NZ623724B2 - Vegf/dll4 binding agents and uses thereof - Google Patents
Vegf/dll4 binding agents and uses thereof Download PDFInfo
- Publication number
- NZ623724B2 NZ623724B2 NZ623724A NZ62372412A NZ623724B2 NZ 623724 B2 NZ623724 B2 NZ 623724B2 NZ 623724 A NZ623724 A NZ 623724A NZ 62372412 A NZ62372412 A NZ 62372412A NZ 623724 B2 NZ623724 B2 NZ 623724B2
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- New Zealand
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- seq
- antibody
- dll4
- vegf
- heavy chain
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Classifications
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Abstract
Disclosed is a bispecific antibody comprising: a) a first antigen-binding site that specifically binds human vascular endothelial growth factor (VEGF), and b) a second antigen-binding site that specifically binds human delta-like 4 ligand (DLL4), comprising the CDRs of the sequences as defined in the specification. Also disclosed is its use in treating cancer. the specification. Also disclosed is its use in treating cancer.
Description
VEGF/DLL4 BINDING AGENTS AND USES THEREOF
FIELD OF THE INVENTION
The t invention generally relates to antibodies and other agents that bind VEGF, DLL4, or
both VEGF and DLL4, ularly anti—VEGF/anti—DLL4 bispecific antibodies, as well as to methods of
using the antibodies or other agents for the treatment of diseases such as cancer.
BACKGROUND OF THE INVENTION
Angiogenesis plays an important role in the pathogenesis of a number of disorders, including
solid tumors and metastasis. The production ofnew blood vessels is essential for providing oxygen and
nutrients for the growth and spread of a tumor, and therefore angiogenesis is a good target for cancer
therapeutics.
Angiogenesis involves a family ofproteins acting as angiogenic activators, including ar
endothelial growth factor (VEGF-A), , VEGF—C, VEGF-E, and their respective receptors
- 1, VEGFR-Z, and VEGFR-3). VEGF—A, also referred to as VEGF or vascular permeability
factor (VPF), exists in several isoforms that arise from alternative splicing ofmRNA of a single VEGF
gene, with VEGF165 being the most biologically relevant isoform.
Anti-VEGF dies have been shown to suppress the growth oftumor cells in vitro and in vivo.
A humanized anti-VEGF monoclonal antibody, bevacizumab IN) has been developed and
approved in the United States as a cancer therapeutic.
The Notch signaling pathway is a universally conserved signal transduction system. It is involved
in cell fate determination during development ing embryonic n formation and post-embryonic
tissue nance. In addition, Notch signaling has been identified as a critical factor in the maintenance
of hematopoietic stem cells.
The Notch pathway has been linked to the pathogenesis of both hematologic and solid tumors and
cancers. us cellular functions and microenvironmental cues associated with tumorigenesis have
been shown to be ted by Notch pathway signaling, including cell proliferation, apoptosis,
adhesion, and angiogenesis (Leong et al., 2006, Blood, 107:2223-2233). In addition, Notch receptors
and/or Notch ligands have been shown to play potential oncogenic roles in a number of human cancers,
including acute myelogenous leukemia, B cell c lymphocytic leukemia, Hodgkin lymphoma,
multiple myeloma, T—cell acute lymphoblastic leukemia, brain cancer, breast cancer, cervical cancer,
colon cancer, lung cancer, atic cancer, prostate cancer, and skin cancer. (Leong et al., 2006, Blood,
107:2223-2233).
Delta-like 4 ligand (DLL4) is an important component of the Notch pathway and has been
identified as a target for cancer therapy. DLL4 is a Notch ligand, characterized by an N-terminal domain,
a Delta/Serrate/Lag-2 (DSL) domain and tandem EGF-like repeats within the extracellular domain. It has
been reported that DLL4 is induced by VEGF and that DLL4 may act as a negative feedback tor for
vascular proliferation.
Anti-DLL4 antibodies have been shown to enhance angiogenic sprouting and branching which
leads to non-productive enesis and decreased tumor growth ra-Troise et al., 2006, Nature,
444:1032-1037). In addition, an anti-DLL4 antibody, 21M18, has been shown to inhibit tumor growth
and reduce the frequency of cancer stem cells in xenograft tumor models (Hoey et al., 2009, Cell Stem
Cell, 5:168-177; U.S. Patent No. 7,750,124).
Although there have been icant strides in development of monoclonal antibodies for use in
cancer treatments, there is still great potential for further improvements. One class of antibody molecules
with the promise of enhanced y and/or reduced side effects (e.g., toxicity) is bispecific antibodies.
Early bispecific molecules were mainly generated using chemical cross-linking of two antibodies,
or were hybrid hybridomas or “quadromas”. One success of the quadroma format is triomabs, which are
mouse/rat combinations that demonstrate a preferential species-specific heavy/light chain pairing. More
recently, advances in dy engineering have provided a wide variety of new antibody formats,
including, but not limited to, tandem scFv (bi-scFv), diabodies, tandem ies (tetra-bodies), single
chain diabodies, and dual variable domain antibodies.
It is one of the objectives of the present invention to provide improved molecules for cancer
treatment, particularly bispecific antibodies that specifically bind human VEGF and human DLL4.
SUMMARY OF THE ION
[0011A] In a first , the t invention es a bispecific antibody comprising:
a) a first antigen-binding site that specifically binds human vascular endothelial growth
factor (VEGF), and
b) a second antigen-binding site that specifically binds human delta-like 4 ligand (DLL4),
n the first antigen-binding site comprises a heavy chain CDR1 comprising NYWMH
(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18),
and a heavy chain CDR3 sing HYDDKYYPLMDY (SEQ ID NO:19);
wherein the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH
(SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising
YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2 is serine,
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asparagine, or glycine, X3 is asparagine or lysine, and X4 is glycine, arginine, or aspartic acid ,
and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and
wherein both the first and second antigen-binding sites comprise a light chain CDR1 comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID
NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
[0011B] In a second aspect, the present invention provides a ific antibody that specifically
binds human VEGF and human DLL4, which comprises:
(a) a heavy chain of SEQ ID NO:7;
(b) a heavy chain of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:56, or SEQ ID NO:62; and
(c) two light chains of SEQ ID NO:8.
[0011C] In a third , the present invention provides a bispecific antibody that specifically
binds human VEGF and human DLL4, which comprises:
(a) a heavy chain encoded by the DNA comprising SEQ ID NO:75, a heavy chain encoded
by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA comprising SEQ
ID NO:34;
(b) a heavy chain encoded by the DNA comprising SEQ ID NO:31, a heavy chain encoded
by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA comprising SEQ
ID NO:34;
(c) a heavy chain encoded by the DNA sing SEQ ID NO:72, a heavy chain encoded
by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA sing SEQ
ID NO:34; or
(d) a heavy chain encoded by the DNA comprising SEQ ID NO:74, a heavy chain encoded
by the DNA comprising SEQ ID NO:33, and a light chain d by the DNA comprising SEQ
ID NO:34.
[0011D] In a fourth aspect, the t invention provides an isolated antibody that specifically
binds human VEGF, which ses:
(a) a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2
comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 comprising
HYDDKYYPLMDY (SEQ ID NO:19); and
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(b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain
CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising
QQSKEVPWTFGG (SEQ ID NO:22).
[0011E] In a fifth aspect, the present invention provides a pharmaceutical composition
comprising the antibody of any of the first to fourth aspects and a pharmaceutically acceptable
carrier.
[0011F] In a sixth aspect, the present invention provides a cell comprising or producing the
antibody of any of the first to fourth aspects, wherein the cell is not within a human body.
[0011G] In a seventh aspect, the present invention provides an isolated polynucleotide molecule
comprising a nucleotide sequence that encodes the antibody of any of the first to fourth aspects.
[0011H] In an eighth aspect, the present invention provides a vector comprising the
polynucleotide of the seventh aspect.
[0011I] In a ninth aspect, the present invention provides a cell comprising the polynucleotide of
the seventh aspect, wherein the cell is not within a human body.
[0011J] In a tenth aspect, the present invention provides use of the dy of any of the first to
fourth aspects in the manufacture of a medicament for treatment of cancer.
[0011K] In an th aspect, the present invention provides use of the antibody of any of the
first to fourth aspects in the manufacture of a ment for ting growth of a tumor.
[0011L] In an twelfth aspect, the t invention provides a method for the production of an
dy, comprising expressing at least one cleotide of the seventh aspect in a cell,
wherein the antibody is not produced within a human body.
The present ion provides binding agents, such as antibodies, that bind VEGF,
DLL4, or both VEGF and DLL4 (VEGF/DLL4-binding agents), as well as itions, such as
pharmaceutical compositions, comprising the binding agents. Binding agents that bind VEGF or
DLL4, as well as at least one additional antigen or target, and ceutical compositions of
such binding agents, are also ed. In certain ments, the binding agents are novel
polypeptides, such as antibodies, antibody nts, and other polypeptides related to such
antibodies. In certain embodiments, the binding agents are antibodies that specifically bind
human VEGF. In some embodiments, the binding agents are antibodies that specifically bind
human DLL4. In some ments, the binding agents are bispecific antibodies that
specifically bind human VEGF and human DLL4. The invention r provides methods of
inhibiting the growth of a tumor by administering the binding agents to a subject with a tumor.
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The invention further provides methods of treating cancer by administering the g agents to
a subject in need thereof. In some embodiments, the methods of treating cancer or inhibiting tumor
growth comprise targeting cancer stem cells with the binding . In certain embodiments, the
methods comprise reducing the frequency of cancer stem cells in a tumor, reducing the number of cancer
stem cells in a
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tumor, reducing the tumorigenicity of a tumor, and/or reducing the tumorigenicity of a tumor by reducing
the number or ncy of cancer stem cells in the tumor.
In one aspect, the invention provides a binding agent, such as an dy, that cally binds
human VEGF. In some embodiments, the binding agent inhibits binding ofVEGF to at least one VEGF
receptor. In some embodiments, the binding agent inhibits binding ofVEGF to VEGFR-l and/or
VEGFR-Z. In some embodiments, the binding agent tes angiogenesis. In certain embodiments,
the antibody or other binding agent further specifically binds to and/or inhibits human DLL4 in addition to
human VEGF.
In some embodiments, the binding agent is an antibody which comprises a heavy chain CDRl
comprising NYWMH (SEQ ID NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR
(SEQ ID NO: 18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO: 19); and a
light chain CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising
AASNQGS (SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the binding agent is an antibody that ses a heavy chain variable
region having at least 80% sequence identity to SEQ ID NO:11; and/or a light chain variable region
having at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the binding agent
comprises a heavy chain variable region having at least 90% sequence ty to SEQ ID NO:11; and/or a
light chain variable region having at least 90% sequence identity to SEQ ID NO: 12. In certain
ments, the g agent comprises a heavy chain variable region having at least 95% ce
identity to SEQ ID NO:11; and/or a light chain variable region having at least 95% sequence ty to
SEQ ID NO:12. In certain embodiments, the binding agent is an antibody that comprises a heavy chain
variable region of SEQ ID NO:11; and/or a light chain variable region of SEQ ID NO:12.
In some embodiments, the g agent is antibody 219R45, 219R45-MB-21M18, 219R45-MB-
21R79, 219R45-MB-21R75, or 219R45-MB—21R83.
In another aspect, the invention provides a binding agent, such as an antibody, that specifically
binds human DLL4. In some embodiments, the binding agent inhibits binding of DLL4 to at least one
Notch receptor. In some embodiments, the binding agent ts binding of DLL4 to Notchl, ,
Notch3, and/or Notch4. In some embodiments, the binding agent inhibits Notch signaling. In some
embodiments, the binding agent promotes unproductive angiogenesis. In certain embodiments, the
antibody or other g agent further cally binds to and/or inhibits human VEGF in addition to
human DLL4.
In some embodiments, the binding agent is an antibody that binds human DLL4 and comprises a
heavy chain CDRl comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain
CDR2 comprising YIXleYX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2
is serine, asparagine, or glycine, X3 is asparagine or lysine, and X4 is glysine, arginine,or aspartic acid
and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID ; and a light chain CDRl
comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ
ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID . In some
embodiments, the antibody comprises a heavy chain CDRl comprising TAYYIH (SEQ ID NO:13) or
AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID
NO:14), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the binding agent is an dy that comprises a heavy chain variable
region having at least 90% or at least 95% sequence identity to SEQ ID NO: 10; and/or a light chain
variable region having at least 90% or at least 95% sequence identity to SEQ ID NO: 12. In n
embodiments, the binding agent is an antibody that comprises a heavy chain variable region of SEQ ID
NO: 10; and a light chain variable region of SEQ ID NO: 12.
In some embodiments, the binding agent is antibody 21R79 or antibody 219R45-MB-21R79.
In some embodiments, the binding agent is an antibody which comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO: 13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising
YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO:16); and a light chain CDRl comprising RASESVDNYGISFMK (SEQ ID , a light
chain CDR2 sing S (SEQ ID NO:21), and a light chain CDR3 comprising
QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the g agent is an antibody that comprises a heavy chain variable
region having at least 90% or at least 95% sequence identity to SEQ ID NOS 8; and/or a light chain
variable region having at least 90% or at least 95% sequence ty to SEQ ID NO: 12. In certain
embodiments, the binding agent is an antibody that comprises a heavy chain variable region of SEQ ID
NOS8; and a light chain variable region of SEQ ID NO:12.
In some embodiments, the binding agent is antibody 21R75 or antibody 219R45-MB-21R75.
In some embodiments, the binding agent is an antibody which comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising
YISNYNRATNYNQKFKG (SEQ ID , and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and a light chain CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light
chain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising
QQSKEVPWTFGG (SEQ ID .
In certain embodiments, the g agent is an antibody that comprises a heavy chain variable
region having at least 90% or at least 95% sequence identity to SEQ ID NO:64; and/or a light chain
variable region having at least 90% or at least 95% ce identity to SEQ ID NO:12. In certain
embodiments, the binding agent is an antibody that comprises a heavy chain variable region of SEQ ID
NO:64; and a light chain variable region of SEQ ID NO: 12.
In some embodiments, the binding agent is antibody 21R83 or dy 219R45-MB-21R83.
In certain embodiments of each of the aforementioned aspects or embodiments, as well as other
s and/or embodiments described elsewhere herein, the binding agent is a ific antibody. In
some embodiments, the bispecific antibody specifically binds human VEGF and a second target. In some
embodiments, the bispecific antibody specifically binds human DLL4 and a second . In some
embodiments, the bispecific antibody specifically binds both human VEGF and human DLL4. In some
embodiments, the ific antibody modulates angiogenesis. In certain embodiments, the bispecific
antibody ts Notch signaling. In some embodiments, the bispecific antibody modulates angiogenesis
and ts Notch ing. In some embodiments, the bispecific antibody reduces the number of
frequency of cancer stem cells. In certain embodiments, the bispecific dy comprises two identical
light chains. In certain embodiments the bispecific antibody is an IgG antibody (e.g., IgG2).
In some embodiments, the bispecific antibody comprises: a first antigen-binding site that
specifically binds human VEGF, wherein the first antigen—binding site comprises a heavy chain CDRl
sing NYWMH (SEQ ID NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR
(SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19), In some
embodiments, the bispecific antibody further comprises: a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the
ific antibody comprises: a first antigen—binding site that specifically binds human VEGF, wherein
the first antigen-binding site comprises (a) a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a
heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3
comprising HYDDKYYPLMDY (SEQ ID NO:19), and (b) a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising S (SEQ ID NO:21),
and a light chain CDR3 sing QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the bispecific antibody ses: a first antigen-binding site that
cally binds human DLL4, wherein the first antigen—binding site comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising
YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2 is serine,
asparagine, or glycine, X3 is asparagine or lysine, and X4 is glysine, arginine,or aspartic acid and a heavy
chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some ments, the
bispecific antibody comprises: a first antigen—binding site that specifically binds human DLL4, wherein
the first antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO: 13), a
heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14),
YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), or
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16). In some embodiments, the bispecific antibody fiirther comprises: a light chain CDR1
comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ
ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the the bispecific antibody comprises: a first antigen-binding site that specifically binds
human DLL4, wherein the first antigen-binding site comprises (a) a heavy chain CDR1 comprising
TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID
NO: 14), YISSYNGATNYNQKFKG (SEQ ID NO: 15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), or
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16), and (b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a
light chain CDR2 comprising S (SEQ ID NO:21), and a light chain CDR3 comprising
QQSKEVPWTFGG (SEQ ID NO:22).
