U.S. patent application number 12/317074 was filed with the patent office on 2010-08-05 for method of inhibition of vascular development using an antibody.
This patent application is currently assigned to DYAX CORP.. Invention is credited to Daniel T. Dransfield, Rene Hoet, Simon E. Hufton, Henk Pieters, Clive R. Wood.
Application Number | 20100196361 12/317074 |
Document ID | / |
Family ID | 46322406 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100196361 |
Kind Code |
A1 |
Wood; Clive R. ; et
al. |
August 5, 2010 |
Method of inhibition of vascular development using an antibody
Abstract
Tie1 and Tie2 are receptor tyrosine kinase proteins that include
a transmembrane domain. Tie1 and Tie2 are present on endothelial
cells. This disclosure describes agents, such as antibodies, that
bind to Tie1, Tie2, and Ang, including ones that inhibit
endothelial cell activity and angiogenesis. The agents can be used
to treat angiogenesis-associated disorders.
Inventors: |
Wood; Clive R.; (Boston,
MA) ; Dransfield; Daniel T.; (Hanson, MA) ;
Pieters; Henk; (Maastricht, NL) ; Hoet; Rene;
(Maastricht, NL) ; Hufton; Simon E.; (Lancs,
GB) |
Correspondence
Address: |
MCCARTER & ENGLISH, LLP BOSTON
265 Franklin Street
Boston
MA
02110
US
|
Assignee: |
DYAX CORP.
Cambridge
MA
|
Family ID: |
46322406 |
Appl. No.: |
12/317074 |
Filed: |
December 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11199739 |
Aug 9, 2005 |
7485297 |
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12317074 |
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11049536 |
Feb 2, 2005 |
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11199739 |
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10916840 |
Aug 12, 2004 |
7348001 |
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11049536 |
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PCT/US04/26116 |
Aug 12, 2004 |
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11199739 |
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60494713 |
Aug 12, 2003 |
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60494713 |
Aug 12, 2003 |
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Current U.S.
Class: |
424/133.1 ;
424/130.1; 530/387.1; 530/387.3 |
Current CPC
Class: |
C07K 2319/30 20130101;
C07K 16/005 20130101; C07K 2317/567 20130101; C07K 2319/00
20130101; C07K 2317/21 20130101; C07K 2317/73 20130101; C07K
16/2863 20130101; C07K 2317/75 20130101; C07K 2317/76 20130101;
C07K 2317/92 20130101; C07K 2317/55 20130101; C07K 2317/565
20130101; A61K 2039/505 20130101; A61P 35/04 20180101 |
Class at
Publication: |
424/133.1 ;
530/387.1; 530/387.3; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; A61P 35/04 20060101
A61P035/04 |
Claims
1-28. (canceled)
29. An isolated human antibody, or antigen-binding fragment
thereof, that binds to human Tie1 with an affinity K.sub.D of less
than 10.sup.-8 M.
30. The isolated human antibody, or antigen-binding fragment
thereof, of claim 29, which binds to human Tie1 with an affinity
K.sub.D of less than 5.times.10.sup.-9 M.
31. The isolated human antibody, or antigen-binding fragment
thereof, of claim 29, which binds to human Tie1 with an affinity
K.sub.D of less than 10.sup.-9 M.
32. An isolated human antibody, or antigen-binding fragment
thereof, that binds to human Tie1 with a K.sub.on rate of at least
6.19.times.10.sup.3 Ms.sup.-1.
33. The isolated human antibody, or antigen-binding fragment
thereof, of claim 32, which binds to human Tie1 with a K.sub.on
rate of at least 7.09.times.10.sup.3 Ms.sup.-1.
34. An isolated human antibody, or antigen-binding fragment
thereof, that binds to human Tie1 and has a K.sub.off rate of
1.02.times.10.sup.-3 s.sup.-1 or less.
35. The isolated human antibody, or antigen-binding fragment
thereof, of claim 34, which binds to human Tie1 and has a K.sub.off
rate of 3.67.times.10.sup.-5 s.sup.-1 or less.
36. The isolated human antibody, or antigen-binding fragment
thereof, of any one of claim 29, 32 or 34, which is a recombinant
antibody.
37. The isolated human antibody, or antigen-binding fragment
thereof, of any one of claim 29, 32 or 34, which is a Fab.
38. The isolated human antibody, or antigen-binding fragment
thereof, of any one of claim 29, 32 or 34, which is an IgG.
39. An isolated human antibody or antigen-binding fragment thereof,
which: induces Tie1 tyrosine phosphorylation; inhibits tube
formation by human umbilical endothelial cells (HUVECs) in vitro;
inhibits angiogenesis in a MATRIGEL.TM. in vivo assay; or
antagonizes the formation of a Tie1, Tie 2 and Ang heteromeric
complex.
40. The isolated human antibody, or antigen-binding fragment
thereof, of claim 39, wherein the antibody, or antigen-binding
fragment thereof, has a heavy chain CDR3 comprising the amino acid
sequence of residues 99-109 of SEQ ID NO:114; and has a light chain
CDR3 comprising the amino acid sequence of residues 89-96 of SEQ ID
NO:116.
41. The isolated human antibody, or antigen-binding fragment
thereof, of claim 39, wherein angiogenesis is inhibited by at least
70% in the MATRIGEL.TM. in vivo assay.
42. The isolated human antibody, or antigen-binding fragment
thereof, of claim 39, which has no deleterious effect on stem
cells.
43. The isolated human antibody, or antigen-binding fragment
thereof, of claim 39, which does not induce platelet agglutination
or aggregation.
44. An isolated human antibody or antigen-binding fragment thereof,
which dissociates from Tie1 with a K.sub.off rate of
1.02.times.10.sup.-3 s.sup.-1 or less and binds to the Tie1
ectodomain.
45. An isolated human antibody or antigen-binding fragment thereof,
with a light chain variable region (LCVR) having a CDR3 domain
comprising the amino acid sequence of residues 89-96 of SEQ ID
NO:116, and with a heavy chain variable region (HCVR) having a CDR3
domain comprising the amino acid sequence of residues 99-109 of SEQ
ID NO:114.
46. The isolated human antibody, or antigen-binding fragment
thereof, of claim 45, wherein the LCVR further has a CDR2 domain
comprising the amino acid sequence of residues 50-56 of SEQ ID
NO:116 and the HCVR further has a CDR2 domain comprising the amino
acid sequence of residues 50-66 of SEQ ID NO:114.
47. The isolated human antibody, or antigen-binding fragment
thereof, of claim 45, wherein the LCVR further has a CDR1 domain
comprising the amino acid sequence of residues 24-34 of SEQ ID
NO:116 and the HCVR further has a CDR1 domain comprising the amino
acid sequence of residues 31-35 of SEQ ID NO:114.
48. A pharmaceutical composition comprising a human antibody, or
antigen binding portion thereof, and a pharmaceutically acceptable
carrier, wherein the human antibody, or antigen-binding portion
thereof, comprises a light chain CDR3 domain comprising the amino
acid sequence of residues 89-96 of SEQ ID NO:116 and a heavy chain
CDR3 domain comprising the amino acid sequence of residues 99-109
of SEQ ID NO:114.
49. A pharmaceutical composition comprising the human antibody, or
antigen-binding portion thereof, of claim 29 and a pharmaceutically
acceptable carrier.
50. A method of inhibiting vascular development in a subject, the
method comprising administering to a subject having or at risk for
a disorder requiring inhibition of vascular development a human
antibody, or antigen-binding fragment thereof, according to claim
29.
51. The method of claim 50, wherein the human antibody, or
antigen-binding fragment thereof, is administered in an amount
effective to decrease vascular development in a subject.
52. The method of claim 50, wherein the subject has a
vascular-dependent cancer or tumor.
53. The method of claim 52, wherein the tumor is a solid tumor.
54. The method of claim 50, further comprising a second therapy
that is an anti-cancer therapy.
55. The method of claim 54, wherein the second therapy is
chemotherapy.
56. The method of claim 54, wherein the second therapy comprises
administering bevacizumab.
57. The method of claim 54, wherein the second therapy comprises
administering an agent selected from the group consisting of 5-FU,
leucovorin, avastin, cyclophosphamide, and/or irinotecan.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/199,739, filed Aug. 9, 2005, which is a continuation-in-part
of U.S. application Ser. No. 11/049,536, filed Feb. 2, 2005, which
is a continuation-in-part of U.S. application Ser. No. 10/916,840,
filed Aug. 12, 2004, which claims priority to U.S. application Ser.
No. 60/494,713, filed on Aug. 12, 2003. This application is also a
continuation-in-part of PCT/US2004/026116, filed Aug. 12, 2004,
which claims priority to U.S. Application Ser. No. 60/494,713,
filed on Aug. 12, 2003. The contents of each of the foregoing
applications are hereby incorporated by reference in their
entirety. This application incorporates by reference ASCII text
file identified by the name 10280-135001.txt, containing 669 KB of
data, and created on Oct. 24, 2005, filed in computer-readable
format (CRF) and encoded on the CD-ROM.
BACKGROUND
[0002] The oxygen and nutrients supplied by the blood vessels are
crucial for tissue development and function. Indeed, the
cardiovascular system is the first organ system to develop in
embryos. During organogenesis and the development of tissues or
tumors, the proximity of the growing cells to the circulatory
system is ensured by the coordinated growth of blood vessels and
organ parenchyma. It may be possible to prevent or treat diseases
by modulating blood vessel development or angiogenesis.
[0003] Blood vessels are composed of an inner layer of endothelial
cells and an outer layer of pericytes or smooth muscle cells. The
first tubular structures are formed by endothelial cells that
subsequently recruit pericytes and smooth muscle cells to ensheath
them. The de novo formation of blood vessels from a dispersed
population of mesodermally derived endothelial precursor cells is
termed vasculogenesis. This primitive network undergoes successive
morphogenetic events including sprouting, splitting, and remodeling
to generate the hierarchical vascular network from large to
branched small vessels. These successive morphogenetic events are
collectively called angiogenesis. Previous studies have identified
a number of endothelial cell specific receptor tyrosine kinases
(RTKs) and their cognate ligands, which mediate the vasculogenic
and angiogenic development of blood vessels. Members of the
vascular endothelial growth factor (VEGF) family and their
receptors function during the formation of the initial embryonic
vascular plexus, whereas angiopoietins (Angs) and their receptor,
Tie2, as well as ephrins and their Eph receptors are implicated in
the subsequent remodeling processes. See, e.g., Jones et al. (2001)
Nat. Rev. Molec. Cell Biol. 2:257 for a review of receptors
involved in angiogenic and lymphangiogenic responses.
[0004] Tie1 and Tie2 are RTKs that are expressed almost exclusively
in endothelial cells and hematopoietic precursor cells. These two
receptors are required for the normal development of vascular
structures during embryogenesis. The two Tie receptors form a RTK
subfamily since, unlike other RTK family members, they include
extracellular EGF-homology domains. See, e.g., Partanen (1992) Mol.
Cell Biol. 12:1698 and WO 93/14124. Targeted disruption of the Tie1
gene in mice results in a lethal phenotype characterized by
extensive hemorrhage and defective microvessel integrity. See,
e.g., Puri et al. (1995) EMBO J. 14:5884. Tie2 null embryos have
defects in vascular remodeling and maturation, resulting from
improper recruitment of periendothelial supporting cells.
Angiopoietins (Ang, e.g., Ang1, Ang2, Ang3, and Ang4) are proteins
that interact with Tie2.
SUMMARY
[0005] In one aspect, the invention features a method of modulating
Tie complex formation, or interactions between Tie complex
components, in a subject. The method includes administering, to a
subject, an agent that binds to Tie1. For example, the agent
promotes Tie1 self-association (e.g., homodimerization) or
antagonizes an association between at least two of the following:
Tie1, Tie2, and an angiopoietin (Ang; such as Ang1, Ang2, Ang3, or
Ang4). In one embodiment, the agent antagonizes formation of a
heteromeric complex of Tie1, Tie2, and Ang. In another embodiment,
the binding of the agent can antagonize the association between
Tie1 and Tie2, between Tie1 and Ang, or between Tie2 and Ang.
[0006] In one embodiment, the agent binds to Tie1. In one
embodiment, the agent antagonizes formation of a heteromeric
complex of Tie1, Tie2, and Ang. In another embodiment, the binding
of the agent can antagonize the association between Tie1 and Tie2,
between Tie1 and Ang, or between Tie2 and Ang. In another
embodiment, the agent enhances Tie1 self-association, e.g.,
homodimerization, and thereby associates Tie1 with Tie1 and
prevents association of Tie1 with Tie2 and/or Ang. The agent can
include at least two valencies for binding to Tie1. In one
embodiment, the agent increases phosphorylation of Tie1, e.g., Tie1
autophosphorylation. This increase can, but need not, depend on
Tie1 self-association.
[0007] In one embodiment, the agent includes a protein, such as an
antibody, that binds to the extracellular domain of human Tie1. For
example, the antibody can be one or more of the following: human,
humanized, non-immunogenic, isolated, monoclonal, and recombinant.
In one embodiment, the antibody can bind to the first Ig-like
C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig
2) of Tie1. In one embodiment, the antibody binds to an EGF-like
domain of Tie1 (e.g., first, second, or third EGF-like domain). In
one embodiment, the antibody binds to the fibronectin type III
repeats region of Tie1. In one embodiment, the antibody binds to
amino acid residues 24-124, 74-174, 124-224, 174-274, 224-324,
274-374, 324-424, 374-474, 424-524, 474-574, 524-624, 574-674,
624-724, 674-759, or 724-759 of SEQ ID NO:2.
[0008] In one embodiment, the agent includes a protein that binds
to a Tie1 ectodomain and includes a heavy chain (HC) immunoglobulin
variable domain sequence and a light chain (LC) immunoglobulin
variable domain sequence. The protein can further include one or
more of the following properties: (1) at least one of the variable
domain sequences includes at least one CDR of the E3 or E3b
antibody (e.g., one, two, or three CDRs of the E3 or E3b antibody);
(2) at least one of the variable domain sequences includes CDR
sequences at least 85% identical, in sum, to the CDRs of the
corresponding variable domain of the E3 or E3b antibody, (3) at
least one of the variable domains is at least 85% identical to the
corresponding immunoglobulin variable domains of the E3 or E3b
antibody, and (4) the protein competes with E3 or E3b for binding
to Tie1 or binds to an epitope that overlaps the epitope bound by
E3 or E3b on Tie1. Example of antibodies that include an antigen
binding site that competes with E3 for binding to Tie1 include
M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01,
M0054H10, M0058F03, and related antibodies.
[0009] In one embodiment, the agent includes the HC and/or LC
variable domain of the E3 or E3b antibody, or a sequence at least
70, 80, 85, 90, 95, 98, 99% identical to the HC and/or LC variable
domains of the E3 or E3b antibody. In one embodiment, the amino
acid sequences of the HC variable domain sequence include CDR1,
CDR2, and CDR3 sequences from the E3 or E3b clone and the LC
variable domain sequence includes CDR1, CDR2, and CDR3 sequences
from the E3 or E3b clone. In one embodiment, the agent comprises
the E3 or E3b antibody. The LC variable domain sequence can include
SEQ ID NO:116. The HC variable domain sequence can include SEQ ID
NO:114. In one embodiment, the HC and LC framework regions are
human. In one embodiment, that agent includes SEQ ID NO:723 and SEQ
ID NO:724.
[0010] In one embodiment, the agent binds to Tie2. In one
embodiment, the agent antagonizes formation of a heteromeric
complex of Tie1, Tie2, and Ang. In another embodiment, the binding
of the agent can antagonize the association between Tie1 and Tie2,
between Tie1 and Ang, or between Tie2 and Ang. In another
embodiment, the agent enhances Tie2 self-association, e.g.,
homodimerization, and, thereby associates Tie2 with Tie2 and
prevents association of Tie2 with Tie1 and/or Ang. In one
embodiment, the agent includes a protein, e.g., an antibody that
binds to the extracellular domain of human Tie2. For example, the
antibody can be one or more of the following: human, humanized,
non-immunogenic, isolated, monoclonal, and recombinant. In one
embodiment, the antibody can bind to the first Ig-like C2-type
domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of
Tie2. In one embodiment, the antibody binds to an EGF-like domain
of Tie2 (e.g., first, second, or third EGF-like domain). In one
embodiment, the antibody binds to the fibronectin type III repeats
region of Tie2. In one embodiment, the antibody binds to amino acid
residues 19-119, 69-169, 119-229, 169-269, 229-329, 269-369,
329-429, 369-469, 429-529, 469-569, 529-629, 569-669, 629-729,
669-745, 729-745 of SEQ ID NO:162.
[0011] In one embodiment, the agent binds to Ang (e.g., Ang1, Ang2,
Ang3, or Ang4). In one embodiment, the agent antagonizes formation
of a heteromeric complex of Tie1, Tie2, and Ang. In another
embodiment, the binding of the agent can antagonize the association
between Tie1 and Tie2, between Tie1 and Ang, or between Tie2 and
Ang. In one embodiment, the agent includes a protein, e.g., an
antibody that binds to Ang. For example, the antibody can be one or
more of the following: human, humanized, non-immunogenic, isolated,
monoclonal, and recombinant. In one embodiment, the antibody binds
to the N-terminal domain of Ang1 (e.g., the N-terminal 50 amino
acids of Ang1). In one embodiment, the antibody binds to the
coiled-coil domain of Ang1. In one embodiment, the antibody binds
to the fibrinogen-like domain of Ang1. In one embodiment, the
antibody binds to amino acid residues 1-100, 50-150, 100-200,
150-250, 200-300, 250-350, 300-400, 350-450, 400-497, or 450-497 of
SEQ ID NO:163.
[0012] In one embodiment, the agent includes a protein that
contains a heavy chain (HC) immunoglobulin variable domain sequence
and a light chain (LC) immunoglobulin variable domain sequence. In
one embodiment, the HC and LC framework regions are human. In one
embodiment, the agent also includes an Fc domain. In one
embodiment, the agent includes the constant domains of a human
IgG1, IgG2, IgG3, or IgG4. In one embodiment, the constant domains
of the heavy chain are f allotype, (a,z) allotype, or any other
allotype.
[0013] In one embodiment, the agent is administered in an amount
effective to decrease vascular development or angiogenesis. In one
embodiment, the subject has an angiogenesis-related disorder. In
other embodiments, the subject has for example: a neoplastic
disorder, metastatic cancer, an angiogenesis-dependent cancer or
tumor, an inflammatory disorder, rheumatoid arthritis, or
psoriasis. In one embodiment, the protein is delivered
systemically.
[0014] In another embodiment, the protein is administered in an
amount effective to reduce one or more of the following activities:
sprouting, splitting, remodeling of blood vessels, vasculogenesis,
and tubule formation. The method can include other features
described herein.
[0015] In one aspect, the invention includes a method of decreasing
or inhibiting endothelial cell activity in the subject, the method
includes administering an agent that decreases or inhibits Tie
complex formation in an amount effective to decrease or inhibit
endothelial cell activity in the subject. The method can include
other features described herein.
[0016] In one aspect, the invention includes a method of decreasing
endothelial cell activity by administering an agent that causes
Tie1 phosphorylation. In one embodiment, the phosphorylation
decreases endothelial cell differentiation, e.g., sprouting,
splitting, and tube formation.
[0017] In another aspect, the invention includes a method of
decreasing endothelial cell activity, the method by administering
an agent that activates a signaling pathway. In one embodiment, the
signaling pathway decreases endothelial cell differentiation, e.g.,
sprouting, splitting, and tube formation. For example, the agent
increases Tie1 autophosphorylation. The method can include other
features described herein.
[0018] In one aspect, the invention includes an antibody for
modulating Tie complex formation in a subject, wherein the antibody
antagonizes an association between at least two of the following:
Tie1, Tie2, and an angiopoietin (Ang). In one embodiment, the
antibody binds to a Tie complex component or to one or more of
Tie1, Tie2, and an Ang. In one embodiment, the antibody antagonizes
formation of a heteromeric complex of Tie1, Tie2, and Ang. In
another embodiment, the antibody can antagonize the association
between Tie1 and Tie2, between Tie1 and Ang, or between Tie2 and
Ang.
[0019] In one embodiment, the antibody binds to Tie1. In one
embodiment, the antibody antagonizes formation of a heteromeric
complex of Tie1, Tie2, and Ang. In another embodiment, the binding
of the antibody can antagonize the association between Tie1 and
Tie2, between Tie1 and Ang, or between Tie2 and Ang. In another
embodiment, the antibody enhances Tie1 self-association, e.g.,
homodimerization, and thereby associates Tie1 with Tie1 and
prevents association of Tie1 with Tie2 or Ang. In another
embodiment, the antibody increases Tie1 phosphorylation and/or
prevents association of Tie1 with Tie2 or Ang. In one embodiment,
the antibody includes an antibody that binds to the extracellular
domain of human Tie1. For example, the antibody can be one or more
of the following: human, humanized, non-immunogenic, isolated,
monoclonal, and recombinant. In one embodiment, the antibody can
bind to the first Ig-like C2-type domain (Ig 1) or to the second
Ig-like C2-type domain (Ig 2) of Tie1. In one embodiment, the
antibody binds to an EGF-like domain of Tie1 (e.g., first, second,
or third EGF-like domain). In one embodiment, the antibody binds to
the fibronectin type III repeats region of Tie1. In one embodiment,
the antibody binds to amino acid residues 24-124, 74-174, 124-224,
174-274, 224-324, 274-374, 324-424, 374-474, 424-524, 474-574,
524-624, 574-674, 624-724, 674-759, or 724-759 of SEQ ID NO:2.
[0020] In one embodiment, the antibody binds to a Tie1 ectodomain
and includes a heavy chain (HC) immunoglobulin variable domain
sequence and a light chain (LC) immunoglobulin variable domain
sequence, the protein further includes one or more of the following
properties: (1) at least one of the variable domain sequences
includes at least one CDR of the E3 or E3b antibody; (2) at least
one of the variable domain sequences includes CDR sequences at
least 85% identical, in sum, to the CDRs of the corresponding
variable domain of the E3 or E3b antibody; (3) at least one of the
variable domains is at least 85% identical to the corresponding
immunoglobulin variable domains of the E3 or E3b antibody, and (4)
the protein competes with E3 or E3b for binding to Tie1 or binds to
an epitope that overlaps the epitope bound by E3 or E3b on Tie1.
For example, the antibody is at least bivalent, e.g., with at least
two antigen binding sites that bind to Tie1. In one embodiment, the
antibody comprises the E3, E3b (e.g., DX-2220), or DX-2240.
[0021] In one embodiment, the antibody includes one or more
variable domains from the E3 or E3b antibody or a variable domain
sequence that is at least 70, 75, 80, 85, 90, 95, 98, or 995
identical to such a variable domain. In one embodiment, the amino
acid sequences of the HC variable domain sequence include CDR1,
CDR2, and CDR3 sequences from the E3 or E3b clone, and the LC
variable domain sequence includes CDR1, CDR2, and CDR3 sequences
from the E3 or E3b clone. In one embodiment, the LC variable domain
sequence includes SEQ ID NO:116. In one embodiment, the HC variable
domain sequence includes SEQ ID NO:114. In one embodiment, the HC
and LC framework regions are human.
[0022] In one embodiment, the antibody binds to Tie2. In one
embodiment, the antibody antagonizes formation of a heteromeric
complex of Tie1, Tie2, and Ang. In another embodiment, the binding
of the antibody can antagonize the association between Tie1 and
Tie2, between Tie1 and Ang, or between Tie2 and Ang. In another
embodiment, the antibody enhances Tie2 self-association, e.g.,
homodimerization, and thereby associates Tie2 with Tie2 and
prevents association of Tie2 with Tie1 or Ang. In one embodiment,
the antibody causes Tie1 phosphorylation. In one embodiment, the
antibody prevents association of Tie1 with Tie2 or Ang. In one
embodiment, the antibody includes an antibody that binds to the
extracellular domain of human Tie2. The antibody may have one or
more of these properties, e.g., the antibody may cause Tie1
phosphorylation and prevent association of Tie1 with Tie2 or Ang,
etc.
[0023] For example, the antibody can be one or more of the
following: human, humanized, non-immunogenic, isolated, monoclonal,
and recombinant. In one embodiment, the antibody can bind to the
first Ig-like C2-type domain (Ig 1) or to the second Ig-like
C2-type domain (Ig 2) of Tie2. In one embodiment, the antibody
binds to an EGF-like domain of Tie2 (e.g., first, second, or third
EGF-like domain). In one embodiment, the antibody binds to the
fibronectin type III repeats region of Tie2. In one embodiment, the
antibody binds to amino acid residues 19-119, 69-169, 119-229,
169-269, 229-329, 269-369, 329-429, 369-469, 429-529, 469-569,
529-629, 569-669, 629-729, 669-745, 729-745 of SEQ ID NO:162.
[0024] In one embodiment, the antibody binds to Ang. In one
embodiment, the antibody antagonizes formation of a heteromeric
complex of Tie1, Tie2, and Ang. In another embodiment, the binding
of the antibody can antagonize the association between Tie1 and
Tie2, between Tie1 and Ang, or between Tie2 and Ang. For example,
the antibody can be one or more of the following: human, humanized,
non-immunogenic, isolated, monoclonal, and recombinant. In one
embodiment, the antibody binds to the N-terminal domain of Ang1
(i.e., the N-terminal 50 amino acids of Ang1). In one embodiment,
the antibody binds to the coiled-coil domain of Ang1. In one
embodiment, the antibody binds to the fibrinogen-like domain of
Ang1. In one embodiment, the antibody binds to amino acid residues
1-100, 50-150, 100-200, 150-250, 200-300, 250-350, 300-400,
350-450, 400-497, or 450-497 of SEQ ID NO:163.
[0025] In one embodiment, the antibody includes a heavy chain (HC)
immunoglobulin variable domain sequence and a light chain (LC)
immunoglobulin variable domain sequence.
[0026] In one embodiment, the HC and LC framework regions are
human. In one embodiment, the antibody also includes an Fe domain.
In one embodiment, the antibody includes the constant domains of a
human IgG1, IgG2, IgG3, or IgG4.
[0027] In one embodiment, the antibody is administered in an amount
effective to decrease vascular development and angiogenesis. In one
embodiment, the antibody is delivered systemically. In one
embodiment, antibody is administered in an amount effective to
reduce one or more of the following activities: sprouting,
splitting, remodeling of blood vessels, vasculogenesis, and tubule
formation.
[0028] In one aspect, the invention includes an isolated protein
that includes one or more variable domains of an antibody described
herein.
[0029] In one aspect, the invention includes a nucleic acid that
includes a coding sequence that encodes a polypeptide that includes
a variable domain of an antibody described herein.
[0030] In one aspect, the invention includes a pharmaceutical
composition that includes an antibody described herein. The
composition and antibody can include other features described
herein.
[0031] In one aspect, the invention includes an antibody described
herein for treatment of an angiogenesis-related disorder. The
antibody and treatment can include other features described
herein.
[0032] In one aspect, the invention includes an antibody described
herein for the manufacture of a medicament for treating an
angiogenesis-related disorder. The medicament and antibody can
include other features described herein.
[0033] In one aspect, the invention includes a method of providing
a first therapy that includes administering a first agent in
combination with a second therapy, e.g., an anti-cancer therapy.
The first agent is an agent that decreases Tie complex formation or
an agent that increases Tie1 homodimerization. For example, the
first agent is a Tie1 binding protein. In one embodiment, the
second therapy includes radiation therapy or surgery. In one
embodiment, the second therapy includes administering a second
agent. For example, the second agent antagonizes or decreases Tie
complex formation or increases Tie1 homodimerization. In one
embodiment, the second agent is an agent that antagonizes signaling
through a VEGF pathway, e.g., a VEGF antagonist antibody, e.g.,
bevacizumab; VEGF-Receptor tyrosine kinase inhibitor, or another
agent that antagonizes VEGF pathway signalling. See also
"Combination Therapies" below.
[0034] In another aspect, the invention includes a composition that
includes an agent that decreases Tie complex formation and an
anti-cancer agent. For example, the anti-cancer agent can be a
second agent that antagonizes Tie complex formation or a second
agent that antagonizes a VEGF pathway.
[0035] In one aspect, the invention features an antibody that
decreases endothelial cell activity by causing Tie1
phosphorylation. For example, the antibody may decrease endothelial
cell differentiation, e.g., sprouting, splitting, and tube
formation.
[0036] In one aspect, the invention features a protein (e.g., an
isolated protein) that includes a heavy chain immunoglobulin
variable domain sequence and a light chain immunoglobulin variable
domain sequence and binds to Tie1 ectodomain. The binding protein
binds to Tie1 ectodomain. For example, the protein binds with an
affinity K.sub.D of less than 10.sup.-8 M, 510.sup.-9 M, 10.sup.-9
M, 10.sup.-10 M, 10.sup.-11 M, or 10.sup.-12 M.
[0037] In one embodiment, one or more of the CDRs of the heavy
and/or light chain variable domain sequence are human, primate,
non-rodent (e.g., non-mouse or non-rat), or synthetic. In one
embodiment, one or more of the framework regions of the heavy
and/or light chain variable domain sequence are human, primate, or
non-rodent (e.g., non-mouse or non-rat).
[0038] In one embodiment, the heavy chain variable domain sequence
includes one or more of the following properties:
[0039] i) a HC CDR1 that includes an amino acid sequence as
follows:
[0040] (AGSR)-Y-(GVK)-M-(GSVF), (SEQ ID NO:117)
[0041] (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO:118), or
[0042] (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO:119);
[0043] ii) a HC CDR2 that includes an amino acid sequence as
follows:
[0044] X-I-Y-P-S-G-G-X-T-X-Y-A-D-S-V-K-G (SEQ ID NO:120), wherein X
is any amino acid,
[0045] (GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY) (SEQ ID NO:121),
[0046] (GSV)-I-(SY)-P-S-G-G-(WNQ)-T-(GY) (SEQ ID NO:160)
[0047] (GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID
NO:122),
[0048] (GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH) (SEQ ID
NO:123), or
[0049] X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO: 722), wherein
X is any amino acid;
[0050] iii) a HC CDR3 that includes an amino acid sequence as
follows:
[0051] V-(four or five residues)-F-D-(I/Y) (SEQ ID NO:124),
[0052] G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO:125),
[0053] (GV)-N-Y-Y-(GYD)-S-(SD)-G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID
NO:126),
[0054]
(GVD)-(AGLN)-(LYR)-(GSTLYH)-(GYD)-(AGSYFP)-(SFD)-(AGYD)-(IY)-(GFD)--
(YDP)-(IP)-A-P-G-L-D-Y (SEQ ID NO:127),
[0055] A-P-R-G-Y-S-Y-G-Y-Y-Y (SEQ ID NO:157)
[0056] VNYYDSSGYGPIAPGLDY (SEQ ID NO:128), or
[0057] G-X-X-G-(AY)-F-D-(YI) (SEQ ID NO:705), wherein X is any
amino acid.
[0058] In one embodiment, the light chain variable domain sequence
includes one or more of the following properties:
[0059] i) a LC CDR1 that includes an amino acid sequence as
follows:
[0060] R-A-S-Q-S-(IV)-S-(SR)-X1-Y-L-(AN) (SEQ ID NO:129),
[0061] R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706),
[0062] T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707),
[0063] (SGQ)-(GS)-(DS)-(NS)-(IL)-(GR)-S-(YKN)-(YS)-(VA) (SEQ ID
NO:708),
[0064] R-A-S-Q-S-V-S-S-X-L (SEQ ID NO:130),
[0065] R-A-S-Q-S-(IV)-S-(SR)-(SY)-(LY)-(ALN) (SEQ ID NO:131),
or
[0066]
R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)-(STIRH)-X1-(SYWNH)-(LV)-(ASN)
(SEQ ID NO:132), wherein X1 can be serine or absent;
[0067] ii) a LC CDR2 that includes an amino acid sequence as
follows:
[0068] X-A-S-X-R-A-T (SEQ ID NO:133), wherein X can be any amino
acid,
[0069] (AGD)-A-S-(STN)-R-A-T (SEQ ID NO:134),
[0070] (DG)-(AV)-S-N-(RL)-(AP)-ST) (SEQ ID NO:709),
[0071] (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:135), or
[0072] (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO:136);
and
[0073] iii) a LC CDR3 that includes an amino acid sequence as
follows:
[0074] Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWRH)-(TIY) (SEQ ID
NO:161),
[0075] Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO:137),
[0076] (LQ)-Q-(SYFR)-(GSYN)-(SKN)-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO:138),
[0077] Q-Q-X-S-(SN)-(WS)-P-X-T-F (SEQ ID NO:710), wherein X is any
amino acid,
[0078] Y-(TG)-(SG)-S-(PGS)-(TN)-X-(VT) (SEQ ID NO:711), wherein X
is any amino acid,
[0079] Q-Q-(YR)-(GS)-S-(SW)-P-R-X1-T (SEQ ID NO:139), wherein X1 is
any amino acid or absent,
[0080] Q-Q-F-N-S-Y-P-H (SEQ ID NO:158)
[0081]
(LQ)-(LQ)-(SYFRD)-(GSYN)-(STRKN)-(STYWFXRP)-(ILMWRH)-(TIY)-(TI)
(SEQ ID NO:140), or
[0082]
(LQ)-(LRQ)-(SYFRD)-(GSYN)-(ASTRKN)-(STYWF)-(SVRP)-(STILMWRH)-(TIY)--
(STI) (SEQ ID NO:141).
[0083] In one embodiment, the light chain variable domain sequence
includes one or more of the following properties:
[0084] i) a LC CDR1 that includes an amino acid sequence as
follows:
[0085] S-X-(ND)-(IV)-(AG)-X1-X2-X3 (SEQ ID NO:142), or
[0086] T-(GR)-(ST)-S-X5-(ND)-(IV)-(AG)-X1-X2-X3-Y-X4-S (SEQ ID
NO:143), wherein X1 is any amino acid (e.g., G or R), X2 is any
amino acid (e.g., Y or N), X3 is any amino acid (e.g., F, N, or K),
X4 is any amino acid (e.g., aliphatic, e.g., V or A);
[0087] ii) a LC CDR2 that includes an amino acid sequence as
follows:
[0088] (DE)-V-N-N-R-P-S (SEQ ID NO:144)
[0089] (DE)-(VD)-(STDN)-(YRDN)-R-P-S (SEQ ID NO:145);
[0090] iii) a LC CDR3 that includes an amino acid sequence as
follows:
[0091] (SQ)-S-(SY)-(ASID)-(GSR)-(ST)-(STRN)-(STYR)-(ATLY)-(SVWQ)
(SEQ ID NO:146).
[0092] In one embodiment, the HC CDR2 includes an amino acid
sequence as follows:
(GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)-D-S-V-K-- G
(SEQ ID NO:147) or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S-V-K-G
(SEQ ID NO:148).
[0093] In one embodiment, the protein includes HC CDR1 and HC CDR2
sequences that are related to the corresponding CDR sequences of
p-F3, E3 or E3b. For example, the protein includes the sequence
MYGM (SEQ ID NO:149), at a position corresponding to HC CDR1. The
sequence can be followed by a small amino acid, e.g., glycine,
alanine, valine, or serine. In another example, the protein the
sequence VISPSGGX.sub.1TX.sub.2YADSAVKG (SEQ ID NO:150), at a
position corresponding to HC CDR2. For example, X.sub.1 can be a
hydrophilic amino acid, e.g., glutamine or asparagine. For example,
X.sub.2 can be a small amino acid, e.g., glycine, alanine, valine,
or serine.
[0094] In one embodiment, the heavy chain variable domain sequence
can have one or more of the following features: the amino acid
residue at Kabat position 31 is A, H, K, N, Q, R, S, or T, e.g., H,
N, R, or S; the amino acid residue at Kabat position 32 is Y; the
amino acid residue at Kabat position 33 is G, K, P, R, or V, e.g.,
K or V; the amino acid residue at Kabat position 34 is M; the amino
acid residue at Kabat position 35 is A, G, H, I, L, M, S, or V,
e.g., G, H, M, or V; the amino acid residue at Kabat position 50 is
G, R, S, or V, e.g., S or V; the amino acid residue at Kabat
position 51 is I; the amino acid residue at Kabat position 52 is S
or Y, e.g., Y; the amino acid residue at Kabat position 52a is P or
S, e.g., P; the amino acid residue at Kabat position 53 is S; the
amino acid residue at Kabat position 54 is G; the amino acid
residue at Kabat position 55 is G; the amino acid residue at Kabat
position 56 is A, F, H, I, Q, W, or Y, e.g., A, W or Y; the amino
acid residue at Kabat position 57 is T; the amino acid residue at
Kabat position 58 is R, S, T, or Y, e.g., Y. In one embodiment, the
length of CDR3 is between 8-18 amino acids, e.g., between 8-12,
8-10, or 15-17 amino acids.
[0095] In one embodiment, two or three of the CDRs of the HC
variable domain sequence match motifs that also match a HC variable
domain of an antibody described herein. Similarly, in one
embodiment, two or three of the CDRs of the LC variable domain
sequence match motifs that also match a LC variable domain of an
antibody described herein. In still another embodiment, the matched
motifs for the CDRs are based on a HC and a LC that are paired in
an antibody described herein.
[0096] In one embodiment, the H1 and H2 hypervariable loops have
the same canonical structure as an antibody described herein. In
one embodiment, the L1 and L2 hypervariable loops have the same
canonical structure as an antibody described herein.
[0097] In one embodiment, the HC CDR1 amino acid sequences have a
length of at least 5 amino acids of which at least 3, 4, or 5 amino
acids are identical to the CDR1 sequence of the HC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the HC CDR2 amino acid sequences have a length of at
least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15,
16, or 17 amino acids are identical to the CDR2 sequence of the HC
of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3,
p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C1, s-C2, s-C7, s-D11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the HC CDR2 amino acid sequences have a length of at
least 17 amino acids of which at least 14, 15, 16, or 17 amino
acids are identical to the CDR2 sequence of the HC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the HC CDR3 amino acid sequences have a length of at
least of at least 7 or 8 amino acids of which at least 5, 6, 7, or
8 amino acids are identical to the CDR3 sequence of the HC of clone
E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4,
p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11,
s-E11, s-G10, s-H4, or another antibody described herein.
[0098] In one embodiment, two or three of the CDRs of the HC
variable domain sequence match motifs described herein such that
the motifs are a set of motifs that match a HC variable domain of a
clone described herein, e.g., E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein. For example, the protein may include SEQ ID
NO:118 and SEQ ID NO:160, e.g., motifs that match the E3 HC
variable domain.
[0099] In one embodiment, the LC CDR1 amino acid sequences have a
length of at least 10, 11, or 12 amino acids of which at least 7,
8, 9, 10, or 11 amino acids are identical to the CDR1 sequence of
the LC of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12,
p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D11, s-E11, s-G10, s-H4, or another antibody described herein. In
one embodiment, the LC CDR2 amino acid sequences have a length of
at least 6 or 7 amino acids of which at least 5, 6, or 7 amino
acids are identical to the CDR2 sequence of the LC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the LC CDR3 amino acid sequences have a length of at
least of at least 8, 9, or 10 amino acids of which at least 7, 8,
9, or 10 amino acids are identical to the CDR3 sequence of the LC
of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3,
p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D11, s-E11, s-G10, s-H4, or another antibody described
herein.
[0100] In one embodiment, two or three of the CDRs of the LC
variable domain sequence match motifs described herein such that
the motifs are a set of motifs that match a LC variable domain of a
clone described herein, e.g., E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein. For example, the protein may include SEQ ID
NO:132, SEQ ID NO:136, and SEQ ID NO:161, e.g., motifs that match
the E3 LC variable domain.
[0101] In one embodiment, the amino acid sequence of the HC
variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequence of the HC
variable domain of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein.
[0102] In one embodiment, the amino acid sequence of the LC
variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequence of the LC
variable domain of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein.
[0103] In one embodiment, the amino acid sequences of the HC and LC
variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequences of the HC and
LC variable domains of a clone selected from the group consisting
of E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4,
p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11,
s-E11, s-G10, s-H4, and any other antibody described herein.
[0104] In one embodiment, the amino acid sequences of one or more
framework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC
and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to corresponding framework regions of the
HC and LC variable domains of clone E3, E3b, G2, p-A1, p-A10, p-B1,
p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein.
[0105] In one embodiment, the amino acid sequences of the HC and LC
variable domain sequences comprise a sequence encoded by a nucleic
acid that hybridizes (e.g., under high stringency) to a nucleic
acid encoding a variable domain of E3, E3b, G2, p-A1, p-A10, p-B1,
p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G 10, s-H4, or another antibody
described herein.
[0106] In one embodiment, the light chain variable domain sequence
is human or non-immunogenic in a human. In one embodiment, the
heavy chain variable domain sequence is human or non-immunogenic in
a human.
[0107] The protein can bind to cells that express Tie1, e.g.,
endothelial cells. In one embodiment, the protein does not
substantially bind (e.g., does not detectably bind) to platelets
(e.g., resting and/or activated platelets).
[0108] In one embodiment, the protein inhibits tube formation by
HUVECs in vitro. For example, the E3 antibody inhibits tube
formation by HUVECs in vitro (e.g., under conditions described in
Example 18). In one embodiment, the protein inhibits angiogenesis
in an in vivo MATRIGEL.TM. plug assay. For example, the E3 antibody
can inhibit angiogenesis in an exemplary assay (see, e.g., an
exemplary assay described in Example 21).
[0109] In one embodiment, the protein recognizes
melanoma-associated structures in a histological section, e.g., not
only melanoma tissue, but antigen in surrounding structures. In one
embodiment, the protein does not stain blood vessels in normal skin
in a histological section.
[0110] In one embodiment, the protein specifically binds to Tie1,
e.g., it binds with at least a 10, 50, 100, 10.sup.3, or 10.sup.4
fold preference for Tie1 relative to another human protein, e.g.,
Tie2, a natural protein other than Tie1 that has a Ig-like domain,
an EGF-like domain, or fibronectin Type III repeat, or human serum
albumin. In one embodiment, the protein binds to a domain of Tie1
described herein.
[0111] In another aspect, the invention features a protein (e.g.,
an isolated protein) that modulates activity of Tie1, e.g., the
Tie1 receptor. For example, the protein is not naturally occurring.
In one embodiment, the protein includes a HC and LC immunoglobulin
variable domain sequence. In one embodiment, one or more of the
CDRs of the heavy and/or light chain variable domain sequence are
human, primate, non-rodent (e.g., non-mouse or non-rat), or
synthetic. In one embodiment, one or more of the framework regions
of the heavy and/or light chain variable domain sequence are human,
primate, or non-rodent (e.g., non-mouse or non-rat). In another
embodiment, the protein is substantially free of an immunoglobulin
variable domain, e.g., the protein includes a peptide that
independently interacts with Tie1 or a polypeptide that does not
include a immunoglobulin variable domain.
[0112] In one embodiment, the protein activates an activity of the
Tie1 protein, e.g., an activity in the Tie1/EpoR chimeric BaF3 cell
assay described in Example 2. A protein that activates in this
assay can behave as antagonists in other conditions, for example,
in vivo.
[0113] In one embodiment, the protein includes the HC and LC
immunoglobulin variable domains of the E3, E3b, or other antibody,
HC and/or LC immunoglobulin variable domain sequences that are at
least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
identical in the CDR regions to the respective CDRs of the E3, E3b
or other antibody described herein. In one embodiment, the protein
competes with E3, E3b, or other antibody described herein for
binding to Tie1 or binds to an epitope that overlaps an epitope
that is recognized by E3, E3b, or other antibody described herein,
or that has at least one, two or three residues in common with an
epitope that is recognized by E3, E3b, or other antibody described
herein.
[0114] In one embodiment, the activating protein enables IL-3
dependent cells that express a chimeric receptor including the Tie1
extracellular domain and the EpoR intracellular domain to survive
in the absence of IL-3.
[0115] In one embodiment, the protein can cause dimerization of
Tie1. In one embodiment, the protein can cause auto-phosphorylation
of the RTK domain of Tie1.
[0116] In one embodiment, the protein synergizes with the E3 or E3b
antibody to activate an activity of Tie, e.g., in the Tie1/EpoR
chimeric BaF3 cell assay. In one embodiment, the protein includes
the HC and LC immunoglobulin variable domains of the G2 or C7
antibody or domains that are at least 70, 80, 85, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions. In
one embodiment, the protein competes with G2 or C7 for binding to
Tie1 or binds to an epitope that overlaps an epitope that is
recognized by G2 or C7 or that has at least one, two or three
residues in common with an epitope that is recognized by G2 or
C7.
[0117] In another embodiment, the protein antagonizes an activity
of the Tie1 protein. For example, the protein can at least
partially inhibit the ability of the E3 or E3b antibody to agonize
the Tie protein. In one embodiment, the protein can at least
partially inhibit the ability of the E3 or E3b antibody to enable
IL-3 dependent cells that express a chimeric receptor including the
Tie1 extracellular domain and the EpoR intracellular domain to
survive in the absence of IL-3.
[0118] In one embodiment, the HC and LC immunoglobulin variable
domain sequences of the protein include the amino acid sequences
that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100% identical to the amino acid sequences of respective
immunoglobulin variable domains of B2 or D11.
[0119] In one embodiment, the Tie1 binding protein includes the HC
and LC immunoglobulin variable domains of an antibody selected from
the group consisting of: B2, D11, A2, A10, P-B1, P-B3, and P-C6 or
immunoglobulin domains that are at least 70, 80, 85, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions to
the CDR regions of the respective antibodies. For example, the
protein binds with an affinity K.sub.D of less than 10.sup.-8 M,
510.sup.-9 M, 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, or
10.sup.-12 M.
[0120] In one embodiment, the protein can at least partially
inhibit the ability of a naturally occurring Tie1 binding protein
from interacting with the Tie protein.
[0121] The protein can include other features described herein.
[0122] In another aspect, the invention features an antibody (e.g.,
an isolated antibody) that binds to the Tie1 ectodomain, but does
not substantially bind to platelets, e.g., as detected by
fluorescence activated cell sorting. For example, the antibody does
not substantially bind to activated platelets and/or resting
platelets. In one embodiment, the antibody binds to endothelial
cells. In one embodiment, the protein is a monoclonal antibody. The
antibody can be provided in a preparation that is free of other
Tie1-binding antibodies that have other specificities, e.g., free
of Tie1 binding antibodies that bind to platelets. The antibody can
include other features described herein.
[0123] In another aspect, the invention features a protein (e.g.,
an isolated protein) that preferentially binds to a Tie1 protein in
a conformation stabilized by the E3 or E3b antibody relative to an
endogenous Tie1 protein in an unstimulated state. In one
embodiment, the protein includes immunoglobulin HC and LC domains.
In another embodiment, the protein includes a peptide (e.g., of
length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids)
that independently binds to Tie1. For example, the peptide can
include one, two, or three disulfide bonds. The protein can include
other features described herein.
[0124] In another aspect, the invention features a protein (e.g.,
an isolated protein) that preferentially binds to a Tie1 protein in
a dimeric conformation relative to a monomeric Tie1 protein. In one
embodiment, the protein includes immunoglobulin HC and LC domains.
In another embodiment, the protein includes a peptide (e.g., of
length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids)
that independently binds to Tie1. For example, the peptide can
include one, two, or three disulfide bonds. The protein can include
other features described herein.
[0125] In another aspect, the invention features a protein (e.g.,
an isolated protein) that preferentially binds to a Tie2 protein in
a conformation that is biased against interaction with Ang or Tie1.
In one embodiment, the protein includes immunoglobulin HC and LC
domains. In another embodiment, the protein includes a peptide
(e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino
acids) that independently binds to Tie2. For example, the peptide
can include one, two, or three disulfide bonds. The protein can
include other features described herein. The invention also
features nucleic acid aptamers that have one or more of these
properties.
[0126] In another aspect, the invention features a protein (e.g.,
an isolated protein) that preferentially binds to an Ang protein,
and modulates (e.g., inhibits) interaction with Tie1 and Tie2. In
one embodiment, the protein includes immunoglobulin HC and LC
domains. In another embodiment, the protein includes a peptide
(e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino
acids) that independently binds to Ang. For example, the peptide
can include one, two, or three disulfide bonds. The protein can
include other features described herein. The invention also
features nucleic acid aptamers that have one or more of these
properties.
[0127] In another aspect, the invention features a protein (e.g.,
an isolated protein) that binds to an epitope of Tie1 ectodomain
with a K.sub.D of less than 2.times.10.sup.-7 M. The epitope
overlaps, is within, or includes an epitope bound by E3, E3b, G2,
p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-A10,
s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11, s-G10,
s-H4, or another antibody described herein or that includes at
least one, two, or three residues in common. For example, the
protein binds with an affinity K.sub.D of less than 10.sup.-8 M,
510.sup.-9 M, 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, or
10.sup.-12 M. In one embodiment, the protein includes
immunoglobulin HC and LC domains. In another embodiment, the
protein includes a peptide (e.g., of length less than 30, 28, 25,
22, 20, 18, 16, or 14 amino acids) that independently binds to
Tie1. For example, the peptide can include one, two, or three
disulfide bonds. The protein can include other features described
herein. The invention also features nucleic acid aptamers that have
one or more of these properties.
[0128] In another aspect, the invention features a protein (e.g.,
an isolated protein) that competitively inhibits binding of E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein to a Tie1
ectodomain. In one embodiment, the protein includes immunoglobulin
HC and LC domains. In another embodiment, the protein includes a
peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or
14 amino acids) that independently binds to Tie1. For example, the
peptide can include one, two, or three disulfide bonds. The protein
can include other features described herein.
[0129] In another aspect, the invention features a protein (e.g.,
an isolated protein) that includes a heavy chain immunoglobulin
variable domain sequence and a light chain immunoglobulin variable
domain sequence and that antagonizes an activity of the Tie1
ectodomain. In one embodiment, CDR1 of the light chain variable
domain sequence includes: Q-S-X-S-S (SEQ ID NO:151) or
R-A-S-Q-S-X-S-S-Y-L-A (SEQ ID NO:152), wherein X is any amino acid
or optionally aliphatic, e.g., isoleucine or valine. In one
embodiment, CDR2 of the light chain variable domain sequence
includes: A-S-X.sub.1-R-X.sub.2-T (SEQ ID NO:153) or
D-A-S-X.sub.1-R-X.sub.2-T (SEQ ID NO:154), wherein X.sub.1 is any
amino acid or optionally a hydrophilic amino acid, e.g., serine or
asparagine, and X.sub.2 is any amino acid or optionally aliphatic
or small aliphatic, e.g., alanine or valine. In one embodiment,
CDR3 of the light chain variable domain sequence includes:
Q-R-S-X.sub.2-W-P-R (SEQ ID NO:155) or
X.sub.1-Q-R-S-X.sub.2-W-P-R-T (SEQ ID NO:156), wherein X.sub.1 is
any amino acid or optionally leucine or glutamine, and X.sub.2 is
any amino acid or optionally lysine or serine.
[0130] In one embodiment, the protein competes with the B2 and/or
D11 antibody for binding to Tie1 or competitively inhibits binding
of B2 and/or D11 to Tie1.
[0131] In one embodiment, the protein antagonizes a Tie1 activity
that is stimulated by the E3 or E3b antibody. In one embodiment,
the protein inhibits dimerization of Tie1. The protein can include
other features described herein.
[0132] In another aspect, the invention features an isolated,
mono-specific protein including a heavy chain immunoglobulin
variable domain sequence and a light chain immunoglobulin variable
domain sequence, wherein the protein binds to Tie1 ectodomain and
includes a human or non-mouse constant domain (e.g., a human IgG1,
IgG2, IgG3, or IgG4 constant domain). The protein can include other
features described herein.
[0133] In another aspect, the invention features an isolated, human
antibody that binds to a Tie1 ectodomain. The protein can include
other features described herein.
[0134] In another aspect, the invention features an isolated
antibody (e.g., an isolated antibody) that binds to a Tie1
ectodomain and contains less than 5, 4, 3, or 2 peptides (of
between 6-9 amino acid length) that are non-human in origin or less
than 5, 4, 3, or 2 peptides that are potential human T cell
epitopes. In one embodiment, the antibody contains no peptide (of
6-9 amino acid length) that is non-human in origin or that is a
potential human T cell epitope.
[0135] In one embodiment, the antibody is obtained by a method that
includes deimmunization. For example, the antibody is deimmunized,
e.g., completely deimmunized. The protein can include other
features described herein.
[0136] In another aspect, the invention features an isolated
antibody that binds to a Tie1 ectodomain and that includes a
modified Fc domain, e.g., a modified human Fc domain. For example,
antibodies may include modifications, e.g., that alter Fc function.
For example, the human IgG1 constant region can be mutated at one
or more residues, e.g., one or more of residues 234 and 237, e.g.,
according to the number in U.S. Pat. No. 5,648,260. Other exemplary
modifications include those described in U.S. Pat. No. 5,648,260.
The protein can include other features described herein.
[0137] In another aspect, the invention features an isolated
protein that binds to the Tie1 receptor with an affinity K.sub.D of
less than 10.sup.-7 M, 10.sup.-8 M, 510.sup.-9 M, 10.sup.-9 M,
10.sup.-10 M, 10.sup.-11 M, or 10.sup.-12 M. The protein can
include other features described herein.
[0138] In another aspect, the invention features an isolated
protein including a heavy chain immunoglobulin variable domain
sequence and a light chain immunoglobulin variable domain sequence,
wherein the protein binds to Tie1 ectodomain and, for example,
includes at least one or more CDRs that are a non-primate CDR
(e.g., a non-mouse or non-rabbit CDR) or a synthetic CDR. The
protein can include other features described herein.
[0139] In another aspect, the invention features an isolated
nucleic acid including a coding sequence that encodes a polypeptide
including an immunoglobulin HC variable domain of an antigen
binding protein that binds to Tie1. The nucleic acid or polypeptide
can include one or more other features described herein. The
nucleic acid can include one or more altered codons. In one
embodiment, the nucleic acid includes SEQ ID NOs:725 and/or 726.
Also featured is a mammalian expression vector that includes SEQ ID
NOs:725 and/or 726.
[0140] In one embodiment, the nucleic acid further includes a
second coding sequence that encodes a polypeptide including an
immunoglobulin HC variable domain, e.g., an HC domain described
herein. In one embodiment, the nucleic acid further includes a
promoter operably linked to the coding sequence.
[0141] In another aspect, the invention features a nucleic acid
that includes one or more coding sequence that encodes one or more
polypeptide chains that collectively include an immunoglobulin HC
or LC variable domain of an antigen binding protein that binds to
Tie1. In one embodiment, the nucleic acid segment encoding at least
one of the variable domains hybridizes to a nucleic acid described
herein, e.g., under stringent conditions (e.g., high stringency
conditions), e.g., it hybridizes to a region encoding a variable
domain and is at least 80, 85, 90, 95, or 98% of the length of such
a region. The nucleic acid can include other features described
herein.
[0142] In another aspect, the invention features a host cell that
contains a first nucleic acid sequence encoding a polypeptide
including a HC variable domain of an antigen binding protein and a
second nucleic acid sequence encoding a polypeptide including a LC
variable domain of the antigen binding protein, wherein the antigen
binding protein binds to Tie1 with a K.sub.D of less than
2.times.10.sup.-7 M. In one embodiment, the HC or LC variable
domain includes at least one human CDR. The antigen binding protein
can include other features described herein.
[0143] In another aspect, the invention features a host cell that
contains a first nucleic acid encoding a polypeptide including a HC
variable region and a second nucleic acid encoding a polypeptide
including a LC variable region, wherein the HC and the LC variable
regions each include at least 70, 80, 85, 90, 92, 95, 97, 98, 99,
or 100% identical to respective amino acid sequences of the HC and
LC variable domains of a clone selected from the group consisting
of E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4,
p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C1, s-C2, s-C7, s-D11,
s-E11, s-G10, and s-H4. The antigen binding protein can include
other features described herein.
[0144] In another aspect, the invention features a pharmaceutical
composition including a protein described herein that interacts
with Tie1 and a pharmaceutically acceptable carrier.
[0145] In another aspect, the invention features a therapeutic
composition including a protein described herein that interacts
with Tie1 wherein the composition is sterile and suitable for
administration to a subject.
[0146] In another aspect, the invention features a method that
includes: providing a signal-dependent or signal-responsive cell
that expresses a chimeric receptor including the Tie1 extracellular
domain and a heterologous intracellular sequence that can produce a
signal; contacting a candidate compound to the cell; and evaluating
a property of the cell that is dependent on the signal. In one
embodiment, the intracellular sequence includes at least a region
of an intracellular sequence of the EpoR protein. The method can be
used, e.g., to evaluate activity of a candidate compound, or a
plurality of compounds.
[0147] In another aspect, the invention features a method that
includes: providing an IL-3 dependent cell that expresses a
chimeric receptor including the Tie1 extracellular domain and the
EpoR intracellular domain; contacting a candidate compound to the
cell under conditions in which the concentration of IL-3 is not
sufficient to sustain viability of the cell; and evaluating a
property of the cell. The method can be used, e.g., to evaluate
activity of a candidate compound, or a plurality of compounds. In
one embodiment, the property is viability. In one embodiment, the
evaluating includes an MTT assay. In one embodiment, the method
further includes administering the candidate compound to a subject.
For example, the candidate compound includes a protein, e.g., a
protein that includes an immunoglobulin variable domain.
[0148] In another aspect, the invention features method of
identifying a compound that modulates Tie1 activity. The method
includes: providing a plurality of candidate compounds; and
evaluating each compound of the plurality using a method described
herein.
[0149] In another aspect, the invention features a culture cell
that expresses a chimeric transmembrane protein including a region
of the Tie1 extracellular domain and a heterologous intracellular
sequence. In one embodiment, the intracellular sequence includes a
region of the EpoR intracellular domain. In one embodiment, the
cell requires IL-3 or Tie1 for viability. For example, the cell is
IL-3 dependent in the absence of the chimeric transmembrane
protein, but is viable in the presence of the E3 or E3b antibody
and the absence of IL-3.
[0150] In another aspect, the invention features a preparation that
includes the isolated mammalian cells (e.g., cells that expresses a
chimeric transmembrane protein including a region of the Tie1
extracellular domain and a heterologous intracellular sequence) and
a Tie1-binding protein, wherein the Tie1-binding protein is
necessary to sustain viability of the cells.
[0151] In another aspect, the invention features a kit including: a
Tie1-binding protein and a culture cell that expresses a chimeric
transmembrane protein including a region of the Tie1 extracellular
domain and a heterologous intracellular sequence.
[0152] In another aspect, the invention features a method of
evaluating a candidate compound. The method includes: providing a
preparation that includes (i) a cell or membrane fraction that
contains (a) an insoluble protein that includes a region of the
Tie1 extracellular domain and a kinase domain and (b) ATP; (ii) a
ligand that alters activity of the kinase domain; and (iii) the
candidate compound; and evaluating the phosphorylation state of the
insoluble protein.
[0153] In another aspect, the invention features a method of
evaluating a candidate compound. The method includes: providing a
preparation that includes (i) a cell or membrane fraction that
includes a Tie1 protein or a transmembrane protein that includes at
least a region of the Tie1 extracellular domain and ATP; (ii) a
ligand that causes autophosphorylation of Tie1 or the transmembrane
protein; and (iii) the candidate compound; and evaluating
phosphorylation state of the Tie1 protein.
[0154] In one embodiment, the ligand is an antibody. In one
embodiment, the ligand includes the HC and LC immunoglobulin
variable domains of the E3 or E3b antibody or domains that are at
least 90% identical in the CDR regions. In one embodiment, the
method further includes administering the candidate compound to a
subject.
[0155] In another aspect, the invention features a method that
includes: providing a preparation that includes (i) a cell or
membrane fraction that includes a transmembrane protein that
includes at least a region of the Tie1 extracellular domain and
ATP; and (ii) a ligand that causes autophosphorylation of Tie1 or
the transmembrane protein; and evaluating phosphorylation state of
the transmembrane protein.
