The new blood vessels provide nutrients to the tumor, facilitating
further growth, and also provide a route for metastasis.
[0007] Numerous angiogenic factors have been identified, including
the particularly potent factor VEGF. VEGF was initially purified
from the conditioned media of folliculostellate cells and from a
variety of cell lines. Various forms of VEGF bind as high affinity
ligands to a suite of VEGF receptors (VEGFRs). VEGFRs are tyrosine
kinase receptors, many of which are important regulators of
angiogenesis. The VEGFR family includes 3 major subtypes: VEGFR1,
VEGFR2 (also known as Kinase Insert Domain Receptor, "KDR", in
humans), and VEGFR3. Among VEGF forms, VEGF-A, VEGF-C and VEGF-D
are known to bind and activate VEGFR2.
[0008] VEGF, acting through its cognate receptors, can function as
an endothelial specific mitogen during angiogenesis. In addition,
there is substantial evidence that VEGF and VEGFRs are up-regulated
in conditions characterized by inappropriate angiogenesis, such as
cancer. As a result, a great deal of research has focused on the
identification of therapeutics that target and inhibit VEGFs or
VEGFRs.
[0009] Therapeutic approaches that target or inhibit VEGFs or
VEGFRs include antibodies, peptides, and small molecule kinase
inhibitors. Of these, antibodies are widely used for in vivo
recognition and inhibition of ligands and cellular receptors.
Highly specific antibodies have been used to block receptor-ligand
interaction, thereby neutralizing the biological activity of the
components, and also to specifically deliver toxic agents to cells
expressing the cognate receptor on its surface. As a result, there
remains a need for effective therapeutics that can specifically
inhibit VEGF/VEGFR pathways as a treatment for disorders
characterized by inappropriate angiogenesis, such as cancer.
[0010] The anti-VEGF antibody "Bevacizumab (BV)", also known as
"rhuMAb VEGF" or "Avastin.RTM." is a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.,
Cancer Res. 57:4593-4599, 1997. It comprises mutated human IgG1
framework regions and antigen-binding complementarity-determining
regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that
blocks binding of human VEGF to its receptors. Approximately 93% of
the amino acid sequence of Bevacizumab, including most of the
framework regions, is derived from human IgG1, and about 7% of the
sequence is derived from the murine antibody A4.6.1. Bevacizumab
has a molecular mass of about 149,000 daltons and is
glycosylated.
SUMMARY
[0011] The anti-VEGFR2 binding proteins described herein may be
used, for example, to detect VEGFR2 in vivo or in vitro.
Additionally, certain VEGFR2 binding proteins described herein may
be used to treat diseases associated with VEGFR2-mediated
biological activity. For example, VEGFR2 mediates the
pro-angiogenic effects of VEGF, and accordingly, certain VEGFR2
binding proteins of the disclosure may be used to inhibit
angiogenesis in a human patient. Certain VEGFR2 binding proteins of
the disclosure may be used to treat disorders such as cancers,
inflammatory diseases, autoimmune diseases and retinopathies. Many
disorders related to the hyperproliferation of cells of a tissue
will include an angiogenic component, and thus it is expected that
certain VEGFR2 binding proteins described herein can be used to
treat such disorders.
[0012] The present disclosure provides a fully human antibody of an
IgG class that binds to a VEGFR2 epitope with a binding affinity of
at least 10.sup.-6M, that has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ
ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.
49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ
ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO.
67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ
ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and combinations thereof,
and that has a light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ
ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO.
52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ
ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.
70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ
ID NO. 80, and combinations thereof. Preferably, the fully human
antibody has both a heavy chain and a light chain wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2
(called VB-A2 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called VB-A3
herein), SEQ ID NO. 5/SEQ ID NO. 6 (called VB-A7 herein), SEQ ID
NO. 7/SEQ ID NO. 8 (called VBV-A7 herein), SEQ ID NO. 9/SEQ ID NO.
10 (called VB-A9 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called
VB-A10 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called VB-B6 herein),
SEQ ID NO. 15/SEQ ID NO. 16 (called VB-B10 herein), SEQ ID NO.
17/SEQ ID NO. 18 (called VB-D5 herein), SEQ ID NO. 19/SEQ ID NO. 20
(called VB-D6 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called VB-D11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called VB-E1 herein), SEQ ID
NO. 25/SEQ ID NO. 26 (called VB-E2 herein), SEQ ID NO. 27/SEQ ID
NO. 28 (called VB-E7 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called
VB-F2 herein), SEQ ID NO. 31/SEQ ID NO. 32 (called VB-F8 herein),
SEQ ID NO. 33/SEQ ID NO. 34 (called VB-G4 herein), SEQ ID NO.
35/SEQ ID NO. 36 (called VB-G6 herein), SEQ ID NO. 37/SEQ ID NO. 38
(called VB-H4 herein), SEQ ID NO. 39/SEQ ID NO. 40 (called VB-H7
herein), SEQ ID NO. 41/SEQ ID NO. 42 (called VB-H9 herein), SEQ ID
NO. 43/SEQ ID NO. 44 (called RV-A9 herein), SEQ ID NO. 45/SEQ ID
NO. 46 (called RV-F8 herein), SEQ ID NO. 47/SEQ ID NO. 48 (called
RV-H2 herein), SEQ ID NO. 49/SEQ ID NO. 50 (called RV-H4 herein),
SEQ ID NO. 51/SEQ ID NO. 52 (called RV-H5 herein), SEQ ID NO.
53/SEQ ID NO. 54 (called C1 herein), SEQ ID NO. 55/SEQ ID NO. 56
(called VR-A2 herein), SEQ ID NO. 57/SEQ ID NO. 58 (called VR-A3
herein), SEQ ID NO. 59/SEQ ID NO. 60 (called VR-A10 herein), SEQ ID
NO. 61/SEQ ID NO. 62 (called VR-B2 herein), SEQ ID NO. 63/SEQ ID
NO. 64 (called VR-B4 herein), SEQ ID NO. 65/SEQ ID NO. 66 (called
VR-B11 herein), SEQ ID NO. 67/SEQ ID NO. 68 (called VR-05 herein),
SEQ ID NO. 69/SEQ ID NO. 70 (called VR-C7 herein), SEQ ID NO.
71/SEQ ID NO. 72 (called VR-C11 herein), SEQ ID NO. 73/SEQ ID NO.
74 (called VR-E3 herein), SEQ ID NO. 75/SEQ ID NO. 76 (called
VR-G11 herein), SEQ ID NO. 77/SEQ ID NO. 78 (called VK-B8 herein),
SEQ ID NO. 79/SEQ ID NO. 80 called VR-H9 herein), SEQ ID NO. 77/SEQ
ID NO. 81 (called VK-B8A herein), and combinations thereof.
[0013] The present disclosure provides a fully human antibody Fab
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.
37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ
ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.
55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ
ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO.
73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40,
SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID
NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58,
SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID
NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76,
SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof. Preferably,
the fully human antibody Fab fragment has both a heavy chain
variable domain region and a light chain variable domain region
wherein the antibody has a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID
NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ
ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.
16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ
ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34,
SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID
NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.
43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID
NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID
NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70,
SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID
NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, and combinations thereof.
[0014] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connecting the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ
ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO.
25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ
ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.
43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ
ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO.
61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ
ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO.
79, SEQ ID NO. 81, and combinations thereof, and that has a light
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10,
SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID
NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28,
SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID
NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,
SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID
NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64,
SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID
NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, and
combinations thereof. Preferably, the fully human single chain
antibody has both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34,
SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID
NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.
43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID
NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID
NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70,
SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID
NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, and combinations thereof.
[0015] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers, comprising
administering an effective amount of an anti-VEGFR2 polypeptide,
wherein the anti-VEGFR2 polypeptide is selected from the group
consisting of a fully human antibody of an IgG class that binds to
a VEGFR2 epitope with a binding affinity of at least 10.sup.-6M, a
fully human antibody Fab fragment, having a variable domain region
from a heavy chain and a variable domain region from a light chain,
a single chain human antibody, having a variable domain region from
a heavy chain and a variable domain region from a light chain and a
peptide linker connecting the heavy chain and light chain variable
domain regions, and combinations thereof;
[0016] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID
NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47,
SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID
NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65,
SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID
NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40,
SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID
NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58,
SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID
NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76,
SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof;
[0017] wherein the fully human antibody Fab fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID
NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,
SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID
NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63,
SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID
NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81,
and combinations thereof, and that has the light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID
NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48,
SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID
NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66,
SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID
NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof;
and
[0018] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.
37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ
ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.
55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ
ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO.
73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and
combinations thereof, and that has the light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40,
SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID
NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58,
SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID
NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76,
SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof.
[0019] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.
10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ
ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID
NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.
37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID
NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46,
SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID
NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO.
55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID
NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64,
SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID
NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO.
73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID
NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81,
and combinations thereof. Preferably, the fully human antibody Fab
fragment has both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called VB-A2 herein), SEQ
ID NO. 3/SEQ ID NO. 4 (called VB-A3 herein), SEQ ID NO. 5/SEQ ID
NO. 6 (called VB-A7 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called
VBV-A7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called VB-A9 herein),
SEQ ID NO. 11/SEQ ID NO. 12 (called VB-A10 herein), SEQ ID NO.
13/SEQ ID NO. 14 (called VB-B6 herein), SEQ ID NO. 15/SEQ ID NO. 16
(called VB-B10 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called VB-D5
herein), SEQ ID NO. 19/SEQ ID NO. 20 (called VB-D6 herein), SEQ ID
NO. 21/SEQ ID NO. 22 (called VB-D11 herein), SEQ ID NO. 23/SEQ ID
NO. 24 (called VB-E1 herein), SEQ ID NO. 25/SEQ ID NO. 26 (called
VB-E2 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called VB-E7 herein),
SEQ ID NO. 29/SEQ ID NO. 30 (called VB-F2 herein), SEQ ID NO.
31/SEQ ID NO. 32 (called VB-F8 herein), SEQ ID NO. 33/SEQ ID NO. 34
(called VB-G4 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called VB-G6
herein), SEQ ID NO. 37/SEQ ID NO. 38 (called VB-H4 herein), SEQ ID
NO. 39/SEQ ID NO. 40 (called VB-H7 herein), SEQ ID NO. 41/SEQ ID
NO. 42 (called VB-H9 herein), SEQ ID NO. 43/SEQ ID NO. 44 (called
RV-A9 herein), SEQ ID NO. 45/SEQ ID NO. 46 (called RV-F8 herein),
SEQ ID NO. 47/SEQ ID NO. 48 (called RV-H2 herein), SEQ ID NO.
49/SEQ ID NO. 50 (called RV-H4 herein), SEQ ID NO. 51/SEQ ID NO. 52
(called RV-H5 herein), SEQ ID NO. 53/SEQ ID NO. 54 (called C1
herein), SEQ ID NO. 55/SEQ ID NO. 56 (called VR-A2 herein), SEQ ID
NO. 57/SEQ ID NO. 58 (called VR-A3 herein), SEQ ID NO. 59/SEQ ID
NO. 60 (called VR-A10 herein), SEQ ID NO. 61/SEQ ID NO. 62 (called
VR-B2 herein), SEQ ID NO. 63/SEQ ID NO. 64 (called VR-B4 herein),
SEQ ID NO. 65/SEQ ID NO. 66 (called VR-B11 herein), SEQ ID NO.
67/SEQ ID NO. 68 (called VR-05 herein), SEQ ID NO. 69/SEQ ID NO. 70
(called VR-C7 herein), SEQ ID NO. 71/SEQ ID NO. 72 (called VR-C11
herein), SEQ ID NO. 73/SEQ ID NO. 74 (called VR-E3 herein), SEQ ID
NO. 75/SEQ ID NO. 76 (called VR-G11 herein), SEQ ID NO. 77/SEQ ID
NO. 78 (called VK-B8 herein), SEQ ID NO. 79/SEQ ID NO. 80 (called
VR-H9 herein), SEQ ID NO. 77/SEQ ID NO. 81 (called VK-B8A herein),
and combinations thereof. Preferably, the fully human single chain
antibody has both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34,
SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID
NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.
43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID
NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID
NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70,
SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID
NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, and combinations
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows SDS-PAGE analysis of VK-B8, an exemplary
anti-VEGFR2 antibody disclosed herein and a commercially available
therapeutic monoclonal antibody.
[0021] FIG. 2 shows how VK-B8 blocks VEGF binding to soluble
VEGFR2-Fc with an IC.sub.50 of about 7.7.times.10.sup.-10 M.
[0022] FIG. 3 shows VK-B8 cell binding to HUVE cells and an
EC.sub.50 of about 1.299.sup.-9 M.
[0023] FIG. 4 shows VEGF-mediated HUVEC proliferation at 100 mg/ml
VEGF. VK-B8 was compared to Bevacizumab (Avastin.RTM.) a marketed
anti-VEGF-A (ligand) antibody. VK-B8 shows comparable efficacy to
Bevacizumab (Avastin.RTM.) in this in vitro model.
[0024] FIG. 5 shows VEGF-stimulated autophosphorylation at 100
ng/ml VEGF of the VEGFR2 receptor in HUVECs. Various anti-VEGFR2
antibodies were compared at an antibody concentration of 10
.mu.g/ml. VK-B8 is the third column from the left.
[0025] FIG. 6 shows overlaid ANSEC (analytical size-exclusion
chromatography) chromatograms of VK-B8 and Avastin.RTM. in PBS
buffer at pH 6.8: STD/standard run (grey dotted line), VK-B8
spectrum (red), Avastin.RTM. spectrum (green).
[0026] FIGS. 7A and 7B show overlaid ANSEC chromatograms (Ultra
Violet trace at 280 nm) of VK-B8 in PBS buffer at pH 6.8
immediately after purification and after 4 months at 4.degree. C.:
VK-B8 immediately after purification spectrum (blue), VK-B8 after 4
months at 4.degree. C. spectrum (green). FIG. 8B is a zoomed in
region of 8A to examine baseline fluctuations.
[0027] FIG. 8 shows an effect of two antibodies on MC38 colon tumor
growth. VK-B8 was compared to Bevacizumab (Avastin.RTM.) a marketed
anti-VEGF-A (ligand) antibody. VK-B8 shows superior efficacy to
Bevacizumab (Avastin) in this in vivo model.
[0028] FIG. 9 shows an effect of two antibodies on A431 epidermoid
carcinoma cell growth. VK-B8 was compared to Bevacizumab (Avastin)
a marketed anti-VEGF-A (ligand) antibody. VK-B8 shows superior
efficacy to Bevacizumab (Avastin.RTM.) in this in vivo model.
[0029] FIG. 10 shows the synergistic combination of an anti-VEGFR2
antibody when administered with an anti-EGFR antibody
(Erbitux.RTM., a humanized anti-EGFR antibody or A6, a fully human
anti-EGFR antibody) inhibiting tumor cell growth in vivo.
[0030] FIG. 11 shows the IC.sub.50 value for inhibition of
VEGF-induced VEGFR2 activation by VK-B8. The IC.sub.50 is 0.12 nM,
that is, inhibition of VEGF-mediated, VEGFR2-activating
autophosphorylation.
[0031] FIG. 12 shows the IC.sub.50 value for the inhibition of
VEGF-induced p44/p42 MAPK (Erk1/2) phosphorylation by VK-B8, or
VEGF-mediated p44/p42 MAPK (Erk1/2) phosphorylation. The IC.sub.50
is 0.08 nM.
[0032] FIG. 13 shows the IC.sub.50 value for the inhibition of
VEGF-induced HUVEC cell proliferation by VK-B8. The IC.sub.50 is
14.9 nM.
[0033] FIG. 14 shows the IC.sub.50 value for the inhibition of
VEGF-induced HUVEC cell migration by VK-B8. The IC.sub.50 is 0.53
nM.
[0034] FIG. 15 shows the IC.sub.50 value for the inhibition of
VEGF-C-induced, VEGFR2 activation by VK-B8 to show VEGF-C-mediated,
VEGFR2-activating autophosphorylation. The IC.sub.50 is 1.9 nM.
DETAILED DESCRIPTION
[0035] The present disclosure provides a fully human antibody of an
IgG class that binds to a VEGFR2 epitope with a binding affinity of
100 nM or less, that has a heavy chain variable domain sequence
that is at least 95% identical to the amino acid sequences selected
from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO.
5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23,
SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID
NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,
SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID
NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,
SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID
NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77,
SEQ ID NO. 79, SEQ ID NO. 81, and combinations thereof, and that
has a light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ
ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.
44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ
ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO.
62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ
ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, and combinations thereof. Preferably, the fully
human antibody has both a heavy chain and a light chain wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2
(called VB-A2 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called VB-A3
herein), SEQ ID NO. 5/SEQ ID NO. 6 (called VB-A7 herein), SEQ ID
NO. 7/SEQ ID NO. 8 (called VBV-A7 herein), SEQ ID NO. 9/SEQ ID NO.
10 (called VB-A9 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called
VB-A10 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called VB-B6 herein),
SEQ ID NO. 15/SEQ ID NO. 16 (called VB-B10 herein), SEQ ID NO.
17/SEQ ID NO. 18 (called VB-D5 herein), SEQ ID NO. 19/SEQ ID NO. 20
(called VB-D6 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called VB-D11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called VB-E1 herein), SEQ ID
NO. 25/SEQ ID NO. 26 (called VB-E2 herein), SEQ ID NO. 27/SEQ ID
NO. 28 (called VB-E7 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called
VB-F2 herein), SEQ ID NO. 31/SEQ ID NO. 32 (called VB-F8 herein),
SEQ ID NO. 33/SEQ ID NO. 34 (called VB-G4 herein), SEQ ID NO.
35/SEQ ID NO. 36 (called VB-G6 herein), SEQ ID NO. 37/SEQ ID NO. 38
(called VB-H4 herein), SEQ ID NO. 39/SEQ ID NO. 40 (called VB-H7
herein), SEQ ID NO. 41/SEQ ID NO. 42 (called VB-H9 herein), SEQ ID
NO. 43/SEQ ID NO. 44 (called RV-A9 herein), SEQ ID NO. 45/SEQ ID
NO. 46 (called RV-F8 herein), SEQ ID NO. 47/SEQ ID NO. 48 (called
RV-H2 herein), SEQ ID NO. 49/SEQ ID NO. 50 (called RV-H4 herein),
SEQ ID NO. 51/SEQ ID NO. 52 (called RV-H5 herein), SEQ ID NO.