In some embodiments, the ific antibody comprises: a) a first antigen-binding site that
specifically binds human VEGF, and b) a second antigen—binding site that specifically binds human
DLL4, wherein the first antigen-binding site comprises a heavy chain CDR1 sing NYWMH (SEQ
ID NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy
chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO: 19); wherein the second antigen-binding site
comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a
heavy chain CDR2 comprising YIXlXZYX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or
alanine, X2 is serine, asparagine, or e, X3 is asparagine or lysine, and X4 is glysine, arginine,or
ic acid and a heavy chain CDR3 sing RDYDYDVGMDY (SEQ ID NO: 16); and a light
chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising
AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In some ments, the bispecific antibody comprises: a) a first antigen—binding site that specifically
binds human VEGF, and b) a second antigen—binding site that specifically binds human DLL4, wherein
the first antigen-binding site ses a heavy chain CDR1 comprising NYWMH (SEQ ID , a
heavy chain CDR2 sing DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3
comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the second antigen-binding site ses a
heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO:16); and wherein both the first and second antigen—binding sites se a light chain
CDR1 sing RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds
human VEGF, and b) a second antigen-binding site that specifically binds human DLL4, wherein the first
antigen-binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy
chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3
comprising HYDDKYYPLMDY (SEQ ID NO: 19); n the second antigen-binding site comprises a
heavy chain CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YISSYNGATNYNQKFKG (SEQ ID NO: 15), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites se a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), and a light chain CDR3 sing QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the a bispecific dy ses: a) a first n-binding site that specifically binds
human VEGF, and b) a second n—binding site that specifically binds human DLL4, wherein the first
antigen-binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO: 17), a heavy
chain CDR2 comprising GRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain CDR3
comprising HYDDKYYPLMDY (SEQ ID ; wherein the second antigen-binding site comprises a
heavy chain CDRl comprising TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising
YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and n both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising S
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds
human VEGF, and b) a second antigen-binding site that specifically binds human DLL4, wherein the first
antigen-binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO: 17), a heavy
chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3
sing HYDDKYYPLMDY (SEQ ID NO: 19); wherein the second antigen-binding site comprises a
heavy chain CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and n both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In some embodiments, the bispecific antibody that specifically binds human VEGF, and
comprises: a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:11, and/or
a light chain variable region having at least 90% sequence identity to SEQ ID NO: 12. In some
embodiments, the bispecific antibody specifically binds human VEGF, and comprises: a heavy chain
variable region having at least 95% ce identity to SEQ ID NO:11, and/or a light chain variable
region having at least 95% sequence identity to SEQ ID NO: 12.
In some embodiments, the bispecific antibody specifically binds human DLL4, and comprises: a
heavy chain variable region having at least 90% sequence ty to SEQ ID NO:9, SEQ ID NO: 10, SEQ
ID N025 8, or SEQ ID NO:64; and/or a light chain variable region having at least 90% ce identity
to SEQ ID NO:12. In some embodiments, the bispecific dy specifically binds human DLL4, and
comprises: a heavy chain variable region having at least 95% sequence identity to SEQ ID NO:9, SEQ ID
NO: 10, SEQ ID N0258, or SEQ ID NO:64; and/or a light chain variable region having at least 95%
ce identity to SEQ ID NO:12.
In some embodiments, the bispecific dy specifically binds human VEGF and human DLL4,
and comprises: (a) a first heavy chain variable region having at least 90% sequence identity to SEQ ID
NO:11; (b) a second heavy chain variable region having at least 90% sequence identity to SEQ ID NO:9,
SEQ ID NO: 10, SEQ ID N025 8, or SEQ ID NO:64; and (c) a first and a second light chain variable region
having at least 90% sequence identity to SEQ ID NO:12. In some embodiments, the VEGF/DLL4
bispecific antibody comprises (a) a first heavy chain variable region having at least 95% sequence identity
to SEQ ID NO:11; (b) a second heavy chain variable region having at least 95% sequence identity to SEQ
ID NO:9; and (c) a first and a second light chain variable region having at least 95% sequence identity to
SEQ ID NO:12. In some embodiments, the VEGF/DLL4 ific antibody comprises (a) a first heavy
chain variable region having at least 95% sequence identity to SEQ ID NO:11; (b) a second heavy chain
variable region having at least 95% sequence identity to SEQ ID NO: 10; and (c) a first and a second light
chain variable region having at least 95% sequence identity to SEQ ID NO:12. In some embodiments, the
VEGF/DLL4 bispecific antibody comprises (a) a first heavy chain variable region having at least 95%
sequence identity to SEQ ID NO:11; (b) a second heavy chain variable region having at least 95%
sequence identity to SEQ ID NO:58; and (c) a first and a second light chain variable region having at least
95% sequence identity to SEQ ID NO:12. In some embodiments, the VEGF/DLL4 bispecific antibody
comprises (a) a first heavy chain variable region having at least 95% sequence identity to SEQ ID NO:11;
(b) a second heavy chain variable region having at least 95% sequence identity to SEQ ID NO:64; and (c)
a first and a second light chain variable region having at least 95% sequence identity to SEQ ID NO:12.
In some embodiments, the VEGF/DLL4-binding agent is a bispecific antibody comprising (a) a
first antigen-binding site that binds human VEGF with a KD between about 0.1 nM and about 1.0 nM and
(b) a second antigen-binding site that cally binds human DLL4 with a KD between about 0.1 nM
and about 20 nM. In certain ments, the ific dy ses two identical light chains.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody selected from the
group consisting of 219R45-MB-21M18, 219R45—MB—21R79, 2 1 9R45-MB-2 1 R75, and 219R45-MB-
21R83.
In certain embodiments of each of the aforementioned aspects, as well as other aspects and/or
embodiments described elsewhere herein, the binding agent or antibody is ed.
In another aspect, the invention provides a polypeptide selected from the group consisting of:
SEQ ID NO: 1, SEQ ID N022, SEQ ID NO:3, SEQ ID NO:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
N027, SEQ ID N028, SEQ ID N029, SEQ ID N0:10, SEQ ID N0:l l, SEQ ID N0:12, SEQ ID N0:46,
SEQ ID N0:47, SEQ ID N0248, SEQ ID NO:49, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ
ID N0:62, SEQ ID N0:63, and SEQ ID N0:64. In some embodiments, the polypeptide is isolated. In
certain embodiments, the polypeptide is substantially pure. In n ments, the polypeptide is an
antibody or part of an antibody, such as an dy fragment.
In another aspect, the invention provides ed polynucleotide molecules comprising a
polynucleotide that encodes the binding agents and/or polypeptides of each of the entioned aspects,
as well as other aspects and/or embodiments described herein. In some embodiments, the polynucleotide
comprises a sequence selected from the group consisting of: SEQ ID N0:29, SEQ ID N0:30, SEQ ID
N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:50, SEQ ID N0:51, SEQ ID
N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, and SEQ ID NO:74. The invention fiirther provides expression vectors that
comprise the polynucleotides, as well as cells that comprise the expression s and/or the
polynucleotides. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell.
In other aspects, the ion provides s of inhibiting growth of a tumor, comprising
ting the tumor with an effective amount of an antibody (or other binding agent) that binds VEGF,
DLL4, or both VEGF and DLL4, including each of those antibodies (or other binding agents) described
herein.
In another aspect, the invention provides a method of inhibiting the growth of a tumor in a
subject, comprising administering to the subject a therapeutically ive amount of an antibody (or
other binding agent) that binds VEGF, DLL4, or both VEGF and DLL4, including each of those
antibodies (or other binding agents) described herein.
In another aspect, the invention provides a method of modulating angiogenesis in a subject,
comprising administering to the subject a therapeutically effective amount of an antibody (or other
binding agent) that binds VEGF, DLL4, or both VEGF and DLL4, including each of those antibodies (or
other g agents) bed herein.
In another aspect, the invention provides a method of reducing the tumorigenicity of a tumor in a
subject, comprising administering to the subject a therapeutically effective amount of an antibody (or
other binding agent) that binds VEGF, DLL4, or both VEGF and DLL4, including each of those
antibodies (or other binding agents) described herein.
In another aspect, the invention provides a method of reducing the tumorigenicity of a tumor in a
subject by reducing the frequency of cancer stem cells in the tumor, comprising administering to the
subject a therapeutically effective amount of an antibody (or other binding agent) that binds VEGF,
DLL4, or both VEGF and DLL4, including each of those antibodies (or other binding agents) bed
In other aspects, the invention provides methods of treating cancer in a subject, comprising
administering to the subject a therapeutically effective amount of an antibody (or other g agent) that
binds VEGF, DLL4, or both VEGF and DLL4, including each of those dies (or other binding
agents) described herein.
ceutical compositions comprising a binding agent (e. g., antibody) described herein and a
ceutically acceptable carrier are further provided, as are cell lines that express and/or produce the
binding agents. Methods of treating cancer and/or inhibiting tumor growth in a t (e. g., a human)
comprising administering to the subject an effective amount of a composition comprising the binding
agents are also provided.
Where aspects or embodiments of the invention are described in terms of a Markush group or
other grouping of alternatives, the present invention encompasses not only the entire group listed as a
whole, but also each member of the group individually and all possible subgroups of the main group, and
also the main group absent one or more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in the claimed ion.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Figure 1. 1A) Heavy chain and light chain CDRs of anti-VEGF/anti-DLL4 bispecific antibodies
219R45-MB-21M18, 219R45-MB-21M79, -MB-21M?5, and -MB-21M83; 1B) Heavy
chain and light chain variable region SEQ ID NOs; 1C) Heavy chain and light chain SEQ ID NOs.
Figure 2. HTRF assay for simultaneous binding of bispecific antibodies to human VEGF and
human DLL4. Results are reported in Relative Fluorescence Units (RFU), which represent the ratio of the
relative fluorescence intensity at 665nm to the relative fluorescence intensity at 620nm. 219R45-MB-
21M18 (); 219R45-MB-21R79 (-I-); 219R45 plus 21M18 (-A-); 219R45 plus 21R79 (43-); 219R45 (-
V-); 21M18 (-0—); 21R79 (—0—); control antibody LZ—l (—A—).
Figure 3. tion of VEGF—induced HUVEC proliferation by anti-VEGF/anti-DLL4 ific
antibodies. Fluorescence intensity is read using an excitation ngth of 530nm and an emission
wavelength of 590. 219R45-MB-21M18 (—0—); 219R45—MB—21R79 (-A-); 219R45 (-l-); Medium with
no VEGF ().
Figure 4. Inhibition of nduced Notch signaling by anti-VEGF/anti-DLL4 bispecific
antibodies. Luciferase activity was measured using a dual luciferase assay kit with firefly luciferase
activity normalized to Renilla luciferase activity. 219R45—MB-21M18 (); 219R45-MB-21R79 (-l-);
21M18 (); 21R79 (-E-).
Figure 5. Inhibition of colon tumor growth in vivo by an EGF/anti-DLL4 bispeciflc
antibody. OMP-C8 colon tumor cells were injected subcutaneously into a human skin graft in ID
mice. Mice were treated with control antibody (—I—), anti—hDLL4 dy 21M18 (-A -), anti-VEGF
antibody bevacizumab (), or anti-VEGF/anti-DLL4 ific antibody 219R45-MB-21M18 (- V-).
Data is shown as tumor volume (photons/sec) over days post-treatment. Antibodies were administered
intraperitoneally at a dose of 25mg/kg once a week.
Figure 6. Tumorigenicity of pancreatic tumor cells after treatment with anti-VEGF/anti-DLL4
bispecific antibodies. 8 tumor cells from mice treated with control antibody, anti-hDLL4
antibody 21M18, anti-VEGF antibody bevacizumab, or anti—VEGF/anti-DLL4 bispecific antibodies
219R45-MB-21M18 or 219R45-MB-21R79 with or without gemcitabine were processed to single cell
suspensions, and serially transplanted into mice. 90 cells from each treatment group were injected
subcutaneously into NOD/SCID mice. Tumors were allowed to grow with no ent. Data is shown
as tumor volume (mm3) on day 55. Tumor frequency is shown as number of tumors over total number of
mice injected in each group.
Figure 7. Bispecific antibody ELISA. Bispecific antibodies 219R45-MB-21M18, 219R45-MB-
2 1 R79, 2 1 9R45-MB-2 1 R75, and 219R45-MB-21R83 were diluted in blocking buffer (1x PBS, 0.1%
gelatin, 0.1% Polysorbate-20, pH 7.4) containing 2ug/ml biotin-DLL4-hFc. The antibodies were serially
diluted 3-fold from SOOng/ml to 0.008ng/ml. The antibody samples were incubated for 2 hours in
blocking buffer containing the biotin-DLL4—hFc. After incubation, the antibody samples were transferred
to a VEGF-coated assay plate (100 l) and incubated for 2 hours. Streptavidin—HRP was added to
each well and incubated for 1 hr. TMB substrate was added to the wells with a 10 minute color
development and the reaction was stopped with 2M sulfuric acid. Absorbance was read at 450—65Onm
and the data analyzed using the 4-parameter fit within the Softmax Pro analysis program.
Figure 8. Imaged capillary isoelectric focusing of anti—VEGF/anti—DLL4 bispecific antibodies.
Figure 9. Inhibition of colon tumor growth by EGF/anti-DLL4 bispecific antibodies in
tumor recurrence model. OMP-C8 colon tumor cells were injected subcutaneously in NOD/SCID mice.
Mice were d with control antibody (-I-), anti-hDLL4 antibody 21M18 (), anti-VEGF antibody
bevacizumab (— A-), a combination of 21M1 8 and bevacizumab (— V -), anti-VEGF/anti-DLL4 ific
antibody 219R45-MB-21M18 (—0—), or anti—VEGF/anti—DLL4 bispecific antibody 219R45-MB-21R79 (-o-
), all in combination with irinotecan. Antibodies 21M18 and bevacizumab were stered
intraperitoneally at a dose of kg once a week, ific antibodies 219R45-MB-21M18 and
219R45-MB-21R79 were administered intraperitoneally at a dose of lSmg/kg once a week, and irinotecan
was administered for the first 4 weeks at a dose of 45mg/kg. Data are shown as tumor volume (mm3) over
days post-treatment.
Figure 10. Tumorigenicity of OMP-C3 colon tumor cells after treatment with anti-VEGF/anti-
DLL4 bispecific dies. Tumors from mice treated with control antibody, anti-hDLL4 antibody
21Ml8, anti-VEGF antibody bevacizumab, a combination of 21M18 and bevacizumab, or anti-
VEGF/anti-DLL4 bispecific antibodies —MB—21M1 8 or 219R45—MB—21R79 with or without
irinotecan were processed to single cell suspensions, and serially transplanted into mice. 150 cells from
each treatment group were injected aneously into NOD/SCID mice. Tumors were allowed to grow
with no treatment. Data are shown as tumor volume (mm3) on day 68.
Figure 11. Inhibition of colon tumor growth in vivo by EGF/anti-DLL4 ific
antibodies. OMP-C8 colon tumor cells were ed subcutaneously into ID mice. Mice were
treated with control antibody (-l—), anti—VEGF antibody bevacizumab (- A -), or anti-VEGF/anti-DLL4
bispecific antibodies 219R45-MB-21M18 (—<>—), 219R45—MB-21R75 (4-), 219R45-MB-21R79 (-o-), or
219R45-MB-21R83 (- V-). Mice were d with antibodies as single agents (Fig. 10A) or in
combination with irinotecan (Fig. 10B). Antibodies were administered intraperitoneally at a dose of
15mg/kg once a week and irinotecan at a dose of 7.5mg/kg one a week. Data are shown as tumor volume
(mm3) over days post-treatment.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel binding agents, including but not d to polypeptides
such as dies, that bind VEGF and/or DLL4 (e.g., a VEGF/DLL4 binding agent). Related
polypeptides and polynucleotides, compositions comprising the VEGF/DLL4-binding agents, and
methods of making the VEGF/DLL4-binding agents are also provided. Methods of using the novel
VEGF/DLL4-binding agents, such as methods of inhibiting tumor growth, methods of treating cancer,
methods of reducing tumorigenicity of a tumor, methods of reducing the frequency of cancer stem cells in
a tumor, and/or methods of modulating angiogenesis, are fiirther provided.
A monoclonal dy that specifically binds human VEGF has been identified, 219R45. This
antibody has a binding affinity for human VEGF of about 0.67nM, and a binding affinity for mouse
VEGF of about 23nM. Several monoclonal dies that specifically bind human DLL4 have been
identified, 21R79, 21R75 and 21R83. Antibody 21R79 has a g affinity for human DLL4 of less
than 0.1nM. Bispecific antibodies that specifically bind human VEGF and human DLL4 have been
ed, 219R45-MB-21M18, 219R45—MB-21R79, 219R45—MB-21R75, and 219R45-MB-21R83 (CDR
ces in Figure 1). As used herein, the “MB” within an antibody name refers to
“monovalent/bispecific”. Bispecific antibody 219R45—MB—21M18 has a binding affinity for human
VEGF of less than 1.0nM and a binding affinity for human DLL4 of about 16nM. Bispecific antibody
219R45-MB-21R79 has a binding affinity for human VEGF of less than 1.0nM and a binding affinity for
human DLL4 of less than 1.0nM. Bispecific antibody 219R45-MB-21R75 has a binding affinity for
human DLL4 of about SnM, while ific antibody —MB-21R83 has a binding affinity for
human DLL4 of about lnM. Bispecific antibodies 219R45—MB—21Ml 8 and 219R45-MB-21R79 bind
mouse VEGF le 1, Table 3). Anti—VEGF/anti—DLL4 bispecific antibodies bind human VEGF and
human DLL4 simultaneously (Example 2, Figure 2). Anti—VEGF/anti—DLL4 bispecific dies inhibit
VEGF-induced proliferation of HUVEC cells (Example 3, Figure 3). Anti-VEGF/anti-DLL4 bispecific
dies inhibit nduced Notch signaling (Example 4, Figure 4). Anti-VEGF/anti-DLL4
bispecific antibodies inhibit tumor growth (Examples 5, 9, 11 and Figures 5, 9, 11). Anti-VEGF/anti-
DLL4 bispecific antibodies inhibit tumorigenicity (Examples 6 and 10 and Figures 6, 10). Anti-
VEGF/anti-DLL4 bispecific antibodies bind both VEGF and DLL4 in a bispecific ELISA (Example 7,
Figure 7). Anti-VEGF/anti-DLL4 bispecific antibodies are isolated and purified to a product comprising
at least 90% heterodimeric antibody (Example 8, Table 7).
1. Definitions
To facilitate an understanding of the present invention, a number of terms and phrases are defined
below.