[0156] In another aspect, the invention features a method that
includes: contacting a mammalian cell with a ligand that (i) can
agonize Tie1 autophosphorylation and/or (ii) can enable an IL-3
dependent cell that expresses a chimeric receptor including the
Tie1 extracellular domain and the EpoR intracellular domain to
remain viable under conditions in which the concentration of IL-3
is not sufficient to sustain viability of the cell; and evaluating
the mammalian cell. In one embodiment, the cell expresses an
endogenous Tie1 protein. In one embodiment, the cell is an
endothelial cell. In one embodiment, the method further includes
contacting the mammalian cell with a test compound, other than the
ligand. For example, the ligand is an antibody. For example, the
ligand includes the HC and LC immunoglobulin variable domains of
the E3 or E3b antibody or domains that are at least 90% identical
in the CDR regions.
[0157] In another aspect, the invention features a method that
includes: contacting a mammalian cell or fraction thereof with an
agent that can modulate the activity of Tie1; and evaluating the
mammalian cell or fraction thereof. In one embodiment, the agent is
contacted to the cell while the cell is living, and the evaluating
includes isolating a fraction of the cell. In one embodiment, the
agent is a protein, e.g., an antibody or a peptide. In one
embodiment, the agent includes the HC and LC immunoglobulin
variable domains of the E3 or E3b antibody or domains that are at
least 90% identical in the CDR regions to the E3 or E3b antibody.
In one embodiment, the agent includes the HC and LC immunoglobulin
variable domains of the B2 or D11 antibody or domains that are at
least 90% identical in the CDR regions to the B2 or D11 antibody.
In one embodiment, the agent includes the HC and LC immunoglobulin
variable domains of the A2, A10, P-B1, P-B3, or P-C6 antibody or
domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% identical% identical in the CDR regions to the
A2, A10, P-B1, P-B3, or P-C6 antibody. In one embodiment, the agent
includes the HC and LC immunoglobulin variable domains of the G2 or
C7 antibody or domains that are at least 90% identical in the CDR
regions to the G2, or C7 antibody. The agent can include other
features described herein.
[0158] In another aspect, the invention features a method of
evaluating a test compound. The method includes evaluating
interaction between an agent that can modulate the activity of Tie1
and a protein that includes at least a region of the Tie1
extracellular domain in the presence of the test compound. In one
embodiment, the agent is a test compound is a small organic
compound with molecular weight less than 8000, 7000, 6000, 5000, or
3000 Daltons. For example, the evaluating includes contacting cells
that include the protein that includes at least a region of the
Tie1 extracellular domain with the agent in the presence of the
test compound. In another example, the evaluating includes forming
a cell-free preparation that includes the protein that includes at
least a region of the Tie1 extracellular domain, the agent, and the
test compound.
[0159] In another aspect, the invention features an artificial
protein complex that includes (i) a protein that includes a Tie1
extracellular domain and (ii) a Tie1 binding protein that can
modulate (e.g., agonize or antagonize) an activity of Tie1. In one
embodiment, the ligand is an antibody (e.g., an antibody described
herein). For example, the ligand includes the HC and LC
immunoglobulin variable domains of an antibody selected from the
group consisting of: E3, E3b, B2, D11, A2, A10, P-B1, P-B3, P-C6,
G2 and C7, or immunoglobulin domains that are at least 90%
identical in the CDR regions to the CDR regions of the respective
antibody. In one embodiment, the complex is present in a membrane
fraction, on a mammalian cell, and/or in a subject.
[0160] In another aspect the invention features a method that
includes: administering a composition that includes a protein that
interacts with Tie1, Tie2, or Ang (e.g., a protein described
herein) to a subject in an amount effective to reduce angiogenesis
in the subject or otherwise treat or prevent a disorder in a
subject. For example, the protein binds to Tie1, Tie2, or Ang with
an affinity K.sub.D of less than 10.sup.-8 M, 510.sup.-9 M,
10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, or 10.sup.-12 M.
[0161] In one embodiment, the protein is a Tie1 binding protein.
The protein can have at least two valencies, each of which binds to
Tie1. For example, at least one, two, or all of the valencies can
be binding sites that competes with E3 for binding to Tie1. In one
embodiment, the protein competes with E3 for binding to Tie1 or
binds to an epitope that overlaps the epitope bound by E3 on
Tie1.
[0162] In one embodiment, the protein comprises a heavy chain
immunoglobulin variable domain sequence and a light chain
immunoglobulin variable domain sequence. The protein further
includes one or more of the following properties: (1) at least one
of the variable domain sequences comprising at least one CDR of the
E3 antibody; (2) at least one of the variable domain sequences
comprising CDR sequences at least 85% identical, in sum, to the
CDRs of the corresponding variable domain of the E3 antibody; (3)
at least one of the variable domains is at least 85% identical to
the corresponding immunoglobulin variable domains of the E3
antibody, and (4) the protein competes with E3 for binding to Tie1
or binds to an epitope that overlaps the epitope bound by E3 on
Tie1.
[0163] In one embodiment, one or more of the CDRs of the heavy
and/or light chain variable domain sequence are human, primate,
non-rodent (e.g., non-mouse or non-rat), or synthetic. In one
embodiment, one or more of the framework regions of the heavy
and/or light chain variable domain sequence are human, primate, or
non-rodent (e.g., non-mouse or non-rat).
[0164] In one embodiment, the heavy chain includes one or more of
the following properties:
[0165] i) a HC CDR1 that includes an amino acid sequence as
follows:
[0166] (AGSR)-Y-(GVK)-M-(GSVF), (SEQ ID NO:117)
[0167] (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO:118), or
[0168] (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO:119);
[0169] ii) a HC CDR2 that includes an amino acid sequence as
follows:
[0170] X-I-Y-P-S-G-G-X-T-X-Y-A-D-S-V-K-G (SEQ ID NO:120), wherein X
is any amino acid,
[0171] (GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY) (SEQ ID NO:121),
[0172] (GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID
NO:122),
[0173] (GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH) (SEQ ID
NO:123); or
[0174] X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO:704), wherein X
is any amino acid;
[0175] iii) a HC CDR3 that includes an amino acid sequence as
follows:
[0176] V-(four or five residues)-F-D-(I/Y) (SEQ ID NO:124),
[0177] G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO:125),
[0178] (GV)-N-Y-Y-(GYD)-S-(SD)-G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID
NO:126),
[0179]
(GVD)-(AGLN)-(LYR)-(GSTLYH)-(GYD)-(AGSYFP)-(SFD)-(AGYD)-(IY)-(GFD)--
(YDP)-(IP)-A-P-G-L-D-Y (SEQ ID NO:127),
[0180] VNYYDSSGYGPIAPGLDY (SEQ ID NO:128), or
[0181] G-X-X-G-(AY)-F-D-(YI) (SEQ ID NO:705), wherein X is any
amino acid.
[0182] In one embodiment, the light chain includes one or more of
the following properties: i) a light chain cdr1 that includes an
amino acid sequence as follows:
[0183] R-A-S-Q-S-(IV)-S-(SR)-X1-Y-L-(AN) (SEQ ID NO:129),
[0184] R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706),
[0185] T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707),
[0186] (SGQ)-(GS)-(DS)-(NS)-(IL)-(GR)-S-(YKN)-(YS)-(VA) (SEQ ID
NO:708),
[0187] R-A-S-Q-S-V-S-S-X-L (SEQ ID NO:130),
[0188] R-A-S-Q-S-(IV)-S-(SR)-(SY)-(LY)-(ALN) (SEQ ID NO:131),
OR
[0189]
R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)-(STIRH)-X1-(SYWNH)-(LV)-(ASN)
(SEQ ID NO:132), wherein X1 can be serine or absent;
[0190] ii) a LC CDR2 that includes an amino acid sequence as
follows:
[0191] X-A-S-X-R-A-T (SEQ ID NO:133), wherein X can be any amino
acid,
[0192] (AGD)-A-S-(STN)-R-A-T (SEQ ID NO:134),
[0193] (DG)-(AV)-S-N-(RL)-(AP)-ST) (SEQ ID NO:709),
[0194] (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:135), OR
[0195] (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO:136);
AND
[0196] iii) a LC CDR3 that includes an amino acid sequence as
follows:
[0197] Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO:137),
[0198] (LQ)-Q-(SYFR)-(GSYN)-(SKN)-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO:138),
[0199] Q-Q-X-S-(SN)-(WS)-P-X-T-F (SEQ ID NO:710), wherein x is any
amino acid,
[0200] Y-(TG)-(SG)-S-(PGS)-(TN)-X-(VT) (SEQ ID NO:711), wherein x
is any amino acid,
[0201] Q-Q-(YR)-(GS)-S-(SW)-P-R-X1-T (SEQ ID NO:139), wherein X1 is
any amino acid or absent,
[0202]
(LQ)-(LQ)-(SYFRD)-(GSYN)-(STRKN)-(STYWF)-(RP)-(ILMWRH)-(TIY)-(TI)
(SEQ ID NO:140), or
[0203]
(LQ)-(LRQ)-(SYFRD)-(GSYN)-(ASTRKN)-(STYWF)-(SVRP)-(STILMWRH)-(TIY)--
(STI) (SEQ ID NO:141).
[0204] In one embodiment, the heavy chain includes one or more of
the following properties:
[0205] i) a HC CDR1 that includes an amino acid sequence as
follows:
[0206] (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO:118), or
[0207] (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO:119);
[0208] ii) a HC CDR2 that includes an amino acid sequence as
follows:
[0209] (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY) (SEQ ID NO:160),
[0210] (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID
NO:122), or
[0211] (GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH) (SEQ ID
NO:123);
[0212] iii) a HC CDR3 that includes an amino acid sequence as
follows:
[0213] APRGYSYGYYY (SEQ ID NO:712).
[0214] In one embodiment, the light chain includes one or more of
the following properties: i) a LC CDR1 that includes an amino acid
sequence as follows:
R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)-(STIRH)-X1-(SYWNH)-(LV)-(ASN)
(SEQ ID NO:132), wherein X1 can be serine or absent; ii) a LC CDR2
that includes an amino acid sequence as follows:
(TAGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:713), or
(AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO:136); and iii) a LC
CDR3 that includes an amino acid sequence as follows:
Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LHWR)-(TIY) (SEQ ID NO:714),
(LQ)-Q-(SYFR)-(GSYN)-(SKN)-(STYW)-(RP)-(LHWR)-(TIY) (SEQ ID
NO:715), or
(LQ)-(LQ)-(SYFRD)-(GSYN)-(STRKN)-(STYWF)-(RP)-(ILMWRH)-(TIY) (SEQ
ID NO:716).
[0215] In one embodiment, the light chain includes one or more of
the following properties: i) a LC CDR1 that includes an amino acid
sequence as follows: S-X-(ND)-(IV)-(AG)-X1-X2-X3 (SEQ ID NO:142),
or T-(GR)-(ST)-S-X5-(ND)-(IV)-(AG)-X1-X2-X3-Y-X4-S (SEQ ID NO:143),
wherein X1 is any amino acid (e.g., G or R), X2 is any amino acid
(e.g., Y or N), X3 is any amino acid (e.g., F, N, or K), X4 is any
amino acid (e.g., aliphatic, e.g., V or A); iii) a LC CDR2 that
includes an amino acid sequence as follows: (DE)-V-N-N-R-P-S (SEQ
ID NO:144); (DE)-(VD)-(STDN)-(YRDN)-R-P-S (SEQ ID NO:145); v) a LC
CDR3 that includes an amino acid sequence as follows:
(SQ)-S-(SY)-(ASID)-(GSR)-(ST)-(STRN)-(STYR)-(ATLY)-(SVWQ) (SEQ ID
NO:146).
[0216] In one embodiment, the HC CDR2 includes an amino acid
sequence as follows:
(GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)-D-S-V-K-- G
(SEQ ID NO:147)or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S-V-K-G
(SEQ ID NO:148).
[0217] In one embodiment, the HC CDR1 amino acid sequences have a
length of at least 5 amino acids of which at least 3, 4, or 5 amino
acids are identical to the CDR1 sequence of the HC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the HC CDR2 amino acid sequences have a length of at
least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15,
16, or 17 amino acids are identical to the CDR2 sequence of the HC
of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3,
p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D11, s-E11, s-G10, s-H4, or another antibody described herein. In
one embodiment, the HC CDR2 amino acid sequences have a length of
at least 17 amino acids of which at least 14, 15, 16, or 17 amino
acids are identical to the CDR2 sequence of the HC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the HC CDR3 amino acid sequences have a length of at
least of at least 7 or 8 amino acids of which at least 5, 6, 7, or
8 amino acids are identical to the CDR3 sequence of the HC of clone
E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4,
p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D11,
s-E11, s-G10, s-H4, or another antibody described herein.
[0218] In one embodiment, the LC CDR1 amino acid sequences have a
length of at least 10, 11, or 12 amino acids of which at least 7,
8, 9, 10, or 11 amino acids are identical to the CDR1 sequence of
the LC of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12,
p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D11, s-E11, s-G10, s-H4, or another antibody described herein. In
one embodiment, the LC CDR2 amino acid sequences have a length of
at least 6 or 7 amino acids of which at least 5, 6, or 7 amino
acids are identical to the CDR2 sequence of the LC of clone E3,
E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3,
s-A10, s-H1, s-A2, s-B2, s-B9, s-C1, s-C2, s-C7, s-D11, s-E11,
s-G10, s-H4, or another antibody described herein. In one
embodiment, the LC CDR3 amino acid sequences have a length of at
least of at least 8, 9, or 10 amino acids of which at least 7, 8,
9, or 10 amino acids are identical to the CDR3 sequence of the LC
of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3,
p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D11, s-E11, s-G10, s-H4, or another antibody described
herein.
[0219] In one embodiment, the amino acid sequence of the HC
variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequence of the HC
variable domain of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C10, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein.
[0220] In one embodiment, the amino acid sequence of the LC
variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequence of the LC
variable domain of clone E3, E3b, G2, p-A1, p-A10, p-B1, p-B3,
p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C1,
s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein.
[0221] In one embodiment, the amino acid sequences of the HC and LC
variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequences of the HC and
LC variable domains of a clone selected from the group consisting
of E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12, p-F3, p-F4,
p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7, s-D 11,
s-E11, s-G10, and s-H4.
[0222] In one embodiment, the amino acid sequences of one or more
framework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC
and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to corresponding framework regions of the
HC and LC variable domains of clone E3, E3b, G2, p-A1, p-A10, p-B1,
p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C1, s-C2, s-C7, s-D11, s-E11, s-G 10, s-H4, or another antibody
described herein.
[0223] In one embodiment, the light chain variable domain sequence
is human or non-immunogenic in a human. In one embodiment, the
heavy chain variable domain sequence is human or non-immunogenic in
a human.
[0224] The protein can bind to cells that express Tie1, e.g.,
endothelial cells. In one embodiment, the protein does not
substantially bind (e.g., does not detectably bind) to
platelets.
[0225] In one embodiment, the protein specifically binds to Tie1,
e.g., it binds with at least a 10, 50, 100, 10.sup.3, or 10.sup.4
fold preference for Tie1 relative to another human protein, e.g.,
Tie2, a natural protein other than Tie1 that has a Ig-like domain,
an EGF-like domain, or fibronectin Type III repeat, or human serum
albumin. In one embodiment, the protein binds to a domain of Tie1
described herein.
[0226] In one embodiment, the protein is delivered locally. In one
embodiment, the protein is delivered systemically.
[0227] In one embodiment, the subject is in need of reduced
angiogenesis, or identified as such. For example, the subject has
an angiogenesis-related disorder. In another example, the subject
has a neoplastic disorder, e.g., a metastatic cancer. For example,
the subject has an angiogenesis-dependent cancer or tumor. The
tumor can be a solid tumor, e.g., a tumor at least 1, 2, 3, 5, 8 or
10 mm in diameter. In one embodiment, the solid tumor has a hypoxic
core. The method can further include administering an
anti-metabolite (e.g., 5-FU, with leucovorin), irinotecan, (or
other topoisomerase inhibitor), doxorubicin, bevacizumab, or all of
these agents. The method can include, prior to administering the
antagonist, evaluating the subject and detecting a solid tumor in
the subject.
[0228] In another embodiment, the subject has an inflammatory
disorder, e.g., rheumatoid arthritis, psoriasis, rheumatoid or
rheumatic inflammatory disease, or other chronic inflammatory
disorders, such as chronic asthma, arterial or
post-transplantational atherosclerosis, and endometriosis. Other
disorders that can be treated include those that have deregulated
or undesired angiogenesis, such as ocular neovascularization, e.g.,
retinopathies (including diabetic retinopathy and age-related
macular degeneration) hemangioblastoma, hemangioma, and
arteriosclerosis.
[0229] In one embodiment, the protein is administered in an amount
effective to reduce one or more of the following activities:
sprouting, splitting and remodeling of blood vessels. In one
embodiment, the protein is administered in an amount effective to
reduce vasculogenesis or tubule formation.
[0230] In one embodiment, the method further includes, prior to the
administering, identifying the subject as a subject in need of
reduced angiogenesis. In one embodiment, the method further
includes administering the protein continuously or in separate
boluses. In one embodiment, the method further includes monitoring
the subject during the course of administration. For example, the
monitoring includes imaging blood vessels (locally or throughout)
the subject. In another example, the monitoring include evaluating
tumor size or tumor load in the subject.
[0231] In another aspect the invention features a method that
includes: administering a composition that includes a protein
described herein (e.g., a protein that reduces a Tie1 activity) to
a subject in an amount effective to reduce a Tie1 activity in the
subject. The method can include other features described
herein.
[0232] In another aspect the invention features a method that
includes: administering a composition that includes a protein
described herein (e.g., a protein that can modulate an activity of
Tie1) to a subject in an amount effective to modulate endothelial
cell activity in the subject. In one embodiment, the protein is
delivered into the circulation.
[0233] In one embodiment, the composition is effective for
sensitizing endothelial cells to a treatment, and providing a
treatment to the subject that inhibits, kills, ablates, or
otherwise arrests the sensitized endothelial cells.
[0234] In another aspect the invention features a method that
includes: (i) contacting the sample (and optionally, a reference,
e.g., control, sample) with a protein that binds to Tie1, e.g., a
protein described herein, under conditions that allow interaction
of the Tie1-binding protein and the Tie1 protein to occur; and (ii)
detecting formation of a complex between the Tie1-binding protein,
and the sample (and optionally, the reference, e.g., control,
sample).
[0235] In another aspect the invention features a method that
includes: (i) administering to a subject (and optionally a control
subject) a Tie1-binding protein (e.g., an antibody or antigen
binding fragment thereof), under conditions that allow interaction
of the Tie1-binding protein and the Tie1 protein to occur; and (ii)
detecting formation of a complex between the Tie1-binding protein
and a Tie1 molecule of the subject or detecting distribution of
Tie1-binding protein or at least one location of the Tie1-binding
protein in the subject. In one embodiment, the Tie1-binding protein
does not modulate the activity of Tie1. The Tie1-binding protein
can be a protein described herein. In one embodiment, the ligand
detects activated Tie1.
[0236] An antibody that binds to Tie1 is preferably monospecific,
e.g., a monoclonal antibody, or antigen-binding fragment thereof.
For example, the antibody can recognize Tie1 on a living cell,
e.g., an endogenous Tie1 molecule or a Tie1 molecule that is
expressed from a heterologous nucleic acid. In one embodiment, the
Tie1-binding protein interacts with primary endothelial cells. The
term "monospecific antibody" refers to an antibody that displays a
single binding specificity and affinity for a particular target,
e.g., epitope. This term includes a "monoclonal antibody" which
refers to an antibody that is produced as a single molecular
species, e.g., from a population of homogenous isolated cells. A
"monoclonal antibody composition" refers to a preparation of
antibodies or fragments thereof of in a composition that includes a
single molecular species of antibody. In one embodiment, a
monoclonal antibody is produced by a mammalian cell. One or more
monoclonal antibody species may be combined.
[0237] The Tie1-binding antibodies can be full-length (e.g., an IgG
(e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2),
IgD, and IgE) or can include only an antigen-binding fragment
(e.g., a Fab, F(ab').sub.2 or scFv fragment), e.g., it does not
include an Fc domain or a CH2, CH3, or CH4 sequence. The antibody
can include two heavy chain immunoglobulins and two light chain
immunoglobulins, or can be a single chain antibody. The antibodies
can, optionally, include a constant region chosen from a kappa,
lambda, alpha, gamma, delta, epsilon or a mu constant region gene.
A Tie1-binding antibody can include a heavy and light chain
constant region substantially from a human antibody, e.g., a human
IgG1 constant region or a portion thereof.
[0238] In one embodiment, the antibody (or fragment thereof) is a
recombinant or modified antibody, e.g., a chimeric, a humanized, a
deimmunized, or an in vitro generated antibody. The term
"recombinant" or "modified" human antibody, as used herein, is
intended to include all antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
expressed using a recombinant expression vector transfected into a
host cell, antibodies isolated from a recombinant, combinatorial
antibody library, antibodies isolated from an animal (e.g., a
mouse) that is transgenic for human immunoglobulin genes or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant antibodies include
humanized, CDR grafted, chimeric, deimmunized, in vitro generated
antibodies, and may optionally include constant regions derived
from human germline immunoglobulin sequences.
[0239] In one embodiment, the antibody binds to an epitope distinct
from an epitope bound by known monoclonal antibodies that bind to
Tie1, e.g., an antibody described in WO 95/26364, e.g., 3C4C7G6 and
10F11G6. In other embodiments, the antibody does not compete with
known monoclonal antibodies that bind to Tie1, e.g., 3C4C7G6 and
10F11G6. In still other embodiments, the antibody does not compete
with ligand described herein, e.g., the E3 antibody.
[0240] Also within the scope of the invention are antibodies or
other agents (e.g., protein or non-protein agents) that bind
overlapping epitopes of, or competitively inhibit the binding of
the proteins disclosed herein, e.g., proteins that bind to Tie1,
Tie2, or Ang. For example, the antibodies or other agents bind
overlapping epitopes of or competitively inhibit the binding of
monospecific antibodies, e.g., E3, E3b, G2, p-A1, p-A10, p-B1,
p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9,
s-C1, s-C2, s-C7, s-D11, s-E11, s-G10, s-H4, or another antibody
described herein to Tie1, or vice versa (e.g., the monospecific
antibodies competitively inhibiting binding of the ligands).
Overlapping epitopes can include at least one amino acid in common.
Agents that competitively inhibit binding of one another do not
necessarily bind to overlapping epitopes. For example, they may
inhibit binding by steric interference or by altering the
conformation of Tie1.
[0241] Any combination of binding proteins is within the scope of
the invention, e.g., two or more antibodies that bind to different
regions of Tie1, Tie2, or Ang, e.g., antibodies that bind to two
different epitopes on the extracellular domain of Tie1, Tie2, or
Ang, e.g., a bispecific antibody.
[0242] In one embodiment, the Tie1-binding antibody or
antigen-binding fragment thereof includes at least one light or
heavy chain immunoglobulin (or preferably, at least one light chain
immunoglobulin and at least one heavy chain immunoglobulin).
Preferably, each immunoglobulin includes a light or a heavy chain
variable region having at least one, two and, preferably, three
complementarity determining regions (CDRs) substantially identical
to a CDR from an anti-Tie1 light or heavy chain variable region,
respectively, i.e., from a variable region of an antibody described
herein, e.g., E3, E3b, G2, p-A1, p-A10, p-B1, p-B3, p-C6, p-D12,
p-F3, p-F4, p-G3, s-A10, s-H1, s-A2, s-B2, s-B9, s-C10, s-C2, s-C7,
s-D 11, s-E11, s-G10, s-H4, or another antibody described
herein.
[0243] In one aspect, the invention features an agent (e.g., an
antibody) that decreases endothelial cell activity by increasing
Tie1 phosphorylation. In one embodiment, the agent decreases
endothelial cell differentiation, e.g., sprouting, splitting, and
tube formation.
[0244] In one aspect, the invention features an agent (e.g., an
antibody) that decreases endothelial cell activity by activating a
signaling pathway. In one embodiment, the antibody decreases
endothelial cell differentiation, e.g., sprouting, splitting, and
tube formation. This agent-induced effect can be independent or
dependent of Tie1 self-association.
[0245] In one aspect, the invention features an isolated protein
that includes a heavy chain immunoglobulin variable domain sequence
and a light chain immunoglobulin variable domain sequence, wherein
the protein binds to Tie1 ectodomain and the heavy chain
immunoglobulin variable domain sequence includes one or more of the
following properties: i) a HC CDR1 that includes an amino acid
sequence of a clone from the group consisting of: M0044-A06;
M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10;
M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06;
M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05;
M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03;
M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07;
M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12;
M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03;
M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09;
M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03;
M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08;
M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03;
M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09;
M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06;
M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10;
M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08;
M0056-B09; M0056-C03; M0056-C004; M0056-E08; M0056-F01; M0056-F02;
M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12;
M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04;
M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06;
M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07;
M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08;
M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06;
and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or
90% identical to such a sequence; ii) a HC CDR2 that includes an
amino acid sequence of a clone from the group consisting of
M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09;
M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03;
M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03;
M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01;
M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01;
M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10;
M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01;
M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05;
M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12;
M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06;
M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08;
M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10;
M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03;
M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12;
M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06;
M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01;
M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08;
M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09;
M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02;
M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06;
M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07;
M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10;
M0062-G06; and M0062-H01, or a sequence that is at least 70, 75,
80, 85, or 90% identical to such a sequence; iii) a HC CDR3 that
includes an amino acid sequence of a clone from the group
consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05;
M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01;
M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07;
M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02;
M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11;
M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11;
M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11;
M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02;
M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03;
M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04;
M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08;
M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01;
M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05;
M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06;
M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04;
M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04;
M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03;
M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05;
M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03;
M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03;
M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12;
M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03;
M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that
is at least 70, 75, 80, 85, or 90% identical to such a
sequence.
[0246] In one embodiment, the protein also includes the light chain
immunoglobulin variable domain sequence which includes one or more
of the following properties: i) a LC CDR1 that includes an amino
acid sequence of a clone from the group consisting of M0044-A06;
M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10;
M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06;
M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05;
M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03;
M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07;
M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12;
M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03;
M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09;
M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03;
M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08;
M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03;
M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09;
M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06;
M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10;
M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08;
M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02;
M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12;
M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04;
M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06;
M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07;
M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08;
M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06;
and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or
90% identical to such a sequence; ii) a LC CDR2 that includes an
amino acid sequence of a clone from the group consisting of:
M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09;
M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03;
M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03;
M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01;
M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01;
M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10;
M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01;
M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05;
M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12;
M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06;
M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08;
M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10;
M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03;
M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12;
M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06;
M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01;
M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08;
M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09;
M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02;
M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06;
M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07;
M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10;
M0062-G06; and M0062-H01, or a sequence that is at least 70, 75,
80, 85, or 90% identical to such a sequence; iii) a LC CDR3 that
includes an amino acid sequence of a clone from the group
consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05;
M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01;
M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07;
M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02;
M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11;
M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11;
M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11;
M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02;
M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03;
M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04;
M0053-F05; M0053-F06; M0053-F08; M0053-C04; M0053-G05; M0054-A08;
M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01;
M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05;
M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06;
M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04;
M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04;
M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-C03;
M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05;
M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03;
M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03;
M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12;
M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03;
M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that
is at least 70, 75, 80, 85, or 90% identical to such a
sequence.
[0247] In one embodiment, the protein includes the amino acid
sequence of the HC variable domain sequence which is at least 85,
90, 95, 98, or 99% identical to the amino acid sequence of the HC
variable domain of clone M0044-A06; M0044-A11; M0044-B04;
M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07;
M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06;
M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09;
M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02;
M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10;
M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10;
M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09;
M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11;
M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08;
M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05;
M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04;
M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12;
M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04;
M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03;
M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03;
M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11;
M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12;
M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03;
M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01;
M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09;
M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02;
M0062-E03; M0062-E11; M0062-F10; M0062-G06; or M0062-H01.
[0248] In one embodiment, the protein includes the amino acid
sequence of the LC variable domain sequence which is at least 85,
90, 95, 98, or 99% identical to the amino acid sequence of the LC
variable domain of clone M0044-A06; M0044-A11; M0044-B04;
M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07;
M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-C06;
M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09;
M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02;
M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10;
M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10;
M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09;
M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11;
M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08;
M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05;
M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04;
M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12;
M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04;
M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03;
M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03;
M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11;
M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12;
M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03;
M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01;
M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09;
M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02;
M0062-E03; M0062-E11; M0062-F10; M0062-G06; or M0062-H01.
[0249] An antibody or other binding protein (e.g., a Tie1-binding
protein, Tie2-binding protein, or Ang binding protein) described
herein can be administered to a subject or used in vitro in
non-derivatized or unconjugated forms. In other embodiments, the
binding protein can be derivatized, modified or linked to another
functional molecule, e.g., another protein (e.g., HSA, an Fc
domain, etc.), a polymer (e.g., PEG) isotope, cell, or insoluble
support. For example, the binding protein can be functionally
linked (e.g., by chemical coupling, genetic fusion, non-covalent
association or otherwise) to one or more other molecular entities,
such as an antibody (e.g., if the protein is an antibody to form a
bispecific or a multi-specific antibody), a toxin, a radioisotope,
a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety,
among others. For example, the binding protein can be coupled to a
radioactive ion (e.g., an .alpha.-, .gamma.-, or .beta.-emitter),
e.g., iodine (.sup.131I or .sup.125I), yttrium (.sup.90Y), lutetium
(.sup.177Lu), actinium (.sup.225Ac), rhenium (.sup.186Re), or
bismuth (.sup.212Bi or .sup.213Bi).
[0250] In another aspect, the invention features a nucleic acid
that includes a coding sequence that encodes a polypeptide
comprising an immunoglobulin heavy or light chain variable domain
that binds to Tie1, e.g., an immunoglobulin heavy or light chain
variable domain described herein. For example, the nucleic acid can
include a particular nucleic acid sequence described herein, a
nucleic acid that is at least 75, 80, 85, 90, 95, 96, 97, 98, or
99% identical to a nucleic acid sequence described herein (e.g., a
particular nucleic acid sequence), or a nucleic acid that
specifically hybridizes (e.g., under conditions described herein,
e.g., high stringency conditions) to a nucleic acid sequence
described herein (e.g., a particular nucleic acid sequence, e.g., a
nucleic acid encoding one or more variable domains of M0044-A06;
M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10;
M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06;
M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05;
M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03;
M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07;
M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12;
M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03;
M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09;
M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03;
M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08;
M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03;
M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09;
M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06;
M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10;
M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08;
M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02;
M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-C08; M0056-G12;
M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04;
M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06;
M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07;
M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08;
M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06;
or M0062-H01), or fragments thereof (e.g., CDR-coding
fragments).
[0251] A nucleic acid described herein can further include a
promoter operably linked to the coding sequence. A nucleic acid can
include a first and second coding sequence, e.g., wherein the first
coding sequence encodes a polypeptide that includes an
immunoglobulin heavy chain variable domain and the second coding
sequence encodes a polypeptide that includes an immunoglobulin
light chain variable domain.
[0252] In another aspect, the invention features a host cell that
contains a first nucleic acid encoding a polypeptide comprising a
heavy chain variable region and a second nucleic acid encoding a
polypeptide comprising a light chain variable region. The heavy
chain variable region and the light chain variable region can
associate to form a Tie1 binding protein. These variable regions
can have one or more properties described herein, e.g., at least
75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to a sequence
described herein. The invention also includes a method of providing
a Tie1-binding antibody. The method can include providing a host
cell described herein; and expressing said first and second nucleic
acids in the host cell under conditions that allow assembly of said
light and heavy chain variable regions to form an antigen binding
protein that interacts with Tie1.
[0253] In another aspect, the invention provides compositions,
e.g., pharmaceutical compositions, which include a pharmaceutically
acceptable carrier, excipient or stabilizer, and at least one of
the Tie1-binding proteins (e.g., antibodies or fragments thereof)
described herein. In one embodiment, the compositions, e.g., the
pharmaceutical compositions, include a combination of two or more
of the aforesaid Tie1-binding proteins.
[0254] In another aspect, the invention features a kit that
includes a Tie1-binding antibody (or fragment thereof), e.g., a
Tie1-binding antibody (or fragment thereof) as described herein,
for use alone or in combination with other therapeutic modalities,
e.g., a cytotoxic or labeling agent, e.g., a cytotoxic or labeling
agent as described herein, along with instructions on how to use
the Tie1 antibody or the combination of such agents to treat,
prevent or detect a Tie1-related disorder, e.g., an endothelial
cell related disorder, e.g., rheumatoid arthritis or metastatic
cancer.
[0255] In another aspect, the binding protein that binds to Tie1 is
a polypeptide that is not an immunoglobulin. For example, the
polypeptide can be of variable length, e.g., 4 to 100 amino acid
residues in length, preferably 5 to 75, 6 to 50, or 7 to 40 amino
acid residues in length, or more preferably 8 to 30 or 10 to 25
amino acid residues in length. In some embodiments, the polypeptide
includes non-standard or synthetic amino acid residues, e.g.,
norleucine, selenocysteine, pyrrolysine, etc. In some embodiments,
the polypeptide includes cross-linking groups, e.g., two cysteine
residues that can form a disulfide bond or some other type of
chemical cross-linking moieties that can be used to cyclize the
peptide. In other preferred embodiments, the polypeptide can be
modified, e.g., using polyethylene glycol or fusion to a soluble
protein, e.g., to increase the solubility or circulatory half-life
of the polypeptide.
[0256] The target-binding protein can be physically associated with
(e.g., fused to) another protein, e.g., a protein that does not
bind to the target, e.g., to the amino or carboxy terminus. For
example, the target-binding protein can be associated with (e.g.,
fused to) a protein that increases serum residence or alters
stability, e.g., an albumin, e.g., a serum albumin, e.g., HSA
(human serum albumin). In another example, the target binding
protein is physically associated with (e.g., fused to) a moiety
that facilitates purification, e.g., a purification tag such as
His, PEG, or to a functional moiety, e.g., Fc.
[0257] In another aspect, the invention features a method of
identifying a protein that specifically binds to Tie1. In preferred
embodiments, the invention includes: providing a Tie1 antigen;
providing a display library (e.g., a phage display library member);
identifying a member present in the library, wherein the member
expresses a protein that specifically binds to the Tie1 antigen.
The term "Tie1 antigen" refers to any antigenic fragment of Tie1
that is at least 8 amino acids in length. For example, a Tie1
antigen can include a fragment of the Tie1 ectodomain, e.g., a
fragment that includes a folded protein domain such as a fragment
described herein. In some embodiments, the Tie1 antigen is of human
origin and includes, e.g., the extracellular domain of human Tie1
or a fragment thereof (e.g., a fragment described herein. The Tie1
antigen can be a recombinant polypeptide optionally fused to
another polypeptide, e.g., a Fc domain, or it can be a cell that
expresses Tie1 on its surface (e.g., an endothelial cell). In other
preferred embodiments, the Tie1 antigen has an activated
conformation, e.g., the Tie1 antigen is a dimeric conformation or a
conformation stabilized by the E3 or E3b antibody described
herein.
[0258] The methods described here are, for example, applicable to
libraries that are based on bacteriophage with a substantially
complete genome (e.g., including a modified gene III) and to
libraries that are based on bacteriophage particles that include a
phagemid nucleic acid. The terms "bacteriophage library member" and
"phage" encompass members of both types of libraries. The term
"bacteriophage particle" refers to a particle formed of
bacteriophage coat proteins that packages a nucleic acid. The
packaged nucleic acid can be a modified bacteriophage genome or a
phagemid, e.g., a nucleic acid that includes a bacteriophage origin
of replication but lacks essential phage genes and cannot propagate
in E. coli without help from "helper phage" or phage genes supplied
in trans.
[0259] In other embodiments, the invention features a method of
identifying a protein that specifically binds to Tie1. The method
includes: providing a Tie1 antigen (e.g., an region of the Tie1
ectodomain); immunizing a non-human animal with the Tie1 antigen;
and isolating a cell that produces a immunoglobulin that interacts
with Tie1. For example, the method can include producing hybridoma
cells from the spleen of the animal (e.g., an immunized mouse); and
identifying individual hybridoma cell lines expressing an antibody
that specifically binds to the Tie1 antigen. For example, the
[0260] In preferred embodiments, the Tie1 antigen is of human
origin and includes, e.g., the extracellular domain of human Tie1
or some fragment thereof, e.g., the HA binding domain of Tie1. The
Tie1 antigen can be a recombinant polypeptide optionally fused to
another polypeptide, e.g., a purification handle, or it can be a
cell that expresses Tie1 (e.g., an endothelial cell) on its
surface. In other preferred embodiments, the Tie1 antigen has an
activated conformation, e.g., dimerized.
[0261] In preferred embodiments, the methods further include
isolating a nucleic acid molecule from the identified phage or
hybridoma, wherein the nucleic acid molecule encodes the
polypeptide or antibody that specifically binds to the Tie1
antigen. The isolated nucleic acid molecules can be used to produce
therapeutic agents, as described herein.
[0262] In another aspect, the invention features nucleic acids that
encode proteins identified by the methods described herein. In
preferred embodiments, the nucleic acids include sequences encoding
a heavy and light chain immunoglobulin or immunoglobulin fragment
described herein. For example, the invention features, a first and
second nucleic acid encoding a heavy and light chain variable
region, respectively, of a Tie1-binding antibody molecule as
described herein. Sequences encoding a heavy and light chain that
function together can be present on separate nucleic acid molecules
or on the same nucleic acid molecule. In another aspect, the
invention features host cells and vectors containing a nucleic acid
described herein.
[0263] In yet another aspect, the invention features a method of
producing a Tie1-binding antibody, or antigen-binding fragment
thereof The method includes: providing a host cell that contains a
first nucleic acid encoding a polypeptide comprising a heavy chain
variable region, e.g., a heavy chain variable region as described
herein; providing a second nucleic acid encoding a polypeptide
comprising a light chain variable region, e.g., a light chain
variable region as described herein; and expressing said first and
second nucleic acids in the host cell under conditions that allow
assembly of said light and heavy chain variable regions to form an
antigen binding protein that interacts with Tie1. The first and
second nucleic acids can be linked or unlinked, e.g., expressed on
the same or different vector, respectively. The first and second
nucleic acids can be components of the same molecule or can reside
on different molecules (e.g., different chromosomes or
plasmids).
[0264] The host cell can be a eukaryotic cell, e.g., a mammalian
cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E.
coli. For example, the mammalian cell can be a cultured cell or a
cell line. Exemplary mammalian cells include lymphocytic cell lines
(e.g., NS0), Chinese hamster ovary cells (CHO), COS cells, HEK294,
oocyte cells, and cells from a transgenic animal, e.g., mammary
epithelial cell. For example, nucleic acids encoding the antibodies
described herein can be expressed in a transgenic animal. In one
embodiment, the nucleic acids are placed under the control of a
tissue-specific promoter (e.g., a mammary specific promoter) and
the antibody is produced in the transgenic animal. For example, the
antibody molecule is secreted into the milk of the transgenic
animal, such as a transgenic cow, pig, horse, sheep, goat or
rodent. To produce a single chain antibody, the nucleic acid is
configured to encode a single polypeptide that comprises both the
heavy and light chain variable domains.
[0265] Tie1 has been found to be overexpressed in association with
a wide range of cancers. Targeting Tie1 on the tumor vasculature
with Tie1-binding proteins (e.g., antibodies) can be used to
inhibit, destroy, or otherwise antagonize the vasculature so that
tumor growth and metastasis is reduced. The proteins can be, for
example, associated with a toxic payload or can mediate direct
functional inhibition. Proteins (e.g., proteins that have an Fc
domain) that can cause ADCC can also be used.
[0266] In another aspect, the invention features a method of
inhibiting an activity of a cell, e.g., an endothelial cell, e.g.,
proliferation, adhesion, growth or survival of a cell, e.g., an
endothelial cell, e.g., an endothelial cell in the vicinity of a
cancer, e.g., a tumor. Exemplary methods include contacting the
cell with a Tie1 binding protein, in an amount sufficient to
inhibit the adhesion, migration, growth or proliferation of the
cell. Methods of administering a Tie1 binding protein can be used,
for example, to treat or prevent a disorder, e.g., an inflammatory
disorder (e.g., rheumatoid arthritis, lupus, restenosis, psoriasis,
graft v. host response, or multiple sclerosis), or a cancerous
disorder (e.g., a malignant or metastatic disorder), by
administering to a subject (e.g., an experimental animal or a human
patient) a Tie1-binding protein in an amount effective to treat or
prevent such disorder.
[0267] A Tie1-binding protein can be used to treat or prevent
angiogenesis-related disorders, particularly angiogenesis-dependent
cancers and tumors. Angiogenesis-related disorders include, but are
not limited to, solid tumors; tumor metastasis; benign tumors
(e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas,
and pyogenic granulomas; rheumatoid arthritis); 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.
[0268] "Angiogenesis-dependent cancers and tumors" are cancers
tumors that require, for their growth (expansion in volume and/or
mass), an increase in the number and density of the blood vessels
supplying then with blood. In one embodiment a Tie1-binding protein
causes regression of such cancers and tumors. "Regression" refers
to the reduction of tumor mass and size, e.g., a reduction of at
least 2, 5, 10, or 25%.
[0269] In addition, Tie1 and Tie2 are also expressed in
hematopoietic cells. (Kukk et al (1997) Br. J. Haematol. 98: 195;
Iwama et al (1993) Biochem. Biophys. Res. Commun. 195: 301).
Accordingly, in another embodiment, a Tie1-binding protein is used
to treat hematopoietic conditions, e.g., hematopoietic cancers.
Examples of hematopoietic cancers include: cancers derived from
hyperplastic/neoplastic cells of hematopoietic origin, e.g., cells
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof. Exemplary cancers include acute promyeloid leukemia
(APML), acute myelogenous leukemia (AML), chronic myelogenous
leukemia (CML), acute lymphoblastic leukemia (ALL), chronic
lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy
cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM),
non-Hodgkin's lymphoma, peripheral T-cell lymphomas, adult T-cell
leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large
granular lymphocytic leukemia (LGF), B cell chronic lymphocytic
leukemia, myelodysplastic syndrome, and Hodgkin's disease.
[0270] In another aspect, the invention features a method of
contacting a cell (in vitro, ex vivo, or in vivo), e.g., an
endothelial cell, e.g., an endothelial cell in the vicinity of a
cancer, e.g., a tumor. The method can include providing an agent
(e.g., a protein) that interacts with Tie1, e.g., a protein
described herein, and contacting the cell with the protein, in an
amount sufficient to form at least one detectable ligand-cell
complex. The protein can include, for example, a label or cytotoxic
entity, e.g., an immunoglobulin Fc domain or a cytotoxic drug.
[0271] In another aspect, the invention features administering the
agent described herein as an adjuvant therapy, e.g., to a subject.
The adjuvant therapy can be a post-operative therapy that is
administered to the subject after the subject has undergone surgery
to remove all or part of a tumor (e.g., after surgery to treat
glioblastoma or colorectal, breast, or lung cancer). For example,
the agent is a protein that inhibits Tie complex formation,
promotes Tie1 homodimerization, or increases Tie1 phosphorylation.
For example, the agent is a protein that binds Tie1 (e.g., an
anti-Tie1 antibody). In one embodiment, the agent is administered
within 6, 12, 24, 48, or 100 hours of surgery. The agent can be
administered before as well as after surgery.
[0272] An exemplary agent is a Tie1 binding agent that includes (a)
a heavy chain variable domain sequence that is at least 85, 90, 95,
98, 99%, or 100% identical to the heavy chain variable domain of
the E3 antibody and a light chain variable domain sequence that is
at least 85, 90, 95, 98, 99%, or 100% identical to the light chain
variable domain of the E3 antibody; (b) a heavy chain variable
domain sequence and a light chain variable domain sequence that
form an antigen binding site that competes with E3 for binding to
Tie1; or (c) one, two, or three, of the CDRs of the heavy chain
variable domain of the E3 antibody, and one, two, or three of the
CDRs of the light chain variable domain of the E3 antibody. Other
Tie1 binding agents described herein can also be used, e.g., a Tie1
binding agent that includes a heavy chain variable domain sequence
that is at least 85, 90, 95, 98, 99%, or 100% identical to the
heavy chain variable domain of M0059A02, M0045A02*, M0054G05,
M0053F05, M0053G05, M0061C06, M0045B01, M0046G12, M0046H11,
M0053A02, M0053A05, M0046B06, M0044B10, M0044B08, M0056G08,
M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, or M0058F03, and
a light chain variable domain sequence that is at least 85, 90, 95,
98, 99%, or 100% identical to the light chain variable domain of
M0059A02, M0045A02*, M0054G05, M0053F05, M0053G05, M0061C06,
M0045B01, M0046G12, M0046H11, M0053A02, M0053A05, M0046B06,
M0044B10, M0044B08, M0056G08, M0045B03, M0053F04, M0055E10,
M0060H01, M0054H10, or M0058F03.
[0273] In another aspect, the invention features a method of
treating, e.g., inhibiting, ablating or killing, a cell or
impairing at least one activity of the cell. The method includes
providing a Tie1-binding protein, e.g. a ligand described herein,
and contacting the cell with the protein, in an amount sufficient
to impair at least one activity of the cell, inhibit, ablate or
kill the cell. The contacting can be in vitro or in vivo. For
example, the cell can be, e.g., an endothelial cell, e.g., an
endothelial cell in the vicinity of a cancer, e.g., a tumor. The
protein can include a cytotoxic entity. Methods of administering a
Tie1 binding protein or other agent described herein can be used,
for example, to treat or prevent a disorder, e.g., a endothelial
cell-based disorder, a blood vessel disorder, wound healing, or a
cancerous disorder (e.g., a malignant or metastatic disorder), by
administering to a subject (e.g., an experimental animal or a human
patient) a Tie1-binding protein in an amount effective to treat or
prevent such disorder.
[0274] A Tie1 binding protein or other agent described herein can
be used on cells in culture, e.g. in vitro or ex vivo. For example,
an endothelial cell, e.g., an endothelial cell in cancer biopsy,
can be cultured in vitro in culture medium and the contacting step
can be effected by adding the Tie1-binding protein to the culture
medium. The method can be performed on cells (e.g., cancerous or
metastatic cells) present in a subject, as part of an in vivo
(e.g., therapeutic or prophylactic) protocol. For in vivo
embodiments, the contacting step is effected in a subject and
includes administering the Tie1-binding protein to the subject
under conditions effective to permit both binding of the protein to
the cell, and the inhibition of adhesion, migration, growth or
proliferation of the cell.
[0275] A Tie1 binding protein or other agent described herein can
be used to treat or prevent cancerous disorders, e.g., including
hematopoietic cancers, solid tumors, soft tissue tumors, and
metastatic lesions, particularly tumors that require a blood supply
or angiogenesis. Examples of solid tumors include malignancies,
e.g., sarcomas, adenocarcinomas, and carcinomas, of the various
organ systems, such as those affecting lung, breast, lymphoid,
gastrointestinal (e.g., colon), and genitourinary tract (e.g.,
renal, urothelial cells), pharynx, as well as adenocarcinomas which
include malignancies such as most colon cancers, rectal cancer,
renal-cell carcinoma, liver cancer, non-small cell carcinoma of the
lung, cancer of the small intestine and cancer of the esophagus.
The subject can be a mammal, e.g., a primate, preferably a higher
primate, e.g., a human (e.g., a patient having, or at risk of, a
disorder described herein, e.g., an endothelial cell-based
disorder, e.g., cancer).
[0276] The Tie1-binding antibody or fragment thereof, e.g., a
Tie1-binding antibody or fragment thereof as described herein, can
be administered to the subject systemically (e.g., orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intraperitoneally, intranasally, transdermally, or by inhalation),
topically, or by application to mucous membranes, such as the nose,
throat and bronchial tubes.
[0277] The methods can further include the step of monitoring the
subject, e.g., for a reduction in one or more of: a reduction in
tumor size; reduction in cancer markers, e.g., levels of cancer
specific antigen; reduction in the appearance of new lesions, e.g.,
in a bone scan; a reduction in the appearance of new
disease-related symptoms; or decreased or stabilization of size of
soft tissue mass; or any parameter related to improvement in
clinical outcome. The subject can be monitored in one or more of
the following periods: prior to beginning of treatment; during the
treatment; or after one or more elements of the treatment have been
administered. Monitoring can be used to evaluate the need for
further treatment with the same Tie1-binding protein or for
additional treatment with additional agents. Generally, a decrease
in one or more of the parameters described above is indicative of
the improved condition of the subject. Information about the
monitoring can be recorded, e.g., in electronic or digital
form.
[0278] The Tie1-binding protein can be used alone in unconjugated
form to thereby inhibit adhesion, migration, or extravasation or
the Tie1-expressing cells, or ablate or kill the Tie1-expressing
cells. If the Tie1-binding protein is an antibody, the ablation or
killing can be mediated, e.g., by an antibody-dependent cell
killing mechanisms such as complement-mediated cell lysis and/or
effector cell-mediated cell killing. In other embodiments, the
Tie1-binding protein can be bound (e.g., physically associated,
either directly or indirectly, covalently or non-covalently) to a
substance, e.g., a cytotoxic agent or moiety, effective to kill or
ablate the Tie1-expressing cells. For example, the Tie1-binding
protein can be coupled to a radioactive ion (e.g., an .alpha.-,
.gamma.-, or .beta.-emitter), e.g., iodine (.sup.131I or
.sup.125I), yttrium (.sup.90Y), lutetium (.sup.177Lu), actinium
(.sup.225Ac), or bismuth (.sup.212Bi or .sup.213Bi).
[0279] The methods and compositions described herein can be used in
combination with other therapeutic modalities. In one embodiment,
the methods include administering to the subject a Tie1-binding
protein, e.g., a Tie1-binding antibody or fragment thereof, in
combination with a cytotoxic agent, in an amount effective to treat
or prevent the disorder. The Tie1-binding protein and the cytotoxic
agent can be administered simultaneously or sequentially. In other
embodiments, a Tie1 binding protein or other agent described herein
is used in combination with surgical and/or radiation
procedures.
[0280] In another aspect, the invention features methods for
detecting the presence of a Tie1 protein or a cell expressing Tie1
(e.g., an endothelial cell) in a sample, in vitro (e.g., a
biological sample, a tissue biopsy, e.g., a cancerous lesion). The
subject method can be used to evaluate, e.g., diagnose or stage a
disorder described herein, e.g., a cancerous disorder. The method
includes: (i) contacting the sample (and optionally, a reference,
e.g., control sample) with a Tie1-binding protein, as described
herein, under conditions that allow interaction of the Tie1-binding
protein and the Tie1 protein to occur; and (ii) detecting formation
of a complex between the Tie1-binding protein, and the sample (and
optionally, the reference, e.g., control, sample). Formation of the
complex is indicative of the presence of Tie1 protein (e.g.,
activated Tie1 protein), and can indicate the suitability or need
for a treatment described herein. For example, a statistically
significant change in the formation of the complex in the sample
relative to the reference sample, e.g., the control sample, is
indicative of the presence of Tie1 (e.g., activated Tie1) in the
sample.
[0281] In yet another aspect, the invention provides a method for
detecting the presence of Tie1 (e.g., activated Tie1) in vivo
(e.g., in vivo imaging in a subject). The subject method can be
used to evaluate, e.g., diagnose, localize, or stage a disorder
described herein, e.g., a cancerous disorder. The method includes:
(i) administering to a subject (and optionally a control subject) a
Tie1-binding protein (e.g., an antibody or antigen binding fragment
thereof), under conditions that allow interaction of the
Tie1-binding protein and the Tie1 protein to occur; and (ii)
detecting formation of a complex between the Tie1-binding protein
and Tie1, wherein a statistically significant change in the
formation of the complex in the subject relative to the reference,
e.g., the control subject or subject's baseline, is indicative of
the presence of the Tie1. The presence of Tie1 in particular
locations within a subject can be indicative of an endothelial-cell
related disorder, e.g., an angiogenesis-related disorder, e.g., a
cancer, e.g., metastatic cancer, or other angiogenesis-related
disorder described herein.
[0282] Tumor cells can express Tie1. In one aspect, the invention
features a method of providing a sample from a subject and
evaluating the Tie1 expression in cells in the sample. In one
embodiment, the result of evaluating Tie1 expression levels is
compared to a reference, e.g., a reference value or reference
quality. For example, the Tie1 expression on the evaluated sample
may have the same, less than, or greater than the reference value.
A reference value or quality can be determined using a control
sample, a statistical value (e.g., an average, median, etc.) or an
arbitrary value. For example, the control sample can be a normal
sample, e.g., a sample devoid of tumor cells from the same or
different subject. A change (e.g., an increase) relative to the
reference can indicate that the sample includes tumor cells, e.g.,
the subject may be indicated as having a tumor.
[0283] In other embodiments, a method of diagnosing or staging a
disorder as described herein (e.g., an inflammatory or cancerous
disorder), is provided. The method includes: (i) identifying a
subject having, or at risk of having, the disorder; (ii) obtaining
a sample of a tissue or cell affected with the disorder; (iii)
contacting said sample or a control sample with a Tie1-binding
protein, under conditions that allow interaction of the binding
agent and the Tie1 protein to occur, and (iv) detecting formation
of a complex. A statistically significant increase in the formation
of the complex between the Tie1-binding protein with respect to a
reference sample, e.g., a control sample, is indicative of the
disorder or the stage of the disorder. For example, the finding of
activated Tie1 on tumor cells located in a solid tumor can indicate
that the tumor is progressing into a metastatic tumor.
[0284] Preferably, the Tie1-binding protein used in the in vivo and
in vitro diagnostic methods is directly or indirectly labeled with
a detectable substance to facilitate detection of the bound or
unbound binding agent. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. In one embodiment,
the Tie1-binding protein is coupled to a radioactive ion, e.g.,
indium (.sup.111In), iodine (.sup.131I or .sup.125I), yttrium
(.sup.90Y), actinium (.sup.225Ac), bismuth (.sup.212Bi or
.sup.213Bi), sulfur (.sup.35S), carbon (.sup.14C), tritium
(.sup.3H), rhodium (.sup.188Rh), or phosphorous (.sup.32P). In
another embodiment, the Tie1-binding protein is labeled with an NMR
contrast agent.
[0285] In one aspect, the invention features a method of imaging
tumor vasculature, the method includes: providing a protein that
binds to Tie1, Tie2, or Ang, e.g., a protein described herein,
wherein the protein is physically associated to an imaging agent;
administering the protein to a patient, e.g., with a tumor; and
imaging the patient, e.g., to detect tumor vasculature.
[0286] In one aspect, the invention features a method of treating a
subject with a blood born neoplastic disorder, the method includes
administering a protein that binds to Tie1, Tie2, or Ang, e.g., a
protein described herein, to a subject with a blood born neoplastic
disorder (e.g., a proliferative disorder of hematopoietic cells,
e.g., leukemia), thereby treating the disorder.
[0287] In one aspect, the invention features a method of diagnosing
and treating a subject, the method includes evaluating a parameter
associated with Tie1, Tie2, or Ang in a subject; and, if the
parameter is altered relative to a reference, administering a
protein described herein to the subject, thereby treating the
subject. In one embodiment, the parameter includes a value
indicative of protein or mRNA levels, e.g., in a tissue of a
subject. In one embodiment, the reference includes a value
determined for a reference subject, e.g., an age/gender matched
subject, e.g., a control or normal subject.
[0288] In one aspect, the invention features a method of treating a
subject, the method includes: administering a protein described
herein to a subject that has elevated Tie1, Tie2, or Ang
biomolecules or activity relative to a reference. The method can
include evaluating the subject, e.g., to determine if the subject
has elevated Tie1, Tie2, or Ang biomolecules or activity relative
to a reference. In one embodiment, the subject has elevated Tie1
protein or mRNA levels.
[0289] The invention also provides polypeptides and nucleic acids
that encompass a range of amino acid and nucleic acid sequences,
e.g., sequences described herein or sequences related to those
described herein. For example, the invention features nucleic acids
that encodes each of the polypeptides described herein. The nucleic
acid can include the cognate codons or any set of codons that can
be translated to produce the respective polypeptide. Such
polypeptides include individual subunits of a multi-chain protein,
e.g., an antibody that includes a plurality of different
polypeptide chains. The nucleic acid may also be a nucleic acid
fragment or vector that is not expressed, but includes a sequence
encoding at least a part of an immunoglobulin variable region
(e.g., including a CDR described herein) or a complement thereof.
Such nucleic acids can be used to prepare useful constructs, cells,
and proteins. In addition, the invention features a host cell that
includes a nucleic acid described herein. The cell can express a
protein described herein, e.g., on its surface. The invention also
includes are proteins that include an amino acid sequence encoded
by a nucleic acid described herein or that hybridize to a nucleic
acid described herein.
[0290] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable domain or
immunoglobulin variable domain sequence. For example, an antibody
can include a heavy (H) chain variable region (abbreviated herein
as VH), and a light (L) chain variable region (abbreviated herein
as VL). In another example, an antibody includes two heavy (H)
chain variable regions and two light (L) chain variable regions.
The term "antibody" encompasses antigen-binding fragments of
antibodies (e.g., single chain antibodies, Fab fragments,
F(ab').sub.2, a Fd fragment, a Fv fragments, and dAb fragments) as
well as complete antibodies.
[0291] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
(CDR), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the framework region and
CDRs has been precisely defined (see, Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
Kabat definitions are used herein. Each VH and VL is typically
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0292] An "immunoglobulin domain" refers to a domain from the
variable or constant domain of immunoglobulin molecules.
Immunoglobulin domains typically contain two .beta.-sheets formed
of about seven .beta.-strands, and a conserved disulphide bond
(see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol.
6:381-405). The canonical structures of hypervariable loops of an
immunoglobulin variable can be inferred from its sequence, as
described in Chothia et al. (1992) J. Mol. Biol. 227:799-817;
Tomlinson et al. (1992) J. Mol. Biol. 227:776-798); and Tomlinson
et al. (1995) EMBO J. 14(18):4628-38.
[0293] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence which can form the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
omit one, two or more N- or C-terminal amino acids, internal amino
acids, may include one or more insertions or additional terminal
amino acids, or may include other alterations. In one embodiment, a
polypeptide that includes immunoglobulin variable domain sequence
can associate with another immunoglobulin variable domain sequence
to form a target binding structure (or "antigen binding site"),
e.g., a structure that interacts with Tie1, e.g., binds to or
inhibits Tie1.
[0294] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains, wherein the heavy and
light immunoglobulin chains are inter-connected by, e.g., disulfide
bonds. The heavy chain constant region includes three domains, CH1,
CH2 and CH3. The light chain constant region includes a CL domain.
The variable region of the heavy and light chains contains a
binding domain that interacts with an antigen. The constant regions
of the antibodies typically mediate the binding of the antibody to
host tissues or factors, including various cells of the immune
system (e.g., effector cells) and the first component (Clq) of the
classical complement system. The term "antibody" includes intact
immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as
subtypes thereof). The light chains of the immunoglobulin may be of
types kappa or lambda. In one embodiment, the antibody is
glycosylated. An antibody can be functional for antibody-dependent
cytotoxicity and/or complement-mediated cytotoxicity.
[0295] One or more regions of an antibody can be human or
effectively human. For example, one or more of the variable regions
can be human or effectively human. For example, one or more of the
CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC
CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC
CDR3 can be human. One or more of the framework regions can be
human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one
embodiment, all the framework regions are human, e.g., derived from
a human somatic cell, e.g., a hematopoietic cell that produces
immunoglobulins or a non-hematopoietic cell. In one embodiment, the
human sequences are germline sequences, e.g., encoded by a germline
nucleic acid. One or more of the constant regions can be human or
effectively human. In another embodiment, at least 70, 75, 80, 85,
90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, and
FR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or the
entire antibody can be human or effectively human. For example,
FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90,
92, 95, 98, or 99% identical to a human sequence encoded by a human
germline V segment of a locus encoding a light or heavy chain
sequence.
[0296] All or part of an antibody can be encoded by an
immunoglobulin gene or a segment thereof. Exemplary human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin light chains
(about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin heavy chains (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g.,
gamma (encoding about 330 amino acids). A light chain refers to any
polypeptide that includes a light chain variable domain. A heavy
chain refers to any polypeptide that a heavy chain variable
domain.