53/SEQ ID NO. 54 (called C1 herein), SEQ ID NO. 55/SEQ ID NO. 56
(called VR-A2 herein), SEQ ID NO. 57/SEQ ID NO. 58 (called VR-A3
herein), SEQ ID NO. 59/SEQ ID NO. 60 (called VR-A10 herein), SEQ ID
NO. 61/SEQ ID NO. 62 (called VR-B2 herein), SEQ ID NO. 63/SEQ ID
NO. 64 (called VR-B4 herein), SEQ ID NO. 65/SEQ ID NO. 66 (called
VR-B11 herein), SEQ ID NO. 67/SEQ ID NO. 68 (called VR-05 herein),
SEQ ID NO. 69/SEQ ID NO. 70 (called VR-C7 herein), SEQ ID NO.
71/SEQ ID NO. 72 (called VR-C11 herein), SEQ ID NO. 73/SEQ ID NO.
74 (called VR-E3 herein), SEQ ID NO. 75/SEQ ID NO. 76 (called
VR-G11 herein), SEQ ID NO. 77/SEQ ID NO. 78 (called VK-B8 herein),
SEQ ID NO. 79/SEQ ID NO. 80 (called VR-H9 herein), SEQ ID NO.
77/SEQ ID NO. 81 (called VK-B8 herein), and combinations
thereof.
[0036] The present disclosure provides a fully human antibody Fab
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.
37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ
ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.
55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ
ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO.
73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40,
SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID
NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58,
SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID
NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76,
SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof. Preferably,
the fully human antibody Fab fragment has both a heavy chain
variable domain region and a light chain variable domain region
wherein the antibody has a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID
NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ
ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.
16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ
ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34,
SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID
NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.
43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID
NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID
NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70,
SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID
NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, and combinations
thereof.
[0037] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ
ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO.
25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ
ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.
43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ
ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO.
61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ
ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO.
79, SEQ ID NO. 81, and combinations thereof, and that has a light
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10,
SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID
NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28,
SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID
NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,
SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID
NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64,
SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID
NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, and
combinations thereof. Preferably, the fully human single chain
antibody has both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34,
SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID
NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.
43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID
NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52,
SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID
NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID NO.
61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID
NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID NO. 70,
SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID
NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID NO.
79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, and combinations
thereof.
[0038] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers, comprising
administering an effective amount of an anti-VEGFR2 polypeptide,
wherein the anti-VEGFR2 polypeptide is selected from the group
consisting of a fully human antibody of an IgG class that binds to
a VEGFR2 epitope with a binding affinity of at least 10.sup.-6M, a
fully human antibody Fab fragment, having a variable domain region
from a heavy chain and a variable domain region from a light chain,
a single chain human antibody, having a variable domain region from
a heavy chain and a variable domain region from a light chain and a
peptide linker connecting the heavy chain and light chain variable
domain regions, and combinations thereof;
[0039] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting SEQ ID NO. 1, SEQ ID
NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ
ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO.
21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ
ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO.
39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ
ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO.
57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ
ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO.
75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and combinations
thereof, and that has a light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID
NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42,
SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID
NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,
SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID
NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78,
SEQ ID NO. 80, and combinations thereof;
[0040] wherein the fully human antibody Fab fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID
NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,
SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID
NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63,
SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID
NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81,
and combinations thereof, and that has the light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID
NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48,
SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID
NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66,
SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID
NO. 76, SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof;
and
[0041] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.
37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ
ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.
55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ
ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO.
73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, and
combinations thereof, and that has the light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40,
SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID
NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58,
SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID
NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76,
SEQ ID NO. 78, SEQ ID NO. 80, and combinations thereof.
[0042] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.
10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ
ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID
NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.
37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID
NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46,
SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID
NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO.
55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID
NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64,
SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID
NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO.
73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID
NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81,
and combinations thereof. Preferably, the fully human antibody Fab
fragment has both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36,
SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID
NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO.
45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID
NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54,
SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID
NO. 59/SEQ ID NO. 60, SEQ ID NO. 61/SEQ ID NO. 62, SEQ ID NO.
63/SEQ ID NO. 64, SEQ ID NO. 65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID
NO. 68, SEQ ID NO. 69/SEQ ID NO. 70, SEQ ID NO. 71/SEQ ID NO. 72,
SEQ ID NO. 73/SEQ ID NO. 74, SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID
NO. 77/SEQ ID NO. 78, SEQ ID NO. 79/SEQ ID NO. 80, SEQ ID NO.
77/SEQ ID NO. 81, and combinations thereof. Preferably, the fully
human single chain antibody has both a heavy chain variable domain
region and a light chain variable domain region, wherein the single
chain fully human antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting SEQ ID NO. 1/SEQ
ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ
ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.
11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID
NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20,
SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID
NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.
29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID
NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38,
SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID
NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.
47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID
NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56,
SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, SEQ ID
NO. 61/SEQ ID NO. 62, SEQ ID NO. 63/SEQ ID NO. 64, SEQ ID NO.
65/SEQ ID NO. 66, SEQ ID NO. 67/SEQ ID NO. 68, SEQ ID NO. 69/SEQ ID
NO. 70, SEQ ID NO. 71/SEQ ID NO. 72, SEQ ID NO. 73/SEQ ID NO. 74,
SEQ ID NO. 75/SEQ ID NO. 76, SEQ ID NO. 77/SEQ ID NO. 78, SEQ ID
NO. 79/SEQ ID NO. 80, SEQ ID NO. 77/SEQ ID NO. 81, and combinations
thereof.
[0043] Preferably, the mammalian cancer to be treated is selected
from the group consisting of ovarian, colon, breast or hepatic
carcinoma cell lines, myelomas, neuroblastic-derived CNS tumors,
monocytic leukemias, B-cell derived leukemias, T-cell derived
leukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast
cell derived tumors, and combinations thereof.
[0044] By "inhibit" is meant a measurable reduction in a
phenomenon, often used herein in reference to any of the following:
the interaction of VEGF with a VEGFR, VEGF- or VEGFR-mediated
angiogenesis, angiogenesis, symptoms of angiogenesis, the viability
of VEGFR-containing cells, the viability of VEGF-dependent Ba/F3
cells, or VEGF- or VEGFR-mediated cellular proliferation as
compared to a control sample not treated with the polypeptide. A
polypeptide will inhibit a VEGF- or VEGFR2 mediated activity if the
reduction in activity or interaction is at least 10%, preferably
20%, 30%, 40%, or 50%, and more preferably 60%, 70%, 80%, 90% or
more.
[0045] By "VEGF biological activity" is meant any function of any
VEGF family member acting through any VEGF receptor, but
particularly signaling through a VEGFR2 receptor. The VEGF ligand
family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental
growth factor (PIGF), as well as various alternatively spliced
forms of VEGF including VEGF121, VEGF145, VEGF165, VEGF189, and
VEGF206 (Tischer et al., J. Biol. Chem, 266:11947-11954, 1991). The
VEGFR family of tyrosine kinase receptors includes VEGFR-1 (also
known as Flt-1), VEGFR2 (also known as KDR (human form) or Flk-1
(mouse form)), and VEGFR-3 (also known as Flt-4). VEGF ligands bind
to the VEGF receptors to induce, for example, angiogenesis,
vasculogenesis, endothelial cell proliferation, vasodilation, and
cell migration. VEGFR2 is believed to be the VEGFR most involved in
angiogenesis. A VEGFR2 or KDR-mediated biological activity is any
biological function in which VEGFR2 or KDR participates in
significantly, such that antagonism of VEGFR2 or KDR causes a
measurable decrease in the biological activity. Methods for
measuring angiogenesis are standard, and are described, for
example, in Jain et al. (Nat. Rev. Cancer 2:266-276, 2002).
Angiogenesis can be assayed by measuring the number of
non-branching blood vessel segments (number of segments per unit
area), the functional vascular density (total length of perfused
blood vessel per unit area), the vessel diameter, the formation of
vascular channels, or the vessel volume density (total of
calculated blood vessel volume based on length and diameter of each
segment per unit area). Exemplary assays for VEGF-mediated
proliferation and angiogenesis can be found in U.S. Pat. No.
6,559,126, the disclosure of which is incorporated by reference
herein, Lyden et al, Nature Medicine 7:1194 (2001), Jacob et al,
Exp. Pathol. 15:1234 (1978) and Bae et al, J. Biol. Chem. 275:13588
(2000). These assays can be performed using either purified
receptor or ligand or both, and can be performed in vitro or in
vivo. These assays can also be performed in cells using a
genetically introduced or the naturally-occurring ligand or
receptor or both. A polypeptide that inhibits the biological
activity of VEGF will cause a decrease of at least 10%, preferably
20%, 30%, 40%, or 50%, and more preferably 60%, 70%, 80%, 90% or
greater decrease in the biological activity of VEGF. The inhibition
of biological activity can also be measured by the IC.sub.50.
Preferably, a polypeptide that inhibits the biological activity of
VEGF or VEGFR2 will have an IC.sub.50 of less than 100 nM, more
preferably less than 10 nM and most preferably less than 1 nM.
[0046] Polypeptides of the present invention can be produced using
any standard methods. In one example, the polypeptides are produced
by recombinant DNA methods by inserting a nucleic acid sequence
(e.g., a cDNA) encoding the polypeptide into a recombinant
expression vector and expressing the DNA sequence under conditions
promoting expression.
[0047] Nucleic acids encoding any of the various polypeptides
disclosed herein may be synthesized chemically. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003
100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002
(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9;
Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al.
Yeast. 1991 7(7):657-78.
[0048] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press,
2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular
Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic updates, herein incorporated by reference. The DNA
encoding the polypeptide is operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, viral, or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences that control the termination of
transcription and translation.
[0049] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a poly-histidine tag, a
FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning
and expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, N.Y., 1985).
[0050] The expression construct is introduced into the host cell
using a method appropriate to the host cell. A variety of methods
for introducing nucleic acids into host cells are known, including,
but not limited to, electroporation; transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent). Suitable host
cells include prokaryotes, yeast, mammalian cells, or bacterial
cells.
[0051] Suitable bacteria include gram negative or gram positive
organisms, for example, E. coli or Bacillus spp. Yeast, preferably
from the Saccharomyces species, such as S. cerevisiae, may also be
used for production of polypeptides. Various mammalian or insect
cell culture systems can also be employed to express recombinant
proteins. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
(Bio/Technology, 6:47, 1988). Examples of suitable mammalian host
cell lines include endothelial cells, COS-7 monkey kidney cells,
CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human
embryonic kidney cells, HeLa, 293, and BHK cell lines. Purified
polypeptides are prepared by culturing suitable host/vector systems
to express the recombinant proteins. For many applications, the
small size of many of the polypeptides disclosed herein would make
expression in E. coli as the preferred method for expression. The
protein is then purified from culture media or cell extracts.
[0052] Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes the nucleic acids
encoding the polypeptide must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system.
[0053] The polypeptide can be purified by isolation/purification
methods for proteins generally known in the field of protein
chemistry. Non-limiting examples include extraction,
recrystallization, salting out (e.g., with ammonium sulfate or
sodium sulfate), centrifugation, dialysis, ultrafiltration,
adsorption chromatography, ion exchange chromatography, hydrophobic
chromatography, normal phase chromatography, reversed-phase
chromatography, gel filtration, gel permeation chromatography,
affinity chromatography, electrophoresis, countercurrent
distribution or any combinations of these. After purification,
polypeptides may be exchanged into different buffers and/or
concentrated by any of a variety of methods known to the art,
including, but not limited to, filtration and dialysis.
[0054] The purified polypeptide is preferably at least 85% pure,
more preferably at least 95% pure, and most preferably at least 98%
pure. Regardless of the exact numerical value of the purity, the
polypeptide is sufficiently pure for use as a pharmaceutical
product.
Post-Translational Modifications of Polypeptides
[0055] In certain embodiments, the binding polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modifications include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. A preferred form of glycosylation
is sialylation, which conjugates one or more sialic acid moieties
to the polypeptide. Sialic acid moieties improve solubility and
serum half-life while also reducing the possible immunogeneticity
of the protein. See, e.g., Raju et al. Biochemistry. 2001 31;
40(30):8868-76. Effects of such non-amino acid elements on the
functionality of a polypeptide may be tested for its antagonizing
role of VEGFR2 or VEGF function, e.g., its inhibitory effect on
angiogenesis or on tumor growth.
[0056] In one specific embodiment, modified forms of the subject
soluble polypeptides comprise linking the subject soluble
polypeptides to nonproteinaceous polymers. In one specific
embodiment, the polymer is polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner as set
forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337. Examples of the modified polypeptide
include PEGylated VK-B8.
[0057] PEG is a water soluble polymer that is commercially
available or can be prepared by ring-opening polymerization of
ethylene glycol according to methods well known in the art (Sandler
and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,
pages 138-161). The term "PEG" is used broadly to encompass any
polyethylene glycol molecule, without regard to size or to
modification at an end of the PEG, and can be represented by the
formula: X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).
[0058] A variety of molecular mass forms of PEG can be selected,
e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to
2300), for conjugating to VEGFR2 binding polypeptides. The number
of repeating units "n" in the PEG is approximated for the molecular
mass described in Daltons. It is preferred that the combined
molecular mass of PEG on an activated linker is suitable for
pharmaceutical use. Thus, in one embodiment, the molecular mass of
the PEG molecules does not exceed 100,000 Da. For example, if three
PEG molecules are attached to a linker, where each PEG molecule has
the same molecular mass of 12,000 Da (each n is about 270), then
the total molecular mass of PEG on the linker is about 36,000 Da
(total n is about 820). The molecular masses of the PEG attached to
the linker can also be different, e.g., of three molecules on a
linker two PEG molecules can be 5,000 Da each (each n is about 110)
and one PEG molecule can be 12,000 Da (n is about 270).
[0059] In a specific embodiment of the invention, a VEGFR2 binding
polypeptide is covalently linked to one poly(ethylene glycol) group
of the formula:
--CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR, with the
--CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an
amide bond with one of the amino groups of the binding polypeptide;
R being lower alkyl; x being 2 or 3; m being from about 450 to
about 950; and n and m being chosen so that the molecular weight of
the conjugate minus the binding polypeptide is from about 10 to 40
kDa. In one embodiment, a binding polypeptide's 6-amino group of a
lysine is the available (free) amino group.
[0060] The above conjugates may be more specifically presented by
formula (II):
P--NHCO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR (II),
wherein P is the group of a binding polypeptide as described
herein, (i.e. without the amino group or amino groups which form an
amide linkage with the carbonyl shown in formula (II); and wherein
R is lower alkyl; x is 2 or 3; m is from about 450 to about 950 and
is chosen so that the molecular weight of the conjugate minus the
binding polypeptide is from about 10 to about 40 kDa. As used
herein, the given ranges of "m" have an orientational meaning. The
ranges of "m" are determined in any case, and exactly, by the
molecular weight of the PEG group.
[0061] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-binding polypeptide conjugates.
N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with
PEG to form active mixed PEG-succinimidyl carbonate that may be
subsequently reacted with a nucleophilic group of a linker or an
amino group of a binding polypeptide (see U.S. Pat. Nos. 5,281,698
and 5,932,462). In a similar type of reaction,
1,1'-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may be
reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed
carbonate (U.S. Pat. No. 5,382,657), respectively.
[0062] In some embodiments, the pegylated binding polypeptide
comprises a PEG molecule covalently attached to the alpha amino
group of the N-terminal amino acid. Site specific N-terminal
reductive amination is described in Pepinsky et al., (2001) JPET,
297, 1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde
for the reductive amination of a protein utilizing other available
nucleophilic amino groups is described in U.S. Pat. No. 4,002,531,
in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow
et al., (1994) Bioconjugate Chem. 5, 133.
[0063] In another embodiment, pegylated binding polypeptide
comprises one or more PEG molecules covalently attached to a
linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the binding polypeptide.
Such an approach is disclosed in U.S. Patent Publication No.
2002/0044921 and in WO094/01451.
[0064] In one embodiment, a binding polypeptide is pegylated at the
C-terminus. In a specific embodiment, a protein is pegylated at the
C-terminus by the introduction of C-terminal azido-methionine and
the subsequent conjugation of a methyl-PEG-triarylphosphine
compound via the Staudinger reaction. This C-terminal conjugation
method is described in Cazalis et al., Bioconjug. Chem. 2004;
15(5):1005-1009.
[0065] The ratio of a binding polypeptide to activated PEG in the
conjugation reaction can be from about 1:0.5 to 1:50, between from
about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous
buffers can be used in the present method to catalyze the covalent
addition of PEG to the binding polypeptide. In one embodiment, the
pH of a buffer used is from about 7.0 to 9.0. In another
embodiment, the pH is in a slightly basic range, e.g., from about
7.5 to 8.5. Buffers having a pKa close to neutral pH range may be
used, e.g., phosphate buffer.
[0066] Conventional separation and purification techniques known in
the art can be used to purify PEGylated binding polypeptide, such
as size exclusion (e.g. gel filtration) and ion exchange
chromatography. Products may also be separated using SDS-PAGE.
Products that may be separated include mono-, di-, tri- poly- and
un-pegylated binding polypeptide, as well as free PEG. The
percentage of mono-PEG conjugates can be controlled by pooling
broader fractions around the elution peak to increase the
percentage of mono-PEG in the composition. About ninety percent of
mono-PEG conjugates represents a good balance of yield and
activity. Compositions in which, for example, at least ninety-two
percent or at least ninety-six percent of the conjugates are
mono-PEG species may be desired. In an embodiment of this invention
the percentage of mono-PEG conjugates is from ninety percent to
ninety-six percent.
[0067] In one embodiment, PEGylated binding polypeptide of the
invention contain one, two or more PEG moieties. In one embodiment,
the PEG moiety(ies) are bound to an amino acid residue which is on
the surface of the protein and/or away from the surface that
contacts the target ligand. In one embodiment, the combined or
total molecular mass of PEG in PEG-binding polypeptide is from
about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to
36,000 Da. In a one embodiment, the PEG in pegylated binding
polypeptide is a substantially linear, straight-chain PEG.
[0068] In one embodiment of the invention, the PEG in pegylated
binding polypeptide is not hydrolyzed from the pegylated amino acid
residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH
6.5) over 8 to 16 hours at room temperature, and is thus stable. In
one embodiment, greater than 80% of the composition is stable
mono-PEG-binding polypeptide, more preferably at least 90%, and
most preferably at least 95%.
[0069] In another embodiment, the pegylated binding polypeptides of
the invention will preferably retain at least 25%, 50%, 60%, 70%
least 80%, 85%, 90%, 95% or 100% of the biological activity
associated with the unmodified protein. In one embodiment,
biological activity refers to its ability to bind to VEGFR2, as
assessed by KD, k.sub.on or k.sub.off. In one specific embodiment,
the pegylated binding polypeptide protein shows an increase in
binding to VEGFR relative to unpegylated binding polypeptide.