The term “antibody” as used herein refers to an immunoglobulin le that recognizes and
cally binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing, through at least one antigen recognition site within the variable region of
the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact
monoclonal antibodies, single chain antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv
fragments), single chain Fv (scFv) antibodies, multispecific antibodies such as bispecific antibodies,
monospecific dies, lent antibodies, ic antibodies, humanized antibodies, human
antibodies, fusion proteins comprising an antigen—binding site of an antibody, and any other modified
immunoglobulin molecule comprising an antigen recognition site (i.e., antigen-binding site) as long as the
antibodies exhibit the desired ical activity. An antibody can be any of the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgGZ, IgG3,
IgG4, IgAl, and IgA2), based on the identity of their heavy chain nt domains referred to as alpha,
delta, epsilon, gamma, and mu, respectively. The ent classes of immunoglobulins have different and
well-known subunit structures and three—dimensional configurations. Antibodies can be naked or
conjugated to other molecules, including but not limited to, toxins and radioisotopes.
The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic
determining variable regions of an intact antibody. Examples of dy fragments include, but are not
limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and
pecific antibodies formed from antibody fragments. “Antibody fragment” as used herein comprises
an antigen-binding site or epitope-binding site.
The term “variable region” of an antibody refers to the variable region of an antibody light chain,
or the le region of an antibody heavy chain, either alone or in combination. The variable regions of
the heavy and light chains each t of four framework regions (FR) connected by three
complementarity ining regions (CDRs), also known as “hypervariable regions”. The CDRs in each
chain are held together in close proximity by the framework regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two
techniques for determining CDRs: (1) an approach based on cross-species ce variability (i.e., Kabat
et al., 1991, ces ofProteins 0fImmunoiogical Interest, 5th Edition, National Institutes of Health,
Bethesda, MD), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-
Lazikani et al., 1997, J. M0]. Biol, 273:927—948). In addition, combinations of these two approaches are
sometimes used in the art to determine CDRs.
The term “monoclonal antibody” as used herein refers to a homogeneous antibody population
involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This
is in contrast to polyclonal antibodies that typically include a mixture of ent antibodies directed
against a y of different antigenic determinants. The term “monoclonal antibody” encompasses both
intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv),
single chain (scFv) antibodies, fusion proteins comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an n recognition site (antigen-binding site). Furthermore,
“monoclonal antibody” refers to such antibodies made by any number of techniques, including but not
limited to, hybridoma production, phage selection, inant expression, and enic animals.
The term “humanized antibody” as used herein refers to forms of non-human (e. g., murine)
antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that
contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in
which residues of the CDRs are replaced by residues from the CDRs of a man species (e.g., mouse,
rat, , or hamster) that have the desired city, affinity, and/or binding capability (Jones et al.,
1986, Nature, 321:522-525; ann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988,
Science, 239: 1534-1536). In some instances, the Fv framework region residues of a human
immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species
that has the desired specificity, y, and/or binding lity. The humanized antibody can be further
modified by the substitution of additional residues either in the Fv framework region and/or within the
replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding
lity. In general, the zed dy will comprise substantially all of at least one, and typically
two or three, variable domains containing all or substantially all of the CDRs that correspond to the non-
human immunoglobulin whereas all or ntially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of
methods used to generate humanized antibodies are described in, for example, US. Patent 5,225,539.
The term “human antibody” as used herein refers to an antibody produced by a human or an
dy having an amino acid sequence corresponding to an antibody produced by a human. A human
antibody may be made using any of the techniques known in the art. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human CDRs.
The term “chimeric antibody” as used herein refers to an antibody wherein the amino acid
sequence of the immunoglobulin le is derived from two or more species. Typically, the variable
region of both light and heavy chains corresponds to the variable region of antibodies derived from one
species of mammals (e. g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding
capability, while the constant regions correspond to sequences in antibodies derived from another species
(usually human).
The phrase “affinity-matured antibody” as used herein refers to an antibody with one or more
alterations in one or more CDRs thereof that result in an improvement in the affinity of the dy for
antigen, compared to a parent antibody that does not possess those alterations(s). The definition also
includes alterations in non-CDR residues made in conjunction with tions to CDR es. Preferred
affinity-matured antibodies will have nanomolar or even lar affinities for the target antigen.
y-matured antibodies are produced by ures known in the art. For example, Marks et al.,
1992, Bio/Technology 10:779-783, describes affinity maturation by VH and VL domain shuffling.
Random mutagenesis of CDR and/or framework residues is described by Barbas et al., 1994, PNAS,
91 :3809-3813; Schier et al., 1995, Gene, 169:147—155; Yelton et al., 1995, J. Immunol. 155: 1994-2004;
Jackson et al., 1995, J. Immunol, 154:3310—9; and Hawkins et al., 1992, J. Mol. Biol, 226:889-896. Site-
ed mutagenesis may also be used to obtain affinity—matured antibodies.
The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that
portion of an antigen capable of being recognized and specifically bound by a particular antibody. When
the n is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous
amino acids juxtaposed by tertiary folding of a n. es formed from contiguous amino acids
(also referred to as linear epitopes) are lly retained upon protein ring, whereas epitopes
formed by tertiary folding (also referred to as conformational epitopes) are lly lost upon protein
ring. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a
unique spatial conformation.
The terms omultimeric molecule” or omultimer” or “heteromultimeric complex” or
“heteromultimeric polypeptide” are used interchangeably herein to refer to a molecule comprising at least
a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid
sequence from the first polypeptide by at least one amino acid residue. The heteromultimeric molecule
can comprise a “heterodimer” formed by the first and second polypeptide or can form higher order tertiary
structures where onal polypeptides are present.
The terms onist” and “antagonistic” as used herein refer to any molecule that partially or
fully blocks, inhibits, reduces, or neutralizes a biological activity of a target and/or signaling pathway
(e.g., the Notch pathway). The term “antagonist” is used herein to include any molecule that partially or
fully blocks, inhibits, reduces, or neutralizes the activity of a protein. Suitable antagonist les
specifically include, but are not d to, antagonist antibodies or antibody fragments.
The terms “modulation” and “modulate” as used herein refer to a change or an tion in a
biological activity. Modulation includes, but is not limited to, stimulating or ting an activity.
Modulation may be an increase or a decrease in activity (e.g., a decrease in angiogenesis or an increase in
angiogenesis), a change in binding teristics, or any other change in the biological, functional, or
immunological properties ated with the activity of a protein, pathway, or other biological point of
interest.
The terms “selectively binds” or “specifically binds” mean that a binding agent or an antibody
reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with
some combination of the above to the epitope, protein, or target le than with alternative substances,
including unrelated proteins. In certain embodiments “specifically binds” means, for instance, that an
antibody binds a n with a KB of about 0.1mM or less, but more y less than about 1 uM. In
certain embodiments, “specifically binds” means that an antibody binds a target at times with a KD of at
least about 0.1uM or less, at other times at least about 0.01pM or less, and at other times at least about
1nM or less. Because of the sequence identity between homologous proteins in different species, specific
binding can include an antibody that recognizes a protein in more than one species (e.g., human VEGF
and mouse VEGF). Likewise, because of homology within n regions of polypeptide sequences of
different proteins, specific binding can include an antibody (or other polypeptide or binding agent) that
recognizes more than one n (e.g., human VEGF-A and human VEGF-B). It is understood that, in
certain embodiments, an antibody or binding moiety that specifically binds a first target may or may not
specifically bind a second . As such, “specific binding” does not necessarily require (although it can
include) exclusive binding, i.e. binding to a single target. Thus, an antibody may, in n embodiments,
specifically bind more than one target. In certain ments, multiple targets may be bound by the
same antigen-binding site on the antibody. For example, an antibody may, in certain instances, comprise
two identical antigen-binding sites, each of which specifically binds the same epitope on two or more
proteins. In certain alternative embodiments, an antibody may be multispecific and comprise at least two
antigen-binding sites with differing specificities. By way of non-limiting example, a bispecific antibody
may comprise one antigen-binding site that recognizes an epitope on one protein (e.g., human VEGF) and
further comprise a second, different antigen—binding site that recognizes a different epitope on a second
protein (e. g., human DLL4). Generally, but not necessarily, reference to binding means c binding.
The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to
polymers of amino acids of any length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino
acid polymer that has been modified naturally or by ention; for example, disulfide bond formation,
ylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling ent. Also included within the definition are, for example,
polypeptides containing one or more s of an amino acid (including, for e, ral amino
acids), as well as other modifications known in the art. It is understood that, because the polypeptides of
this invention may be based upon antibodies, in certain embodiments, the polypeptides can occur as single
chains or associated chains.
The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to
polymers of nucleotides of any length, and e DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any
substrate that can be incorporated into a polymer by DNA or RNA polymerase.
“Conditions of high ency” may be identified by those that: (1) employ low ionic th
and high temperature for washing, for example 15mM sodium chloride/1.5mM sodium citrate/0. 1%
sodium dodecyl sulfate at 50°C; (2) employ during hybridization a ring agent, such as formamide,
for example, 50% (v/v) formamide with 0.1% bovine serum n/0. 1% Ficoll/0. 1%
polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 in 5x SSC (0.75M NaCl, 75mM sodium
citrate) at 42°C; or (3) employ during hybridization 50% formamide in 5x SSC, 50mM sodium phosphate
(pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's on, sonicated salmon sperm DNA (50ug/ml),
0.1% SDS, and 10% dextran e at 42°C, with washes at 42°C in 0.2x SSC and 50% formamide,
followed by a high-stringency wash consisting of 0. 1x SSC containing EDTA at 55°C.
The terms “identical” or percent “identity” in the context of two or more nucleic acids or
polypeptides, refer to two or more sequences or subsequences that are the same or have a specified
percentage of nucleotides or amino acid residues that are the same, when compared and aligned
(introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino
acid substitutions as part of the sequence identity. The percent identity may be measured using sequence
comparison software or algorithms or by Visual inspection. Various algorithms and software that may be
used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These
include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and
variations thereof. In some embodiments, two nucleic acids or polypeptides of the invention are
substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue
identity, when compared and aligned for maximum correspondence, as measured using a sequence
comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the
sequences that is at least about 10, at least about 20, at least about 40—60 residues, at least about 60-80
es in length or any integral value therebetween. In some embodiments, identity exists over a longer
region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the
sequences are substantially identical over the full length of the sequences being compared, such as the
coding region of a nucleotide sequence.
A “conservative amino acid tution” is one in which one amino acid residue is replaced with
another amino acid residue having a similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, including basic side chains (e.g., , arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), ar side chains (e. g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, cine) and aromatic side chains (e.g., ne, phenylalanine, tryptophan,
histidine). For example, substitution of a phenylalanine for a tyrosine is a vative substitution.
ably, conservative substitutions in the sequences of the ptides and antibodies of the invention
do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the
n to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid
conservative substitutions which do not eliminate antigen binding are well-known in the art.
The term “vector” as used herein means a uct, which is capable of delivering, and usually
expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but
are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage
vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA
expression vectors ulated in liposomes.
A ptide, antibody, cleotide, vector, cell, or composition which is “isolated” is a
ptide, dy, polynucleotide, vector, cell, or composition which is in a form not found in nature.
Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which
have been purified to a degree that they are no longer in a form in which they are found in nature. In
some embodiments, a polypeptide, antibody, cleotide, vector, cell, or composition which is isolated
is substantially pure.
The term “substantially pure” as used herein refers to al which is at least 50% pure (i.e.,
free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
The terms “cancer” and “cancerous” as used herein refer to or describe the physiological
condition in mammals in which a population of cells are characterized by unregulated cell growth.
Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic
cancers such as lymphoma and ia.
The terms “tumor” and “neoplasm” as used herein refer to any mass of tissue that results from
excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including
pre-cancerous lesions.
The term “metastasis” as used herein refers to the process by which a cancer spreads or transfers
from the site of origin to other s of the body with the development of a r cancerous lesion at a
new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring
cells and migrates via the bloodstream or lymph from the y site of disease to invade neighboring
body structures.
The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumor initiating cell” are
used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative
capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell
progeny wherein the entiated cells have d proliferative or developmental ial; and (3) are
capable of ric cell divisions for self-renewal or self-maintenance. These ties confer on the
cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an
immunocompromised host (e.g., a mouse) compared to the majority oftumor cells that fail to form
tumors. Cancer stem cells undergo enewal versus differentiation in a chaotic manner to form tumors
with abnormal cell types that can change over time as ons occur.
The terms “cancer cell” and “tumor cell” refer to the total population of cells derived from a
cancer or tumor or pre-cancerous lesion, including both non—tumorigenic cells, which comprise the bulk of
the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the terms
“cancer cell” or “tumor cell” will be modified by the term “non—tumorigenic” when referring solely to
those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer
stem cells.
The term “tumorigenic” as used herein refers to the functional features of a cancer stem cell
including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and
proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor
cells).
The term “tumorigenicity” as used herein refers to the ability of a random sample of cells from the
tumor to form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice).
This definition also includes enriched and/or isolated populations of cancer stem cells that form palpable
tumors upon serial transplantation into immunocompromised hosts (e. g., mice).
The term “subject” refers to any animal (e.g., a ), including, but not limited to, humans,
non-human primates, canines, felines, rodents, and the like, which is to be the ent of a particular
treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a
human subject.
The term “pharmaceutically acceptable” refers to a product or compound approved (or
approvable) by a regulatory agency of the Federal government or a state government or listed in the US.
Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
The terms “pharmaceutically acceptable excipient, carrier or adjuvant” or “acceptable
pharmaceutical carrier” refer to an excipient, carrier or adjuvant that can be administered to a subject,
together with at least one binding agent (e.g., an antibody) of the present disclosure, and which does not
destroy the activity of the g agent. The excipient, carrier or adjuvant should be nontoxic when
administered with a binding agent in doses sufficient to deliver a therapeutic effect.
The terms tive amount” or “therapeutically ive amount” or “therapeutic effect” refer
to an amount of a binding agent, an antibody, polypeptide, polynucleotide, small organic molecule, or
other drug effective to ” a e or disorder in a subject or mammal. In the case of cancer, the
therapeutically ive amount of a drug (e.g., an dy) has a eutic effect and as such can
reduce the number of cancer cells; se tumorigenicity, tumorigenic frequency or tumorigenic
capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer
cell population; inhibit and/or stop cancer cell infiltration into peripheral organs including, for example,
the spread of cancer into soft tissue and bone; inhibit and/or stop tumor or cancer cell metastasis; inhibit
and/or stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated
with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
To the extent the agent, for example an antibody, prevents growth and/or kills existing cancer cells, it can
be referred to as cytostatic and/or xic.
The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1)
therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed
pathologic condition or disorder and 2) lactic or preventative measures that t or slow the
development of a targeted pathologic condition or disorder. Thus those in need of treatment include those
y with the disorder; those prone to have the disorder; and those in whom the disorder is to be
prevented. In some embodiments, a subject is successfully “treated” according to the methods of the
present invention if the t shows one or more of the following: a reduction in the number of or
complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell
ration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition
of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer ; relief of
one or more symptoms associated with the specific cancer; reduced morbidity and mortality; ement
in quality of life; reduction in genicity; reduction in the number or frequency of cancer stem cells;
or some combination of effects.
As used in the present disclosure and claims, the singular forms EL 99 L:
a an” and “the” include plural
forms unless the context clearly dictates otherwise.
It is understood that wherever embodiments are bed herein with the language “comprising”
otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of”
are also provided. It is also understood that wherever embodiments are described herein with the
language “consisting essentially of” otherwise analogous embodiments described in terms of “consisting
of” are also provided.
The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A
and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B,
and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C;
A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
II. Antibodies
The present invention provides agents that specifically bind human VEGF proteins and/or human
DLL4 proteins. These agents are referred to herein as “VEGF/DLL4-binding agents”. The phrase
DLL4-binding agent” encompasses agents that bind only VEGF, agents that bind only DLL4, and
bispecific agents that bind both VEGF and DLL4. In certain embodiments, in addition to specifically
binding VEGF and/or DLL4, the VEGF/DLL4—binding agents further cally bind at least one
onal target or antigen. In some embodiments, the VEGF/DLL4-binding agent is an antibody. In
some embodiments, the VEGF/DLL4-binding agent is a polypeptide. In certain embodiments, the
VEGF/DLL4-binding agent specifically binds human VEGF. In n ments, the VEGF/DLL4-
binding agent specifically binds human DLL4. In certain embodiments, the LL4-binding agent is
a bispecific antibody. In certain embodiments, the VEGF/DLL4—binding agent is a bispecific dy
that specifically binds human VEGF and human DLL4. The full—length amino acid (aa) sequences for
human VEGF (VEGF-A) and human DLL4 are known in the art and are provided herein as SEQ ID
NO:27 (VEGF) and SEQ ID NO:23 (DLL4).