[0297] The term "antigen-binding fragment" of a full-length
antibody (or simply "antibody portion," or "fragment"), as used
herein, refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a target of
interest. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of a full length antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
including two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR) that retains
functionality. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules known as single
chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883.
[0298] Antibody fragments can be obtained using any appropriate
technique including conventional techniques known to those with
skill in the art. The term "monospecific antibody" refers to an
antibody that displays a single binding specificity and affinity
for a particular target, e.g., epitope. This term includes a
"monoclonal antibody" or "monoclonal antibody composition," which
as used herein refer to a preparation of antibodies or fragments
thereof of single molecular composition. As used herein, "isotype"
refers to the antibody class (e.g., IgM or IgG1) that is encoded by
heavy chain constant region genes.
[0299] In one embodiment, the HC or LC of an antibody includes
sequences that correspond to an amino acid sequence encoded by a
human germline sequence, e.g., the framework regions and/or in the
CDRs. For example, the antibody can include sequences from the
human DP47 antibody. In one embodiment, one or more codons for the
antibody are altered relative to the germline nucleic acid
sequence, but are chosen to encode the same amino acid sequence.
Codons can be selected, e.g., to optimize expression in a
particular system, create restriction enzyme sites, create a silent
fingerprint, etc.
[0300] In one embodiment, CDR2 of the antibody HC includes at least
11, 12, 13, 14, or 15 amino acid positions that are identical to
the amino acids found in CDR2 of DP47.
[0301] A "humanized" immunoglobulin variable region is an
immunoglobulin variable region that includes sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. Descriptions of "humanized" immunoglobulins include, for
example, U.S. Pat. No. 6,407,213 and U.S. Pat. No. 5,693,762.
[0302] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. An "effectively human" antibody is an antibody that includes
a sufficient number of human amino acid positions such that the
antibody does not elicit an immunogenic response in a normal
human.
[0303] As used herein, "Tie complex" refers to a heteromeric
complex that includes Tie1, Tie2, and an angiopoietin (Ang). The
Tie complex is formed in part by association of the extracellular
domains of Tie1 and Tie2 and also includes Ang. As used herein,
"complex members" refers to the proteins that are included in a
heteromeric Tie complex. Accordingly, Tie1, Tie2, and Ang are all
complex members. The term "Ang" includes all angiopoietins, such as
Ang1, Ang2, Ang3, and Ang4. The heteromeric Tie complex can include
other proteins in addition to Tie1, Tie2, and Ang. A protein or
ligand that antagonizes complex formation inhibits or decreases the
association of Tie1, Tie2, or Ang with at least one other member of
the complex and thereby decreases Tie2 signaling and downstream
effects such as angiogenesis. Angiogenesis includes all stages of
vessel development (e.g., blood or lymphatic vessel development),
including initial vessel formation and later vessel remodeling and
morphological changes.
[0304] As used herein, the terms "agonist" and "antagonist"
describe properties in context of a particular activity or effect.
For example, the E3 or E3b antibody can be an agonist in the
context of promoting Tie1 self-association (e.g.,
homodimerization), yet an antagonist in the context of decreasing
or inhibiting Tie complex formation and tube formation by HUVECs.
Likewise, an agent that is an agonist in the context of a Tie1
signaling pathway can be an antagonist in the context of
endothelial cell sprouting, splitting, and tube formation.
[0305] The term "Tie1 ectodomain" refers to an extracellular region
of a Tie1 protein, e.g., a region that includes about amino acids
25-759 of SEQ ID NO:2. Other exemplary regions are regions that
include one or more EGF-like domains (e.g., 214-256, 258-303,
303-345, 214-303, 258-345, or 214-345 of SEQ ID NO:2); one or more
Ig-Like C2-type domains (e.g., 43-105, 43-426, 372-426); one or
more Fibronectin Type III repeats (e.g., 446-540, 543-639, 643-744,
446-639, 543-744, or 446-744 of SEQ ID NO:2); and combinations
thereof. The terms "first Ig-like C2-type domain" and "Ig 1" refer
to the immunoglobulin-like domain in Tie1 or Tie2 that is located
closest to the amino terminus of the protein relative to the other
Ig-like C2-type domain (the second such domain). For example, for
Tie1, the first Immunoglobulin-like C2-type domain is located at
about residue 43 to about residue 105 and the second Ig-like
C2-type domain is located at about residue 372 to about residue
426.
[0306] As used herein, "binding affinity" refers to the apparent
association constant or K.sub.a. The K.sub.a is the reciprocal of
the dissociation constant (K.sub.d). A ligand may, for example,
have a binding affinity of at least 10.sup.5, 10.sup.6, 10.sup.7 or
10.sup.8 M.sup.-1 for a particular target molecule. Higher affinity
binding of a ligand to a first target relative to a second target
can be indicated by a higher K.sub.a (or a smaller numerical value
K.sub.d) for binding the first target than the K.sub.a (or
numerical value K.sub.d) for binding the second target. In such
cases the ligand has specificity for the first target relative to
the second target. Differences in binding affinity (e.g., for
specificity or other comparisons) can be at least 1.5, 2, 5, 10,
50, 100, or 1000-fold. For example, a Tie1 -binding protein may
preferentially bind to Tie1 at least 1.5, 2, 5, 10, 50, 100, or
1000-fold better than to another antigen, e.g., Tie2, EGF,
fibronectin, or human serum albumin. A Tie1-binding protein may
also be species-specific or species-general (e.g., can bind to a
Tie1 protein from more than one species).
[0307] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, surface plasmon resonance, or spectroscopy
(e.g., using a fluorescence assay). These techniques can be used to
measure the concentration of bound and free ligand as a function of
ligand (or target) concentration. The concentration of bound ligand
([Bound]) is related to the concentration of free ligand ([Free])
and the concentration of binding sites for the ligand on the target
where (N) is the number of binding sites per target molecule by the
following equation:
[Bound]=N[Free]/((1/Ka)+[Free])
[0308] Although quantitative measurements of Ka are routine, it is
not always necessary to make an exact determination of K.sub.a,
though, since sometimes it is sufficient to obtain a qualitative
measurement of affinity, e.g., determined using a method such as
ELISA or FACS analysis, is proportional to K.sub.a, and thus can be
used for comparisons, such as determining whether a higher affinity
is, e.g., 2, 5, 10, 20, or 50 fold higher than a reference. Binding
affinity is typically evaluated in 0.01 M HEPES pH 7.4, 0.15 M
NaCl, 3 mM EDTA and 0.005% (v/v) surfactant P20.
[0309] An "isolated composition" refers to a composition that is
removed from at least 90% of at least one component of a natural
sample from which the isolated composition can be obtained.
Compositions produced artificially or naturally can be
"compositions of at least" a certain degree of purity if the
species or population of species of interests is at least 5, 10,
25, 50, 75, 80, 90, 95, 98, or 99% pure on a weight-weight
basis.
[0310] An "epitope" refers to the site on a target compound that is
bound by a ligand, e.g., an antigen-binding protein (e.g., a Fab or
antibody). In the case where the target compound is a protein, for
example, an epitope may refer to the amino acids that are bound by
the ligand. Overlapping epitopes include at least one common amino
acid residue.
[0311] As used herein, the term "substantially identical" (or
"substantially homologous") is used herein to refer to a first
amino acid or nucleotide sequence that contains a sufficient number
of identical or equivalent (e.g., with a similar side chain, e.g.,
conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have
similar activities. In the case of antibodies, the second antibody
has the same specificity and has at least 50% of the affinity of
the same.
[0312] Sequences similar or homologous (e.g., at least about 85%
sequence identity) to the sequences disclosed herein are also part
of this application. In some embodiment, the sequence identity can
be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher. Alternatively, substantial identity exists when the nucleic
acid segments will hybridize under selective hybridization
conditions (e.g., highly stringent hybridization conditions), to
the complement of the strand. The nucleic acids may be present in
whole cells, in a cell lysate, or in a partially purified or
substantially pure form.
[0313] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In a preferred embodiment, the length of
a reference sequence aligned for comparison purposes is at least
30%, preferably at least 40%, more preferably at least 50%, even
more preferably at least 60%, and even more preferably at least
70%, 80%, 90%, 100% of the length of the reference sequence. The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0314] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a sequence identity or
homology limitation described herein) are a Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0315] As used herein, the term "homologous" is synonymous with
"similarity" and means that a sequence of interest differs from a
reference sequence by the presence of one or more amino acid
substitutions (although modest amino acid insertions or deletions)
may also be present. Presently preferred means of calculating
degrees of homology or similarity to a reference sequence are
through the use of BLAST algorithms (available from the National
Center of Biotechnology Information (NCBI), National Institutes of
Health, Bethesda Md.), in each case, using the algorithm default or
recommended parameters for determining significance of calculated
sequence relatedness. The percent identity between two amino acid
or nucleotide sequences can also be determined using the algorithm
of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0316] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described
in that reference and either can be used. Specific hybridization
conditions referred to herein are as follows: 1) low stringency
hybridization conditions in 6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); 2) medium stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C.; and 4) very high stringency hybridization conditions
are 0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one
or more washes at 0.2.times.SSC, 1% SDS at 65.degree. C.
[0317] It is understood that the proteins described herein may have
mutations relative to a particular protein described herein (e.g.,
a conservative or non-essential amino acid substitutions), which do
not have a substantial effect on function. Whether or not a
particular substitution will be tolerated, i.e., will not adversely
affect desired biological properties, such as binding activity can
be determined as described in Bowie, et al. (1990) Science
247:1306-1310. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). It is
possible, for example, for framework and CDR amino acid residues to
include one or more conservative substitutions.
[0318] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of the binding agent, e.g.,
the antibody, without abolishing or more preferably, without
substantially altering a biological activity, whereas an
"essential" amino acid residue results in such a change.
[0319] Generally, where "X" is used to represent an amino acid
residue, any amino acid (e.g., any of the twenty naturally
occurring amino acids) can be used at that position, or at least a
subset thereof (e.g., any of the nineteen non-cysteine amino
acids).
[0320] The terms "polypeptide" or "peptide" (which may be used
interchangeably) refer to a polymer of three or more amino acids
linked by a peptide bond, e.g., between 3 and 30, 12 and 60, or 30
and 300, or over 300 amino acids in length. The polypeptide may
include one or more unnatural amino acids. Typically, the
polypeptide includes only natural amino acids. A "protein" can
include one or more polypeptide chains. Accordingly, the term
"protein" encompasses polypeptides. A protein or polypeptide can
also include one or more modifications, e.g., a glycosylation,
amidation, phosphorylation, and so forth. The term "small peptide"
can be used to describe a polypeptide that is between 3 and 30
amino acids in length, e.g., between 8 and 24 amino acids in
length.
[0321] Statistical significance can be determined by any art known
method. Exemplary statistical tests include: the Students T-test,
Mann Whitney U non-parametric test, and Wilcoxon non-parametric
statistical test. Some statistically significant relationships have
a P value of less than 0.05, or 0.02. Particular ligands may show a
difference, e.g., in specificity or binding, that are statistically
significant (e.g., P value <0.05 or 0.02).
[0322] Other features and advantages of the instant invention will
become more apparent from the following detailed description and
claims. Embodiments of the invention can include any combination of
features described herein. In no case does the term "embodiment"
necessarily exclude one or more other features disclosed herein,
e.g., in another embodiment. The contents of all references, patent
applications and patents, cited throughout this application are
hereby expressly incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0323] FIG. 1 illustrates a bivariant FACS plot showing labelling
with the platelet specific marker CD42 with Tie1 and labelling with
the E3 antibody. Only a background number of CD42 positive cells
are labeled by the E3 antibody.
[0324] FIGS. 2A, 2B, 2C, and 2D are plots of the number of
branching points versus antibody concentration comparing germlined
E3 (2C and 2D) with parental E3 (2A and 2B).
[0325] FIG. 3 depicts a graph of blood vessel density in matrigels
that were stained with fluorescein-lectin from an in vivo assay
using MATRIGEL.TM. and evaluating the germlined E3 antibody.
[0326] FIG. 4 depicts results of tube formation in HUVECs using the
parental E3 and E3b (germlined) proteins.
[0327] FIG. 5 depicts graphically the results from animal studies
in which nu/nu mice were implanted with SW-480 colorectal cancer
cells and treated with DX-2220 (10 mg/kg), cisplatin (4 mg/kg), or
a control. Control conditions were: no treatment, PBS vehicle
alone, or a non-specific, isotype-matched IgG1 antibody (A2-SV) (10
mg/kg). Tumor weight is plotted on the y axis; days after tumor
cell injection is plotted on the x axis.
[0328] FIG. 6 depicts graphically the results from animal studies
in which nu/nu mice were implanted with LNM35 lung cancer cells and
treated with DX-2220 (20 mg/kg) or a non-specific, isotype-matched
IgG1 antibody (A2-SV) (20 mg/kg). Tumor volume (mm.sup.3) is
plotted on the y axis; days after tumor cell injection is plotted
on the x axis.
[0329] FIGS. 7A and 7B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-A1, respectively.
[0330] FIGS. 8A and 8B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-A5, respectively.
[0331] FIGS. 9A and 9B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-A6, respectively.
[0332] FIGS. 10A and 10B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-Al 0, respectively.
[0333] FIGS. 11A and 11B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-B1, respectively.
[0334] FIGS. 12A and 12B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-B3, respectively.
[0335] FIGS. 13A and 13B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-C6, respectively.
[0336] FIGS. 14A and 14B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-D6, respectively.
[0337] FIGS. 15A and 15B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-D10, respectively.
[0338] FIGS. 16A and 16B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-D12, respectively.
[0339] FIGS. 17A and 17B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-F3, respectively.
[0340] FIGS. 18A and 18B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-F4, respectively.
[0341] FIGS. 19A and 19B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-G3, respectively.
[0342] FIGS. 20A and 20B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-A2, respectively.
[0343] FIGS. 21A and 21B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-Al 0, respectively.
[0344] FIGS. 22A and 22B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-B2, respectively.
[0345] FIGS. 23A and 23B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-B9, respectively.
[0346] FIGS. 24A and 24B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-C2, respectively.
[0347] FIGS. 25A and 25B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-C7, respectively.
[0348] FIGS. 26A and 26B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-C10, respectively.
[0349] FIGS. 27A and 27B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-D11, respectively.
[0350] FIGS. 28A and 28B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-E11, respectively.
[0351] FIGS. 29A and 29B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-G4, respectively.
[0352] FIG. 30 lists the amino acid sequence of the light chain
variable domain of clone s-G9.
[0353] FIGS. 31A and 31B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-G10, respectively.
[0354] FIGS. 32A and 32B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-H1, respectively.
[0355] FIGS. 33A and 33B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
s-H4, respectively.
[0356] FIGS. 34A and 34B list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
G2, respectively.
[0357] FIGS. 35 and 36 list the amino acid sequence of the heavy
chain variable domain and the light chain variable domain of clone
p-A1, respectively.
[0358] FIG. 37 provides Table 5, a summary of heavy chain
sequences.
[0359] FIG. 38 provides Table 6, a summary of light chain
sequences.
[0360] FIG. 39 provides Table 9, characteristics of some exemplary
Tie1 binding antibodies.
DETAILED DESCRIPTION
[0361] This disclosure provides, inter alia, agents (also referred
to as binding proteins and ligands) that bind to components of a
Tie complex, e.g., Tie1, Tie2, and Ang. Examples of such agents
include proteins, for example, a small peptide (e.g., a cyclic or
linear peptide, e.g., of between 7 and 25 amino acids), a
polypeptide (e.g., a polypeptide of at least 20 amino acids), or a
multi-chain protein (e.g., including at least two peptides or
polypeptides). An example of a multi-chain protein is an IgG
full-length antibody that has separate heavy and light chains. An
example of a polypeptide is a single chain antibody.
[0362] Agents can be selected that have particular properties,
e.g., ability to antagonize Tie1/Tie2/Ang complex formation,
ability to promote Tie1 homodimerization, and ability to promote
Tie1 phosphorylation. For example, agents that bind to Tie1, Tie2,
or Ang can be tested for their ability to antagonize formation of
heteromeric Tie complexes. Antagonism of this complex decreases
Tie2 signaling and its downstream effects, such as promoting
angiogenesis.
[0363] Tie1 is a receptor tyrosine kinase protein that includes a
transmembrane domain. Tie1 is present almost exclusively on
endothelial cells. Accordingly, a Tie1-binding protein can be used,
e.g., to specifically recognize or target an endothelial cell. Some
Tie1-binding proteins can also be used to agonize or antagonize
endothelial cells. In some embodiments, these Tie1-binding proteins
have an affinity for particular structural features (e.g., a
feature listed below), a combination of features listed below,
and/or an epitope that includes at least one amino acid in a
structural feature listed below (The sequence is relative to the
amino acid sequence provided in SEQ ID NO:2, Example 1, below):
TABLE-US-00001 Key From To Length Description SIGNAL 1 24 24
POTENTIAL. CHAIN 25 1138 1114 TYROSINE-PROTEIN KINASE RECEPTOR
TIE1. DOMAIN 25 759 735 EXTRACELLULAR (POTENTIAL). TRANSMEM 760 784
25 POTENTIAL. DOMAIN 785 1138 354 CYTOPLASMIC (POTENTIAL). DOMAIN
43 105 63 IG-LIKE C2-TYPE 1. DOMAIN 214 256 43 EGF-LIKE 1. DOMAIN
258 303 46 EGF-LIKE 2. DOMAIN 305 345 41 EGF-LIKE 3. DOMAIN 372 426
55 IG-LIKE C2-TYPE 2. DOMAIN 446 540 95 FIBRONECTIN TYPE-III 1.
DOMAIN 543 639 97 FIBRONECTIN TYPE-III 2. DOMAIN 643 744 102
FIBRONECTIN TYPE-III 3. DOMAIN 839 1118 280 PROTEIN KINASE. NP_BIND
845 853 9 ATP (BY SIMILARITY). BINDING 870 870 ATP (BY SIMILARITY).
ACT_SITE 979 979 BY SIMILARITY. CARBOHYD 83 83 N-LINKED (GLCNAC . .
. ) (POTENTIAL). CARBOHYD 161 161 N-LINKED (GLCNAC . . . )
(POTENTIAL). CARBOHYD 503 503 N-LINKED (GLCNAC . . . ) (POTENTIAL).
CARBOHYD 596 596 N-LINKED (GLCNAC . . . ) (POTENTIAL). CARBOHYD 709
709 N-LINKED (GLCNAC . . . ) (POTENTIAL). MOD_RES 1007 1007
PHOSPHORYLATION (AUTO-) (BY SIMILARITY).
[0364] Tie2 is a receptor tyrosine kinase protein that includes a
transmembrane domain. Tie2 is present almost exclusively on
endothelial cells. Accordingly, a Tie2-binding protein can be used,
e.g., to specifically recognize or target an endothelial cell. Some
Tie2-binding proteins can also be used to modulate (e.g., agonize
or antagonize) an activity of an endothelial cell. In some
embodiments, these Tie2-binding proteins have an affinity for
particular structural features, a combination of features, and/or
an epitope that includes at least one amino acid in a structural
feature. Exemplary structural features of Tie2 include: two Ig-like
domains, three EGF-like domains, and three fibronectin type III
domains.
[0365] The angiopoietins are a family of ligands that bind to Tie2.
Some Ang-binding proteins (e.g., antibodies or artificial
Ang-binding proteins) can be used to agonize or antagonize
endothelial cells. In some embodiments, these Ang-binding proteins
have an affinity for particular structural features, a combination
of features, and/or an epitope that includes at least one amino
acid in a structural feature. Exemplary structural features
include: the N-terminal region of about 50 amino acids, the
coiled-coil domain, or the fibrinogen-like domain.
[0366] Examples of Ang-binding proteins include proteins that
inhibit Ang multimerization (e.g., ability of Ang proteins to form
tetramers), proteins that inhibit Ang-Tie2 interactions, and
proteins that inhibit a ternary complex of Tie1-Tie2-Ang.
Inhibitory proteins can function by disrupting existing
interactions or by preventing interactions from occurring.
[0367] Tie1 and Tie2 can associate through their extracellular
domains and form a heteromeric complex with an angiopoietin (Ang),
such as Ang1, Ang2, Ang3, and Ang4. This heteromeric complex
activates the intracellular signaling cascade mediated by Tie2.
Thus, antagonizing formation of this heteromeric complex provides a
novel approach to inhibiting Tie2 signaling and its downstream
effects, such as angiogenesis. Complex formation can be antagonized
by proteins that bind to the extracellular domains of Tie1 or Tie2
or that bind to Ang so as to prevent its recruitment into the
complex or to prevent its multimerization.
[0368] One method for identifying proteins that bind to Tie1
includes: providing a library and selecting from the library one or
more members that encode a protein that binds to the Tie1 antigen
or a fragment thereof (e.g., the extracellular domain, an EGF
domain, a fibronectin repeat, or an Ig-superfamily domain (e.g., a
Ig-like C2-type 2 domain)). The selection can be performed in a
number of ways. For example, the library can be a display library.
The Tie1 can be tagged and recombinantly expressed. The Tie1 is
purified and attached to a support, e.g., to affinity beads, or
paramagnetic beads or other magnetically responsive particles. The
Tie1 can also be expressed on the surface of a cell. Members of the
display library that specifically bind to the cell, e.g., only if
the Tie1 is activated, can be selected. Analogous procedures can be
performed to identify proteins that bind to Tie2 or a fragment
thereof (e.g., the extracellular domain, an EGF domain, a
fibronectin repeat, or an Ig-superfamily domain (e.g., a Ig-like
C2-type 2 domain)). Analogous procedures can also be performed to
identify proteins that bind to Ang or a fragment thereof (e.g., the
N-terminal domain, the coiled-coil domain, or the fibrinogen-like
domain).
[0369] Proteins identified as being capable of binding a Tie
complex member can be tested for their ability to antagonize
heteromeric complex formation, ability to promote Tie1
phosphorylatoin, and/or ability to promote Tie1 homodimerization,
as described in the examples below. Proteins identified as
antagonizing formation of the heteromeric complex can be used in
pharmaceutical compositions to treat a subject in need of such
treatment, for example, a subject with an angiogenesis-dependent
cancer or tumor or other angiogenesis-related disorders.
[0370] Exemplary Tie1 Modulators
[0371] In one embodiment, a Tie1-binding protein can modulate a
Tie1 activity. For example, a Tie1 -binding protein can function as
a Tie1 agonist or antagonist in the Tie1/EpoR chimeric BaF3 cell
assay described in Example 2. Tie1 agonists in this Tie1/EpoR
chimeric BaF3 cell assay can stimulate certain activity of an
endothelial cell under particular conditions, e.g., the conditions
of the Tie1/EpoR chimeric BaF3 cell assay.
[0372] Some Tie1 binding proteins increase phosphatidyl inositol
3-kinase (PI3 kinase) activity in an endothelial cell and/or Akt
kinase activity. Kontos et al. suggest that the cytoplasmic domain
of Tie1 can associate with the p85 subunit of PI3 kinase and
activate PI3 kinase activity. Kontos et al. (2002) Mol. Cell Biol.
22:1704-1713. The Tie1 cytoplasmic domain may also associate with a
protein tyrosine phosphatase Shpt. See, e.g., Marron et al. (2000)
Adv. Exp. Med. Biol. 476:35-46.
[0373] Some Tie binding proteins may increase dimerization, and/or
tyrosine phosphorylation (e.g., as a result of
auto-phosphorylation) of the Tie1 cytoplasmic domain, e.g., the
tyrosine in the motif YVN at about amino acid 1117.
[0374] Tie1-binding protein can be evaluated in a cell assay (e.g.,
in the Tie1/EpoR chimeric BaF3 cell assay as described below in
Example 2). An exemplary cell assay uses a growth factor dependent
cell in which a chimeric receptor that includes the Tie1 ectodomain
fused to the intracellular domain of the growth factor receptor is
expressed. Cells are evaluated for ability to grow in the absence
of the essential growth factor, but in the presence of a test
compound, e.g., a Tie1-binding protein. If the Tie1-binding protein
agonizes Tie1 in the Tie1/EpoR chimeric BaF3 cell assay, a
signalling activity of the Tie1 chimera can substitute for
stimulation by the required growth factor thorough its cognate
receptor. Thus, survival of the cell in the absence of the required
growth factor can be used as an indication that the Tie1-binding
protein interacts with the Tie1 ectodomain.
[0375] Tie1 agonists in the Tie1/EpoR chimeric BaF3 cell assay may
behave as inhibitors of Tie1 activity under other conditions, e.g.,
in vivo, and, irrespective of in vitro properties, may be useful as
inhibitors of angiogenesis in vivo.
[0376] Tie1 binding proteins can be used, e.g., to reduce an
activity of an endothelial cell. For example, some Tie1 binding
proteins can be used to decrease phosphatidyl inositol 3-kinase
(PI3 kinase) activity in an endothelial cell, Shp2 activity, and/or
Akt kinase activity. Some Tie1 binding proteins may also reduce
dimerization, and/or tyrosine phosphorylation (e.g., as a result of
auto-phosphorylation) of the Tie1 cytoplasmic domain, e.g., the
tyrosine in the motif YVN at about amino acid 1117.
[0377] Tie1 -binding protein can be evaluated for activity in a
cell assay. For example, the binding protein can be assayed for
ability to prevent another ligand, e.g., the E3 antibody, from
modulating a Tie1 activity in a cell assay described herein (e.g.,
the Tie1/EpoR chimeric BaF3 cell assay as described below in
Example 2).
[0378] Display Libraries
[0379] A number of methods can be used to identify proteins that
bind to Tie1, Tie2, Ang, fragments thereof, complexes that include
one or more of these proteins or fragments thereof. In one
embodiment, a display library is used to identify such proteins. A
display library is a collection of entities; each entity includes
an accessible protein component and a recoverable component that
encodes or identifies the protein component. The protein component
can be of any length, e.g. from three amino acids to over 300 amino
acids. In a selection, the protein component of each member of the
library is probed with a target, e.g., Tie1 protein, and if the
protein component binds to the target, the display library member
is identified, e.g., by retention on a support. The method can be
adapted for other targets, such as Tie2, Ang, fragments thereof,
complexes that include one or more of these proteins or fragments
thereof.
[0380] Retained display library members are recovered from the
support and analyzed. The analysis can include amplification and a
subsequent selection under similar or dissimilar conditions. For
example, positive and negative selections can be alternated. The
analysis can also include determining the amino acid sequence of
the protein component and purification of the protein component for
detailed characterization. A variety of formats can be used for
display libraries. Examples include the following.
[0381] Phage Display. One format utilizes viruses, particularly
bacteriophages. This format is termed "phage display." The protein
component is typically covalently linked to a bacteriophage coat
protein. The linkage results form translation of a nucleic acid
encoding the protein component fused to the coat protein. The
linkage can include a flexible peptide linker, a protease site, or
an amino acid incorporated as a result of suppression of a stop
codon. Phage display is described, for example, in Ladner et al.,
U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO
92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO
92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol.
Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology
4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et
al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins
et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al.
(1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods
Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0382] Phage display systems have been developed for filamentous
phage (phage fl, fd, and M13) as well as other bacteriophage (e.g.
T7 bacteriophage and lambdoid phages; see, e.g., Santini (1998) J.
Mol. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6;
Houshmet al. (1999) Anal Biochem 268:363-370). The filamentous
phage display systems typically use fusions to a minor coat
protein, such as gene III protein, and gene VIII protein, a major
coat protein, but fusions to other coat proteins such as gene VI
protein, gene VII protein, gene IX protein, or domains thereof can
also been used (see, e.g., WO 00/71694). In one embodiment, the
fusion is to a domain of the gene III protein, e.g., the anchor
domain or "stump," (see, e.g., U.S. Pat. No. 5,658,727 for a
description of the gene III protein anchor domain). It is also
possible to physically associate the protein being displayed to the
coat using a non-peptide linkage, e.g., a non-covalent bond or a
non-peptide covalent bond. For example, a disulfide bond and/or
c-fos and c-jun coiled-coils can be used for physical associations
(see, e.g., Crameri et al. (1993) Gene 137:69 and WO 01/05950).
[0383] Bacteriophage displaying the protein component can be grown
and harvested using standard phage preparatory methods, e.g., PEG
precipitation from growth media. After selection of individual
display phages, the nucleic acid encoding the selected protein
components, by infecting cells using the selected phages.
Individual colonies or plaques can be picked, the nucleic acid
isolated and sequenced.
[0384] Cell-based Display. In still another format the library is a
cell-display library. Proteins are displayed on the surface of a
cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic
cells include E. coli cells, B. subtilis cells, and spores (see,
e.g., Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryotic
cells include yeast (e.g., Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Hanseula, or Pichia pastoris). Yeast
surface display is described, e.g., in Boder and Wittrup (1997)
Nat. Biotechnol. 15:553-557 and WO 03/029456, which describes a
yeast display system that can be used to display immunoglobulin
proteins such as Fab fragments and the use of mating to generate
combinations of heavy and light chains.
[0385] Ribosome Display. RNA and the polypeptide encoded by the RNA
can be physically associated by stabilizing ribosomes that are
translating the RNA and have the nascent polypeptide still
attached. Typically, high divalent Mg.sup.2+ concentrations and low
temperature are used. See, e.g., Mattheakis et al. (1994) Proc.
Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat
Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol.
328:404-30; and Schaffitzel et al. (1999) J Immunol Methods.
231(1-2):119-35.
[0386] Polypeptide-Nucleic Acid Fusions. Another format utilizes
polypeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions
can be generated by the in vitro translation of mRNA that include a
covalently attached puromycin group, e.g., as described in Roberts
and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302, and
U.S. Pat. No. 6,207,446. The mRNA can then be reverse transcribed
into DNA and crosslinked to the polypeptide.
[0387] Other Display Formats. Yet another display format is a
non-biological display in which the protein component is attached
to a non-nucleic acid tag that identifies the polypeptide. For
example, the tag can be a chemical tag attached to a bead that
displays the polypeptide or a radiofrequency tag (see, e.g., U.S.
Pat. No. 5,874,214).
[0388] Display technology can also be used to obtain binding
proteins, e.g., antibodies that interact with particular epitopes
of a target. This can be done, for example, by using competing
non-target molecules that lack the particular epitope or are
mutated within the epitope, e.g., with alanine. Such non-target
molecules can be used in a negative selection procedure as
described below, as competing molecules when binding a display
library to the target, or as a pre-elution agent, e.g., to capture
in a wash solution dissociating display library members that are
not specific to the target.
[0389] Iterative Selection. In one preferred embodiment, display
library technology is used in an iterative mode. A first display
library is used to identify one or more binding proteins for a
target. These proteins are then varied, e.g., using a mutagenesis
method, to form a second display library. Higher affinity binding
proteins are then selected from the second library, e.g., by using
higher stringency or more competitive binding and washing
conditions.
[0390] In some implementations, the mutagenesis is targeted to
regions known or likely to be at the binding interface. If, for
example, the identified binding proteins are antibodies, then
mutagenesis can be directed to the CDR regions of the heavy or
light chains as described herein. Further, mutagenesis can be
directed to framework regions near or adjacent to the CDRs, e.g.,
framework regions, particular within ten, five, or three amino
acids of a CDR junction. In the case of antibodies, mutagenesis can
also be limited to one or a few of the CDRs, e.g., to make precise
step-wise improvements.
[0391] Some exemplary mutagenesis techniques include: error-prone
PCR (Leung et al. (1989) Technique 1:11-15), recombination (see,
e.g., U.S. Ser. No. 10/279,633), DNA shuffling using random
cleavage (Stemmer (1994) Nature 389-391; termed "nucleic acid
shuffling"), RACHITT.TM. (Coco et al. (2001) Nature Biotech.
19:354), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids
Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991)
Methods Enzymol. 208:564-586) and incorporation of degenerate
oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245).
[0392] In one example of iterative selection, the methods described
herein are used to first identify a binding protein from a display
library that binds a Tie1 with at least a minimal binding
specificity for a target or a minimal activity, e.g., an
equilibrium dissociation constant for binding of greater than 1 nM,
10 nM, or 100 nM. The nucleic acid sequence encoding the initial
identified binding protein is used as a template nucleic acid for
the introduction of variations, e.g., to identify a second binding
protein that has enhanced properties (e.g., binding affinity,
kinetics, or stability) relative to the initial binding
protein.
[0393] Off-Rate Selection. Since a slow dissociation rate can be
predictive of high affinity, particularly with respect to
interactions between polypeptides and their targets, the methods
described herein can be used to isolate binding proteins with a
desired kinetic dissociation rate (i.e. reduced) for a binding
interaction to a target.
[0394] To select for slow dissociating binding proteins from a
display library, the library is contacted to an immobilized target.
The immobilized target is then washed with a first solution that
removes non-specifically or weakly bound biomolecules. Then the
immobilized target is eluted with a second solution that includes a
saturation amount of free target, i.e., replicates of the target
that are not attached to the particle. The free target binds to
biomolecules that dissociate from the target. Rebinding is
effectively prevented by the saturating amount of free target
relative to the much lower concentration of immobilized target.
[0395] The second solution can have solution conditions that are
substantially physiological or that are stringent. Typically, the
solution conditions of the second solution are identical to the
solution conditions of the first solution. Fractions of the second
solution are collected in temporal order to distinguish early from
late fractions. Later fractions include biomolecules that
dissociate at a slower rate from the target than biomolecules in
the early fractions. It is also possible to recover display library
members that remain bound to the target even after extended
incubation. These can either be dissociated using chaotropic
conditions or can be amplified while attached to the target. For
example, phage bound to the target can be contacted to bacterial
cells.
[0396] Selecting and Screening for Specificity. "Selection" refers
to a process in which many members of a display library are allowed
to contact the target and those that bind are recovered and
propagated. The selection can be from a library having numerous
members, e.g., more than 10.sup.10 members. "Screening" refers to a
process in which isolated members of the library are tested singly
for binding to the target. Through automation, thousands of
candidates may be screened in a highly parallel process. The
display library selection methods described herein can include a
selection process that discards display library members that bind
to a non-target molecule. Examples of non-target molecules include,
e.g., extracellular domains of molecules that include an
immunoglobulin super-family domain or an EGF domain and receptor
tyrosine kinases other than Tie1, e.g., Tie2, or other than Tie2,
e.g., Tie1, or other than Tie1 and Tie2. In one implementation, a
so-called "negative selection" step is used to discriminate between
the target and related non-target molecule and a related, but
distinct non-target molecules. The display library or a pool
thereof is contacted to the non-target molecule. Members of the
sample that do not bind the non-target are collected and used in
subsequent selections for binding to the target molecule or even
for subsequent negative selections. The negative selection step can
be prior to or after selecting library members that bind to the
target molecule.
[0397] In another implementation, a screening step is used. After
display library members are isolated for binding to the target
molecule, each isolated library member is tested for its ability to
bind to a non-target molecule (e.g., a non-target listed above).
For example, a high-throughput ELISA screen can be used to obtain
this data. The ELISA screen can also be used to obtain quantitative
data for binding of each library member to the target. The
non-target and target binding data are compared (e.g., using a
computer and software) to identify library members that
specifically bind to Tie1, Tie2, Ang, fragments thereof, or a
complex comprising one or more such components.
[0398] The display library selection and screening methods
described herein can include a selection or screening process that
selects for display library members that bind to specific sites on
the target molecule. For example, elution with high concentration
of an antibody described herein can be used to select for phage
that bind to an epitope that is near or overlaps with the epitope
bound by the antibody used for elution. Accordingly, one can screen
for a phage that binds to the E3-binding site of Tie1 by performing
ELISAs with and without E3 antibody in the buffer.
[0399] The following description provides one exemplary method for
identifying antibodies that bind to Tie1 using a phagemid Fab
library. For example, three rounds of selection can be performed
with decreasing amounts of target protein (e.g., 100, 50 and 50
.mu.g for first, second, and third rounds, respectively). The
target is immobilized on streptavidin coated magnetic beads
(Dynal). The library is depleted against streptavidin coated
magnetic beads prior to each round of selection and optionally
against an unrelated protein which may include a common
purification handle. For example, if the target is produced as a
fusion to a Fc domain, the library can be depleted against soluble
Trail-Fc (a commercially available Fc fusion protein). The
depletion process removes Fc binders.
[0400] Each round of selection can include, e.g., two cycles of
streptavidin magnetic bead depletion, a cycle of binding of phage
to Tie1-coated beads, ten cycles of washes, elution of bound phage,
and propagation of enriched phage for the next round. Phage bound
to Tie1 -coated beads after ten washes can be directly amplified or
eluted before amplification. After three rounds of selection,
individual clones may be grown in 96-well microtiter plates and
individually screened for Tie1 binding activity by phage ELISA.
ELISAs can include evaluations of binding to Tie1, specificity
controls, and unrelated controls. Isolates can be DNA fingerprinted
to determine the diversity emerging from the selection process. For
example, positive isolates can be PCR amplified with the
oligonucleotide primers M13-reverse and geneIII-forward (see, e.g.,
Marks et al. (1991), J. Mol. Biol. 222:581). The products can be
analyzed by BstNI fingerprinting.
[0401] An exemplary method for performing ELISA's with phage that
display a binding protein is as follows. Individual clones can be
grown and rescued as described previously (Marks et al. (1991), J.
Mol. Biol. 222:581). For ELISAs, 96-well Immulon 2 HB plates
(Thermo Labsystems) are coated with 1 .mu.g/well ImmunoPure.TM.
streptavidin (Pierce) in PBS and incubated overnight at 4.degree.
C. After three washes with PBS, 100 .mu.L of biotinylated Tie1
protein is allowed to bind to the immobilized streptavidin for
30-60 minutes at room temperature. Then, Tie1-coated wells are
blocked with 300 .mu.L of 2% milk/1.times.PBS/0.05% Tween (2%
MPBST) for two hours at 37.degree. C. The wells are incubated with
100 .mu.L of phage culture supernatant that had been blocked with
2% MPBST for one hour at room temperature. The wells are washed
five times with 1.times.PBS/Tween 0.1% (PBST), and incubated with
100 .mu.L of anti-M13-HRP secondary antibody at a 1:5,000 dilution
for one hour at room temperature. The wells are washed five times
with PBST before developing with TMB-solution and read at 630
nm.
[0402] For the cell ELISAs, cells are washed once in PBS and
resuspended at a concentration of 1.times.10.sup.6 to
2.times.10.sup.6 cells/mL of PBS. A final concentration of
1-2.times.10.sup.5 cells per well of a 96-well tissue culture plate
(Falcon, VWR) can be used. The cells are fixed by adding an equal
volume of 0.2% glutaraldehyde (Sigma-Aldrich) and incubating at
37.degree. C. for 12 minutes. They are then washed three times with
PBS using an automated plate washer (Bio-Tek Instruments, Inc.) and
blocked with 200 .mu.L of 2% MPBST for one hour at room
temperature. The rest of the ELISA procedure can be performed as
described above except that 1.times.PBS/Tween 0.05% is used for the
washes and incubations.
[0403] Germlining Antibodies
[0404] It is possible to modify an antibody that binds Tie1, Tie2,
or Ang, e.g., an antibody described herein, in order to make the
variable regions of the antibody more similar to one or more
germline sequences. For example, an antibody can include one, two,
three or more amino acid substitutions, e.g., in a framework or CDR
region, to make it more similar to a reference germline sequence.
One exemplary germlining method can include: identifying one or
more germline sequences that are similar (e.g., most similar in a
particular database) to the sequence of the isolated antibody. Then
mutations (at the amino acid level) can be made in the isolated
antibody, either incrementally, in combination, or both. For
example, a nucleic acid library that includes sequences encoding
some or all possible germline mutations is made. The mutated
antibodies are then evaluated, e.g., to identify an antibody that
has one or more additional germline residues relative to the
isolated antibody and that is still useful (e.g., has a functional
activity). In one embodiment, as many germline residues are
introduced into an isolated antibody as possible.
[0405] In one embodiment, mutagenesis is used to substitute or
insert one or more germline residues into a CDR region. For
example, the germline CDR residue can be from a germline sequence
that is similar (e.g., most similar) to the variable region being
modified. After mutagenesis, activity (e.g., binding or other
functional activity) of the antibody can be evaluated to determine
if the germline residue or residues are tolerated. Similar
mutagenesis can be performed in the framework regions.
[0406] Selecting a germline sequence can be performed in different
ways. For example, a germline sequence can be selected if it meets
a predetermined criteria for selectivity or similarity, e.g., at
least a certain percentage identity, e.g., at least 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The
selection can be performed using at least 2, 3, 5, or 10 germline
sequences. In the case of CDR1 and CDR2, identifying a similar
germline sequence can include selecting one such sequence. In the
case of CDR3, identifying a similar germline sequence can include
selecting one such sequence, but may including using two germline
sequences that separately contribute to the amino-terminal portion
and the carboxy-terminal portion. In other implementations more
than one or two germline sequences are used, e.g., to form a
consensus sequence.
[0407] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at at least 30, 40, 50, 60,
70, 80, 90, 95 or 100% of the CDR amino acid positions that are not
identical to residues in the reference CDR sequences, residues that
are identical to residues at corresponding positions in a human
germline sequence (i.e., an amino acid sequence encoded by a human
germline nucleic acid).
[0408] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at at least 30, 50, 60, 70,
80, 90 or 100% of the FR regions are identical to FR sequence from
a human germline sequence, e.g., a germline sequence related to the
reference variable domain sequence.
[0409] Accordingly, it is possible to isolate an antibody which has
similar activity to a given antibody of interest, but is more
similar to one or more germline sequences, particularly one or more
human germline sequences. For example, an antibody can be at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% identical to a
germline sequence in a region outside the CDRs (e.g., framework
regions). Further an antibody can include at least 1, 2, 3, 4, or 5
germline residues in a CDR region, the germline residue being from
a germline sequence of similar (e.g., most similar) to the variable
region being modified. Germline sequences of primary interest are
human germline sequences. The activity of the antibody (e.g., the
binding activity) can be within a factor or 100, 10, 5, 2, 0.5,
0.1, and 0.001 of the original antibody.
[0410] Exemplary germline reference sequences for Vkappa include:
O12/O2, O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23,
L9, L24, L11, L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11,
L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See, e.g.,
Tomlinson et al. (1995) EMBO J. 14(18):4628-3.
[0411] A germline reference sequence for the HC variable domain can
be based on a sequence that has particular canonical structures,
e.g., 1-3 structures in the H1 and H2 hypervariable loops. The
canonical structures of hypervariable loops of an immunoglobulin
variable domain can be inferred from its sequence, as described in
Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al.
(1992) J. Mol. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO
J. 14(18):4628-38. Exemplary sequences with a 1-3 structure
include: DP-1, DP-8, DP-12, DP-2, DP-25, DP-15, DP-7, DP-4, DP-31,
DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-46, DP-47,
DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54.
[0412] Diversity
[0413] Display libraries and other libraries include variation at
one or more positions in the displayed polypeptide. The variation
at a given position can be synthetic or natural. For some
libraries, both synthetic and natural diversity are included.
[0414] Synthetic Diversity. Libraries can include regions of
diverse nucleic acid sequence that originate from artificially
synthesized sequences. Typically, these are formed from degenerate
oligonucleotide populations that include a distribution of
nucleotides at each given position. The inclusion of a given
sequence is random with respect to the distribution. One example of
a degenerate source of synthetic diversity is an oligonucleotide
that includes NNN wherein N is any of the four nucleotides in equal
proportion.
[0415] Synthetic diversity can also be more constrained, e.g., to
limit the number of codons in a nucleic acid sequence at a given
trinucleotide to a distribution that is smaller than NNN. For
example, such a distribution can be constructed using less than
four nucleotides at some positions of the codon. In addition,
trinucleotide addition technology can be used to further constrain
the distribution. So-called "trinucleotide addition technology" is
described, e.g., in Wells et al. (1985) Gene 34:315-323, U.S. Pat.
No. 4,760,025 and U.S. Pat. No. 5,869,644.
[0416] Natural Diversity. Libraries can include regions of diverse
nucleic acid sequence that originate (or are synthesized based on)
from different naturally-occurring sequences. An example of natural
diversity that can be included in a display library is the sequence
diversity present in immune cells (see also below). Nucleic acids
are prepared from these immune cells and are manipulated into a
format for polypeptide display.
[0417] Antibody Display Libraries
[0418] In one embodiment, the display library presents a diverse
pool of proteins, each of which includes an immunoglobulin domain,
e.g., an immunoglobulin variable domain. Display libraries are
particular useful, for example for identifying human or "humanized"
antibodies that recognize human antigens. Such antibodies can be
used as therapeutics to treat human disorders such as
endothelial-related disorders, e.g., metastatic cancer. Since the
constant and framework regions of the antibody are human, these
therapeutic antibodies may avoid themselves being recognized and
targeted as antigens. The constant regions are also optimized to
recruit effector functions of the human immune system. The in vitro
display selection process surmounts the inability of a normal human
immune system to generate antibodies against self-antigens.
[0419] A typical antibody display library displays a polypeptide
that includes a VH domain and a VL domain. An "immunoglobulin
domain" refers to a domain from the variable or constant domain of
immunoglobulin molecules. Immunoglobulin domains typically contain
two .beta.-sheets formed of about seven .beta.-strands, and a
conserved disulphide bond (see, e.g., A. F. Williams and A. N.
Barclay 1988 Ann. Rev Immunol. 6:381-405). The canonical structures
of hypervariable loops of an immunoglobulin variable can be
inferred from its sequence, as described in Chothia et al. (1992)
J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol.
227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.
The display library can display the antibody as a Fab fragment
(e.g., using two polypeptide chains) or a single chain Fv (e.g.,
using a single polypeptide chain). Other formats can also be
used.
[0420] As in the case of the Fab and other formats, the displayed
antibody can include a constant region as part of a light or heavy
chain. In one embodiment, each chain includes one constant region,
e.g., as in the case of a Fab. In other embodiments, additional
constant regions are displayed.
[0421] Antibody libraries can be constructed by a number of
processes (see, e.g., de Haard et al. (1999) J. Biol. Chem
274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20. and
Hoogenboom et al. (2000) Immunol Today 21:371-8). Further, elements
of each process can be combined with those of other processes. The
processes can be used such that variation is introduced into a
single immunoglobulin domain (e.g., VH or VL) or into multiple
immunoglobulin domains (e.g., VH and VL). The variation can be
introduced into an immunoglobulin variable domain, e.g., in the
region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4,
referring to such regions of either and both of heavy and light
chain variable domains. In one embodiment, variation is introduced
into all three CDRs of a given variable domain. In another
preferred embodiment, the variation is introduced into CDR1 and
CDR2, e.g., of a heavy chain variable domain. Any combination is
feasible. [0324] In one process, antibody libraries are constructed
by inserting diverse oligonucleotides that encode CDRs into the
corresponding regions of the nucleic acid. The oligonucleotides can
be synthesized using monomeric nucleotides or trinucleotides. For
example, Knappik et al. (2000) J. Mol. Biol. 296:57-86 describe a
method for constructing CDR encoding oligonucleotides using
trinucleotide synthesis and a template with engineered restriction
sites for accepting the oligonucleotides.
[0422] In another process, an animal, e.g., a non-human animal,
e.g., a rodent, is immunized with the Tie1. The animal is
optionally boosted with the antigen to further stimulate the
response. Then spleen cells are isolated from the animal, and
nucleic acid encoding VH and/or VL domains is amplified and cloned
for expression in the display library. The non-human animal can
include one or more human immunoglobulin gene sequences. For
example, the animal can include a complete human immunoglobulin
locus. The animal may also have an inactivated endogenous
immunoglobulin locus.
[0423] In yet another process, antibody libraries are constructed
from nucleic acid amplified from na{dot over (i)}ve germline
immunoglobulin genes (e.g., human genes). The amplified nucleic
acid includes nucleic acid encoding the VH and/or VL domain.
Sources of immunoglobulin-encoding nucleic acids are described
below. Amplification can include PCR, e.g., with primers that
anneal to the conserved constant region, or another amplification
method.
[0424] Nucleic acid encoding immunoglobulin domains or fragments
thereof can be obtained from the immune cells of, e.g., a human, a
primate, mouse, rabbit, camel, or rodent. In one example, the cells
are selected for a particular property. B cells at various stages
of maturity can be selected. In another example, the B cells are
na{dot over (i)}ve.
[0425] In one embodiment, fluorescent-activated cell sorting (FACS)
is used to sort B cells that express surface-bound IgM, IgD, or IgG
molecules. Further, B cells expressing different isotypes of IgG
can be isolated. In another preferred embodiment, the B or T cell
is cultured in vitro. The cells can be stimulated in vitro, e.g.,
by culturing with feeder cells or by adding mitogens or other
modulatory reagents, such as antibodies to CD40, CD40 ligand or
CD20, phorbol myristate acetate, bacterial lipopolysaccharide,
concanavalin A, phytohemagglutinin or pokeweed mitogen.
[0426] In still another embodiment, the cells are isolated from a
subject that has an immunological disorder, e.g., systemic lupus
erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren
syndrome, systemic sclerosis, or anti-phospholipid syndrome. The
subject can be a human, or an animal, e.g., an animal model for the
human disease, or an animal having an analogous disorder. In yet
another embodiment, the cells are isolated from a transgenic
non-human animal that includes a human immunoglobulin locus.
[0427] In one preferred embodiment, the cells have activated a
program of somatic hypermutation. Cells can be stimulated to
undergo somatic mutagenesis of immunoglobulin genes, for example,
by treatment with anti-immunoglobulin, anti-CD40, and anti-CD38
antibodies (see, e.g., Bergthorsdottir et al. (2001) J Immunol.
166:2228). In another embodiment, the cells are na{dot over
(i)}ve.
[0428] The nucleic acid encoding an immunoglobulin variable domain
can be isolated from a natural repertoire by the following
exemplary method. First, RNA is isolated from the immune cell. Full
length (i.e., capped) mRNAs are separated (e.g. by
dephosphorylating uncapped RNAs with calf intestinal phosphatase).
The cap is then removed with tobacco acid pyrophosphatase and
reverse transcription is used to produce the cDNAs.
[0429] The reverse transcription of the first (antisense) strand
can be done in any manner with any suitable primer. See, e.g., de
Haard et al. (1999) J. Biol. Chem 274:18218-30. The primer binding
region can be constant among different immunoglobulins, e.g., in
order to reverse transcribe different isotypes of immunoglobulin.
The primer binding region can also be specific to a particular
isotype of immunoglobulin. Typically, the primer is specific for a
region that is 3` to a sequence encoding at least one CDR. In
another embodiment, poly-dT primers may be used (and may be
preferred for the heavy-chain genes).
[0430] A synthetic sequence can be ligated to the 3' end of the
reverse transcribed strand. The synthetic sequence can be used as a
primer binding site for binding of the forward primer during PCR
amplification after reverse transcription. The use of the synthetic
sequence can obviate the need to use a pool of different forward
primers to fully capture the available diversity.
[0431] The variable domain-encoding gene is then amplified, e.g.,
using one or more rounds. If multiple rounds are used, nested
primers can be used for increased fidelity. The amplified nucleic
acid is then cloned into a display library vector.
[0432] Any method for amplifying nucleic acid sequences may be used
for amplification. Methods that maximize and do not bias diversity
are preferred. A variety of techniques can be used for nucleic acid
amplification. The polymerase chain reaction (PCR; U.S. Pat. Nos.
4,683,195 and 4,683,202, Saiki, et al. (1985) Science 230,
1350-1354) utilizes cycles of varying temperature to drive rounds
of nucleic acid synthesis. Transcription-based methods utilize RNA
synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. No
6,066,457; U.S. Pat. No 6,132,997; U.S. Pat. No 5,716,785; Sarkar
et. al., Science (1989) 244: 331-34 ; Stofler et al., Science
(1988) 239: 491). NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and
5,554,517) utilizes cycles of transcription, reverse-transcription,
and RNaseH-based degradation to amplify a DNA sample. Still other
amplification methods include rolling circle amplification (RCA;
U.S. Pat. Nos. 5,854,033 and 6,143,495) and strand displacement
amplification (SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825).
[0433] Secondary Screening Methods
[0434] After selecting candidate display library members that bind
to a target, each candidate display library member can be further
analyzed, e.g., to further characterize its binding properties for
the target. Similarly candidate binding proteins (e.g., by
immunization, etc.) obtained by other methods can also be analyzed.
Each candidate binding protein can be subjected to one or more
secondary screening assays. The assay can be for a binding
property, a catalytic property, a physiological property (e.g.,
cytotoxicity, renal clearance, immunogenicity), a structural
property (e.g., stability, conformation, oligomerization state) or
another functional property. The same assay can be used repeatedly,
but with varying conditions, e.g., to determine pH, ionic, or
thermal sensitivities.
[0435] As appropriate, the assays can use the display library
member directly, a recombinant polypeptide produced from the
nucleic acid encoding a displayed polypeptide, or a synthetic
peptide synthesized based on the sequence of a displayed
polypeptide. Exemplary assays for binding properties include the
following.
[0436] ELISA. Proteins encoded by a display library can also be
screened for a binding property using an ELISA assay. For example,
each protein is contacted to a microtitre plate whose bottom
surface has been coated with the target, e.g., a limiting amount of
the target. The plate is washed with buffer to remove
non-specifically bound polypeptides. Then the amount of the protein
bound to the plate is determined by probing the plate with an
antibody that can recognize the polypeptide, e.g., a tag or
constant portion of the polypeptide. The antibody is linked to an
enzyme such as alkaline phosphatase, which produces a colorimetric
product when appropriate substrates are provided. The protein can
be purified from cells or assayed in a display library format,
e.g., as a fusion to a filamentous bacteriophage coat.
Alternatively, cells (e.g., live or fixed) that express the target
molecule, e.g., Tie1, Tie2, or Ang, can be plated in a microtitre
plate and used to test the affinity of the peptides/antibodies
present in the display library or obtained by selection from the
display library.
[0437] Cell Binding Assays. Binding proteins (e.g., Tie1, Tie2, or
Ang binding proteins) can be evaluated for their ability to
interact with one or more cell types, e.g., endothelial cells or
platelets. Fluorescent activated cell sorting (FACS) is one
exemplary method for testing an interaction between a protein and a
cell. The binding protein is labeled directly or indirectly with a
fluorophore, before or after, binding to the cells, and then cells
are counted in a FACS sorter.
[0438] For example, the following method can be used to evaluate
whether a Tie1 binding protein interacts with platelets or other
cell types.
[0439] Isolation of Platelets. Human blood can be obtained from
informed healthy volunteers. For example, venous blood is collected
into one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g citric
acid, and 2.5 g glucose in 100 ml dH.sub.2O). The blood is
centrifuged at 800.times.g for 15 min at room temperature and the
platelet-rich plasma is removed and incubated for 60 min at
37.degree. C. in the presence of 1 mM acetylsalicylic acid followed
by centrifugation at 1000.times.g for 10 min at room temperature.
The platelet pellet can be resuspended at a density of
2.times.10.sup.8 cells/ml with HEPES-buffered Tyrode's solution
(137 mM NaCl, 2.7 mM KCl, 1 mM MgCl.sub.2, 3 mM NaH.sub.2PO.sub.4,
5 mM glucose, 10 mM HEPES pH 7.4, 0.2% bovine serum albumin, and
0.05 U/mL apyrase). See also, e.g., Komecki et al. (1990) J Biol
Chem. 265:10,042-10,048 and Naik et al. (1995) Biochem J.
310:155-162).
[0440] FACS. For example, for FACS analysis of platelets, cells can
be resuspended in 0.1% BSA/PBS (4.times.10.sup.5 cells/sample) in
the presence of PGE1 (1 mg/mL) and incubated with a candidate Tie1
binding protein (e.g., at about 5 .mu.g/mL) or with a control.
After a 1-hour incubation at 22.degree. C., the cells are washed
with 0.1% BSA/PBS, treated with 50 .mu.L 1/100 diluted FITC-labeled
secondary antibody, incubated for 30 minutes on ice, washed, and
resuspended in 0.1% BSA/PBS. The samples are analyzed using an
Immunocytometry Systems flow cytometer (FACSORT.TM., Becton
Dickinson, San Jose, Calif.). See also, e.g., Malgorzata et al.
(2000) Blood, Vol. 95 No. 8 (April 15 pp. 2600-2609.
[0441] In addition, it is possible to evaluate platelets by
Westerns analysis of SDS-page separated proteins from isolated
platelets and by immunoprecipitation. Still other methods involve
binding cells to surfaces to which the Tie1-binding protein is
attached (e.g., coated to).
[0442] Other cell types can be prepared for FACS by methods known
in the art.
[0443] Homogeneous Binding Assays. The binding interaction of
candidate polypeptide with a target can be analyzed using a
homogenous assay, i.e., after all components of the assay are
added, additional fluid manipulations are not required. For
example, fluorescence resonance energy transfer (FRET) can be used
as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat.
No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A
fluorophore label on the first molecule (e.g., the molecule
identified in the fraction) is selected such that its emitted
fluorescent energy can be absorbed by a fluorescent label on a
second molecule (e.g., the target) if the second molecule is in
proximity to the first molecule. The fluorescent label on the
second molecule fluoresces when it absorbs to the transferred
energy. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, the spatial
relationship between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. A binding event that is configured for monitoring by FRET
can be conveniently measured through standard fluorometric
detection means well known in the art (e.g., using a fluorimeter).
By titrating the amount of the first or second binding molecule, a
binding curve can be generated to estimate the equilibrium binding
constant.
[0444] Surface Plasmon Resonance (SPR). The binding interaction of
a molecule isolated from a display library and a target can be
analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA)
detects biospecific interactions in real time, without labeling any
of the interactants. Changes in the mass at the binding surface
(indicative of a binding event) of the BIA chip result in
alterations of the refractive index of light near the surface (the
optical phenomenon of surface plasmon resonance (SPR)). The changes
in the refractivity generate a detectable signal, which are
measured as an indication of real-time reactions between biological
molecules. Methods for using SPR are described, for example, in
U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer
Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345;
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line
resources provide by BlAcore International AB (Uppsala,
Sweden).
[0445] Information from SPR can be used to provide an accurate and
quantitative measure of the equilibrium dissociation constant
(K.sub.d), and kinetic parameters, including K.sub.on and
K.sub.off, for the binding of a biomolecule to a target. Such data
can be used to compare different biomolecules. For example,
different proteins can be compared to identify individuals that
have high affinity for the target or that have a slow K.sub.off.
This information can also be used to develop structure-activity
relationships (SAR). For example, the kinetic and equilibrium
binding parameters of matured versions of a parent protein can be
compared to the parameters of the parent protein. Variant amino
acids at given positions can be identified that correlate with
particular binding parameters, e.g., high affinity and slow
K.sub.off. This information can be combined with structural
modeling (e.g., using homology modeling, energy minimization, or
structure determination by crystallography or NMR). As a result, an
understanding of the physical interaction between the protein and
its target can be formulated and used to guide other design
processes.
[0446] Protein Arrays. Proteins identified from the display library
can be immobilized on a solid support, for example, on a bead or an
array. For a protein array, each of the polypeptides is immobilized
at a unique address on a support. Typically, the address is a
two-dimensional address. Protein arrays are described below (see,
e.g., Diagnostics). It is also possible to use a protein array to
evaluate any plurality of proteins, e.g., for interaction with
Tie1, Tie2, or Ang.
[0447] Cellular Assays. Candidate proteins can be selected from a
library by transforming the library into a host cell; the library
could have been previously identified from a display library. For
example, the library can include vector nucleic acid sequences that
include segments that encode the polypeptides and that direct
expression, e.g., such that the proteins are produced within the
cell, secreted from the cell, or attached to the cell surface. The
cells can be screened or selected for proteins that bind to the
Tie1, Tie2, or Ang, e.g., as detected by a change in a cellular
phenotype or a cell-mediated activity. For example, in the case of
an antibody that binds to Tie1, the activity may be
autophosphorylation, activation of PI3 Kinase, activation of AKT,
or a change in endothelial cell activity (e.g., proliferation).