[0070] The serum clearance rate of PEG-modified polypeptide may be
decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even
90%, relative to the clearance rate of the unmodified binding
polypeptide. The PEG-modified polypeptide may have a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. The half-life of PEG-binding polypeptide may be
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified binding
polypeptide. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half-life, such as the half-life of the protein in the serum or
other bodily fluid of an animal.
Therapeutic Formulations and Modes of Administration
[0071] The present disclosure features methods for treating
conditions or preventing pre-conditions which respond to an
inhibition of VEGF biological activity. Preferred examples are
conditions that are characterized by inappropriate angiogenesis.
Techniques and dosages for administration vary depending on the
type of specific polypeptide and the specific condition being
treated but can be readily determined by the skilled artisan. In
general, regulatory agencies require that a protein reagent to be
used as a therapeutic is formulated so as to have acceptably low
levels of pyrogens. Accordingly, therapeutic formulations will
generally be distinguished from other formulations in that they are
substantially pyrogen free, or at least contain no more than
acceptable levels of pyrogen as determined by the appropriate
regulatory agency (e.g., FDA).
[0072] Therapeutic compositions of the present disclosure may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Administration may be parenteral
(e.g., intravenous, subcutaneous), oral, or topical, as
non-limiting examples. In addition, any gene therapy technique,
using nucleic acids encoding the polypeptides of the invention, may
be employed, such as naked DNA delivery, recombinant genes and
vectors, cell-based delivery, including ex vivo manipulation of
patients' cells, and the like.
[0073] The composition can be in the form of a pill, tablet,
capsule, liquid, or sustained release tablet for oral
administration; or a liquid for intravenous, subcutaneous or
parenteral administration; gel, lotion, ointment, cream, or a
polymer or other sustained release vehicle for local
administration.
[0074] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott
Williams & Wilkins, Philadelphia, Pa.). Formulations for
parenteral administration may, for example, contain excipients,
sterile water, saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
used to control the release of the compounds. Nanoparticulate
formulations (e.g., biodegradable nanoparticles, solid lipid
nanoparticles, liposomes) may be used to control the
biodistribution of the compounds. Other potentially useful
parenteral delivery systems include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. The concentration of the compound in the formulation
varies depending upon a number of factors, including the dosage of
the drug to be administered, and the route of administration.
[0075] A therapeutically effective dose refers to a dose that
produces the therapeutic effects for which it is administered. The
exact dose will depend on the disorder to be treated, and may be
ascertained by one skilled in the art using known techniques. In
general, the polypeptide is administered at about 0.01 mg/kg to
about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per
day, and most preferably 0.1 mg/kg to about 20 mg/kg per day. The
polypeptide may be given daily (e.g., once, twice, three times, or
four times daily) or preferably less frequently (e.g., weekly,
every two weeks, every three weeks, monthly, or quarterly). In
addition, as is known in the art, adjustments for age as well as
the body weight, general health, sex, diet, time of administration,
drug interaction, and the severity of the disease may be
necessary.
Exemplary Uses
[0076] The VEGFR2 binding proteins described herein and their
related variants are useful in a number of therapeutic and
diagnostic applications. These include the inhibition of the
biological activity of VEGF by competing for or blocking the
binding to a VEGFR2.
[0077] On the basis of their efficacy as inhibitors of VEGF
biological activity, the polypeptides of the invention are
effective against a number of conditions associated with
inappropriate angiogenesis, including but not limited to autoimmune
disorders (e.g., rheumatoid arthritis, inflammatory bowel disease
or psoriasis); cardiac disorders (e.g., atherosclerosis or blood
vessel restenosis); retinopathies (e.g., proliferative
retinopathies generally, diabetic retinopathy, age-related macular
degeneration or neovascular glaucoma), renal disease (e.g.,
diabetic nephropathy, malignant nephrosclerosis, thrombotic
microangiopathy syndromes; transplant rejection; inflammatory renal
disease; glomerulonephritis; mesangioproliferative
glomerulonephritis; haemolytic-uraemic syndrome; and hypertensive
nephrosclerosis); hemangioblastoma; hemangiomas; thyroid
hyperplasias; tissue transplantations; chronic inflammation;
Meigs's syndrome; pericardial effusion; pleural effusion;
autoimmune diseases; diabetes; endometriosis; chronic asthma;
undesirable fibrosis (particularly hepatic fibrosis) and cancer, as
well as complications arising from cancer, such as pleural effusion
and ascites. Preferably, the VEGFR2-binding polypeptides of the
invention can be used for the treatment or prevention of
hyperproliferative diseases or cancer and the metastatic spread of
cancers. Non-limiting examples of cancers include bladder, blood,
bone, brain, breast, cartilage, colon kidney, liver, lung, lymph
node, nervous tissue, ovary, pancreatic, prostate, skeletal muscle,
skin, spinal cord, spleen, stomach, testes, thymus, thyroid,
trachea, urogenital tract, ureter, urethra, uterus, or vaginal
cancer. Additional treatable conditions can be found in U.S. Pat.
No. 6,524,583, incorporated by reference herein. Other references
describing uses for VEGFR2 binding polypeptides include: McLeod et
al., Invest. Ophthalmol. Vis. Sci. 2002; 43(2):474-82; Watanabe et
al., Exp. Dermatol. 2004; 13(11):671-81; Yoshiji et al., Gut. 2003
52(9):1347-54; Verheul et al., Oncologist. 2000; 5 Suppl 1:45-50;
and Boldicke et al., Stem Cells. 2001 19(1):24-36.
[0078] As described herein, angiogenesis-associated diseases
include, but are not limited to, angiogenesis-dependent cancer,
including, for example, solid tumors, blood born tumors such as
leukemias, and tumor metastases; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; inflammatory disorders such as immune and
non-immune inflammation; chronic articular rheumatism and
psoriasis; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial
angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; and wound granulation and wound
healing; telangiectasia psoriasis scleroderma, pyogenic granuloma,
coronary collaterals, ischemic limb angiogenesis, corneal diseases,
rubeosis, arthritis, diabetic neovascularization, fractures,
vasculogenesis, hematopoiesis.
[0079] A VEGFR2 binding polypeptide can be administered alone or in
combination with one or more additional therapies such as
chemotherapy, radiotherapy, immunotherapy, surgical intervention,
or any combination of these. Long-term therapy is equally possible
as is adjuvant therapy in the context of other treatment
strategies, as described above.
[0080] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0081] In certain embodiments, the subject anti-VEGFR2 antibodies
agents of the invention can be used alone. Alternatively, the
subject agents may be used in combination with other conventional
anti-cancer therapeutic approaches directed to treatment or
prevention of proliferative disorders (e.g., tumor). For example,
such methods can be used in prophylactic cancer prevention,
prevention of cancer recurrence and metastases after surgery, and
as an adjuvant of other conventional cancer therapy. The present
disclosure recognizes that the effectiveness of conventional cancer
therapies (e.g., chemotherapy, radiation therapy, phototherapy,
immunotherapy, and surgery) can be enhanced through the use of a
subject polypeptide therapeutic agent.
[0082] A wide array of conventional compounds has been shown to
have anti-neoplastic activities. These compounds have been used as
pharmaceutical agents in chemotherapy to shrink solid tumors,
prevent metastases and further growth, or decrease the number of
malignant cells in leukemic or bone marrow malignancies. Although
chemotherapy has been effective in treating various types of
malignancies, many anti-neoplastic compounds induce undesirable
side effects. It has been shown that when two or more different
treatments are combined, the treatments may work synergistically
and allow reduction of dosage of each of the treatments, thereby
reducing the detrimental side effects exerted by each compound at
higher dosages. In other instances, malignancies that are
refractory to a treatment may respond to a combination therapy of
two or more different treatments.
[0083] When a polypeptide therapeutic agent of the present
invention is administered in combination with another conventional
anti-neoplastic agent, either concomitantly or sequentially, such
therapeutic agent may be found to enhance the therapeutic effect of
the anti-neoplastic agent or overcome cellular resistance to such
anti-neoplastic agent. This allows decrease of dosage of an
anti-neoplastic agent, thereby reducing the undesirable side
effects, or restores the effectiveness of an anti-neoplastic agent
in resistant cells.
[0084] Pharmaceutical compounds that may be used for combinatory
anti-tumor therapy include, merely to illustrate:
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
mechlorethamine, medroxyprogesterone, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,
paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0085] Certain chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into, for example,
following groups: anti-metabolites/anti-cancer agents, such as
pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,
gemcitabine and cytarabine) and purine analogs, folate antagonists
and related inhibitors (mercaptopurine, thioguanine, pentostatin
and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes-dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic
compounds (TNP-470, genistein) and growth factor inhibitors (e.g.,
VEGF inhibitors, fibroblast growth factor (FGF) inhibitors);
angiotensin receptor blocker; nitric oxide donors; anti-sense
oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors
and differentiation inducers (tretinoin); mTOR inhibitors,
topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,
camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,
etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),
corticosteroids (cortisone, dexamethasone, hydrocortisone,
methylpednisolone, prednisone, and prenisolone); growth factor
signal transduction kinase inhibitors; mitochondrial dysfunction
inducers and caspase activators; and chromatin disruptors.
[0086] In certain embodiments, pharmaceutical compounds that may be
used for combinatory anti-angiogenesis therapy include: (1)
inhibitors of release of "angiogenic molecules," such as bFGF
(basic fibroblast growth factor); (2) neutralizers of angiogenic
molecules, such as an anti-.beta.FGF antibodies; and (3) inhibitors
of endothelial cell response to angiogenic stimuli, including
collagenase inhibitor, basement membrane turnover inhibitors,
angiostatic steroids, fungal-derived angiogenesis inhibitors,
platelet factor 4, thrombospondin, arthritis drugs such as
D-penicillamine and gold thiomalate, vitamin D.sub.3 analogs,
alpha-interferon, and the like. For additional proposed inhibitors
of angiogenesis, see Blood et al., Bioch. Biophys. Acta.
1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990),
Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos.
5,092,885; 5,112,946; 5,192,744; 5,202,352; and 6,573,256. In
addition, there are a wide variety of compounds that can be used to
inhibit angiogenesis, for example, endostatin protein or
derivatives, lysine binding fragments of angiostatin, melanin or
melanin-promoting compounds, plasminogen fragments (e.g., Kringles
1-3 of plasminogen), tropoin subunits, antagonists of vitronectin
.alpha..sub.v.beta., peptides derived from Saposin B, antibiotics
or analogs (e.g., tetracycline, or neomycin), dienogest-containing
compositions, compounds comprising a MetAP-2 inhibitory core
coupled to a peptide, the compound EM-138, chalcone and its
analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.
6,395,718; 6,462,075; 6,465,431; 6,475,784; 6,482,802; 6,482,810;
6,500,431; 6,500,924; 6,518,298; 6,521,439; 6,525,019; 6,538,103;
6,544,758; 6,544,947; 6,548,477; 6,559,126; and 6,569,845).
[0087] Depending on the nature of the combinatory therapy,
administration of the polypeptide therapeutic agents may be
continued while the other therapy is being administered and/or
thereafter. Administration of the polypeptide therapeutic agents
may be made in a single dose, or in multiple doses. In some
instances, administration of the polypeptide therapeutic agents is
commenced at least several days prior to the conventional therapy,
while in other instances, administration is begun either
immediately before or at the time of the administration of the
conventional therapy.
[0088] The VEGFR2 binding proteins described herein can also be
detectably labeled and used to contact cells expressing VEGFR2 for
imaging applications or diagnostic applications. For diagnostic
purposes, the polypeptide of the invention is preferably
immobilized on a solid support. Preferred solid supports include
columns (for example, affinity columns, such as agarose-based
affinity columns), microchips, or beads.
[0089] In one example of a diagnostic application, a biological
sample, such as serum or a tissue biopsy, from a patient suspected
of having a condition characterized by inappropriate angiogenesis
is contacted with a detectably labeled polypeptide of the invention
to detect levels of VEGFR2. The levels of VEGFR2 detected are then
compared to levels of VEGFR2 detected in a normal sample also
contacted with the labeled polypeptide. An increase of at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in the levels of the
VEGFR2 may be considered a diagnostic indicator of a condition
characterized by inappropriate angiogenesis.
[0090] In certain embodiments, the VEGFR2 binding polypeptides of
the invention are further attached to a label that is able to be
detected (e.g., the label can be a radioisotope, fluorescent
compound, enzyme or enzyme co-factor). The active moiety may be a
radioactive agent, such as: radioactive heavy metals such as iron
chelates, radioactive chelates of gadolinium or manganese, positron
emitters of oxygen, nitrogen, iron, carbon, or gallium, .sup.43K,
.sup.52Fe, .sup.57Co, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.123I,
.sup.125I, .sup.131I, .sup.132I, or .sup.99Tc. A binding agent
affixed to such a moiety may be used as an imaging agent and is
administered in an amount effective for diagnostic use in a mammal
such as a human and the localization and accumulation of the
imaging agent is then detected. The localization and accumulation
of the imaging agent may be detected by radio scintigraphy, nuclear
magnetic resonance imaging, computed tomography or positron
emission tomography. Immunoscintigraphy using VEGFR2 binding
polypeptides directed at VEGFR2 may be used to detect and/or
diagnose cancers and vasculature. For example, any of the binding
polypeptide against the VEGFR2 marker labeled with
.sup.99Technetium, .sup.111Indium, or .sup.125Iodine may be
effectively used for such imaging. As will be evident to the
skilled artisan, the amount of radioisotope to be administered is
dependent upon the radioisotope. Those having ordinary skill in the
art can readily formulate the amount of the imaging agent to be
administered based upon the specific activity and energy of a given
radionuclide used as the active moiety. Typically a person skilled
in the art administers 0.1-100 millicuries per dose of imaging
agent, preferably 1-10 millicuries, most often 2-5 millicuries.
Thus, compositions according to the present invention useful as
imaging agents comprising a targeting moiety conjugated to a
radioactive moiety comprise 0.1-100 millicuries, in some
embodiments preferably 1-10 millicuries, in some embodiments
preferably 2-5 millicuries, in some embodiments more preferably 1-5
millicuries.
[0091] The VEGFR2 binding polypeptides can also be used to deliver
additional therapeutic agents (including but not limited to drug
compounds, chemotherapeutic compounds, and radiotherapeutic
compounds) to a cell or tissue expressing VEGFR2. In one example,
the VEGFR2 binding polypeptide is fused to a chemotherapeutic agent
for targeted delivery of the chemotherapeutic agent to a tumor cell
or tissue expressing VEGFR2.
[0092] The VEGFR2 binding polypeptides are useful in a variety of
applications, including research, diagnostic and therapeutic
applications. For instance, they can be used to isolate and/or
purify receptor or portions thereof, and to study receptor
structure (e.g., conformation) and function.
[0093] In certain aspects, the various binding polypeptides can be
used to detect or measure the expression of VEGFR2, for example, on
endothelial cells (e.g., venous endothelial cells), or on cells
transfected with a VEGFR2 gene. Thus, they also have utility in
applications such as cell sorting and imaging (e.g., flow
cytometry, and fluorescence activated cell sorting), for diagnostic
or research purposes.
[0094] In certain embodiments, the binding polypeptides of
fragments thereof can be labeled or unlabeled for diagnostic
purposes. Typically, diagnostic assays entail detecting the
formation of a complex resulting from the binding of a binding
polypeptide to VEGFR2. The binding polypeptides or fragments can be
directly labeled, similar to antibodies. A variety of labels can be
employed, including, but not limited to, radionuclides,
fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors and ligands (e.g., biotin, haptens). Numerous
appropriate immunoassays are known to the skilled artisan (see, for
example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and
4,098,876). When unlabeled, the binding polypeptides can be used in
assays, such as agglutination assays. Unlabeled binding
polypeptides can also be used in combination with another (one or
more) suitable reagent which can be used to detect the binding
polypeptide, such as a labeled antibody reactive with the binding
polypeptide or other suitable reagent (e.g., labeled protein
A).
[0095] In one embodiment, the binding polypeptides of the present
invention can be utilized in enzyme immunoassays, wherein the
subject polypeptides are conjugated to an enzyme. When a biological
sample comprising a VEGFR2 protein is combined with the subject
binding polypeptides, binding occurs between the binding
polypeptides and the VEGFR2 protein. In one embodiment, a sample
containing cells expressing a VEGFR2 protein (e.g., endothelial
cells) is combined with the subject antibodies, and binding occurs
between the binding polypeptides and cells bearing a VEGFR2 protein
recognized by the binding polypeptide. These bound cells can be
separated from unbound reagents and the presence of the binding
polypeptide-enzyme conjugate specifically bound to the cells can be
determined, for example, by contacting the sample with a substrate
of the enzyme which produces a color or other detectable change
when acted on by the enzyme. In another embodiment, the subject
binding polypeptides can be unlabeled, and a second, labeled
polypeptide (e.g., an antibody) can be added which recognizes the
subject binding polypeptide.
[0096] In certain aspects, kits for use in detecting the presence
of a VEGFR2 protein in a biological sample can also be prepared.
Such kits will include a VEGFR2 binding polypeptide which binds to
a VEGFR2 protein or portion of said receptor, as well as one or
more ancillary reagents suitable for detecting the presence of a
complex between the binding polypeptide and the receptor protein or
portions thereof. The polypeptide compositions of the present
invention can be provided in lyophilized form, either alone or in
combination with additional antibodies specific for other epitopes.
The binding polypeptides and/or antibodies, which can be labeled or
unlabeled, can be included in the kits with adjunct ingredients
(e.g., buffers, such as Tris, phosphate and carbonate, stabilizers,
excipients, biocides and/or inert proteins, e.g., bovine serum
albumin). For example, the binding polypeptides and/or antibodies
can be provided as a lyophilized mixture with the adjunct
ingredients, or the adjunct ingredients can be separately provided
for combination by the user. Generally these adjunct materials will
be present in less than about 5% weight based on the amount of
active binding polypeptide or antibody, and usually will be present
in a total amount of at least about 0.001% weight based on
polypeptide or antibody concentration. Where a second antibody
capable of binding to the binding polypeptide is employed, such
antibody can be provided in the kit, for instance in a separate
vial or container. The second antibody, if present, is typically
labeled, and can be formulated in an analogous manner with the
antibody formulations described above.