In certain embodiments, the VEGF/DLL4-binding agent or antibody binds VEGF and/or DLL4
with a dissociation constant (KD) of about luM or less, about 100nM or less, about 40nM or less, about
20nM or less, about 10nM or less, about lnM or less, or about 0.1nM or less. In some embodiments, a
VEGF/DLL4-binding agent or dy binds VEGF and/or DLL4 with a KD of about 20nM or less. In
some embodiments, a LL4-binding agent or antibody binds VEGF and/or DLL4 with a KD of
about 10nM or less. In some embodiments, a VEGF/DLL4—binding agent or antibody binds VEGF and/or
DLL4 with a KD of about lnM or less. In some embodiments, a VEGF/DLL4-binding agent or antibody
binds VEGF and/or DLL4 with a KD of about 0.1nM or less. In some embodiments, the VEGF/DLL4-
binding agent binds both human VEGF and mouse VEGF with a KD of about 100nM or less. In some
embodiments, the VEGF/DLL4-binding agent binds both human VEGF and mouse VEGF with a KD of
about SOnM or less. In some ments, a LL4—binding agent binds both human DLL4 and
mouse DLL4 with a KB of about 100nM or less. In some embodiments, a VEGF/DLL4-binding agent
binds both human DLL4 and mouse DLL4 with a KD of about SOnM or less. In some embodiments, the
iation nt of the binding agent (e.g., an antibody) to VEGF is the iation constant
determined using a VEGF fusion protein comprising at least a n ofVEGF lized on a Biacore
chip. In some embodiments, the dissociation constant of the binding agent (e. g., an antibody) to DLL4 is
the dissociation constant determined using a DLL4—fusion protein comprising at least a portion of DLL4
immobilized on a Biacore chip.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
first antigen-binding site that specifically binds VEGF and a second antigen-binding site that specifically
binds DLL4. In some embodiments, a VEGF/DLL4—binding agent or antibody binds both VEGF and
DLL4 with a KD of about 100nM or less. In some embodiments, a VEGF/DLL4-binding agent or
antibody binds both VEGF and DLL4 with a KD of about SOnM or less. In some embodiments, a
VEGF/DLL4-binding agent or antibody binds both VEGF and DLL4 with a KD of about 20nM or less. In
some embodiments, a VEGF/DLL4-binding agent or antibody binds both VEGF and DLL4 with a KD of
about 10nM or less. In some embodiments, a VEGF/DLL4-binding agent or antibody binds both VEGF
and DLL4 with a KD of about lnM or less. In some embodiments, the affinity of one of the antigen-
binding sites may be weaker than the affinity of the other antigen-binding site. For example, the KD of
one antigen binding site may be about lnM and the KD of the second antigen-binding site may be about
10nM. In some embodiments, the difference in affinity between the two antigen-binding sites may be
about 2-fold or more, about 3-fold or more, about 5—fold or more, about 8-fold or more, about 10-fold or
more, about d or more, about 20-fold or more, about 30—fold or more, about 50-fold or more, or
about 100-fold or more. Modulation of the ies of the two n—binding sites may affect the
biological activity of the bispecific antibody. For example, decreasing the affinity of the antigen-binding
site for DLL4 or VEGF, may have a desirable effect, for example decreased toxicity of the binding agent
or increased therapeutic index.
By way of non-limiting example, the bispecific antibody may comprise (a) a first antigen-binding
site that binds human VEGF with a KD between about 0.1 nM and about 1.0 nM, and (b) a second antigen-
binding site that specifically binds human DLL4 with a KD n about 0.1 nM and about 20 nM,
between about 0.5nM and about 20nM, between about 1.0 nM and 10nM. In n embodiments, the
bispecific antibody comprises two identical light chains.
In certain embodiments, the VEGF/DLL4—binding agent (e.g., an dy) binds VEGF and/or
DLL4 with a half maximal effective concentration (EC50) of about lnM or less, about lOOnM or less,
about 40nM or less, about 20nM or less, about lOnM or less, about lnM or less, or about 0.1nM or less.
In certain embodiments, a VEGF/DLL4—binding agent (e.g., an antibody) binds VEGF and/or DLL4 with
a half maximal effective concentration (EC50) of about 1 uM or less, about lOOnM or less, about 40nM or
less, about 20nM or less, about lOnM or less, about lnM or less, or about 0.1nM or less.
In certain embodiments, the VEGFXDLL4—binding agent is an antibody. In some embodiments,
the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In
some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a
humanized antibody. In some embodiments, the antibody is a human antibody. In certain embodiments,
the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In certain embodiments, the antibody is an IgGl
dy. In certain embodiments, the antibody is an IgG2 antibody. In certain embodiments, the
antibody is an antibody fragment comprising an n—binding site. In some embodiments, the antibody
is a bispeciflc dy. In some embodiments, the antibody is monovalent, eciflc, bivalent, or
multispeciflc. In some embodiments, the antibody is conjugated to a cytotoxic moiety. In some
embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.
The VEGF/DLL4-binding agents (e.g., antibodies) of the present invention can be assayed for
specific binding by any method known in the art. The immunoassays which can be used include, but are
not limited to, competitive and non-competitive assay systems using techniques such as e analysis,
FACS analysis, fluorescence, immunocytochemistry, n blot analysis, radioimmunoassay,
ELISA, “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion
precipitin reaction, immunodiffusion assay, agglutination assay, complement-fixation assay,
immunoradiometric assay, fluorescent immunoassay, homogeneous time-resolved fluorescence assay
(HTRF), and protein A assay. Such assays are routine and well-known in the art (see, e.g.,
Ausubel et al., Editors, l994-present, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,
New York, NY).
For example, the specific binding of an dy to human VEGF and/or human DLL4 may be
determined using ELISA. An ELISA assay comprises preparing antigen, coating wells of a 96 well
microtiter plate with antigen, adding the antibody or other binding agent conjugated to a detectable
compound such as an enzymatic substrate (e.g. adish peroxidase or alkaline phosphatase) to the
well, incubating for a period of time, and detecting the presence of the binding agent bound to the antigen.
In some embodiments, the g agent or dy is not ated to a detectable nd, but
instead a second antibody that izes the binding agent or antibody (e.g., an anti-Fc antibody) and is
conjugated to a detectable compound is added to the well. In some embodiments, instead of coating the
well with the antigen, the binding agent or antibody can be coated to the well and a second antibody
conjugated to a detectable compound can be added following the addition of the antigen to the coated
well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase
the signal detected as well as other variations of ELISAs known in the art.
In another example, the specific binding of an antibody to human VEGF and/or human DLL4
may be determined using FACS. A FACS screening assay may comprise generating a cDNA construct
that expresses an antigen as a fusion protein, ecting the construct into cells, expressing the antigen
on the e of the cells, mixing the binding agent or antibody with the transfected cells, and incubating
for a period of time. The cells bound by the binding agent or antibody may be identified by using a
secondary antibody conjugated to a detectable compound (e.g., jugated anti-Fe antibody) and a
flow cytometer. One of skill in the art would be knowledgeable as to the parameters that can be modified
to ze the signal detected as well as other variations of FACS that may enhance screening (e.g.,
screening for blocking antibodies).
The binding affinity of an antibody or other binding—agent to an n (e.g., VEGF or DLL4)
and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One
example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I), or fragment or variant thereof, with the antibody of interest in the presence of
increasing amounts of unlabeled n followed by the detection of the antibody bound to the labeled
antigen. The affinity of the antibody for the n and the binding off-rates can be determined from the
data by Scatchard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the
binding on and off rates of antibodies or agents that bind an antigen (e.g., VEGF or DLL4). Biacore
kinetic analysis comprises analyzing the binding and iation of dies from chips with
immobilized antigen (e.g., VEGF or DLL4) on their surface.
In certain embodiments, the ion provides a VEGF-binding agent (e. g., an antibody) that
specifically binds human VEGF, wherein the VEGF-binding agent (e.g., an dy) comprises one, two,
three, four, five, and/or six of the CDRs of dy 219R45 (see Table 1). In some embodiments, the
VEGF-binding agent comprises one or more of the CDRs of 219R45, two or more of the CDRs of
219R45, three or more of the CDRs of 2 l9R45, four or more of the CDRs of 219R45, five or more of the
CDRs of 219R45, or all six of the CDRs of 219R45. In some ments, the VEGF-binding agent
binds human VEGF and mouse VEGF.
Table 1
NYWMH
HC CDRI
(SEQ ID NO:17)
DINPSNGRTSYKEKFKR
HC CDRZ
(SEQ ID NO: 18)
HYDDKYYPLMDY
HC CDR3
(SEQ ID NO: 19)
RASESVDNYGISFMK
LC CDR]
(SEQ ID NO:20)
AASN GSQ
LC CDR2
(SEQ ID NO:21)
QQSKEVPWTFGG
LC CDR3
(SEQ ID NO:22)
In certain embodiments, the invention provides a VEGF-binding agent (e.g., an antibody) that
cally binds human VEGF, wherein the VEGF-binding agent comprises a heavy chain CDRI
sing NYWMH (SEQ ID , a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR
(SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19). In some
embodiments, the VEGF-binding agent further comprises a light chain CDRl comprising
DNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the
VEGF-binding agent comprises: (a) a heavy chain CDRl comprising NYWMH (SEQ ID NO: 17), a heavy
chain CDR2 sing DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain CDR3
comprising HYDDKYYPLMDY (SEQ ID NO:19), and (b) a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the invention provides a VEGF-binding agent (e.g., an antibody) that
specifically binds human VEGF, wherein the VEGF—binding agent comprises: (a) a heavy chain CDRl
comprising NYWMH (SEQ ID NO:17), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (b) a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), or a
t f comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 sing
HYDDKYYPLMDY (SEQ ID NO:19), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (d) a light chain CDRl sing RASESVDNYGISFMK (SEQ ID NO:20), or a t
thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (l) a light
chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or a variant thereof comprising 1, 2, 3, or
4 amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative
substitutions.
In certain ments, the invention provides a inding agent (e. g., an antibody) that
specifically binds VEGF, wherein the VEGF—binding agent comprises a heavy chain variable region
having at least about 80% sequence identity to SEQ ID NO:11, and a light chain variable region having at
least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the VEGF-binding agent
comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%,
at least about 97%, or at least about 99% sequence identity to SEQ ID NO:11. In certain embodiments,
the VEGF-binding agent comprises a light chain le region having at least about 85%, at least about
90%, at least about 95%, at least about 97%, or at least about 99% sequence ty to SEQ ID NO: 12.
In certain embodiments, the VEGF-binding agent comprises a heavy chain variable region having at least
about 95% sequence identity to SEQ ID NO:1 l, and a light chain variable region having at least about
95% sequence identity to SEQ ID NO:12. In certain embodiments, the VEGF—binding agent comprises a
heavy chain variable region comprising SEQ ID N02] 1, and a light chain variable region comprising SEQ
ID NO:12. In certain ments, the inding agent comprises a heavy chain le region
consisting essentially of SEQ ID NO:11, and a light chain variable region consisting essentially of SEQ
ID NO:12. In some embodiments, the VEGF—binding agent ses a heavy chain comprising SEQ ID
NO:49, and a light chain sing SEQ ID NO:8. In some embodiments, the VEGF-binding antibody
or other agent comprises a heavy chain comprising SEQ IDNO:7, and a light chain comprising SEQ ID
NO:8.
In some embodiments, the VEGF-binding agent binds VEGF with a KD of about 10nM or less. In
some embodiments, the VEGF-binding agent binds VEGF with a KD of about 1nM or less. In some
embodiments, the VEGF-binding agent binds VEGF with a KD of about 0.1nM or less. In some
ments, the VEGF-binding agent binds VEGF with a KD of about 0.01nM or less. In some
embodiments, at least one amino acid residue in at least one CDR of the VEGF-binding agent is
substituted with a different amino acid so that the affinity of the VEGF-binding agent for VEGF is altered.
In some embodiments, the affinity of the VEGF—binding agent is increased. In some embodiments, the
affinity of the VEGF-binding agent is decreased. In some embodiments, the inding agent binds
human VEGF. In some embodiments, the VEGF—binding agent binds human VEGF and mouse VEGF.
In certain embodiments, the VEGF—binding agent comprises the heavy chain variable region and
light chain variable region of the 219R45 dy. In certain embodiments, the VEGF-binding agent
comprises the heavy chain and light chain of the 219R45 antibody (with or without the leader sequence).
In certain embodiments, a VEGF-binding agent is the 219R45 antibody.
In certain embodiments, a VEGF—binding agent comprises, consists essentially of, or consists of,
the antibody 219R45.
In certain embodiments, a VEGF-binding agent (e.g., an antibody) binds the same epitope, or
essentially the same epitope, on VEGF as an antibody of the invention. In another embodiment, a VEGF-
binding agent is an antibody that binds an epitope on VEGF that overlaps with the e on VEGF
bound by an antibody of the invention. In certain embodiments, a VEGF-binding agent (e.g., an antibody)
binds the same e, or essentially the same epitope, on VEGF as antibody 219R45. In another
embodiment, the VEGF-binding agent is an antibody that binds an epitope on VEGF that overlaps with
the epitope on VEGF bound by antibody 219R45.
In some embodiments, the VEGF—binding agent inhibits binding ofVEGF to at least one VEGF
receptor. In certain embodiments, the inding agent inhibits binding of human VEGF to VEGFR-
l or VEGFR-Z. In some embodiments, the inding agent specifically binds VEGF and modulates
angiogenesis. In some ments, the VEGF-binding agent specifically binds VEGF and inhibits
angiogenesis. In some embodiments, the VEGF—binding agent specifically binds VEGF and inhibits
tumor growth.
In certain embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4-binding agent (e. g., an antibody) comprises one, two,
three, four, five, and/or Six of the CDRs of antibody 21R79 (see Table 2). In some embodiments, the
DLL4-binding agent ses one or more of the CDRs of 21R79, two or more of the CDRs of 21R79,
three or more of the CDRs of 21R79, four or more of the CDRs of 21R79, five or more of the CDRs of
21R79, or all six of the CDRs of 21R79. In certain embodiments, the ion provides a DLL4-binding
agent (e.g., an antibody) that specifically binds human DLL4, wherein the DLL4-binding agent (e.g., an
antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 21R75 (see Table 2).
In some embodiments, the DLL4-binding agent comprises one or more of the CDRs of 21R75, two or
more ofthe CDRs of21R75, three or more ofthe CDRs of21R75, four or more ofthe CDRs of21R75,
five or more of the CDRs of 21R75, or all six of the CDRs of 21R75. In certain embodiments, the
invention provides a DLL4-binding agent (e.g., an antibody) that specifically binds human DLL4, wherein
the DLL4-binding agent (e.g., an dy) comprises one, two, three, four, five, and/or six of the CDRs
of antibody 21R83 (see Table 2). In some embodiments, the DLL4-binding agent ses one or more
ofthe CDRs of21R83, two or more ofthe CDRs of21R83, three or more ofthe CDRs of21R83, four or
more ofthe CDRs 3, five or more ofthe CDRs of21R83, or all six ofthe CDRs of21R83. In
some embodiments, the DLL4-binding agent binds human DLL4 and mouse DLL4.
Table 2
_ 21R79 21R75 21R83
TAYYIH TAYYIH TAYYIH
HC CDRl
(SEQ ID NO: 13) (SEQ ID NO: l3) (SEQ ID NO: 13)
YIANYNRATNYNQKFKG YIAGYKDATNYNQKFKG YISNYNRATNYNQKFKG
HC CDR2
(SEQ ID NO: 14) (SEQ ID NO:59) (SEQ ID NO:65)
VGMDY RDYDYDVGMDY RDYDYDVGMDY
HC CDR3
(SEQ ID NO: 16) (SEQ ID NO: 16) (SEQ ID NO: 16)
RASESVDNYGISFMK RASESVDNYGISFMK RASESVDNYGISFMK
LC CDRI
(SEQ ID NO:20) (SEQ ID NO:20) (SEQ ID NO:20)
S AASNQGS AASNQGS
LC CDR2
(SEQ ID NO:21) (SEQ ID NO:21) (SEQ ID NO:21)
(SEQ ID NO:22) (SEQ ID NO:22) (SEQ ID NO:22)
In certain ments, the heavy chain CDRl of the DLL4—binding dy is a minimal HC
CDRl comprising AYYIH (SEQ ID .
In some embodiments, the binding agent is an antibody that binds human DLL4 and comprises a
heavy chain CDRl comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain
CDR2 comprising YX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2
is serine, asparagine, or glycine, X3 is asparagine or lysine, and X4 is glysine, arginine,or aspartic acid
and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDRI
comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ
ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In certain embodiments, the invention es a DLL4-binding agent (e.g., an antibody) that
specifically binds human DLL4, n the DLL4—binding agent comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG
(SEQ ID NO:14), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In some
embodiments, the DLL4-binding agent further comprises a light chain CDRl comprising
DNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID N021),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In n embodiments, the
DLL4-binding agent comprises: (a) a heavy chain CDRl comprising TAYYIH (SEQ ID NO:13), a heavy
chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3
comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) a light chain CDRl sing
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID N021),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In n embodiments, the ion provides a DLL4—binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4—binding agent comprises: (a) a heavy chain CDRl
comprising TAYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (b) a heavy chain CDR2 sing YIANYNRATNYNQKFKG (SEQ ID NO:14), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising
RDYDYDVGMDY (SEQ ID NO: 16), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (d) a light chain CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), or a variant
thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light
chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or a variant thereof comprising 1, 2, 3, or
4 amino acid tutions. In n embodiments, the amino acid substitutions are conservative
substitutions.
In certain embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds DLL4, wherein the inding agent comprises a heavy chain variable region
having at least about 80% sequence identity to SEQ ID NO:10, and a light chain variable region having at
least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the DLL4-binding agent
comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%,
at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 10. In certain embodiments,
the DLL4-binding agent comprises a light chain variable region having at least about 85%, at least about
90%, at least about 95%, at least about 97%, or at least about 99% ce identity to SEQ ID NO: 12.