[0448] In another embodiment, the library of cells is in the form
of a cellular array. The cellular array can likewise be screened
for any appropriate detectable activity. In other embodiments,
competition binding assays are used to identify proteins that are
compete with a reference protein for binding to Tie1. Similarly,
epitope mapping can be used to identify proteins that bind to a
particular epitope of Tie. Fragments and mutants of Tie1 can be
also be used in the binding protein-identification process, e.g.,
in one or more of characterization, screening, or immunization.
[0449] Methods for Obtaining Target-Binding Antibodies
[0450] In addition to the use of display libraries, other methods
can be used to obtain a target-binding antibody or in combination
with the use of display libraries. For example, the Tie1 ectodomain
or a region thereof can be used as an antigen in a non-human
animal, e.g., a rodent. Similarly, Tie2 or Ang, or a region thereof
can be used as an antigen in a non-human animal, e.g., a
rodent.
[0451] In one embodiment, the non-human animal includes at least a
part of a human immunoglobulin gene. For example, it is possible to
engineer mouse strains deficient in mouse antibody production with
large fragments of the human Ig loci. Using the hybridoma
technology, antigen-specific Mabs derived from the genes with the
desired specificity may be produced and selected. See, e.g.,
XenoMouse.TM., Green et al. Nature Genetics 7:13-21 (1994), U.S.
20030070185, WO 96/34096, published Oct. 31, 1996, and PCT
Application No. PCT/US96/05928, filed Apr. 29, 1996.
[0452] In another embodiment, a monoclonal antibody is obtained
from the non-human animal, and then modified, e.g., humanized or
deimmunized. Winter describes a CDR-grafting method that may be
used to prepare the humanized antibodies (UK Patent Application GB
2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539.
All of the CDRs of a particular human antibody may be replaced with
at least a portion of a non-human CDR or only some of the CDRs may
be replaced with non-human CDRs. It is only necessary to replace
the number of CDRs required for binding of the humanized antibody
to a predetermined antigen.
[0453] Humanized antibodies can be generated by replacing sequences
of the Fv variable region that are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,
BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089,
U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762. Those methods
include isolating, manipulating, and expressing the nucleic acid
sequences that encode all or part of immunoglobulin Fv variable
regions from at least one of a heavy or light chain. Sources of
such nucleic acid are well known to those skilled in the art and,
for example, may be obtained from a hybridoma producing an antibody
against a predetermined target, as described above. The recombinant
DNA encoding the humanized antibody, or fragment thereof, can then
be cloned into an appropriate expression vector.
[0454] A target-binding antibody may also be modified by specific
deletion of human T cell epitopes or "deimmunization" by the
methods disclosed in WO 98/52976 and WO 00/34317, the contents of
which are specifically incorporated by reference herein. Briefly,
the heavy and light chain variable regions of an antibody can be
analyzed for peptides that bind to MHC Class II; these peptides
represent potential T-cell epitopes (as defined in WO 98/52976 and
WO 00/34317). For detection of potential T-cell epitopes, a
computer modeling approach termed "peptide threading" can be
applied, and in addition a database of human MHC class II binding
peptides can be searched for motifs present in the V.sub.H and
V.sub.L sequences, as described in WO 98/52976 and WO 00/34317.
These motifs bind to any of the 18 major MHC class II DR allotypes,
and thus constitute potential T cell epitopes. Potential T-cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable regions, or preferably, by
single amino acid substitutions. As far as possible conservative
substitutions are made, often but not exclusively, an amino acid
common at this position in human germline antibody sequences may be
used. Human germline sequences are disclosed in Tomlinson, I. A. et
al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995)
Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al. (1992) J.
Mol. Bio. 227:799-817. The V BASE directory provides a
comprehensive directory of human immunoglobulin variable region
sequences (compiled by Tomlinson, I. A. et al. MRC Centre for
Protein Engineering, Cambridge, UK). After the deimmunizing changes
are identified, nucleic acids encoding V.sub.H and V.sub.L can be
constructed by mutagenesis or other synthetic methods (e.g., de
novo synthesis, cassette replacement, and so forth). Mutagenized
variable sequence can, optionally, be fused to a human constant
region, e.g., human IgG1 or .kappa. constant regions.
[0455] In some cases a potential T cell epitope will include
residues which are known or predicted to be important for antibody
function. For example, potential T cell epitopes are usually biased
towards the CDRs. In addition, potential T cell epitopes can occur
in framework residues important for antibody structure and binding.
Changes to eliminate these potential epitopes will in some cases
require more scrutiny, e.g., by making and testing chains with and
without the change. Where possible, potential T cell epitopes that
overlap the CDRs were eliminated by substitutions outside the CDRs.
In some cases, an alteration within a CDR is the only option, and
thus variants with and without this substitution should be tested.
In other cases, the substitution required to remove a potential T
cell epitope is at a residue position within the framework that
might be critical for antibody binding. In these cases, variants
with and without this substitution should be tested. Thus, in some
cases several variant deimmunized heavy and light chain variable
regions were designed and various heavy/light chain combinations
tested in order to identify the optimal deimmunized antibody. The
choice of the final deimmunized antibody can then be made by
considering the binding affinity of the different variants in
conjunction with the extent of deimmunization, i.e., the number of
potential T cell epitopes remaining in the variable region.
Deimmunization can be used to modify an antibody that includes a
non-human sequence, e.g., a murine antibody or other non-human
monoclonal antibody. Deimmunization can be used to modify an
antibody isolated from a display library.
[0456] Endothelial Cell Assays
[0457] A target-binding protein or a candidate binding protein can
be characterized using a cellular assay, e.g., to evaluate a change
in a cellular phenotype or other activity when the binding protein
is contacted to the cell. Typically the cell is expresses a protein
that includes at least part of the ectodomain of Tie. In some
embodiments, the cell expresses Tie1, e.g., a full-length, mature
Tie1 protein, Tie2, and/or is contacted with Ang.
[0458] Endothelial cell proliferation. A candidate target-binding
protein can be tested for endothelial proliferation inhibiting
activity using a biological activity assay such as the bovine
capillary endothelial cell proliferation assay, the chick CAM
assay, the mouse corneal assay, and evaluating the effect of the
binding protein on implanted tumors. The chick CAM assay is
described, e.g., by O'Reilly, et al. in "Angiogenic Regulation of
Metastatic Growth" Cell, vol. 79 (2), Oct. 21, 1994, pp. 315-328.
Briefly, three day old chicken embryos with intact yolks are
separated from the egg and placed in a petri dish. After three days
of incubation a methylcellulose disc containing the protein to be
tested is applied to the CAM of individual embryos. After 48 hours
of incubation, the embryos and CAMs are observed to determine
whether endothelial growth has been inhibited. The mouse corneal
assay involves implanting a growth factor-containing pellet, along
with another pellet containing the suspected endothelial growth
inhibitor, in the cornea of a mouse and observing the pattern of
capillaries that are elaborated in the cornea.
[0459] Angiogenesis. Angiogenesis may be assayed , e.g., using
various human endothelial cell systems, such as umbilical vein,
coronary artery, or dermal cells. Suitable assays include Alamar
Blue based assays (available from Biosource International) to
measure proliferation; migration assays using fluorescent
molecules, such as the use of Becton Dickinson Falcon HTS
FLUOROBLOCK.TM. cell culture inserts to measure migration of cells
through membranes in presence or absence of angiogenesis enhancer
or suppressors; and tubule formation assays based on the formation
of tubular structures by endothelial cells on MATRIGEL.TM. (Becton
Dickinson) or collagen I.
[0460] Cell adhesion. Cell adhesion assays measure adhesion of
cells to purified adhesion proteins or adhesion of cells to each
other, in presence or absence of candidate target-binding proteins.
Cell-protein adhesion assays measure the ability of agents to
modulate the adhesion of cells to purified proteins. For example,
recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and
used to coat the wells of a microtiter plate. The wells used for
negative control are not coated. Coated wells are then washed,
blocked with 1% BSA, and washed again. Compounds are diluted to 2 x
final test concentration and added to the blocked, coated wells.
Cells are then added to the wells, and the unbound cells are washed
off. Retained cells are labeled directly on the plate by adding a
membrane-permeable fluorescent dye, such as calcein-AM, and the
signal is quantified in a fluorescent microplate reader.
[0461] Cell-cell adhesion assays can be used to measure the ability
of candidate target-binding proteins to modulate binding of cells
to each other. These assays can use cells that naturally or
recombinantly express an adhesion protein of choice. In an
exemplary assay, cells expressing the cell adhesion protein are
plated in wells of a multiwell plate together with other cells
(either more of the same cell type, or another type of cell to
which the cells adhere). The cells that can adhere are labeled with
a membrane-permeable fluorescent dye, such as BCECF, and allowed to
adhere to the monolayers in the presence of candidate binding
proteins. Unbound cells are washed off, and bound cells are
detected using a fluorescence plate reader. High-throughput cell
adhesion assays have also been described. See, e.g., Falsey J R et
al., Bioconjug Chem. May-June 2001;12(3):346-53.
[0462] Tubulogenesis. Tubulogenesis assays can be used to monitor
the ability of cultured cells, generally endothelial cells, to form
tubular structures on a matrix substrate, which generally simulates
the environment of the extracellular matrix. Exemplary substrates
include MATRIGEL.TM. (Becton Dickinson), an extract of basement
membrane proteins containing laminin, collagen IV, and heparin
sulfate proteoglycan, which is liquid at 4.degree. C. and forms a
solid gel at 37.degree. C. Other suitable matrices comprise
extracellular components such as collagen, fibronectin, and/or
fibrin. Cells are stimulated with a pro-angiogenic stimulant, and
their ability to form tubules is detected by imaging. Tubules can
generally be detected after an overnight incubation with stimuli,
but longer or shorter time frames may also be used. Tube formation
assays are well known in the art (e.g., Jones M K et al., 1999,
Nature Medicine 5:1418-1423). These assays have traditionally
involved stimulation with serum or with the growth factors FGF or
VEGF. In one embodiment, the assay is performed with cells cultured
in serum free medium. In one embodiment, the assay is performed in
the presence of one or more pro-angiogenic agents, e.g.,
inflammatory angiogenic factors such as TNF-.alpha., or FGF, VEGF,
phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
[0463] Cell Migration. An exemplary assay for endothelial cell
migration is the human microvascular endothelial (HMVEC) migration
assay. See, e.g., Tolsma et al. (1993) J. Cell Biol 122, 497-511.
Migration assays are known in the art (e.g., Paik J H et al., 2001,
J Biol Chem 276:11830-11837). In one example, cultured endothelial
cells are seeded onto a matrix-coated porous lamina, with pore
sizes generally smaller than typical cell size. The lamina is
typically a membrane, such as the transwell polycarbonate membrane
(Corning Costar Corporation, Cambridge, Mass.), and is generally
part of an upper chamber that is in fluid contact with a lower
chamber containing pro-angiogenic stimuli. Migration is generally
assayed after an overnight incubation with stimuli, but longer or
shorter time frames may also be used. Migration is assessed as the
number of cells that crossed the lamina, and may be detected by
staining cells with hemotoxylin solution (VWR Scientific.), or by
any other method for determining cell number. In another exemplary
set up, cells are fluorescently labeled and migration is detected
using fluorescent readings, for instance using the Falcon HTS
FLUOROBLOK.TM. (Becton Dickinson). While some migration is observed
in the absence of stimulus, migration is greatly increased in
response to pro-angiogenic factors. The assay can be used to test
the effect of a target-binding protein on endothelial cell
migration.
[0464] Sprouting assay. An exemplary sprouting assay is a
three-dimensional in vitro angiogenesis assay that uses a
cell-number defined spheroid aggregation of endothelial cells
("spheroid"), embedded in a collagen gel-based matrix. The spheroid
can serve as a starting point for the sprouting of capillary-like
structures by invasion into the extracellular matrix (termed "cell
sprouting") and the subsequent formation of complex anastomosing
networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58). In an
exemplary experimental set-up, spheroids are prepared by pipetting
400 human umbilical vein endothelial cells into individual wells of
a nonadhesive 96-well plates to allow overnight spheroidal
aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).
Spheroids are harvested and seeded in 900 .mu.l of
methocel-collagen solution and pipetted into individual wells of a
24 well plate to allow collagen gel polymerization. Test agents are
added after 30 min by pipetting 100 .mu.l of 10-fold concentrated
working dilution of the test substances on top of the gel. Plates
are incubated at 37.degree. C. for 24 h. Dishes are fixed at the
end of the experimental incubation period by addition of
paraformaldehyde. Sprouting intensity of endothelial cells can be
quantitated by an automated image analysis system to determine the
cumulative sprout length per spheroid.
[0465] In some embodiments, a target-binding protein has a
statistically significant effect on an assay described herein,
e.g., a cellular assay described herein.
[0466] Protein Production
[0467] Standard recombinant nucleic acid methods can be used to
express a binding protein that binds to Tie1, Tie2, or Ang. See,
for example, the techniques described in Sambrook & Russell,
Molecular Cloning: A Laboratory Manual, 3.sup.rd Edition, Cold
Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al., Current
Protocols in Molecular Biology (Greene Publishing Associates and
Wiley Interscience, N.Y. (1989). Generally, a nucleic acid sequence
encoding the binding proteinis cloned into a nucleic acid
expression vector. If the protein includes multiple polypeptide
chains, each chain can be cloned into an expression vector, e.g.,
the same or different vectors, that are expressed in the same or
different cells. Methods for producing antibodies are also provided
below.
[0468] Antibody Production. Some antibodies, e.g., Fabs, can be
produced in bacterial cells, e.g., E. coli cells. For example, if
the Fab is encoded by sequences in a phage display vector that
includes a suppressible stop codon between the display entity and a
bacteriophage protein (or fragment thereof), the vector nucleic
acid can be shuffled into a bacterial cell that cannot suppress a
stop codon. In this case, the Fab is not fused to the gene III
protein and is secreted into the media.
[0469] Antibodies can also be produced in eukaryotic cells. In one
embodiment, the antibodies (e.g., scFv's) are expressed in a yeast
cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol
Methods. 251:123-35), Hanseula, or Saccharomyces.
[0470] In one embodiment, antibodies are produced in mammalian
cells. Preferred mammalian host cells for expressing the clone
antibodies or antigen-binding fragments thereof include Chinese
Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in
Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in Kaufman
and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines,
e.g., NSO myeloma cells, SP2 cells, COS cells, HEK 293T cells, and
a cell from a transgenic animal, e.g., a transgenic mammal. For
example, the cell is a mammary epithelial cell.
[0471] In addition to the nucleic acid sequence encoding the
immunoglobulin domain, the recombinant expression vectors may carry
additional sequences, such as sequences that regulate replication
of the vector in host cells (e.g., origins of replication) and
selectable marker genes. The selectable marker gene facilitates
selection of host cells into which the vector has been introduced
(see e.g., U.S. Pats. Nos. 4,399,216, 4,634,665 and 5,179,017). For
example, typically the selectable marker gene confers resistance to
drugs, such as G418, hygromycin or methotrexate, on a host cell
into which the vector has been introduced. Preferred selectable
marker genes include the dihydrofolate reductase (DHFR) gene (for
use in dhfr.sup.- host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).
Another exemplary expression system is the glutamine synthase (GS)
vector system available from Lonza Group Ltd. CH (see, e.g., Clark
et al. (2004) BioProcess International 2(4):48-52; Barnes et al.
(2002) Biotech Bioeng. 81(6):631-639).
[0472] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, a recombinant
expression vector encoding both the antibody heavy chain and the
antibody light chain is introduced into dhfr- CHO cells by calcium
phosphate-mediated transfection. Within the recombinant expression
vector, the antibody heavy and light chain genes are each
operatively linked to enhancer/promoter regulatory elements (e.g.,
derived from SV40, CMV, adenovirus and the like, such as a CMV
enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G.
[0473] The codon usage can adapted to the codon bias of the host
cell, e.g., for CHO cells it can be adapted for the codon bias
Cricetulus griseus genes. In addition, regions of very high
(>80%) or very low (<30%) GC content can be avoid avoided
where possible. During the optimization process following
cis-acting sequence motifs were avoided: internal TATA-boxes;
chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence
stretches; ARE, INS, CRS sequence elements; repeat sequences and
RNA secondary structures; and (cryptic) splice donor and acceptor
sites, branch points. Two STOP codons can be used to ensure
efficient termination. The codon optimization of the sequence can
be evaluated according to Sharp, P. M., Li, W. H., Nucleic Acids
Res. 15 (3), 1987). The standard codon adaptation index (CAI) can
be used. Rare codons include those with a quality class between
0-40.
[0474] The invention features isolated nucleic acid molecules that
are altered relative to a sequence described herein, e.g., to
include improved codons or sequence features, include an isolated
nucleic acid molecule that comprises a heavy or light chain coding
sequence. For example, at least 30, 40, 45, 50, 60, 65, 70, 75, or
80% of the codons in the heavy or light chain coding sequence are
non-rare or frequent codons in a mammalian cell or the heavy or
light chain coding sequence includes fewer than 50, 45, 40, 35, 30,
25, 20, 15, 10% rare codons in a mammalian cell, e.g., a Chinese
hamster cell (Cricetulus griseus). In one embodiment, the codon
adaptation index is greater than 0.6, 0.7, 0.8, 0.85, 0.90, 0.92,
0.94, 0.95, 0.96, 0.97, or 0.98.
[0475] In one embodiment, the heavy chain coding sequence encodes
(i) a polypeptide comprising an antibody heavy chain described
herein (e.g., an E3 heavy chain as set forth in SEQ ID NO:723),
(ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99%
identical to an antibody heavy chain coding sequence described
herein (e.g., SEQ ID NO:723), or (iii) a polypeptide that comprises
a heavy chain variable domain sequence having the CDRs of an
antibody heavy chain variable domain described herein (e.g., an E3
heavy chain variable domain). In one embodiment, the heavy chain
coding sequence differs from SEQ ID NO:703 at at least 2, 3, 5, 6,
8, 9, 10, or 15 codons.
[0476] In one embodiment, the light chain coding sequence encodes
(i) a polypeptide comprising an antibody light chain described
herein (e.g., an E3 light chain as set forth in SEQ ID NO:724),
(ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99%
identical to an antibody light chain coding sequence described
herein (e.g., SEQ ID NO:724), or (iii) a polypeptide that comprises
a light chain variable domain sequence having the CDRs of an
antibody light chain variable domain described herein (e.g., an E3
light chain variable domain). In one embodiment, the light chain
coding sequence differs from SEQ ID NO:702 at at least 3, 5, 6, 8,
9, 10, or 15 codons.
[0477] In one embodiment, for example, one or more of the ala-GCG
codons can be changed to GCC; one or more of the arg-CGT codons are
changed to CGC; one or more of the pro-CCG codons are changed to
CCC, CCT, or CCA; one or more of the ser-TCG codons are changed to
TCC; and/or one or more of the thr-ACG codons are changed to
ACC.
[0478] Codon-altered (e.g., codon-optimized) sequences can be used
to produce an antibody. An exemplary method includes providing a
mammalian cell that includes an antibody-coding nucleic acid and
expressing the nucleic acid in the cell, e.g., maintaining the cell
under conditions in which the protein is expressed. The
antibody-coding nucleic acid can be providing in a mammalian
expression vector, e.g., a vector that is introduced into the cell.
The cell can be a non-human mammalian cell, e.g., a CHO cell.
[0479] For antibodies that include an Fc domain, the antibody
production system preferably synthesizes antibodies in which the Fc
region is glycosylated. For example, the Fc domain of IgG molecules
is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fey receptors and
complement Clq (Burton and Woof (1992) Adv. Immunol. 51:1-84;
Jefferis et al. (1998) Immunol. Rev. 163:59-76). In a preferred
embodiment, the Fc domain is produced in a mammalian expression
system that appropriately glycosylates the residue corresponding to
asparagine 297. The Fc domain can also include other eukaryotic
post-translational modifications.
[0480] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method of expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
[0481] It is also possible to produce antibodies that bind to Tie1,
Tie2, or Ang by immunization, e.g., using an animal, e.g., with
natural, human, or partially human immunoglobulin loci. Such an
antibody can be of any allotype, e.g., a,z allotype, f allotype, or
non-A allotype. Non-human antibodies can also be modified to
include substitutions for human immunoglobulin sequences, e.g.,
consensus human amino acid residues at particular positions, e.g.,
at one or more of the following positions (preferably at least
five, ten, twelve, or all): (in the FR of the variable domain of
the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L,
64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or
(in the FR of the variable domain of the heavy chain) 2H, 4H, 24H,
36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H,
74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according to the Kabat
numbering). See, e.g., U.S. Pat. No. 6,407,213.
[0482] Tie1 production. Methods for producing Tie1 ectodomain
protein, Tie1 protein, or Tie1 liposomes are known in the art. See,
e.g., WO 93/14124. Methods for producing Tie2 and Ang are similarly
known. See e.g., U.S. Pat. Nos. 6,521,424, 6,376,653; WO 96/11269;
WO 96/31598.
[0483] Biotinylation Methods. A variety of methods are available to
biotinylate proteins, e.g., an immunoglobulin protein or a target
protein. For example, the protein can be incubated with a 5-fold
molar excess of sulfo-NHS--SS-biotin in 50 mM HEPES, pH 8.0, 100 mM
NaCl overnight at 4.degree. C. Free biotin is removed by buffer
exchange into PBS, 0.01% Tween 20, e.g., using a BIOMAX.RTM. device
with a 10 kDa molecular weight cut-off membrane or by dialysis. The
number of biotin molecules incorporated per mole of protein can be
determined using the HABA assay as described by the manufacturer
(Pierce).
[0484] Pharmaceutical Compositions
[0485] In another aspect, the invention provides compositions,
e.g., pharmaceutically acceptable compositions, which include an
agent that binds to Tie1, Tie2, or Ang, e.g., an antibody molecule,
other polypeptide or peptide identified as binding to Tie1, Tie2,
or Ang, or described herein, formulated with a pharmaceutically
acceptable carrier. Pharmaceutical compositions encompass labeled
binding proteins (e.g., for in vivo imaging) as well as therapeutic
compositions.
[0486] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the binding protein, may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0487] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0488] Compositions may be in a variety of forms. These include,
for example, liquid, semi-solid and solid dosage forms, such as
liquid solutions (e.g., injectable and infusible solutions),
dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions,
such as compositions similar to those used for administration of
humans with antibodies. The preferred mode of administration is
parenteral (e.g., intravenous, subcutaneous, intraperitoneal,
intramuscular). In a preferred embodiment, the target-binding
protein is administered by intravenous infusion or injection. In
another preferred embodiment, the target-binding protein is
administered by intramuscular or subcutaneous injection.
[0489] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and infrasternal injection and
infusion.
[0490] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration. Sterile injectable solutions
can be prepared by incorporating the binding protein in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying that yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0491] The binding proteins described herein can be administered by
a variety of methods known in the art, although for many
applications, the preferred route/mode of administration is
intravenous injection or infusion. For example, for therapeutic
applications, the target-binding protein can be administered by
intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5,
or 1 mg/min to reach a dose of about 1 to 100 mg/m.sup.2 or 7 to 25
mg/m.sup.2. The route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0492] In certain embodiments, the binding protein may be orally
administered, for example, with an inert diluent or an assimilable
edible carrier. The compound (and other ingredients, if desired)
may also be enclosed in a hard or soft shell gelatin capsule,
compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
described herein by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0493] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
pharmaceutical can be administered with a needleless hypodermic
injection device, such as the devices disclosed in U.S. Pat. Nos.
5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,
or 4,596,556. Examples of implants and modules include: U.S. Pat.
No. 4,487,603, which discloses an implantable micro-infusion pump
for dispensing medication at a controlled rate; U.S. Pat. No.
4.,486,194, which discloses a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system. Of
course, many other such implants, delivery systems, and modules are
also known.
[0494] In certain embodiments, a binding protein described herein
can be formulated to ensure proper distribution in vivo. For
example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic protein
crosses the BBB (if desired), it can be formulated, for example, in
liposomes. For methods of manufacturing liposomes, see, e.g., U.S.
Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may
include one or more moieties which are selectively transported into
specific cells or organs, thus enhance targeted drug delivery (see,
e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
[0495] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms can be dictated by and
directly dependent on (a) the unique characteristics of the active
compound and the particular therapeutic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0496] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody described herein
is 0.1-20 mg/kg, more preferably 1-10 mg/kg. The target-binding
antibody can be administered by intravenous infusion at a rate of
less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to
100 mg/m.sup.2 or about 5 to 30 mg/m.sup.2. For binding proteins
smaller in molecular weight than an antibody, appropriate amounts
can be proportionally less. It is to be noted that dosage values
may vary with the type and severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0497] The pharmaceutical compositions may be prepared using a
"therapeutically effective amount" or a "prophylactically effective
amount" of an target-binding protein described herein. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the composition may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the binding protein to elicit a desired response in the individual.
A therapeutically effective amount is also one in which any toxic
or detrimental effects of the composition are outweighed by the
therapeutically beneficial effects. A "therapeutically effective
dosage" preferably inhibits a measurable parameter, e.g.,
inflammation or tumor growth rate by at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. The ability of a compound to inhibit a
measurable parameter, e.g., cancer, can be evaluated in an animal
model system predictive of efficacy in human tumors. Alternatively,
this property of a composition can be evaluated by examining the
ability of the compound to inhibit, such inhibition in vitro by
assays known to the skilled practitioner.
[0498] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0499] Also within the scope of the invention are kits including
the binding protein that binds to Tie1, Tie2, or Ang and
instructions for use, e.g., treatment, prophylactic, or diagnostic
use. In one embodiment, the instructions for diagnostic
applications include the use of the target-binding protein (e.g.,
antibody or antigen-binding fragment thereof, or other polypeptide
or peptide) to detect Tie1, Tie2, or Ang, in vitro, e.g., in a
sample, e.g., a biopsy or cells from a patient having an
inflammatory disorder or a cancer or neoplastic disorder, or in
vivo. In another embodiment, the instructions for therapeutic
applications include suggested dosages and/or modes of
administration in a patient with a cancer or neoplastic disorder.
The kit can further contain at least one additional reagent, such
as a diagnostic or therapeutic agent, e.g., a diagnostic or
therapeutic agent as described herein, and/or one or more
additional target-binding proteins, formulated as appropriate, in
one or more separate pharmaceutical preparations.
[0500] In one embodiment, target binding proteins (such as the Tie1
antibodies described herein) can be produced from gene-based
vectors, such as transgenes or via adenoviral delivery.
[0501] Stabilization and Retention
[0502] In one embodiment, a target-binding agent (e.g., a Tie1
-binding protein, polypeptide, antibody, or aptamer described
herein) is physically associated with a moiety that improves its
stabilization and/or retention in circulation, e.g., in blood,
serum, lymph, or other tissues.
[0503] For example, a target-binding agent can be associated with a
polymer, e.g., a substantially non-antigenic polymers, such as
polyalkylene oxides or polyethylene oxides. Suitable polymers will
vary substantially by weight. Exemplary polymers include polymers
having molecular number average weights ranging from about 200 to
about 35,000, from about 1,000 to about 15,000, and 2,000 to about
12,500.
[0504] For example, an target-binding agent can be conjugated to a
water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.
polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0505] Other compounds can also be attached to the same polymer,
e.g., a cytotoxin, a label, or another targeting agent, e.g.,
another target-binding agent or an unrelated agent. Mono-activated,
alkoxy-terminated polyalkylene oxides (PAO's), e.g.,
monomethoxy-terminated polyethylene glycols (mPEG's); C.sub.1-4
alkyl-terminated polymers; and bis-activated polyethylene oxides
(glycols) can be used for crosslinking. See, e.g., U.S. Pat. No.
5,951,974.
[0506] In its most common form poly(ethylene glycol), PEG, is a
linear or branched polyether terminated with hydroxyl groups and
having the general structure:
HO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
PEG can be synthesized by anionic ring opening polymerization of
ethylene oxide initiated by nucleophilic attack of a hydroxide ion
on the epoxide ring. Particularly useful for polypeptide
modification is monomethoxy PEG, mPEG, having the general
structure:
CH.sub.3O--(CH.sub.2CH.sub.2O)--.sub.n--CH.sub.2CH.sub.2--OH
For further description, see, e.g., Roberts et al. (2002) Advanced
Drug Delivery Reviews 54:459-476.
[0507] In one embodiment, the polymer prior to cross-linking need
not be, but preferably is, water soluble. Generally, after
crosslinking, the product is water soluble, e.g., exhibits a water
solubility of at least about 0.01 mg/ml, and more preferably at
least about 0.1 mg/ml, and still more preferably at least about 1
mg/ml. In addition, the polymer should not be highly immunogenic in
the conjugate form, nor should it possess viscosity that is
incompatible with intravenous infusion or injection if the
conjugate is intended to be administered by such routes.
[0508] In one embodiment, the polymer contains only a single group
which is reactive. This helps to avoid cross-linking of protein
molecules. However, it is within the scope herein to maximize
reaction conditions to reduce cross-linking, or to purify the
reaction products through gel filtration or ion exchange
chromatography to recover substantially homogenous derivatives. In
other embodiments, the polymer contains two or more reactive groups
for the purpose of linking multiple agents to the polymer backbone.
Again, gel filtration or ion exchange chromatography can be used to
recover the desired derivative in substantially homogeneous
form.
[0509] The molecular weight of the polymer can range up to about
500,000 D, and preferably is at least about 20,000 D, or at least
about 30,000 D, or at least about 40,000 D. The molecular weight
chosen can depend upon the effective size of the conjugate to be
achieved, the nature (e.g. structure, such as linear or branched)
of the polymer, and the degree of derivatization.
[0510] The covalent crosslink can be used to attach a
target-binding agent (e.g., a protein) to a polymer, for example,
crosslinking to the N-terminal amino group and epsilon amino groups
found on lysine residues, as well as other amino, imino, carboxyl,
sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may
be covalently bonded directly to the target-binding protein without
the use of a multifunctional (ordinarily bifunctional) crosslinking
agent. Covalent binding to amino groups is accomplished by known
chemistries based upon cyanuric chloride, carbonyl diimidazole,
aldehyde reactive groups (PEG alkoxide plus diethyl acetal of
bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG
chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated
succinimidyl esters, activated dithiocarbonate PEG,
2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate
activated PEG.) Carboxyl groups can be derivatized by coupling
PEG-amine using carbodiimide. Sulfhydryl groups can be derivatized
by coupling to maleimido-substituted PEG (e.g. alkoxy-PEG amine
plus sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) WO 97/10847 or
PEG-maleimide commercially available from Shearwater Polymers,
Inc., Huntsville, Ala.). Alternatively, free amino groups on the
binding protein (e.g. epsilon amino groups on lysine residues) can
be thiolated with 2-imino-thiolane (Traut's reagent) and then
coupled to maleimide-containing derivatives of PEG, e.g., as
described in Pedley et al., Br. J. Cancer, 70: 1126-1130
(1994).
[0511] Functionalized PEG polymers that can be attached to a
target-binding agent (e.g., protein) are available, e.g., from
Shearwater Polymers, Inc. (Huntsville, Ala.). Such commercially
available PEG derivatives include, e.g., amino-PEG, PEG amino acid
esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated
PEG, PEG-propionic acid, PEG amino acids, PEG succinimidyl
succinate, PEG succinimidyl propionate, succinimidyl ester of
carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl
esters of amino acid PEGs, PEG-oxycarbonylimidazole,
PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether,
PEG-aldehyde, PEG vinylsulfone, PEG-maleimide,
PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinyl
derivatives, PEG silanes, and PEG phospholides. The reaction
conditions for coupling these PEG derivatives may vary depending on
the Tie1-binding protein, the desired degree of PEGylation, and the
PEG derivative utilized. Some factors involved in the choice of PEG
derivatives include: the desired point of attachment (such as
lysine or cysteine R-groups), hydrolytic stability and reactivity
of the derivatives, stability, toxicity and antigenicity of the
linkage, suitability for analysis, etc. Specific instructions for
the use of any particular derivative are available from the
manufacturer.
[0512] The conjugates of an target-binding agent (e.g., a Tie1
binding protein) and a polymer can be separated from the unreacted
starting materials, e.g., by gel filtration or ion exchange
chromatography, e.g., HPLC. Heterologous species of the conjugates
are purified from one another in the same fashion. Resolution of
different species (e.g., containing one or two PEG residues) is
also possible, e.g., due to the difference in the ionic properties
of unreacted amino acids. See, e.g., WO 96/34015.
[0513] A target binding protein can also be physically associated
with a protein that provides a stabilizing or retention function,
e.g., an albumin, e.g., human serum albumin. US 20040171794
describes exemplary methods for physically associating a protein
with serum albumin. For exemplary, human albumin sequences or
fragments thereof, see EP 201 239, EP 322 094 WO 97/24445,
WO95/23857 especially the mature form of human albumin as shown in
SEQ ID NO:18 of US 20040171794 and WO 01/79480 or albumin from
other vertebrates or fragments thereof, or analogs or variants of
these molecules or fragments thereof. Other exemplary human serum
albumin proteins can include one or both of the following sets of
point mutations Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and
Arg-410 to Ala; or Arg-410 to Ala, Lys-413 to Gln, and Lys-414 to
Gln (see, e.g., International Publication No. WO95/23857, with
reference to SEQ ID NO:18 of US 20040171794).
[0514] Aptamers
[0515] In one embodiment, the invention also features target
protein-binding agents such as aptamers. The term nucleic acid
"aptamer," as used herein, refers to a nucleic acid molecule which
has a conformation that includes an internal non-duplex nucleic
acid structure of at least 5 nucleotides. An aptamer can be a
single-stranded nucleic acid molecule which has regions of
self-complementarity. Exemplary aptamers include nucleic acid
molecules that bind to a target molecule other than a nucleic acid,
e.g., to Tie1, Tie2, or Ang. Particular aptamers may also modulate
formation of a Tie complex or have one or more properties of a
target binding agent described herein and can be used in place of a
target binding protein.
[0516] Aptamers can be screened in vitro since a selected aptamer
can be recovered by standard nucleic acid amplification procedures.
The method can be enhanced, e.g., in later rounds of selection, by
splitting selected aptamers into pools and modifying each aptamer
in the pool with a detectable label such as a fluorophore. Pools
having aptamers that functionally alter the properties of the label
can be identified. Such pools can be repeatedly split and
reanalyzed to identify the individual aptamers with the desired
properties (see, e.g., Jhaveri et al. Nature Biotechnol.
18:1293).
[0517] In addition, aptamers can be screened for activity in vivo.
For example, shuffled nucleic acids can be cloned into an
expression vector that is introduced into cells. RNA aptamers
resulting from the expressed shuffled nucleic acids can be screened
for a biological activity. Cells having the activity can be
isolated and the expression vector for the selected RNA aptamer
recovered.
[0518] An important feature of therapeutic oligomers (e.g.,
aptamers) is the design of the backbone of the administered
oligomer. In some embodiments, the backbone contains
internucleoside linkages that are stable in vivo and is structured
such that the oligomer is resistant to endogenous nucleases, such
as nucleases that attack the phosphodiester linkage. At the same
time, the oligomer retains its ability to hybridize to the target
DNA or RNA (Agarwal, K. L. et al. (1979) Nucleic Acids Res. 6:3009;
Agarwal, S. et al. (1988) Proc. Natl. Acad. Sci USA 85:7079).
Modified oligonucleotides can be constructed using alternate
internucleotide linkages. Several of these exemplary linkages are
described in Uhlmann, E. and Peyman, A. (1990) Chemical Reviews
90:543-584. Among these are methylphosphonates (wherein one of the
phosphorus-linked oxygens has been replaced by methyl);
phosphorothioates (wherein sulphur replaces one of these oxygens)
and various amidates (wherein NH.sub.2 or an organic amine
derivative, such as morpholidates or piperazidates, replace an
oxygen). These substitutions confer enhanced stability. WO 91/15500
teaches various oligonucleotide analogs in which one or more of the
internucleotide linkages are replaced by a sulfur based linkage,
typically sulfamate diesters, which are isosteric and isoelectric
with the phosphodiester. WO 89/12060 similarly discloses linkages
containing sulfides, sulfoxides, and sulfones. WO 86/05518 suggests
a variant of stereoregular polymeric 3',5'linkages. U.S. Pat. No.
5,079,151 discloses a msDNA molecule of branched RNA linked to a
single strand DNA via a 2',5' phosphodiester linkage. U.S. Pat. No.
5,264,562 describes modified linkages of the formula
--Y'CX'.sub.2Y'-- wherein Y' is independently O or S and wherein
each X' is a stabilizing substituent and independently chosen.
Morpholino-type internucleotide linkages are described in U.S. Pat.
No. 5,034,506 and in some cases give rise to an increased affinity
of the oligomer for complementary target sequences. U.S. Pat. Nos.
5,264,562 5,596,086 disclose modified oligonucleotides having
modified nucleoside linkages which are capable of strong
hybridization to target RNA and DNA.
[0519] Treatments
[0520] Binding agents that bind to Tie1, Tie2, or Ang have
therapeutic and prophylactic utilities. For example, these binding
agents can be administered to cells in culture, e.g. in vitro or ex
vivo, or can be administered to a subject, e.g., in vivo, to treat,
prevent, and/or diagnose a variety of disorders, such as
endothelial cell disorders, blood vessel development disorders,
wound healing, inflammatory diseases and cancers, particularly
metastatic cancers. The term "treat" or "treatment" refers to the
application or administration of an agent, alone or in combination
with one or more other agents (e.g., a second agent) to a subject,
e.g., a patient, e.g., a patient who has a disorder (e.g., a
disorder as described herein), a symptom of a disorder or a
predisposition for a disorder, e.g., to cure, heal, alleviate,
relieve, alter, remedy, ameliorate, improve or affect the disorder,
the symptoms of the disorder or the predisposition toward the
disorder. Treating a cell refers to a reduction in an activity of a
cell, e.g., ability of an endothelial cell to form tubes or
vessels. A reduction does not necessarily require a total
elimination of activity, but a reduction, e.g., a statistically
significant reduction, in the activity or the number of the
cell.
[0521] As used herein, an amount of a target binding agent
effective to treat a disorder, or a "therapeutically effective
amount" refers to an amount of the binding agent which is
effective, upon single or multiple-dose administration to a
subject, in treating a cell, e.g., an endothelial cell (e.g., a
Tie1-expressing endothelial cell) or cancer cell (particularly a
metastatic cell thereof), or in prolonging curing, alleviating,
relieving or improving a subject with a disorder as described
herein beyond that expected in the absence of such treatment. In
some cases, a therapeutically effective amount can be ascertained
by evaluating the ability of the binding agent to reduce tumor size
of a xenograft in a nude mouse model relative to an untreated
control mouse. As used herein, "inhibiting the growth" of a tumor
or other neoplasm refers to slowing, interrupting, arresting or
stopping its growth and metastases and does not necessarily
indicate a total elimination of the neoplastic growth.
[0522] As used herein, an amount of an target-binding agent
effective to prevent a disorder, or a "a prophylactically effective
amount" of the binding agent refers to an amount of a target
binding agent, e.g., a Tie1-binding protein, e.g., a Tie1-binding
antibody described herein, which is effective, upon single- or
multiple-dose administration to the subject, for preventing or
delaying the occurrence of the onset or recurrence of a disorder,
e.g., an endothelial cell-related disorder, a blood vessel
development disorder, an inflammatory disease or a cancer.
[0523] Subjects that can be treated include human and non-human
animals. For example, the human can be a human patient having a
disorder characterized by abnormal cell proliferation or cell
differentiation. The term "non-human animals" includes all
vertebrates, e.g., non-mammals (such as chickens, amphibians,
reptiles) and mammals, such as non-human primates, sheep, dog, cow,
pig, etc.
[0524] A binding agent described herein can be used to reduce
angiogenesis in a subject, e.g., to treat a cancer (e.g., a solid
tumor) or an angiogenesis-associated disorder. The method includes
administering the binding to the subject, e.g., in an amount
effective to modulate angiogenesis, a symptom of the disorder, or
progression of the disorder. The agent (e.g., a Tie1-binding
protein, e.g., an anti-Tie1 antibody, e.g., E3) may be administered
multiple times (e.g., at least two, three, five, or ten times)
before a therapeutically effective amount is attained.
[0525] The binding agent, e.g., a Tie1 binding protein, can be used
to treat or prevent cancer. In one embodiment, reduction in Tie1
activity by a Tie1-binding protein can reduce or prevent
angiogenesis near and around the tumor, thereby reducing or
preventing tumor growth. In another embodiment, the neoplasia
includes endothelial or hematopoietic cells that are proliferating
abnormally. A Tie1-binding protein can be used to modulate the
cells of a cancer themselves, e.g., to kill or ablate a neoplastic
cell that expresses Tie1. For example, the cell is a hematopoietic
cell.
[0526] Examples of cancers that can be treated include, but are not
limited to, solid tumors, soft tissue tumors, and metastatic
lesions. Examples of solid tumors include malignancies, e.g.,
sarcomas, adenocarcinomas, and carcinomas, of the various organ
systems, such as those affecting lung, breast, lymphoid,
gastrointestinal (e.g., colon), and genitourinary tract (e.g.,
renal, urothelial cells), pharynx, prostate, ovary as well as
adenocarcinomas which include malignancies such as most colon
cancers, rectal cancer, renal-cell carcinoma, liver cancer,
non-small cell carcinoma of the lung, cancer of the small intestine
and so forth. Metastatic lesions of the aforementioned cancers can
also be treated or prevented using the Tie1 binding proteins and
other agents described herein.
[0527] Still further examples of solid tumors that can be treated
include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
gastrointestinal system carcinomas, colon carcinoma, pancreatic
cancer, breast cancer, genitourinary system carcinomas, ovarian
cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical cancer, endocrine system carcinomas, testicular tumor,
lung carcinoma, small cell lung carcinoma, non-small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0528] A Tie1-binding protein can also be used to inhibit the
proliferation of hyperplastic/neoplastic cells of hematopoietic
origin, e.g., cells arising from myeloid, lymphoid or erythroid
lineages, or precursor cells thereof, particularly such cells that
express Tie1 For instance, the binding proteins described herein
can be used for the treatment of various myeloid disorders
including, but not limited to, acute promyeloid leukemia (APML),
acute myelogenous leukemia (AML) and chronic myelogenous leukemia
(CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol.
11:267-97). Lymphoid malignancies which may be treated include, but
are not limited to acute lymphoblastic leukemia (ALL), which
includes B-lineage ALL and T-lineage ALL, chronic lymphocytic
leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia
(HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of
malignant lymphomas include non-Hodgkin's lymphoma and variants
thereof, peripheral T-cell lymphomas, adult T-cell
leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large
granular lymphocytic leukemia (LGF) and Hodgkin's disease. As Tie1
has been shown to be upregulated in acute myelogenous leukemia and
myelodysplastic syndrome (Verstovsek et al., 2001, Leuk, Lymphoma),
B cell chronic lymphocytic leukemia (Aguayo et al, 2001. Leukemia
Research 25(4):279-85.), binding proteins that interact with Tie1
can be used to detect, treat, or prevent these diseases.
[0529] Accordingly, a subject having or at risk for a hematopoietic
disorder, e.g., a hematopoietic cancer, can be treated by
administering a Tie1 binding protein, e.g. a Tie1 binding protein
that increases Tie1 homodimerization, or a binding protein that
antagonizes Tie complex formation. For example, the Tie1 binding
protein can be an anti-Tie1 antibody, e.g., an antibody described
herein. The administration of the binding protein can include
multiple administrations, e.g., to achieve a therapeutic
concentration using more than one dose. For example, the
administrations can be about once a week, every second or third
day, etc.
[0530] Methods of administering Tie1-binding proteins and other
agents are also described in "Pharmaceutical Compositions".
Suitable dosages of the molecules used will depend on the age and
weight of the subject and the particular drug used. The binding
proteins can be used as competitive agents to inhibit, reduce an
undesirable interaction, e.g., between a natural or pathological
agent and the Tie1.
[0531] In one embodiment, the Tie1-binding proteins are used to
inhibit (e.g., inhibit at least one activity of, reduce
proliferation, migration, growth or viability) of a cell, e.g., an
endothelial cell in vivo. The binding proteins can be used by
themselves or conjugated to an agent, e.g., a cytotoxic drug,
cytotoxin enzyme, or radioisotope. This method includes:
administering the binding protein alone or attached to a cytotoxic
drug, to a subject requiring such treatment.
[0532] Since the Tie1-binding proteins recognize Tie1-expressing
endothelial cells and can bind to endothelial cells that are
associated with (e.g., in proximity of or intermingled with) cancer
cells, e.g., cancerous lung, liver, colon, breast, ovarian,
epidermal, laryngeal, and cartilage cells, and particularly
metastatic cells thereof, Tie1-binding proteins can be used to
inhibit (e.g., inhibit at least one activity, reduce growth and
proliferation, or kill) any such cells and inhibit angiogenesis.
Reducing endothelial cell activity near a cancer can indirectly
inhibit (e.g., inhibit at least one activity, reduce growth and
proliferation, or kill) the cancer cells which may be dependent on
the endothelial cells for nutrients, growth signals and so
forth.
[0533] Alternatively, the binding proteins bind to cells in the
vicinity of the cancerous cells, but are sufficiently close to the
cancerous cells to directly or indirectly inhibit (e.g., inhibit at
least one activity, reduce growth and proliferation, or kill) the
cancers cells. Thus, the Tie1-binding proteins (e.g., modified with
a toxin, e.g., a cytotoxin) can be used to selectively inhibit
(e.g., kill or ablate cells in cancerous tissue (including the
cancerous cells themselves and endothelial cells associated with or
invading the cancer).
[0534] The binding proteins may be used to deliver a variety of
cytotoxic drugs including therapeutic drugs, a compound emitting
radiation, molecules of plants, fungal, or bacterial origin,
biological proteins, and mixtures thereof. The cytotoxic drugs can
be intracellularly acting cytotoxic drugs, such as toxins
short-range radiation emitters, e.g., short-range, high-energy
.alpha.-emitters.
[0535] To kill or ablate normal, benign hyperplastic, or cancerous
cells, a first binding protein is conjugated with a prodrug which
is activated only when in close proximity with a prodrug activator.
The prodrug activator is conjugated with a second binding protein,
preferably one which binds to a non-competing site on the target
molecule. Whether two binding proteins bind to competing or
non-competing binding sites can be determined by conventional
competitive binding assays. Exemplary drug-prodrug pairs are
described in Blakely et al., (1996) Cancer Research,
56:3287-3292.
[0536] The Tie1-binding proteins can be used directly in vivo to
eliminate antigen-expressing cells via natural complement-dependent
cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity
(ADCC). The binding proteins described herein can include
complement binding effector domain, such as the Fc portions from
IgG1, -2, or -3 or corresponding portions of IgM which bind
complement. In one embodiment, a population of target cells is ex
vivo treated with a binding agent described herein and appropriate
effector cells. The treatment can be supplemented by the addition
of complement or serum containing complement. Further, phagocytosis
of target cells coated with a binding protein described herein can
be improved by binding of complement proteins. In another
embodiment target, cells coated with the binding protein which
includes a complement binding effector domain are lysed by
complement.
[0537] Use of the therapeutic methods described herein to treat
cancers has a number of benefits. Tie1 expression may be induced in
response to hypoxic signals that can arise within the interior of a
tumor to stimulate changes in vasculature, including blood and
lymphatic vessels so as to increase nutrient and oxygen supply to
the tumor. Certain Tie1-binding antibodies (e.g., E3 and related
antibodies) may be particularly effective because they can inhibit
changes to tumor vasculature and may cause a decrease in
intra-tumor pressure. These agents may also be well suited as
therapeutics in situations in which conventional agents have
difficulty in penetrating into a tumor. Furthermore, Tie1 binding
proteins may leave hematopoiesis unaffected. Treatment can be
effectively monitored with clinical parameters. Alternatively,
these parameters can be used to indicate when such treatment should
be employed.
[0538] A Tie1 binding protein, e.g. a Tie1 binding protein that
increases Tie1 homodimerization, or a binding protein that
antagonizes Tie complex formation can be administered to a subject
to treat or prevent an inflammatory disorder, e.g., psoriasis or
rheumatoid arthritis.
[0539] Psoriasis. Psoriasis is a chronic skin disease,
characterized by scaling and inflammation. When psoriasis develops,
typically patches of skin thicken, redden, and become covered with
silvery scales, referred to as plaques. Psoriasis most often occurs
on the elbows, knees, scalp, lower back, face, palms, and soles of
the feet. The disease also may affect the fingernails, toenails,
and the soft tissues inside the mouth and genitalia. About 10
percent of people with psoriasis have joint inflammation that
produces symptoms of arthritis. Patients can be evaluated using a
static Physician Global Assessment (sPGA), and receive a category
score ranging from six categories between clear and very severe.
The score is based on plaque, scaling, and erythema. The
therapeutic methods herein can be used to achieve an improvement
for at least one of these indicia.
[0540] Rheumatoid arthritis ("RA") is a chronic inflammatory
disease that causes pain, swelling, stiffness, and loss of
function, primarily the joints. RA frequently begins in the
synovium, the membrane that surrounds a joint creating a protective
sac. In many individuals suffering from RA, leukocytes infiltrate
from the circulation into the synovium causing continuous abnormal
inflammation (e.g., synovitis). Consequently, the synovium becomes
inflamed, causing warmth, redness, swelling, and pain. The collagen
in the cartilage is gradually destroyed, narrowing the joint space
and eventually damaging bone. The inflammation causes erosive bone
damage in the affected area. During this process, the cells of the
synovium grow and divide abnormally, making the normally thin
synovium thick and resulting in a joint that is swollen and puffy
to the touch. RA can be assessed by a variety of clinical measures.
Some exemplary indicia include the total Sharp score (TSS), Sharp
erosion score, and the HAQ disability index. The therapeutic
methods herein can be used to achieve an improvement for at least
one of these indicia.
[0541] A Tie1 binding protein (e.g. a Tie1 binding protein that
increases Tie1 homodimerization) or a binding protein that
antagonizes Tie complex formation can be administered to a subject
to treat or prevent a retinal disorder, e.g., a proliferative
retinopathy, such as diabetic retinopathy, ischemic retinopathy, or
retinopathy of prematurity; choroidal neovascularization; lens
neovasculation; corneal neovascularization; iridial
neovascularization; or conjunctival neovascularization. The binding
protein can be used to reduce the risk of retinal detachment
associated with pathological ocular neovascularization. In some
cases, the binding protein is administered by subconjunctival
administration.
[0542] Combination Therapies
[0543] Binding proteins described herein can be administered in
combination with one or more of the other therapies for treating
cancers, including, but not limited to: surgery; radiation therapy,
and chemotherapy. For example, proteins that antagonize Tie complex
formation or that modulate Tie signalling activity (including,
e.g., proteins that promote Tie1 homodimerization and/or
phosphorylation) can also be used in combination with other
anti-cancer therapies, such as radiation therapy, chemotherapy,
surgery, or administration of a second agent. For example, the
second agent can be one that targets or negatively regulates the
VEGF signaling pathway. Examples of this latter class include VEGF
antagonists (e.g., anti-VEGF antibodies such as bevacizumab) and
VEGF receptor antagonists (e.g., anti-VEGF receptor antibodies).
One particularly combination includes bevacizumab. The combination
can further include 5-FU and leucovorin, and/or irinotecan.
[0544] The term "combination" refers to the use of the two or more
agents or therapies to treat the same patient, wherein the use or
action of the agents or therapies overlap in time. The agents or
therapies can be administered at the same time (e.g., as a single
formulation that is administered to a patient or as two separate
formulations administered concurrently) or sequentially in any
order. Sequential administrations are administrations that are
given at different times. The time between administration of the
one agent and another agent can be minutes, hours, days, or weeks.
The use of a Tie1 binding protein described herein can also be used
to reduce the dosage of another therapy, e.g., to reduce the
side-effects associated with another agent that is being
administered, e.g., to reduce the side-effects of an anti-VEGF
antibody such as bevacizumab. Accordingly, a combination can
include administering a second agent at a dosage at least 10, 20,
30, or 50% lower than would be used in the absence of the Tie1
binding protein.
[0545] In addition, a subject can be treated for an
angiogenesis-associated disorder by administering to the subject a
first and second agent. For example, the first agent modulates
early stage angiogenesis and the second agent modulates a
subsequent stage of angiogenesis or also modulates early stage
angiogenesis. The first and second agents can be administered using
a single pharmaceutical composition or can be administered
separately. In one embodiment, the first agent is a VEGF pathway
antagonist (e.g., an inhibitor of a VEGF (e.g., VEGF-A, -B, or -C)
or a VEGF receptor (e.g., KDR or VEGF receptor III (Flt4)) or a
bFGF pathway antagonist (e.g., an antibody that binds to bFGF or a
bFGF receptor). Other VEGF pathway antagonists are also described,
herein and elsewhere. In one embodiment, the second agent inhibits
or decreases assembly and stabilization of the blood vessels,
disrupts maintenance of blood or lymphatic vessels, or alters
distribution of lymphatic vessels in tumors. For example, the
second agent comprises inhibits a Tie complex formation or promotes
Tie1 homodimerization. For example, the second agent is a Tie1
binding protein described herein.
[0546] Once a tumor reaches a certain size (e.g., .about.1-2 mm),
the tumor requires new vasculature prior to increasing its mass. An
early stage of tumor angiogenesis can include a signal from the
tumor, e.g., secretion of VEGF, to stimulate the growth of new
blood vessels from the host and infiltration of the tumor by the
vessels. VEGF can, for example, stimulate proliferation of
endothelial cells that are then assembled into blood vessels. A
late stage of tumor angiogenesis can include a signal that leads to
the assembly and stabilization of the blood vessels. This assembly
and stabilization may involve interaction between the endothelial
cells and the pericytes that surround the endothelial cells of the
vessels. Tie1, for example, may play a role in the assembly and
stabilization of the vessels and in maintaining the association
between the pericytes and endothelial cells. Thus, an effective
therapy to treat angiogenesis-related disorders can involve a
combination of an agent that modulates an early stage angiogenesis
(e.g., VEGF pathway antagonists, e.g., anti-VEGF (e.g.,
bevacizumab) or anti-VEGF receptor (e.g., anti-KDR) antibodies; or
antagonists of other pro-angiogenic pathways, e.g., anti-bFGF
antibodies or anti-bFGF receptor (e.g., anti-bFGF receptor-1, -2,
-3) antibodies) and an agent that modulates a late stage of tumor
angiogenesis (e.g., antagonists of Tie1 (e.g., anti-Tie1 antibodies
(e.g., an antibody disclosed herein, e.g., an E3 antibody)), of
Tie2 (e.g., anti-Tie2 antibodies), or of Angs (e.g., anti-Ang
antibodies (e.g., anti-Ang2 antibodies) or anti-Ang2 peptides
(e.g., inhibitory Ang2 peptides)). One or more of these agents can
be used in combination. One or more of these agents may also be
used in combination with other anti-cancer therapies, such as
radiation therapy or chemotherapy.
[0547] Exemplary VEGF receptor antagonists include inhibitors of
VEGF receptor tyrosine kinase activity.
4-[4-(1-Amino-1-methylethyl)phenyl]-2-[4-(2-morpholin-4-yl-ethyl)phenylam-
ino]pyrimidine-5-carbonitrile (JNJ-17029259) is one of a structural
class of 5-cyanopyrimidines that are orally available, selective,
nanomolar inhibitors of the vascular endothelial growth factor
receptor-2 (VEGF-R2). Additional examples include:
PTK-787/ZK222584(Astra-Zeneca), SU5416, SU11248 (Pfizer), and
ZD6474
([N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy-
]quinazolin-4-amine]). Still other agents that can be used in
combination with Tie1-binding proteins are broad specificity
tyrosine kinase inhibitors, e.g., SU6668. See, e.g., Bergers, B. et
al. (2003) J. Clin. Invest. 111, 1287-1295.
[0548] The second agent or therapy can also be another anti-cancer
agent or therapy. Nonlimiting examples of anti-cancer agents
include, e.g., anti-microtubule agents, topoisomerase inhibitors,
antimetabolites, mitotic inhibitors, alkylating agents,
intercalating agents, agents capable of interfering with a signal
transduction pathway, agents that promote apoptosis, radiation, and
antibodies against other tumor-associated antigens (including naked
antibodies, immunotoxins and radioconjugates). Examples of the
particular classes of anti-cancer agents are provided in detail as
follows: antitubulin/antimicrotubule, e.g., paclitaxel,
vincristine, vinblastine, vindesine, vinorelbin, taxotere;
topoisomerase I inhibitors, e.g., irinotecan, topotecan,
camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin,
idarubicin, teniposide, amsacrine, epirubicin, merbarone,
piroxantrone hydrochloride; antimetabolites, e.g., 5-fluorouracil
(5-FU), methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine
phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin,
alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA,
pentostatin, 5-azacitidine, 5-Aza 2'-deoxycytidine, ara-A,
cladribine, 5-fluorouridine, FUDR, tiazofurin,
N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]--
2-thenoyl]-L-glutamic acid; alkylating agents, e.g., cisplatin,
carboplatin, mitomycin C, BCNU=Carmustine, melphalan, thiotepa,
busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide
phosphate, cyclophosphamide, nitrogen mustard, uracil mustard,
pipobroman, 4-ipomeanol; agents acting via other mechanisms of
action, e.g., dihydrolenperone, spiromustine, and desipeptide;
biological response modifiers, e.g., to enhance anti-tumor
responses, such as interferon; apoptotic agents, such as
actinomycin D; and anti-hormones, for example anti-estrogens such
as tamoxifen or, for example antiandrogens such as
4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluorometh-
yl)propionanilide.
[0549] A combination therapy can include administering an agent
that reduces the side effects of other therapies. The agent can be
an agent that reduces the side effects of anti-cancer treatments.
For example, the agent can be leucovorin.
[0550] Combination therapies that include administering a Tie1
binding protein or other binding protein described herein can also
be used to treat a subject having or at risk for another
angiogenesis related disorder (e.g., a disorder other than cancer,
e.g., disorders that include undesired endothelial cell
proliferation or undesirable inflammation, e.g., rheumatoid
arthritis.
[0551] Diagnostic Uses
[0552] Binding proteins that bind to Tie1 (e.g., antibodies, e.g.,
an antibody described herein) have in vitro and in vivo diagnostic,
therapeutic and prophylactic utilities.
[0553] In one aspect, the invention provides a diagnostic method
for detecting the presence of a Tie1, in vitro (e.g., a biological
sample, such as tissue, biopsy, e.g., a cancerous tissue) or in
vivo (e.g., in vivo imaging in a subject). The method includes: (i)
contacting a sample with Tie1-binding protein; and (ii) detecting
formation of a complex between the Tie1-binding protein and the
sample. The method can also include contacting a reference sample
(e.g., a control sample) with the binding protein, and determining
the extent of formation of the complex between the binding protein
and the sample relative to the same for the reference sample. A
change, e.g., a statistically significant change, in the formation
of the complex in the sample or subject relative to the control
sample or subject can be indicative of the presence of Tie1 in the
sample. The Tie1-binding protein can be directly or indirectly
labeled with a detectable substance to facilitate detection of the
bound or unbound antibody. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials.
[0554] Complex formation between the Tie1-binding protein and Tie1
can be detected by measuring or visualizing either the binding
protein bound to the Tie1 or unbound binding protein. Conventional
detection assays can be used, e.g., an enzyme-linked immunosorbent
assays (ELISA), a radioimmunoassay (RIA) or tissue
immunohistochemistry. Further to labeling the Tie1-binding protein,
the presence of Tie1 can be assayed in a sample by a competition
immunoassay utilizing standards labeled with a detectable substance
and an unlabeled Tie1-binding protein. In one example of this
assay, the biological sample, the labeled standards and the Tie1
binding agent are combined and the amount of labeled standard bound
to the unlabeled binding protein is determined. The amount of Tie1
in the sample is inversely proportional to the amount of labeled
standard bound to the Tie1 binding agent.
[0555] Fluorophore and chromophore labeled binding proteins can be
prepared. Since antibodies and other proteins absorb light having
wavelengths up to about 310 nm, the fluorescent moieties should be
selected to have substantial absorption at wavelengths above 310 nm
and preferably above 400 nm. A variety of suitable fluorescers and
chromophores are described by Stryer (1968) Science, 162:526 and
Brand, L. et al. (1972) Annual Review of Biochemistry, 41:843-868.