[0097] Similarly, the present disclosure also provides a method of
detecting and/or quantitating expression of VEGFR2, wherein a
composition comprising a cell or fraction thereof (e.g., membrane
fraction) is contacted with a binding polypeptide which binds to a
VEGFR2 or portion of the receptor under conditions appropriate for
binding thereto, and the binding is monitored. Detection of the
binding polypeptide, indicative of the formation of a complex
between binding polypeptide and VEGFR2 or a portion thereof,
indicates the presence of the receptor. Binding of a polypeptide to
the cell can be determined by standard methods, such as those
described in the working examples. The method can be used to detect
expression of VEGFR2 on cells from an individual. Optionally, a
quantitative expression of VEGFR2 on the surface of endothelial
cells can be evaluated, for instance, by flow cytometry, and the
staining intensity can be correlated with disease susceptibility,
progression or risk.
[0098] The present disclosure also provides a method of detecting
the susceptibility of a mammal to certain diseases. To illustrate,
the method can be used to detect the susceptibility of a mammal to
diseases which progress based on the amount of VEGFR2 present on
cells and/or the number of VEGFR2-positive cells in a mammal. In
one embodiment, the invention relates to a method of detecting
susceptibility of a mammal to a tumor. In this embodiment, a sample
to be tested is contacted with a binding polypeptide which binds to
a VEGFR2 or portion thereof under conditions appropriate for
binding thereto, wherein the sample comprises cells which express
VEGFR2 in normal individuals. The binding and/or amount of binding
is detected, which indicates the susceptibility of the individual
to a tumor, wherein higher levels of receptor correlate with
increased susceptibility of the individual to a tumor.
[0099] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0100] The terms "VEGFR2 inhibitor" and "VEGFR2 antagonist" are
used interchangeably. Each is a molecule that detectably inhibits
at least one function of VEGFR2. Conversely, a "VEGFR2 agonist" is
a molecule that detectably increases at least one function of
VEGFR2. The inhibition caused by a VEGFR2 inhibitor need not be
complete so long as it is detectable using an assay. Any assay of a
function of VEGFR2 can be used, examples of which are provided
herein. Examples of functions of VEGFR2 that can be inhibited by a
VEGFR2 inhibitor, or increased by a VEGFR2 agonist, include cancer
cell growth or apoptosis (programmed cell death), and so on.
Examples of types of VEGFR2 inhibitors and VEGFR2 agonists include,
but are not limited to, VEGFR2 binding polypeptides such as antigen
binding proteins (e.g., VEGFR2 inhibiting antigen binding
proteins), antibodies, antibody fragments, and antibody
derivatives.
[0101] The terms "peptide," "polypeptide" and "protein" each refers
to a molecule comprising two or more amino acid residues joined to
each other by peptide bonds. These terms encompass, e.g., native
and artificial proteins, protein fragments and polypeptide analogs
(such as muteins, variants, and fusion proteins) of a protein
sequence as well as post-translationally, or otherwise covalently
or non-covalently, modified proteins. A peptide, polypeptide, or
protein may be monomeric or polymeric.
[0102] A "variant" of a polypeptide (for example, an antibody)
comprises an amino acid sequence wherein one or more amino acid
residues are inserted into, deleted from and/or substituted into
the amino acid sequence relative to another polypeptide sequence.
Disclosed variants include, for example, fusion proteins.
[0103] A "derivative" of a polypeptide is a polypeptide (e.g., an
antibody) that has been chemically modified, e.g., via conjugation
to another chemical moiety (such as, for example, polyethylene
glycol or albumin, e.g., human serum albumin), phosphorylation, and
glycosylation. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and muteins thereof, examples of which are
described below.
[0104] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. See, for example, Korndorfer
et al., 2003, Proteins: Structure, Function, and Bioinformatics,
Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.
20:639-654. In addition, peptide antibody mimetics ("PAMs") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronectin components as a scaffold.
[0105] An antigen binding protein can have, for example, the
structure of a naturally occurring immunoglobulin. An
"immunoglobulin" is a tetrameric molecule. In a naturally occurring
immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Preferably, the anti-VEGFR2 antibodies disclosed herein are
characterized by their variable domain region sequences in the
heavy V.sub.H and light V.sub.L amino acid sequences. The preferred
antibody is VK-B8, which is a kappa IgG antibody. Within light and
heavy chains, the variable and constant regions are joined by a "J"
region of about 12 or more amino acids, with the heavy chain also
including a "D" region of about 10 more amino acids. See generally,
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). The variable regions of each light/heavy chain pair
form the antibody binding site such that an intact immunoglobulin
has two binding sites.
[0106] The variable regions of naturally occurring immunoglobulin
chains exhibit the same general structure of relatively conserved
framework regions (FR) joined by three hypervariable regions, also
called complementarity determining regions or CDRs. From N-terminus
to C-terminus, both light and heavy chains comprise the domains
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino
acids to each domain is in accordance with the definitions of Kabat
et al. in Sequences of Proteins of Immunological Interest, 5.sup.th
Ed., US Dept. of Health and Human Services, PHS, NIH, NIH
Publication no. 91-3242, 1991. Other numbering systems for the
amino acids in immunoglobulin chains include IMGT.RTM.
(International ImMunoGeneTics information system; Lefranc et al.,
Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and
Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).
[0107] An "antibody" refers to an intact immunoglobulin or to an
antigen binding portion (Fab) thereof that competes with the intact
antibody for specific binding, unless otherwise specified. Antigen
binding portions may be produced by recombinant DNA techniques or
by enzymatic or chemical cleavage of intact antibodies. Antigen
binding portions include, inter alia, Fab, Fab', F(ab').sub.2, Fv,
domain antibodies (dAbs), and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), chimeric
antibodies, diabodies, triabodies, tetrabodies, and polypeptides
that contain at least a portion of an immunoglobulin that is
sufficient to confer specific antigen binding to the
polypeptide.
[0108] A Fab fragment is a monovalent fragment having the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; a F(ab').sub.2 fragment is a
bivalent fragment having two Fab fragments linked by a disulfide
bridge at the hinge region; a Fd fragment has the V.sub.H and
C.sub.H1 domains; an Fv fragment has the V.sub.L and V.sub.H
domains of a single arm of an antibody; and a dAb fragment has a
V.sub.H domain, a V.sub.L domain, or an antigen-binding fragment of
a V.sub.H or V.sub.L domain (U.S. Pat. Nos. 6,846,634 and
6,696,245, the disclosures of which are incorporated by reference
herein).
[0109] A single-chain antibody (scFv) is an antibody in which a
V.sub.L and a V.sub.H region are joined via a linker (e.g., a
synthetic sequence of amino acid residues) to form a continuous
protein chain wherein the linker is long enough to allow the
protein chain to fold back on itself and form a monovalent antigen
binding site (Bird et al., 1988, Science 242:423-26 and Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
[0110] Diabodies are bivalent antibodies comprising two polypeptide
chains, wherein each polypeptide chain comprises V.sub.H and
V.sub.L domains joined by a linker that is too short to allow for
pairing between two domains on the same chain, thus allowing each
domain to pair with a complementary domain on another polypeptide
chain (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the
two polypeptide chains of a diabody are identical, then a diabody
resulting from their pairing will have two identical antigen
binding sites. Polypeptide chains having different sequences can be
used to make a diabody with two different antigen binding sites.
Similarly, tribodies and tetrabodies are antibodies comprising
three and four polypeptide chains, respectively, and forming three
and four antigen binding sites, respectively, which can be the same
or different.
[0111] Complementarity determining regions (CDRs) and framework
regions (FR) of a given antibody may be identified (Sequences of
Proteins of Immunological Interest, 5.sup.th Ed., US Dept. of
Health and Human Services, PHS, NIH, NIH Publication no. 91-3242,
1991). Other numbering systems for the amino acids in
immunoglobulin chains include IMGT.RTM. (International
ImMunoGeneTics information system; Lefranc et al., Dev. Comp.
Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol.
Biol. 309(3):657-670; 2001). One or more CDRs may be incorporated
into a molecule either covalently or noncovalently to make it an
antigen binding protein. An antigen binding protein may incorporate
the CDR(s) as part of a larger polypeptide chain, may covalently
link the CDR(s) to another polypeptide chain, or may incorporate
the CDR(s) noncovalently. The CDRs permit the antigen binding
protein to specifically bind to a particular antigen of
interest.
[0112] An antigen binding protein may have one or more binding
sites. If there is more than one binding site, the binding sites
may be identical to one another or may be different. For example, a
naturally occurring human immunoglobulin typically has two
identical binding sites, while a "bispecific" or "bifunctional"
antibody has two different binding sites.
[0113] The term "human antibody" includes all antibodies that have
each and every variable and constant regions derived from human
immunoglobulin sequences. In one embodiment, fully human antibody,
all of the variable and constant domains are derived from human
immunoglobulin sequences (a fully human antibody).
[0114] A humanized antibody has a sequence that differs from the
sequence of an antibody derived from a non-human species by one or
more amino acid substitutions, deletions, and/or additions, such
that the humanized antibody is less likely to induce an immune
response, and/or induces a less severe immune response, as compared
to the non-human species antibody, when it is administered to a
human subject. In one embodiment, certain amino acids in the
framework and constant domains of the heavy and/or light chains of
the non-human species antibody are mutated to produce the humanized
antibody. In another embodiment, the constant domain(s) from a
human antibody are fused to the variable domain(s) of a non-human
species. In another embodiment, one or more amino acid residues in
one or more CDR sequences of a non-human antibody are changed to
reduce the likely immunogenicity of the non-human antibody when it
is administered to a human subject, wherein the changed amino acid
residues either are not critical for immuno specific binding of the
antibody to its antigen, or the changes to the amino acid sequence
that are made are conservative changes, such that the binding of
the humanized antibody to the antigen is not significantly worse
than the binding of the non-human antibody to the antigen. Examples
of how to make humanized antibodies may be found in U.S. Pat. Nos.
6,054,297; 5,886,152; and 5,877,293.
[0115] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies.
[0116] Further, the framework regions may be derived from one of
the same anti-VEGFR2 antibodies, from one or more different
antibodies, such as a human antibody, or from a humanized antibody.
In one example of a chimeric antibody, a portion of the heavy
and/or light chain is identical with, homologous to, or derived
from an antibody from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with, homologous to, or derived from an
antibody (-ies) from another species or belonging to another
antibody class or subclass. Also included are fragments of such
antibodies that exhibit the desired biological activity (U.S. Pat.
No. 4,816,567)
[0117] A "neutralizing antibody" or an "inhibitory antibody" is an
antibody that inhibits the activation of VEGFR2 when an excess of
the anti-VEGFR2 antibody reduces the amount of activation or
inhibition by at least about 20% using an assay such as those
described herein in the Examples. In various embodiments, the
antigen binding protein reduces the amount of amount of activation
of VEGFR2 by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 99%, and 99.9%.
[0118] Fragments or analogs of antibodies can be readily prepared
by those of ordinary skill in the art following the teachings of
this specification and using techniques well-known in the art.
Preferred amino- and carboxy-termini of fragments or analogs occur
near boundaries of functional domains. Structural and functional
domains can be identified by comparison of the nucleotide and/or
amino acid sequence data to public or proprietary sequence
databases. Computerized comparison methods can be used to identify
sequence motifs or predicted protein conformation domains that
occur in other proteins of known structure and/or function.
[0119] A "CDR grafted antibody" is an antibody comprising one or
more CDRs derived from an antibody of a particular species or
isotype and the framework of another antibody of the same or
different species or isotype.
[0120] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0121] An antigen binding protein "specifically binds" to an
antigen (e.g., human VEGFR2) if it binds to the antigen with a
dissociation constant of 1 nM or less.
[0122] An "antigen binding domain, "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0123] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0124] The "percent homology" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3 (Accelrys, San Diego, Calif.)) using its
default parameters.
[0125] A "host cell" is a cell that can be used to express a
nucleic acid. A host cell can be a prokaryote, for example, E.
coli, or it can be a eukaryote, for example, a single-celled
eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a
tobacco or tomato plant cell), an animal cell (e.g., a human cell,
a monkey cell, a hamster cell, a rat cell, a mouse cell, or an
insect cell) or a hybridoma. Examples of host cells include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells or their derivatives such as
Veggie CHO and related cell lines which grow in serum-free media
(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain
DX-B11, which is deficient in DHFR (Urlaub et al., 1980, Proc.
Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10)
cell lines, the CV1/EBNA cell line derived from the African green
monkey kidney cell line CV1 (ATCC CCL 70) (McMahan et al., 1991,
EMBO J. 10:2821), human embryonic kidney cells such as 293,293 EBNA
or MSR 293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a
cultured cell that can be transformed or transfected with a
polypeptide-encoding nucleic acid, which can then be expressed in
the host cell. The phrase "recombinant host cell" can be used to
denote a host cell that has been transformed or transfected with a
nucleic acid to be expressed. A host cell also can be a cell that
comprises the nucleic acid but does not express it at a desired
level unless a regulatory sequence is introduced into the host cell
such that it becomes operably linked with the nucleic acid. It is
understood that the term host cell refers not only to the
particular subject cell but also to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to, e.g., mutation or environmental
influence, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
Antigen Binding Proteins
[0126] Antigen binding proteins (e.g., antibodies, antibody
fragments, antibody derivatives, antibody muteins, and antibody
variants) are polypeptides that bind to VEGFR2, (preferably, human
VEGFR2). Antigen binding proteins include antigen binding proteins
that inhibit a biological activity of VEGFR2.
[0127] Oligomers that contain one or more antigen binding proteins
may be employed as VEGFR2 antagonists. Oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers. Oligomers comprising two or more antigen binding
proteins are contemplated for use, with one example being a
homodimer. Other oligomers include
[0128] One embodiment is directed to a dimer comprising two fusion
proteins created by fusing a VEGFR2 binding fragment of an
anti-VEGFR2 antibody to the Fc region of an antibody. The dimer can
be made by, for example, inserting a gene fusion encoding the
fusion protein into an appropriate expression vector, expressing
the gene fusion in host cells transformed with the recombinant
expression vector, and allowing the expressed fusion protein to
assemble much like antibody molecules, whereupon interchain
disulfide bonds form between the Fc moieties to yield the
dimer.
[0129] The term "Fc polypeptide" includes native and mutein forms
of polypeptides derived from the Fc region of an antibody.
Truncated forms of such polypeptides containing the hinge region
that promotes dimerization also are included. Fusion proteins
comprising Fc moieties (and oligomers formed therefrom) offer the
advantage of facile purification by affinity chromatography over
Protein A or Protein G columns.
[0130] Another method for preparing oligomeric antigen binding
proteins involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., 1988, Science
240:1759), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
oligomeric proteins are described in WO 94/10308, and the leucine
zipper derived from lung surfactant protein D (SPD) described in
Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.,
1994, Semin. Immunol. 6:267-78. In one approach, recombinant fusion
proteins comprising an anti-VEGFR2 antibody fragment or derivative
fused to a leucine zipper peptide are expressed in suitable host
cells, and the soluble oligomeric anti-VEGFR2 antibody fragments or
derivatives that form are recovered from the culture
supernatant.
[0131] The present disclosure provides a VEGFR2 antigen binding
protein (for example, an anti-VEGFR2 antibody), that has one or
more of the following characteristics: binds to both human and
murine VEGFR2, inhibits the activation of human VEGFR2, inhibits
the activation of murine VEGFR2, and binds to or near the ligand
binding domain of VEGFR2.
[0132] Antigen-binding fragments of antigen binding proteins of the
invention may be produced by conventional techniques. Examples of
such fragments include, but are not limited to, Fab and
F(ab').sub.2 fragments.
[0133] The present disclosure provides monoclonal antibodies that
bind to VEGFR2. Monoclonal antibodies may be produced using any
technique known in the art, e.g., by immortalizing spleen cells
harvested from the transgenic animal after completion of the
immunization schedule. The spleen cells can be immortalized using
any technique known in the art, e.g., by fusing them with myeloma
cells to produce hybridomas. Myeloma cells for use in
hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas). Examples of suitable cell lines for use in
mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4
1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0
Bul; examples of cell lines used in rat fusions include R210.RCY3,
Y3-Ag 1.2.3, IR983F and 48210. Other cell lines useful for cell
fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.
[0134] Antigen binding proteins directed against VEGFR2 can be
used, for example, in assays to detect the presence of VEGFR2
polypeptides, either in vitro or in vivo. The antigen binding
proteins also may be employed in purifying VEGFR2 proteins by
immunoaffinity chromatography. Those antigen binding proteins that
additionally can block ligand binding-mediated activation of VEGFR2
may be used to inhibit a biological activity that results from such
binding. Blocking antigen binding proteins can be used in the
methods disclosed herein. Such antigen binding proteins that
function as VEGFR2 antagonists may be employed in treating any
VEGFR2-induced condition, including but not limited to various
cancers.
[0135] Antigen binding proteins may be employed in an in vitro
procedure, or administered in vivo to inhibit a VEGFR2-induced
biological activity. Disorders caused or exacerbated (directly or
indirectly) by the activation of VEGFR2, examples of which are
provided herein, thus may be treated. In one embodiment, the
present invention provides a therapeutic method comprising in vivo
administration of a VEGFR2 blocking antigen binding protein to a
mammal in need thereof in an amount effective for reducing a
VEGFR2-induced biological activity.
[0136] Antigen binding proteins include fully human monoclonal
antibodies that inhibit a biological activity of VEGFR2, such as
angiogenesis.
[0137] Antigen binding proteins may be prepared by any of a number
of conventional techniques. For example, they may be purified from
cells that naturally express them (e.g., an antibody can be
purified from a hybridoma that produces it), or produced in
recombinant expression systems, using any technique known in the
art. See, for example, Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.), Plenum
Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow
and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1988).
[0138] Any expression system known in the art can be used to make
the recombinant polypeptides of the invention. In general, host
cells are transformed with a recombinant expression vector that
comprises DNA encoding a desired polypeptide. Among the host cells
that may be employed are prokaryotes, yeast or higher eukaryotic
cells. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include insect cells and established cell lines of mammalian
origin. Examples of suitable mammalian host cell lines include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC
CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC
CRL 10) cell lines, and the CV1/EBNA cell line derived from the
African green monkey kidney cell line CV1 (ATCC CCL 70) as
described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by Pouwels et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
[0139] The transformed cells can be cultured under conditions that
promote expression of the polypeptide, and the polypeptide
recovered by conventional protein purification procedures. One such
purification procedure includes the use of affinity chromatography,
e.g., over a matrix having all or a portion (e.g., the
extracellular domain) of VEGFR2 bound thereto. Polypeptides
contemplated for use herein include substantially homogeneous
recombinant mammalian anti-VEGFR2 antibody polypeptides
substantially free of contaminating endogenous materials.