In certain embodiments, the DLL4—binding agent comprises a heavy chain variable region having at least
about 95% sequence identity to SEQ ID NO:10, and a light chain variable region having at least about
95% sequence ty to SEQ ID NO:12. In certain embodiments, the DLL4-binding agent comprises a
heavy chain variable region comprising SEQ ID NO:10, and a light chain le region comprising SEQ
ID NO:12. In certain embodiments, the DLL4—binding agent comprises a heavy chain variable region
consisting essentially of SEQ ID NO: 10, and a light chain le region consisting essentially of SEQ
ID NO:12. In some embodiments, the DLL4-binding agent comprises a heavy chain comprising SEQ ID
NO:48, and a light chain comprising SEQ ID NO:8. In some embodiments, the DLL4-binding dy
or other agent comprises a heavy chain comprising SEQ ID N026, and a light chain sing SEQ ID
NO:8. In some embodiments, the antibody is a bispecific dy.
In n embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4—binding agent comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG
(SEQ ID NO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO: 16). In some
embodiments, the DLL4-binding agent further comprises a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In certain ments, the
DLL4-binding agent comprises: (a) a heavy chain CDRl comprising TAYYIH (SEQ ID NO: 13), a heavy
chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3
comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising PWTFGG (SEQ ID NO:22).
In certain embodiments, the invention provides a DLL4—binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4—binding agent comprises: (a) a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (b) a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising
RDYDYDVGMDY (SEQ ID NO: 16), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (d) a light chain CDRl comprising RASESVDNYGISFMK (SEQ ID , or a variant
thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), or a variant thereof comprising 1, 2, 3, or 4 amino acid tutions; and (f) a light
chain CDR3 comprising QQSKEVPWTFGG (SEQ ID , or a t thereof comprising 1, 2, 3, or
4 amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative
substitutions.
In certain embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds DLL4, wherein the DLL4—binding agent comprises a heavy chain variable region
having at least about 80% sequence identity to SEQ ID NO:58, and a light chain variable region having at
least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the DLL4-binding agent
comprises a heavy chain le region having at least about 85%, at least about 90%, at least about 95%,
at least about 97%, or at least about 99% sequence identity to SEQ ID NO:58. In certain embodiments,
the DLL4-binding agent comprises a light chain variable region having at least about 85%, at least about
90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 12.
In n embodiments, the DLL4-binding agent comprises a heavy chain variable region having at least
about 95% ce identity to SEQ ID NO:58, and a light chain variable region having at least about
95% sequence identity to SEQ ID NO:12. In certain embodiments, the DLL4-binding agent comprises a
heavy chain variable region comprising SEQ ID NO:58, and a light chain variable region comprising SEQ
ID NO:12. In n embodiments, the inding agent comprises a heavy chain variable region
consisting essentially of SEQ ID N025 8, and a light chain variable region consisting essentially of SEQ
ID NO:12. In some embodiments, the DLL4—binding agent comprises a heavy chain comprising SEQ ID
NO:56, and a light chain comprising SEQ ID N08
In n embodiments, the invention provides a DLL4—binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4—binding agent comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG
(SEQ ID NO:65), and a heavy chain CDR3 comprising VGMDY (SEQ ID NO: 16). In some
embodiments, the DLL4-binding agent r comprises a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the
DLL4-binding agent comprises: (a) a heavy chain CDRl comprising TAYYIH (SEQ ID NO: 13), a heavy
chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3
comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) a light chain CDRl comprising
DNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID N021),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID .
In certain embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds human DLL4, wherein the DLL4—binding agent comprises: (a) a heavy chain CDRl
comprising TAYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (b) a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising
RDYDYDVGMDY (SEQ ID NO: 16), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (d) a light chain CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), or a variant
f comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), or a variant f comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light
chain CDR3 comprising PWTFGG (SEQ ID , or a variant thereof sing 1, 2, 3, or
4 amino acid substitutions. In certain embodiments, the amino acid substitutions are vative
substitutions.
In certain embodiments, the invention provides a DLL4-binding agent (e.g., an antibody) that
specifically binds DLL4, wherein the DLL4-binding agent comprises a heavy chain variable region
having at least about 80% sequence identity to SEQ ID NO:64, and a light chain variable region having at
least 80% sequence identity to SEQ ID NO:12. In n embodiments, the DLL4-binding agent
comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%,
at least about 97%, or at least about 99% sequence identity to SEQ ID NO:64. In certain embodiments,
the DLL4-binding agent ses a light chain variable region having at least about 85%, at least about
90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:12.
In certain ments, the DLL4-binding agent comprises a heavy chain variable region having at least
about 95% sequence identity to SEQ ID NO:64, and a light chain variable region having at least about
95% sequence identity to SEQ ID NO: 12. In certain embodiments, the DLL4-binding agent comprises a
heavy chain variable region comprising SEQ ID NO:64, and a light chain variable region comprising SEQ
ID NO: 12. In certain ments, the DLL4—binding agent comprises a heavy chain variable region
consisting essentially of SEQ ID NO:64, and a light chain variable region consisting essentially of SEQ
ID NO:12. In some embodiments, the DLL4-binding agent comprises a heavy chain comprising SEQ ID
NO:62, and a light chain comprising SEQ ID NO:8.In some embodiments, the agent is a bispecific
antibody.
In some embodiments, the DLL4—binding agent is an antibody that comprises a heavy chain
comprising SEQ ID N025, and a light chain comprising SEQ ID NO:8. In some embodiments, the
antibody is a bispecific antibody.
In some embodiments, the inding agent binds DLL4 with a KD of 25nM or less. In some
embodiments, the DLL4-binding agent binds DLL4 with a KD of 10nM or less. In some embodiments,
the DLL4-binding agent binds DLL4 with a KD of about lnM or less. In some embodiments, the DLL4-
binding agent binds DLL4 with a KB of about 0.1nM or less. In some embodiments, the DLL4-binding
agent binds DLL4 with a KD of about 0.01nM or less. In some embodiments, at least one amino acid
residue in at least one CDR of the DLL4—binding agent is substituted with a different amino acid so that
the affinity of the DLL4-binding agent for DLL4 is altered. In some embodiments, the affinity of the
DLL4-binding agent is increased. In some embodiments, the affinity of the DLL4-binding agent is
decreased.
In certain ments, the DLL4-binding agent comprises the heavy chain variable region and
the light chain variable region of the 21R79 antibody. In certain embodiments, the inding agent
comprises the heavy chain and light chain of the 21R79 antibody (with or without the leader sequence).
In certain embodiments, the inding agent is the 21R79 antibody.
In certain embodiments, a DLL4—binding agent comprises, consists essentially of, or consists of,
the antibody 21R79.
In certain embodiments, the DLL4-binding agent comprises the heavy chain variable region and
the light chain variable region of the 21R75 antibody. In certain embodiments, the DLL4-binding agent
comprises the heavy chain and light chain of the 21R75 antibody (with or without the leader sequence).
In certain embodiments, the DLL4-binding agent is the 21R75 antibody.
In certain embodiments, a DLL4-binding agent comprises, consists essentially of, or ts of,
the antibody 21R75.
In certain embodiments, the DLL4-binding agent comprises the heavy chain variable region and
the light chain variable region of the 21R83 antibody. In certain ments, the DLL4-binding agent
comprises the heavy chain and light chain of the 21R83 antibody (with or without the leader sequence).
In certain embodiments, the DLL4-binding agent is the 21R83 antibody.
In certain ments, a DLL4-binding agent comprises, ts essentially of, or consists of,
the antibody 21R83.
In some embodiments, a DLL4—binding agent binds an N—terminal fragment of human DLL4
(amino acids 1-191 of SEQ ID NO:24). In some embodiments, the DLL4-binding agent binds an e
comprising amino acids 40-47 of SEQ ID NO:25. In some embodiments, the inding agent binds
an e comprising amino acids 113-120 of SEQ ID NO:25. In some embodiments, the DLL4-binding
agent binds an epitope comprising amino acids 40—47 of SEQ ID NO:25 and amino acids 0 of SEQ
ID NO:25.
In certain embodiments, a DLL4—binding agent (e.g., an antibody) binds the same epitope, or
essentially the same epitope, on DLL4 as an antibody of the invention. In another embodiment, a DLL4-
binding agent is an antibody that binds an epitope on DLL4 that overlaps with the epitope on DLL4 bound
by an antibody of the invention. In certain embodiments, a DLL4-binding agent (e.g., an antibody) binds
the same epitope, or essentially the same epitope, on DLL4 as antibody 21R79. In another embodiment,
the DLL4-binding agent is an antibody that binds an e on DLL4 that overlaps with the epitope on
DLL4 bound by dy 21R79. In certain ments, a DLL4—binding agent (e.g., an antibody)
binds the same epitope, or essentially the same epitope, on DLL4 as antibody 21R75. In another
embodiment, the DLL4-binding agent is an antibody that binds an epitope on DLL4 that overlaps with the
epitope on DLL4 bound by antibody 21R75. In n embodiments, a DLL4-binding agent (e. g., an
antibody) binds the same epitope, or essentially the same epitope, on DLL4 as antibody 21R83. In
another embodiment, the DLL4-binding agent is an antibody that binds an epitope on DLL4 that overlaps
with the epitope on DLL4 bound by antibody 21R83.
In some embodiments, the inding agent inhibits g of DLL4 to at least one Notch
receptor. In certain embodiments, the Notch receptor is Notch] or Notch4. In some
, Notch2, Notch3,
embodiments, the DLL4-binding agent specifically binds DLL4 and inhibits DLL4 activity. In some
embodiments, the inding agent specifically binds DLL4 and ts Notch signaling. In some
embodiments, the DLL4-binding agent specifically binds DLL4 and modulates angiogenesis. In some
embodiments, the DLL4-binding agent specifically binds DLL4 and ts tumor growth. In some
embodiments, the DLL4-binding agent specifically binds DLL4 and inhibits tumorigenicity. In some
embodiments, the DLL4-binding agent specifically binds DLL4 and reduces the number or frequency of
CSCs in a tumor.
In certain embodiments, the invention es a VEGF/DLL4-binding agent that is a ific
antibody. In some embodiments, the VEGF/DLL4 g agent is a bispecific antibody sing a
first antigen-binding site that specifically binds human VEGF. In some embodiments, the VEGF/DLL4
binding agent is a bispecific antibody comprising a first antigen-binding site that specifically binds human
VEGF and a second antigen-binding site that binds a tumor—associated target. In some embodiments, the
LL4-binding agent is a ific antibody comprising: a first antigen-binding site that
specifically binds human VEGF, wherein the first antigen—binding site comprises a heavy chain CDRl
comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR
(SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO: 19). In some
embodiments, the bispecific antibody further comprises: a light chain CDR] comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody comprising: a first antigen-binding site that
specifically binds human VEGF, wherein the first antigen—binding site comprises (a) a heavy chain CDRl
comprising NYWMH (SEQ ID NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR
(SEQ ID NO: 18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO: 19), and (b) a
light chain CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising
AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising PWTFGG (SEQ ID NO:22).
In some embodiments, the VEGFKDLL4 g agent is a bispecific antibody comprising a first
heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:1 1. In some
embodiments, the bispecific antibody r comprises a light chain variable region having at least 80%
sequence identity to SEQ ID NO: 12. In n embodiments, the bispecific VEGF/DLL4-binding agent
comprises a first heavy chain variable region having at least about 85%, at least about 90%, at least about
95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:1 1, and a light chain
variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at
least about 99% sequence identity to SEQ ID NO:12.
In certain embodiments, the invention es a VEGF/DLL4-binding agent that is a bispecific
antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a
first antigen-binding site that specifically binds human DLL4. In some embodiments, the VEGF/DLL4
binding agent is a bispecific antibody comprising a first antigen-binding site that specifically binds human
DLL4 and a second antigen-binding site that binds a tumor—associated . In some ments, the
VEGF/DLL4-binding agent is a bispecific antibody sing: a first antigen-binding site that
specifically binds human DLL4, wherein the first antigen—binding site comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO: 13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising
YIXleYX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2 is serine,
asparagine, or glycine, X3 is asparagine or lysine, and X4 is glysine, arginine,or aspartic acid and a heavy
chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID N021),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the
LL4-binding agent is a bispecific antibody comprising: a first antigen-binding site that
specifically binds human DLL4, wherein the first antigen—binding site comprises a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG
(SEQ ID , YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID
NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising
RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific dy comprises a first
n-binding site comprising a heavy chain CDRl comprising TAYYIH (SEQ ID NO: 13), a heavy
chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO: 14), and a heavy chain CDR3
comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific antibody
comprises a first antigen-binding site comprising a heavy chain CDRl comprising TAYYIH (SEQ ID
NO:13), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO: 15), and a heavy
chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific
antibody ses a first antigen-binding site comprising a heavy chain CDRl comprising TAYYIH
(SEQ ID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a
heavy chain CDR3 comprising VGMDY (SEQ ID NO:16). In some embodiments, the
ific antibody comprises a first antigen—binding site comprising a heavy chain CDRl comprising
TAYYIH (SEQ ID NO:13), a heavy chain CDR2 sing YISNYNRATNYNQKFKG (SEQ ID
NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO: 16). In some
embodiments, the bispecific antibody further comprises: a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising S (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID . In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody comprising: a first antigen-binding site that
specifically binds human DLL4, wherein the first antigen—binding site comprises (a) a heavy chain CDRl
comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG
(SEQ ID NO: 14), YISSYNGATNYNQKFKG (SEQ ID NO: 15), YIAGYKDATNYNQKFKG (SEQ ID
NO:59), or YISNYNRATNYNQKFKG (SEQ ID N0265), and a heavy chain CDR3 comprising
VGMDY (SEQ ID NO:16), and (b) a light chain CDRl comprising RASESVDNYGISFMK
(SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID N021), and a light chain CDR3
sing QQSKEVPWTFGG (SEQ ID NO:22).
In some embodiments, the VEGF/DLL4 g agent is a bispecific antibody comprising a first
heavy chain variable region having at least about 80% ce identity to SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:58, or SEQ ID NO:64. In some embodiments, the bispecific antibody further
comprises a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12. In
certain embodiments, the bispecific LL4—binding agent comprises a first heavy chain le
region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least
about 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64; and/or
a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least
about 97%, or at least about 99% sequence identity to SEQ ID NO:12.
In certain embodiments, the invention provides a VEGF/DLL4—binding agent (e. g., a bispecific
antibody) that specifically binds human VEGF and human DLL4. In some embodiments, the bispecific
antibody comprises: a) a first antigen-binding site that specifically binds human VEGF, and b) a second
antigen-binding Site that specifically binds human DLL4, wherein the first antigen-binding site comprises
a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising
DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY
(SEQ ID NO:19); wherein the second antigen—binding site ses a heavy chain CDRl comprising
TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID N0279), a heavy chain CDR2 comprising
YIXleYX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2 is serine,
asparagine, or glycine, X3 is asparagine or lysine, and X4 is glysine, arginine,or aspartic acid and a heavy
chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO: 16); and a light chain CDRl comprising
RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing S (SEQ ID NO:21),
and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, a
bispecific antibody comprises a first antigen—binding site that specifically binds human VEGF, and a
second antigen-binding site that cally binds human DLL4, wherein the first antigen-binding site
comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising
DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 sing HYDDKYYPLMDY
(SEQ ID NO: 19), and the second antigen-binding site comprises a heavy chain CDRl comprising
TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID
NO: 14), GATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), or
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO:16); and wherein both the first and second antigen—binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing AASNQGS
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
In some embodiments, the bispecific dy comprises a first antigen-binding site that
specifically binds human VEGF, and a second n—binding site that specifically binds human DLL4,
wherein the first antigen-binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID
NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain
CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19), and the second antigen-binding site comprises
a heavy chain CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3 sing RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing S
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the bispecific antibody is 219R45—MB—21R?9.
In some embodiments, the bispecific antibody comprises a first antigen—binding site that
specifically binds human VEGF, and a second antigen-binding site that specifically binds human DLL4,
wherein the first antigen-binding site comprises a heavy chain CDRl sing NYWMH (SEQ ID
NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain
CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19), and the second antigen-binding site ses
a heavy chain CDRl comprising TAYYIH (SEQ ID N0213), a heavy chain CDR2 comprising
YISSYNGATNYNQKFKG (SEQ ID NO:15), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen—binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
ments, the bispecific antibody is 219R45—MB-21M18.
In some embodiments, the bispecific dy comprises a first antigen-binding site that
specifically binds human VEGF, and a second antigen—binding site that specifically binds human DLL4,
n the first antigen-binding site which comprises a heavy chain CDRl comprising NYWMH (SEQ
ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy
chain CDR3 comprising HYDDKYYPLMDY (SEQ ID N0219), and the second antigen-binding site
comprises a heavy chain CDRl comprising TAYYIH (SEQ ID NO: 1 3), a heavy chain CDR2 comprising
YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO:16); and wherein both the first and second antigen—binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID N0220), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID . In some
embodiments, the bispecific antibody is —MB—21R75.
In some embodiments, the bispecific antibody comprises a first antigen-binding site that
specifically binds human VEGF, and a second antigen—binding site that specifically binds human DLL4,
n the first antigen-binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID
NO: 17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 1 8), and a heavy chain
CDR3 comprising HYDDKYYPLMDY (SEQ ID NO: 19), and the second n-binding site comprises
a heavy chain CDRl sing TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 sing RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the bispecific antibody is 219R45—MB-21R83.