The binding proteins can be labeled with fluorescent chromophore
groups by conventional procedures such as those disclosed in U.S.
Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. One group of
fluorescers having a number of the desirable properties described
above is the xanthene dyes, which include the fluoresceins and
rhodamines. Another group of fluorescent compounds are the
naphthylamines. Once labeled with a fluorophore or chromophore, the
binding protein can be used to detect the presence or localization
of the Tie1 in a sample, e.g., using fluorescent microscopy (such
as confocal or deconvolution microscopy).
[0556] Histological Analysis. Immunohistochemistry can be performed
using the binding proteins described herein. For example, in the
case of an antibody, the antibody can synthesized with a label
(such as a purification or epitope tag), or can be detectably
labeled, e.g., by conjugating a label or label-binding group. For
example, a chelator can be attached to the antibody. The antibody
is then contacted to a histological preparation, e.g., a fixed
section of tissue that is on a microscope slide. After an
incubation for binding, the preparation is washed to remove unbound
antibody. The preparation is then analyzed, e.g., using microscopy,
to identify if the antibody bound to the preparation. The method
can be used to evaluate an endothelial cell or tissue formed by
endothelial cells, e.g., blood vessels. The antibody (or other
polypeptide or peptide) can be unlabeled at the time of binding.
After binding and washing, the antibody is labeled in order to
render it detectable.
[0557] Protein Arrays. The Tie1-binding protein can also be
immobilized on a protein array. The protein array can be used as a
diagnostic tool, e.g., to screen medical samples (such as isolated
cells, blood, sera, biopsies, and the like). Of course, the protein
array can also include other binding proteins, e.g., that bind to
Tie1 or to other target molecules, such as hyaluronic acid.
[0558] Methods of producing polypeptide arrays are described, e.g.,
in De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et
al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res.
28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763;
WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be
spotted at high speed, e.g., using commercially available robotic
apparati. The array substrate can be, for example, nitrocellulose,
plastic, glass, e.g., surface-modified glass. The array can also
include a porous matrix, e.g., acrylamide, agarose, or another
polymer.
[0559] For example, the array can be an array of antibodies, e.g.,
as described in De Wildt, supra. Cells that produce the binding
proteins can be grown on a filter in an arrayed format. Polypeptide
production is induced, and the expressed polypeptides are
immobilized to the filter at the location of the cell. A protein
array can be contacted with a labeled target to determine the
extent of binding of the target to each immobilized polypeptide. If
the target is unlabeled, a sandwich method can be used, e.g., using
a labeled probed, to detect binding of the unlabeled target.
Information about the extent of binding at each address of the
array can be stored as a profile, e.g., in a computer database. The
protein array can be produced in replicates and used to compare
binding profiles, e.g., of a target and a non-target.
[0560] FACS. (Fluorescent Activated Cell Sorting). The
target-binding protein can be used to label cells, e.g., cells in a
sample (e.g., a patient sample). The binding protein can also be
attached (or attachable) to a fluorescent compound. The cells can
then be sorted using fluorescent activated cell sorted (e.g., using
a sorter available from Becton Dickinson Immunocytometry Systems,
San Jose Calif.; see also U.S. Pat. No. 5,627,037; 5,030,002; and
5,137,809). As cells pass through the sorter, a laser beam excites
the fluorescent compound while a detector counts cells that pass
through and determines whether a fluorescent compound is attached
to the cell by detecting fluorescence. The amount of label bound to
each cell can be quantified and analyzed to characterize the
sample.
[0561] The sorter can also deflect the cell and separate cells
bound by the binding protein from those cells not bound. The
separated cells can be cultured and/or characterized.
[0562] In Vivo Imaging. In still another embodiment, the invention
provides a method for detecting the presence of a Tie1-expressing
cancerous tissues in vivo. The method includes: administering the
Tie1-binding protein to a subject; and detecting the Tie1-binding
protein in the subject. The detecting can include determining
location or time of formation of the complex. The method can
include scanning or otherwise imaging the subject, e.g., a region
of the subject's body. Another method includes (i) administering to
a subject (e.g., a patient having a cancer or neoplastic disorder)
a Tie1-binding antibody, conjugated to a detectable marker; (ii)
exposing the subject to a means for detecting said detectable
marker to the Tie1-expressing tissues or cells. For example, the
method can be used visualize blood vessels or the location of
endothelial cells, e.g., Tie1-expressing endothelial cells. The
subject can be imaged, e.g., by NMR or other tomographic means.
[0563] Examples of labels useful for diagnostic imaging include
radiolabels such as .sup.131I, .sup.111In, .sup.123I, .sup.99mTc,
.sup.32P, .sup.125I, .sup.3H, .sup.14.sub.C, and .sup.188Rh,
fluorescent labels such as fluorescein and rhodamine, nuclear
magnetic resonance active labels, positron emitting isotopes
detectable by a positron emission tomography ("PET") scanner,
chemiluminescers such as luciferin, and enzymatic markers such as
peroxidase or phosphatase. Short-range radiation emitters, such as
isotopes detectable by short-range detector probes can also be
employed. The binding protein can be labeled with such reagents
using known techniques. For example, see Wensel and Meares (1983)
Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. for
techniques relating to the radiolabeling of antibodies and D.
Colcher et al. (1986) Meth. Enzymol. 121: 802-816.
[0564] A radiolabeled binding protein can also be used for in vitro
diagnostic tests. The specific activity of an isotopically-labeled
protein depends upon the half-life, the isotopic purity of the
radioactive label, and how the label is incorporated into the
protein.
[0565] Effective imaging agents for tumor-associated
neo-vasculature are needed. Tie1 is up regulated on
tumor-associated vasculature. The binding proteins described herein
can be used to image such vasculature.
The binding proteins described herein can be used for imaging in
several ways. A binding protein can be physically associated, e.g.,
coupled to a chelator for imaging agents such as .sup.99mTc,
.sup.186Re, or .sup.188Re. .sup.99mTc and .sup.188Re emit gamma
rays suitable for single photon emission computer tomography
(SPECT) imaging. Radioactive fluorine (.sup.18F), indium
(.sup.111In), iodine (.sup.123I, .sup.131I), gallium (.sup.68Ga,
.sup.67Ga), carbon (.sup.11C), thallium (.sup.201Tl), and other
elements may be used as imaging agents.
[0566] The binding proteins can also be attached, covalently or
non-covalently, to a particle, e.g., a nano-particle, that includes
a radionuclide or spin labels suitable for use as an imaging agent.
The binding proteins can be linked to a spin label that would allow
imaging through MRI. Botnar et al. (Circulation. (2004)
109:2023-2029.) describe MRI imaging using an exemplary
gadolinium-labeled peptide. The binding proteins described herein
can be similarly labeled for imaging.
[0567] Chen et al. (J. Nucl. Med., (2004) 45:1776-1783) showed that
coupling a small PEG molecule (average molecular weight 3.4 KDa)
improved that pharmacodynamics of an
.alpha..sub.v.beta..sub.3-binding peptide. Binding proteins (e.g.,
Tie1, Tie2, or Ang binding proteins) can be coupled to PEG
molecules to adjust the clearance rate and pathway.
[0568] Positron Emission Tomography (PET) can be used with imaging
agents such as positron emitters such as .sup.64Cu and .sup.18F.
These isotopes are becoming more readily available. .sup.64Cu can
be captured in the chelator DOTA. DOTA derivatives can be
covalently linked to proteins. In one embodiment, one or more DOTA
derivatives are attached to a binding protein (e.g., a Fab) through
a lysine side group.
[0569] Fabs are useful binding agents for imaging because they: a)
clear from the system fairly raipdly, allowing imaging within a few
hours of injection, and b) penetrate tumors efficiently.
[0570] Fabs that bind to Tie1, Tie2, or Ang can be produced, e.g.,
in E. coli or in eukaryotic cells. The Fabs can be purified by
chromatography over protein A. Ion exchange chromatography can also
be used. For use in imaging, covalent attachment of a chelating
group suitable to the desired radionuclide or other imaging agent
allows the Fab to be labeled at the time of use. The Fabs can also
have spin labels attached to allow MRI imaging. Fabs can also be
attached to particles (e.g., nano-particles) that include a
radionuclide or spin label suitable for imaging. In particular
embodiments, Fabs may be coupled to PEG molecules to adjust the
rate and pathway of clearance. In other embodiments, the Fabs are
not coupled to PEG, e.g., to maintain their rapid clearance
properties.
[0571] Procedures for labeling polypeptides with the radioactive
isotopes (such as .sup.14C, .sup.3H, .sup.35S, .sup.125I, .sup.32P,
.sup.131I) are generally known. For example, tritium labeling
procedures are described in U.S. Pat. No. 4,302,438. Iodinating,
tritium labeling, and .sup.35S labeling procedures, e.g., as
adapted for murine monoclonal antibodies, are described, e.g., by
Goding, J. W. (Monoclonal antibodies: principles and practice:
production and application of monoclonal antibodies in cell
biology, biochemistry, and immunology 2nd ed. London; Orlando:
Academic Press, 1986. pp 124-126) and the references cited therein.
Other procedures for iodinating polypeptides, such as antibodies,
are described by Hunter and Greenwood (1962) Nature 144:945, David
et al. (1974) Biochemistry 13:1014-1021, and U.S. Pat. Nos.
3,867,517 and 4,376,110. Radiolabeling elements which are useful in
imaging include .sup.123I, .sup.131I, .sup.111In, and .sup.99mTc,
for example. Procedures for iodinating antibodies are described by
Greenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J.
(1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971)
Immunochemistry 289-297. Procedures for .sup.99mTc-labeling are
described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor
Imaging: The Radioimmunochemical Detection of Cancer, New York:
Masson 111-123 (1982) and the references cited therein. Procedures
suitable for .sup.111In-labeling antibodies are described by
Hnatowich, D. J. et al. (1983) J. Immul. Methods, 65:147-157,
Hnatowich, D. et al. (1984) J. Applied Radiation, 35:554-557, and
Buckley, R. G. et al. (1984) F.E.B.S. 166:202-204.
[0572] In the case of a radiolabeled binding protein, the binding
protein is administered to the patient, is localized to the tumor
bearing the antigen with which the binding protein reacts, and is
detected or "imaged" in vivo using known techniques such as
radionuclear scanning using e.g., a gamma camera or emission
tomography. See e.g., A. R. Bradwell et al., "Developments in
Antibody Imaging", Monoclonal Antibodies for Cancer Detection and
Therapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press
1985). Alternatively, a positron emission transaxial tomography
scanner, such as designated Pet VI located at Brookhaven National
Laboratory, can be used where the radiolabel emits positrons (e.g.,
.sup.11C, .sup.18F, .sup.15O, and .sup.13N).
[0573] MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses
NMR to visualize internal features of living subject, and is useful
for prognosis, diagnosis, treatment, and surgery. MRI can be used
without radioactive tracer compounds for obvious benefit. Some MRI
techniques are summarized in EP-A-0 502 814. Generally, the
differences related to relaxation time constants T1 and T2 of water
protons in different environments are used to generate an image.
However, these differences can be insufficient to provide sharp
high resolution images.
[0574] The differences in these relaxation time constants can be
enhanced by contrast agents. Examples of such contrast agents
include a number of magnetic agents paramagnetic agents (which
primarily alter T1) and ferromagnetic or superparamagnetic (which
primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA
chelates) can be used to attach (and reduce toxicity) of some
paramagnetic substances (e.g., Fe.sup.+3, Mn.sup.+2, Gd.sup.+3).
Other agents can be in the form of particles, e.g., less than 10
.mu.m to about 10 nM in diameter). Particles can have
ferromagnetic, antiferromagnetic or superparamagnetic properties.
Particles can include, e.g., magnetite (Fe.sub.3O.sub.4),
.gamma.-Fe.sub.2O.sub.3, ferrites, and other magnetic mineral
compounds of transition elements. Magnetic particles may include:
one or more magnetic crystals with and without nonmagnetic
material. The nonmagnetic material can include synthetic or natural
polymers (such as sepharose, dextran, dextrin, starch and the
like
[0575] The target-binding proteins can also be labeled with an
indicating group containing of the NMR-active .sup.19F atom, or a
plurality of such atoms inasmuch as (i) substantially all of
naturally abundant fluorine atoms are the .sup.19F isotope and,
thus, substantially all fluorine-containing compounds are
NMR-active; (ii) many chemically active polyfluorinated compounds
such as trifluoracetic anhydride are commercially available at
relatively low cost, and (iii) many fluorinated compounds have been
found medically acceptable for use in humans such as the
perfluorinated polyethers utilized to carry oxygen as hemoglobin
replacements. After permitting such time for incubation, a whole
body MRI is carried out using an apparatus such as one of those
described by Pykett (1982) Scientific American, 246:78-88 to locate
and image cancerous tissues.
[0576] Information obtained from evaluating an target-binding
protein, e.g., a binding protein described herein, can be recorded
on machine-compatible media, e.g., computer readable or computer
accessible media. The information can be stored as a computer
representation, e.g., in a database (e.g., in the case of imaging
using a binding protein, a database of images for one or a
plurality of subjects). The term "computer representation" refers
to information which is in a form that can be manipulated by a
computer. The act of storing a computer representation refers to
the act of placing the information in a form suitable for
manipulation by a computer.
[0577] Also within the scope of the invention are kits including
the binding protein that binds to Tie1 and instructions for
diagnostic use, e.g., the use of the target-binding protein (e.g.,
antibody or antigen-binding fragment thereof, or other polypeptide
or peptide) to detect Tie1, in vitro, e.g., in a sample, e.g., a
biopsy or cells from a patient having a cancer or neoplastic
disorder, or in vivo, e.g., by imaging a subject. The kit can
further contain a least one additional reagent, such as a label or
additional diagnostic agent. For in vivo use the binding protein
can be formulated as a pharmaceutical composition.
[0578] The following examples are not to be construed as
limiting.
EXAMPLES
Example 1
Tie1 Sequences
[0579] An exemplary Tie1 amino acid sequence (SEQ ID NO:2) is as
follows:
TABLE-US-00002 MVWRVPPFLLPILFLASHVGAAVDLTLLANLRLTDPQRFFLTCVSGEAGA
GRGSDAWGPPLLLEKDDRIVRTPPGPPLRLARNGSHQVTLRGFSKPSDLV
GVFSCVGGAGARRTRVIYVHNSPGAHLLPDKVTHTVNKGDTAVLSARVHK
EKQTDVIWKSNGSYFYTLDWHEAQDGRFLLQLPNVQPPSSGIYSATYLEA
SPLGSAFFRLIVRGCGAGRWGPGCTKECPGCLHGGVCHDHDGECVCPPGF
TGTRCEQACREGRFGQSCQEQCPGISGCRGLTFCLPDPYGCSCGSGWRGS
QCQEACAPGHFGADCRLQCQCQNGGTCDRFSGCVCPSGWHGVHCEKSDRI
PQILNMASELEFNLETMPRINCAAAGNPFPVRGSIELRKPDGTVLLSTKA
IVEPEKTTAEFEVPRLVLADSGFWECRVSTSGGQDSRRFKVNVKVPPVPL
AAPRLLTKQSRQLVVSPLVSFSGDGPISTVRLHYRPQDSTMDWSTIVVDP
SENVTLMNLRPKTGYSVRVQLSRPGEGGEGAWGPPTLMTTDCPEPLLQPW
LEGWHVEGTDRLRVSWSLPLVPGPLVGDGFLLRLWDGTRGQERRENVSSP
QARTALLTGLTPGTHYQLDVQLYHCTLLGPASPPAHVLLPPSGPPAPRHL
HAQALSDSEIQLTWKHPEALPGPISKYVVEVQVAGGAGDPLWIDVDRPEE
TSTIIRGLNASTRYLFRMRASIQGLGDWSNTVEESTLGNGLQAEGPVQES
RAAEEGLDQQLILAVVGSVSATCLTILAALLTLVCIRRSCLHRRRTFTYQ
SGSGEETILQFSSGTLTLTRRPKLQPEPLSYPVLEWEDITFEDLIGEGNF
GQVIRAMIKKDGLKMNAAIKMLKEYASENDHRDFAGELEVLCKLGHHPNI
INLLGACKNRGYLYIAIEYAPYGNLLDFLRKSRVLETDPAFAREHGTAST
LSSRQLLRFASDAANGMQYLSEKQFIHRDLAARNVLVGENLASKIADFGL
SRGEEVYVKKTMGRLPVRWMAIESLNYSVYTTKSDVWSFGVLLWEIVSLG
GTPYCGMTCAELYEKLPQGYRMEQPRNCDDEVYELMRQCWRDRPYERPPF
AQIALQLGRMLEARKAYVNMSLFENFTYAGIDATAEEA
[0580] An exemplary nucleic acid sequence (SEQ ID NO:1) that
encodes Tie1 is as follows:
TABLE-US-00003 atggtctggc gggtgccccc tttcttgctc cccatcctct
tcttggcttc tcatgtgggc 60 gcggcggtgg acctgacgct gctggccaac
ctgcggctca cggaccccca gcgcttcttc 120 ctgacttgcg tgtctgggga
ggccggggcg gggaggggct cggacgcctg gggcccgccc 180 ctgctgctgg
agaaggacga ccgtatcgtg cgcaccccgc ccgggccacc cctgcgcctg 240
gcgcgcaacg gttcgcacca ggtcacgctt cgcggcttct ccaagccctc ggacctcgtg
300 ggcgtcttct cctgcgtggg cggtgctggg gcgcggcgca cgcgcgtcat
ctacgtgcac 360 aacagccctg gagcccacct gcttccagac aaggtcacac
acactgtgaa caaaggtgac 420 accgctgtac tttctgcacg tgtgcacaag
gagaagcaga cagacgtgat ctggaagagc 480 aacggatcct acttctacac
cctggactgg catgaagccc aggatgggcg gttcctgctg 540 cagctcccaa
atgtgcagcc accatcgagc ggcatctaca gtgccactta cctggaagcc 600
agccccctgg gcagcgcctt ctttcggctc atcgtgcggg gttgtggggc tgggcgctgg
660 gggccaggct gtaccaagga gtgcccaggt tgcctacatg gaggtgtctg
ccacgaccat 720 gacggcgaat gtgtatgccc ccctggcttc actggcaccc
gctgtgaaca ggcctgcaga 780 gagggccgtt ttgggcagag ctgccaggag
cagtgcccag gcatatcagg ctgccggggc 840 ctcaccttct gcctcccaga
cccctatggc tgctcttgtg gatctggctg gagaggaagc 900 cagtgccaag
aagcttgtgc ccctggtcat tttggggctg attgccgact ccagtgccag 960
tgtcagaatg gtggcacttg tgaccggttc agtggttgtg tctgcccctc tgggtggcat
1020 ggagtgcact gtgagaagtc agaccggatc ccccagatcc tcaacatggc
ctcagaactg 1080 gagttcaact tagagacgat gccccggatc aactgtgcag
ctgcagggaa ccccttcccc 1140 gtgcggggca gcatagagct acgcaagcca
gacggcactg tgctcctgtc caccaaggcc 1200 attgtggagc cagagaagac
cacagctgag ttcgaggtgc cccgcttggt tcttgcggac 1260 agtgggttct
gggagtgccg tgtgtccaca tctggcggcc aagacagccg gcgcttcaag 1320
gtcaatgtga aagtgccccc cgtgcccctg gctgcacctc ggctcctgac caagcagagc
1380 cgccagcttg tggtctcccc gctggtctcg ttctctgggg atggacccat
ctccactgtc 1440 cgcctgcact accggcccca ggacagtacc atggactggt
cgaccattgt ggtggacccc 1500 agtgagaacg tgacgttaat gaacctgagg
ccaaagacag gatacagtgt tcgtgtgcag 1560 ctgagccggc caggggaagg
aggagagggg gcctgggggc ctcccaccct catgaccaca 1620 gactgtcctg
agcctttgtt gcagccgtgg ttggagggct ggcatgtgga aggcactgac 1680
cggctgcgag tgagctggtc cttgcccttg gtgcccgggc cactggtggg cgacggtttc
1740 ctgctgcgcc tgtgggacgg gacacggggg caggagcggc gggagaacgt
ctcatccccc 1800 caggcccgca ctgccctcct gacgggactc acgcctggca
cccactacca gctggatgtg 1860 cagctctacc actgcaccct cctgggcccg
gcctcgcccc ctgcacacgt gcttctgccc 1920 cccagtgggc ctccagcccc
ccgacacctc cacgcccagg ccctctcaga ctccgagatc 1980 cagctgacat
ggaagcaccc ggaggctctg cctgggccaa tatccaagta cgttgtggag 2040
gtgcaggtgg ctgggggtgc aggagaccca ctgtggatag acgtggacag gcctgaggag
2100 acaagcacca tcatccgtgg cctcaacgcc agcacgcgct acctcttccg
catgcgggcc 2160 agcattcagg ggctcgggga ctggagcaac acagtagaag
agtccaccct gggcaacggg 2220 ctgcaggctg agggcccagt ccaagagagc
cgggcagctg aagagggcct ggatcagcag 2280 ctgatcctgg cggtggtggg
ctccgtgtct gccacctgcc tcaccatcct ggccgccctt 2340 ttaaccctgg
tgtgcatccg cagaagctgc ctgcatcgga gacgcacctt cacctaccag 2400
tcaggctcgg gcgaggagac catcctgcag ttcagctcag ggaccttgac acttacccgg
2460 cggccaaaac tgcagcccga gcccctgagc tacccagtgc tagagtggga
ggacatcacc 2520 tttgaggacc tcatcgggga ggggaacttc ggccaggtca
tccgggccat gatcaagaag 2580 gacgggctga agatgaacgc agccatcaaa
atgctgaaag agtatgcctc tgaaaatgac 2640 catcgtgact ttgcgggaga
actggaagtt ctgtgcaaat tggggcatca ccccaacatc 2700 atcaacctcc
tgggggcctg taagaaccga ggttacttgt atatcgctat tgaatatgcc 2760
ccctacggga acctgctaga ttttctgcgg aaaagccggg tcctagagac tgacccagct
2820 tttgctcgag agcatgggac agcctctacc cttagctccc ggcagctgct
gcgtttcgcc 2880 agtgatgcgg ccaatggcat gcagtacctg agtgagaagc
agttcatcca cagggacctg 2940 gctgcccgga atgtgctggt cggagagaac
ctagcctcca agattgcaga cttcggcctt 3000 tctcggggag aggaggttta
tgtgaagaag acgatggggc gtctccctgt gcgctggatg 3060 gccattgagt
ccctgaacta cagtgtctat accaccaaga gtgatgtctg gtcctttgga 3120
gtccttcttt gggagatagt gagccttgga ggtacaccct actgtggcat gacctgtgcc
3180 gagctctatg aaaagctgcc ccagggctac cgcatggagc agcctcgaaa
ctgtgacgat 3240 gaagtgtacg agctgatgcg tcagtgctgg cgggaccgtc
cctatgagcg accccccttt 3300 gcccagattg cgctacagct aggccgcatg
ctggaagcca ggaaggccta tgtgaacatg 3360 tcgctgtttg agaacttcac
ttacgcgggc attgatgcca cagctgagga ggcctga 3417
Example 2
Selection and Primary Screening
[0581] We have used phage display to select Tie1-specific
antibodies from a very large phage library that displays
immunoglobulins as Fab fragments. To isolate antibodies specific to
Tie1, a phage displayed Fab antibody library was selected against
the Tie1 extracellular domain fused to human Fc or to a histidine
purification tag.
[0582] Selection in solution was done using biotin labelled antigen
which was captured on streptavidin coated magnetic beads
(M-280-DYNAL.RTM.). Selection on cells expressing Tie1 was
performed using a KINGFISHER.TM. automated magnetic bead capture
device. Selection on immobilized antigen was performed using
Tie1-Fc coated onto immunotubes. Several selection strategies were
used :
[0583] Strategy 1: Round 1 (500 mM biotin labelled Tie1/magnetic
beads), Round 2 (1.times.10.sup.7 Tie1 expressing
cells/Kingfisher), Round 3 (1.times.10.sup.7 Tie1 expressing
cells/Kingfisher)
[0584] Strategy 2: Round 1 (500 mM biotin labelled Tie1/magnetic
beads), Round 2 (1.times.10.sup.7 Tie1 expressing
cells/KINGFISHER.TM.), (300 mM biotin labelled Tie1/magnetic
beads)
[0585] Strategy 3: Round 1 (Tie1 Fc coated immunotubes at 5
.mu.g/ml), Round 2 (Tie1-Fc coated immunotubes), Round 3 (Tie1 Fc
coated immunotubes plus depletion with human IgG).
[0586] Library members recovered from the selection strategies were
tested for antigen binding in phage ELISA. Each isolate was tested
for binding to coated Tie1 Fc. Strategy 1 did not identify any
binding clones whereas strategy 2 identified 13 positive clones
(n=95). Strategy 3 identified 86 binding clones (n=95).
[0587] Sequence analysis of the selected clones were grouped on the
basis of the CDR3 selected of the heavy chain and resulted in 23
different antibodies with unique VH-CDR3 sequences.
[0588] We reformatted the selected Fabs as completely human
antibodies by recloning the VH and VL coding sequences from the
display library vector into two vectors of a mammalian expression
vector system. These vectors contain the human kappa constant
domain and the human gamma-1 heavy chain constant region. The
vectors were co-transfected into mammalian CHO-K1 cells for
expression and production of the corresponding complete IgGs. These
antibodies were characterized using several assays as described
below, including: 1. Western blotting and immunoprecipitation of
Tie1 transfected cells and primary human endothelial cells; 2.
Immunofluorescence of Tie1 transfected cells and primary human
endothelial cells; 3. Stimulation and inhibition of Tie1 in BaF3
cells and primary human endothelial cells; and 4. Immunostaining of
human tissues.
[0589] We identified 23 antibodies that interact with Tie1. See
FIGS. 7-36. After sequence confirmation of the reformatted clones
they were used in a transient transfection of HEK293T cells. After
growth the IgG was purified from culture supernatants using a
protein A column. The quality of purified IgG1 was determined using
SDS-PAGE.
[0590] The specificity of the Tie1 specific IgG's can be determined
in a whole cell ELISA on mouse lung microvascular endothelial cells
(LEII) and LEII-Tie1 cells transfected with a Tie1 expression
construct. Cells are seeded into 96 well plates at a density of
10,000 cells/well and were fixed using 4% paraformaldehyde.
Staining and detection of binding of IgG1 to LEII cells are
detected using standard labelling with a HRP conjugated rabbit anti
human HRP and TMB staining. Binding of purified IgG1 to LEII-Tie1
transfected cells can also be corrected for Tie1 protein that is
expressed endogenously. Alternatively cells that have little or no
endogenous Tie1 can be used for the analysis.
[0591] At least one of the binding antibodies--E3--functions as a
Tie1 activating antibody in the BaF3 cell bioassay. We studied Tie1
phosphorylation in response to E3 IgG treatment in transiently
transfected COSI cells and human primary endothelial cells. Our
results indicate that E3 IgG activates the Tie1 receptor. The BaF3
cell bioassay (also referred to as the "Tie1/EpoR chimericBAF cell
assay" may provide an indication of a ligand's ability to
cross-link the Tie1 receptor. Because the assay is artificial,
crosslinking of the non-naturally occurring Tie-Epo fusion proteins
may or may not be predictive of a ligand's ability to modulate in
vivo function.
[0592] E3 can be used, instead of possible natural ligands to
characterize several functions of Tie1 in vitro and in vivo. The
region of Tie1 which interacts with E3 can be the target for small
molecular weight compounds for Tie1 activation or inhibition.
[0593] Although E3 functions in one particular Tie1 activating
assay, E3 and other positives in this assay may also have
inhibitory effect as to other functions or in other contexts. For
example, E3 can inhibit tube formation by HUVEC cells. See
below.
[0594] In addition, we found two antibodies that inhibit the
survival effect conferred by E3 in the BaF3 cell bioassay. These
two antibodies may inhibit dimerization of Tie1 induced by E3 in
the BaF3 assay. Two antibodies, B2 and D11, completely blocked the
viability of Tie1/EpoR cells when used in combination with E3.
[0595] Methods
[0596] Cell culture. COS1 cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum (FCS), glutamine and antibiotics. The murine BaF3 pre-B
lymphocytes were cultured in DMEM supplemented with 10% FCS,
glutamine, antibiotics and 2 ng/ml interleukin-3 (Calbiochem).
Human dermal microvascular endothelial cells (HDMVECs), obtained
from PromoCell (Heidelberg, Germany) were cultured in endothelial
cell medium provided by the supplier and used at passages 4-7.
[0597] Western blotting and immunoprecipitation. COSI cells were
transfected with pcDNA3-Tie1-V5 (1 .mu.g DNA per 10 cm cell culture
plate) using FUGENE 6 (Roche) according to manufacturer's
instruction and incubated for 48 h before stimulation. For
immunoprecipitation, Tie1 transfected cells and HMVEC cells were
lysed in DOC-RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl,
1% Triton-X-100, 0.1% SDS, 1% DOC, 10 mM EDTA) supplemented with
aprotinin, leupeptin, PMSF and sodium vanadate. Immunoprecipitation
was carried out from equal amount of cell lysates by incubating
with polyclonal anti-human Tie1 antibodies (R&D), monoclonal
anti-V5 antibodies (Invitrogen) or altogether 23 anti-Tie1
antibodies (1 .mu.g/ml) for 1 to 2 h followed by incubation with
protein G-Sepharose (Amersham Pharmacia Biotech AB) for 1 h. The
immunoprecipitates were washed twice with PBS-T and twice with PBS,
followed by elution with the Laemmli buffer and separation in 8%
SDS-PAGE. The blots were probed with the 23 anti-Tie1 antibodies (5
.mu.g/ml) and subsequently anti-human Fc antibodies conjugated with
HRP.
[0598] Immunofluorescence staining. COS1 cells on the glass
coverslips were transiently transfected with pcDNA3-Tie1-V5 (the
V5-epitope was added to the 3` terminus of pcDNA3-Tie1) (1 .mu.g
DNA per 10 cm cell culture plate) using FUGENE.TM. 6 (Roche)
according to manufacturer's instruction and incubated for 48 h
before staining. Cells were fixed in 4% paraformaldehyde for 10 min
at 4.degree. C. If required, the cells were permeabilized with 0.2%
Triton X-100 in PBS for 5 min. Unspecific binding sites were
blocked by incubation with 1% BSA in PBS for 30 min. The cells were
then stained with anti-Tie1 antibodies (5 .mu.g/ml) and anti-V5
antibodies for 1 h at room temperature, followed by incubation with
FITC-conjugated anti-human antibodies (DAKO, 40 .mu.g/ml) and
TRITC-conjugated anti-mouse antibodies (DAKO, 15 .mu.g/ml) for 30
min. Hoechst 33258 fluorochrome (Sigma, 0.5 .mu.g/ml) was used for
the staining of the nuclei.
[0599] BaF3 bioassay. To generate Tie1-EpoR expressing BaF3 cells
for the bioassay, BaF3 pre-B cells were stably transfected with a
nucleic acid that expresses chimeric receptor containing the
extracellular domain of human Tie1 fused with the transmembrane and
cytoplasmic domains of the mouse erythropoietin receptor. The
nucleic acid used was a Tie1-EpoR chimeric cDNA in a pEF-BOS
expression vector. The nucleic acid encoding the chimeric receptor
was constructed by cloning the PCR amplified extracellular part of
human Tie1 (bp 37-2316 of X60975) as EcoRI-BglII fragment into
mEpoR-pcDNA vector. The cDNA encoding for the chimeric receptor
consisting of the extracellular part of Tie1 fused with the
transmembrane and intracellular domains of EpoR was subcloned into
the pEF-BOS expression vector. Vector was linearized and
co-transfected into BaF3 cells with pcDNA3.1(+) Zeo vector
(Invitrogen). Stable cell pools were generated by selection with
250 .mu.g/ml Zeocin. The expression of Tie1/EpoR fusion protein in
several clones was analyzed by Western blotting with an antibody
against EpoR.
[0600] To perform the assays, BaF3 cells expressing the Tie1-EpoR
chimera were split in 96-well microtiter plates at 50 000
cells/well in the presence of the indicated concentrations of
anti-Tie1 antibodies. The E3 antibody used in this study was the
germ-lined E3 antibody (DX-2220). As controls, Zeocin resistant
pools not expressing the Tie1-EpoR were used. After 48 h, the
viability of the cells was determined by adding MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(Sigma), 0.5 mg/ml), followed by further 2 h of culture, addition
of an equal volume of cell lysis solution (10% SDS, 10 mM HCl) and
incubation overnight at 37.degree. C. Absorbance was measured at
540 nm.
[0601] Tie1 phosphorylation assay. COS1 cells were transfected with
pcDNA3-Tie1-V5. After 24 h of transfection, the cells were serum
starved for 8 h and then treated with E3 IgG. For the Tie1
phosphorylation assay, HDMVECs were cultured on 10 cm dishes to
near confluence, starved (8-16 h) in serum free medium and
stimulated as indicated. After the stimulations, the cells were
lysed in lysis buffer (RIPA-DOC: 50 mM Tris-HCl pH 8.0, 150 mM
NaCl, 1% Triton-X-100, 0.1% SDS, 0.5% DOC, 10 mM EDTA, supplemented
with aprotinin, leupeptin, PMSF and sodium vanadate). Clarified
lysates from transfected COS 1 cells or HDMVECs were
immunoprecipitated with anti-V5 or anti-Tie1 B9, respectively.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose
and immunoblotted using the anti-phosphotyrosine and anti-Tie1
(R&D systems) antibodies.
[0602] Immunostaining of human tissues. To evaluate reactivity of
anti-Tie1 antibodies in immunohistochemistry, 5 .mu.m cryosections
of human kidney and lung were dried at room temperature for 30 min
and fixed with cold acetone for 10 min. Slides were washed with PBS
and treated with 0.03% H.sub.2O.sub.2 in PBS for 15 min to reduce
endogenous peroxidase activity. TNB (30 min at room temperature)
was used to block non-specific binding and sections were incubated
with Tie1 antibodies at concentration of 10 .mu.g/ml overnight at
+4.degree. C. After several washings with PBS, biotinylated anti
human antibody (1:300, Zymed) was added to the tissues. Signal was
amplified by using a TSA kit and detected with AEC staining.
[0603] Results
[0604] Western blotting, immunoprecipitation and immunofluorescence
of Tie1 transfected cells and primary human endothelial cells (see
Table 1).
TABLE-US-00004 TABLE 1 Assay Summary WB: Tie1- WB: IP: Tie1- IP:
IF: Tie1- IF: BaF3 Clone transfected HDMEC transfected HDMEC
transfected HDMEC assay E3 + + ND - + + + G2 + + ++ + + ++ - A2 + +
++ + + ++ - A10 + + ++ + + + - B2 + + + - + + - B9 + + ++ ++ ++ + -
C2 + + ++ ++ + ++ - C7 + + ++ + + + - C10 + + ++ ++ ++ + - D11 + +
+ - + ++ - E11 + + ++ + + ++ - G10 + + ++ + ++ + - H1 + + ++ + ++ +
- H4 + + ++ + + + - P-A1 + + ++ ++ + ++ - P-A10 + + ++ - + + - P-B1
+ + + - weak + - P-B3 + + - - + + - P-C6 + + + - + ++ - P-D12 + + +
- + + - P-F3 + + - - ++ ++ - P-F4 + + ++ - cross ++ - P-G3 + + ++ +
+ + - PH1 - - - - - - -
[0605] To confirm the binding ability of the 23 selected anti-Tie1
antibodies, we first performed western blotting and
immunoprecipitation using COS 1 cells transfected with
pcDNA3-Tie1-V5 (V5 tagged) and primary endothelial cells. Next, to
find out if the anti-Tie1 antibodies recognize Tie1 in living
cells, those cells were studied by immunofluorescence staining. All
the antibodies analyzed recognized both transfected and endogenous
Tie1, although differences were detected in the binding affinity as
shown in Table 1.
[0606] Stimulation and inhibition of Tie1 in Tie1-EpoR transfected
BaF3 cells and human primary endothelial cells. Although no ligand
for Tie1 has been identified, we used the following efficient
screening method for Tie1-binding proteins. Interleukin-3 dependent
pre-B-lymphocyte (BaF3) cells were transfected with a construct
that expresses a Tie1-EpoR fusion protein. Since BaF3 cells are
IL-3 dependent, they die unless IL-3 is provided. However, Tie-EpoR
receptor expressing BaF3 cells can survive and proliferate if the
medium contains a Tie1-binding protein, either a natural ligand or
an artificial mimetic. Cell survival can be quantitated, e.g., by
colorimetric MTT-assay, which measures mitochondrial activity.
[0607] The results from the BaF3 cell assays indicated that, of the
23 different monoclonal antibodies tested, only E3 IgG was able to
promote survival of Tie1-EpoR cells whereas the viability of EpoR
BaF3 cells used as a control was not affected by E3 IgG. The IgG
part of the immunoglobulin molecule was needed for the survival
effect of E3 IgG, as the E3 Fab fragment had no effect on the
viability of Tie1-EpoR cells. A concentration of 50 ng/ml of E3 IgG
gave almost maximal viability in Tie1-EpoR cell survival assays and
the viability was dose dependent.
[0608] To test if the E3 IgG binding to the extracellular region of
Tie1 induces autophosphorylation of Tie1, the Tie1 receptor
phosphorylation level in response to E3 IgG treatment was studied
in transiently transfected COS 1 cells and human primary
endothelial cells. COS 1 cells were transfected with an expression
vector containing a V5-tagged full length Tie1 cDNA, and, after
serum starvation, the cells were treated with E3 IgG (200 ng/ml).
Cell lysates were extracted at several time points and Tie1 was
immunoprecipitated with anti-V5 followed by western blotting using
anti-phosphotyrosine and anti-Tie1 antibodies. The results
indicated that Tie1 is tyrosine phosphorylated after 10 to 30 min
of E3 IgG stimulation. To determine if E3 IgG induces Tie1
phosphorylation in primary endothelial cells, HDMVEC cells were
serum starved and stimulated with several concentrations of E3 for
60 min. Tie1 was then immunoprecipitated from cell lysates and
subjected to anti-phosphotyrosine blotting analysis, which showed
receptor phosphorylation following E3 IgG stimulation at 50-200
ng/ml. Also higher concentrations of E3 (500-1000 ng/ml) induced
Tie1 phosphorylation but the response was more rapid and was most
prominent after 5 min of stimulation.
[0609] To study the kinetics of E3 IgG induced Tie1 activation,
cells were stimulated with E3 IgG (200 ng/ml) and receptor
phosphorylation was studied at various time points. Tie1
phosphorylation was highest 15-30 min after E3 IgG treatment but
phosphorylation persisted for up to 1 h.
[0610] To determine if any of the other monoclonal antibodies
tested inhibit the survival effect of E3 IgG in Tie1-EpoR BaF3
assay, antibodies were studied in combination with E3 IgG. A
concentration of 100 ng/ml of E3 IgG together with 100 (1:1) or 500
(1:5) ng/ml of the other antibodies were used and the viability of
Tie1-EpoR cells was measured. The results from both combinations of
E3 IgG and the test antibody (in 1:1 and 1:5 ratios) were similar
and indicated that two of the 23 antibodies (B2 and D11) blocked
completely the survival effect of E3 IgG. Several antibodies (A2,
A10, P-B1, P-B3 and P-C6) inhibited the viability effect of E3 IgG
to some extent and two of the antibodies (G2 and C7) promoted the
survival of Tie1/EpoR BaF3 cells in combination with E3 IgG.
[0611] Immunostaining of human tissues. The anti-Tie1 antibodies
react with human Tie1 in cultured cells. It is also possible to
determine whether they could stain human tissue samples from lung
and kidney as well as from tumors by using biotinylated anti-Tie1
antibodies and detecting bound antibodies using labeled
streptavidin or avidin.
Example 3
Exemplary Sequences
[0612] Sequences of exemplary immunoglobulin variable domains are
shown in FIGS. 7-36.
Example 4
Inhibition of Tube Formation by HUVEC Cells Using Anti Tie1
E3-IgG
[0613] To demonstrate the ability of E3 to inhibit angiogenesis in
vitro, purified E3 was tested for its ability to inhibit tube
formation by human umbilical cord endothelial cells (HUVECS). Human
Umbilical vein endothelial cells (HUVEC) were obtained by treating
fresh human umbilical cord veins with Trypsin-EDTA (1.times.)
(Gibco/Invitrogen) for 20-25 minutes at 37.degree. C. The cells
were cultured in a T-25 flask coated with attachment factor (AF),
(Cascade Biologics) in RPMI 1640 medium supplemented with 10% FCS,
0.4% BBE, 1% 1-glutamin, 1% penicillin/streptomycin. Primary
cultures were detached with warm Trypsin-EDTA and used when
confluent at the second or third passage. The cells were maintained
in a proliferative state by culturing them in a split ratio 1:2 at
an approximate density of the monolayer of about 60-80%. To
dissociate the cells, HUVEC monolayers were treated with
trypsin/EDTA (500 .mu.l/dish) at 37.degree. C. for 3 min. Trypsin
activity was stopped by adding 3 volumes of complete RPMI medium.
The cells were carefully scraped, separated by repeated pipetting,
and finally washed with PBS.
[0614] After 2 passages HUVECs were seeded in their culture medium
(40.times.10.sup.3/50 .mu.l/well of a 96-well plate) on a collagen
gel (50 .mu.l of collagen I 1.5 mg/ml) prepared by mixing 7.5
volumes of 2 mg/ml collagen (Collagen R; Serva, Heidelberg,
Germany), 1 volume of 10.times. MEM, 1.5 volume of NaHCO.sub.3
(15.6 mg/ml) and .about.1 volume of NaOH to adjust the pH to 7.4.
After 1.5h, the culture medium was then discarded and the cells
were covered with a new layer of collagen (1.5 mg/ml, new
preparation, 50 .mu.l/well). After polymerization of the gel,
culture medium was added to each well in presence or in absence of
E3 antibody (1 ng/ml to 10 .mu.g/ml). The assay was performed with
a streptavidin antibody used as a control (from 1 ng/ml to 10
.mu.g/ml): The total length of the tube network on the culture
surface was quantified at 40.times. magnification by the
METAVUE.TM. Software (Universal Imaging Corporation). Results from
triplicate wells were expressed as mean vessel area per field
.+-.SEM (relative units). Each assay was performed at least three
times.
[0615] E3 is a potent inhibitor of tube formation by HUVECS even at
a concentration of 10 ng/ml. The control anti-streptavidin has no
effect on the ability of HUVECS to form tubes. This results
indicates that E3 can inhibit at least one aspect of
angiogenesis.
Example 5
Immunohistochemical Analysis of E3 Binding to Matched Tumor and
Normal Tissue Sections
[0616] To evaluate the binding of E3 to Tie1 in primary tumor and
normal tissue the antibody was produced as an IgG and biotin
labeled. The E3 antibody and two other anti Tie1 antibodies B2 and
D11 were reformatted as full length IgG molecules. Nucleic acids
encoding these IgGs were transiently transfected into HEK293T
cells. Plasmid preparations for transient cell transfections were
performed using the HP-GENELUTE.TM. MIDI prep kit (Sigma, cat. no.
NA0200). HEK293T cells (GenHunter Corp. cat. no. Q401) were seeded
24 hours before transfection; 6.times.10.sup.6 cells were plated
per 10-cm culture dish. Transfections were carried out using
LIPOFECTAMINE.TM. 2000 reagent (Invitrogen, cat. no. 11668019)
following the manufacturer's instructions. Five micrograms of
plasmid DNA was used per 10-cm dish. Cells were cultured in DMEM
(Invitrogen, cat. no. 31966021) supplemented with 10% "ultra-low
IgG" fetal calf serum (Invitrogen, cat. no. 16250078), at
37.degree. C., 5% CO.sub.2, in a water saturated atmosphere.
Conditioned media were harvested 72 hours and 144 hours after
transfection, pooled and sterile filtered.
[0617] One hundred microliters of Protein A beads (rProtein A
Sepharose 4 Fast Flow, Amersham Biosciences, cat. no. 17-1279-01)
equilibrated in PBS were added to the cell culture supernatants,
and these were rotated overnight at 4.degree. C., e.g., in 50 ml
tubes. The beads were collected by centrifugation, transferred to a
96-well filter plate (UNI-FILTER 800 GF/B, Whatman, cat. no.
7700-2803) and washed extensively with PBS using a vacuum manifold
(Macherey Nagel, cat. no. 760681). Elution of the antibodies was
achieved by resuspending the beads in 400 .mu.l of 12.5 mM citric
acid. After a 30 to 60 second incubation, the bead eluates were
collected, using the vacuum manifold, into the wells of a 96-well
collection plate (UNIPLATE 750, Whatman, cat. no. 7701-5750). Each
well of the collection plate contained 60 .mu.l of 1 M HEPES pH 7.5
buffer to immediately neutralize the eluted fractions. The elution
step was performed twice to maximize antibody recovery. The eluted
samples were then dialyzed against PBS using dialysis cassettes
(Slide-A-Lyser Dialysis Cassettes, MWCO 10,000, Pierce, cat. no.
66380) and protein concentration was determined from the absorbance
at 280 nm assuming that a 1 mg/ml solution has an absorbance of
1.35. The quality of the preparations was analyzed by reducing and
non-reducing SDS-PAGE.
[0618] The Tie1 antibodies were biotinylated using the EZ-link
Sulfo-NHS-SS-Biotin (Pierce, Cat. 21331). For Tie1/Fc and Tie1-His,
the reaction was performed for 2 hours on ice in 50 mM sodium
carbonate buffer, pH 9.6, in the presence of a 5-fold molar excess
of biotinylating agent. For the antibodies, the reaction was
performed for 2 hours on ice in PBS, in the presence of a 15-fold
molar excess of EZ-link Sulfo-NHS-SS-Biotin. The reaction was
stopped by the addition of Tris-HCl, pH 7.5 (50 mM final
concentration) followed by a 1-hour incubation on ice. Samples were
then dialyzed against PBS.
[0619] Various normal and tumor tissue sections were stained with
biotinylated antibodies. A mouse monoclonal anti-Tie1 antibody
(7e8) (Alitalo laboratory, University of Helsinki) was used as a
positive control. Sections without primary antibody served as
negative control. All samples were fresh frozen tissues and
staining was performed with the TSA-kit (Perkin-Elmer Life
Sciences). After acetone fixation (10-20 min, -20.degree. C.) the
slides were treated with 0.73% H.sub.2O.sub.2 for 10 min to reduce
endogenous peroxidase activity followed by blocking for 30 min with
TNB buffer. Sections (5-10 mm thick) were incubated with primary
antibodies (10 .mu.g/ml) overnight at 4.degree. C. Sections with
the mouse monoclonal anti-tie1 antibody (7e8) were treated with
biotinylated anti-mouse antibodies (VectaStain) before the addition
of streptavidin-HRP. Signal was amplified by using a TSA kit and
the visualized by AEC (235 ml NaAc,15 ml AEC (stock solution: 1600
mg 3-amino-9-ethyl-carbazole and 480 ml N-dimethylformamide), 250
.mu.l H.sub.2O.sub.2).
[0620] In general, Tie1 expression was upregulated in tumor tissue
when compared with matching normal tissue. However, in the tumor
tissues the anti Tie1 antibodies stained other structures in
addition to the vessels. Furthermore, some tissue specificity in
the expression of certain epitopes was observed. For example, the
E3 antibody stained vessels in the lung and kidney but not in the
skin while the B2 antibody stained vessels very faintly in other
normal tissues than in the breast. Shedding of the ectodomain of
Tie1 into the tumor tissues can explain observed differences.
[0621] In skin tissue, the E3, B2, and D11 antibodies stained blood
vessels very faintly whereas the murine 7e8 control antibody gave a
clear staining in the normal skin. In melanoma tissue, the 7e8
antibody stained vessels only but the E3, B2, and D11 antibodies
also stained other surrounding structures. The staining pattern was
similar with all three of the E3, B2, and D11 antibodies.
[0622] In lung tissue, we observed that the E3 antibody stained
especially clearly the large veins in the lung, whereas D11 and 7e8
gave a faint staining. B2 did not stain the same veins. The
expression of Tie1 was dramatically upregulated in lung carcinoma
and all the antibodies stained vessels more strongly in samples
with lung carcinoma than in samples from normal lung. In the lung
tumors, the E3, B2, and D11 antibodies stained structures other
than vessels.
[0623] In kidney, the E3 and D11 antibodies stained kidney tubules
in addition to the vessels. B2 gave only very faint staining of
either tubules or vessels while 7e8 stained only vessels. In
hypernephroma tissue, only the E3 antibody gave a clear
staining.
[0624] In breast, E3 gave the brightest staining in the veins and
capillaries of the mammary tissue, B2 and 7e8 gave a similar
staining while D11 stained those structures rather faintly. In
breast carcinoma the Tie1 expression was substantially upregulated,
and the E3, B2, and D11 antibodies stained also other structures in
addition to vessels.
Example 6
Binding to Mouse Endothelial Cell Lines of Anti Tie1 E3-IgG Using
Flow Cytometry
[0625] We evaluated if E3 cross reacts with mouse Tie1 in situ and
thus if we can evaluate E3 activity in mouse tumor xenograft models
binding to mouse endothelial cells was tested and compared with
human and transfected cell lines.
[0626] Specific binding of the Tie1 antibodies and of control Mabs
to mouse endothelial cells was measured by flow cytometry analysis
(FACSscan, Becton Dickinson, Oxnard, Epics, Coulter). Mouse
endothelial cell lines MS1, Le-2, Bend3, SVEC (ATCC, Rockville) and
Tie1 transfected Le-2 cells were stained. Cell staining was
modified from existing protocols. About 200,000 cells were used in
each experiment: after trypsinization, cells were washed one time
in PBS and resuspended PBS, 10% heat inactivated human serum
(incubation buffer). To test specificity, antibodies were incubated
at different dilutions for 1 h at room temperature. Cells were spun
down by centrifugation for 3 min at 611 g. Between incubations
cells were washed twice with PBS. Then relevant biotinylated
antibodies (A2 against streptavidin, E3 against Tie1, were added
and incubated for 1 h at room temperature). The E3 DX-2210
antibody, in which the light chain has been germlined, was used for
these studies. This was followed by incubation with
Strepatvidin-R-phycoerythrin (Dako, Glostrup, Denmark) for 1 hour
at room temperature in incubation buffer. After the final
incubation step bound antibodies were detected by means of flow
cytometry on a FACSCan and Epics Altra (Becton Dickinson, Oxnard,
Coulter) and results analyzed.
[0627] Intracellular Tie1 was measured as described above, except
for the addition of Saponin to the incubation buffer to a final
concentration of 0.1% during incubations. The anti-Tie1 antibody E3
binds to mouse endothelial cell lines indicating a cross reactivity
of E3 with mouse and human Tie1 in situ. The binding pattern in
mouse cell lines detected by flow cytometry is different from the
binding pattern in HUVEC in that in mouse cells there is a greater
cell surface staining than that compared to primary human
endothelial cell lines. DX-2210 stained positively both mouse
endothelial cell lines as well as the HUVEC control cells. There
was a shift in the fluorescent signal when the cells were treated
with saponin, indicating a significant intracellular pool of
sequestered Tie1.
Example 7
Determination of anti Tie1 E3-IgG Binding to Human Platelets Using
Flow Cytometry
[0628] Binding experiments with a purified polyclonal goat
antiserum against Tie1 (R&D systems) had showed binding to
human platelets in a previous study (Tsiamis et al., (2000) J.
Vasc. Res. 37:437-42). The conclusion form this study was that
platelets represent a large pool of Tie1 immunoreactivity which
could present a problem for development of Tie1 as a therapeutic
target. To determine if the antibody E3 binds to platelets we
performed flow cytometric analysis on both activated and
inactivated platelets and compared the staining pattern with the
purified anti Tie1 polyclonal serum.
[0629] To avoid platelet activation, human platelets were isolated
from plasma of healthy donors using the platelet GelSep kit
(Biocytex, Marseille, France) kit according to the guidelines of
the manufacturer. Platelets were activated by the addition of
thrombin to a final concentration of 0.8 U/ml. To distinguish
activated from non-activated platelets double staining was
performed with Tie1 antibodies/control antibodies and antibody CD42
(total platelets) or CD62 (activated platelets).
[0630] After preparation, platelets were resuspended in buffer 2 of
the GelSep kit, 10% heat inactivated human serum (incubation
buffer) and incubated for 1 hour. To test specificity, biotinylated
antibodies human anti-Tie1(E3), human anti-streptavidin (A2-SV, an
antibody that does not bind Tie1), human anti-FITC and goat
anti-Tie (R&D systems) were incubated with 500 000 platelets
per test for 1 hour at different dilutions (2 .mu.g/ml, 10
.mu.g/ml) for 1 h at room temperature. Platelets were spun down by
centrifugation for 10 min at 611 g. Between incubations platelets
were washed twice with Buffer 1 of the GelSep kit. Then,
Strepatvidin-R-phycoerythrin together with anti-CD42-PercP or
anti-CD62-PercP were incubated for 30 minutes at room temperature
in incubation buffer After the last incubation and washing
detection of bound antibodies was performed by means of flow
cytometry on a FACSscan and Epics Altra (Becton Dickinson, Oxnard,
Coulter,) and results analyzed. Cells were gated on SSC and
anti-CD42-PercP for the total platelets in case non-activated
platelets were used and on SSC and anti-CD62-PercP for the
activated platelets.
[0631] The polyclonal goat anti-Tie1 antibody indeed binds to
platelets under the conditions tested. This binding is lower when
platelets are activated. In contrast, the human anti-Tie1 antibody
E3 shows no significant binding to total platelets, nor to
activated platelets (FIG. 1).
Example 10
Assessment of Tie1 Immunoreactivity in Human Platelets Using
Immunoprecipitation with Anti Tie! E3-IgG
[0632] A previous study with a purified polyclonal goat antiserum
against Tie1 (R&D Systems) had showed binding to human
platelets (Tsiamis et al., 2000). The conclusion from this study
was that platelets represent a large pool of Tie1 immunoreactivity
which could present a problem for development of Tie1 as a
therapeutic target. To exclude the possibility that the antibody E3
binds to platelets immunoprecipitation of lysates prepared from
platelets and HUVECS were performed. Both activated and inactivated
platelets were tested. Anti-Tie1 antibodies B2, D11, E3, the goat
polyclonal AF619 (R&D) and negative control antibodies
anti-FITC and anti-Streptavidin were used. HUVECS were retrieved
from culture dishes by trypsinization and platelets were prepared
with the platelet GelSep kit (Biocytex, Marseille, France) kit
according to the guidelines of the manufacturer. Per
immunoprecipitation experiment 3-5.times.10.sup.6 and
3.times.10.sup.8 cells platelets were used for each antibody
tested. Platelets and cells were washed with PBS and spun down at
1400 rpm for 4 minutes and supernatant was removed. Then cells were
lysed in 1 ml lysis buffer containing 50 mM Tris HCL pH 7.5, 150 mM
NaCl, 0.5% Deoxycholic acid (DOC) and 0.5% NP-40 for 5 minutes. The
lysed cells were spin down for 10 minutes at 14.000 rpm and 5
.mu.g/ml antibody was added to the supernatant and incubated at
4.degree. C. on a rotator. 100 .mu.l/sample protein A beads
(Uppsala, Sweden) were washed 3 times with lysis buffer
(centrifugation speed: 15 seconds, 2000 rpm) then cell lysates
incubated with antibody were added for 30 minutes 4.degree. C. Then
beads were washed three times with washing buffer containing 50 mM
Tris HCL pH 7.5, 400 mM NaCl, 0.5% DOC, 0.5% NP-40. Finally, beads
are spun down and the pellets was resuspended in an equal amount in
sample buffer to perform SDS-page and Western blotting. In Western
blotting Tie1 was detected with the polyclonal goat anti-Tie1
antibody. The conclusions of this study are that E3 is able to
immunoprecipitate Tie1 in HUVEC but not in platelets.
Example 8
Distribution of Tie1 in HUVEC Cells Determined by Staining with
Anti Tie1 E3-IgG
[0633] We analyzed the staining pattern of E3 in HUVECS using
confocal microscopy. HUVEC were trypsinised, washed with PBS and
spotted at a density of 60 000 cells on a gelatine coated
microscope slide and incubated for 24 hours in a humidified
incubator at 37.degree. C. Cells were air dried and fixed with 4%
paraformaldehyde for 20 minutes at room temperature. The slides
were washed with PBS. The slides were incubated with 10% Heat
inactivated human serum (incubation buffer).
[0634] For measuring specific binding to Tie1, biotinylated
antibody E3 and biotinylated negative control antibody A2 were used
at a concentration of 10 .mu.g/ml and incubated for 1 hour at room
temperature. Slides were washed twice with PBS. Then,
Strepatvidin-R-phycoerythrin (Dako, Glostrup, Denmark) was added
and incubated for 1 hour at room temperature. After the last
incubation and washing detection of bound antibodies was performed
by means of confocal microscopy.
[0635] E3 binds specifically to HUVEC as detected by confocal
microscopy. The staining is pre-dominantly located inside of the
cell which suggests a large intracellular pool of Tie1 relative to
a smaller pool of cell surface localized Tie1. The localization of
E3 was consistent with co-localization of Tie1 with a cytoskeletal
protein.
Example 9
Conversion of Somatic Mutations Positioned in the Framework Region
of Anti Tie1 E3 to Germline Residues
[0636] To reduce potential immunogenicity of E3 in humans, all non
germline amino acid residues in the LC framework regions were
corrected back to germline. An initial analysis was performed which
aligned the LC of E3 with a database containing all kappa and
lambda light chain germline genes. The LC of E3 was shown to have
closest homology to DPK4 and three substitutions in E3 relative to
the germline framework regions were identified.
[0637] We constructed a germlined version of E3 in which the LC
framework regions were altered to include sequences identical to
the DPK4 germline framework regions. The germlined E3 antibody was
constructed by engineering a nucleic acid encoding the desired
sequence. Changes to nucleic acids encoding the E3 LC variable
domain were made by PCR and other standard molecular biological
techniques and verified by nucleic acid sequencing.
[0638] An exemplary germlined light chain variable domain E3
sequence includes:
DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLIYTASTLQSGVP
SRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHTFGQGTRLEIK (SEQ ID NO: 159).
The altered positions are underscored.
[0639] We produced the germlined version of the E3 antibody as both
a soluble Fab and as an IgG. The Fab cassette of the positive
sFAB-expressing clone was PCR amplified with oligonucleotides,
ligated into a mammalian expression vector containing the human
IgG4 Fc region and electroporated into XL1 Blue MRF' cells. The
prokaryotic ribosomal binding sequence and gene three leader
sequence were replaced with a mammalian internal ribosomal entry
and heavy chain leader sequences. Reformatted antibody clones were
sequenced to confirm accuracy following the cloning procedure.
Endotoxin-free DNA was prepared and used for transient transfection
studies.
Example 10
Production and Testing of Germlined Anti Tie1 E3--Fab for Binding
to Recombinant Tie1-Fc in ELISA
[0640] To evaluate if the conversion of any of the somatic
mutations in the framework of E3 back to germline residues had any
effect on binding activity the soluble Fabs were produced. The
soluble expression vector containing the parental E3 Fab and the
germlined E3 Fab construct were grown overnight at 30.degree. C. in
2.times.TY broth containing 100 .mu.g/ml ampicillin and 2% glucose
and use 4 ml of this overnight culture to inoculate 400 ml of
2.times.TY broth containing 100 .mu.g/ml ampicillin and 0.1%
glucose. Cells were grow at 37.degree. C. until an OD.sub.600 of
0.8-1.0, 1 mM IPTG was added and the culture was maintained at
30.degree. C. for 4 hours. The cultures were spun down at 4,000 rpm
for 15 min at 4 C. The supernatants were discarded and resuspend
the pellets resuspended in 4.8 ml of ice cold TES buffer (0.2 M
Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose, pH 8.0) containing proteases
inhibitors (protease inhibitor cocktail tablets [Roche]: dissolve 1
tablet in 1 ml of water and dilute 50-times in TES buffer).