[0140] Antigen binding proteins may be prepared, and screened for
desired properties, by any of a number of known techniques. Certain
techniques involve isolating a nucleic acid encoding a polypeptide
chain (or portion thereof) of an antigen binding protein of
interest (e.g., an anti-VEGFR2 antibody), and manipulating the
nucleic acid through recombinant DNA technology. The nucleic acid
may be fused to another nucleic acid of interest, or altered (e.g.,
by mutagenesis or other conventional techniques) to add, delete, or
substitute one or more amino acid residues, for example.
[0141] Single chain antibodies may be formed by linking heavy and
light chain variable domain (Fv region) fragments via an amino acid
bridge (short peptide linker), resulting in a single polypeptide
chain. Such single-chain Fvs (scFvs) have been prepared by fusing
DNA encoding a peptide linker between DNAs encoding the two
variable domain polypeptides (V.sub.L and V.sub.H). The resulting
polypeptides can fold back on themselves to form antigen-binding
monomers, or they can form multimers (e.g., dimers, trimers, or
tetramers), depending on the length of a flexible linker between
the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423;
Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different
V.sub.L and V.sub.H-comprising polypeptides, one can form
multimeric scFvs that bind to different epitopes (Kriangkum et al.,
2001, Biomol. Eng. 18:31-40). Techniques developed for the
production of single chain antibodies include those described in
U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989,
Nature 334:544, de Graaf et al., 2002, Methods Mol. Biol.
178:379-87.
[0142] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen-binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures, e.g., DNA
encoding the constant domain of an antibody of the desired isotype
(Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover, if
an IgG4 is desired, it may also be desired to introduce a point
mutation (CPSCP->CPPCP) in the hinge region (Bloom et al., 1997,
Protein Science 6:407) to alleviate a tendency to form intra-H
chain disulfide bonds that can lead to heterogeneity in the IgG4
antibodies.
[0143] In particular embodiments, antigen binding proteins of the
present invention have a binding affinity (K.sub.a) for VEGFR2 of
at least 10.sup.6 nM. In other embodiments, the antigen binding
proteins exhibit a K.sub.a of at least 10.sup.7, at least 10.sup.8,
at least 10.sup.9, or at least 10.sup.10 M. In another embodiment,
the antigen binding protein exhibits a K.sub.a substantially the
same as that of an antibody described herein in the Examples.
[0144] In another embodiment, the present disclosure provides an
antigen binding protein that has a low dissociation rate from
VEGFR2. In one embodiment, the antigen binding protein has a
K.sub.off of 1.times.10.sup.-4 to 1.times.10.sup.-1M or lower. In
another embodiment, the K.sub.off is 5.times.10.sup.-5 to
5.times.10.sup.-1M or lower. In another embodiment, the K.sub.off
is substantially the same as an antibody described herein in the
Examples. In another embodiment, the antigen binding protein binds
to VEGFR2 with substantially the same K.sub.off as an antibody
described herein in the Examples.
[0145] In another aspect, the present disclosure provides a VEGFR2
membrane binding protein. In one embodiment, the antigen binding
protein has an IC.sub.50 of 1000 nM or lower. In another
embodiment, the IC.sub.50 is 100 nM or lower; in another
embodiment, the IC.sub.50 is 10 nM or lower. In another embodiment,
the IC.sub.50 is substantially the same as that of an antibody
described herein in the Examples. In another embodiment, the
antigen binding protein inhibits an activity of VEGFR2 with
substantially the same IC.sub.50 as an antibody described herein in
the Examples.
[0146] In another aspect, the present disclosure provides an
antigen binding protein that binds to human VEGFR2 expressed on the
surface of a cell and, when so bound, inhibits VEGFR2 signaling
activity in the cell. Any method for determining or estimating the
amount of VEGFR2 on the surface and/or in the interior of the cell
can be used. In other embodiments, binding of the antigen binding
protein to the VEGFR2-expressing cell causes less than about 75%,
50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, or 0.1% of the cell-surface
VEGFR2 to be internalized.
[0147] In another aspect, the present disclosure provides an
antigen binding protein having a half-life of at least one day in
vitro or in vivo (e.g., when administered to a human subject). In
one embodiment, the antigen binding protein has a half-life of at
least three days. In another embodiment, the antigen binding
protein has a half-life of four days or longer. In another
embodiment, the antigen binding protein has a half-life of eight
days or longer. In another embodiment, the antigen binding protein
is derivatized or modified such that it has a longer half-life as
compared to the underivatized or unmodified antigen binding
protein. In another embodiment, the antigen binding protein
contains one or more point mutations to increase serum half life,
such as described in WO00/09560, incorporated by reference
herein.
[0148] The present disclosure further provides multi-specific
antigen binding proteins, for example, bispecific antigen binding
protein, e.g., antigen binding protein that binds to two different
epitopes of VEGFR2, or to an epitope of VEGFR2 and an epitope of
another molecule, via two different antigen binding sites or
regions. Moreover, bispecific antigen binding protein as disclosed
herein can comprise a VEGFR2 binding site from one of the
herein-described antibodies and a second VEGFR2 binding region from
another of the herein-described antibodies, including those
described herein by reference to other publications. Alternatively,
a bispecific antigen binding protein may comprise an antigen
binding site from one of the herein described antibodies and a
second antigen binding site from another VEGFR2 antibody that is
known in the art, or from an antibody that is prepared by known
methods or the methods described herein.
[0149] Numerous methods of preparing bispecific antibodies are
known in the art. Such methods include the use of hybrid-hybridomas
as described by Milstein et al., 1983, Nature 305:537, and chemical
coupling of antibody fragments (Brennan et al., 1985, Science
229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S. Pat. No.
6,010,902). Moreover, bispecific antibodies can be produced via
recombinant means, for example by using leucine zipper moieties
(i.e., from the Fos and Jun proteins, which preferentially form
heterodimers; Kostelny et al., 1992, J. Immunol. 148:1547) or other
lock and key interactive domain structures as described in U.S.
Pat. No. 5,582,996. Additional useful techniques include those
described in U.S. Pat. Nos. 5,959,083; and 5,807,706.
[0150] In another aspect, the antigen binding protein comprises a
derivative of an antibody. The derivatized antibody can comprise
any molecule or substance that imparts a desired property to the
antibody, such as increased half-life in a particular use. The
derivatized antibody can comprise, for example, a detectable (or
labeling) moiety (e.g., a radioactive, colorimetric, antigenic or
enzymatic molecule), a detectable bead (such as a magnetic or
electrodense (e.g., gold bead), a molecule that binds to another
molecule (e.g., biotin or streptavidin), a therapeutic or
diagnostic moiety (e.g., a radioactive, cytotoxic, or
pharmaceutically active moiety), or a molecule that increases the
suitability of the antibody for a particular use (e.g.,
administration to a subject, such as a human subject, or other in
vivo or in vitro uses). Examples of molecules that can be used to
derivatize an antibody include albumin (e.g., human serum albumin)
and polyethylene glycol (PEG). Albumin-linked and PEGylated
derivatives of antibodies can be prepared using techniques well
known in the art. In one embodiment, the antibody is conjugated or
otherwise linked to transthyretin (TTR) or a TTR variant. The TTR
or TTR variant can be chemically modified with, for example, a
chemical selected from the group consisting of dextran,
poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
Example 1
[0151] This example illustrates production runs and how to make
anti-VEGFR2 antibody VK-B8. The expression cassette coding for the
heavy chain and dihydrofolate reductase (DHFR) was driven by the
CMV major immediate early promoter. The expression cassette coding
for the light chain and neomycin phosphotransferase was driven by
the CMV major immediate early promoter. Transfected cells were
cultured in CD OptiCHO medium (Life Technologies) without
nucleosides prior to Fluorescence-Activated Cell Sorting (FACS).
Cells were "sorted" once using FACS to enrich for high producers.
After FACS enrichment, the cell pool was cultured and passaged
until the cell viability was greater than 90%. For production of
VK-B8, cells were scaled to a desirable density and volume at
37.degree. C. and then placed at 28.degree. C. for the duration of
the production run.
[0152] VK-B8 was produced in Chinese hamster ovary (CHO) cells
stably transfected with expression vectors containing the human
VK-B8 mAb IgG1 heavy and kappa light chain structural genes. The
host cell line used for the construction of the cell line was a
Chinese hamster ovary dihydrofolate reductase (dhfr)-deficient host
cell line, CHO-DG44 (Life Technologies). The cell line was
maintained in shake flasks and routinely passaged every 3 to 4 days
using DG44 (Life Technologies) medium and supplemented with
PLURONIC F-68 (Life Technologies) and GlutaMAX I-CTS (Life
Technologies). Two plasmids were used to generate the stable pool;
the heavy chain and light chain expression vectors
pIRES.VEGFR2.VKB8.HC and pIRES.VEGFR2.VKB8.LC, respectively. The
plasmid coding for the heavy chain also coded for dhfr as a second
cistron which was used as a selectable and amplifiable marker. The
plasmid coding for the light chain also contains neomycin
phosphotransferase as a second cistron which was used as a
selectable marker. The vectors were co-transfected into the host
cell line using the cationic lipid Freestyle Max and selected for
DHFR and neomycin phosphotransferase expression.
Example 2
[0153] This example illustrates a differential scanning calorimetry
study measuring thermodynamic stability of the anti-VEGFR2
monoclonal antibody VK-B8, compared with Bevacizumab
(Avastin.RTM.), using MicroCal.TM. DSC (GE Healthcare). A
thermogram for each antibody (0.48-0.55 mg/mL) was obtained in PBS
buffer, pH 7.4 from 25.degree. C. to 85.degree. C., using a scan
rate of 1.degree. C. per minute, unless otherwise mentioned. The
VK-B8 melting temperature profile was monitored in PBS buffer at
both pH 7.4 and 6.
TABLE-US-00001 Melting IgG Temperature in .degree. C. Avastin 71.23
VK-B8 74.78
[0154] An Analytical Size-Exclusion Chromatography (ANSEC) (Water's
Breeze-HPLC) analysis of the monoclonal antibodies VK-B8 and
Bevacizumab (Avastin.RTM.) was performed in PBS buffer, pH 6.8 at
0.5 mL/min flow rate using TSKgel Supper SW3000 column (TOSOH
Biosciences). Aggregate formation of VK-B8 in PBS buffer at
4.degree. C. was monitored for 4 months using the same system. IgG
samples were compared with the BIO-RAD gel filtration protein
standard (Cat #, 151-1901: Thyroglobi=670 KDa; Gamma-globulin=158
KDa; Ovalbumin=44 KDa; Myoglobin=17 KDa; Vitamin B12=1.35 KDa). The
results are shown in FIG. 7, which provide overlaid ANSEC
chromatograms (Ultra Violet trace at 280 nm) of VK-B8 and
Bevacizumab (Avastin) in PBS buffer at pH 6.8; STD/standard run
(grey dotted line), VK-B8 spectrum (red), Bevacizumab (Avastin)
spectrum (green).
[0155] ANSEC analysis of the VK-B8 IgG sample at 4.degree. C. after
0 days and 4 months is shown in FIGS. 7A and 7B which show overlaid
ANSEC analysis (Ultra Violet trace at 280 nm0 days (blue) and 4
months (green)) A: representative full spectra, B: Enlarged version
of A indicating that VK-B8 remained intact after 4 months.
[0156] Further, a Biacore analysis of four antibodies was done for
binding.
Biacore analysis of several VEGFR-2 antibodies, including VK-B8
TABLE-US-00002 mAb ka (1/Ms) kd (1/s) Kd (M) RV-H5 2.76E+06
2.25E-03 8.15E-10 VB-A3 3.88E+06 1.20E-02 3.09E-09 VB-A9 1.64E+05
4.96E-04 3.02E-09 VK-B8 7.24E+05 8.73E-05 1.21E-10
Example 3
[0157] This example illustrates in vivo data comparing a marketed
anti-VEGF (ligand) humanized monoclonal antibody (Bevacizumab
(Avastin.RTM.)) to VK-B8, a disclosed herein fully human
anti-VEGFR2 (receptor) antibody, for effects on MC38 colon cancer
growth measured by solid tumor volume. C57BL6/6 mice were injected
subcutaneously with 2.times.10.sup.6 MC38 colon tumor cells. After
4 days, a treatment protocol was initiated. Test antibody was
administered at 0.2 mg/mouse ip 3.times. per week in a volume of
0.1 ml. The tumor volume was measured as W.times.L.times.L/2
wherein W is tumor width and L is tumor length. FIG. 8 shows that
the disclosed antibody VK-B8 displays greater efficacy than the
marketed antibody Bevacizumab (Avastin.RTM.; anti-VEGF ligand
humanized antibody).
Example 4
[0158] This example illustrates in vivo data comparing a marketed
anti-VEGF (ligand) humanized monoclonal antibody (Bevacizumab
(Avastin.RTM.)) to VK-B8, a disclosed herein fully human
anti-VEGFR2 (receptor) antibody, for effects on A431 epidermoid
carcinoma cell growth measured by solid tumor volume. Nude mice
were injected subcutaneously with 2.times.10.sup.6 A431 epidermoid
carcinoma cells. After 4 days, a treatment protocol was initiated.
Test antibody was administered at 0.2 mg/mouse ip 3.times. per week
in a volume of 0.1 nil. The tumor volume was measured as
W.times.L.times.L/2 wherein W is tumor width and L is tumor length.
FIG. 9 shows that the disclosed antibody, VK-B8, shows similar
efficacy compared to the marketed antibody Bevacizumab
(Avastin.RTM.; anti-VEGF ligand humanized antibody).
Example 5
[0159] This example illustrates in vivo data comparing a marketed
anti-VEGF (ligand) humanized monoclonal antibody (Bevacizumab
(Avastin.RTM.)) to (1) Erbitux.RTM. (cetuximab), a humanized
anti-EGFR antibody, (2) A6, a fully human anti-EGFR antibody, (3)
combination of A6 plus Bevacizumab (Avastin.RTM.), (4) combination
of A6 plus Erbitux, and (5) A6 plus VK-B8 wherein VK-B8 is a
disclosed herein fully human anti-VEGFR2 (receptor) antibody for
effects on A431 epidermoid carcinoma cell growth by measuring solid
tumor volume. Nude mice were injected subcutaneously with
2.times.10.sup.6 A431 epidermoid carcinoma cells. After 4 days, a
treatment protocol was initiated. Test antibody was administered at
0.2 mg/mouse ip 3.times. per week in a volume of 0.1 nil. The tumor
volume was measured as W.times.L.times.L/2 wherein W is tumor width
and L is tumor length. FIG. 10 shows similar efficacy for each of
the treatment groups with the combination of A6+Avastin.RTM. and
A6+VK-B8 showing the highest efficacy for tumor growth
inhibition.
Example 6
[0160] This example illustrates in vitro data for VK-B8 cellular
EC.sub.50 measurements. Protocol: 50,000 HUVECs were aliquoted into
the wells of a 96-well, v-bottom plate in 100 ul FACS Buffer
(PBS+2% FBS). A twelve point, 3.times. dilution curve of VK-B8 was
made in FACS Buffer starting at 50 .mu.g/ml (3.33.times.10.sup.-7
M). Cells were spun down, washed 1.times. with FACS Buffer, and
then resuspended in 25 .mu.l of antibody solution in triplicate.
After 0.5 hr. incubation, cells were washed 1.times. with FACS
Buffer and resuspended in 50 .mu.l PE-conjugated, goat anti-human
IgG (.gamma.-chain specific) secondary antibody (Southern Biotech
Cat #2040-09). Cells were further incubated for 0.5 hr. and then
washed 1.times. with FACS Buffer. Cells were resuspended in 25
.mu.l FACS Buffer and the median fluorescence intensity in the
FL2-H channel was determined using the Intellicyt HTFC flow
cytometer.
[0161] Results: The cell binding EC.sub.50 for VK-B8 on HUVECs was
determined to be 1.3 nM. Data was analyzed and plotted in Graph Pad
Prizm using non-linear regression fit. Data points are shown (FIG.
4) as the median fluorescence intensity (MFI) of positively labeled
cells +/- Std Error.
Example 7
[0162] This example illustrates in vitro data for cell binding as
part of an initial screen for several disclosed antibodies,
including VK-B8. Protocol: 100,000 HUVECs were aliquoted into tubes
in 100 .mu.l FACS Buffer (PBS+2% FBS). Cells were spun down and
then resuspended in 100 .mu.l of FACS Buffer plus 10 .mu.g/ml of
the indicated antibody in triplicate. After 0.5 hr incubation,
cells were washed 1.times. with FACS Buffer and resuspended in 100
.mu.l PE-conjugated, goat anti-human IgG (.gamma.-chain specific)
secondary antibody (Southern Biotech Cat #2040-09). Cells were
further incubated for 0.5 hr and then washed 1.times. with FACS
Buffer. Cells were resuspended in 300 .mu.l FACS Buffer and the
median fluorescence intensity in the FL2-H channel was determined
using the FACS Aria flow cytometer (BD).
[0163] Results: The cell binding for the anti-VEGFR2 antibodies
disclosed (FIG. 3) on HUVECs was strong compared to background
staining (control). The histograms shown depict the number events
with specific fluorescence intensities. Blue histograms are the
VEGFR2-specific antibody while the red histograms are the
background control. Data shown is representative of multiple
experiments.
Example 8
[0164] This example illustrates in vitro data showing inhibition of
VEGF-mediated HUVEC proliferation by VK-B8 (anti-VEGFR2) versus FDA
approved anti-VEGF Bevacizumab (Avastin.RTM.) Protocol: 5000 HUVECs
were plated into the wells of a 96-well white opaque cell culture
cluster in 100 .mu.l EBM-2 media supplemented with the growth
factors etc. (Lonza). 24 hr later, media was removed, cells washed
1.times. with PBS, and then starved for 18 hr in 50 .mu.l
non-supplemented (basal) EBM-2 media. Antibodies were diluted to
2.times. the indicated concentration in 50 .mu.l non-supplemented
(basal) EBM-2 media then added to the cells after removal of the
starvation media. After 1 hr incubation, VEGF was added at a
concentration of 200 ng/ml in 50 .mu.l (final concentration of VEGF
is 100 ng/ml). Cells were then incubated for 48 hr. after which the
Promega Cell Titer Glo kit was used to evaluate proliferation.
Luminescence output is directly proportional to cell number.
[0165] Results: VK-B8 inhibited VEGF-stimulated HUVEC proliferation
(FIG. 7). Proliferation inhibition was equal to that conferred by
Bevacizumab (Avastin.RTM.) at the same dose. Data shown is the mean
relative light units of triplicate samples +/- Std Error
Example 9
[0166] This example illustrates in vitro data showing VEGF
stimulated auto-phosphorylation of VEGFR2 receptor in HUVECs.