In some embodiments, the VEGF/DLL4 binding agent (e.g., a bispecific antibody) comprises a
first heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:11, a second
heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:9, SEQ ID
NO: 10, SEQ ID N025 8, or SEQ ID NO:64, and a first and a second light chain le region having at
least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4-
binding agent comprises a first heavy chain variable region having at least about 85%, at least about 90%,
at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:11; a
second heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at
least about 97%, or at least about 99% sequence identity to SEQ ID NO:9, SEQ ID NO: 10, SEQ ID
NOS8, or SEQ ID NO:64; and a first and a second light chain variable region having at least about 85%,
at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ
ID NO: 12. In certain embodiments, the bispecific LL4-binding agent comprises a first heavy
chain variable region having at least about 95% sequence identity to SEQ ID NO:11, a second heavy
chain variable region having at least about 95% sequence identity to SEQ ID NO:9, and a first and a
second light chain variable region having at least about 95% sequence identity to SEQ ID NO:12. In
certain embodiments, the bispecific VEGFKDLL4—binding agent comprises a first heavy chain variable
region having at least about 95% sequence identity to SEQ ID NO:1 1, a second heavy chain variable
region having at least about 95% sequence identity to SEQ ID NO: 10, and a first and a second light chain
le region having at least about 95% ce identity to SEQ ID NO:12. In certain embodiments,
the bispecific VEGF/DLL4-binding agent comprises a first heavy chain variable region having at least
about 95% sequence identity to SEQ ID NO:1 1, a second heavy chain variable region having at least
about 95% sequence ty to SEQ ID NO:58, and a first and a second light chain variable region having
at least about 95% sequence identity to SEQ ID NO:12. In certain embodiments, the bispecific
VEGF/DLL4-binding agent ses a first heavy chain variable region having at least about 95%
sequence identity to SEQ ID NO:11, a second heavy chain variable region having at least about 95%
sequence identity to SEQ ID NO:64, and a first and a second light chain variable region having at least
about 95% sequence ty to SEQ ID NO:12. In certain embodiments, the bispecific LL4-
binding agent comprises a first heavy chain variable region comprising SEQ ID NO:11, a second heavy
chain variable region comprising SEQ ID NO:9, and a first and a second light chain variable region
comprising SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4-binding agent comprises
a first heavy chain variable region comprising SEQ ID NO:11, a second heavy chain le region
comprising SEQ ID NO:10, and a first and a second light chain variable region comprising SEQ ID
NO:12. In certain embodiments, the bispecific LL4-binding agent comprises a first heavy chain
variable region sing SEQ ID NO:11, a second heavy chain variable region sing SEQ ID
NOS8, and a first and a second light chain le region comprising SEQ ID NO:12. In certain
embodiments, the bispecific VEGF/DLL4—binding agent comprises a first heavy chain variable region
comprising SEQ ID N021 1, a second heavy chain variable region comprising SEQ ID NO:64, and a first
and a second light chain le region comprising SEQ ID NO:12. In certain embodiments, the
bispecific VEGF/DLL4-binding agent comprises a first heavy chain variable region consisting essentially
of SEQ ID NO:11, a second heavy chain variable region consisting essentially of SEQ ID NO:9, and a
first and a second light chain variable region consisting essentially of SEQ ID NO:12. In certain
embodiments, the bispecific VEGF/DLL4—binding agent comprises a first heavy chain variable region
consisting essentially of SEQ ID N011 1, a second heavy chain variable region consisting essentially of
SEQ ID NO: 10, and a first and a second light chain variable region consisting essentially of SEQ ID
NO:12. In certain embodiments, the bispecific VEGF/DLL4—binding agent comprises a first heavy chain
variable region consisting ially of SEQ ID NO:1 1, a second heavy chain variable region consisting
essentially of SEQ ID N025 8, and a first and a second light chain variable region consisting essentially of
SEQ ID NO: 12. In certain embodiments, the bispecific VEGF/DLL4-binding agent comprises a first
heavy chain variable region consisting essentially of SEQ ID NO: 1 l, a second heavy chain variable region
consisting essentially of SEQ ID NO:64, and a first and a second light chain variable region consisting
essentially of SEQ ID NO:12.
In some ments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
heavy chain variable region from the anti-VEGF antibody 219R45. In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy chain variable region from
the anti-DLL4 dy 21M18. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
antibody which comprises a heavy chain variable region from the anti-DLL4 antibody 21R79. In some
embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a heavy chain
variable region from the anti-DLL4 antibody 21R75. In some embodiments, the VEGF/DLL4-binding
agent is a bispecific antibody which comprises a heavy chain variable region from the anti-DLL4 antibody
21R83. In some ments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
heavy chain variable region from the anti-VEGF antibody 219R45, a heavy chain variable region from the
anti-DLL4 antibody 21R79 and two identical light chain variable regions. In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy chain variable region from
the anti-VEGF antibody 219R45, a heavy chain variable region from the LL4 antibody 21Ml8 and
two identical light chain variable regions. In some embodiments, the VEGF/DLL4-binding agent is a
bispecific dy which comprises a heavy chain variable region from the anti-VEGF antibody 219R45,
a heavy chain variable region from the anti-DLL4 antibody 21R75 and two identical light chain variable
regions. In some ments, the LL4—binding agent is a bispecific antibody which comprises
a heavy chain variable region from the anti—VEGF antibody 219R45, a heavy chain variable region from
the anti-DLL4 antibody 21R83 and two identical light chain variable regions.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
first CH3 domain and a second CH3 domain, each of which is d to promote formation of
heteromultimers. In some embodiments, the first and second CH3 domains are modified using a knobs-
into-holes technique. In some ments, the first and second CH3 domains comprise changes in
amino acids that result in altered electrostatic ctions. In some ments, the first and second
CH3 domains comprise changes in amino acids that result in altered hydrophobic/hydrophilic ctions.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises
heavy chain constant regions selected from the group consisting of: (a) a first human IgG1 nt
region, wherein the amino acids at positions 253 and 292 are substituted with glutamate or aspartate, and a
second human IgG1 constant region, wherein the amino acids at positions 240 and 282 are substituted
with lysine; (b) a first human IgG2 constant region, wherein the amino acids at positions 249 and 288 are
tuted with glutamate or aspartate, and a second human IgG2 constant region wherein the amino
acids at ons 236 and 278 are substituted with lysine; (c) a first human IgG3 constant region, wherein
the amino acids at positions 300 and 339 are substituted with glutamate or aspartate, and a second human
IgG3 constant region wherein the amino acids at positions 287 and 329 are substituted with lysine; and (d)
a first human IgG4 constant region, wherein the amino acids at positions 250 and 289 are substituted with
glutamate or aspartate, and a second IgG4 constant region wherein the amino acids at positions 237 and
279 are substituted with lysine.
In some embodiments, the VEGF/DLL4-binding agent is a bispecific dy which ses a
first human IgG1 constant region with amino acid tutions at positions 253 and 292, wherein the
amino acids are glutamate or aspartate, and a second human IgG1 constant region with amino acid
tutions at positions 240 and 282, wherein the amino acids are lysine. In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody which comprises a first human IgG2 constant region
with amino acid substitutions at positions 249 and 288, n the amino acids are glutamate or
aspartate, and a second human IgG2 constant region with amino acid substitutions at positions 236 and
278, wherein the amino acids are lysine. In some embodiments, the VEGF/DLL4-binding agent is a
bispecific antibody which comprises a first human IgG3 constant region with amino acid substitutions at
positions 300 and 339, wherein the amino acids are glutamate or ate, and a second human IgG2
constant region with amino acid substitutions at positions 287 and 329, n the amino acids are
. In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
first human IgG4 constant region with amino acid substitutions at positions 250 and 289, wherein the
amino acids are glutamate or aspartate, and a second human IgG4 constant region with amino acid
substitutions at ons 237 and 279, wherein the amino acids are lysine.
In some embodiments, the VEGF/DLL4—binding agent is a ific antibody which comprises a
first human IgG2 constant region with amino acid substitutions at positions 249 and 288, wherein the
amino acids are glutamate, and a second human IgG2 constant region with amino acid substitutions at
positions 236 and 278, wherein the amino acids are lysine. In some embodiments, the VEGF/DLL4-
binding agent is a bispecific antibody which ses a first human IgG2 constant region with amino
acid substitutions at positions 249 and 288, wherein the amino acids are asparate, and a second human
IgG2 constant region with amino acid substitutions at positions 236 and 278, wherein the amino acids are
lysine.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
heavy chain of SEQ ID N017. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
antibody which comprises a heavy chain of SEQ ID N025. In some embodiments, the VEGF/DLL4-
binding agent is a bispecific antibody which comprises a heavy chain of SEQ ID NO:56. In some
embodiments, the VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy chain of
SEQ ID N0:62. In some embodiments, the bispecific antibody further ses a light chain of SEQ ID
NO: 12. In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which comprises a
heavy chain of SEQ ID N027, a heavy chain of SEQ ID N0:5, and two light chains of SEQ ID N0:8. In
some embodiments, the VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy
chain of SEQ ID N027, a heavy chain of SEQ ID N0:6, and two light chains of SEQ ID N0:8. In some
embodiments, the VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy chain of
SEQ ID N027, a heavy chain of SEQ ID N0256, and two light chains of SEQ ID N0:8. In some
embodiments, the VEGF/DLL4-binding agent is a bispecific antibody which comprises a heavy chain of
SEQ ID N027, a heavy chain of SEQ ID N0262, and two light chains of SEQ ID N0:8.
In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody which binds
VEGF with a KD of about SOnM or less, about 25nM or less, about 10nM or less, about 1nM or less, or
about 0.1nM or less. In some embodiments, the VEGF/DLL4—binding agent is a bispecific antibody
which binds DLL4 with a KB of about SOnM or less, about 25nM or less, about 10nM or less, about 1nM
or less, or about 0.1nM or less. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
dy which binds VEGF with a KD of about SOnM or less and binds DLL4 with a KD of about SOnM
or less. In some embodiments, the bispecific antibody binds VEGF with a KD of about 25nM or less and
binds DLL4 with a KD of about 25nM or less. In some ments, the bispecific antibody binds VEGF
with a KD of about 10nM or less and binds DLL4 with a KD of about 10nM or less. In some
embodiments, the bispecific antibody binds VEGF with a KD of about 1nM or less and binds DLL4 with a
KD of about 1nM or less.
In some embodiments, the LL4—binding agent is a bispecific antibody which comprises
one antigen-binding site with a binding affinity that is weaker than the binding affinity of the second
antigen-binding site. For example, in some ments, the bispecific antibody may bind VEGF with a
KD ranging from about 0.1nM to 1nM and may bind DLL4 with a KD ranging from about 1nM to 10nM.
Or the ific antibody may bind VEGF with a KD ranging from about lnM to 10nM and may bind
DLL4 with a KD ranging from about 0.1nM to 1nM. In some embodiments, the bispecific dy may
bind DLL4 with a KD ranging from about 0.1nM to 1nM and may bind VEGF with a KD ranging from
about 1nM to 10nM. Or the ific antibody may bind DLL4 with a KD ranging from about 1nM to
10nM and may bind VEGF with a KD ranging from about 0.1nM to lnMIn some embodiments, the
ence in affinity between the two antigen-binding sites may be about 2-fold or more, about 3-fold or
more, about 5-fold or more, about 8—fold or more, about 10—fold or more, about 15-fold or more, about 30-
fold or more, about 50-fold or more, or about lOO—fold or more. In some embodiments, at least one amino
acid residue in at least one CDR of the antigen—binding site for VEGF is substituted with a different amino
acid so that the affinity of the VEGF-binding site is altered. In some embodiments, the affinity of the
VEGF-binding site is increased. In some embodiments, the affinity of the VEGF-binding site is
decreased. In some embodiments, at least one amino acid residue in at least one CDR of the antigen-
binding site for DLL4 is substituted with a different amino acid so that the affinity of the DLL4-binding
site is altered. In some ments, the affinity of the DLL4—binding site is increased. In some
embodiments, the affinity of the DLL4-binding site is decreased. In some embodiments, the affinities of
both the VEGF and DLL4 antigen-binding sites are altered.
The invention provides ptides, ing but not limited to antibodies, that cally bind
VEGF and/or DLL4. In some embodiments, a polypeptide binds human VEGF. In some embodiments, a
ptide binds human DLL4. In some embodiments, a polypeptide binds human VEGF and mouse
VEGF. In some ments, a polypeptide binds human DLL4 and mouse DLL4.
In some embodiments, a VEGF—binding agent comprises a polypeptide comprising a sequence
selected from the group consisting of: SEQ ID N023, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID N0:11, SEQ ID NO: 12, SEQ ID NO:47, and SEQ ID NO:49.
In some embodiments, a DLL4—binding agent comprises a polypeptide comprising a ce
selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID N0:5, SEQ
N0 ID:6, SEQ ID NO:8, SEQ ID N0:9, SEQ ID N0:10, SEQ ID N0:12, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0258, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID
NO:64.
In some embodiments, a VEGF/DLL4-binding agent comprises a polypeptide comprising a
sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID N0:3, SEQ ID
NO:4, SEQ ID N0:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, SEQ ID N0:10, SEQ
ID N0:11, SEQ ID NO: 12, SEQ ID NO:46, SEQ ID NO:47, SEQ ID N0:48, SEQ ID NO:49, SEQ ID
N0:56, SEQ ID N0:57, SEQ ID N0258, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64.
In some ments, a VEGF/DLL4—binding agent comprises a polypeptide comprising a
sequence selected from the group ting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID N0:5, SEQ ID
N026, SEQ ID N029, SEQ ID N0:10, SEQ ID NO:46, SEQ ID N0z48, SEQ ID N0256, SEQ ID N0:57,
SEQ ID N0258, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some embodiments, the
VEGF/DLL4 binding agent further ses a polypeptide comprising a sequence selected from the
group consisting of: SEQ ID N023, SEQ ID NO:7, SEQ ID N0:1 1, SEQ ID NO:47, and SEQ ID NO:49.
In some embodiments, the VEGF/DLL4 g agent further comprises a polypeptide comprising a
sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:8, and SEQ ID N0:12.
In some embodiments, a VEGF/DLL4—binding agent comprises a polypeptide comprising a
sequence selected from the group consisting of: SEQ ID N0:3, SEQ ID NO:7, SEQ ID N0:11, SEQ ID
NO:47, and SEQ ID NO:49. In some embodiments, the VEGF/’DLL4 binding agent further comprises a
polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some
embodiments, the VEGF/DLL4 binding agent fiirther comprises a polypeptide comprising a sequence
selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO: 12.
In certain embodiments, a VEGFXDLL4—binding agent (e.g., antibody) competes for specific
binding to VEGF with an antibody that comprises a heavy chain variable region comprising SEQ ID
NO:11 and a light chain variable region comprising SEQ ID NO:12. In certain embodiments, a
VEGF/DLL4-binding agent competes with antibody 219R45 for c binding to human VEGF. In
some embodiments, a VEGF/DLL4-binding agent or antibody competes for c binding to VEGF in
an in vitro competitive binding assay. In some embodiments, the VEGF is human VEGF. In some
embodiments, the VEGF is mouse VEGF.
In certain embodiments, a VEGF—DLL4—binding agent (e.g., an antibody) binds the same epitope,
or essentially the same epitope, on VEGF as an antibody of the invention. In another embodiment, a
VEGF/DLL4-binding agent is an antibody that binds an epitope on VEGF that ps with the epitope
on VEGF bound by an antibody of the ion. In certain embodiments, a VEGF/DLL4-binding agent
(e.g., an antibody) binds the same epitope, or essentially the same epitope, on VEGF as antibody 219R45.
In r embodiment, the VEGF/DLL4-binding agent is an antibody that binds an e on VEGF
that overlaps with the epitope on VEGF bound by antibody 219R45.
In n embodiments, the LL4—binding agent is an agent that es for specific
binding to VEGF with the antibody 219R45 (e.g., in a competitive g assay).
In certain embodiments, a VEGF/DLL4—binding agent (e.g., antibody) competes for specific
binding to DLL4 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:9
SEQ ID NO:10, SEQ ID N025 8, or SEQ ID NO:64 and a light chain variable region comprising SEQ ID
NO:12. In certain embodiments, a VEGF/DLL4—binding agent competes with antibody 21R79 for
specific binding to human DLL4. In n embodiments, a VEGF/DLL4-binding agent competes with
antibody 21R75 for specific binding to human DLL4. In certain embodiments, a VEGF/DLL4-binding
agent competes with dy 21R83 for specific binding to human DLL4. In some embodiments, a
LL4-binding agent or antibody competes for specific binding to DLL4 in an in vitro competitive
binding assay. In some embodiments, the DLL4 is human DLL4. In some embodiments, the DLL4 is
mouse DLL4.
In certain embodiments, a VEGF/DLL4-binding agent (e.g., an antibody) binds the same epitope,
or essentially the same epitope, on DLL4 as an antibody of the invention. In another embodiment, a
VEGF/DLL4-binding agent is an dy that binds an epitope on DLL4 that overlaps with the epitope
on DLL4 bound by an antibody of the invention. In certain embodiments, a VEGF/DLL4-binding agent
binds the same epitope, or essentially the same epitope, on DLL4 as antibody 21R79. In certain
embodiments, a LL4-binding agent binds the same epitope, or essentially the same epitope, on
DLL4 as antibody 21R75. In certain embodiments, a LL4-binding agent binds the same epitope,
or essentially the same epitope, on DLL4 as antibody 21R83. In another embodiment, the VEGF/DLL4-
binding agent is an antibody that binds an epitope on DLL4 that overlaps with the epitope on DLL4 bound
by antibody 21R79. In another embodiment, the VEGF/DLL4—binding agent is an antibody that binds an
epitope on DLL4 that ps with the epitope on DLL4 bound by antibody 21R75. In another
embodiment, the VEGF/DLL4-binding agent is an antibody that binds an epitope on DLL4 that overlaps
with the epitope on DLL4 bound by dy 21R83.