Transfer to 50 ml Falcon tubes and place on ice for 5-10 min.
During this incubation, wash the centrifugation bottles with 5.25
ml TES:H.sub.2O (1:3) containing proteases inhibitors and add this
to the cells. Incubate for 20 more min on ice. Spin at 3000 g for
15 min at 4.degree. C. and transfer the supernatants into new
centrifugation tubes. Resuspend the cell pellets in 6 ml TES
containing 15 mM MgSO.sub.4 and proteases inhibitors and incubate
on ice for 15 min. Centrifuge at 3000 g for 15 min at 4.degree. C.
Transfer the supernatants into the centrifugation tubes and spin at
8000 g for 20 min at 4.degree. C. Collect the supernatants and
dialyze against PBS. The Fabs were purified by metal chelate
chromatography. Incubate the dialyzed periplasmic extracts with 1
ml of TALON.TM. Metal Affinity Resin (Clontech) and rotate at room
temperature for 2 hours. Transfer the beads into empty gravity
column (Poly-Prep chromatography columns, Bio-Rad, Cat. 731-1550).
Wash the beads with 5 mM imidazole in PBS and elute the Fabs with
150 mM imidazole in PBS. Dialyze against PBS using dialysis
cassettes (SLIDE-A-LYSER.TM. Dialysis Cassettes, MWCO 10,000,
Pierce, cat. no. 66380) and determine the protein concentration
from the absorbance at 280 nm assuming that a 1 mg/ml solution has
an absorbance of 0.86. The quality of the preparations can be
analyzed by reducing and non-reducing SDS-PAGE.
[0641] Wells of an IMMULON.TM. 2 HB plate coated overnight with 500
ng or 50 ng of purified recombinant human Tie1-Fc target antigen
per 100 microliters 0.1 M sodium bicarbonate buffer, pH 8.5.
Parental E3, E3 germlined (E3g) or a negative control soluble Fab
were loaded into wells at either 5 micrograms or 1 microgram per
100 microliters of PBST. Recombinant human Tie1-Fc target antigen
is dissolved in an appropriate amount of acetic acid and
subsequently diluted into 0.1 M sodium bicarbonate buffer, pH 9.6
at final concentrations of 500 ng and 50 ng per 100 microliters.
After addition of the target antigen to the wells the microtitre
plate is incubated overnight at 4.degree. C. The plate is
subsequently washed 5 times with PBST and blocked with 1% BSA in
PBS at 37.degree. C. for 2 hours. The plate is again washed plate
times with wash buffer, PBST and 100 microliters per well of
purified Fab at 5 or 1 micrograms per 100 microliter PBST was added
followed by incubation at room temperature for 1 hour. After
washing plate 7 times with PBST 100 microliters of a 1:5000
dilution of anti-sFab-HRP in PBST was added (Pierce Product
#31414). After washing the wells seven times 100 microliters
TMB-H.sub.2O.sub.2 solution was added to each well and the plate
read at 630 nm in an ELISA. Both E3 and germlined E3 bound to the
recombinant human Tie1-Fc target antigen by this assay.
Example 11
Production and Testing of Germlined Anti Tie1-E3--Fab for Binding
to Recombinant Human Tie1 in BIAcore
[0642] Recombinant purified human Tie1-Fc antigen (Stock 2.45
mg/ml) was biotinylated using the EZ-link Sulfo-NHS-SS-Biotin
(Pierce, Cat. 21331). The reaction was performed for 2 hours on ice
in 50 mM sodium carbonate buffer, pH 9.6, in the presence of a
5-fold molar excess of biotinylating agent and was stopped by the
addition of Tris-HCl, pH 7.5 (50 mM final concentration) followed
by a 1-hour incubation on ice. Samples were then dialyzed against
PBS. The antigen was then diluted 1/100 fold in HBS and was then
captured onto a streptavidin chip. This was coated to a density of
830RU (resonance units). All analysis was performed in HBS buffer.
The parental Fab E3 and germlined E3 Fab were prepared as described
above. A stock solution of 0.587 mg/ml (11740 nM) was diluted 1/587
in HBS+BSA to obtain a stock of 20 nM and the germlined Fab E3
0.025 mg/ml (500 nM) was diluted 1/25 in HBS+BSA to obtain a stock
of 20 nM. Serial dilutions were made of each Fab preparation to
obtain 10 nM, 5 nM, 2.5 nM, and 1.25 nM solutions. For the
association phase samples were injected at 30 .mu.l/min for 4
minutes using kinject program. This was followed by a 10 minutes
dissociation phase, any remaining sample was stripped from the Tie1
Fc surface at a flow of 50 .mu.l/min with a single injection of 5
mM NaOH+1M NaCl for 18 seconds. All samples were run and analyzed
in duplicate.
[0643] Sensorgrams were analyzed using the simultaneous ka/kd
fitting program with 1:1 model in the BIAEVALUATION.TM. software
3.1. From the analysis we can see that the germlining of the E3
antibody has had minimal effect on the binding activity of the
antibody.
TABLE-US-00005 TABLE 2 Comparison of the binding affinity of
parental and germlined E3 Fab E3 Fab Tie1 Fc k.sub.on (1/Ms)
k.sub.off (1/s) K.sub.D(1) nM parental Human 3.00E+05 6.10E-04 2.0
germlined Human 3.00E+05 1.02E-03 3.4
Example 12
Comparison of Affinity of Germlined Anti Tie1 E3--IgG to Parental
Anti Tie1 E3 for Binding to Recombinant Human Tie1 Using
BIAcore
[0644] In order to evaluate if the binding behavior had been
affected in any way by the conversion of the somatic mutations back
to germline residues, the germlined antibody was produced and
tested as an IgG. The germlined E3-IgG construct used to
transiently transfect HEK293T cells and purified.
[0645] The germlined E3 IgG1 stock solution 0.63 mg/ml was diluted
1/50 in a buffer of pH4.5 and the parental E3 IgG1 stock solution
0.56 mg/ml (2143-001) was diluted 1/50 in a buffer of pH 4.5. The
IgG were directly coated onto a CM5 chip. The surface of the chips
was activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the
IgG was flowed over until 780RU germlined E3-IgG and 728 non
germlined E3 IgG was coated onto the surface. All flow cells were
subsequently deactivated with a 7 minute pulse of 1M ethanolamine
hydrochloride pH 8.5. All analysis was performed in HBS buffer.
Purified recombinant human Tie1 Fc was diluted 1/28.7 in HBS to
obtain a 400 nM stock solution. Serial dilutions were made to
obtain 200 nM, 100 nM, 50 nM and 25 nM Tie1 Fc stocks. For analysis
of the association phase samples were injected at 30 .mu.l/min for
8.3 minutes using kinject program. This was followed by a 40
minutes dissociation phase. Any antigen remaining associated to the
surface was stripped from the IgG surface at a flow of 50 .mu.l/min
with two injections of 10 mM glycine pH 1.5 for 30 seconds. All
samples were run and analyzed in duplicate
[0646] Sensorgrams were analyzed using the simultaneous ka/kd
fitting program with 1:1 model in the BIAEVALUATION.TM. software
3.1. Germlining had minimal impact on the binding activity of the
E3 IgG with respect to human Tie1 Fc.
TABLE-US-00006 TABLE 3 Comparison of the binding affinity of
parental and germlined E3 IgG E3 IgG Tie1 Fc k.sub.on (1/Ms)
k.sub.off (1/s) K.sub.D(1) nM parental Human 6.19E+03 3.61E-05 5.83
germlined Human 7.09E+03 3.67E-05 5.17
Example 13
Production and Testing of Germlined Anti Tie1-E3--Fab for Binding
to Recombinant Mouse Tie1 in BIAcore
[0647] Mouse Tie 1-Fc antigen (0.5 mg/ml stock) was biotinylated
using established procedures and after dilution 1/100 fold in HBS
this was then used for capturing to a streptavidin chip. This was
coated to a resonance value of 740RU. All analysis was performed in
HBS buffer. The parental Fab E3 0.587 mg/ml (11740 nM) was diluted
1/587 in HBS+BSA to obtain a stock of 20 nM and the germlined Fab
E3 0.025 mg/ml (500 nM) was diluted 1/25 in HBS+BSA to obtain a
stock of 20 nM. Serial dilutions were made of each Fab preparation
to obtain 10 nM, 5 nM, 2.5 nM, and 1.25 nM. For the association
phase samples were injected at 30 .mu.l/min for 4 minutes using
kinject program. This was followed by a 10 minutes dissociation
phase, any remaining sample was stripped from the Tie1 Fc surface
at a flow of 50 .mu.l/min with a single injection of 50 mM NaOH+1 M
NaCl for 18 seconds. All samples were run and analyzed in
duplicate.
[0648] Sensorgrams were analyzed using the simultaneous ka/kd
fitting program with 1:1 model in the BIAEVALUATION.TM. software
3.1. The germlining of the E3 antibody has had minimal effect on
the binding activity of the antibody.
TABLE-US-00007 TABLE 4 Comparison of the binding affinity of
parental and germlined E3 Fab E3 Fab Tie1 Fc kon (1/Ms) koff (1/s)
KD(1) nM parental Mouse 2.46E+05 9.50E-04 3.9 germlined Mouse
3.40E+05 1.04E-03 3.1
Example 14
Comparison of Affinity of Germlined Anti Tie1 E3--IgG to Parental
Anti Tie1 E3 for Binding to Recombinant Mouse Tie1 Using
BIAcore
[0649] In order to evaluate if the binding behavior had been
affected in any way by the conversion of the somatic mutations back
to germline, the germlined antibody was produced and tested as an
IgG. The germlined E3 was reformatted to an IgG as described. This
was then used to transiently transfect HEK293T cells using
established procedures. The IgG was purified from the culture
supernatant using protein A column chromatography using established
procedures and the subsequent IgG was then tested for binding
activity using surface plasmon resonance (BIAcore). The germlined
E3 IgG1 stock solution 0.63 mg/ml (2146-002) was diluted 1/50 in a
buffer of pH 4.5 and the parental E3 IgG1 stock solution 0.56 mg/ml
(2143-001) was diluted 1/50 in a buffer of pH 4.5. The IgG were
directly coated via onto a CM5 chip. The surface of the chips was
activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the IgG
was flowed over until 780RU germlined E3-IgG and 728 non germlined
E3 IgG was coated onto the surface. All flow cells were
subsequently deactivated with a 7 minute pulse of 1M ethanolamine
hydrochloride pH8,5. All analysis was performed in HBS buffer.
Purified recombinant mouse Tie1 Fc was diluted 1/6,5 in HBS to
obtain a 400 nM stock solution. Serial dilutions were made to
obtain 200 nM, 100 nM, 50 nM and 25 nM Tie1 Fc stocks. For analysis
of the association phase samples were injected at 30 .mu.l/min for
8.3 minutes using kinject program. This was followed by a 40
minutes dissociation phase. Any antigen remaining associated to the
surface was stripped from the IgG surface at a flow of 50 .mu.l/min
with two injections of 10 mM glycine pH1.5 for 30 seconds. All
samples were run and analyzed in duplicate
[0650] Sensorgrams were analyzed using the simultaneous ka/kd
fitting program with 1:1 model in the BIAEVALUATION.TM. software
3.1. The germlining process had minimal impact on the binding
activity of the E3 IgG with respect to mouse Tie1-Fc.
TABLE-US-00008 TABLE 5 Comparison of the binding affinity of
parental and germlined E3 IgG E3 IgG Tie1 Fc kon (1/Ms) koff (1/s)
KD(1) nM parental Mouse 6.17E+03 9.20E-05 14.9 germlined Mouse
6.00E+03 8.99E-05 15
Example 15
Comparison of IC.sub.50 of Germlined Anti Tie1-E3 and Parental Anti
Tie1-E3 in Tube Formation Assays using HUVEC Cells
[0651] Germlined E3 (DX-2220) and its parental antibody (DX-2200)
were evaluated in the tube formation assay in a collagen type-I
matrix. Human Umbilical vein endothelial cells (HUVEC) (freshly
isolated) were obtained by treating human umbilical cord veins with
Trypsin-EDTA (1.times.) (Gibco/Invitrogen) for 20-25 minutes at
37.degree. C. The cells were then cultured in a T-25 flask coated
with attachment factor (AF), (Cascade Biologics) in RPMI 1640
medium supplemented with 10% FCS, 0.4% BBE, 1% 1-glutamin, 1%
penicillin/streptomycin. Primary cultures were detached with warm
Trypsin-EDTA and used when confluent at the second or third
passage. During culturing, the cells were kept in a proliferative
state by culturing them in a split ratio 1:2 at an approximate
density of the monolayer of about 60-80%. HUVEC monolayers were
treated with trypsin/EDTA (500 .mu./dish) at 37.degree. C. for 3
min. Trypsin activity was stopped by adding 3 volumes of complete
RPMI medium. The cells were carefully scraped, separated by
repeated pipetting, and finally washed with PBS. HUVECs (passage 2)
were seeded in their culture medium (40.times.10.sup.3/50
.mu.l/well of a 96-well plate) on a collagen gel (50 .mu.l of
collagen I 1.5 mg/ml) prepared by mixing 7.5 volumes of 2 mg/ml
collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of
10.times. MEM, 1.5 volume of NaHCO3 (15.6 mg/ml) and .about.1
volume of NaOH to adjust the pH to 7.4. After 1 h 30 min., the
culture medium was then discarded and the cells were covered with a
new layer of collagen (1.5 mg/ml, new preparation, 50 .mu.l/well).
After polymerization of the gel, culture medium was added to each
well in presence or in absence of E3 antibody (DX-2200) or
germlined E3 antibody (DX-2220) (0.1 ng/ml to 100 ng/ml). The total
length of the tube network on the culture surface was quantified at
40.times. magnification by the METAVUE.TM. Software (Universal
Imaging Corporation). Results from triplicate wells were expressed
as mean vessel area per field .+-.SEM (relative units). Each assay
was performed at least three times. The conclusions are that
conversion of the three somatic mutations to germline amino acids
in E3 has had little effect on the potency of E3. Both parental E3
(FIGS. 2A and 2B) and germlined E3 (FIGS. 2C and 2D) inhibit tube
formation in vitro with an IC.sub.50 less than 10 ng/ml, i.e. 66
pM.
[0652] Preliminary studies demonstrated that a monovalent Fab
version of DX-2240 Fab was unable to inhibit tube formation in
HUVECs. Thus, in this assay, bivalency is required to elicit an
effect in a cell-based assay.
Example 16
Analysis of Germlined Anti Tie1-E3 in Tube Formation Assays with
Mouse Endothelial Cells
[0653] In order to assess mouse Tie1 cross-reactivity and
biological activity on mouse Tie1, both E3 and germlined E3 were
evaluated for their ability to inhibit tube formation in vitro
using mouse endothelial cell line (LEII).
[0654] LEII lung mouse endothelial cell line (ATCC) was cultured in
a T-25 flask in MEM medium with GLUTAMAX.TM. (Life Technologies
Ltd., Paisley, Scotland) supplemented with 10% FCS, and 1%
penicillin/streptomycin. During culturing, the cells were kept in a
proliferative state by culturing them in a split ratio 1:5 at an
approximate density of the monolayer of about 80%. LEII monolayers
were treated with trypsin/EDTA (500 .mu.l/dish) at 37.degree. C.
for 3 min. Trypsin activity was stopped by adding 3 volumes of
complete MEM medium. The cells were carefully scraped, separated by
repeated pipetting, and finally washed with PBS.LEII cells were
seeded in their culture medium (20-40.times.103/50 .mu.l/well of a
96-well plate) on a basement membrane (BIOCOAT.TM. Angiogenesis
System; Becton Dickinson). After polymerization of the MATRIGEL.TM.
(30 min at 37.degree. C., 5% CO.sub.2 environment) the endothelial
cell suspension resuspended in complete culture medium in the
presence of the desired molecules (4.105 cells/ml; 50 .mu.l/well)
was added to each well. The angiogenesis assay plate was then
incubated for 16 to 18 hours at 37.degree. C., 5% CO.sub.2
atmosphere. The total length of the tube network was then
quantified at 40.times. magnification by the METAVUE.TM. Software
(Universal Imaging Corporation). Results from triplicate wells were
expressed as mean vessel area per field .+-.SEM (relative units).
Each assay was performed at least two times. Germlined E3 is a
potent inhibitor of tube formation in mouse endothelial cells.
Example 17
Immunohistochemical Analysis of Mouse Tumor Tissue Sections Using
Anti Tie1-E3 IgG
[0655] We determined if antibody E3 binds to mouse endothelial
cells in mouse xenographs. Immunohistochemistry was performed with
biotinylated E3 IgG1 (a,z allotype) antibody and control antibodies
anti-CD31 (endothelial cell specific marker) and anti-PCNA
(proliferating cell nuclear antigen). Formalin-fixed tumor tissues
from a mouse-xenograph containing SW480 cells (ATCC) were tested
for the binding pattern of the human anti-Tie1 antibody E3. 5 .mu.m
sections of paraffin embedded tissues were deparaffinized,
rehydrated and pretreated with warm the citrate buffer (0.01 M
sodium citrate, pH6 at 95.degree. C.) for 45 min. The slides were
cooled down in fresh citrate buffer for 20 min and rinsed with
distilled water. The slides were hydrogen peroxide treated, (0.3%
H.sub.2O.sub.2 in PBS), and preincubated with PBS, 5% FCS, 5% heat
inactivated human serum (HS) for 1 hour. Between antibody
incubations slides were washed 3 times 5 minutes in PBS.
Biotinylated antibodies E3 and A2-SV were diluted to a
concentration of 10 .mu.g/ml in PBS, 10% HS and incubated for 1
hour at RT. Slides were then incubated with an avidin-HRP (Dako)
for 30 minutes at room temperature. Staining was detected by AEC
(Vector Laboratories, Burlingame) and H.sub.2O.sub.2. The
peroxidase reaction was stopped with water and slides were
counter-stained with haematoxylin. The tissues were evaluated for
their binding reactivity. The staining pattern was consistent with
staining of mouse endothelial cell Tie1 and also with Tie1
expressed by the E3 binds to Tie1 expressed by SW480 tumor cells in
a mouse xenograft.
Example 18
E3 Activity in a MATRIGEL.TM. Plug Assay
[0656] The germlined variant of the E3 IgG antibody was evaluated
in an in vivo assay for angiogenesis induced by bFGF in
MATRIGEL.TM. plugs. Growth factor reduced MATRIGEL.TM. (BD
Biosciences, catalog #354230) was supplemented with 80 ng/ml of
bFGF (R&D Systems, catalog #234-FSE). The A2-SV or an IgG4 E3
antibody (10 .mu.g/ml) or PBS was injected subcutaneously into the
abdominal area of NMRI nu/nu mice (150 .mu.l of Matrigel/plug).
[0657] In the first assay, two mice were injected with MATRIGEL.TM.
supplemented with bFGF and soluble VEGFR-1 (10 .mu.g/ml) as a
positive control for an angiogenic inhibitor. At day 7
post-implantation mice were anesthetized and perfused through heart
with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS).
MATRIGEL.TM. plugs and a piece of liver were removed and embedded
in paraffin. Sections were cut and stained with hematoxylin and
eosin (H&E).
[0658] The staining revealed modification of MATRIGEL.TM. and
formation of vessel-like structures in the PBS and A2-SV antibody
treated plugs. Even though the E3 antibody and soluble VEGFR1
supplemented plugs contained single cells, there were neither
modification of the matrix nor organization of the cells observed
in these plugs. This results indicates that the germlined E3
antibody inhibits angiogenesis in MATRIGEL.TM. in vivo.
[0659] In a second assay, mice were treated as described above, and
then anesthetized eight days post-implantation and injected with
fluorescein-conjugated tomato (lycopersicon esculentum) lectin (100
.mu.g in 200 .mu.l of PBS; Vector, catalog #FL-1171) into the tail
vein. After five min circulation the animals were perfused through
the heart with 10 ml of PBS followed by 10 ml of 4% PFA in PBS.
MATRIGEL.TM. plugs and pieces of kidney and liver were removed and
frozen in OCT (Tissue-Tek). Nuclei were visualized on sections by
using VECTASHIELD.RTM. mounting medium containing DAPI (Vector) and
analyzed under fluorescence microscopy.
[0660] Staining of the MATRIGEL.TM. with fluorescein lectin
revealed stain-positive material for the PBS and control antibody
(A2-SV) containing plugs, but no staining could be detected in the
E3 antibody containing plugs. As a control, the blood vessels of
the kidney and liver from the same mice showed nice staining with
the fluorescent lectin.
[0661] Results from these two experiments suggest that the
anti-Tie1 antibody E3 can inhibit bFGF-induced angiogenesis in
vivo.
[0662] To assess further the potential anti-angiogenic activity of
the E3 antibody, a third assay examining the effect of DX-2210 (E3
antibody with germlined light chain) on bFGF-induced endothelial
cell tube formation in MATRIGEL.TM. plugs was performed. Growth
factor reduced MATRIGEL.TM. supplemented with HUVECs, bFGF, and
DX-2210, A2-SV (negative control IgG), or PBS were injected
subcutaneously into the abdominal area of Balb/c nu/nu mice (150
.mu.l MATRIGEL.TM./plug). At day 8 post-implantation, mice were
anesthetized and injected with fluorescein-conjugated tomato
(Lycopercicon esculentum) lectin into the tail vein. After a five
minute circulation period, the MATRIGEL.TM. plugs and liver and
kidneys were removed and frozen in OCT media. In order to
quantitate the amount of blood vessels in the MATRIGEL.TM. plugs,
sections were cut and either stained for endothelial cell content
using an anti-CD-31 antibody or analyzed under fluorescence
microscopy to assess the amount of functional blood vessels (tomato
lectin staining) (data not shown). In addition, the amount of blood
vessels per unit area was quantitated. These results demonstrated
that DX-2210 inhibits bFGF-induced angiogenesis by 70% in the
MATRIGEL.TM. assay (FIG. 3).
Example 19
Evaluating Effects of Ligands on Complex Formation
[0663] A candidate protein (for example, E3 or E3b antibody) that
binds a complex member, such as Tie1, Tie2, or an angiopoietin is
tested for its ability to antagonize formation of a heteromeric
complex that includes Tie1, Tie2, and Ang, by inhibiting its
formation or disrupting the heteromeric complex once it forms.
[0664] To test the ability of a candidate protein to disrupt
complex formation, cells expressing Tie1 and Tie2 are treated with
Ang for a period of time sufficient to allow binding of Ang to Tie1
and/or Tie2. The cells are contacted with the candidate protein for
a period of time sufficient to allow disruption of the complex. The
cells are treated with a membrane non-permeable cross-linker, such
as DTSSP, to chemically cross-link the proteins. Cell lysates are
prepared and subjected to immunoprecipitation with an antibody
specific to a complex member. The immunoprecipitated proteins are
separated by SDS-PAGE electrophoresis and immunoblotted with
antibodies specific to the complex members. A positive control
immunoprecipitation-immunoblot is also performed in which cells
expressing Tie1 and Tie2 are treated with Ang but not with the
candidate protein or are treated with a nonspecific protein. If
treatment with the candidate protein decreases the amount of a
complex member--that is not bound by the immunoprecipitating
antibody-associated with the immunoprecipitated member as compared
to the positive control, the candidate protein is an antagonist of
complex formation.
[0665] To determine if a candidate protein inhibits complex
formation, a similar experiment is performed, except that the cells
expressing Tie1 and Tie2 are treated with the candidate protein
prior to treatment of the cells with Ang. The cells are incubated
for a period of time sufficient to allow complex formation in the
absence of candidate protein. As described above, a positive
control in which the cells are not treated with a candidate protein
or are treated with a nonspecific protein is performed. The treated
cells are then lysed and immunoprecipitations and immunoblots are
performed as described above.
[0666] Candidate proteins that antagonize complex formation, by
inhibiting complex formation or by disrupting complexes, are then
tested for their effects on angiogenesis in an assay described
herein.
Example 20
Tie2 Amino Acid Sequence
[0667] An exemplary Tie2 amino acid sequence is as follows:
TABLE-US-00009 SWISS PROT ACCESSION NUMBER: Q02763 (SEQ ID NO: 162)
MDSLASLVLC GVSLLLSGTV EGAMDLILIN SLPLVSDAET SLTCIASGWR 50
PHEPITIGRD FEALMNQHQD PLEVTQDVTR EWAKKVVWKR EKASKINGAY 100
FCEGRVRGEA IRIRTMKMRQ QASFLPATLT MTVDKGDNVN ISFKKVLIKE 150
EDAVIYKNGS FIHSVPRHEV PDILEVHLPH AQPQDAGVYS ARYIGGNLFT 200
SAFTRLIVRR CEAQKWGPEC NHLCTACMNN GVCHEDTGEC ICPPGFMGRT 250
CEKACELHTF GRTCKERCSG QEGCKSYVFC LPDPYGCSCA TGWKGLQCNE 300
ACHPGFYGPD CKLRCSCNNG EMCDRFQGCL CSPGWQGLQC EREGIPRMTP 350
KIVDLPDHIE VNSGKFNPIC KASGWPLPTN EEMTLVKPDG TVLHPKDFNH 400
TDHFSVAIFT IHRILPPDSG VWVCSVNTVA GMVEKPFNIS VKVLPKPLNA 450
PNVIDTGHNF AVINISSEPY FGDGPIKSKK LLYKPVNHYE AWQHIQVTNE 500
IVTLNYLEPR TEYELCVQLV RRGEGGEGHP GPVRRFTTAS IGLPPPRGLN 550
LLPKSQTTLN LTWQPIFPSS EDDFYVEVER RSVQKSDQQN IKVPGNLTSV 600
LLNNLHPREQ YVVRARVNTK AQGEWSEDLT AWTLSDILPP QPENIKISNI 650
THSSAVISWT ILDGYSISSI TIRYKVQGKN EDQHVDVKIK NATIIQYQLK 700
GLEPETAYQV DIFAENNIGS SNPAFSHELV TLPESQAPAD LGGGKMLLIA 750
ILGSAGMTCL TVLLAFLIIL QLKRANVQRR MAQAFQNVRE EPAVQFNSGT 800
LALNRKVKNN PDPTIYPVLD WNDIKFQDVI GEGNFGQVLK ARIKKDGLRM 850
DAAIKRMKEY ASKDDHRDFA GELEVLCKLG HHPNIINLLG ACEHRGYLYL 900
AIEYAPHGNL LDFLRKSRVL ETDPAFAIAN STASTLSSQQ LLHFAADVAR 950
GMDYLSQKQF IHRDLAARNI LVGENYVAKI ADFGLSRGQE VYVKKTMGRL 1000
PVRWMAIESL NYSVYTTNSD VWSYGVLLWE IVSLGGTPYC GMTCAELYEK 1050
LPQGYRLEKP LNCDDEVYDL MRQCWREKPY ERPSFAQILV SLNRMLEERK 1100
TYVNTTLYEK FTYAGIDCSA EEAA 1124
Example 21
Ang1 Amino Acid Sequence
[0668] An exemplary Ang1 amino acid sequence is as follows:
TABLE-US-00010 NCBI ACCESSION NUMBER: AAM92271; gi: 22203641 (SEQ
ID NO: 163) 1 MTVFLSFAFL AAILTHIGCS NQRRSPENSG RRYNRIQHGQ
CAYTFILPEH DGNCRESTTD 61 QYNTNALQRD APHVEPDFSS QKLQHLEHVM
ENYTQWLQKL ENYIVENMKS EMAQIQQNAV 121 QNHTATMLEI GTSLLSQTAE
QTRKLTDVET QVLNQTSRLE IQLLENSLST YKLEKQLLQQ 181 TNEILKIHEK
NSLLEHKILE MEGKHKEELD TLKEEKENLQ GLVTRQTYII QELEKQLNRA 241
TTNNSVLQKQ QLELMDTVHN LVNLCTKEVL LKGGKREEEK PFRDCADVYQ AGFNKSGIYT
301 IYINNMPEPK KVFCNMDVNG GGWTVIQHRE DGSLDFQRGW KEYKMGFGNP
SGEYWLGNEF 361 IFAITSQRQY MLRIELMDWE GNRAYSQYDR FHIGNEKQNY
RLYLKGHTGT AGKQSSLILH 421 GADFSTKDAD NDNCMCKCAL MLTGGWWFDA
CGPSNLNGMF YTAGQNHGKL NGIKWHYFKG 481 PSYSLRSTTM MIRPLDF
Example 22
Conversion of a Mutation Positioned in the Framework 3 Region of
Anti-Tie1 E3 Heavy Chain to Germline Residue
[0669] In order to limit the risk of potential immunogenicity of
the E3 antibody after administration to patients, all non-germline
mutations in framework regions were corrected back to germline
amino acid residues. The anti-Tie1 E03 antibody was isolated from
Dyax Fab 200 library. In this library, the HC framework regions are
unique and correspond to the DP47 germline segment (V3-23). Since
the construction of the synthetic HC-CDR1-CDR2 sublibrary was made
through the assembling of overlapping oligonucleotides, followed by
some PCR cycles, mutations may have been introduced by one of those
2 steps. Therefore, an analysis was performed which aligned the HC
of E03 antibody with the DP47 germline gene sequence. One
non-germline mutation positioned in framework region number 3 was
identified where a methionine residue has been replaced by a valine
residue.
[0670] A strategy was designed to repair this mutation. The
introduction of the germlined residue was facilitated by the
presence of internal restriction sites in the framework flanking
regions of the CDRs. Indeed, the design of the HC-CDR1-CDR2
sublibrary, present in FAB 310 library, was made in such a way that
the shuffling of every CDR is allowed by the presence of unique
restriction sites in the framework flanking regions. Since the
valine residue to be corrected is located in FR3 region, 3' near
the XbaI site, a primer was designed containing both the XbaI
sequence and the corrected methionine germline residue. The changes
were introduced by PCR using the Top XbaI -M forward primer
combined with a 3' reverse primer (CJ-lift Nhe REV) annealing in
the CHI region. A PCR fragment of .about.180 bp was then generated.
The germlining PCR primers were:
TABLE-US-00011 Top XbaI-M primer: (SEQ ID NO: 717) 5'
TTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCA GaTGAAC 3' (SEQ ID NO:
718) F T I S R D N S K N T L Y L Q M N CJ-lift Nhe REV: (SEQ ID NO:
719) 5' GGAGGGTGCTAGCGGGAAGACCG 3'
Example 23
Cloning of Germlined Heavy Chain Tie1 E3
[0671] In order to determine if the germlined residue introduced
into E3 heavy chain had affected the biological activity of the
antibody, a soluble Fab expression vector containing the germlined
E3 antibody (termed "E3b" or DX-2220) was constructed. The PCR
fragment from Example 22 was digested overnight with 50 U/.mu.g
XbaI restriction enzyme, followed by a 5 hours digestion with 25
U/.mu.g BstEII. The cleaved PCR product was then purified on a 1%
TAE-agarose preparative gel. Ligation into the similarly-digested
phagemid expression vector (pMID1) containing the Tie1 E3 germlined
light chain sequence was performed for three hours at room
temperature. Five nanograms of the newly-ligated material were
electroporated into TG1 bacterial cells. Verification of the
correction of the mutation was performed by sequence determination
of the heavy chain of 20 randomly picked isolates. The resulting
coding construct contained sequences that encode a germlined HC and
a germlined LC sequence in a Fab format (termed Fab E3b).
Example 24
Production and Purification of E3b (DX-2220)
[0672] The E3b Fab antibody was reformatted to a human IgG1. This
construct was then used to transiently transfect HEK293T cells.
Plasmid preparations for transient cell transfections were obtained
using the Qiagen filter Plasmid Maxi kit (Qiagen, cat. no. 12263).
HEK293T cells (GenHunter Corp., cat. no. Q401) were seeded 24 hours
before transfection; 220.times.10.sup.6 cells were plated per
CELLSTACK.RTM. culture vessel (CellSTACK.RTM.-10 Chamber, Corning,
cat. no. 3271). Transfections were carried out using the
GeneJuice.RTM. reagent (VWR, cat. no. novg70967-3) following the
manufacturer's instructions. 650 micrograms of plasmid DNA was used
per CELLSTACK.RTM.. Cells were cultured in DMEM (Invitrogen, cat.
no. 31966021) supplemented with 10% "ultra-low IgG" fetal calf
serum (Invitrogen, cat. no. 16250078), at 37.degree. C., 5%
CO.sub.2, in a water saturated atmosphere. Conditioned media were
harvested 72, 144 and 216 hours after transfection, pooled and
sterile filtered.
[0673] Cell culture supernatants were loaded on a 25-ml rProteinA
FF column (GE Healthcare, cat. no. 17-1279-02) equilibrated against
PBS containing 0.5 M NaCl. The column was washed with PBS
containing 0.5 M NaCl, then with 0.1 M sodium acetate pH 5.0 to
remove bovine IgGs and the antibody was eluted with 12.5 mM citric
acid. Fractions (5 ml) containing the antibody were neutralized by
addition of 150 .mu.l of 1 M Tris-HCl, pH 9.0.
[0674] The E3b antibody was further purified by cation exchange.
The antibody was dialyzed against 50 mM sodium citrate, pH 5.0, and
loaded on a HiLoad 26/10 SP Sepharose HP column (GE Healthcare,
cat. no. 17-1138-01) equilibrated in the same buffer. The antibody
was eluted with 50 mM sodium citrate, pH 5.0, containing 1 M NaCl
(linear gradient on 10 column volumes). Fractions containing the
antibody were pooled and dialyzed against PBS. Antibody
concentration was calculated from the absorbance at 280 nm,
assuming that a protein concentration of 1 mg/ml has an absorbance
of 1.36.
Example 25
Testing of E3b-IgG1 Binding to TIE-1/Fc in BIAcore
[0675] The germlined E3b IgG1 stock solution (0.56 mg/ml) and the
parental E3 IgG1 stock solution (0.41 mg/ml) were diluted 50-fold
in 10 mM sodium acetate, pH 4.5. The IgGs were directly coated on a
CM5 chip. The surface of the chip was activated with a 7-minute
pulse of 0.05 M NHS/0.2 M EDC and the IgG was run over the chip
until 823 RU of germlined E3b and 788 RU of parental E3 were coated
on the surface. All flow cells were subsequently deactivated with a
7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5. All
further experiments were performed in HBS buffer.
[0676] Purified recombinant human Tie1/Fc was diluted in HBS to
final concentrations of 200, 100, 50, 25 and 12.5 nM. Samples were
injected at 30 .mu.l/min for 8.3 minutes using the kinject program.
This was followed by a 50-minute dissociation phase. Any remaining
antigen was stripped from the surface with two 30-sec injections of
10 mM glycine, pH 1.5.
[0677] The sensorgrams obtained with this approach are shown below.
Visual analysis shows that the dissociation (k.sub.off) is
extremely slow (only a very small fraction of Tie1/Fc dissociated
despite the long dissociation time), which suggests a very tight
interaction. Interestingly, the dissociation rates for the IgGs as
measured here are much slower that those of the corresponding Fabs
(see Example 29 below), indicating that there is a significant
increase in the affinity when going from the monovalent Fab to the
bivalent IgG (avidity).
Example 26
Testing of E3b--Fab Binding to Tie1/Fc in BIAcore
[0678] To evaluate if the binding behaviour had been affected in
any way by the conversion of the somatic mutations back to germline
residues, the parental and the germlined E3b antibodies were
produced and tested in Biacore as Fab fragments. Here, by contrast
to what was done for the IgGs (see Example 28), and in order to
measure a monovalent interaction, the Fabs were run over the
antigen directly coated onto the surface.
[0679] Recombinant human Tie1/Fc was coated on a CM5 chip. The
surface of the chip was first activated with a 7-minute pulse of
0.05 M NHS/0.2 M EDC, then Tie1/Fc (2 .mu.g/ml in 10 mM sodium
acetate, pH 4.0) was run over the chip surface until 750 RUs were
coated on the surface. All flow cells were subsequently deactivated
with a 7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5.
All further experiments were performed in HBS buffer.
[0680] The parental and the germlined E3b Fabs were prepared. A
series of dilution (50, 25, 12.5, 6.25 and 3.125 nM) was prepared
in HBS buffer. Samples were injected at 30 .mu.l/min for 5.3
minutes using the KINJECT.TM. program. This was followed by a
10-minute dissociation phase, and the remaining Fab was stripped
from the surface with a single 18-sec injection of 50 mM NaOH/1 M
NaCl.
[0681] Sensorgrams were analyzed using the simultaneous ka/kd
fitting program from the BIAEVALUATION.TM. software 3.1 assuming a
1:1 model. This analysis proved that the germlining of the E3
antibody has little or no effect on the affinity against
Tie1/Fc:
TABLE-US-00012 E3 Fab Tie1 Fc kon (1/Ms) koff (1/s) KD (nM)
Parental Human 8.81e4 1.05e-03 12 germlined Human 1.36e5 1.01e-03 7
(E3b)
Example 27
Testing of E3b--IgG for Biological Activity in Tube Formation
Assays
[0682] The purpose of this study was to determine if the correction
of HC mutation back to germline in the parental E3 has any effect
on the biological activity. Human umbilical vein endothelial cells
(HUVEC) (freshly isolated) were obtained by treating human
umbilical cord veins with Trypsin-EDTA (1.times.)
(Gibco/Invitrogen) for 20-25 minutes at 37.degree. C. The cells
were then cultured in a T-25 flask coated with attachment factor
(AF), (Cascade Biologics) in RPMI 1640 medium supplemented with 10%
FCS, 0.4% BBE, 1% 1-glutamin, 1% penicillin/streptomycin. Primary
cultures were detached with warm Trypsin-EDTA and used when
confluent at the second or third passage. During culturing, the
cells were kept in a proliferative state by culturing them in a
split ratio 1:2 at an approximate density of the monolayer of about
60-80%. HUVEC monolayers were treated with trypsin/EDTA (500
.mu.l/dish) at 37.degree. C. for 3 min. Trypsin activity was
stopped by adding 3 volumes of complete RPMI medium. The cells were
carefully scraped, separated by repeated pipetting, and finally
washed with PBS. HUVECs (passage 3) were seeded in their culture
medium (40.times.10.sup.3/50 .mu.l/well of a 96-well plate) on a
collagen gel (50 .mu.l of collagen I 1.5 mg/ml) prepared by mixing
7.5 volumes of 2 mg/ml collagen (Collagen R; Serva, Heidelberg,
Germany), 1 volume of 10.times. MEM, 1.5 volume of NaHCO.sub.3
(15.6 mg/ml) and .about.1 volume of NaOH to adjust the pH to 7.4.
After 1 h 30 min, the culture medium was then discarded and the
cells were covered with a new layer of collagen (1.5 mg/ml, new
preparation, 50 .mu.l/well). After polymerization of the gel,
culture medium was added to each well in presence or in absence of
E3b-IgG1 or parental E3 antibody (1 pg/ml to 10 ng/ml). Endothelial
tube formation was assessed with an inverted photomicroscope.
Microphotographs of the centre of each well at low power
(.times.40) were taken with a Nikon camera with the aid of
imaging-capture software. Tube formation in the microphotographs
was quantitatively analysed (total tube length) with METAVUE.RTM.
software (data not shown). Tube formation by untreated HUVECs in
full endothelial cell growth medium was used as control. Results
from triplicate wells were expressed as mean vessel area per field
.+-.SEM (relative units) (FIG. 4). The conclusions are that
E3b-IgG1 inhibits tube formation. Correction of HC mutation had no
significant effect on biological activity.
Example 28
Exemplary Tie1 Binding Sequences
[0683] The following are exemplary sequences of immunoglobulin
light chain and heavy chain variable domains:
TABLE-US-00013 1. 806C-M0044-A06 L-Variable (AA): (SEQ ID NO: 164)
QSELTQPPSASGTPGQRVTISCSGSSSSIGLNPVNWYQQLPGTAPKVVIHSNDQRPSGV
PDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPAFGGGTKLTVL L-Variable
(DNA): (SEQ ID NO: 165)
CAGAGCGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCAT
CTCTTGTTCTGGAAGCAGCTCCAGCATCGGACTTAATCCTGTAAACTGGTACCAGCAGCTCCCAG
GAACGGCCCCCAAAGTAGTCATCCATAGTAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTC
TCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGACTCCAGTCTGAGGATGAGGC
TGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTCCGGCATTCGGCGGAGGGACCAAGC
TGACCGTCCTAG H-Variable (AA): (SEQ ID NO: 166)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMMWVRQAPGKGLEWVSRIYPSGGITQYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVYRAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 167)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACGTTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCATTACTCAGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATGTCTACAGGGCTTTTGATATCTGGGGCC
AAGGGACAATGGTCACCGTCTCAAGC 2. 806C-M0044-A11 L-Variable (AA): (SEQ
ID NO: 168)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPGGTFGQGTKVEIK L-Variable (DNA):
(SEQ ID NO: 169)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGT
GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGT
GTATTACTGTCAGCAGTATGGTAGCTCACCTCCGGGGGGAACGTTCGGCCAAGGGACCAAGGTGG
AAATCAAA H-Variable (AA): (SEQ ID NO: 170)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMHWVRQAPGKGLEWVSSIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSHHFHFWGDYYFLEYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 171)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTATATTACTGTGCGAGAGATAGCCATCATTTCCATTTTTGGGGTGACT
ATTATTTTCTAGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 3.
806C-M0044-B04 L-Variable (AA): (SEQ ID NO: 172)
QDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 173)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTA
CTACTGTCAACAGAGTTACAGTACCCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 174)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYLMFWVRQAPGKGLEWVSYIYPSGGWTMYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQNYYDSSGYYYRGFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 175)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACCTTATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCTGGACTATGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGGCAAAATTACTATGATAGTAGTGGTTATTACT
ATCGTGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 4.
806C-M0044-B05 L-Variable (AA): (SEQ ID NO: 176)
DIHMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCQQRSNWPPGITFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 177)
GACATCCATATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTC
CTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTC
CCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGT
GGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTA
CTGTCAGCAGCGTAGCAACTGGCCTCCGGGGATCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 178)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWVRQAPGKGLEWVSSIYPSGGWTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVLLHYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 179)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCT
TTCTTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTCATTATGCTGACTCC
GTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAG
CTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCAAGAGTACTACTACACTACTTTGACTACT
GGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 5. 806C-M0044-B08 L-Variable (AA):
(SEQ ID NO: 180)
QDIQMTQSPSFLSASVGDRVTISCRASQYISIYLNWYQQRPGEAPKLLINAASSLQSGDPSRFSG
SGSGTDFTLTINSLQPDDFATYYCQQYKSYPLTFGEGTKVEIK L-Variable (DNA): (SEQ
ID NO: 181)
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCCGCATCTGTAGGAGACAGAGTCACCAT
CTCTTGCCGGGCAAGTCAGTACATCAGCATATATTTGAATTGGTATCAGCAGAGACCAGGGGAAG
CCCCTAAACTCCTGATCAATGCTGCATCCAGTTTGCAAAGTGGGGACCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGATGATTTTGCAACTTA
TTACTGCCAACAGTATAAGAGTTACCCCCTCACTTTCGGCGAGGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 182)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSVISPSGGQTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTALYYCAGGDRYGPLHYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 183)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACGGTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCTTGTATTACTGTGCGGGAGGGGACAGGTATGGACCCTTGCACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCAAGC 6. 806C-M0044-B09 L-Variable (AA):
(SEQ ID NO: 184)
QDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYHASNLETGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCLQYKSYPRLFGQGTKVEVK L-Variable (DNA): (SEQ
ID NO: 185)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTACCATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGA
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTA
TTACTGTCTTCAGTATAAAAGTTACCCTCGATTGTTCGGCCAAGGGACCAAGGTGGAAGTCAAA
H-Variable (AA): (SEQ ID NO: 186)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSVIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGYYDSSGYSRFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 187)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCTGTGTATTACTGTGCGAGTGGTTACTATGATAGTAGTGGTTACTCCCGAT
TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 7. 806C-M0044-B10
L-Variable (AA): (SEQ ID NO: 188)
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPQLMIYEGSKRPSGLSNRFS
GSKSDNTASLTISGLQAEDEADYYCCSYAGSSTLVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 189)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCCAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGACTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGACAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCTGCTCATATGCAGGTAGTAGCACTTTAGTATTCGGCGGAGGGACCAAGCTGACCG
TCCTA H-Variable (AA): (SEQ ID NO: 190)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMGWVRQAPGKGLEWVSSIYPSGGPTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARSEVGAPDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 191)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCCTACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCATGTATTACTGTGCGAGAAGCGAAGTGGGAGCCCCCGACTACTGGGGCC
AGGGAACCCTGGTCACCGTCTCAAGC 8. 806C-M0044-B12 L-Variable (AA): (SEQ
ID NO: 192)
QDIQMTQSPSTLSASVGDTVTMTCRASQSISGWLAWYQQKPGKAPNLLIFKASTLKSGVPSRFRG
SGSGTEFTLTISSLQPDDFATYYCQQYNSYSQTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 193)
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTTTCTGCATCTGTAGGAGACACCGTCACCAT
GACTTGCCGGGCCAGTCAGAGTATTAGTGGGTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAACCTCCTGATCTTTAAGGCGTCTACTTTAAAAAGTGGGGTCCCGTCAAGGTTTCGCGGC
AGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTA
TTACTGCCAACAATATAATAGTTATTCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 194)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMHWVRQAPGKGLEWVSSIYPSGGYTVYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATDRWSSGGYGVDFWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 195)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCCACAGACCGG
TGGAGCAGTGGCGGGTACGGTGTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 9.
806C-M0044-C07 L-Variable (AA): (SEQ ID NO: 196)
QDIQMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPQFGQGTKVEIK L-Variable (DNA):
(SEQ ID NO: 197)
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCAT
CTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGC
AGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCT
GACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGA
GGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCAGTTCGGCCAAGGGACCAAGG
TGGAAATCAAG H-Variable (AA): (SEQ ID NO: 198)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYDMSWVRQAPGKGLEWVSYIYPSGGPTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDWASRFATWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 199)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGATATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCCCTACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGGCGATTGGGCTTCTCGTTTTGCCACCTGGG
GCCAGGGGACCACGGTCACCGTCTCAAGC 10. 806C-M0044-D01 L-Variable (AA):
(SEQ ID NO: 200)
QYELTQPPSVSVAPGQTARITCGGNNIGIKSVNWYQQKPGQAPVLVVYDDSGRPSGIPQRFSGSN
SGNTATLTINRVEAGDEADYYCQVWDSGSDHWVFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 201)
CAGTACGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTG
TGGGGGAAACAACATTGGAATTAAAAGTGTGAACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTG
TGCTGGTCGTCTATGATGAT
AGTGGCCGGCCCTCAGGGATCCCTCAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCT
GACCATCAACAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTGGTA
GTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA H-Variable (AA): (SEQ
ID NO: 202)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMGWVRQAPGKGLEWVSSIYPSGGFTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNFVESSHYYHDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 203)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTTTACTCGTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCCAGAAATTTCGTTGAAAGTAGTCATTATTACCATG
ACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 11. 806C-M0044-E03
L-Variable (AA): (SEQ ID NO: 204)
QSELTQPPSVSVAPGQTAVITCGGSNIGGKSVHWYQQKSGQAPVLVVFDDRDRPSGIPERFSGSN
SGNTATLTITRVEVGDEADYYCQVWDSGTDHRVFGGGTRLTAL L-Variable (DNA): (SEQ
ID NO: 205)
CAGAGCGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCGTGATTACCTG
TGGGGGGAGCAACATTGGAGGTAAAAGTGTACACTGGTACCAGCAGAAGTCAGGCCAGGCCCCTG
TGCTGGTCGTCTTTGATGATCGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAAC
TCCGGGAACACGGCCACCCTGACCATCACCAGGGTCGAAGTCGGGGATGAGGCCGACTATTACTG
TCAGGTGTGGGATAGTGGAACTGATCATCGGGTGTTCGGCGGAGGGACCAGGCTGACCGCCCTA
H-Variable (AA): (SEQ ID NO: 206)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMFWVRQAPGKGLEWVSGIYPSGGHTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSGGYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 207)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCCATACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGACGAGGC
TCGGGGGGCTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 12.
806C-M0044-F03 L-Variable (AA): (SEQ ID NO: 208)
QSALTQDPAVSVALGQTVRITCRGDRLRSYYSSWYQQKPRQAPVLVMFGRNNRPSGIPDRFSGST
SGSTASLTITATQADDEADYFCSSRDGSGNFLFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 209)
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGGCAGACAGTCAGGATCACATG
CCGAGGAGACAGACTCAGAAGTTATTATTCAAGTTGGTACCAGCAGAAGCCACGACAGGCCCCTG
TTCTTGTCATGTTTGGTAGAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCACC
TCAGGAAGCACAGCTTCCTTGACCATCACTGCGACTCAGGCGGACGATGAGGCTGACTATTTCTG
TAGTTCCCGGGACGGCAGTGGTAATTTCCTCTTCGGCGGAGGGACCAAACTGACCGTCCTT
H-Variable (AA): (SEQ ID NO: 210)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMIWVRQAPGKGLEWVSSIYPSGGTTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSDLGSGWYSAEYFQHWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 211)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCACTACTTCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCTGTGTATTACTGTGCGAGAAGCGACCTAGGCAGTGGCTGGTATAGCGCTG
AATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 13. 806C-M0044-F06
L-Variable (AA): (SEQ ID NO: 212)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSGNLLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTITRLEPEDFAVYFCQQYGGSPPVTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 213)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCGGCAACCTCTTAGCCTGGTATCAGCAGAAACCTGGCC
AGGCTCCCAGACTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCACCAGACTGGAGCCTGAAGATTTTGCAGTGTATTTCTGTCAGCAGTATG
GTGGCTCACCTCCGGTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 214)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYLMIWVRQAPGKGLEWVSRIYPSGGGTEYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTYYYDSSGYQPAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 215)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACCTTATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCGGTACTGAGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTCACGTATTACTATGATAGTAGTGGTTATC
AACCCGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 14.
806C-M0044-F09 L-Variable (AA): (SEQ ID NO: 216)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLIISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 217)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCATCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTATTGTCAGCAGCGTAGCAACTGGCCTCGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 218)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMTWVRQAPGKGLEWVSVIGPSGGNTMYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVWGAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 219)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGGTATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCGGTCCTTCTGGTGGCAATACTATGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTATGGGGTGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACCGTCTCAAGC 15. 806C-M0044-G06 L-Variable (AA): (SEQ ID
NO: 220)
QDIQMTQSPATLSVSPGERATLSCRASQSVYNNLAWYQQKPGQAPRLLIYDASTTATGIPARFSG
SGSGTDFTLTITSLEPEDFAVYYCQQRSNWPSLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 221)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAACGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTTACAACAACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCACCACGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCACCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCCTCGCTCACTTTC GGCGGAGGGACCAAGGTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 222)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWVRQAPGKGLEWVSSIYPSGGWTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVLLHYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 223)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTCATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACTGCAGTCTACTATTGTGCAAGAGTACTACTACACTACTTTGACTACTGGGGCC
AGGGAACCCTGGTCACCGTCTCAAGC 16. 806C-M0044-G07 L-Variable (AA): (SEQ
ID NO: 224)
QDIQMTQSPSFLSASLGDRVTITCRATQGIGTFLAWYQQKAGRAPKLLIYGASTLQSGVPSRFSG
SGSGTEFTLTISSLQPEDFATYYCQQPNSFFGQGTKLEIK L-Variable (DNA): (SEQ ID
NO: 225)
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTTTAGGAGACAGAGTCACCAT
CACTTGTCGGGCCACTCAGGGCATCGGCACTTTTTTAGCCTGGTATCAGCAAAAAGCAGGGAGAG
CCCCTAAACTCCTGATCTATGGTGCTTCCACTTTGCAGAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATTCACTCTCACAATAAGCAGCCTGCAGCCTGAAGATTTTGCAACTTA
TTACTGTCAACAGCCTAATAGTTTTTTTGGGCAGGGGACCAAGCTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 226)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWVRQAPGKGLEWVSSIYPSGGWTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVLLHYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 227)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTCATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCT
ACTATTGTGCAAGAGTACTA
CTACACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 17.
806C-M0044-G11 L-Variable (AA): (SEQ ID NO: 228)
QDIQMTQSPSSVSASVGDRVTITCRASQDISSWLVWYQQKPGKAPKLLIHDASNLQSGVPSRFSG
SGSGTDFTLTINSLQPEDFATYYCQQANSFPVTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 229)
CAAGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCAT
TACTTGTCGGGCGAGTCAGGATATTAGCAGTTGGTTAGTCTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCCATGATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGGTCTGGGACAGATTTTACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTA
CTATTGTCAACAGGCTAACAGTTTCCCGGTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 230)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYPMLWVRQAPGKGLEWVSSISPSGGATAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYSDYGVFESWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 231)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACCCTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCGCTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAAAGGCTCA
TACAGTGATTACGGGGTCTTTGAGTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 18.
806C-M0044-H03 L-Variable (AA): (SEQ ID NO: 232)
QRVLTQPPSASGTPGQRVTISCSGSSSNVGSNNVNWYQQLPGQAPKLLIDSNNHRPSGVPDRFSG
SKSGTSASLALSGLQSEDEADYYCATWDDNLIAPVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 233)
CAGAGGGTCTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTG
TTCTGGAAGCAGCTCCAATGTCGGAAGTAATAATGTAAACTGGTATCAGCAGCTCCCAGGACAGG
CCCCCAAACTCCTCATCGATAGTAATAATCACCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGC
TCCAAGTCTGGCACCTCAGCCTCCCTGGCCCTCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTA
TTATTGTGCGACATGGGACGACAACCTGATTGCCCCGGTATTCGGCGGAGGGACCAAGCTGACCG
TCCTA H-Variable (AA): (SEQ ID NO: 234)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMSWVRQAPGKGLEWVSGIVPSGGWTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNYYDFWSGYYISRFGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 235)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGTATCGTTCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGATAACTATTACGATTTTTGGAGTGGTTATT
ATATTTCTCGATTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 19.
806C-M0044-H05 L-Variable (AA): (SEQ ID NO: 236)
QYELTQPASVSGSPGQSITISCTGSSSDVSGYNYVSWYQHHPGKAPKLMLYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 237)
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGATCCAGCAGTGACGTTAGTGGTTATAACTATGTCTCCTGGTACCAACACCACCCAGGCA
AAGCCCCCAAACTCATGCTTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCAGCTCATATACAAGCAGCAGCACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCG
TCCTA
H-Variable (AA): (SEQ ID NO: 238)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTVPLDSGSYYFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 239)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTACGGTACCCCTTGATAGTGGGAGCTACT
ACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 20. 806C-M0044-H07
L-Variable (AA): (SEQ ID NO: 240)
QDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCLQDYNYPWTFGQGTNVEIK L-Variable (DNA): (SEQ
ID NO: 241)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTAT
GCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTT
CACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTACA
ATTACCCGTGGACGTTCGGC CAAGGGACCAATGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 242)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYLMTWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCAREMYYDFWSGYYRGFDIWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 243)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACCTTATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCACATATTACTGTGCGAGAGAGATGTATTACGATTTTTGGAGTGGTTATT
ATCGAGGTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 21.
806C-M0044-H09 L-Variable (AA): (SEQ ID NO: 244)
QDIQMTQSPSTLSASIGDRVTITCRASQRVSTWVAWYQQKPGRAPKLLIYMASRLESGVPSRFSG
SGSGTEFTLTISSLQPDDFATYWCQQYNFYPRTFGQGTKVDIK L-Variable (DNA): (SEQ
ID NO: 245)
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATAGGAGACAGAGTCACCAT
CACTTGCCGGGCCAGTCAGCGTGTTAGTACTTGGGTGGCCTGGTATCAGCAGAAACCAGGGAGAG
CCCCAAAACTCTTGATCTATATGGCGTCTAGGTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAGTTCACTCTCACCATAAGCAGCCTGCAGCCTGATGATTTTGCTACTTA
TTGGTGCCAACAATATAATTTTTATCCTCGGACGTTCGGCCAAGGGACCAAGGTGGACATCAAA
H-Variable (AA): (SEQ ID NO: 246)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMNWVRQAPGKGLEWVSSISPSGGQTPYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGGAYIPDSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 247)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTGGTACGGTATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCCAGACTCCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGCGAGATCTC
GGTGGGGCCTACATACCTGACTCCTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 22.
806C-M0045-A02 L-Variable (AA): (SEQ ID NO: 248)
QDIQMTQSPSFLSASVGDRVTITCRASQGISNYLAWYQQEPGKAPKLLIYSASTLQTGVPSRFSG
SGSGTEFTLTISSLQPEDFATYYCQQFNSYPRTFGHGTKVEFK L-Variable (DNA): (SEQ
ID NO: 249)
CAAGACATCCAGATGACCCAGTCTCCTTCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCACCAT
CACTTGCCGGGCCAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAAGAACCAGGGAAAG
CCCCTAAGCTCCTCATCTATTCTGCGTCCACTTTGCAAACTGGAGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTA
TTACTGTCAACAGTTTAACAGTTACCCTCGAACGTTCGGCCACGGGACCAAGGTGGAATTCAAA
H-Variable (AA): (SEQ ID NO: 250)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYPMMWVRQAPGKGLEWVSVISPSGGQTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGGRLNAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 251)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTACTTACCCTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCGTGTATTACTGTACGAGAGGGGGGAGGCTGAATGCTTTTGATATCTGGG
GCCAAGGGACAATGGTCACCGTCTCAAGC 23. 806C-M0045-A04 L-Variable (AA):
(SEQ ID NO: 252)
QSALTQDPAVSVALGQTVRFTCQGDSLRNYHPSWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSS
SGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGTGTKVTVL L-Variable (DNA): (SEQ
ID NO: 253)
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGTTCACTTG
CCAAGGAGACAGCCTCAGAAATTATCATCCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTG
TACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGC
TCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTG
TAACTCCCGGGACAGCAGTGGTAACCATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA
H-Variable (AA): (SEQ ID NO: 254)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYQMGWVRQAPGKGLEWVSRIYPSGGVTKYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFGPGDLWSGYYDAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 255)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATTTACCAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCGTTACTAAGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTTCGGTCCGGGCGATTTATGGAGTGGTT
ATTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 24.