Protocol: 100,000 HUVECs were plated in the wells of a 12-well cell
culture cluster in 2.5 ml EBM-2 media supplemented with the growth
factors etc. (Lonza). 24 hr. later, media were removed and the
cells washed 1.times. with PBS, and then starved for 18 hr. in 1 ml
non-supplemented (basal) EBM-2 media. Antibodies were diluted to
2.times. the indicated concentration in 1 ml non-supplemented
(basal) EBM-2 media then added to the cells after removal of
starvation media. After 0.5 hr. incubation, VEGF was added at a
concentration of 200 ng/ml in 1 ml basal media (final VEGF
concentration is 100 ng/ml). Cells were then incubated for 30 min.
Cells were washed with PBS and lysed in 1.times. Cell Lysis Buffer
(Cell Signaling). Phosphorylation of VEGFR2 was detected using the
Cell Signaling Path Scan ELISA Antibody Pair (#7824) according to
manufacturer's protocol with modification of the volumes to support
half-area plates (all volumes cut in half).
[0167] Results: HUVECs were treated with 100 ng/ml VEGF to
stimulate activating auto-phosphorylation of VEGFR2. Pre-treatment
of cells with VK-B8 and other clones blocked this activation of
VEGFR2. Bevacizumab (Avastin.RTM.) treatment shows expected
inhibition of VEGFR2 auto-phosphorylation. Data shown (FIG. 6) is
the mean absorbance at 450 nm of triplicate samples +/- Std
Error.
Example 10
[0168] This example illustrates in vitro data showing the
inhibition of VEGF stimulated auto-phosphorylation of the VEGFR2
receptor in HUVECs by VK-B8. Upon binding of VEGF, VEGFR2 undergoes
activating auto-phosphorylation at Tyrosine1175 leading to
downstream signaling events and the activation of cellular events
such as proliferation and cell migration. To assess the ability of
VK-B8 to inhibit activation of VEGFR2 by VEGF, 50,000 HUVECs were
plated in the wells of a 96-well cell culture cluster in 100 .mu.l
EGM-2 media supplemented with growth factors (Lonza). 24 hr. later,
the medium was removed and the cells washed 1.times. with PBS
followed by starvation for 18 hr. in 100 .mu.l non-supplemented
(basal) EGM-2 media. Cells were then preincubated with a serial
dilution curve of VK-B8 for 15 minutes, followed by incubation with
VEGF at a final concentration of 50 ng/ml for 5 minutes. Cells were
washed with PBS and lysed in 1.times. Cell Lysis Buffer (Cell
Signaling). Phosphorylation of VEGFR2 at Tyr1175 was detected using
the Cell Signaling Path Scan ELISA Antibody Pair (#7824) according
to manufacturer's protocol with modification of the volumes to
support half-area plates (all volumes cut in half). Absorbance at
450 nm was plotted vs. antibody concentration and non-linear
regression was used to determine the IC.sub.50 value (FIG. 11). The
IC.sub.50 for VK-B8 inhibition of VEGF-induced VEGFR2
auto-phosphorylation was 0.12 nM.
Example 11
[0169] This example illustrates in vitro data showing the
inhibition of VEGF stimulated phosphorylation of p44/p42 MAPK
(Erk1/2) by VK-B8 in HUVECs. Upon binding of VEGF, VEGFR2 undergoes
activating auto-phosphorylation, establishing a signaling cascade
which results in the activating phosphorylation of p44/p42 MAPK
(Erk1/2). To assess the ability of VK-B8 to inhibit this
activation, 50,000 HUVECs were plated in the wells of a 96-well
cell culture cluster in 100 ul EGM-2 media supplemented with growth
factors (Lonza). 24 hr. later, the medium was removed and the cells
washed 1.times. with PBS followed by starvation for 18 hr. in 100
.mu.l non-supplemented (basal) EBM-2 media. Cells were then
preincubated with a serial dilution curve of VK-B8 for 15 minutes,
followed by incubation with VEGF at a final concentration of 50
ng/ml for 5 minutes. Cells were washed with PBS and lysed in
1.times. Cell Lysis Buffer (Cell Signaling). Phosphorylation of
p44/p42 MAPK (Erk1/2) was detected using a Cell Signaling Path Scan
ELISA Antibody Pair (#7246) according to manufacturer's protocol
with modification of the volumes to support half-area plates (all
volumes cut in half). Absorbance at 450 nm was plotted vs. antibody
concentration and non-linear regression was used to determine the
IC.sub.50 value (FIG. 12). The IC.sub.50 for VK-B8 inhibition of
VEGF-induced p44/p42 MAPK (Erk1/2) phosphorylation was 0.08 nM.
Example 12
[0170] This example illustrates in vitro data showing inhibition of
VEGF-mediated HUVEC proliferation by VK-B8. The effect of VEGF
activation of VEGFR2 on endothelial cells is to trigger
proliferation. In oncology, a tumor will secrete VEGF to manipulate
the vascular micro-environment, leading to proliferation and
expansion of the vasculature and finally invasion of the
vasculature into the tumor. Inhibition of the process has been
shown to functionally starve the tumor. To assess the inhibition of
VEGF-induced endothelial cell proliferation by VK-B8, 5000 HUVECs
were plated into the wells of a 96-well cell culture cluster in 100
.mu.l EGM-2 media supplemented with growth factors (Lonza). 24 hr
later, media was removed, cells washed 1.times. with PBS, and then
starved for 18 hr. in 100 .mu.l non-supplemented (basal) EGM-2
media. Cells were then preincubated with a serial dilution curve of
VK-B8 for 15 minutes, followed by incubation with VEGF at a final
concentration of 50 ng/ml for 48 hr. To measure proliferation, the
Promega Cell Titer 96 Non-Radioactive Cell Proliferation kit was
used. Absorbance at 570 nm was directly proportional to cell
number. Abs570 nm was plotted vs. antibody concentration and
non-linear regression was used to determine the IC.sub.50 for this
effect (FIG. 13). The IC.sub.50 value for the inhibition of
proliferation by VK-B8 was 14 nM.
Example 13
[0171] This example illustrates in vitro data showing inhibition of
VEGF-mediated HUVEC migration by VK-B8. An effect of VEGF
activation of VEGFR2 on endothelial cells is to trigger cell
migration. In oncology, a tumor will secrete VEGF to manipulate the
vascular micro-environment, leading to migration and invasion of
the vasculature into the tumor. Inhibition of this process has been
shown to functionally starve the tumor. To assess the inhibition of
VEGF-induced endothelial cell migration by VK-B8, 80,000 HUVECs per
sample were preincubated with a serial dilution curve of VK-B8 for
20 minutes and then plated onto the sites of the upper plate of a
96-well modified Boyden Chamber (NeuroProbe, 8 .mu.m pores). The
lower chamber of the plate contained VEGF at a final concentration
of 5 ng/ml. Twenty hours after the plating of cells, the number of
cells which migrated through the membrane to the lower chamber were
counted and plotted vs. the antibody concentration (FIG. 14). The
IC.sub.50 value for the inhibition of VEGF-induced HUVEC migration
by VK-B8 was calculated using non-linear regression and determined
to be 0.5 nM.
Example 14
[0172] This example illustrates in vitro data showing the
inhibition of VEGF-C stimulated auto-phosphorylation of the VEGFR2
receptor in HUVECs by VK-B8. In addition to VEGF (VEGF-A), other
members of this growth factor family can bind and activate VEGFR2
leading to functional outcomes. The growth factor VEGF-C can bind
and activate VEGFR2 and VEGFR3. Here we demonstrate the ability of
VK-B8 to block the activation of VEGFR2 by VEGF-C. To assess the
ability of VK-B8 to inhibit activation of VEGFR2 by VEGF-C, 50,000
HUVECs were plated in the wells of a 96-well cell culture cluster
in 100 .mu.l EGM-2 media supplemented with growth factors (Lonza).
24 hr. later, the medium was removed and the cells washed 1.times.
with PBS followed by starvation for 18 hr. in 100 .mu.l
non-supplemented (basal) EGM-2 media. Cells were then preincubated
with a serial dilution curve of STI-A0168 (VK-B8) for 15 minutes,
followed by incubation with VEGF-C at a final concentration of 1
.mu.g/ml for 5 minutes. Cells were washed with PBS and lysed in
1.times. Cell Lysis Buffer (Cell Signaling). Phosphorylation of
VEGFR2 at Tyr1175 was detected using the Cell Signaling Path Scan
ELISA Antibody Pair (#7824) according to manufacturer's protocol
with modification of the volumes to support half-area plates (all
volumes cut in half). Absorbance at 450 nm was plotted vs. antibody
concentration and non-linear regression was used to determine the
IC.sub.50 value (FIG. 15). The IC.sub.50 for VK-B8 inhibition of
VEGF-C-induced VEGFR2 auto-phosphorylation was 1.9 nM.
Example 15
[0173] This example provides a summary table of affinity
measurements, EC.sub.50 measurement, IC.sub.50 measurements of the
identified antibodies.
TABLE-US-00003 mAb VB-A2 VB-A9 VB-E1 VB-F2 VR-B2 RV-H5 VR-B4 VR-E3
ka 1.44E+6 3.71E+4 1.80E+5 6.16E+4 3.36E+5 5.39E+5 2.04E+5 3.75E+4
kd 1.55E-3 2.35E-3 1.62E-3 2.13E-3 3.19E-3 5.71E-3 2.54E-3 8.20E-4
Kd 1.08E-9 6.34E-8 9.02E-9 3.46E-8 9.50E-9 1.06E-8 1.25E-8 2.19E-8
EC50 0.50 0.25 0.5 weak 0.747 0.323 24.3 1.29 nM IC50, 25.3 2.05
weak weak -- 5.79 -- 9.32 nM VK-A1 VK-B7 VK-B8 VK-C1 VK-E5 VK-G9
1121* ka 6.57E+4 7.56E+4 1.27E+5 1.71E+6 1.30E+5 1.10E+5 4.79E+5 kd
2.42 1.92E-3 7.75E-4 2.01E-3 1.16E-3 1.91E-3 2.5E-5 KD 3.55E-9
3.20E-8 6.11E-9 1.17E-8 8.97E-9 1.75E-8 5.0E-11 EC50 0.109 1.89
0.25 2.0 0.175 0.653 0.25 nM IC50 3.57 7.33 2.04 57.0 3.03 5.57 0.8
nM *published data in the literature: Lu, D., et al., 278:
43496-43507, JBC (2003)
TABLE-US-00004 Sequence Listing: Binder VH VL VB-A2
MAQVQLVQXGAEVKKPGASVKVSCKASGYTFTSYY
AIQLTQSPSSLSASVGDRVTITCRASQGISNY MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG
LAWYQQKPGKVPKLLIYAASTLQSGVPSRFS RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRG
GSGSGTDFTLTISSLQPEDFATYYCQQSYLTP MLRHWGMDVWGQGTTVTVSS WTFGQGTKVEIK
SEQ ID NO. 1 SEQ ID NO. 2 VB-A3 MAQVQLVQSGAEVKKPGASVKVSCKASGHTFNYYY
SYELTQPPSVSVAPGKTASITCGGNNIGSKS MHWVRQAPGQGLEWMGQTNLNSGGTNYAPKFQ
VHWYQQKPGQAPVLVIYYDRDRPSGIPERF GRVTMTRDTSISTAYMELSSLRSDDTAVYYCANLNS
SGSNSGNTATLTISRVEAGDEADYYCQVWD GWFHFENWGQGTLVTVSS SSSYHPVFGGGTKLTVL
SEQ ID NO. 3 SEQ ID NO. 4 VB-A5 MAQMQLVQSGAEVKKPGSSVKVSCEASGGTFSSFA
QPVLTQPPSASGTPGQRVTIFCSGSTSNIGS ISWVRQAPGQGLEWMGRVIPVFGTANYAQTFQG
NTVNWYHHLPGTAPKLLIYSNNQRPSGVPD
RVTITADKSTSTMFMELSSLRSEDTAVYYCARESGDY
RFSGSKSGTSASLAISGLQSADEADYYCAAW YDGSRYVDAFDIWGQGTMVTVSS
DDNLNGWVFGGGTKLTVL SEQ ID NO. 5 SEQ ID NO. 6 VB-A7
MAQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYY
SYVLTQPASVSGSPGQSITISCAGTSSDIGGY MHWVRQAPGQGLQWMGIINPSGGSTSYAQNFQ
NSVSWYQQNPGKAPKLMIYEGSKRPSGVS GRVTMTRDTSTSTVYMELSSLISQDTAVYYCARSGY
NRFSGSKSGTTASLTISGLQAEDEADYYCSSY SSSWLSYGMDVWGQGTTVTVSS
TNSDTWVFGGGTKLTVL SEQ ID NO. 7 SEQ ID NO. 8 VB-A9
MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSALTQPRSVSASPGQSVTISCTGTSSDVGG
MNWVRQAPGKGLEWVSSISSGSSYIYYADSLKGRF YDYVSWYQQHPGKAPKLMIYEVSNRPSGV
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDFGNW SNRFSGSKSGNTASLSISGLQAEDEADYYCS
GQGTLVTVSS SYTSTSSPVVFGGGTKLTVL SEQ ID NO. 9 SEQ ID NO. 10 VB-A10
MAEVQLVESGGGLVQPGGSLRLSCAVSGFTLSSYE QPVLTQPPSASGTPGQRVTISCSGSSSNLGS
MMWVRQAPGKGLEWVSYISDSGGLIYYSDSVKGR NYVYWYQHLPGTAPKLLIYRNKQRPSGVPD
FTISRDSAKNSLYLQMNSLRDEDTAVYYCARDEYSS
RFSGSKSGTSASLAISGLRSEDEADYYCAVW GMDVWGQGTTVTVSS DGSLSGYVFGTGTKLTVL
SEQ ID NO. 11 SEQ ID NO. 12 VB-B6
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFSSYY QSVLTQPRSVSGSPGQSVTISCTGTSSDVGG
MHWVRQAPGQGLEWMGIINPSAGSTNYAQKFQG YNYVSWYQQHPDKAPKLMLYDVSKRPSGV
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYYY
SNRFSGSKSGNTASLTIXALQAEDEADYYCS DTSGYHGYFKYWGQGTLVTVSS
SYTSSTNLGVFGGGTKVTVL SEQ ID NO. 13 SEQ ID NO. 14 VB-610
MAEVQLLESGGGLVQPGRSLRLSCAASGFSFDDYA QSALTQPPSASGTPGQRVTISCSGSSSNIGT
MHWVRQAPGKGLEWVSSISWNSGSRGYADSVKG NTVNWYQQLPGTAPRLLIYFNNQRPSGVP
RFTISRDNAKNSLFLQMNSLRTEDTALYYCAKGVVY
DRFSGSKSATSASLAISGLQSEDEADYYCSA SSPYYGMDVWGQGTTVTVSS
WDDILNDPVFGGGTKLTVL SEQ ID NO. 15 SEQ ID NO. 16 VB-D5
MAQVQLVQSGAEVKKPGASVKISCKASGYIFNTYSI
QAVLTQPPSASGTPGQRVTISCSGSSSNIGS HWVRQAPGQSFEWMGWSSAGDDNTKYSDDFHH
NTVNWYQQLPGTAPKLLIYSNNQRPSGVP RLTIARDTSASTVYMELRGLTSDDTAIYYCARGYELD
DRFSGSKSGTSASLAISGLQSEDEADYYCAA FWGQGTLVTVSS WDDSLNGDVVFGGGTKVTVL
SEQ ID NO. 17 SEQ ID NO. 18 VB-D6
MAEVQLVESGGDLVKPGGSXRLSCAASGFSFSDYY
QSALTQPASVSGSPGQTITISCAGTPSDIGLY
MSWIRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTI
NYVAWFQHHPGKAPKLIIYDVTKRPSGTSN SRDNAKNSLYLQMNSLRAEDTAVYYCARDAPRYG
RFSGSKSGNTASLTISGLQADDEADYFCSSY MDVWGQGTTVTVSS TTSNSFVLFGEGTKLTVL
SEQ ID NO. 19 SEQ ID NO. 20 VB-D11
MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA QSVVTQQPSVSAAPGQKVTISCSGGSSNIG
ISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGR NNYVSWYQQLPGTAPKLLIYDNNKRPSGIP
VTITADESTSTAYMELSSLRSEDTAVYYCARGSERVY
DRFSGSKSGSSASLAITGLQAEDEADYYCQS SSHTYYGMDVWGQGTTVTVSS
YDSSLSGYVFGTGTKLTVL SEQ ID NO. 21 SEQ ID NO. 22 VB-E1
MAQVQLVESGAEVKKPGASVKVSCKASGYTFTNYYI
DIVMTQSPDSLAVSLGERATINCKTSQSVLY HWVRQAPGQGLEWMGIINPSSGSTNYAQKFQGR
NANNKNYLNWYQQKPGQPPKLLIYWASAR VTMTRDTSTSTVYMELSSLRSEDTAVYYCARQRWD
ESGVPDRFSGSGSGTDFTLTIRSLQPDDFAT LLDDAFDIWGQGTMVTVSS
YYCQQAISFPLTFGGGTKVEIK SEQ ID NO. 23 SEQ ID NO. 24 VB-E2
MAEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG SYELMQPPSVSEAPGMTAQITCGGNNIGSK
MHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG SVHWYQQKPGQAPVLVIYYDSERPSGIPDR
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDWS FSGSNSGNTASLTINRVEAGDEADYYCQVW
GPITLWGQGTLVTVSS DSSSDH HVVFGGGTKLTVL SEQ ID NO. 25 SEQ ID NO. 26
VB-E7 MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYE
QSALTQPPSMSAAPGQKVTISCSGTSSNIG MNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRF
NHYVSWYQRLPDTAPKLLIYDNNRRPSGIP TISRDNAKNSLYLQMNGLRAEDTAVYYCAREGDSS
DRFSGSKSGTSATLGITGLQTGDEADYYCGT GFDYWGQGTLVTVSS WDSSLSGYVFGTGTKVTVL
SEQ ID NO. 27 SEQ ID NO. 28 VB-F2
MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSDHY
DIVMTQTPLFLPVTLGQPASISCRSSQSLVHS MDWVRQAPGKGLEWVGRIRNKPNSYTSEYAASVK
DGNTYLNWFQQRPGQSPRRLIYKVSIRDSG GRFTISRDDSKNSLYLQMNSLRAEDTAVYYCASRSG
VPGRFSGSGSGTDFTLKISSVEAEDIGVYYC SYYDHMDVWGQGTTVTVSS
MQGTDRPYTFGQGTKLEIK SEQ ID NO. 29 SEQ ID NO. 30 VB-F8
MAQMQLVQSGGGVVQPGRSLRLSCVVSGFTFNDY QAGLTQSPSVSAAPGQRVTISCTGSSSNIGA
PMHWVRQAPGKGLEWVALLSYDGTSAYYADSVEG GYDVHWYQQLPGTAPKLLIYGNSNRPSGVP
RFTISRDNSKNTLYLQMNTLRTEDTAVYYCASEGSP
DRFSGSKSGTSASLAITGLQAEDEADYYCQS DAFDIWGQGTMVTVSS
YDSSLSGSVFGGGTKVTVL SEQ ID NO. 31 SEQ ID NO. 32 VB-G4
MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI
QSVVTQPASVSGSPGQSITISCTGTTTDVGA GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ
YNYVSWYQHHPGKAPKLIIYDLNNRPSGISN
VTISADKSISTAYLQWSSLKASDTAMYYCARLDYDSS
RFSGFKSGNTASLTISGLQAEDEADYYCSSYT GYYGAFDIWGQGTMVTVSS
SSSTWVFGGGTKLTVL SEQ ID NO. 33 SEQ ID NO. 34 VB-G6
MAQVQLVESGAEVKKPGASVKVSCKASGYTFTSYY QAGLTQPPSASGSPGQSVTISCTGTSSDVG
MHWVRQAPGQGLQWMGIINPSGGSTSYAQNFQ GYNYVSWYQQHPGKAPKLMIYEVSKRPSG
GRVTMTRDTSTSTVYMELSSLISEDTAVYYCARSGYS
VPDRFSGSKSGNTASLTVSGLQAEDEADYY SSWLSYGMDVWGQGTTVTVSS
CSSFTTSSTWVFGGGTQLTVL SEQ ID NO. 35 SEQ ID NO. 36 VB-H4
MAEVQLVQSGAEVKKPGASVKLSCKASGYTFNNYA QPVLTQPPSASGTPGQRVTIYCSGSNSNIGG
TIWVRQAPEQGLEYVGWISAYSGHTNYAQKLQGR NSVNWYQQLPGTAPKLLIYHNNQRPSGVP
VSMTTDTSTTTAYMELRSLRSDDTAVYYCARWSG DRFSGSRSGTSASLAISGLQSGDEADYSCAA
WGSYHLLGMDVWGQGTTVTVSS WDDSLRGYVFGTGTKVTVL SEQ ID NO. 37 SEQ ID NO.