In n embodiments, the VEGF/DLL4-binding agent is an agent that competes for specific
binding to DLL4 with the antibody 21R79 (e.g., in a competitive binding assay). In certain embodiments,
the VEGF/DLL4—binding agent is an agent that competes for specific g to DLL4 with the antibody
21R75 (e. g., in a competitive binding assay). In certain embodiments, the VEGF/DLL4-binding agent is
an agent that es for specific binding to DLL4 with the antibody 21R83 (e. g., in a itive
binding assay). In certain embodiments, the VEGF/DLL4—binding agent is an agent that competes for
specific binding to DLL4 with the antibody 21M18 (e.g., in a competitive binding assay).
In certain embodiments, the VEGF/DLL4—binding agent is an agent that es for c
binding to VEGF and/or DLL4 with the ific antibody 219R45-MB-21M18 (e.g., in a competitive
binding . In certain embodiments, the VEGF/DLL4—binding agent is an agent that competes for
specific binding to VEGF and/or DLL4 with the bispecific antibody 219R45-MB-21M79 (e.g., in a
competitive binding . In certain embodiments, the VEGF/DLL4-binding agent is an agent that
competes for specific binding to VEGF and/or DLL4 with the bispecific antibody 219R45-MB-21M75
(e.g., in a competitive binding assay). In certain embodiments, the VEGF/DLL4-binding agent is an agent
that competes for c binding to VEGF and/or DLL4 with the bispecific antibody 219R45-MB-
21M83 (e. g., in a itive binding assay).
In certain ments, the VEGFXDLL4-binding agent (e.g., an antibody) described herein binds
VEGF and modulates VEGF activity. In some embodiments, the VEGF/DLL4-binding agent is a VEGF
antagonist and inhibits VEGF activity. In some embodiments, the VEGF/DLL4-binding agent is a VEGF
antagonist and modulates enesis. In some embodiments, the VEGF/DLL4—binding agent is a
VEGF antagonist and inhibits angiogenesis. In some embodiments, the VEGF/DLL4-binding agent is a
VEGF antagonist and inhibits tumor growth.
In certain embodiments, a VEGF/DLL4-binding agent (e.g., an antibody) described herein binds
human DLL4 and modulates DLL4 activity. In some embodiments, a VEGF/DLL4-binding agent is a
DLL4 antagonist and inhibits DLL4 activity. In some embodiments, a VEGF/DLL4-binding agent is a
DLL4 antagonist and inhibits Notch activity. In some embodiments, a VEGF/DLL4-binding agent is a
DLL4 antagonist and inhibits Notch signaling. In some embodiments, a VEGF/DLL4-binding agent is a
DLL4 nist and modulates angiogenesis. In some embodiments, a VEGF/DLL4-binding agent is a
DLL4 antagonist and es aberrant angiogenesis. In some ments, a LL4-binding
agent is a DLL4 antagonist and inhibits tumor growth.
In n embodiments, a VEGFXDLL4—binding agent (e.g., an antibody) described herein is a
bispecific antibody that binds human VEGF and modulates VEGF activity. In certain embodiments, a
VEGF/DLL4-binding agent (e.g., an antibody) described herein is a bispecific antibody that binds human
DLL4 and tes DLL4 activity. In certain embodiments, a VEGF/DLL4—binding agent (e.g., an
antibody) described herein is a bispecific antibody that binds human VEGF and human DLL4 and
modulates both VEGF and DLL4 activity. In some embodiments, the bispecific dy is a VEGF
antagonist and a DLL4 antagonist and inhibits both VEGF activity and DLL4 activity. In some
embodiments, the bispecific dy is a VEGF antagonist and a DLL4 antagonist and inhibits VEGF
activity and Notch activity. In some embodiments, the bispecific antibody is a VEGF nist and a
DLL4 antagonist and inhibits VEGF activity and Notch signaling. In some embodiments, the bispecific
antibody is a VEGF antagonist and a DLL4 antagonist and modulates angiogenesis. In some
embodiments, the bispecific antibody is a VEGF antagonist and a DLL4 antagonist and promotes aberrant
angiogenesis. In some embodiments, the bispecific antibody is a VEGF antagonist and a DLL4 antagonist
and inhibits angiogenesis. In some embodiments, the bispecific antibody is a VEGF antagonist and a
DLL4 antagonist and inhibits tumor growth.
In certain embodiments, the VEGF/DLL4—binding agent (e.g., an antibody or a bispecific
dy) is an nist ofVEGF. In some embodiments, the VEGF/DLL4-binding agent is an
antagonist ofVEGF and ts VEGF activity. In certain embodiments, the VEGF/DLL4-binding agent
inhibits VEGF activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%,
at least about 75%, at least about 90%, or about 100%. In certain embodiments, a LL4-binding
agent that inhibits human VEGF ty is antibody 219R45. In certain embodiments, a VEGF/DLL4-
binding agent that inhibits human VEGF activity is a bispecific antibody comprising the antigen-binding
site of 219R45. In certain embodiments, a VEGF/DLL4—binding agent that inhibits human VEGF activity
is the bispecific antibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4-binding agent
that inhibits human VEGF activity is the bispecific antibody 219R45—MB—21R79. In certain
embodiments, a VEGF/DLL4-binding agent that inhibits human VEGF activity is the bispecific antibody
219R45-MB-21R75. In certain embodiments, a VEGF/DLL4-binding agent that inhibits human VEGF
activity is the bispecific antibody 219R45-MB-21R83.
In certain embodiments, the VEGF/DLL4—binding agent (e.g., an antibody) is an antagonist of
DLL4. In some embodiments, the VEGF/DLL4—binding agent is an antagonist of DLL4 and inhibits
DLL4 activity. In n embodiments, the VEGF/DLL4—binding agent inhibits DLL4 activity by at least
about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about
90%, or about 100%. In certain embodiments, a VEGF/DLL4-binding agent that inhibits human DLL4
ty is antibody 21R79. In certain embodiments, a VEGF/DLL4-binding agent that inhibits human
DLL4 activity is antibody 21R75. In certain embodiments, a VEGF/DLL4-binding agent that inhibits
human DLL4 activity is antibody 21R83. In certain embodiments, a VEGF/DLL4-binding agent that
inhibits human DLL4 activity is a bispecific antibody comprising the antigen-binding site of 21R79. In
certain embodiments, a VEGF/DLL4-binding agent that inhibits human DLL4 activity is a bispecific
antibody comprising the antigen-binding site of 21R75. In certain embodiments, a VEGF/DLL4-binding
agent that inhibits human DLL4 activity is a bispecific dy comprising the antigen-binding site of
21R83. In certain embodiments, a VEGF/DLL4-binding agent that inhibits human DLL4 ty is the
bispecific antibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4-binding agent that
inhibits human DLL4 activity is the bispecific antibody —MB-21R79. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits human DLL4 activity is the bispecific antibody 219R45-MB-
21R75. In n embodiments, a VEGF/DLL4—binding agent that inhibits human DLL4 activity is the
bispecific antibody 2 1 9R45-MB-2 1 R83.
In certain embodiments, the VEGF/DLL4—binding agent (e.g., antibody) is an antagonist h
signaling. In certain embodiments, the VEGF/DLL4—binding agent inhibits Notch signaling by at least
about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about
90%, or about 100%. In certain embodiments, a VEGF/DLL4-binding agent that inhibits Notch ing
is antibody 21R79. In certain embodiments, a LL4-binding agent that ts Notch signaling is
antibody 21R75. In certain embodiments, a VEGF/DLL4-binding agent that inhibits Notch signaling is
antibody 21R83. In n embodiments, a VEGF/DLL4-binding agent that inhibits Notch ing is a
bispecific antibody comprising the antigen-binding site of 21R79. In certain ments, a
VEGF/DLL4-binding agent that ts Notch ing is a bispecific antibody comprising the antigen-
binding site of 21R75. In certain embodiments, a VEGF/DLL4-binding agent that inhibits Notch
signaling is a ific antibody comprising the antigen—binding site of 21R83. In certain embodiments,
a VEGF/DLL4-binding agent that inhibits Notch signaling is the bispecific antibody 219R45-MB-21M18.
In certain embodiments, a VEGF/DLL4—binding agent that inhibits Notch signaling is the bispecific
antibody -MB-21R79. In certain embodiments, a VEGF/DLL4—binding agent that inhibits Notch
signaling is the bispecific antibody 219R45-MB-21R75. In certain embodiments, a VEGF/DLL4-binding
agent that inhibits Notch signaling is the bispecific antibody 219R45-MB-21R83.
In certain embodiments, the VEGF/DLL4-binding agent (e.g., antibody) inhibits binding of
VEGF to at least one receptor. In some ments, the VEGF/DLL4-binding agent inhibits binding of
VEGF to VEGFR-l or VEGFR—2. In certain embodiments, the VEGF/DLL4-binding agent inhibits
binding ofVEGF to at least one VEGF receptor by at least about 10%, at least about 25%, at least about
50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits binding of human VEGF to at least one VEGF receptor is
antibody 219R45. In certain embodiments, a VEGF/DLL4-binding agent that inhibits binding of human
VEGF to at least one VEGF receptor is a bispeciflc antibody comprising the n-binding site of
2 l 9R45. In certain embodiments, a VEGFXDLL4—binding agent that inhibits binding of human VEGF to
at least one VEGF receptor is the bispecific antibody 219R45—MB—21M18. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits binding of human VEGF to at least one VEGF or is the
bispecific antibody 219R45-MB-21R79. In certain ments, a VEGF/DLL4—binding agent that
ts g of human VEGF to at least one VEGF receptor is the bispecific antibody 219R45-MB-
21R75. In certain embodiments, a VEGF/DLL4-binding agent that inhibits binding of human VEGF to at
least one VEGF or is the bispecific dy 219R45-MB-21R83.
In certain embodiments, the VEGF/DLL4—binding agent (e.g., antibody) inhibits binding of DLL4
protein to at least one Notch receptor. In some embodiments, the LL4-binding agent inhibits
g of DLL4 to , Notch2, Notch3, and/or Notch4. In certain embodiments, the VEGF/DLL4-
binding agent inhibits binding of DLL4 to at least one Notch receptor by at least about 10%, at least about
%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain
embodiments, a VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one Notch
receptor is antibody 21R79. In certain embodiments, a VEGF/DLL4-binding agent that inhibits binding
of human DLL4 to at least one Notch or is antibody 21R75. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one Notch receptor is
antibody 21R83. In certain embodiments, a VEGF/DLL4-binding agent that inhibits binding of human
DLL4 to at least one Notch receptor is a bispecific antibody comprising the antigen-binding site of 21R79.
In certain embodiments, a VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one
Notch receptor is a bispecific antibody comprising the antigen-binding site of 21R75. In certain
embodiments, a VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one Notch
receptor is a bispecific antibody comprising the antigen—binding site of 21R83. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one Notch receptor is the
bispecific antibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4-binding agent that
inhibits binding of human DLL4 to at least one Notch receptor is the bispecific antibody 219R45-MB-
21R79. In certain embodiments, a LL4-binding agent that ts binding of human DLL4 to at
least one Notch or is the bispeciflc antibody 219R45-MB-21R75. In certain embodiments, a
VEGF/DLL4-binding agent that inhibits binding of human DLL4 to at least one Notch receptor is the
bispecific dy —MB—21R83.
In vivo and in vitro assays for determining whether a VEGF/DLL4-binding agent (or candidate
VEGF/DLL4-binding agent) inhibits VEGF or affects angiogenesis are known in the art. In vitro assays
of enesis include but are not limited to, HUVEC proliferation assays, endothelial cell tube
formation assays, sprouting (or sprout ion) assays, HUVEC cell migration assays, and invasion
assays. In some embodiments, cells in the presence ofVEGF and the presence of a VEGF/DLL4-binding
agent are compared to cells in the presence ofVEGF without the LL4-binding agent present, and
ted for effects on angiogenesis (or biological effects associated with angiogenesis). In vivo assays
of angiogenesis include, but are not limited to, matrigel plug assays, corneal micropocket assays, and
n allantoic membrane (CAM) assays.
In vivo and in vitro assays for determining whether a VEGF/DLL4—binding agent (or candidate
VEGF/DLL4-binding agent) inhibits Notch activation or signaling are known in the art. For example,
ased, rase reporter assays utilizing a c reporter vector containing multiple copies of the
TCF-binding domain upstream of a firefly luciferase reporter gene may be used to measure Notch
signaling levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959—66; TOPflash, Millipore, Billerica MA).
In some embodiments, a ased, luciferase reporter assay ing a CBF/Luc reporter vector
containing multiple copies of the CBF—binding domain upstream of a firefly luciferase report genes may
be used. The level ofNotch signaling in the presence of one or more Notch ligands (e.g., DLL4 expressed
on the surface of transfected cells or soluble DLL4—F0 fusion protein) and in the presence of a
VEGF/DLL4-binding agent is compared to the level ofNotch signaling without the VEGF/DLL4-binding
agent present.
In certain embodiments, the VEGF/DLL4-binding agents have one or more of the following
effects: inhibit proliferation of tumor cells, t tumor growth, reduce the tumorigenicity of a tumor,
reduce the frequency of cancer stem cells in a tumor, trigger cell death of tumor cells, prevent metastasis
of tumor cells, decrease survival of tumor cells, modulate angiogenesis, inhibit angiogenesis, inhibit
productive angiogenesis, or promote aberrant angiogenesis.
In certain embodiments, the VEGF/DLL4—binding agents are capable of inhibiting tumor growth.
In certain embodiments, the VEGF/DLL4—binding agents are capable of inhibiting tumor growth in vivo
(e.g., in a aft mouse model, and/or in a human having cancer). In certain embodiments, tumor
growth is inhibited at least about two-fold, about three—fold, about five—fold, about ten-fold, about 50-fold,
about 100-fold, or about IOOO-fold as compared to an untreated tumor.
In certain embodiments, the VEGF/DLL4—binding agents are capable of reducing the
tumorigenicity of a tumor. In n embodiments, the VEGF/DLL4-binding agent or antibody is capable
of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a
mouse xenograft model. In n ments, the VEGF/DLL4-binding agent or antibody is capable
of reducing the tumorigenicity of a tumor by decreasing the number or ncy of cancer stem cells in
the tumor. In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by
at least about two-fold, about three—fold, about five—fold, about ld, about 50-fold, about 100-fold, or
about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is
determined by limiting dilution assay using an animal model. Additional examples and guidance
regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer
stem cells in a tumor can be found, e.g., in International Publication Number ; US.
Patent Publication No. 2008/0064049; and US. Patent Publication No. 2008/0178305.
In certain embodiments, the LL4—binding agents are capable of modulating angiogenesis.
In certain embodiments, the LL4-binding agents are capable of modulating angiogenesis in vivo
(e.g., in a xenograft mouse model, and/or in a human having cancer). In certain ments,
VEGF/DLL4-binding agents are capable of inhibiting angiogenesis. In certain ments,
LL4-binding agents are capable ofpromoting aberrant angiogenesis. In certain embodiments,
LL4-binding agents are e of inhibiting angiogenesis and/or promoting nt
enesis, leading to unproductive vascularization.
In certain embodiments, the VEGF/DLL4—binding agents described herein have a circulating half-
life in mice, cynomolgus monkeys, or humans of at least about 2 hours, at least about 5 hours, at least
about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2
weeks. In certain embodiments, the VEGF/DLL4—binding agent is an IgG (e.g., IgGl or IgG2) antibody
that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 2 hours, at least
about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week,
or at least about 2 weeks. Methods of increasing (or decreasing) the half-life of agents such as
polypeptides and antibodies are known in the art. For example, known methods of increasing the
circulating half-life of IgG dies include the introduction of mutations in the Fc region which
increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0 (see, e. g.,
US. Patent Publication Nos. 2005/0276799, 2007/0148164, and 2007/0122403). Known methods of
increasing the circulating half-life of antibody fragments lacking the Fc region include such techniques as
PEGylation.
In some embodiments, the VEGFXDLL4—binding agents are antibodies. Polyclonal dies can
be ed by any known method. In some embodiments, polyclonal antibodies are produced by
immunizing an animal (e.g, a rabbit, rat, mouse, goat, donkey) with an antigen of interest (e.g., a purified
peptide fragment, full-length recombinant protein, or fiision protein) by multiple subcutaneous or
intraperitoneal injections. The antigen can be optionally conjugated to a r such as keyhole limpet
hemocyanin (KLH) or serum albumin. The antigen (with or without a carrier protein) is diluted in sterile
saline and usually ed with an adjuvant (e.g., Complete or Incomplete Freund’s Adjuvant) to form a
stable emulsion. After a sufficient period of time, polyclonal antibodies are recovered from the
immunized animal, y from blood or ascites. The polyclonal dies can be purified from serum
or ascites according to standard s in the art including, but not limited to, affinity chromatography,
ion-exchange chromatography, gel electrophoresis, and dialysis.
In some embodiments, the VEGF/DLL4—binding agents are monoclonal antibodies. Monoclonal
antibodies can be prepared using hybridoma s known to one of skill in the art (see e. g., Kohler and
Milstein, 1975, Nature, 5-497). In some embodiments, using the hybridoma method, a mouse,
hamster, or other appropriate host animal, is immunized as described above to elicit from lymphocytes the
production of antibodies that cally bind the immunizing antigen. In some embodiments,
lymphocytes can be immunized in vitro. In some embodiments, the immunizing antigen can be a human
protein or a portion thereof. In some embodiments, the immunizing antigen can be a mouse protein or a
portion thereof.