806C-M0045-B01 L-Variable (AA): (SEQ ID NO: 256)
QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFS
GSKSATTASLTVSGLQAEDEADYYCSSYAGSNNLIFGGGTKVTVL L-Variable (DNA):
(SEQ ID NO: 257)
CAGAGCGCTTTGACTCAGCCTGCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGCCACCAC
GGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATG
CAGGCAGCAACAATTTGATATTCGGCGGGGGGACCAAGGTGACCGTCCTA H-Variable (AA):
(SEQ ID NO: 258)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYQMQWVRQAPGKGLEWVSVIYPGGYTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLQFYGSSAAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 259)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACCAGATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGT
CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGC
TGAGGACACGGCCGTGTATTACTGTGCAAGACTCCAGTTCTACGGTTCCTCTGCTGCTTTTGACA
TCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 25. 806C-M0045-B03 L-Variable
(AA): (SEQ ID NO: 260)
QDIQMTQSPDTLSLSPGERATLSCRASQSISRYLAWYQQRPGQAPSLLIYDASERAAGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRGNWPLTFGGGTKVDIR L-Variable (DNA): (SEQ
ID NO: 261)
CAAGACATCCAGATGACCCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTATTAGTAGATACTTAGCCTGGTACCAACAAAGACCTGGCCAGG
CTCCCAGCCTCCTCATCTAT
GATGCATCCGAAAGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTGGCA
ACTGGCCGCTCACTTTCGGC GGAGGGACCAAGGTGGACATCAGA H-Variable (AA): (SEQ
ID NO: 262)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMIWVRQAPGKGLEWVSVISPSGGHTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIQYYGGAFDIWGQGKMVTVSS
H-Variable (DNA): (SEQ ID NO: 263)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACCCTATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCATACTTCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAATCCAGTACTACGGTGGGGCTTTTGATATCT
GGGGCCAAGGGAAAATGGTCACCGTCTCAAGC 26. 806C-M0045-B11 L-Variable
(AA): (SEQ ID NO: 264)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPHTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 265)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTACTGTCAGCAGCGTAGCAACTGGCCTCACACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 266)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYGMLWVRQAPGKGLEWVSVISPSGGQTFYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGAEKGMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 267)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCCTTACGGTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTTTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGGCTAGGTGCGGAAAAAGGTATGGACGTCTGGG
GCCAAGGGACCACGGTCACCGTCTCAAGC 27. 806C-M0045-C02 L-Variable (AA):
(SEQ ID NO: 268)
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQRPGQAPRLLIYGASSRATGIPDRFSG
SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 269)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGTATGGTAGCTCACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 270)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMGWVRQAPGKGLEWVSSIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPHCSGGSCYGGYYYYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 271)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAAAGATTCC
CCGCATTGTAGTGGTGGTAGCTGCTACGGGGGCTACTACTACTACGGTATGGACGTCTGGGGCCA
AGGGACCACGGTCACCGTCTCAAGC 28. 806C-M0045-C11 L-Variable (AA): (SEQ
ID NO: 272)
QSELTQPASVSGSPGQSITISCTGTNRDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQADDEAEYYCSSYTSSGTRVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 273)
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAACAGAGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGACGAGGCTGA
GTATTACTGCAGCTCATATACAAGCAGCGGCACTCGAGTCTTCGGAACTGGGACCAAGGTCACCG
TCCTA H-Variable (AA): (SEQ ID NO: 274)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMVWVRQAPGKGLEWVSSIYPSGGVTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVAGALDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 275)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACATTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATGTT
GCCGGAGCTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 29.
806C-M0045-C12 L-Variable (AA): (SEQ ID NO: 276)
QYELTQPASVSGSPGQSITISCTGTSTDVGGYNYVSWYQKHPGKAPKLMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTNTITVVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 277)
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCACTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAAAAACACCCAGGCA
AAGCCCCCAAACTCATGATTTATGATGTCAGTAACCGGCCCTCTGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
CTATTACTGCAGCTCATATACAAACACCATCACCGTGGTGTTCGGCGGAGGGACCAAGCTGACCG
TCCTA H-Variable (AA): (SEQ ID NO: 278)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYWMHWVRQAPGKGLEWVSSIYSSGGRTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAHTDSSTWYRWYFDLWGRGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 279)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACTGGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATTCTTCTGGTGGCCGTACTCATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCCTAAG
GGCTGAGGACACCGCCATGTATTACTGTGCACACACTGATAGCAGCACCTGGTACCGGTGGTACT
TCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC 30. 806C-M0045-D01
L-Variable (AA): (SEQ ID NO: 280)
QDIQMTQSPSTLSSSVGDRVTITCRASQSVSNWLAWYQQKPGKAPKVLIYKASTLESGVPSRFSG
SGSGTEFTLTISSLQPDDFATYYCQHYHRYSRTFGQGTKVEIK L-Variablie (DNA): (SEQ
ID NO: 281)
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTTCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCCAGTCAGAGTGTTAGTAACTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGGTCCTAATCTATAAGGCGTCTACTTTAGAAAGTGGGGTCCCGTCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTA
TTACTGCCAACATTATCATCGTTATTCTCGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 282)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMTWVRQAPGKGLEWVSSIYPSGGWTWYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWQGGAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 283)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTGGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAACTGGCAGGGCGGTGCTTTTGACATCT
GGGGCCAAGGGACAATGGTCACCGTCTCAAGC 31. 806C-M0045-D07 L-Variable
(AA): (SEQ ID NO: 284)
QDIQMTQSPGTLSLSPGERATLSCRASQSVNSNQLAWYQQKPGQAPRLLIYGASNRATGIPARFS
GSGSGTDFTLTISSLEPEDFAVYYCQQRSNFWTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 285)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAACAGCAACCAGTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGTA
GCAACTTTTGGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 286)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYLMMWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARVAPYDSSGSVNYAFDPWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 287)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCTTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCATGTATTACTGTGCCAGAGTCGCCCCCTATGATAGTAGTGGTTCGGTAA
ATTACGCGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 32.
806C-M0045-G01 L-Variable (AA): (SEQ ID NO: 288)
QDIQMTQSPSSLSASVGDRVTITCRASQNINIYLNWYQQKPGRAPSLLIYTQSNLRSGVPSRFSG
SGYGTDFTLTISGLQPEDFATYYCQQSHSAPRTFGQGTRVEIK L-Variable (DNA): (SEQ
ID NO: 289)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGAACATTAACATCTATTTGAATTGGTATCAGCAGAAGCCAGGGAGAG
CCCCTAGCCTCCTGATTTAT
ACTCAATCCAATTTGCGAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATATGGCACAGATTT
CACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTCACA
GTGCCCCCCGGACGTTCGGC CAGGGGACCAGGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 290)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMVWVRQAPGKGLEWVSVIYPSGGWTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREMIDTISPGWHFDLWGRGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 291)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGAAATG
ATTGACACTATTTCGCCCGGCTGGCACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTC
AAGC 33. 806C-M0045-G10 L-Variable (AA): (SEQ ID NO: 292)
QSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGKNNRPSGIPDRFSGSS
SGNTASLTITGAQAEDEADYYCQSRGSSSGNHYVFGTGTKVTVL L-Variable (DNA): (SEQ
ID NO: 293)
CAGAGCGAATTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATG
CCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTG
TACTTGTCATGTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGT
TCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTG
TCAGTCCCGGGGCAGCAGCAGTGGTAACCATTATGTCTTC
GGAACTGGGACCAAGGTCACCGTCCTA H-Variable (AA): (SEQ ID NO: 294)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYQMMWVRQAPGKGLEWVSSIYPSGGFTRYADSVK
GRFTISRDNSKNILYLQMNSLRAEDTAVYYCAKSYYYGSGTYHYSYYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 295)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCAGATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTTTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATATTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTAT
ATTACTGTGCGAAATCATAT
TACTATGGGTCGGGGACCTATCATTACTCTTACTACGGTATGGACGTCTGGGGCCAAGGGACCAC
GGTCACCGTCTCAAGC 34. 806C-M0046-A11 L-Variable (AA): (SEQ ID NO:
296)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFT
GSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 297)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCTTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGTAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCACT
GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGT
GTATTACTGTCAGCACTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 298)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMDWVRQAPGKGLEWVSGIYPSGGHTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCARLYLWGSYPTQVAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 299)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCCATACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCACGTATTACTGTGCGAGACTTTACCTTTGGGGGAGTTATCCCACCCAGG
TTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 35. 806C-M0046-B06
L-Variable (AA): (SEQ ID NO: 300)
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 301)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 302)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYPMLWVRQAPGKGLEWVSSIYPSGGMTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGYYDSSGWTFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 303)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATGTACCCTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCATGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAAGGTTACTATGATAGTAGTGGGTGGACCT
TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 36. 806C-M0046-B10
L-Variable (AA): (SEQ ID NO: 304)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK L-Varible (DNA): (SEQ
ID NO: 305)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCGCTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA H-Variable (AA): (SEQ
ID NO: 306)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMNWVRQAPGKGLEWVSGIYSSGGYIYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCARRHFNGVGFDLWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 307)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGTATCTATTCTTCTGGTGGCTATATTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCACATATTACTGTGCGAGAAGACATTTCAACGGGGTTGGTTTTGATCTCT
GGGGCCAAGGGACAATGGTCACCGTCTCAAGC 37. 806C-M0046-G12 L-Variable
(AA): (SEQ ID NO: 308)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFS
GSGSGTEFTLTISSLQSEDFAVYYCQLYKTFGGGTKVEIK L-Variable (DNA): (SEQ ID
NO: 309)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT
GGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGT
TTATTACTGTCAGCTGTATAAGACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 310)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSVIYPSGGGTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGYSSGWFLFYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 311)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGGTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCTGTGT
ATTACTGTGCGAGAGTCGGG
TATAGCAGTGGCTGGTTTCTCTTTTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGT
CTCAAGC 38. 806C-M0046-H03 L-Variable (AA): (SEQ ID NO: 312)
QSALTQPRSVSGSPGQSVTISCTGSNTDVGRYNFVSWYQQKPGKAPKLIIYDVYKRPSGVPDRFS
GSKSGNTASLTISGLQADDEADYYCCSYARASTFSYVFGIGTEVTVL L-Variable (DNA):
(SEQ ID NO: 313)
CAGAGCGCTTTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTG
CACTGGATCCAATACTGATGTTGGTCGATACAATTTTGTTTCCTGGTACCAACAAAAGCCAGGCA
AAGCCCCCAAACTCATAATTTATGATGTCTATAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGATGAGGCTGA
TTATTACTGCTGCTCATATGCTCGCGCCTCCACTTTCTCTTATGTCTTCGGAATTGGGACCGAAG
TCACCGTCCTT H-Variable (AA): (SEQ ID NO: 314)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMVWVRQAPGKGLEWVSSIYPSGGHTPYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQTGGYAHFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 315)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCATACTCCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAGACGGGTGGCTACGCCCACTTTGATTACT
GGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 39. 806C-M0046-H10 L-Variable
(AA): (SEQ ID NO: 316)
QDIQMTQSPSSLSASVGDRVTMTCRASQGIGTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG
SGSGTDFTLTISSLQPEDVATYYCQKYNSAPRPFGQGTQVEIK L-Variable (DNA): (SEQ
ID NO: 317)
CAAGACATCCAGATGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
GACTTGCCGGGCGAGTCAGGGCATTGGCACTTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAG
TTCCTAAGCTCCTGATCTATGCTGCGTCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGC
AGTGGATCTGGGACGGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTA
TTACTGTCAAAAGTATAACAGTGCCCCTCGTCCGTTCGGCCAAGGGACCCAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 318)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVSSIYPSGGWTLYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVGPFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 319)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACGTTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTCTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGCAGTG
GGACCTTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 40. 806C-M0046-H11
L-Variable (AA): (SEQ ID NO: 320)
QYELIQPPSVSGIPGQRVTISCSGNNSNFGSNTVTWYQQLPGTAPKLLIYSDSRRPSGVPDRFSG
SRSDTSASLAISGLQSEDEAEYHCAAWDDSLNGVFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 321)
CAGTACGAATTGATTCAGCCACCCTCAGTGTCTGGGATCCCCGGACAGAGGGTCACCATCTCTTG
TTCTGGAAACAACTCCAACTTCGGAAGTAATACTGTAACCTGGTACCAGCAGCTCCCAGGAACGG
CCCCCAAACTCCTCATCTAT
AGTGATAGTCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGACACCTCAGC
CTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGAGTATCACTGTGCAGCATGGGATG
ACAGCCTAAATGGGGTGTTC GGCGGAGGGACCAAGCTGACCGTCCTA H-Variable (AA):
(SEQ ID NO: 322)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMEWVRQAPGKGLEWVSVIYPSGGHTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYYDILTGYYKYYFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 323)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGGAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCCATACTAATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGAGGCTATTACGATATTTTGACTGGTTATT
ATAAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 41.
806C-M0047-B03 L-Variable (AA): (SEQ ID NO: 324)
QDIQMTQSPSPLSASVGDSVTITCRASQRIGSYLNWYQQNPGKAPKLLIYGASNLESGVPSRFSG
RGSGTEFTLTITSLQPEDFATYFCQQTSSVSPLTFGQGTRLDIK L-Variable (DNA): (SEQ
ID NO: 325)
CAAGACATCCAGATGACCCAGTCTCCATCCCCCCTGTCTGCATCTGTAGGAGACAGTGTCACCAT
CACTTGTCGGGCAAGTCAGAGGATTGGCAGCTACTTGAATTGGTATCAGCAGAATCCAGGCAAAG
CCCCAAAACTCCTGATCTAC
GGTGCATCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCCGTGGATCTGGGACAGAGTT
CACACTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTATTTCTGTCAACAGACCTCCA
GTGTCTCCCCGCTCACCTTC GGCCAAGGGACACGACTGGACATTAAA H-Variable (AA):
(SEQ ID NO: 326)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMSWVRQAPGKGLEWVSVIYPSGGWTWYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMMYYYDSSGYLRADAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 327)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTGGACTTGGTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAATGATGTATTACTATGATAGTAGTGGTTACC
TAAGGGCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 42.
806C-M0047-D01 L-Variable (AA): (SEQ ID NO: 328)
QDIQMTQSPGTLSTSIGDRVTITCRASQSINEWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPALTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 329)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTCTCTACATCTATAGGAGACAGAGTCACCAT
CACTTGCCGGGCCAGTCAGAGTATTAATGAGTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGTATGGTAGCTCACCCGCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 330)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMMWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTALYYCARSMGYGDAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 331)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGTTTAAG
GGCTGAGGACACCGCCTTGTATTACTGTGCGAGATCAATGGGCTATGGTGATGCTTTTGATATCT
GGGGCCAAGGGACAATGGTCACCGTCTCAAGC 43. 806C-M0047-D03 L-Variable
(AA): (SEQ ID NO: 332)
QDIQMTQSPSSLSASVGDRVTITCRASQTIRSYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSYSMSSWTFGQGTNLEIK L-Variable (DNA): (SEQ
ID NO: 333)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACAAT
CACTTGCCGGGCAAGTCAGACCATTAGAAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTA
CTACTGTCAACAGAGTTACAGTATGTCGTCGTGGACTTTTGGCCAGGGGACCAACCTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 334)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYPMAWVRQAPGKGLEWVSWISPGGKTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTATYYCARGSRHYDKFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 335)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGTTTACCCTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTGGATCTCTCCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAAGGT
CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGC
TGAGGACACAGCCACGTATTACTGTGCGAGAGGGAGCCGCCACTATGATAAGTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCAAGC 44. 806C-M0047-E10 L-Variable (AA):
(SEQ ID NO: 336)
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKVMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLLAEDEADYYCSSYTSTATYVLGTGTRVTVV L-Variable (DNA):
(SEQ ID NO: 337)
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTACAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAAGTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCGGGGCTCCTGGCTGAGGACGAAGCTGA
TTATTACTGCAGCTCATATACAAGTACAGCCACCTATGTC
CTCGGAACTGGGACCAGGGTCACCGTCGTA H-Variable (AA): (SEQ ID NO: 338)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMAWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALPGGYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 339)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGGGCCTTA
CCGGGGGGCTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 45.
806C-M0047-G09 L-Variable (AA): (SEQ ID NO: 340)
QDIQMTQSPGTLSLSPGERATLACRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFS
GSGSDTDFTLKISRVEAEDVGTYYCMQATFWPYAFGQGTKLEIK L-Variable (DNA): (SEQ
ID NO: 341)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CGCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGACAGATTCAGCGGCAGTGGGTCAGACACTGA
TTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGACTTATTACTGCATGCAAGCTA
CATTCTGGCCGTACGCTTTT GGCCAGGGGACCAAGCTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 342)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYRMVWVRQAPGKGLEWVSGIYPSGGFTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYDSSGYYFRGGFDPWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 343)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTGGTACCGTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTTTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTGTAT
TACTATGATAGTAGTGGTTATTATTTCCGTGGGGGGTTCGACCCCTGGGGCCAGGGCACCCTGGT
CACCGTCTCAAGC 46. 806C-M0053-A02 L-Variable (AA): (SEQ ID NO: 344)
QSVLTQPPSVSGIPGQRVTISCSGNNSNFGSNTVTWYQQLPGTAPKLLIYSDSRRPSGVPDRFSG
SRSDTSASLAISGLQSEDEAEYHCAAWDDSLNGVFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 345)
CAGAGCGTCTTGACTCAGCCACCCTCAGTGTCTGGGATCCCCGGACAGAGGGTCACCATCTCTTG
TTCTGGAAACAACTCCAACTTCGGAAGTAATACTGTAACCTGGTACCAGCAGCTCCCAGGAACGG
CCCCCAAACTCCTCATCTAT
AGTGATAGTCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGACACCTCAGC
CTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGAGTATCACTGTGCAGCATGGGATG
ACAGCCTAAATGGGGTGTTC GGCGGAGGGACCAAGCTGACCGTCCTA H-Variable (AA):
(SEQ ID NO: 346)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYLMQWVRQAPGKGLEWVSSIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATRKDGYSRSAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 347)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACCTTATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAACAAGGAAG
GATGGCTACAGTCGAAGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 47.
806C-M0053-A03 L-Variable (AA): (SEQ ID NO: 348)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQRGNWPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 349)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCGTG
GCAACTGGCCCCGGACGTTC GGCCAAGGGACCAAGGTGGAAATCAAA H-Variable (AA):
(SEQ ID NO: 350)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMWWVRQAPGKGLEWVSGIYPSGWTVYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLGGTRAFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 351)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTTGGACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGA
CAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATT
ACTGTGCGAAAGATCTGGGG
GGGACCCGTGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 48.
806C-M0053-A05 L-Variable (AA): (SEQ ID NO: 352)
QSELTQPASVSGSPGQSITISCTGTSSDDVGGYNYVSWYQQHPGKAPKLLIYDVSDRPSGVSNRF
SGSKSGNTASLTISGLLAEDEADYYCGSYRVTSVSRSYVFGTETK L-Variable (DNA):
(SEQ ID NO: 353)
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAG
GCAAAGCCCCCAAACTCCTG
ATTTATGATGTCAGTGATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCTGGCTGAGGACGAGGCTGATTATTATTGCGGCTCAT
ATCGCGTCACCAGCGTCAGC AGATCCTATGTCTTCGGAACTGAGACCAAG H-Variable
(AA): (SEQ ID NO: 354)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYPMTWVRQAPGKGLEWVSRIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRIAALDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 355)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACCCTATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGGGGTCGT
ATAGCAGCTCTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 49.
806C-M0053-A09 L-Variable (AA): (SEQ ID NO: 356)
QSALTQGPTVSVALGQTVRITCQGDTLRYFSASWYQQKPGQAPVLVIFGANNRPSGIPDRFSGSR
SGVTASLTITGAQAEDEAEYYCNSRDGSGNWLFGGGTKLSVL L-Variable (DNA):
(SEQ ID NO: 357)
CAGAGCGCTTTGACTCAGGGCCCTACTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATG
TCAAGGAGACACCCTCAGATACTTTTCTGCAAGTTGGTACCAGCAGAAGCCGGGACAGGCCCCTG
TCCTTGTCATCTTTGGGGCA
AACAATCGGCCCTCAGGGATCCCAGACCGGTTCTCTGGCTCCAGGTCAGGAGTCACCGCTTCCTT
GACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAGTATTACTGTAACTCCCGGGACGGCAGTG
GTAATTGGCTGTTCGGCGGA GGGACCAAGCTGTCCGTCCTC H-Variable (AA): (SEQ ID
NO: 358)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMHWVRQAPGKGLEWVSVIYPSGGATLYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGQYSSGWYTEGWFDPWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 359)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTCTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGCCAG
TATAGCAGTGGCTGGTACACGGAGGGCTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGT
CTCAAGC 50. 806C-M0053-B09 L-Variable (AA): (SEQ ID NO: 360)
QYELTQPPSASGTPGQRVTISCSGSSSNIGSNNVNWYQQLPGTAPKLLIYSNDQRPSGVPDRFSG
SKSATSASLAISGLQSEDEADYHCAAWDDSLNGPVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 361)
CAGTACGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTG
TTCTGGAAGCAGCTCCAACATCGGAAGTAATAATGTCAACTGGTACCAGCAACTCCCAGGAACGG
CCCCCAAACTCCTCATCTAC
AGTAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGCCACCTCAGC
CTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATCACTGTGCAGCATGGGATG
ACAGCCTGAATGGTCCGGTG TTCGGCGGAGGGACCAAGCTGACCGTCCTA H-Variable
(AA): (SEQ ID NO: 362)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMQWVRQAPGKGLEWVSSIYPSGGITYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRGTTRAFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 363)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCATTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGACGA
GGAACGACGCGGGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 51.
806C-M0053-B11 L-Variable (AA): (SEQ ID NO: 364)
QYELTQPPSVSVAPGQTAKILCGGNDIGRKFVHWYQQKPGQAPVLVVFDDSDRPSGIPERFSGSN
SGSTATLTISGVEAGDEADYFCQVWDLSSDHWVFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 365)
CAGTACGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAAGATTCTCTG
TGGGGGAAACGACATTGGAAGAAAGTTTGTTCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTG
TGCTGGTCGTCTTTGATGAT
AGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAATTCTGGGAGCACGGCCACCCT
GACCATCAGCGGGGTCGAAGCCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGATCTTAGTA
GTGATCATTGGGTGTTCGGC GGAGGGACCAAGCTGACCGTCCTA H-Variable (AA): (SEQ
ID NO: 366)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMHWVRQAPGKGLEWVSRIGSSGGHTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCATDYYYDSSGYYYPAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 367)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGATTACGCTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCGGTTCTTCTGGTGGCCATACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCGACTGACTAT
TACTATGATAGTAGTGGTTATTACTACCCTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCAC
CGTCTCAAGC 52. 806C-M0053-D03 L-Variable (AA): (SEQ ID NO: 368)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 369)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATG
GTAGCTCACCTCTGTTCGGC GGAGGGACCAAGGTGGAGATCAAA H-Variable (AA): (SEQ
ID NO: 370)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMMWVRQAPGKGLEWVSSIYPSGGSTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVQGGAGAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 371)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGCTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTACAG
GGGGGGGCGGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 53.
806C-M0053-D06 L-Variable (AA): (SEQ ID NO: 372)
QDIQMTQSPSSLSASVGDRVTITCRASQSINTYLNWYQHKPGKAPELLISAASSLQSGVPSRFSG
SGSGTDFTLTISSLRPEDFATYYCQQSHSISTFTFGPGTKVDVK L-Variable (DNA): (SEQ
ID NO: 373)
CAAGACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGAGCATTAACACCTATTTAAATTGGTATCAGCACAAACCAGGGAAGG
CCCCTGAGCTCCTGATCTCT
GCTGCATCTAGCTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT
CACTCTCACCATCAGCAGTCTGCGACCTGAAGATTTTGCGACTTACTACTGTCAACAGAGTCACA
GTATATCCACATTCACTTTC GGCCCTGGGACCAAAGTGGATGTCAAG H-Variable (AA):
(SEQ ID NO: 374)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMHWVRQAPGKGFEWVSSIVPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMYYYDSSGYYVGRFDIWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 375)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTTGAGTGGGTTTCTTCT
ATCGTTCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGACAAATG
TATTACTATGATAGTAGTGGTTATTATGTCGGGCGTTTTGATATCTGGGGCCAAGGGACCACGGT
CACCGTCTCAAGC 54. 806C-M0053-D12 L-Variable (AA): (SEQ ID NO: 376)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPRITFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 377)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCTCCCCGGATCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 378)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTVPLDSGSYYFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 379)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTTACG
GTACCCCTTGATAGTGGGAGCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC
AAGC 55. 806C-M0053-E03 L-Variable (AA): (SEQ ID NO: 380)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPQLTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 381)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATG
GTAGCTCACCCCAGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 382)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMWWVRQAPGKGLEWVSSIYPSGGWTQYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGGGGFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 383)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTCAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCCGTGT
ATTACTGTGCGAAAGATGTT
GGGGGGGGTGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 56.
806C-M0053-E04 L-Variable (AA): (SEQ ID NO: 384)
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSG
SGSGTEFTLTISSLQSEDFAVYYCLTRVTFGGGTKVELK L-Variable (DNA): (SEQ ID
NO: 385)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATT
CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCTAACACGAGTCA
CTTTCGGCGGAGGGACCAAG GTTGAGCTCAAG H-Variable (AA): (SEQ ID NO: 386)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMGWVRQAPGKGLEWVSSIYPSGGWTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSPLVVPAAIKSGAYYYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 387)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATTCC
CCCCTAGTAGTACCAGCTGCTATTAAGAGCGGGGCCTACTACTACGGTATGGACGTCTGGGGCCA
AGGGACCACGGTCACCGTCTCAAGC 57. 806C-M0053-E08 L-Variable (AA): (SEQ
ID NO: 388)
QSVLTQPPSASGTPGQRVSISCSGSSYNIGVYDVYWYQQLPGTAPKLLIYTNNQRPSGVPDRFSG
SKSGTSASLSISGLRSEDEADYYCAAWDDSLAGWVFGGGTKVTVL L-Variable (DNA):
(SEQ ID NO: 389)
CAGAGCGTCTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAGTATCTCTTG
TTCTGGAAGCAGCTACAACATCGGAGTTTATGATGTATACTGGTACCAGCAGCTCCCAGGAACGG
CCCCCAAACTCCTCATCTAT
ACCAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGC
CTCCCTGTCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCCTGGGATG
ACAGCCTGGCTGGTTGGGTG TTCGGCGGAGGGACCAAGGTGACCGTCCTA H-Variable
(AA): (SEQ ID NO: 390)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMLWVRQAPGKGLEWVSVIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVLRAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 391)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGGGTA
CTAAGAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 58.
806C-M0053-F04 L-Variable (AA): (SEQ ID NO: 392)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK L-Variable (DNA): (SEQ
ID NO: 393)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCGGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGTCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCGATCACCTTCGGC CAAGGGACACGACTGGAGATTAAA H-Variable (AA): (SEQ
ID NO: 394)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYGMYWVRQAPGKGLEWVSVISPSGGYTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYSSGWYLDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 395)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGGTTACGGTATGTATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCTATACTCATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGCGTAT
AGCAGTGGCTGGTACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 59.
806C-M0053-F05 L-Variable (AA): (SEQ ID NO: 396)
QSVLTQPPSLSVSPGQTARIACSGDNLGSRYISWYQQKSGQSPVVVLYQDYRRPSGIPERISGSN
SGNTATLTISGTQAVDEADYYCQAWDRSTAVFGGGTRLTVL L-Variable (DNA): (SEQ ID
NO: 397)
CAGAGCGTCTTGACTCAGCCACCCTCACTGTCCGTGTCCCCAGGGCAGACAGCCCGCATCGCCTG
CTCTGGAGATAATTTGGGGAGTAGATATATTTCCTGGTATCAGCAGAAGTCAGGCCAGTCTCCTG
TGGTGGTCCTCTATCAAGAC
TACAGACGGCCCTCAGGGATCCCTGAGCGAATCTCTGGCTCCAACTCTGGGAACACAGCCACTCT
GACCATCAGCGGGACTCAGGCTGTGGATGAGGCGGACTATTATTGTCAGGCGTGGGACAGAAGCA
CTGCGGTGTTCGGCGGAGGG ACCAGGCTGACCGTCCTA H-Variable (AA): (SEQ ID
NO: 398)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTVPLDSGSYYFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 399)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTTACG
GTACCCCTTGATAGTGGGAGCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC
AAGC 60. 806C-M0053-F06 L-Variable (AA): (SEQ ID NO: 400)
QDIQMTQSPDTLSLSPGERATLSCRASHSVTNNRLAWYQQKPGQSPRLLIYGASNRAAGIPARFS
GSGSGTDFTLTISSLEPEDFAVYYCQQRSHWLYTFGQGTKLEIK L-Variable (DNA): (SEQ
ID NO: 401)
CAAGACATCCAGATGACCCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGAGAAAGAGCCACCCT
CTCATGCAGGGCCAGTCACAGTGTTACTAACAACCGCTTAGCCTGGTACCAGCAGAAACCTGGCC
AGTCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCGCTGGCATCCCTGCCAGGTTCAGTGGCAGTGGCTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGCGTA
GCCACTGGCTTTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 402)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMIWVRQAPGKGLEWVSSIYPSGGQTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARKNGYNNVFDVWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 403)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGGCTGTGT
ATTACTGTGCAAGAAAAAAT
GGCTACAATAATGTATTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 61.
806C-M0053-F08 L-Variable (AA): (SEQ ID NO: 404)
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 405)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATG
CAGGTAGTAGCACTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 406)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMLWVRQAPGKGLEWVSSIYPSGGWTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTPTHNWNDDPDAFDIWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 407)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAGCCGTGT
ATTACTGTACCACCCCTACC
CACAACTGGAACGATGACCCTGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTC
AAGC 62. 806C-M0053-G04 L-Variable (AA): (SEQ ID NO: 408)
QSVLTQPPSVSVAPGQTATITCGGNNIGTKSVHWYQQKPGQAPVFVYDDNDRPSGIPERFSGSNS
GNTATMTISRVEAGDEADYYCQVWDPTGDQYVFGSGTKVTVL L-Variable (DNA): (SEQ
ID NO: 409)
CAGAGCGTCTTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCACGATTACCTG
TGGGGGAAACAACATTGGAACTAAAAGTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTG
TCTTCGTCTATGATGATAAT
GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCCGGGAACACGGCCACCATGAC
CATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATCCTACTGGTG
ATCAGTATGTCTTCGGAAGT GGGACCAAGGTCACCGTCCTA H-Variable (AA): (SEQ ID
NO: 410)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMLWVRQAPGKGLEWVSVIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVVVPAFYYYYYMDVWGKGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 411)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTACTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTAGTA
GTACCAGCTTTCTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTC
AAGC 63. 806C-M0053-G05 L-Variable (AA): (SEQ ID NO: 412)
QSELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLGGVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 413)
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATA
CAAGCAGCAGCACTCTCGGG GGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA
H-Variable (AA): (SEQ ID NO: 414)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMDWVRQAPGKGLEWVSSIYPSGGFTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKMATMDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 415)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGGATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGAGAAG
ATGGCTACAATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 64.
806C-M0054-A08 L-Variable (AA): (SEQ ID NO: 416)
QYELTQPASVSGSPGQSITISCTGTSSDVGGCNYVSWYQQHPGKAPQLLIYDVSYRPSGVSNRFS
GSKSGNTASLTISGLQADDEADYYCSSCTSSSTLFGTGTKVTVL L-Variable (DNA): (SEQ
ID NO: 417)
CAGTACGAATTGACTCAACCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTGTAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCCAACTCTTGATT
TATGATGTCAGTTATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGACGAGGCTGATTACTACTGCAGCTCATGTA
CAAGTAGCAGCACTCTCTTC GGAACTGGGACCAAGGTCACCGTCCTA H-Variable (AA):
(SEQ ID NO: 418)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMHWVRQAPGKGLEWVSRIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAGESNGMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 419)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTGGCT
GGGGAGTCGAACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 65.
806C-M0054-B06 L-Variable (AA): (SEQ ID NO: 420)
QDIQMTQSPSSLSASIGDRVTVTCRTSQSIDTYLNWYQQKPGQAPNLLIYGASSLESGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSYTTSYTFGRGTTLEIQ L-Variable (DNA): (SEQ
ID NO: 421)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCAGCATCTATAGGAGACAGAGTCACCGT
CACTTGCCGGACAAGTCAGAGCATTGACACCTATTTAAATTGGTATCAGCAAAAACCAGGGCAAG
CCCCTAACCTCCTGATCTAT
GGTGCATCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT
CACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACA
CTACCTCCTACACTTTTGGC CGGGGGACCACGCTGGAGATCCAA H-Variable (AA): (SEQ
ID NO: 422)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYKMQWVRQAPGKGLEWVSSIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQTYYYDSSGYFRNAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 423)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATTTACAAGATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGACAAACG
TATTACTATGATAGTAGTGGTTATTTCCGCAATGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCAAGC 66. 806C-M0054-B08 L-Variable (AA): (SEQ ID NO: 424)
QSVLTQAASVSGSPGQSITLSCTGATRDVSWYQQHPGKAPKLVLYEVNSRPSDVSDRFSGSMSGN
TASLTISGLQAEDEADYYCSSTTSRAPRVIFGGGTKLTVL L-Variable (DNA): (SEQ ID
NO: 425)
CAGAGCGTCTTGACTCAGGCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCCTCTCCTG
CACTGGAGCCACCAGGGACGTCTCCTGGTACCAACAACACCCAGGCAAGGCCCCCAAACTCGTCC
TTTATGAAGTCAATAGTCGC
CCCTCAGACGTTTCCGATCGCTTCTCTGGCTCCATGTCTGGCAACACGGCCTCCCTGACCATCTC
TGGACTCCAGGCTGAAGACGAGGCTGATTATTACTGCTCCTCAACCACAAGTCGCGCCCCTCGCG
TGATTTTCGGCGGAGGGACC AAACTGACCGTCCTA H-Variable (AA): (SEQ ID NO:
426)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMVWVRQAPGKGLEWVSWIYPSGGWTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSNYYDSAATLDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 427)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTGG
ATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGGTCAAAT
TACTATGATAGTGCTGCGACTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 67.
806C-M0054-C03 L-Variable (AA): (SEQ ID NO: 428)
QDIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKPGKAPKLLISAASTLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSYSTPSFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 429)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGACCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTCT
GCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT
CACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACA
GTACCCCCTCGTTCGGCCAA GGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ ID
NO: 430)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYQMLWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGYSSGWYALTSKTFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 431)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACCAGATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTGGGG
TATAGCAGTGGCTGGTACGCGTTGACTTCAAAGACTTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCAAGC 68. 806C-M0054-C07 L-Variable (AA): (SEQ ID NO: 432)
QDIQMTQSPATLSLSPGDRAILSCRASHNIDNFLAWYQQKPGQAPRLLIYDASHRATGIPPRFSG
SGSGTDFTLTISSLEPEDFAVYFCQQRTNWLFGGGTKVEIK L-Variable (DNA): (SEQ ID
NO: 433)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCGGGGGATCGAGCCATCCT
CTCCTGTAGGGCCAGTCACAATATTGACAACTTCTTAGCCTGGTATCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCTCATAGGGCCACTGGCATCCCCCCCCGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAACCTGAAGATTTTGCTGTGTATTTCTGTCAACAACGGACCA
ACTGGCTTTTCGGCGGAGGG ACCAAGGTGGAGATCAAA H-Variable (AA): (SEQ ID
NO: 434)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMNWVRQAPGKGLEWVSRIWPSGGSTVYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSRYFDVWGRGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 435)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTGGCCTTCTGGTGGCTCTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATTCT
TCTCGATACTTCGATGTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC 69.
806C-M0054-E04 L-Variable (AA): (SEQ ID NO: 436)
QDIQMTQSPATLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIYGTSTRATGIPARFSG
SGSGTEFTLTISSLQSEDFVVYYCQQYKDWPLTFGGGTTVEIK L-Variable (DNA): (SEQ
ID NO: 437)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTATTAGCAGTAATTTAGCCTGGTACCAACAAAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GGTACATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACCGAGTT
CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGTAGTTTATTACTGTCAGCAGTATAAAG
ACTGGCCTCTCACTTTCGGC GGAGGGACCACGGTGGAGATCAAG H-Variable (AA): (SEQ
ID NO: 438)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMHWVRQAPGKGLEWVSVIYPSGGVTEYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQYSGHDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 439)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGTTACTGAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATCAA
TACAGTGGCCATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 70.
806C-M0054-G01 L-Variable (AA): (SEQ ID NO: 440)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRYSWPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 441)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTTACA
GCTGGCCTCTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAG H-Variable (AA): (SEQ
ID NO: 442)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYQMIWVRQAPGKGLEWVSYIVPSGGFTAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNYYGMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 443)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGAGTACCAGATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTATATCGTTCCTTCTGGTGGCTTTACTGCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTGAACTACTACGGTATGGACGTCTGGGGCC
AAGGGACCACGGTCACCGTCTCAAGC 71. 806C-M0054-G05 L-Variable (AA): (SEQ
ID NO: 444)
QSALTQPASVSGSPGQSISISCTGTNTDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 445)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCAGCATCTCCTG
CACTGGAACCAACACTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCAGCTCATATACAAGTAGTAGCACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCG
TCCTA H-Variable (AA): (SEQ ID NO: 446)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYLMEWVRQAPGKGLEWVSGIYPSGGKTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNVISVAGTGYYYYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 447)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACCTTATGGAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTGAAC
GTTATATCAGTGGCTGGTACTGGCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCAC
GGTCACCGTCTCAAGC 72. 806C-M0054-H10 L-Variable (AA): (SEQ ID NO:
448)
QDIQMTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATDIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSSWPITFGLGTRLEIK L-Variable (DNA): (SEQ
ID NO: 449)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCATCTACTTAGCCTGGTACCAACAGAAACCTGGTCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGACATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTAGCA
GCTGGCCGATCACCTTCGGC CTTGGGACACGACTGGAGATTAAA H-Variable (AA): (SEQ
ID NO: 450)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMIWVRQAPGKGLEWVSVISPSGGHTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIQYYGGAFDIWGQGKMVTVSS H-Variable
(DNA): (SEQ ID NO: 451)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACCCTATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCATACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAATCCAG
TACTACGGTGGGGCTTTTGATATCTGGGGCCAAGGGAAAATGGTCACCGTCTCAAGC 73.
806C-M0055-A09 L-Variable (AA): (SEQ ID NO: 452)
QDIQMTQSPSSLSASVGDGVTITCRASQSINNHLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 453)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACGGAGTCACCAT
CACTTGCCGGGCAAGTCAGAGCATTAACAACCATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGGTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTA
CTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
H-Variable (AA): (SEQ ID NO: 454)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYRMSWVRQAPGKGLEWVSGIYPSGGGTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPTYYYDSSGYYYSGPIDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 455)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACCGTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCGGTACTACTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGACCCACGTATTACTATGATAGTAGTGGTTATT
ACTACTCGGGGCCTATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 74.
806C-M0055-B11 L-Variable (AA): (SEQ ID NO: 456)
QYELTQPASVSGSPGQSITISCTGTNTDVGGYNLVSWYQQHPGKAPKLIIYEVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEVDYYCGSYTSSSTHVFGSGTKVTVL L-Variable (DNA):
(SEQ ID NO: 457)
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAACACTGACGTTGGTGGTTATAACCTTGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATAATT
TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGTTGATTATTATTGCGGCTCATATA
CAAGCAGCAGTACTCATGTC TTCGGAAGTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 458)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMHWVRQAPGKGLEWVSVIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTAGWFDPWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 459)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGGGACT
GCAGGGTGGTTCGACCCTTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 75.
806C-M0055-B12 L-Variable (AA): (SEQ ID NO: 460)
QSELTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 461)
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCTGCTCATATGCAGGTAGTAGCACTTATGTCTTCGGAACTGGGACCAAGGTCACCG
TCCTA H-Variable (AA): (SEQ ID NO: 462)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMTWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARQEDGGYGTWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 463)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCGAGACAGGAG
GATGGTGGCTACGGGACTTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 76.
806C-M0055-C05 L-Variable (AA): (SEQ ID NO: 464)
QSVLTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPVLVIYGENNRPSGIPDRFSGSS
SGNTGSLTITGAQAEDEADYYCNSRDTSGSHLLFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 465)
CAGAGCGTCTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATG
CCAAGGAGACAGCCTCAGAAGCTATTATGCAACCTGGTACCAACAGAAGCCAGGACAGGCCCCTG
TACTTGTCATCTATGGTGAA
AACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGTTCAGGAAACACAGGTTCCTT
GACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACACCAGTG
GTAGTCATCTATTATTCGGC GGAGGGACCAAGCTGACCGTCCTG H-Variable (AA): (SEQ
ID NO: 466)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYKMLWVRQAPGKGLEWVSSIYPSGGWTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASYYDSGGYYRENFQFWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 467)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACAAGATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCCTCTTACTATGATAGTGGAGGTTATTACC
GAGAAAACTTCCAGTTTTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 77.
806C-M0055-C07 L-Variable (AA): (SEQ ID NO: 468)
QDIQMTQSPSSLSASVGDRVTIICRASQSISIYLNWYQQKPGKAPKVLIYDASSLQSGVPSRFSG
SGSGTDFSLTITSLQPEDFATYYCQQSYSTPPMYTFGQGTKLEIK L-Variable (DNA):
(SEQ ID NO: 469)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CATTTGCCGGGCAAGTCAGAGCATCAGCATCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGGTCCTGATATATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCAGTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTA
CTACTGTCAACAGAGTTACAGTACCCCTCCCATGTACACTTTTGGCCAGGGGACCAAGCTGGAGA
TCAAA H-Variable (AA): (SEQ ID NO: 470)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMHWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKGLDFWSGPDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 471)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCTGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCACATATTACTGTGCAAAAGGGCTCGATTTTTGGAGTGGCCCGGACTACT
GGGGCCAGGGCACCCTGGTCACCGTCTCAAGC 78. 806C-M0055-D03 L-Variable
(AA): (SEQ ID NO: 472)
QDIQMTQSPSSLSASVGDRVTITCWASQDIRTSLAWYQQKPGKPPKLLIFAASTLQGGVPSRFSG
SGSGTEFTLTISGLQPEDFATYYCQHLNGYPLTFGDGTKVEIR L-Variable (DNA): (SEQ
ID NO: 473)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCTGGGCCAGTCAGGATATTCGCACTTCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAC
CCCCTAAACTCCTCATCTTTGCTGCGTCTACTTTGCAAGGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATTCACTCTCACAATCTCCGGCCTGCAGCCTGAGGATTTTGCGACTTA
TTACTGTCAGCACCTTAATGGTTACCCGCTCACTTTCGGC GATGGGACCAAGGTGGAGATCAGA
H-Variable (AA): (SEQ ID NO: 474)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYVMQWVRQAPGKGLEWVSVIYPSGGMTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCARIRGDTRAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 475)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACGTTATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCATGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAGCCACGT
ATTACTGTGCACGGATACGC
GGTGACACCAGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 79.
806C-M0055-D06 L-Variable (AA): (SEQ ID NO: 476)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDLAVYYCQLFGSSPRITFGQGTRLEIK L-Variable (DNA):
(SEQ ID NO: 477)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTGGCAGTATATTACTGTCAGCTGTTTG
GAAGCTCTCCTCGGATCACC TTCGGCCAGGGGACGCGGCTGGAAATTAAA H-Variable
(AA): (SEQ ID NO: 478)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMWWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSLGCSSTSCYDAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 479)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGGTCTTCT
CTAGGGTGTAGTAGTACCAGCTGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCAC
CGTCTCAAGC 80. 806C-M0055-D12 L-Variable (AA): (SEQ ID NO: 480)
QDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG
SGSGTDFTLTISSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 481)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAG
TTCCTAAGCTCCTGATCTAT
GCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTT
CACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACA
GTGCCCCCTGGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 482)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMWWVRQAPGKGLEWVSSISSGGSTVYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTTVTGNYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 483)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTACTTACGGTATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTCTTCTGGTGGCTCTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGA
CAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATT
ACTGTGCGAGAGATCTGACT
ACGGTGACGGGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 81.
806C-M0055-E04 L-Variable (AA): (SEQ ID NO: 484)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSQLAWYQHKRGQPPRLLIYGASSRATGIPDRFS
GSGSGTDYILTISRLEPEDFAVYYCQHFGSSPPATFGQGTKVEIK L-Variable (DNA):
(SEQ ID NO: 485)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTATCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTTCCAGCAGCCAGTTAGCCTGGTACCAGCATAAACGTGGCC
AGCCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTACATTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCATTTTG
GTAGTTCACCTCCGGCGACG TTCGGCCAAGGGACCAAGGTGGAAATCAAA H-Variable
(AA): (SEQ ID NO: 486)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMVWVRQAPGKGLEWVSSIYPSGGVTIYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSSSGWYNPRRAFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 487)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATGGA
AGTAGCAGTGGCTGGTACAATCCCCGTAGGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCAC
CGTCTCAAGC 82. 806C-M0055-E06 L-Variable (AA): (SEQ ID NO: 488)
QYELTQPPSLSVSPGQTVKITCSAEKLSEKYVAWYQQRPGQSPVMVIYQDSRRPSGIPERFSGSN
SGNTATLTISGTQPMDEADYYCQAWFSDSLPFGSGTKVTVL L-Variable (DNA): (SEQ ID
NO: 489)
CAGTACGAATTGACTCAGCCACCCTCTCTGTCCGTGTCCCCAGGACAGACAGTCAAGATCACCTG
CTCTGCAGAGAAGTTGAGTGAGAAATATGTTGCTTGGTATCAACAGAGGCCGGGCCAGTCCCCTG
TCATGGTCATCTATCAAGAT
AGTAGGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCT
GACCATCAGCGGGACCCAGCCCATGGATGAGGCTGACTACTATTGTCAGGCGTGGTTTAGCGACA
GTCTCCCCTTTGGAAGTGGG ACCAAGGTCACCGTCCTA
H-Variable (AA): (SEQ ID NO: 490)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMIWVRQAPGKGLEWVSSIYPSGGHTIYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAREGGGATSFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 491)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGATCTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCATACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCGAGAGAGGGC
GGGGGAGCTACCTCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 83.
806C-M0055-E10 L-Variable (AA): (SEQ ID NO: 492)
QDIQMTQSPATLSLSPGERATLSCRASQSVRTYLGWYQQKHGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 493)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGGACCTATTTAGGCTGGTACCAACAGAAACATGGCCAGG
CTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 494)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYPMFWVRQAPGKGLEWVSVISPSGGQTSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSFSGLAALDFWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 495)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACCCTATGTTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAAATCATTC
TCAGGCTTAGCAGCTCTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 84.
806C-M0055-E12 L-Variable (AA): (SEQ ID NO: 496)
QDIQMTQSPGTLSLSPGERATLSCRASQTVSSGSLAWYQQKPGLAPRLLIYGASRRGTGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSTLPLTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 497)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGACAGTGAGCAGCGGCTCCTTAGCCTGGTACCAGCAGAAACCTGGCC
TGGCTCCCAGGCTCCTCATC
TATGGTGCATCCCGTAGGGGCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGTCAGCAGTATG
GTAGTACACTCCCGCTCACT TTCGGCGGAGGGACCAAGGTCGAGATCAAA H-Variable
(AA): (SEQ ID NO: 498)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYTMYWVRQAPGKGLEWVSSIYPSGGWTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARGRGGSKAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 499)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACACTATGTATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGGCTGTGT
ATTACTGTGCGAGAGGCCGT
GGTGGTAGCAAAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 85.
806C-M0055-F10 L-Variable (AA): (SEQ ID NO: 500)
QSELTQPASVSGSPGQSITISCTGTTSDVGGYNYVSWYQQDPGKVPKLIIYEVYNRPSGVSNRFS
GSKSGNTASLTISGLRAEDEADYYCSSKTSSVTYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 501)
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCACCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTATCAACAGGACCCAGGCA
AAGTCCCCAAACTCATAATT
TATGAGGTCTATAATCGGCCCTCAGGGGTTTCAAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCGGGCTGAGGACGAGGCTGATTATTACTGCAGCTCAAAAA
CAAGCAGCGTCACTTATGTC TTTGGAACTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 502)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMSWVRQAPGKGLEWVSRIYPSGGGTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEAGGSYFLDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 503)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCGGTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAAAGAGGCG
GGTGGGAGCTACTTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 86.
806C-M0055-G02 L-Variable (AA): (SEQ ID NO: 504)
QSELTQPRSVSGSLGQSVTISCTGTTSDVGRYNFVSWYQQYPGRAPKLIIHDVTRRPSGVSDRFS
GSKSGNTASLTISGLQAEDEADYYCCSYAGSFYVFGSGTQVTVL L-Variable (DNA): (SEQ
ID NO: 505)
CAGAGCGAATTGACTCAGCCTCGCTCAGTGTCCGGGTCTCTTGGACAGTCAGTCACCATCTCCTG
CACTGGAACCACCAGTGATGTTGGTCGTTATAACTTTGTCTCCTGGTACCAACAGTATCCAGGCA
GAGCCCCCAAACTCATCATT
CATGATGTCACTCGGCGGCCCTCCGGGGTATCTGATCGCTTCTCTGGCTCCAAGTCCGGCAACAC
GGCCTCCCTGACCATCTCTGGTCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATG
CAGGCAGCTTTTATGTCTTC GGATCTGGGACCCAGGTCACCGTCTTG H-Variable (AA):
(SEQ ID NO: 506)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMIWVRQAPGKGLEWVSGIYPSGGATGYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCARDGGDIVVPDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 507)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCGCTACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAGCCACGT
ATTACTGTGCGAGAGATGGG
GGGGATATTGTAGTGCCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 87.
806C-M0055-G03 L-Variable (AA): (SEQ ID NO: 508)
QYELTQPPSASGTPGQRVTISCSGSSSNIGTNTVYWYQQLPGTAPKLLIYTNVQRPSGVPDRFSG
SKSGTSASLAISGLQSEDEADYYCQSYDGSLSSAVFGGGTQLTVL L-Variable (DNA):
(SEQ ID NO: 509)
CAGTACGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTG
TTCTGGAAGCAGCTCCAACATCGGAACTAATACTGTATACTGGTACCAGCAGCTCCCAGGAACGG
CCCCCAAACTCCTCATCTAT
ACTAATGTCCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGC
CTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACG
GCAGCCTGAGTTCTGCTGTG TTCGGAGGAGGCACCCAGCTGACCGTCCTC H-Variable
(AA): (SEQ ID NO: 510)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYHMGWVRQAPGKGLEWVSSIYSSGGITQYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRVGGWSLFNWFDPWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 511)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACCATATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATTCTTCTGGTGGCATTACTCAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGGCCGA
GTCGGTGGCTGGTCCCTTTTTAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTC
AAGC 88. 806C-M0055-H04 L-Variable (AA): (SEQ ID NO: 512)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK
L-Variable (DNA): (SEQ ID NO: 513)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCTCGGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 514)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMYWVRQAPGKGLEWVSRIVPSGGWTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKGDWYFDLWGRGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 515)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACCCTATGTATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCGTTCCTTCTGGTGGCTGGACTAACTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGATAAG
GGGGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC 89.
806C-M0056-A01 L-Variable (AA): (SEQ ID NO: 516)
QDIQMTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAIYYCQQRSNWPPALTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 517)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTCTGTCTCCAGGGGAGAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGGTACTTAGCCTGGTATCAACAAAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAATTTATTACTGTCAGCAGCGTAGCA
ACTGGCCTCCGGCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA H-Variable
(AA): (SEQ ID NO: 518)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMGWVRQAPGKGLEWVSWIYPSGGITSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARITYFDTSVIDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 519)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGCTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTGG
ATCTATCCTTCTGGTGGCATTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCACGGATTACG
TATTTTGATACCAGCGTTATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 90.
806C-M0056-A06 L-Variable (AA): (SEQ ID NO: 520)
QSVLTQPASVSGSPGQSITISCTGTSSNVGNYNLVSWYQQHPGKAPKLMIYEDNKRPSGVSNRFS
VSKSGNTASLTISGLQTEDEAEYYCCSYAGSGTWCFGRRGTRVTV L-Variable (DNA):
(SEQ ID NO: 521)
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTAATGTTGGGAATTATAACCTTGTCTCCTGGTACCAGCAGCACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGACAATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGTGTCCAAGTCTGGCAACAC
GGCCTCCCTGACAATCTCTGGGCTCCAGACTGAGGACGAGGCTGAATATTACTGCTGCTCATATG
CAGGTAGTGGCACTTGGTGT TTCGGGCGGAGGGGAACCAGAGTGACCGTC H-Variable
(AA): (SEQ ID NO: 522)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYPMEWVRQAPGKGLEWVSRIVPSGGWTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASRVVTTYLDYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 523)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACCCTATGGAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCGTTCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGTCGGGTG
GTAACTACGTACTTAGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 91.
806C-M0056-B08 H-Variable (AA): (SEQ ID NO: 524)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYVMSWVRQAPGKGLEWVSSIYPSGGGTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTATYYCARRKAAAGYLDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 525)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGTTTACGTTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCGGTACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCACATATTACTGTGCGAGACGAAAAGCAGCAGCAGGTTACCTTGACTACT
GGGGCCAGGGAACCCTGGTCACCGTCTCAAGC L-Variable (AA): (SEQ ID NO: 526)
QSALTQPASVSGSPGQSITISCTGTSSDIGAYKHVSWYQQHPGKAPKLMIYEVTNRPSGISNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSRNTWVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 527)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATTTCCTG
CACTGGAACTAGCAGTGACATTGGTGCTTATAAACATGTCTCCTGGTATCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGTCACTAATCGGCCCTCAGGGATTTCTAATCGTTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGTTCATATA
CAAGCCGTAACACTTGGGTA TTTGGCGGAGGGACCAAGCTGACCGTCCTA 92.
806C-M0056-B09 L-Variable (AA): (SEQ ID NO: 528)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGRSPSFGPGTKVDIK L-Variable (DNA): (SEQ
ID NO: 529)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGATGCATCCAGTAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATG
GTAGGTCACCCTCTTTCGGC CCTGGGACCAAAGTGGATATCAAA H-Variable (AA): (SEQ
ID NO: 530)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMSWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARDRPGAFDVWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 531)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGGCTGTGT
ATTACTGTGCAAGAGATCGG
CCTGGAGCTTTTGATGTTTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 93.
806C-M0056-C03 L-Variable (AA): (SEQ ID NO: 532)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPDDSATYYCQQYNSYPITFGQGTRLEIK L-Variable (DNA): (SEQ
ID NO: 533)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGATGATTCTGCAACCTATTACTGCCAACAATATA
ATAGTTATCCGATCACCTTC GGCCAAGGGACACGACTGGAGATTAAA H-Variable (AA):
(SEQ ID NO: 534)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMWWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIGAVGGFDSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 535)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGGATC
GGAGCAGTGGGCGGGTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 94.
806C-M0056-C04 L-Variable (AA): (SEQ ID NO: 536)
QDIQMTQSPSSLSASVGDRVTIACRASHDISDNLNWYQQKPGRAPKVVISDAFNLEAGVPSRFSG
SRSGTYFTFTINSLQPEDVATYYCQQFNNVPYTFGQGTKLEIK L-Variable (DNA): (SEQ
ID NO: 537)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CGCTTGCCGGGCGAGTCACGACATTAGTGACAATTTAAATTGGTATCAGCAAAAACCAGGGAGAG
CCCCTAAGGTCGTGATCTCCGATGCATTCAATTTGGAAGCAGGGGTCCCATCAAGGTTCAGTGGA
AGTAGATCTGGGACATATTTTACTTTCACCATCAACAGCCTGCAGCCTGAAGATGTTGCAACATA
TTACTGTCAACAATTTAATAATGTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
H-Variable (AA): (SEQ ID NO: 538)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMAWVRQAPGKGLEWVSRIYPSGGKTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGGGGRAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 539)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACATTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAGGGTGGTGGTGGGCGTGCTTTTGATATCT
GGGGCCAAGGGACAATGGTCACCGTCTCAAGC 95. 806C-M0056-E08 L-Variable
(AA): (SEQ ID NO: 540)
QSALTQDPAVSVALGQTVKITCQGDSLRNYYASWYQQKPGQAPIVVIYGKNNRPSGIPDRFSGSR
SGSTASLTITGAQAVDEADYYCSSRDTTNYRMEFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 541)
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATCACATG
CCAAGGAGACAGTCTCAGAAATTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTA
TAGTTGTCATCTATGGTAAA
AACAACCGGCCCTCAGGGATCCCAGACCGTTTCTCTGGCTCCAGGTCAGGAAGCACAGCTTCCTT
GACCATCACTGGGGCTCAGGCGGTAGATGAGGCTGACTATTACTGTAGTTCCCGGGACACTACTA
ATTACCGCATGGAATTCGGC GGAGGGACCAAGCTGACTGTCCTA H-Variable (AA): (SEQ
ID NO: 542)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMAWVRQAPGKGLEWVSGIYPSGGFTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIAGGAYHLDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 543)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTTTACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCTGTGT
ATTACTGTGCGAAAATTGCA
GGGGGAGCCTACCACCTTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 96.
806C-M0056-F01 L-Variable (AA): (SEQ ID NO: 544)
QDIQMIQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 545)
CAAGACATCCAGATGATCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTACTGTCAGCAGCGTAGCAACTGGCCTCCGGCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGA
TCAAA H-Variable (AA): (SEQ ID NO: 546)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMEWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSGRYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 547)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGGTATGGAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGGGTAGTGGCCGGTACTTTGACTACTGGG
GCCAGGGCACCCTGGTCACCGTCTCAAGC 97. 806C-M0056-F02 L-Variable (AA):
(SEQ ID NO: 548)
QSELTQPPSASGSPGQSVTITCTGTSSDVGYYNYVSWYQQHPGKAPKLMIFEVSNRPSGVPDRFS
GSKSGNTASLTVSGLQAEDEAHYYCSSYAGSDNFVFGSGTKVTVL L-Variable (DNA):
(SEQ ID NO: 549)
CAGAGCGAATTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCACCTG
CACTGGAACCAGCAGTGACGTTGGTTATTATAACTATGTCTCCTGGTATCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATTTTTGAGGTCAGTAATCGGCCCTCAGGGGTCCCTGATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTCA
TTATTACTGCAGCTCATATGCAGGCAGCGACAATTTTGTCTTCGGAAGTGGGACCAAGGTCACCG
TCTTA H-Variable (AA): (SEQ ID NO: 550)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYVMGWVRQAPGKGLEWVSSIYPSGGYTWYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGGGGRAFDIWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 551)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATTTACGTTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTTGGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGACAGGGA
GGAGGCGGTCGTGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 98.
806C-M0056-F10 L-Variable (AA): (SEQ ID NO: 552)
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLFYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 553)
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCAGCTCATATACAAGCAGCAGCACTCTCTTTTATGTCTTCGGAACTGGGACCAAGG
TCACCGTCCTA H-Variable (AA): (SEQ ID NO: 554)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMMWVRQAPGKGLEWVSYIVPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDYYDFWSGYWWSGGYGMDVWGQGTTVTVSS
H-Variable (DNA): (SEQ ID NO: 555)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTATATCGTTCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTGACTATTACGATTTTTGGAGTGGTTATT
GGTGGTCGGGGGGGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 99.
806C-M0056-F11 L-Variable (AA): (SEQ ID NO: 556)
QDIQMTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYATSTLQSGVPSRFSG
SGSGTEFTLAISTLQPEDFATYYCQQLNSYPITFGQGTRLEIK L-Variable (DNA): (SEQ
ID NO: 557)
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCCAGTCAGGGCATAAGCACTTATTTAGCCTGGTATCAGCAAAAGCCAGGGAAAG
CCCCTAAGCTCTTGATCTAT
GCTACATCCACTTTGCAAAGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATT
CACTCTCGCAATCAGCACCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAACTCAATA
GTTACCCGATCACTTTCGGC CAAGGGACGCGACTGGAGATTAAA H-Variable (AA): (SEQ
ID NO: 558)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMLWVRQAPGKGLEWVSVIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVLRAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 559)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGGGTA
CTAAGAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 100.
806C-M0056-G03 L-Variable (AA): (SEQ ID NO: 560)
QNIQMTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQVPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISRLEPEDFAVYYCQQYGSLPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 561)
CAAAACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTATTAGCAGTTACTTAGCCTGGTATCAACAGAAACCTGGCCAGG
TTCCCAGGCTCCTCATCTAT
GATGCATCCAATAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATGGTA
GTTTACCTCGGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 562)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMHWVRQAPGKGLEWVSVIYPSGGKTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREMGGSGWYDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 563)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCATCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGAAATG
GGTGGTAGTGGCTGGTACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 101.
806C-M0056-G04 L-Variable (AA): (SEQ ID NO: 564)
QDIQMTQSPATLSLSPGARATLSCRASQSVSSYLAWYQQRPGQTPRLLIYGASSRATGIPDRFSG
SGSGTDFTLTISRLEPEDFAVYYCQQYGSSRHTFGQGTKLEIK L-Variable (DNA): (SEQ
ID NO: 565)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGCAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAGACCTGGCCAGA
CTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTA
GCTCACGACACACTTTTGGC CAGGGGACCAAGCTGGAGATCAAA H-Variable (AA): (SEQ
ID NO: 566)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMRWVRQAPGKGLEWVSGIYPSGGWTTYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATVAAAAGAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 567)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACGTTATGCGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAACAGTGGCA
GCAGCTGCGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 102.