38 VB-H7 MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY
QAVLTQPASVSGSPGQSITISCTGTSSDVGG MHWVRQATGQGLEWLGWMNPKTGVTGYAQKF
YNYVSWYQQHPGKAPKLMIYEVNNRPSGV QGRVTMTRNTSINTAYMELNSLTSEDTADYYCARG
SNRFSGSKSGNTASLTISGLQAEDEADYYCT DYGGPQDMDVWGQGTTVTVSS
SYTSSNTRMFGGGTKLTVL SEQ ID NO. 39 SEQ ID NO. 40 VB-H9
MAEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY QAVLTQPRSVSGSPGQSVTISCTGTRSDVGT
MHWVRQAPGQGLEWMGWINPNSGGTNYAQKF YNYVSWYQQLPGKAPKLMIHDVSKRPSGV
QGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDL
PDRLSGSKSGNTASLTISGLQSEDEADYYCIS QGITIFGVANSPPYYYGMDVWGQGTTVTVSS
YTLHRTWMFGGGTKLTVL SEQ ID NO. 41 SEQ ID NO. 42 RV-A9
MAEVQLVQSGAEVKKPGASVKISCKASGYIFNTYSIH
QPVLTQPPSVSGTPGQRVSISCSGSSSNIGS WVRQAPGQSFEWMGWSSAGDDNTKYSDDFHHR
NSVNWYQQLPGTAPRLLIYNNDQRPSGVP LTIARDTSASTVYMELRGLTSDDTAIYYCARGYELDF
DRFSASKSGTSASLAIGGLQSEDEGDYYCSA WGQGTLVTVSS WDDSLNGPWVFGGGTKLTVL
SEQ ID NO. 43 SEQ ID NO. 44 RV-F8
MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSVLTQPPSVSAAPGQKVTISCSGSTSNIAN
MNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFT NFVSWYQQLPGTAPKLLIYDNNKRPSGIPD
ISRDNAKNSLYLQMNSLRAEDTAVYYCARVGATMG RFSGSKSGTSATLGITGLQAGDEADYFCGT
DYWGQGTLVTVSS WDSSLSASYVFGTGTKVTVL SEQ ID NO. 45 SEQ ID NO. 46
RV-H2 MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWI
DIVMTQSPSSVSAFVGDRVTITCRASQDVG GWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ
SWLVWYQQKPGKVPKLLIYGASTLQSGVPS VTISADKSTSTAYLQWSSLKASDTAMYYCARLGSSG
RFSGGGSGTDFTLTISSLQPEDFATYYCQQA WYDAFDIWGQGTMVTVSS KSLPYTFGQGTKLE I
K SEQ ID NO. 47 SEQ ID NO. 48 RV-H4
MAQVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYS LPVLTQPASVSGSPGQSITISCTGTDSDVGG
MNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFT YNYVSWYQQHPGKAPKLIISDVSNRPSGVS
ISRDNAKNSLYLQMNSLRAEDTAVYYCARVGATMG NRFSGSKSGNTASLTISGLQADDEADYYCNS
DYWGQGTLVTVSS YTVHATVLFGGGTKVTVL SEQ ID NO. 49 SEQ ID NO. 50 RV-H5
MAEVQLLESGGGLVKPRGSLRLSCAASGFTFSNAW QAGLTQPPSVSVSPGQTASITCSGDKLGDKY
MSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVK ISWYQQKPGQSPVMVIFQDTKRPSGIPERF
GRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTVWR SGSNSGNTATLTISGTQAMDEADYYCQAW
FGELFRWGQGTLVTVSS DTSTVFGGGTKLTVL SEQ ID NO. 51 SEQ ID NO. 52 C1
MAEVQLVQSGSELKKPGASVKISCKASGYTLTNHAL QPVLTQPPSVSVAPGQTARITCGGNNIGSK
NWVRQAPGQGLEWMAWMNTNTGNPTYAQDFT LVHWYQQKPGQAPVLVVYDDSDRPSGIPE
GRFVFSLDTSVSTAYLEISSLKAEDTAIYYCAREPRDA
RFSGSNSGNTATLTISRVEAGDEADYYCQV DAFDIWGQGTMVTVSS WDSSSDLVVFGGGTKLTVL
SEQ ID NO. 53 SEQ ID NO. 54 VR-A2
MAQVQLVQSGTEVKKPGESLRISCRSSGYKFTNYWI
QSVVTQPPSVSAAPRQKVTISCSGSSSNIGN GWVRQLPGQGLEWMGVILPGDSDTRYGPSFQGH
NYVSWYQQLPGTAPKLLIYDNNRRPSGIPD VSISVDKSISTVYLEWESLKASDTAMYYCASWDNFD
RFSGSKSGTSATLGITGLQTGDEADYYCGT HWGQGTLVTVSS WDSSLSAGVFGTGTKVTVL SEQ
ID NO. 55 SEQ ID NO. 56 VR-A3 MAEVQLVQSGAEVKKPGTSVTISCKTSGYTFTTYYIH
AIRMTQSPDSLAVSLGERATINCKSSQSVLY WVRQAPGQGLEWMGIILPSGGNTNYAPNFQGRV
SSNNKNYLAWYQQKPGQPPKLLIYWASTRE TMTRDTSTSTVNMELSSLTSDDTAVYYCVREYRGGY
SGVPDRFSGSGSGTDFTLTISSLQAEDVAVY FDYWGQGTLVTVSS
YCQQYYSTPYTFGQGTKLEIK SEQ ID NO. 57 SEQ ID NO. 58 VR-A10
MAQVQLVESGAEVKKPGASVKVSCKASGYTFTSYY QSVVTQPPSVSGAPGQRVTISCTGSSSNIGA
MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG GYDVHWYKQLPGTAPKLLIYGNNNRPSGV
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAN
PDRFSGSKSGTSASLAITGLQAEDEADYYCQ APNSYYYMDVWGKGTTVTVSS
SYDSSLSEGVFGTGTKVTVL SEQ ID NO. 59 SEQ ID NO. 60 VR-B2
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA DIQLTQSPPSVSASVGDRVTITCRASQDIST
MHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG WLAWYQQKPGSAPKVLIYAASTFQSGVPSR
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGA
FRGSGSGTYFTLTISGLQPEDFATYYCQQGN ASYWYFDLWGRGTLVTVSS SFPPTFGQGTKLEIK
SEQ ID NO. 61 SEQ ID NO. 62 VR-B4
MAQVQLVQSGAEVKKPGSSVKVSCKAYGGTFGSY SYELTQPASVSGSPGQSITISCTGSSSDVGGY
GVSWVRRAPGQGLEWMGRLIPIFGTRDYAQKFQG NFVSWYRQHAGKAPKLMIYDVTNRPSGVS
RVTLTADESTNTAYMELSSLRSEDTAVYYCARDGDY
TRFSGSKSGTTASLTISGLQPDDEAHYYCSSY YGSGSYYGMDVWGQGTTVTVSS
TTTSTWVFGGGTKLTVL SEQ ID NO. 63 SEQ ID NO. 64 VR-B11
MAEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG QSVVTQPPSVSAAPGQRVTISCTGSSSNIGA
MHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG GYDVHWYQQLPGTAPKLLIYANNNRPSGV
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGYS
PDRFSGSKTGTSASLAITGLQADDEADYFCQ YGGGFDYWGQGTLVTVSS
SYDSSLSGWVFGGGTKLTVL SEQ ID NO. 65 SEQ ID NO. 66 VR-05
MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSVLTQPASVSGSPGQSITISCTGTSSDVGG
MNWVRQAPGKGLEWVSSISSGSSYIYYADSLKGRF YNYVSWYQQHPGKAPKLMIYDVSNRPSGV
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDFGNW SNRFSGSKSGNTASLTISGLQAEDEADYYCS
GQGTLVTVSS SYTSSSTPYVFGTGTKVTVL SEQ ID NO. 67 SEQ ID NO. 68 VR-C7
MAQVQLVESGGGLVQPGGSLRLSCAASGFNFSSYE
SYELMQPHSVSESPGKTVTISCTGSSGSIASN MNWVRQAPGKGLEWVSYISSSGSTKHYADSVKGR
YVQWYQQRPGSAPTTVIYEDDQRPSGVPD FTISRDNAKNSLYLQMNSLRAEDTAVYYCAREHYNS
RFSGSIDSSSNSAALTISGLKTEDEADYYCQS WYFDLWGRGTLVTVSS YDSANVVFGGGTKLTVL
SEQ ID NO. 69 SEQ ID NO. 70 VR-C11
MAEVQLLESGGGWVKPGGSLRLSCAASGFPFSDYY LPVLTQPPSVSAAPGQKVTIPCSGTYSNIVN
MTWVRQAPGKGLEWVSYITTGGRIIYSADSVRGRF NYVSWYQQLPGTAPKLLIYDNNKRPSGIPD
TISRDNAKNSVYLQMNSLRAEDTAVYYCARPLRELS RFSGSKSGTSATLGITGLQTGDEADYYCGT
PGGFDLWGRGTMVTVSS WDNSLRRWVFGEGTKLTVL SEQ ID NO.71 SEQ ID NO. 72
VR-E3 MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS
QPVLTQPPSASGSPGQSVTISCTGTSSDVGG MNWVRQAPGKGLEWVSSISSASSYIYYADSVKGRF
YNYVSWYQQHPGKAPKLMIYDVNNRPSGV TISRDNAKKSLYLQLNSLTVEDTAVYYCAREYWGSP
PDRFSGSKSGNTASLTISGLQAEDEADYYCS DYWGRGTLVTVSS SYTSSSTRVFGGGTKLTVL
SEQ ID NO. 73 SEQ ID NO. 74 VR-G11
MAEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS QSVVTQPPSVSAAPGQKVTISCSGSSSNIGN
MNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFT
NYVSWYQQLPGTAPKLLIYDYNKRPSGIPDR ISRDNAKNSLYLQMNSLRAEDTAVYYCARGGVSPG
FSGSQSGTSATLGITGLQTGDEADYYCGTW DYWGQGTLVTVSS DSSLTLYVFGTGTKLTVL SEQ
ID NO. 75 SEQ ID NO. 76 VK-B8 MAQVQLVQSGAEVKKPGSSVKVSCKAYG
ETTLTQSPATLSVSPGERATVSCRASQSLGS
GTFGSYGVSWVRRAPGQGLEWMGRLIPI NLGWFQQKPGQAPRLLIYGASTRATGIPAR
FGTRDYAQKFQGRVTLTADESTNTAYMEL FSGSGSGTEFTLTISSLQSEDFAVYFCQQYN
SSLRSEDTAVYYCARDGDYYGSGSYYGMD DWPITFGQGTRLEIK VWGQGTLVTVSS SEQ ID
NO. 78 SEQ ID NO. 77 VR-H9 MAQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSY
QPVLTQPPSVSKDLRQTATLTCTGNGNNVG AISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGR
YQGAAWLQQHQGHPPKLLSYRNNNRPSGI VTITADKSTSTAYMELSSLRSEDTAVYYCARGHDYYG
SERFSASRSGNTASLTISGLQPEDEADYFCSA SGNNQEDYFDPWGQGTLVTVSS
WDNSLSAWVFGGGTKLTVL SEQ ID NO. 79 SEQ ID NO. 80 VK-B8A
MAQVQLVQSGAEVKKPGSSVKVSCKAYG ETTLTQSPATLSVSPGERATVSCRASQSLGS
GTFGSYGVSWVRRAPGQGLEWMGRLIPI NLGWFQQKPGQAPRLLIYGASTRATGIPAR
FGTRDYAQKFQGRVTLTADESTNTAYMEL FSGSGSGTEFTLTISSLQSEDFAVYFCQQYN
SSLRSEDTAVYYCARDGDYYGSGSYYGMD DWPITFGQGTKLEIK VWGQGTLVTVSS SEQ ID
NO. 81 SEQ ID NO. 77
Sequence CWU 1
1
811123PRThomo Sapiansmisc_feature(9)..(9)Xaa can be any naturally
occurring amino acid 1Met Ala Gln Val Gln Leu Val Gln Xaa Gly Ala
Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Ser Tyr Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Ile Ile
Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln 50 55 60 Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr 65 70 75 80 Val
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Asp Arg Gly Met Leu Arg His Trp Gly Met Asp Val
100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
2107PRThomo sapians 2Ala Ile Gln Leu 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 Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln 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 Ser Tyr Leu Thr Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 3121PRThomo
sapians 3Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
His Thr Phe Asn 20 25 30 Tyr Tyr Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Gln Thr Asn Leu Asn
Ser Gly Gly Thr Asn Tyr Ala Pro 50 55 60 Lys Phe Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr 65 70 75 80 Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Asn Leu Asn Ser Gly Trp Phe His Phe Glu Asn Trp Gly 100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 4108PRThomo sapians
4Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1
5 10 15 Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser
Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
Val Ile Tyr 35 40 45 Tyr Asp Arg Asp Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys
Gln Val Trp Asp Ser Ser Ser Tyr His 85 90 95 Pro Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 5128PRThomo sapians 5Met Ala
Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15
Gly Ser Ser Val Lys Val Ser Cys Glu Ala Ser Gly Gly Thr Phe Ser 20
25 30 Ser Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Met Gly Arg Val Ile Pro Val Phe Gly Thr Ala Asn
Tyr Ala Gln 50 55 60 Thr Phe Gln Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr 65 70 75 80 Met Phe Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Ser Gly
Asp Tyr Tyr Asp Gly Ser Arg Tyr Val 100 105 110 Asp Ala Phe Asp Ile
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 6110PRThomo
sapians 6Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Phe Cys Ser Gly Ser Thr Ser Asn
Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr His His Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Ala Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu 85 90 95 Asn Gly
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
7125PRThomo sapians 7Met Ala Gln Val Gln Leu Val Gln Ser Gly Pro
Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Ser Tyr Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Gln 35 40 45 Trp Met Gly Ile Ile
Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln 50 55 60 Asn Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr 65 70 75 80 Val
Tyr Met Glu Leu Ser Ser Leu Ile Ser Gln Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Ser Gly Tyr Ser Ser Ser Trp Leu Ser Tyr Gly Met
100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125 8110PRThomo sapians 8Ser Tyr Val Leu Thr Gln Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ala
Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp
Tyr Gln Gln Asn Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr
Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70
75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Asn
Ser 85 90 95 Asp Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 110 9115PRThomo sapians 9Met Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ser
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp
Val Ser Ser Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55
60 Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Phe Gly Asn Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser 115 10111PRThomo sapians
10Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Ala Ser Pro Gly Gln 1
5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Ser Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Thr 85 90 95 Ser Ser Pro Val Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 11120PRThomo
sapians 11Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Phe Thr Leu Ser 20 25 30 Ser Tyr Glu Met Met Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Asp Ser
Gly Gly Leu Ile Tyr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Ser Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Asp Glu Tyr Ser Ser Gly Met Asp Val Trp Gly Gln 100 105 110
Gly Thr Thr Val Thr Val Ser Ser 115 120 12110PRThomo sapians 12Gln
Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Leu Gly Ser Asn
20 25 30 Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Arg Asn Lys Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr
Cys Ala Val Trp Asp Gly Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 13126PRThomo sapians
13Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1
5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ser 20 25 30 Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu 35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Ala Gly Ser
Thr Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr 65 70 75 80 Val Tyr Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly
Tyr Tyr Tyr Asp Thr Ser Gly Tyr His Gly Tyr 100 105 110 Phe Lys Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
14111PRThomo sapiansmisc_feature(78)..(78)Xaa can be any naturally
occurring amino acid 14Gln Ser Val Leu Thr Gln Pro Arg Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Asp Lys Ala Pro Lys Leu 35 40 45 Met Leu Tyr Asp Val
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Xaa Ala Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90
95 Thr Asn Leu Gly Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100
105 110 15124PRThomo sapians 15Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ser Phe Asp 20 25 30 Asp Tyr Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser
Ser Ile Ser Trp Asn Ser Gly Ser Arg Gly Tyr Ala Asp 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70
75 80 Leu Phe Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
Tyr 85 90 95 Tyr Cys Ala Lys Gly Val Val Tyr Ser Ser Pro Tyr Tyr
Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 16110PRThomo sapians 16Gln Ser Ala Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Thr Asn 20 25 30 Thr Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu 35 40 45 Ile Tyr
Phe Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60
Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65
70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Asp
Ile Leu 85 90 95 Asn Asp Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 17117PRThomo sapians 17Met Ala Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Asn 20 25 30 Thr Tyr
Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Phe Glu 35 40 45
Trp Met Gly Trp Ser Ser Ala Gly Asp Asp Asn Thr Lys Tyr Ser Asp 50
55 60 Asp Phe His His Arg Leu Thr Ile Ala Arg Asp Thr Ser Ala Ser
Thr 65 70 75 80 Val Tyr Met Glu Leu Arg Gly Leu Thr Ser Asp Asp Thr
Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Gly Tyr Glu Leu Asp Phe Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
18117PRThomo sapaians 18Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ile Phe Asn 20 25 30 Thr Tyr Ser Ile His Trp Val
Arg Gln Ala Pro Gly Gln Ser Phe Glu 35 40 45 Trp Met Gly Trp Ser
Ser Ala Gly Asp Asp Asn Thr Lys Tyr Ser Asp 50 55 60 Asp Phe His
His Arg Leu Thr Ile Ala Arg Asp Thr Ser Ala Ser Thr 65 70 75 80 Val
Tyr Met Glu Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala Ile Tyr 85 90
95 Tyr Cys Ala Arg Gly Tyr Glu Leu Asp Phe Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 19120PRThomo
sapiansmisc_feature(20)..(20)Xaa can be any naturally occurring
amino acid 19Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu
Val Lys Pro 1 5 10 15 Gly Gly Ser Xaa Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Phe Ser 20 25 30 Asp Tyr Tyr Met Ser Trp Ile Arg Gln
Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Ser
Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr
Cys Ala Arg Asp Ala Pro Arg Tyr Gly Met Asp Val Trp Gly Gln 100 105
110 Gly Thr Thr Val Thr Val Ser Ser 115
120 20111PRThomo sapians 20Gln Ser Ala Leu Thr Gln Pro Ala Ser Val
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Ile Thr Ile Ser Cys Ala Gly
Thr Pro Ser Asp Ile Gly Leu Tyr 20 25 30 Asn Tyr Val Ala Trp Phe
Gln His His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Ile Ile Tyr Asp
Val Thr Lys Arg Pro Ser Gly Thr Ser Asn Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80
Gln Ala Asp Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Thr Ser 85
90 95 Asn Ser Phe Val Leu Phe Gly Glu Gly Thr Lys Leu Thr Val Leu
100 105 110 21127PRThomo sapians 21Met Ala Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser 20 25 30 Ser Tyr Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln 50 55 60
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr 65
70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Ser Glu Arg Val Tyr Ser Ser
His Thr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 125 22110PRThomo sapians 22Gln Ser Val
Val Thr Gln Gln Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys
Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Asn Asn 20 25
30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ser Ser Ala Ser Leu Ala Ile
Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Thr Gly
Thr Lys Leu Thr Val Leu 100 105 110 23123PRThomo sapians 23Met Ala
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 Asn Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Ser Gly Ser Thr Asn
Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr 65 70 75 80 Val Tyr Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gln Arg Trp
Asp Leu Leu Asp Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 24113PRThomo sapians 24Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Lys Thr Ser Gln Ser Val Leu Tyr Asn 20 25
30 Ala Asn Asn Lys Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser
Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 65 70 75 80 Ile Arg Ser Leu Gln Pro Asp Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 85 90 95 Ala Ile Ser Phe Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys 25119PRThomo
sapians 25Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Lys Asp Trp Ser Gly Pro Ile Thr Leu Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 26109PRThomo sapians 26Ser Tyr Glu
Leu Met Gln Pro Pro Ser Val Ser Glu Ala Pro Gly Met 1 5 10 15 Thr
Ala Gln Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25
30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45 Tyr Asp Ser Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Ser Leu Thr Ile Asn Arg
Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Asp Ser Ser Ser Asp His 85 90 95 His Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 27120PRThomo sapians 27Met Ala Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30
Ser Tyr Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35
40 45 Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Gly Leu Arg Ala Glu
Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Asp Ser Ser
Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 28110PRThomo sapians 28Gln Ser Ala Leu Thr Gln Pro Pro
Ser Met Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys
Ser Gly Thr Ser Ser Asn Ile Gly Asn His 20 25 30 Tyr Val Ser Trp
Tyr Gln Arg Leu Pro Asp Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Asp Asn Asn Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65
70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser
Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
Val Leu 100 105 110 29110PRThomo sapians 29Gln Ser Ala Leu Thr Gln
Pro Pro Ser Met Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile
Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly Asn His 20 25 30 Tyr Val
Ser Trp Tyr Gln Arg Leu Pro Asp Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Asp Asn Asn Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu
Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp
Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu 100 105 110 30112PRThomo sapians 30Asp Ile Val Met Thr
Gln Thr Pro Leu Phe Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp
Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40
45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Ile Arg Asp Ser Gly Val Pro
50 55 60 Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp Ile Gly Val Tyr Tyr
Cys Met Gln Gly 85 90 95 Thr Asp Arg Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 31120PRThomo sapians 31Met Ala
Gln Met Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15
Gly Arg Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe Asn 20
25 30 Asp Tyr Pro Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Val Ala Leu Leu Ser Tyr Asp Gly Thr Ser Ala Tyr
Tyr Ala Asp 50 55 60 Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu Arg
Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ser Glu Gly Ser
Pro Asp Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr
Val Ser Ser 115 120 32111PRThomo sapians 32Gln Ala Gly Leu Thr Gln
Ser Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser 85 90 95 Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys
Val Thr Val Leu 100 105 110 33125PRThomo sapians 33Met Ala Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Glu
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr 20 25 30
Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu 35
40 45 Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser
Pro 50 55 60 Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr 65 70 75 80 Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Arg Leu Asp Tyr Asp Ser
Ser Gly Tyr Tyr Gly Ala Phe 100 105 110 Asp Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 125 34110PRThomo sapians 34Gln Ser
Val Val Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Thr Thr Asp Val Gly Ala Tyr 20
25 30 Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys
Leu 35 40 45 Ile Ile Tyr Asp Leu Asn Asn Arg Pro Ser Gly Ile Ser
Asn Arg Phe 50 55 60 Ser Gly Phe Lys Ser Gly Asn Thr Ala Ser Leu
Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 110 35125PRThomo sapians 35Met
Ala Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro 1 5 10
15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Gln 35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr
Ser Tyr Ala Gln 50 55 60 Asn Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr 65 70 75 80 Val Tyr Met Glu Leu Ser Ser Leu
Ile Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ser Gly
Tyr Ser Ser Ser Trp Leu Ser Tyr Gly Met 100 105 110 Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 36110PRThomo
sapians 36Gln Ala Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Lys Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Thr Thr Ser 85 90 95 Ser Thr
Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110
37125PRThomo sapians 37Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Asn 20 25 30 Asn Tyr Ala Thr Ile Trp Val
Arg Gln Ala Pro Glu Gln Gly Leu Glu 35 40 45 Tyr Val Gly Trp Ile
Ser Ala Tyr Ser Gly His Thr Asn Tyr Ala Gln 50 55 60 Lys Leu Gln
Gly Arg Val Ser Met Thr Thr Asp Thr Ser Thr Thr Thr 65 70 75 80 Ala
Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Trp Ser Gly Trp Gly Ser Tyr His Leu Leu Gly Met
100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125 38110PRThomo sapians 38Gln Pro Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Tyr Cys Ser
Gly Ser Asn Ser Asn Ile Gly Gly Asn 20 25 30 Ser Val Asn Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr His
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly
Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70
75 80 Ser Gly Asp Glu Ala Asp Tyr Ser Cys Ala Ala Trp Asp Asp Ser
Leu 85 90 95 Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val
Leu 100 105 110 39122PRThomo sapians 39Met Ala Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Gly Tyr Tyr
Met His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu
Glu 35 40 45 Trp Leu Gly Trp Met Asn Pro Lys Thr Gly Val Thr Gly
Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asn
Thr Ser Ile Asn Thr 65 70 75 80 Ala Tyr Met Glu Leu Asn Ser Leu Thr
Ser Glu Asp Thr Ala Asp Tyr 85 90 95 Tyr Cys Ala Arg Gly Asp Tyr
Gly Gly Pro Gln Asp Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 40110PRThomo sapians 40Gln Ala Val Leu
Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Glu Val Asn Asn Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Thr
Ser Tyr Thr Ser Ser 85 90 95 Asn Thr Arg Met Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110 41133PRThomo sapians 41Met Ala Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25
30 Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
35 40 45 Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr
Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Ile Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Leu Gln Gly
Ile Thr Ile Phe Gly Val Ala Asn 100 105 110 Ser Pro Pro Tyr Tyr Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr 115 120 125 Val Thr Val Ser
Ser 130 42110PRThomo sapians 42Gln Ala Val Leu Thr Gln Pro Arg Ser
Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr
Gly Thr Arg Ser Asp Val Gly Thr Tyr 20 25 30 Asn Tyr Val Ser Trp
Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile His
Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Leu 50 55 60 Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70
75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ile Ser Tyr Thr Leu
His 85 90 95 Arg Thr Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 110 43117PRThomo sapians 43Met Ala Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Asn 20 25 30 Thr Tyr Ser
Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Phe Glu 35 40 45 Trp
Met Gly Trp Ser Ser Ala Gly Asp Asp Asn Thr Lys Tyr Ser Asp 50 55
60 Asp Phe His His Arg Leu Thr Ile Ala Arg Asp Thr Ser Ala Ser Thr
65 70 75 80 Val Tyr Met Glu Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala
Ile Tyr 85 90 95 Tyr Cys Ala Arg Gly Tyr Glu Leu Asp Phe Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 44111PRThomo
sapians 44Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro
Gly Gln 1 5 10 15 Arg Val Ser Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asn Asn Asp Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu
Gly Asp Tyr Tyr Cys Ser Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly
Pro Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
45119PRThomo sapians 45Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ser Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile
Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Val Gly Ala Thr Met Gly Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 46111PRThomo sapians
46Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1
5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Ala Asn
Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr
Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr
Phe Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Ser Tyr Val
Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 47123PRThomo
sapians 47Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro 1 5 10 15 Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Ser Phe Thr 20 25 30 Ser Tyr Trp Ile Gly Trp Val Arg Gln Met
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Met Gly Ile Ile Tyr Pro Gly
Asp Ser Asp Thr Arg Tyr Ser Pro 50 55 60 Ser Phe Gln Gly Gln Val
Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr 65 70 75 80 Ala Tyr Leu Gln
Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 Tyr Cys
Ala Arg Leu Gly Ser Ser Gly Trp Tyr Asp Ala Phe Asp Ile 100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 48107PRThomo
sapians 48Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Phe
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Val Gly Ser Trp 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly 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 Ala Lys Ser Leu Pro Tyr 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 49119PRThomo sapians
49Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro 1
5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30 Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr
Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Val
Gly Ala Thr Met Gly Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 50110PRThomo sapians 50Leu Pro Val Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile
Ser Cys Thr Gly Thr Asp Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr
Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45
Ile Ile Ser Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr
Thr Val His 85 90 95 Ala Thr Val Leu Phe Gly Gly Gly Thr Lys Val
Thr Val Leu 100 105 110 51122PRThomo sapians 51Met Ala Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Arg Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Asn
Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40
45 Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr
50 55 60 Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys 65 70 75 80 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Thr Thr Val Trp Arg Phe
Gly Glu Leu Phe Arg Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 52105PRThomo sapians 52Gln Ala Gly Leu Thr Gln Pro
Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr
Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ile 20 25 30 Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Val Met Val Ile Phe 35 40 45 Gln
Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Thr Ser Thr
Val Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
53121PRThomo sapians 53Met Ala Glu Val Gln Leu Val Gln Ser Gly Ser
Glu Leu Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Leu Thr 20 25 30 Asn His Ala Leu Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Ala Trp Met
Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln 50 55 60 Asp Phe Thr
Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr 65 70 75 80 Ala
Tyr Leu Glu Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr 85 90
95 Tyr Cys Ala Arg Glu Pro Arg Asp Ala Asp Ala Phe Asp Ile Trp Gly
100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120
54108PRThomo sapians 54Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly Ser Lys Leu Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Leu 85 90
95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
55117PRThomo sapians 55Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr
Glu Val Lys Lys Pro 1 5 10 15 Gly Glu Ser Leu Arg Ile Ser Cys Arg
Ser Ser Gly Tyr Lys Phe Thr 20 25 30 Asn Tyr Trp Ile Gly Trp Val
Arg Gln Leu Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Val Ile
Leu Pro Gly Asp Ser Asp Thr Arg Tyr Gly Pro 50 55 60 Ser Phe Gln
Gly His Val Ser Ile Ser Val Asp Lys Ser Ile Ser Thr 65 70 75 80 Val
Tyr Leu Glu Trp Glu Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90
95 Tyr Cys Ala Ser Trp Asp Asn Phe Asp His Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 56110PRThomo sapians 56Gln Ser
Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Arg Gln 1 5 10 15
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20
25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Asp Asn Asn Arg Arg Pro Ser Gly Ile Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly
Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys
Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Gly Val Phe Gly Thr
Gly Thr Lys Val Thr Val Leu 100 105 110 57120PRThomo sapians 57Met
Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10
15 Gly Thr Ser Val Thr Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr
20 25 30 Thr Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu 35 40 45 Trp Met Gly Ile Ile Leu Pro Ser Gly Gly Asn Thr
Asn Tyr Ala Pro 50 55 60 Asn Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr 65 70 75 80 Val Asn Met Glu Leu Ser Ser Leu
Thr Ser Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg Glu Tyr
Arg Gly Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser 115 120 58113PRThomo sapians 58Ala Ile Arg Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser
Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr
Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile 100 105 110 Lys 59125PRThomo sapians 59Met
Ala Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro 1 5 10
15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu 35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr
Ser Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr 65 70 75 80 Val Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly
Ala Asn Ala Pro Asn Ser Tyr Tyr Tyr Met 100 105 110 Asp Val Trp Gly
Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 60111PRThomo
sapians 60Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Lys Gln Leu Pro
Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser
Glu Gly Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110
61123PRThomo sapians 61Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Asp Leu Gly Ala Ala Ser Tyr Trp Tyr Phe Asp Leu
100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120
62107PRThomo sapians 62Asp Ile Gln Leu Thr Gln Ser Pro Pro Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Ser Ala Pro Lys Val Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Phe Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser
Gly Thr Tyr Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Phe Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 63126PRThomo
sapians 63Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Tyr Gly
Gly Thr Phe Gly 20 25 30 Ser Tyr Gly Val Ser Trp Val Arg Arg Ala
Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Leu Ile Pro Ile
Phe Gly Thr Arg Asp Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Ser Thr Asn Thr 65 70 75 80 Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Asp Gly Asp Tyr Tyr Gly Ser Gly Ser Tyr Tyr Gly 100 105 110
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
64110PRThomo sapians 64Ser Tyr Glu Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Phe Val Ser Trp Tyr Arg
Gln His Ala Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val
Thr Asn Arg Pro Ser Gly Val Ser Thr Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Pro Asp Asp Glu Ala His Tyr Tyr Cys Ser Ser Tyr Thr Thr Thr 85 90
95 Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 65122PRThomo sapians 65Met Ala Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val
Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Ala Lys Asp Gly Tyr Ser Tyr Gly Gly Gly Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
66111PRThomo sapians 66Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Asn
Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Thr Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln
Ala Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser Ser 85 90
95 Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 67115PRThomo sapians 67Met Ala Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ser Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser
Ser Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser
Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Arg Asp Phe Gly Asn Trp Gly Gln Gly Thr
Leu Val Thr 100 105 110 Val Ser Ser 115 68111PRThomo sapians 68Gln
Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Pro Tyr Val Phe
Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 69121PRThomo
sapians 69Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Asn Phe Ser 20 25 30 Ser Tyr Glu Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Ser Ser
Gly Ser Thr Lys His Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Glu His Tyr Asn Ser Trp Tyr Phe Asp Leu Trp Gly 100 105 110
Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 70110PRThomo sapians
70Ser Tyr Glu Leu Met Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1
5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser
Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro
Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser
Ala Ala Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ala Asn Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 71123PRThomo
sapians 71Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Trp Val
Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Pro Phe Ser 20 25 30 Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Thr Thr Gly
Gly Arg Ile Ile Tyr Ser Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Pro Leu Arg Glu Leu Ser Pro Gly Gly Phe Asp Leu 100 105 110
Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 72110PRThomo
sapians 72Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Gln 1 5 10 15 Lys Val Thr Ile Pro Cys Ser Gly Thr Tyr Ser Asn
Ile Val Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asn Ser Leu 85 90 95 Arg Arg
Trp Val Phe Gly Glu Gly Thr Lys Leu Thr Val Leu 100 105 110
73119PRThomo sapians 73Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ser Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile
Ser Ser Ala Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser 65 70 75 80 Leu
Tyr Leu Gln Leu Asn Ser Leu Thr Val Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Arg Glu Tyr Trp Gly Ser Pro Asp Tyr Trp Gly Arg Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 74110PRThomo sapians
74Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1
5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Asn Asn Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 75119PRThomo
sapians 75Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser 20 25 30 Ser Tyr Ser Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Ser Ser
Ser Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Gly Gly Val Ser Pro Gly Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 76110PRThomo sapians 76Gln Ser Val
Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25
30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gln Ser Gly Thr Ser Ala Thr Leu Gly Ile
Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly
Thr Trp Asp Ser Ser Leu 85 90 95 Thr Leu Tyr Val Phe Gly Thr Gly
Thr Lys Leu Thr Val Leu 100 105 110 77126PRThomo sapians 77Met Ala
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15
Gly Ser Ser Val Lys Val Ser Cys Lys Ala Tyr Gly Gly Thr Phe Gly 20
25 30 Ser Tyr Gly Val Ser Trp Val Arg Arg Ala Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Met Gly Arg Leu Ile Pro Ile Phe Gly Thr Arg Asp
Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Leu Thr Ala Asp
Glu Ser Thr Asn Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Gly Asp
Tyr Tyr Gly Ser Gly Ser Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 78107PRThomo
sapians 78Glu Thr Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser
Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Val Ser Cys Arg Ala Ser
Gln Ser Leu Gly Ser Asn 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp
Phe Ala Val Tyr Phe Cys Gln Gln Tyr Asn Asp Trp Pro Ile 85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 79128PRThomo
sapians 79Met Ala Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser 20 25 30 Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Ile Ile Pro Ile
Leu Gly Ile Ala Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr 65 70 75 80 Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Gly His Asp Tyr Tyr Gly Ser Gly Asn Asn Gln Glu 100 105 110
Asp Tyr Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125 80110PRThomo sapians 80Gln Pro Val Leu Thr Gln Pro Pro Ser
Val Ser Lys Asp Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr
Gly Asn Gly Asn Asn Val Gly Tyr Gln 20 25 30 Gly Ala Ala Trp Leu
Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Tyr Arg
Asn Asn Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60 Ala
Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln 65 70
75 80 Pro Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ala Trp Asp Asn Ser
Leu 85 90 95 Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 110 81107PRThomo sapains 81Glu Thr Thr Leu Thr Gln Ser
Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Val
Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Asn 20 25 30 Leu Gly Trp
Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Asn Asp Trp
Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105
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