806C-M0056-G08 L-Variable (AA): (SEQ ID NO: 568)
QDIQMTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLLYGTSNRATGIPDRFS
GSGSGTDFTLTISRLEPEDFALYYCQQRYKWPLTFGPGTKVDFK L-Variable (DNA): (SEQ
ID NO: 569)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCCGGCTCCTCCTC
TATGGTACATCCAACAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGACTTTGCACTTTATTACTGTCAGCAGCGTT
ACAAGTGGCCTCTCACTTTC GGCCCTGGGACCAAGGTGGATTTCAAA H-Variable (AA):
(SEQ ID NO: 570)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMWWVRQAPGKGLEWVSVISPSGGQTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGQIHGGNLASWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 571)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGGTATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCCGTGT
ATTACTGTGCCAAAGGGCAA
ATCCACGGTGGTAATCTTGCCTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 103.
806C-M0056-G12 L-Variable (AA): (SEQ ID NO: 572)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMISDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 573)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACTAGCAGCGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATGATT
TCTGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATA
CAAGCAGCAGCACTCTGTAT GTCTTCGGAACTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 574)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARVGYSSSWDPHFDYWGQGTLVTVSS
H-Variable (DNA): (SEQ ID NO: 575)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCGAGAGTCGGG
TATAGCAGCAGCTGGGACCCCCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAG C
104. 806C-M0056-H04 L-Variable (AA): (SEQ ID NO: 576)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTEFTLTISSLQSEDFGVYYCQQYKDWPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 577)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
GTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGGAGTTTATTATTGTCAGCAGTATA
AGGACTGGCCTCGAACGTTC GGCCAAGGGACCAAGGTGGAAATCAAA H-Variable (AA):
(SEQ ID NO: 578)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYRMVWVRQAPGKGLEWVSSIYPSGGPTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWSYYYDSSGYYPVSGPFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 579)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACCGTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCCTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGATGGTCG
TATTACTATGATAGTAGTGGTTATTACCCCGTGAGTGGGCCTTTTGATATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCAAGC 105. 806C-M0056-H12 L-Variable (AA): (SEQ ID
NO: 580)
QDIQMTQSPGTLSLSPGERATLSCRASQGVRSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSQGFTFGPGTKVDIK L-Variable (DNA):
(SEQ ID NO: 581)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGGGTGTTAGAAGTACCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATG
GTAGCTCACAGGGTTTCACT TTCGGCCCTGGGACCAAAGTGGATATCAAA H-Vairable
(AA): (SEQ ID NO: 582)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMHWVRQAPGKGLEWVSVIYPSGGITAYADSVK
GRFTISRDNSKNTLYLQMNSLRADDTAVYYCTREVMGPSDYWGQGTLVTVSS H-Vairable
(DNA): (SEQ ID NO: 583)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCATTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGATGACACAGCCGTGT
ATTACTGTACTAGAGAGGTT
ATGGGACCATCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 106.
806C-M0057-B05 L-Vairable (AA): (SEQ ID NO: 584)
QDIQMTQSPATLSVSPGERATLSCRSSQSLSNNLAWYQQKPGQAPRLLIYGASTRATGIPARFSG
SGSGTEFTLTISSLQSEDFATYYCQQANSFPRTFGQGTKLEIK L-Vairable (DNA): (SEQ
ID NO: 585)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGTCCAGTCAGAGTCTTAGCAACAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GGTGCATCCACCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT
CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACA
GTTTCCCTCGAACTTTTGGC CAGGGGACCAAGCTGGAGATCAAA H-Vairable (AA): (SEQ
ID NO: 586)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYVMHWVRQAPGKGLEWVSSIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSTTYSSRPFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 587)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAAGTACGTTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGACCTCTACG
ACTTATAGCAGCAGGCCCTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 107.
806C-M0057-H07 L-Vairable (AA): (SEQ ID NO: 588)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAASKLEDGVPSRFSG
SGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK L-Vairable (DNA): (SEQ
ID NO: 589)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCAT
CACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAGGGAAAG
CCCCTAAACTCCTGATCTAT
GCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGGGACAGATTT
CACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGAGCTACT
CTAGTCCAGGGATCACTTTC GGCCCTGGGACCAAGGTGGAGATCAAA H-Vairable (AA):
(SEQ ID NO: 590)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMMWVRQAPGKGLEWVSVIYSSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSRGIYYAMDVWGQGTTVTVSS H-Vairable
(DNA): (SEQ ID NO: 591)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATTCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGTATCT
CGCGGGATCTACTACGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 108.
806C-M0058-A09 L-Vairable (AA): (SEQ ID NO: 592)
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFVVYYCQQYGRSRYTFGQGTKLEIK L-Vairable (DNA): (SEQ
ID NO: 593)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGTAGTGTATTACTGTCAGCAGTATG
GTAGGTCACGGTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA H-Vairable (AA):
(SEQ ID NO: 594)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMHWVRQAPGKGLEWVSSIYPSGGPTHYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGYSSGWYIHWYFDLWGRGTLVTVSS
H-Vairable (DNA): (SEQ ID NO: 595)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCCTACTCATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGGGTATAGCAGTGGCTGGTACATTCACT
GGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC 109. 806C-M0058-D04
L-Variable (AA): (SEQ ID NO: 596)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSG
SGSGTHFTFTISSLQPEDFATYYCQQADSFPITFGQGTRLEIK L-Variable (DNA): (SEQ
ID NO: 597)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCAT
CACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAGCTCCTGATCTAC
GATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACACACTT
TACCTTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTGACA
GTTTCCCGATCACCTTCGGC CAAGGGACACGACTGGAGATTAAA H-Variable (AA): (SEQ
ID NO: 598)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYFMTWVRQAPGKGLEWVSGISPSGGITSYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYSDYGVFNSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 599)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACTTTATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTCTCCTTCTGGTGGCATTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAAAGGCTCA
TACAGTGATTACGGGGTCTTTAATTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 110.
806C-M0058-E09 L-Variable (AA): (SEQ ID NO: 600)
QDIQMTQSPATLSVSPGERATLSCRASQSISSSLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 601)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCGCTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA H-Variable (AA): (SEQ
ID NO: 602)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYVMAWVRQAPGKGLEWVSVIYPSGGATYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRLAVTHFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 603)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTAATTACGTTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTACGAGACTGGCG
GTTACTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 111.
806C-M0058-F03 L-Variable (AA): (SEQ ID NO: 604)
QDIQMTQSPSTLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYGASNLQSGVSSRFSG
SGSATDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 605)
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAAG
TTCCTAAACTCCTGATCTAT
GGTGCATCTAATTTGCAGTCAGGGGTCTCATCGCGGTTCAGTGGCAGTGGATCTGCGACAGATTT
CACCCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATA
GTTACCCTCTGACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA H-Variable (AA): (SEQ
ID NO: 606)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSVISPSGGQTAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATVRWFGAFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 607)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGATTACGGTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCTGTGT
ATTACTGTGCCACAGTTAGA
TGGTTCGGGGCATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 112.
806C-M0058-G03 L-Variable (AA): (SEQ ID NO: 608)
QDIQMTQSPGTLSLSPGERATLSCRASQSVTSSFLSWYQHRPGQAPRLLIYATSTRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQHYHTSPPTYTFGQGTKLEIK L-Variable (DNA):
(SEQ ID NO: 609)
CAAGACATCCAGATGACCCAGTCTCCAGGCACGCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAAAGTGTGACCAGCAGCTTCTTATCCTGGTACCAGCACAGACCTGGCC
AGGCTCCCAGGCTCCTCATCTATGCTACATCCACCAGGGCCACAGGCATCCCAGACAGGTTCAGT
GGCAGTGGGTCTGGGACAGACTTCACTCTCACTATCAGCAGACTGGAGCCTGAAGATTTTGCAGT
GTATTACTGTCAGCACTATCATACCTCACCTCCCACTTACACTTTTGGCCAGGGGACCAAGCTGG
AGATCAAA H-Variable (AA): (SEQ ID NO: 610)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYLMYWVRQAPGKGLEWVSVIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARGYYYGMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 611)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCTTTACCTTATGTATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGT
ATTACTGTGCGAGAGGCTAC
TACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 113.
806C-M0058-H01 L-Variable (AA): (SEQ ID NO: 612)
QSALTQPPSVSVAPGETAEITCGGENIGSKSVHWYQQKPGQAPVLVIYYDNDRPSGIPERFSGSN
FGSTATLTISRVEAGDEADYYCQVWDSGSEHYVFGTETKVTVLGQ L-Variable (DNA):
(SEQ ID NO: 613)
CAGAGCGCTTTGACTCAGCCACCCTCAGTCTCAGTGGCCCCAGGGGAGACGGCCGAAATTACCTG
TGGGGGCGAGAACATTGGAAGTAAAAGTGTCCATTGGTACCAGCAGAAGCCAGGCCAGGCCCCAG
TGCTGGTCATCTATTATGATAACGACCGCCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAAC
TTTGGGAGCACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTG
TCAGGTCTGGGATAGTGGCAGTGAGCACTATGTCTTCGGAACTGAGACCAAGGTCACCGTCCTAG
GTCAG H-Variable (AA): (SEQ ID NO: 614)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYIMMWVRQAPGKGLEWVSSIYPSGGHTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWYYGMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 615)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGGTTACATTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGATGGTAT
TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 114.
806C-M0059-A02 L-Variable (AA): (SEQ ID NO: 616)
QSALTQPASVSGSPGQSITISCTGTNSDVGGYNYVSWYQQHPGKAPKLIIFDVTNRPSGVSNRFS
GSKAGNTASLTISGLQAEDEADYYCSSYSSTSPRFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 617)
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCAGGGTCTCCTGGACAGTCGATCACCATTTCCTG
CACTGGAACCAACAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATAATTTTTGATGTCACTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCT
GGCTCCAAGGCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGA
TTATTACTGCAGCTCATATTCAAGTACCAGCCCTCGCTTCGGCGGAGGGACCAAGCTGACCGTCC
TG H-Variable (AA): (SEQ ID NO: 618)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYQMQWVRQAPGKGLEWVSRIYPSGGWTVYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRITYDSSGYYDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 619)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATGTACCAGATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTGGACTGTTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCGTGTATTACTGTACACGGATCACGTATGATAGTAGTGGTTATTACGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 115. 806C-M0059-A06 L-Variable
(AA): (SEQ ID NO: 620)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAASKLEDGVPSRFSG
SGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK L-Variable (DNA): (SEQ
ID NO: 621)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCAT
CACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAGGGAAAG
CCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGC
AGTGGAACTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTA
TTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCACTTTCGGCCCTGGGACCAAGGTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 622)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYKMIWVRQAPGKGLEWVSGIYPSGGWTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARLLPALRGAVMDVWGQGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 623)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCCTTACAAGATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCATGTATTACTGTGCGAGACTGTTACCAGCCTTGCGGGGAGCCGTGATGG
ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 116. 806C-M0060-B02
L-Variable (AA): (SEQ ID NO: 624)
QSVLTQDPTVSVALGQTVRITCRGDRLRSYYSSWYQQKPRQAPVLVMFGRNNRPSGIPDRFSGST
SGSTASLTITATQADDEADYFCSSRDGSGNFLFGGGTKLTVL L-Variable (DNA): (SEQ
ID NO: 625)
CAGAGCGTCTTGACTCAGGACCCTACTGTGTCTGTGGCCTTGGGGCAGACAGTCAGGATCACATG
CCGAGGAGACAGACTCAGAAGTTATTATTCAAGTTGGTACCAGCAGAAGCCACGACAGGCCCCTG
TTCTTGTCATGTTTGGTAGAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCACC
TCAGGAAGCACAGCTTCCTTGACCATCACTGCGACTCAGGCGGACGATGAGGCTGACTATTTCTG
TAGTTCCCGGGACGGCAGTGGTAATTTCCTCTTCGGCGGAGGGACCAAACTGACCGTCCTT
H-Variable (AA): (SEQ ID NO: 626)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMHWVRQAPGKGLEWVSSIYPSGGITRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTALYYCARQRGSGWHDSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 627)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATTTACCCTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCATTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCTTGT
ATTACTGTGCGAGACAACGG
GGCAGTGGCTGGCATGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 117.
806C-M0060-H01 L-Variable (AA): (SEQ ID NO: 628)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPVTFGQGTRLEIK L-Variable (DNA): (SEQ
ID NO: 629)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCGGTCACCTTCGGC CAAGGGACACGACTGGAGATTAAA H-Variable (AA): (SEQ
ID NO: 630)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYPMVWVRQAPGKGLEWVSVIVPSGGFTAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARKRPGNAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 631)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTATTACCCTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCGTTCCTTCTGGTGGCTTTACTGCTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCATGTATTACTGTGCGAGAAAGCGACCTGGAAATGCTTTTGATATCTGGG
GCCAAGGGACAATGGTCACCGTCTCAAGC 118. 806C-M0061-A03 L-Variable (AA):
(SEQ ID NO: 632)
QDIQMTQSPSFLSASVGDSVAITCRASQDISRFLAWYQQRPGKAPKLLIFSASTLQSGVPSRFSG
SGSGTEFTLTINALQPEDFATYYCQQLSRYSTFGQGTKLEIK L-Variable (DNA): (SEQ
ID NO: 633)
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGTGTCGCCAT
CACTTGCCGGGCCAGTCAGGACATTAGTCGTTTTTTAGCCTGGTATCAGCAAAGACCAGGGAAAG
CCCCTAAACTCCTGATTTTT
TCTGCTTCCACTTTACAAAGTGGGGTCCCATCCAGGTTCAGCGGCAGTGGATCTGGGACAGAATT
TACTCTCACAATCAACGCCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAACTTAGTC
GTTATTCGACGTTCGGCCAAGGCACCAAACTGGAAATCAAA H-Variable (AA): (SEQ ID
NO: 634)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYKMWWVRQAPGKGLEWVSSISPGGWTHYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPVSSGGDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 635)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTATTACAAGATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTCTCCTGGTGGCTGGACTCATTATGCTGACTCCGTTAAAGGT
CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGC
TGAGGACACGGCCGTGTATTACTGTGCTAGAGGCCCTGTCAGTAGTGGTGGGGACTACTGGGGCC
AGGGAACCCTGGTCACCGTCTCAAGC 119. 806C-M0061-C05 L-Variable (AA):
(SEQ ID NO: 636)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 637)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCTCCGCTCACTTTC GGCGGAGGGACCAAGGTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 638)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMMWVRQAPGKGLEWVSSIYPSGGQTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIAGGAYHLDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 639)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACGTTATGATGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCAGACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAAAATTGCAGGGGGAGCCTACCACCTTGATTACT
GGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 120. 806C-M0061-C06 L-Variable
(AA): (SEQ ID NO: 640)
QYELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLTIFDVTKRPSGVSDRFS
GSKSDNTASLTISGLQAEDEADYYCGSYTSSGSRVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 641)
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCA
AAGCCCCCAAACTCACGATT
TTTGATGTCACTAAACGGCCCTCAGGGGTTTCTGATCGCTTCTCTGGCTCCAAGTCTGACAATAC
GGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAAGACGAAGCTGATTATTACTGCGGCTCATATA
CAAGCAGCGGCTCTCGGGTC TTCGGAACTGGGACCAAGGTCACCGTCCTC H-Variable
(AA): (SEQ ID NO: 642)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMGWVRQAPGKGLEWVSRIYPSGGFTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRIREGYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 643)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTTTACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTACGAGGATAAGGGAAGGGTACTTTGACTACTGGGGCC
AGGGAACCCTGGTCACCGTCTCAAGC 121. 806C-M0061-F07 L-Variable (AA):
(SEQ ID NO: 644)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYAASKLEDGVPSRFSG
SGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK L-Variable (DNA): (SEQ
ID NO: 645)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCAT
CACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAG
CCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGC
AGTGGAACTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTA
TTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCACTTTCGGCCCTGGGACCAAGGTGGAGATCA
AA H-Variable (AA): (SEQ ID NO: 646)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMTWVRQAPGKGLEWVSSIYPSGGFTAYADSVT
GRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAKSTYYYEGSGYYRAFDIWGQGTMVTVSS
H-Variable (DNA): (SEQ ID NO: 647)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGACTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTTTACTGCTTATGCTGACTCCGTTACA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCATGTATTACTGTGCGAAATCGACT
TATTACTATGAGGGTAGTGGTTATTACCGCGCTTTTGATATCTGGGGCCAAGGGACAATGGTCAC
CGTCTCAAGC 122. 806C-M0061-G12 L-Variable (AA): (SEQ ID NO: 648)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPARFS
GSGSGTDFTLTISGLEPEDFVVYYCQKYGSSSLTFGGGTKVETK L-Variable (DNA): (SEQ
ID NO: 649)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTCTATCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGTGGCCTGGAGCCTGAAGATTTTGTAGTGTATTACTGTCAGAAGTATG
GTAGTTCATCGCTCACTTTC GGCGGAGGGACCAAGGTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 650)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYKMWWVRQAPGKGLEWVSVIYPSGGVTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISYSPVGAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 651)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCAGTACAAGATGTGGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGATCTCGTAT
AGTCCCGTGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 123.
806C-M0061-H09 L-Variable (AA): (SEQ ID NO: 652)
QSALTQPPSVSGSPGQSVTISCTGTSSDVGSYNRVSWYRQPPGTAPKVIIYDINNRPSGVPDRFS
GSRSGDTAYLTISGLQVEDEADYYCSSFTSSSTYIFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 653)
CAGAGCGCTTTGACTCAGCCTCCCTCCGTGTCCGGGTCTCCTGGACAGTCAGTCACCATTTCCTG
CACTGGAACCAGCAGTGACGTTGGTAGTTATAACCGTGTCTCCTGGTACCGGCAGCCCCCAGGCA
CAGCCCCCAAAGTCATCATT
TATGACATCAATAATCGGCCCTCAGGTGTCCCTGATCGCTTCTCTGGGTCCAGGTCTGGCGACAC
GGCCTACCTGACCATCTCTGGGCTCCAGGTGGAGGACGAGGCTGATTATTACTGTAGCTCATTTA
CAAGCAGCAGCACCTATATC TTCGGAACTGGGACCAAGGTCACCGTCCTG H-Variable
(AA): (SEQ ID NO: 654)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYKMYWVRQAPGKGLEWVSVIYPSGGYTDYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQLPMSYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 655)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGTTTACAAGATGTATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTATACTGATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGCGGCAGCTGCCCATGTCGTACTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCAAGC 124. 806C-M0062-A12 L-Variabe (AA):
(SEQ ID NO: 656)
QDIQMTQSPLSLPVTPGEPASMSCRSSQSLLQSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTWTFGQGTKVEIK L-Variable (DNA):
(SEQ ID NO: 657)
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCAT
GTCCTGCAGGTCTAGTCAGAGCCTCCTGCAAAGTAATGGATACAACTATTTGGATTGGTACCTGC
AGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCT
GACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGA
GGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTTGGACGTTCGGCCAAGGGACCAAGG
TGGAAATCAAA H-Variable (AA): (SEQ ID NO: 658)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMVWVRQAPGKGLEWVSRIYPSGGFTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKTAHMDVWGKGTTVTVSS H-Variable
(DNA): (SEQ ID NO: 659)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCGCGTATCTATCCTTCTGGTGGCTTTACTAATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAAGACAGCCCACATGGACGTCTGGGGCA
AAGGGACCACGGTCACCGTCTCAAGC 125. 806C-M0062-B05 L-Variable (AA):
(SEQ ID NO: 660)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIK L-Variable (DNA): (SEQ
ID NO: 661)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTA
TTACTGTCAGCAGCGTAGCAGCTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AG H-Variable (AA): (SEQ ID NO: 662)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMNWVRQAPGKGLEWVSSIYPSGGWTNYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRYGDYVRHWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 663)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGAATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCCAGAGGGGGG
AGATACGGTGACTACGTGCGTCACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 126.
806C-M0062-B07 L-Variable (AA): (SEQ ID NO: 664)
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQHKPGQAPRLLIYGASIRATGIPARFSG
SGSGTEFTLTISSLQSEDFGVYYCQQYKDWPRTFGQGTKVEIK L-Variable (DNA): (SEQ
ID NO: 665)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACTCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCACAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GGTGCATCCATCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT
CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGGAGTTTATTATTGTCAGCAGTATAAGG
ACTGGCCTCGAACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA H-Variable (AA): (SEQ
ID NO: 666)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMAWVRQAPGKGLEWVSSIYPSGGVTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSIAAAGTAYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 667)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGATCTT
AGTATAGCAGCAGCTGGTACTGCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 127.
806C-M0062-C08 L-Variable (AA): (SEQ ID NO: 668)
QDIQMTQSPGTLSLSPGERATLSCRASQSFVGSRNLAWYQQKPGQPPRLLIYGAFNRATGIPGRF
SGSGSGTDFTLTISRLEPEDFAVYYCQQYGTSPRTFGGGTKVEIK L-Variable (DNA):
(SEQ ID NO: 669)
CAAGACATCCAGATGACCCAGTCTCCAGGCACGCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTTTTGTCGGCAGCAGAAACTTAGCCTGGTACCAGCAAAAACCTG
GCCAGCCTCCCAGGCTCCTCATCTATGGTGCATTCAACAGGGCCACTGGCATCCCAGGCAGGTTT
AGTGGCAGTGGCTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC
AGTGTATTACTGTCAGCAGTATGGTACGTCACCTCGGACTTTCGGCGGAGGGACCAAAGTGGAGA
TCAAA H-Variable (AA): (SEQ ID NO: 670)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMQWVRQAPGKGLEWFSSIYPSGGATIYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGIPGYFDSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 671)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCAGTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGTTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAAGGGGA
ATTCCGGGCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 128.
806C-M0062-D04 L-Variable (AA): (SEQ ID NO: 672)
QDIQMTQSPLSLSASIGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYATSTLQSGVPSRFSG
SGSGTEFILTISGLQPEDFATYYCQQFNFYPLTLGGGTRVEIKRT L-Variable (DNA):
(SEQ ID NO: 673)
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGTCTGCATCTATAGGAGACAGAGTCACCAT
CACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAGCCAGGGAAAG
CCCCTAAACTCCTGATCTAT
GCAACTTCCACTTTACAGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATT
CATTCTCACAATCAGCGGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATT
TTTATCCTCTCACTCTCGGC GGAGGGACCAGGGTGGAGATCAAACGAACT H-Variable
(AA): (SEQ ID NO: 674)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMVWVRQAPGKGLEWVSSISPSGGNTGYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGNGGFDSWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 675)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTTCTTACGGTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCAATACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAGGAAAT
GGTGGCTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 129.
806C-M0062-E02 L-Variable (AA): (SEQ ID NO: 676)
QSVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 677)
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCACCCAGGCA
AAGCCCCCAAACTCATGATT
TATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC
GGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATG
CAGGTAGTAGCACTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 678)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMSWVRQAPGKGLEWVSVIYPSGGWTGYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVATTSFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 679)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGGGTG
GCAACTACTAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 130.
806C-M0062-E03 L-Variable (AA): (SEQ ID NO: 680)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRSITFGQGTRLEIK L-Variable (DNA):
(SEQ ID NO: 681)
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT
CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCA
ACTGGCCTCGATCGATCACC TTCGGCCAAGGGACACGACTGGAGATTAAA H-Variable
(AA): (SEQ ID NO: 682)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYLMRWVRQAPGKGLEWVSGIYPSGGITAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSYYNYYFDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 683)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACCTTATGCGTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCATTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGCTTCG
GGGAGTTATTATAATTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC
131. 806C-M0062-E11 L-Variable (AA): (SEQ ID NO: 684)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAASKLEDGVPSRFSG
SGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK L-Variable (DNA): (SEQ
ID NO: 685)
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCAT
CACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAGGGAAAG
CCCCTAAACTCCTGATCTAT
GCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGGGACAGATTT
CACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGAGCTACT
CTAGTCCAGGGATCACTTTC GGCCCTGGGACCAAGGTGGAGATCAAA H-Variable (AA):
(SEQ ID NO: 686)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMHWVRQAPGKGLEWVSRIYPSGGITYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGILTGPNYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 687)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGCATTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCATTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGGGATT
TTGACTGGCCCAAACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 132.
806C-M0062-F10 L-Variable (AA): (SEQ ID NO: 688)
QSALTQSPSASASLGASVKLTCSLSSGHSSYAIAWHQQQPEKGPQYLMKVNSDGSHTKGDGIPDR
FSGSSSGAERYLTISSLQSEDEADYYCQTWGTGSWVFGGGTKLTVL L-Variable (DNA):
(SEQ ID NO: 689)
CAGAGCGCTTTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTG
CAGTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATGGCATCAGCAGCAGCCAGAGAAGGGCC
CCCAGTACTTAATGAAGGTTAACAGTGATGGCAGCCACACCAAGGGGGACGGGATCCCTGATCGC
TTCTCAGGCTCCAGCTCTGGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGA
GGCTGACTATTACTGTCAGACCTGGGGCACTGGCTCTTGGGTGTTCGGCGGAGGGACCAAGCTGA
CCGTCCTA H-Variable (AA): (SEQ ID NO: 690)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMSWVRQAPGKGLEWVSYIYPSGGHTEYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREREGTPDYWGQGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 691)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGTCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTAT
ATCTATCCTTCTGGTGGCCATACTGAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGT
ATTACTGTGCGAGAGAAAGG
GAAGGGACCCCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 133.
806C-M0062-G06 L-Variable (AA): (SEQ ID NO: 692)
QSVLTQPASVSGSPGQSITISCTGTSSDDVGGYNYVSWYQQHPGKAPKLLIYDVINRPSGVSNRF
SGSKSGNTASLTISGLQAEDEADYYCSSYASSGARVFGTGTKVTVL L-Variable (DNA):
(SEQ ID NO: 693)
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTG
CACTGGAACCAGCAGTGACGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAG
GCAAAGCCCCCAAACTCCTG
ATTTATGATGTCATTAATCGGCCCTCAGGAGTTTCTAATCGCTTCTCTGGGTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCAT
ATGCAAGCAGCGGCGCTCGA GTCTTCGGAACTGGGACCAAGGTCACCGTCCTA H-Variable
(AA): (SEQ ID NO: 694)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMIWVRQAPGKGLEWVSVIYPSGGHTRYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRVYSSGSAYFDLWGRGTLVTVSS H-Variable
(DNA): (SEQ ID NO: 695)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATTTACCCTATGATTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCCATACTCGTTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACAGCCGTGTATTACTGTACGAGACGGGTATATAGTAGTGGTTCTGCGTACTTCG
ATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC 134. 806C-M0062-H01
L-Variable (AA):
(SEQ ID NO: 696)
QDIQMTQSPSTLSASVGDRVTITCRASQSVAGLLAWFQQKPGKAPKLLISKASILETGVPSRFSG
SGSGTEFTLTITSLQPDDFATYYCQQYSFNSGTFGQGTRVEMK L-Variable (DNA): (SEQ
ID NO: 697)
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTGGGAGACAGAGTCACCAT
CACCTGCCGGGCCAGCCAGAGTGTTGCTGGCTTGTTGGCCTGGTTTCAGCAGAAACCGGGCAAAG
CCCCTAAACTCCTCATCTCTAAGGCGTCTATTTTAGAGACTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATTCACTCTCACCATCACCAGCCTGCAGCCTGATGATTTCGCAACTTA
TTACTGCCAACAATATAGTTTCAATTCTGGGACATTCGGCCAAGGGACCAGGGTGGAAATGAAA
H-Variable (AA): (SEQ ID NO: 698)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMAWVRQAPGKGLEWVSYIYPSGGYTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTALYYCARVRDSAFDIWGQGTMVTVSS H-Variable
(DNA): (SEQ ID NO: 699)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTG
CGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGGCTTGGGTTCGCCAAGCTCCTGGTAAAG
GTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAA
GGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAG
GGCTGAGGACACCGCCTTGTATTACTGTGCGAGAGTAAGGGATTCCGCTTTTGATATCTGGGGCC
AAGGGACAATGGTCACCGTCTCAAGC
Example 29
Exemplary Tie1 Antibodies
[0684] Tables 5 (FIGS. 37) and 6 (FIG. 38) list CDR regions of
exemplary light and heavy chain variable regions which are listed
herein. FIG. 39 (Table 9) list properties of some of the exemplary
antibodies
[0685] Some antibodies described herein include related variable
domains. The same variable domain can function with a different
partner variable domain. For example, M0044-G06 and M0044-B05 share
a HC variable domain, but have different LC variable domains, as do
M0044-G07 and M0044-B05. Other antibodies that have the same HC
variable domain include: HC 54(M0053-D12) and 19(M0044-H05); HC
59(M0053-F05) and 19(M0044-H05); HC 72(M0054-H10) and
25(M0045-B03); and HC 98(M0056-F11) and 57(M0053-E08). Some
antibodies that have the same LC variable domain include: LC
114(M0059-A06) and 106(M0057-H07); LC 130(M0062-E11) and
106(M0057-H07); and LC 115(M0060-B02) and 12(M0044-F03). Some
antibodies have the same CDR3. For example, the CDR3 sequence,
QGGGGRAFDI, is present in M0056-004 and M0056-F02. The CDR3
sequence IAGGAYHLDY is present in M0056-E08 and M0061-C05.
[0686] In some cases, an antibody can include a non-germline
residue. One or more of such non-germline residues can be modified,
e.g., to restore the germline residue. Exemplary non-germline
residues include: L45F (see, e.g., M0053-D06); V48F (see, e.g.,
M0062-008); delta S53 (see, e.g., M0045-B01; M0047-D03; M0055-D12;
M0061-A03); delta G54 (see, e.g., M0053-A03); T57I (see, e.g.,
M0046-B10); E85D (see, e.g., M0056-H12); T87M (see, e.g.,
M0053-F06; M0055-E12; M0056-B08); V89L (see, e.g., M0044-B08;
M0047-D01; M0060-B02; M0062-H01); V89M (see, e.g., M0044-B10;
M0045-C12; M0045-D07; M0053-B11; M0055-B12; M0055-E06; M0056-A01;
M0056-G12; M0058-G03; M0059-A06; M0060-H01; M0061-F07); V89T (see,
e.g., M0044-H07; M0046-A11; M0046-B10; M0047-D03; M0055-007;
M0055-D03; M0055-G02); A93T (see, e.g., M0045-A02; M0053-F08;
M0056-H12; M0058-E09; M0059-A02; M0061-C06; M0062-G06); and T107K
(see, e.g., M0045-B03).
Example 30
Sequence of DX-2220 Antibody
[0687] DX-2220 is a full length, IgG1, germlined human anti-Tie1
antibody E3b. The sequence of DX-2220 is as follows:
TABLE-US-00014 DX-2220 Light Chain Amino Acid Sequence: (SEQ ID NO:
700) DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLIYT
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHTFGQG
TRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
DX-2220 Heavy Chain Amino Acid Sequence: (SEQ ID NO: 701)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYGMVWVRQAPGKGLEWV
SVISPSGGNTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
APRGYSYGYYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK An exemplary
DX-2220 Light Chain Nucleotide Sequence: (SEQ ID NO: 702)
ggcgtgcactctgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtca
ccatcacttgccgggcgagtcagggcattggccattatttagcctggtatcagcagaaaccagggaaagt
tcctaagctcctgatctatactgcatccactttgcaatcaggggtcccatctcggttcagtggcagtgga
tctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaac
agtttaatagttaccctcacaccttcggccaagggacacgactggagattaaacgaactgtggctgcacc
atctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctg
aataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactccc
aggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaa
agcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca
aagagcttcaacaggggagagtgttaataa An exemplary DX-2220 Heavy Chain
Nucleotide Sequence: (SEQ ID NO: 703)
gaagttcaattgttagagtctggtggcggtcttgttcagcctggtggttctttacgtcttt
cttgcgctgcttccggattcactttctctatgtacggtatggtttgggttcgccaagctcctggtaaagg
tttggagtgggtttctgttatctctccttctggtggcaatactggttatgctgactccgttaaaggtcgc
ttcactatctctagagacaactctaagaatactctctacttgcagatgaacagcttaagggctgaggaca
ctgcagtctactattgtgcgagagccccacgtggatacagctatggttactactactggggccagggaac
cctggtcaccgtctcaagcgcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagc
acctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgt
ggaactcaggcgccctgaccagcggcgtccacaccttcccggctgtcctacagtcctccggactctactc
cctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcac
aagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccac
cgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccct
catgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaag
ttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaaca
gcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg
caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga
gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc
tggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta
caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag
agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc
agaagagcctctccctgtctccgggtaaatga
Example 31
DX-2220 Slows Colorectal Cancer Xenograft Tumor Progression in Nude
Mice
[0688] Mice (nu/nu) were implanted subcutaneously with
5.times.10.sup.6 SW-480 (colorectal cancer) cells. After 12 days,
when tumors reached approximately 100-200 mg, the mice were
separated into 5 groups and treated with the following agents (or
left untreated):
[0689] 1--Untreated
[0690] 2--Vehicle (PBS)
[0691] 3--Cisplatin (4 mg/kg/, IV, q2d.times.5 times)
[0692] 4--A2-SV (negative control antibody@10 mg/kg, IP,
q2d.times.14 times)
[0693] 5--DX-2220 (anti-Tie-1 antibody@10 mg/kg, IP, q2d.times.14
times)
[0694] Throughout the study, the length (L) and width (W) of any
tumors that developed were measured in millimeters using calibrated
vernier calipers, where L is the longer of the two dimensions. When
applicable, tumor weight (M) in milligrams was calculated by using
the formula associated with a prolate ellipsoid:
M=(L.times.W.sup.2)/2. Table 7 shows the average weights (in mg) of
the tumors for each of the groups. A2-SV is an isotype matched
(IgG1) negative control antibody that binds strepavidin.
TABLE-US-00015 TABLE 7 Tumor Weight (mg) Days after Group 1 Group 2
Group 3 Group 4 Group 5 Cell Injection Untreated Vehicle Cisplatin
A2-SV DX-2220 5 57 95 48 111 112 9 88 117 69 120 137 12 118 139 137
149 139 15 153 203 185 159 145 19 202 309 207 308 186 22 316 431
235 350 224 26 403 532 310 405 292 28 449 587 363 526 328
[0695] The results from the animal study shown in Table 7 are
depicted graphically in FIG. 5. DX-2220 slowed tumor progression by
44% when compared to vehicle (PBS)-treated control animals. In
addition, DX-2220 was as efficacious as the chemotherapeutic
control (cisplatin).
Example 32
Production and Testing of Germlined Anti Tie1 E3 Fab and IgG for
Binding to Human and Mouse Tie1 in BIACore
[0696] Expression and Purification. Fabs were produced in the E.
coli strain, TG1, using an expression vector containing a Pe1B
leader sequence for secretion into the periplasm. Under the
conditions used for induction (overnight incubation at 30.degree.
C. in the presence of 1 mM IPTG), the majority of the secreted Fab
was localized in the culture medium rather than the periplasm. The
secreted Fab was recovered by adding protein A resin to the
clarified culture medium. This protein A resin was then packed into
a column to facilitate washing, with PBS, and elution with 50 mM
sodium phosphate, 150 mM NaCl, pH 2.5. The pH was brought to
approximately neutral by addition of one half volume of 1 M HEPES
before buffer exchange into PBS The concentration of the purified
germlined E3 (DX-2220) Fab was determined using OD.sub.280 1.4=1.0
mg/ml.
[0697] The IgG was produced transiently in HEK293T cells using
either LIPOFECTAMINE.TM. 2000 or GENEJUICE.TM. as the transfection
reagent. Antibody could be produced from cells harvested at 72,
144, and 216 hours post transfection. Purification of the IgG from
the conditioned culture media essentially followed the same
protocol outlined above for the Fab purification. The concentration
of the purified IgG was determined using OD.sub.280 1.4=1.0
mg/ml.
[0698] For preclinical animal studies, IgG were purified using a
two-step purification procedure, initially with protein A
chromatography subsequently followed by ion exchange chromatography
(IEX). Purified IgGs were subjected to biochemical analyses to
assess endotoxin levels, leached protein A, DNA content, and host
cell proteins.
[0699] Biochemical Analysis
[0700] Affinity analysis of the Fab and IgGs was performed using
surface plasmon resonance using a BIAcore 3000 instrument. For this
analysis both dimeric (Tie1-Fc fusion protein) and monomeric
(Tie1-HIS) versions of the extracellular domain of the Tie1 were
used. The sensor chips used in these experiments were CM5
(dextran-coated) which allow immobilization of proteins to the chip
via standard amine coupling chemistries. The concentration of
flowed antibody was determined using a surface plasmon resonance
based method. Using a high-density protein A sensor chip, under
mass transport limited conditions, the response signal is dependent
only on the concentration of the antibody in the sample under test.
This approach allows a precise determination of the antibody
concentration, a parameter important for accurate determination of
the K.sub.D.
[0701] 470 RUs of Tie1 HIS protein were coated on a CM5 sensor chip
and 5, 20, 100, and 500 nM of the Fab flowed over the chip at four
different flow rates (10, 30, 50 and 80 .mu.l/s), rates in which
the system is not mass transport limited. Kinetic data was
typically determined using a range of analyte concentrations and
three different chip coating densities. The data from the lowest
coating density that gave a good signal was typically chosen, this
was often in a range from 50-100 RUs. Using such low coating
densities allowed the sensor curves to be fit using
BIAEVALUATION.TM. 3.0 software to a 1:1 model, often even when
using a bivalent analyte (IgG or Tie1-Fc fusion protein). For
bivalent analytes, when fit to a 1:1 model was not possible, the
curves were fit using a 2:1 model. The generated data is shown in
Table 8.
TABLE-US-00016 TABLE 8 Kinetic data for binding of DX-2220 Fab and
IgG to human Tie1-Fc fusion protein Human Tie1-Fc K.sub.on Fab
(1/Ms) K.sub.off(1/s) K.sub.D (nM) Fab Parental 8.26E+03 4.47E-05
5.4 Fab Germlined 9.30E+03 4.41E-05 4.7 IgG Parental 6.19E+03
3.61E-05 5.8 IgG Germlined 7.09E+03 3.67E-05 5.2
[0702] The anti-Tie1 antibodies described here bind to both human
and mouse Tie1 molecules. The binding of the anti-Tie1 Fab to mouse
Tie1-Fc fusion protein was compared with the binding of the
anti-Tie1 Fab to human Tie1-Fc fusion protein. In both of these
experiments the Tie1-Fc fusion protein was immobilized on the
sensor chip and the anti Tie-1 Fab served as the analyte. Under
these experimental conditions the anti-Tie1 Fab has very similar
K.sub.D (.about.3 nM) values for both the human and mouse Tie1-Fc
fusion proteins (Table 9). Under conditions that are likely to
mimic those used in the animal efficacy experiments, i.e. Tie1-Fc
immobilized to the CM5 sensor chip and anti-Tie1 used as the
analyte, the measured K.sub.D was 0.2 nM. This greater than 10 fold
increase in affinity represents an avidity effect that results from
a bivalent molecule (anti-Tie1) binding to a multivalent surface
(immobilized Tie1-Fc).
Example 33
Sequence of DX-2240: Germlined F Allotyped E3 Antibody
TABLE-US-00017 [0703] DX-2240 (Light, heavy - variable, constant).
Variable region: DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLIYT
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHTFGQ GTRLEIK Light
constant: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC SEQ ID
NO: 724 light chain (variable + constant) DX-2240 Heavy variable:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYGMVWVRQAPGKGLEWVSV
ISPSGGNTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAP
RGYSYGYYYWGQGTLVTVSS Heavy constant (CH1, Hinge, CH2, CH3):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 723 heavy chain (variable
+ constant)
[0704] The light chain can optionally further include the following
signal sequence: Light signal sequence: MGWSCIILFLVATATGVHS (SEQ ID
NO:720) The heavy chain can optionally further include the
following signal sequence MGWSCIILFLVATATGAHS (SEQ ID NO:721).
Example 34
Characterization of DX-2240 from a GS-CHO Cell Line
[0705] The anti-Tie1 antibody DX-2240 (light and heavy chain
germlined and f-allotype) antibodies was produced in HEK293T cells.
A stable CHO cell line expressing DX-2240 was generated. Using
standard molecular biology cloning techniques, the light and heavy
chains from DX-2240 was inserted into glutamine synthase (GS)
vector system available from Lonza Group Ltd. CH (see, e.g., Clark
et al. (2004) BioProcess International 2(4):48-52; Barnes et al.
(2002) Biotech Bioeng. 81(6):631-639). The single vector constructs
containing the light and heavy chains respectively, were then
combined to create a single, double gene vector. This DNA construct
was then used to generate stable CHO cell lines, grown under MSX
selection pressure. One of these clones was then expanded and a
single 40L stirred bioreactor seeded and run over the course of 12
days. Following the completion of this run, 36 liters of clarified
CHO culture supernatant was loaded onto a 200 ml Protein A XK50
column. The column was first washed with PBS pH 7.4, followed by a
PBS+0.4M NaCl pH7.4 wash, and then with a final wash of PBS pH 7.4
prior to the low pH elutions. DX-2240 IgG1 was eluted first with
0.1M NaCitrate pH 3.5 followed by the same buffer at pH 3.0. A
sharp protein peak eluted at pH 3.0. The pH 3.0 elution contained a
predominant peak representing DX-2240, with a low level of
contaminants eluting shortly thereafter. A high degree of purity
(>95%) of DX-2240 was obtained.
Example 35
Sequence Optimization of Nucleic Acid Encoding DX-2240 Antibody
[0706] To improve expression of DX-2240 in CHO cells, a synthetic
gene with optimized codons and sequences was engineered. The
strategies include codon optimization, CpG island and splice site
analysis. An optimized DX-2240 sequence has been synthesized and
reformatted into the glutamine synthase (GS) vector system. The
exemplary codon optimized sequence is as follows:
TABLE-US-00018 DX-2240-heavy chain (SEQ ID NO: 725) Signal sequence
ATGGGCTGGTCCTGTATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGCTCACTCT
GAGGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGA
CTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCC ATGTACGGCATGGTG TGGGTGAGGCAG
GCCCCTGGCAAGGGCCTGGAGTGGGTGTCC GTGATCTCTCCTTCTGGCGGCAATACCGGC
TACGCCGACTCTGTGAAGGGC CGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTG
TACCTGCAGATGAACTCCCTGAGAGCCGAGGATACCGCCGTGTACTACTGTGCCAGA GCC
CCTAGAGGCTACTCCTACGGCTACTACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCC
TCTGCTTCTACCAAGGGCCCTTCCGTGTTTCCTCT GGCCCCTTCCTCCAAGTCTACCTCT
GGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCCGTGACAGTG
TCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCTGTGCTGCAGTCC
TCCGGCCTGTACTCTCTGTCCTCCGTGGTGACAGTGCCTTCCTCTTCTCTGGGCACCCAG
ACCTACATCTGTAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAG
CCTAAGTCCTGTGACAAGACCCACACCTGCCCTCCTTGTCCTGCCCCTGAGCTGCTGGGC
GGACCTTCTGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCTCCAGGACC
CCTGAGGTGACCTGTGTGGTGGTGGACGTGTCTCACGAGGATCCCGAGGTGAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGGGAGGAGCAGTAC
AACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC
AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAAAAGACCATC
TCCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCAGGGAG
GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGAT
ATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCT
GTGCTGGACTCTGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGA
TGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC
ACCCAGAAGTCCCTGTCTCTGTCCCCCGGCAAGTGATGAGAATTC DX-2240 Light chain
(SEQ ID NO: 726): Signal sequence
ATGGGCTGGTCCTGTATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGTGCACTCT
GACATCCAGATGACCCAGTCCCCTTCCTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCAC
CTGTAGAGCCTCTCAGGGCATCGGCCACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGTGCCCAAGC
TGCTGATCTACACCGCCTCCACCCTGCAGTCTGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACC
GATTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATGTGGCCACCTACTACTGC
CAGCAGTTCAACTCCTACCCCCACACC TTCGGCCAGGGCACCAGACTGGAGATCAAG
AGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCTC
TGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGC
AGTCCGGCAATTCCCAGGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACC
CTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC
CTCTCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGATGAGAATTC
Example 36
Pharmacokinetic and Biodistribution Studies in Mice
[0707] The in vivo pharmacokinetics and stability of DX-2240
(produced in HEK293T cells) was determined by iodinating the
protein on available tyrosine residues and measuring plasma
clearance and stability in mice after a single intravenous dose.
Samples were radio-iodinated by the indirect method using the
IODO-GEN.TM. reagent (method from Pierce, and described by
Chizzonite et al. ((1991) J. Immunol. 147:1548; (1992) J. Immunol.
148: 3117). Samples were incubated with the .sup.125I-NaI solution
for 9 min at which time tyrosine (10 mg/mL, a saturated solution)
was added to quench the reaction. After about 15 min a 5 .mu.l
aliquot was removed as a standard for counting. For each labeling
reaction, the .sup.125I-labeled material (approx. 0.6 mL) was
purified using a single 5 mL D-salt 1800 polyacrylamide column
(Pierce). Columns were washed with 25 mM Tris, 0.4 M NaCl, pH 7.5
containing 2.5% HSA to block nonspecific sites then extensively
with the same buffer minus the HSA. Samples were applied in and
columns were eluted with a series of 0.3 mL aliquots. Recovery of
applied activity in all protein fractions was >75% and the total
recovery of applied activity was >90%. The fractions containing
peak levels of labeled protein were pooled for animal injections.
To prepare the injectate, the pool was diluted with Tris buffer (pH
7.5) so that the 100 .mu.l injection volume contained about 10
.mu.g of labeled material.
[0708] Solutions containing the radio-labeled compounds were
administered to all mice by injection into the tail vein. At
predetermined times post-administration animals were sacrificed and
blood samples were taken for analyses. Time points tested after
injection of radio-labeled compounds were: approximately 0, 7, 15,
30 and 90 minutes, 4 h, 8 h, 16 h, 24 h, 48 h and 72 h after
injection. Four animals were sacrificed for each time point. At
sacrifice, 0.5 mL aliquots of blood were collected into
anticoagulant (0.02 mL EDTA) tubes. Plasma was separated from cells
by centrifugation and the plasma fraction was divided into two
aliquots, one frozen and one stored at 4.degree. C. for immediate
analysis.
[0709] Analyses included gamma counting of all samples. In this
single dose i.v. study, DX-2240 exhibited a relatively short-half
life in mice of less than 5 hours. Analysis of the biodistribution
of DX-2240 in these mice revealed some accumulation of this
antibody, at 30 minutes, in the lungs (12.85% ID/g), spleen (7.44%
ID/g), kidney (8.34% ID/g), liver (5.42% ID/g) and heart (4.04%
ID/g). DX-2240, due to its interaction with Tie1 on the surface of
endothelial cells, may accumulate in areas of high vascularization
such as the lung. Therefore, multiple administrations of DX-2240
may be required to achieve an effective steady-state level of this
antibody in the serum of mice. ELISA on ocular bleeds following
three every other day dosings, as well as terminal bleed samples
from tumor-bearing mice treated with DX-2220, were performed. In
each case, levels of DX-2220 in the serum averaged 500 .mu.g/ml,
suggesting that despite a short serum half-life in mice, an
effective steady-state level of this antibody can be achieved
following just three doses of DX-2220.
[0710] In addition, SEC-HPLC analysis of plasma samples to assess
the in vivo stability of DX-2240 was performed. Instability of
DX-2240 in mouse could have contributed to the fast clearance of
this compound from the serum. SEC-HPLC analysis was performed for
two plasma samples at time points at 0 min, 30 min, 90 min, 24 h
and 72 h. The analysis of the radio-labeled DX-2240 showed that
this compound is stable in vivo both to degradation and to
interactions with plasma components. Therefore, the relatively
rapid half-life of DX-2240 in mice is not due to degradation of
this compound.
Example 37
DX-2220 Slows Lung Cancer Xenograft Tumor Progression in Nude
Mice
[0711] The effect of DX-2220 on tumor growth in mice bearing human
LNM35 lung cancer xenografts was tested. For these studies, LNM35
cells were injected subcutaneously in the lateral thorax of athymic
nude mice. Four days after tumor cell implantation, treatment was
initiated with DX-2220 or A2-SV (negative control antibody) at a
dosage of 20 mg/kg, three times a week. Tumor sizes were measured
at day 6, 8 and 10 post antibody treatment.
[0712] As shown in FIG. 6, DX-2220 significantly slowed
(.about.60%) tumor progression in this mouse xenograft model
(p=0.037). In addition, mice treated with DX-2220 did not exhibit
any significant loss in body weight. These data coupled demonstrate
that DX-2220 possesses significant tumor growth inhibitory activity
in vivo.
Example 38
DX-2220 Slows Tumor Progression in Nude Mice
[0713] In addition to the in vivo studies presented above, four
additional mouse xenografts studies were performed. These studies
were conducted either according to the protocols used in the SW-480
or LNM35 study. The results from these studies are listed
below.
TABLE-US-00019 LLC (mouse lung carcinoma) 20% inhibition @ day 14
after start of treatment PC-3 (human prostate cancer) 24%
inhibition @ day 28 after start of treatment LNM35 #3 (human lung
carcinoma) 30% inhibition @ day 21 after start of treatment Colo205
(human colorectal cancer) no effect
[0714] These results suggest that the E3 anti-Tie1 antibody has an
effect on a variety of tumor types, indicating broad therapeutic
applicability.
Example 39
Immunohistochemical Analysis of Normal Tissues
[0715] A series of immunohistochemical analyses on a series of
non-malignant normal human tissues to assess potential areas of
immunoreactivity of the E3 anti-Tie1 antibody was performed.
Antibody titration experiments were conducted on both cryostat and
paraformaldehyde fixed sections of select normal human tissues with
biotinylated DX-2220 and an IgG isotype control antibody to
determine the preferred tissue preservation conditions as well as
optimal concentration of the antibody that would result in minimal
background and maximal detection of signal. A concentration of 20
.mu.g/ml for the primary antibody was selected for the study with
biotinylated DX-2220 and the biotinylated IgG isotype control
antibody used as the primary antibodies, and the principal
detection system consisting of Streptavidin HRP with DAB as the
chromagen. Tissues also were stained with the positive control
antibodies (anti-CD31 and anti-vimentin) to ensure that the tissue
antigens were preserved and accessible for immunohistochemical
analysis. Only tissues that were positive for CD31 and vimentin
staining were selected for the remainder of the study. The negative
control consisted of performing the entire immunohistochemical
procedure on adjacent sections in the absence of primary antibody.
Slides were imaged with a DVC 1310C digital camera coupled to a
Nikon microscope.
[0716] The negative control (no primary antibody) slides showed
occasional faint background staining within renal tubular
epithelium and occasional granulocytes, but was uniformly negative
in all other cell types, including the positive control cell line
(HMEC, Human Microvascular Endothelial Cells) and positive control
colon cancer. The IgG isotype control antibody showed faint
background staining of granulocytes, macrophages, adrenal cortex,
renal tubular epithelium, fallopian tube epithelium, hepatocytes,
Leydig cells, and thyroid. The positive control cell line (HMEC)
and positive control colon cancer sample showed either no staining
or background staining.
[0717] DX-2220 demonstrated moderate membrane staining within the
HMEC cell line, and staining of macrophages, some carcinoma cells,
and endothelial cells within the colon cancer positive control
samples. Within normal tissues, the antibody showed faint to
moderate staining of macrophages, microglia in the brain, squamous
epithelium of the cervix, faint staining of skeletal muscle, islets
of Langerhans, and placental endothelium. These observations are
consistent with low level expression of Tie1 in some endothelial,
hematopoietic and epithelial tissues, as anticipated from previous
reports. The islets of Langerhans staining were unexpected and
should be investigated further. Most other faint staining was
similar to that seen with the IgG isotype control. If the
background from the IgG isotype control is subtracted from the
analysis of DX-2220, the majority of tissues were negative,
including adrenal, bladder, blood, bone marrow, neurons, breast,
colon, endothelium, eye, fallopian tube, heart, kidney, liver,
lung, lymphocytes, ovary, exocrine pancreas, pituitary, prostate,
skin, spinal cord, spleen, seminiferous epithelium of the testis,
thymic lymphocytes, ureter, and uterus.
Example 40
Platelet Studies
[0718] It has been reported that Tie1 is expressed on platelets
(Tsiamis et al. (2000) J. Vasc. Res. 37(6):437). The possibility of
platelet immunoreactivity with the E3 anti-Tie1 antibody was
investigated by FACs analysis and immunoprecipitation studies.
DX-2200 did not show significant binding to platelets, nor did it
immunoprecipitate Tie1 from platelet extracts. In addition, the
effect of DX-2200 and DX-2210 on platelet agglutination and
aggregation was investigated. Ristocetin, a cofactor that induces
platelet agglutination by mediating the binding of von Willebrand
factor (vWF) to platelet membrane glycoprotein GPIb (CD42), was
used as a positive control for platelet agglutination. Antibodies
to CD9 were used as a positive control to activate platelets and
induce platelet aggregation, with kinetics and extent comparable to
physiological agonists such as thrombin (reference). Neither the
DX-2200 nor its light chain germlined variant DX-2210 induced
platelet agglutination or aggregation.
Example 41
Chord Blood Stem Cell Studies
[0719] To evaluate the binding characteristics to blood progenitor
cells (stem cells), FACS analysis with the anti-Tie1 antibody (or
the appropriate negative control antibody) on G-CSF mobilized
peripheral blood cells and with bone marrow cells was performed.
Briefly, cells were blocked with 10% heat-inactivated human AB
serum/2% mouse serum. Binding was initiated with biotinylated
DX-2220 or biotinylated A2 negative control antibody. After washing
the cells, primary antibodies were detected using FITC-labeled
streptavidin. Following an additional 30 minute incubation period,
remaining erythrocytes were lysed, and the resulting cell pellet
after centrifugation was resuspended in PBS prior to FACS analysis.
Data acquisition was performed on a FACSCanto.TM.
(Becton-Dickinson) using FacsDiva.TM. software. Active gating on
SSC/CD45 was used. Progenitor cells were gated on CD45.sup.+
CD34.sup.+ cells and were acquired automatically with at least
100,000 CD45.sup.+ CD34.sup.+ counted.
[0720] While the expression of Tie1 has been reported on certain
hematopoietic malignancies, this experiment demonstrated that
neither the negative control IgG A02, nor the E3 anti-Tie1 antibody
DX-2220, positively stained CD45.sup.+ CD34.sup.+ blood progenitor
cells. This finding supports the hypothesis that targeting Tie1
with E3 should have no deleterious effects on stem cells, unlike
certain chemotherapeutic agents.
Example 42
In Vitro Hematopoiesis Studies
[0721] The effect of anti-Tie1 antibodies (DX-2220 and DX-2240) on
human myeloid and erythroid progenitors was evaluated using
methylcellulose-based in vitro colony assays and megakaryocyte
progenitors using collagen-based in vitro assays. Neither DX-2220
nor DX-2240 inhibited colony formation in the particular conditions
of this in vitro assay at concentrations up to 100 .mu.g/ml.
[0722] The effect of the E3 anti-Tie1 antibody DX-2240 on the
recovery of the mouse hematopoietic system using an in vivo
myeloablation model was evaluated. Mice were injected with 5-FU on
day 0 and then received either DX-2240 or a negative control
antibody. At various time points following injection (days 2, 4, 6,
8, 10, 12 and 14), 4 mice were sacrificed from control and treated
groups and peripheral blood and femurs were harvested. The
peripheral blood and femoral cells were analyzed to determine the
following: [0723] Total nucleated cells per femur [0724] Frequency
of bone marrow colony forming cells for both myeloid and erythroid
progenitors [0725] Total hematopoietic CFC per femur [0726] Total
megakaryocytic CFC per femur [0727] Total white blood cell count
and differential analysis of mature cells
[0728] DX-2240 had no effect on the recovery of the mouse
hematopoietic system following 5-FU administration. This supports
the in vitro findings that DX-2240 possesses no hematological
toxicities under these assay conditions. Thus, it is particularly
useful as a therapeutic as it will not interfere with normal
hematopoietic functions required to maintain red cell and
lymphocyte production.
[0729] The anti-Tie1 antibody was also evaluated for its effect on
implanted tumors. Tumor cells were injected subcutaneously into the
abdominal region of mice (Balb/C nu/nu female mice, 5-6 weeks old).
The following tumor cells were tested: a lung carcinoma (mouse
syngeneic Lewis lung carcinoma (LLC)); human lung carcinoma LNM35;
an aggressive human colon carcinoma clone (SW480R). Treatments with
anti-Tie1 antibody were initiated 4-6 days post-implantation. The
anti-Tie1 antibodies or a control antibody (the A2
anti-streptavidin antibody) were administered intraperitoneally at
20 mg/kg every second day. Tumor volume was measured every second
day and calculated as 0.5.times.height.times.width.times.depth.
[0730] The E3 anti-Tie1 antibody (also termed DX-2240) inhibits
primary tumor growth of LLC (about a 20% effect, p=0.078; ANOVA,
single factor test) and LNM35. In one study, this anti-Tie1
antibody inhibited primary tumor growth by 60% (most responsive).
(Doubling of the antibody dose with administration only twice a
week rather than every second day resulted in only a modest effect
on primary tumor growth). The SW480R tumor was not responsive to
the antibody treatment under these conditions. As described in
Example 34, the antibody was effective for inhibiting tumor growth
of SW480 cells (i.e., rather than the derivative SW480R cells).
[0731] Histological analysis of tumor sections from the experiment
in which 60% inhibition was observed indicated that blood vessels
in anti-Tie1 antibody treated tumors have a distinct morphology
even though blood vessel density may not be altered. In anti-Tie
antibody treated tumors, the vessels form septa-like structures in
between lobuli of tumor cells. These tumors also have more necrosis
than control antibody treated tumors. The distribution of smooth
muscle cells (as detected by anti-smooth muscle actin antibody
staining) was also altered. The lymphatic vessels in anti-Tie1
antibody treated tumors were also more dispersed, somewhat dilated
and in several instances composed of adjacent lumina clustered
together in a string. These observations indicate that this
anti-Tie antibody has a distinctive effect on tumor necrosis and
vessel organization within the tumor.
Example 43
Evaluating Combination Therapies
[0732] An animal model can be used to evaluate combination
therapies. For example, the combinations (provided
intraperitoneally) can be tested for ability to modulate tumor
growth in female NCr nu/nu mice with xenografts of HT29, COLO205,
or PC3. The following are some exemplary test regimes:
TABLE-US-00020 Dose for HT29 and Compound COLO205 xenografts
Schedule PBS 0.2 ml/20 g Q2D .times. 14; D4 DX-2230 10 mg/kg/inj
Q2D .times. 14; D4 DX-2230 + avastin 10 mg/kg/inj; 2.5 mg/kg/inj
Q2D .times. 14; D4, Q3D .times. 3; D3 avastin 2.5 mg/kg/inj Q3D
.times. 3; D4 A2-SV (control) 10 mg/kg/inj Q2D .times. 14; D4
[0733] Another regime is as follows:
TABLE-US-00021 Compound Dose for PC3 xenografts Schedule PBS 0.2
ml/20 g Q2D .times. 14; D4 DX-2230 10 mg/kg/inj Q2D .times. 14; D4
DX-2230 + 10 mg/kg/inj; Q2D .times. 14; D4, QD .times. 1; D4
cyclophosphamide 150 mg/kg/inj cyclophosphamide 150 mg/kg/inj QD
.times. 1; D4 A2-SV (control) 10 mg/kg/inj Q2D .times. 14; D4
[0734] A2-SV is the control anti-streptavidin antibody. Twelve
animals can be used in each group. Clinical signs, mean group
weights are evaluated every day. Individual body weights and tumor
burden are evaluated twice weekly. At study termination, tissue
samples can be obtained from tumors, liver, lung, spleen, heart,
axial node, kidney, and uterus.
[0735] Other embodiments are within the following claims:
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100196361A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100196361A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References