Following zation, lymphocytes are isolated and fused with a suitable myeloma cell line
using, for example, polyethylene . The hybridoma cells are selected using specialized media as
known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen may be
identified by a y of methods including, but not limited to, immunoprecipitation, immunoblotting,
and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay). The
hybridomas can be propagated either in in vitro culture using rd methods (J.W. , 1996,
Monoclonal Antibodies: Principles and Practice, 3rd Edition, ic Press, San Diego, CA) or in vivo
as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or
ascites fluid according to standard methods in the art including, but not limited to, affinity
chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques
as known to one skilled in the art. The polynucleotides encoding a monoclonal antibody are isolated from
mature B-cells or hybridoma cells, such as by RT—PCR using oligonucleotide primers that specifically
y the genes encoding the heavy and light chains of the antibody, and their sequence is determined
using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then
cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into
host cells such as E. coli, simian COS cells, Chinese r ovary (CHO) cells, or a cells that do
not otherwise produce immunoglobulin proteins.
In certain other ments, recombinant monoclonal antibodies, or fragments thereof, can be
isolated from phage display libraries expressing variable domains or CDRs of a d s (see e.g.,
McCafferty et al., 1990, Nature, 2-554; Clackson et al., 1991, , 352:624-628; and Marks et
al., 1991, J. Mol. Biol, 222:581-597).
The polynucleotide(s) encoding a monoclonal antibody can be modified, for example, by using
recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant
domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted
for those regions of, for example, a human antibody to generate a chimeric antibody, or for a non-
globulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions
are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-
directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity,
etc. of a monoclonal antibody.
In some embodiments, a monoclonal antibody against VEGF and/or DLL4 is a zed
antibody. Typically, humanized antibodies are human immunoglobulins in which residues from the
CDRs are replaced by residues from a CDR of a non—human s (e.g., mouse, rat, rabbit, hamster,
etc.) that have the desired specificity, affinity, and/or binding capability using methods known to one
skilled in the art. In some embodiments, the Fv ork region residues of a human immunoglobulin
are replaced with the corresponding residues in an antibody from a non-human species that has the desired
specificity, affinity, and/or binding capability. In some embodiments, a humanized antibody can be
further modified by the tution of additional residues either in the FV framework region and/or within
the replaced non-human residues to refine and optimize antibody specificity, y, and/or capability.
In general, a humanized antibody will comprise substantially all of at least one, and typically two or three,
variable domain regions containing all, or substantially all, of the CDRs that correspond to the non-human
immunoglobulin whereas all, or substantially all, of the framework regions are those of a human
immunoglobulin consensus sequence. In some embodiments, a humanized antibody can also comprise at
least a portion of an globulin nt region or domain (Fc), typically that of a human
immunoglobulin. In certain embodiments, such humanized antibodies are used eutically because
they may reduce antigenicity and HAMA (human anti—mouse antibody) responses when administered to a
human subject. One skilled in the art would be able to obtain a functional humanized antibody with
d immunogenicity following known ques (see e.g., US. Patent Nos. 5,225,539; 089;
,693,761; and 5,693,762).
In certain embodiments, the VEGF/DLL4—binding agent is a human dy. Human antibodies
can be directly prepared using various techniques known in the art. In some embodiments, human
antibodies may be generated from immortalized human B lymphocytes zed in vitro or from
lymphocytes isolated from an immunized individual. In either case, cells that produce an antibody
directed t a target antigen can be ted and isolated (see, e.g., Cole et al., 1985, onal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boemer et al., 1991, J. Immunol, -95; and
US. Patent Nos. 5,750,373; 5,567,610; and 5,229,275). In some embodiments, the human antibody can
be selected from a phage library, where that phage library expresses human antibodies (Vaughan et al.,
1996, Nature Biotechnology, 14:309—3 14; Sheets et al., 1998, PNAS, 95:6157-6162; Hoogenboom and
Winter, 1991, J. Mol. Biol, 2272381; Marks et al., 1991, J. Mol. Biol, 222:581). Alternatively, phage
display technology can be used to produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable domain gene repertoires from unimmunized donors. Techniques for the
generation and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969,108;
6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068;
6,706,484; and 7,264,963; and Rothe et al., 2008, J. Mol. Bio., 376:1182-1200. Once antibodies are
identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling
(Marks et al., 1992, Bio/Technology, 102779—783) and site—directed mutagenesis, may be employed to
generate high affinity human antibodies.
In some embodiments, human dies can be made in transgenic mice that contain human
immunoglobulin loci. Upon immunization these mice are e of producing the full repertoire of
human antibodies in the absence of endogenous immunoglobulin production. This approach is described
in U.S. Patent Nos. 5,545,807; 5,545,806; 825; 5,625,126; 5,633,425; and 5,661,016.
This invention also encompasses bispecific antibodies. Bispecific antibodies are capable of
specifically izing and binding at least two different antigens or epitopes. The different epitopes can
either be within the same molecule (e. g., two epitopes on a single protein) or on ent molecules (e. g.,
one epitope on a protein and one epitope on a second protein). In some embodiments, a bispecific
antibody has enhanced potency as compared to an individual antibody or to a combination ofmore than
one antibody. In some ments, a ific antibody has reduced toxicity as compared to an
individual antibody or to a combination ofmore than one antibody. It is known to those of skill in the art
that any g agent (e.g., antibody) may have unique pharmacokinetics (PK) (e. g., circulating half-
life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active
binding agents wherein the two individual binding agents have different PK profiles. In some
embodiments, a ific dy has the ability to concentrate the actions of two binding agents (e.g.,
antibodies) in a common area (e. g., a tumor and/or tumor environment). In some embodiments, a
bispecific antibody has the ability to concentrate the actions of two binding agents (e. g., antibodies) to a
common target (e. g., a tumor or a tumor cell). In some embodiments, a bispecific antibody has the ability
to target the actions of two binding agents (e.g., antibodies) to more than one biological pathway or
function.
In certain embodiments, the bispecific antibody cally binds VEGF and a second target. In
certain embodiments, the bispecific dy specifically binds DLL4 and a second target. In certain
embodiments, the bispecific antibody specifically binds VEGF and DLL4. In some embodiments, the
bispecific antibody cally binds human VEGF and human DLL4. In some embodiments, the
bispecific dy is a monoclonal human or a humanized antibody. In some embodiments, the
bispecific antibody inhibits angiogenesis and reduces cancer stem cell number or frequency. In some
embodiments, the bispecific antibody inhibits blood vessel growth and inhibits blood vessel maturation.
In some embodiments, the ific antibody prevents endothelial roliferation. In some
embodiments, the bispecific antibody has decreased toxicity and/or side effects. In some embodiments,
the ific antibody has decreased toxicity and/or side effects as compared to a mixture of the two
individual antibodies or the antibodies as single agents. In some ments, the bispeciflc antibody has
an increased eutic index. In some embodiments, the bispecific antibody has an increased
therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single
agents.
In some embodiments, the bispecific antibody can specifically recognize and bind a first antigen
target, (e.g., DLL4) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g.,
CD2, CD3, CD28, or B7) or a PC receptor (e.g., CD64, CD32, or CD16) so as to focus cellular defense
mechanisms to the cell expressing the first antigen target. In some embodiments, the bispecific dies
can be used to direct cytotoxic agents to cells which express a particular target antigen. These antibodies
possess an antigen—binding site (e. g., to human DLL4) and a second site which binds a cytotoxic agent or
a radionuclide or, such as EOTUBE, DPTA, DOTA, or TETA.
Techniques for making bispecific antibodies are known by those skilled in the art, see for
example, Millstein et al., 1983, Nature, 305:537—539; Brennan et al., 1985, Science, 229:81; Suresh et al.,
1986, Methods in Enzymol., 121 : 120; Traunecker et al., 1991, EMBO J., 10:3655-3659; Shalaby et al.,
1992, J. Exp. Med, 175:217-225; Kostelny et al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994,
J. Immunol., 152:5368; U.S. Patent No. 5,731,168; International Publication No. ; and
US. Patent ation No. 2011/0123532. In some embodiments, the bispecific antibodies comprise
heavy chain constant regions with modifications in the amino acids which are part of the interface
between the two heavy chains. In some embodiments, the bispecific antibodies can be generated using a
“knobs-into-holes” strategy (see. e.g., US. Patent No. 5,731,168; Ridgway et. al., 1996, Prat. Engin,
9:617-621). At times the “knobs” and “holes” terminology is replaced with the terms “protuberances” and
“cavities”. In some embodiments, the bispecific antibodies may comprise variant hinge regions incapable
of g de linkages between the heavy chains (see, e.g., ). In some
embodiments, the modifications may comprise changes in amino acids that result in altered ostatic
interactions. In some embodiments, the modifications may comprise changes in amino acids that result in
altered hobic/hydrophilic interactions.
Bispecific antibodies can be intact antibodies or antibody fragments sing antigen-binding
sites. Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies
can be prepared (Tutt et al., 1991, J. l, 147:60). Thus, in certain embodiments the antibodies to
VEGF and/or DLL4 are multispecific.
In certain ments, the antibodies (or other ptides) described herein may be
monospecific. In certain embodiments, each of the one or more n-binding sites that an antibody
contains is capable of binding (or binds) a homologous epitope on different proteins.
In certain embodiments, the VEGF/DLL4—binding agent is an antibody fragment. Antibody
fragments may have ent functions or capabilities than intact antibodies; for example, antibody
nts can have increased tumor penetration. Various techniques are known for the production of
antibody fragments including, but not limited to, proteolytic digestion of intact antibodies. In some
embodiments, antibody fragments include a F(ab')2 nt produced by pepsin digestion of an antibody
molecule. In some embodiments, dy fragments include a Fab fragment generated by reducing the
disulfide bridges of an F(ab')2 fragment. In other embodiments, antibody fragments include a Fab
fragment generated by the treatment of the antibody molecule with papain and a reducing agent. In
certain embodiments, antibody fragments are produced recombinantly. In some embodiments, antibody
fragments e Fv or single chain Fv (scFv) fragments. Fab, Fv, and scFv antibody fragments can be
expressed in and secreted from E. coli or other host cells, ng for the production of large amounts of
these fragments. In some embodiments, antibody fragments are isolated from dy phage libraries as
discussed herein. For example, methods can be used for the construction of Fab sion libraries
(Huse et al., 1989, Science, 246: 1275—1281) to allow rapid and effective identification of monoclonal Fab
fragments with the desired specificity for VEGF and/or DLL4 or derivatives, fragments, analogs or
homologs thereof. In some embodiments, antibody fragments are linear antibody fragments. In certain
embodiments, antibody fragments are monospecific or bispecific. In certain embodiments, the
LL4-binding agent is a scFv. Various techniques can be used for the production of single-chain
dies specific to VEGF or DLL4 (see, e.g., U.S. Patent No. 4,946,778).
It can further be ble, especially in the case of dy fragments, to modify an antibody in
order to alter (e.g., increase or decrease) its serum half-life. This can be achieved, for example, by
incorporation of a salvage receptor g epitope into the antibody fragment by mutation of the
riate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then
fused to the dy nt at either end or in the middle (e.g., by DNA or peptide synthesis).
Heteroconjugate dies are also within the scope of the present invention. Heteroconjugate
antibodies are composed of two ntly joined antibodies. Such antibodies have, for example, been
proposed to target immune cells to unwanted cells (see, e.g., U.S. Patent No. 4,676,980). It is also
contemplated that the heteroconjugate antibodies can be ed in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins
can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of
suitable reagents for this purpose include iminothiolate and methylmercaptobutyrimidate.
For the purposes of the present invention, it should be appreciated that modified dies can
se any type of variable region that provides for the association of the antibody with the target (i.e.,
human VEGF or human DLL4). In this regard, the variable region may comprise or be derived from any
type of mammal that can be induced to mount a humoral response and generate immunoglobulins against
the desired antigen. As such, the variable region of the modified antibodies can be, for example, of
human, murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.) or rabbit origin. In some
embodiments, both the variable and constant regions of the modified globulins are human. In
other embodiments, the variable regions of compatible antibodies (usually derived from a non-human
source) can be engineered or specifically tailored to improve the binding properties or reduce the
immunogenicity of the molecule. In this respect, variable s usefiil in the present invention can be
humanized or otherwise altered through the inclusion of imported amino acid sequences.
In certain embodiments, the le domains in both the heavy and light chains are d by at
least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement
and sequence modification and/or tion. Although the CDRs may be d from an antibody of the
same class or even subclass as the antibody from which the framework regions are derived, it is envisaged
that the CDRs may be derived from an antibody of different class and often from an antibody from a
different species. It may not be necessary to replace all of the CDRs with all of the CDRs from the donor
variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may
only be necessary to transfer those residues that are required to maintain the activity of the antigen-
binding site.
Alterations to the le region notwithstanding, those skilled in the art will iate that the
modified antibodies of this invention will comprise antibodies (e. g., full-length antibodies or
immunoreactive fragments thereof) in which at least a fraction of one or more of the nt region
domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as
increased tumor localization or increased serum half—life when compared with an antibody of
approximately the same genicity comprising a native or unaltered constant region. In some
embodiments, the constant region of the modified antibodies will comprise a human constant region.
Modifications to the constant region compatible with this ion comprise additions, deletions or
substitutions of one or more amino acids in one or more domains. The d antibodies disclosed
herein may comprise alterations or modifications to one or more of the three heavy chain constant
domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, one
or more domains are lly or entirely deleted from the constant s of the modified antibodies. In
some ments, the modified antibodies will comprise domain deleted constructs or variants wherein
the entire CH2 domain has been removed (ACH2 ucts). In some embodiments, the omitted constant
region domain is replaced by a short amino acid spacer (e.g., 10 amino acid residues) that es some
of the molecular flexibility typically imparted by the absent constant region.
In some embodiments, the modified antibodies are engineered to fuse the CH3 domain directly to
the hinge region of the antibody. In other embodiments, a e spacer is inserted between the hinge
region and the modified CH2 and/or CH3 s. For example, constructs may be expressed wherein
the CH2 domain has been deleted and the ing CH3 domain (modified or unmodified) is joined to
the hinge region with a 5-20 amino acid spacer. Such a spacer may be added to ensure that the regulatory
elements of the constant domain remain free and accessible or that the hinge region remains e.
However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and
elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any
spacer added to the construct will be relatively non—immunogenic so as to maintain the d biological
qualities of the modified antibodies.
In some embodiments, the modified antibodies may have only a partial deletion of a constant
domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino
acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby
se cancer cell localization and/or tumor penetration. rly, it may be desirable to simply delete
the part of one or more constant region domains that l a specific effector function (e.g. complement
Clq binding) to be modulated. Such partial deletions of the constant regions may improve selected
characteristics of the antibody (serum half—life) while leaving other desirable functions associated with the
subject constant region domain intact. Moreover, as alluded to above, the constant regions of the
disclosed dies may be modified through the mutation or substitution of one or more amino acids
that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity
provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration
and immunogenic profile of the modified antibody. In certain embodiments, the modified dies
comprise the addition of one or more amino acids to the constant region to enhance desirable
characteristics such as decreasing or increasing or fimction or provide for more cytotoxin or
carbohydrate attachment sites.
It is known in the art that the constant region mediates l effector functions. For example,
binding of the Cl component of complement to the Fc region of IgG or IgM antibodies (bound to antigen)
activates the complement system. Activation of complement is important in the opsonization and lysis of
cell pathogens. The activation of complement also stimulates the inflammatory response and can also be
involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell
expressing a PC receptor (FcR). There are a number of Fc receptors which are specific for different
s of antibody, including IgG (gamma receptors), IgE on receptors), IgA (alpha receptors) and
IgM (mu receptors). Binding of antibody to Fc receptors on cell es triggers a number of important
and diverse biological responses ing ment and destruction of antibody-coated particles,
clearance of immune complexes, lysis of antibody—coated target cells by killer cells (called antibody-
dependent cell cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and l
of immunoglobulin production.
In certain embodiments, the modified antibodies e for altered effector functions that, in
turn, affect the biological profile of the administered antibody. For example, in some ments, the
deletion or inactivation (through point mutations or other means) of a constant region domain may reduce
Fc receptor binding of the circulating modified antibody thereby increasing cancer cell localization and/or
tumor penetration. In other embodiments, the constant region modifications se the serum ife
of the antibody. In other embodiments, the constant region modifications reduce the serum half-life of the
dy. In some embodiments, the constant region is modified to eliminate disulfide linkages or
oligosaccharide moieties. Modifications to the constant region in accordance with this invention may
easily be made using well known biochemical or molecular engineering techniques known to those of
skill in the art.
In certain embodiments, a VEGF/DLL4—binding agent that is an antibody does not have one or
more effector ons. For ce, in some embodiments, the antibody has no ADCC activity, and/or
no complement-dependent cytotoxicity (CDC) ty. In certain embodiments, the antibody does not
bind an Fc receptor, and/or ment factors. In certain embodiments, the antibody has no effector
function.
The present invention further embraces variants and equivalents which are substantially
gous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth
herein. These can contain, for example, conservative substitution mutations, i.e. the substitution of one or
more amino acids by similar amino acids. For example, conservative substitution refers to the substitution
of an amino acid with another amino acid within the same general class such as, for example, one acidic
amino acid with another acidic amino acid, one basic amino acid with another basic amino acid or one
neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid
substitution is well known in the art and described herein.
Thus, the present invention provides methods for producing an antibody that binds VEGF and/or
DLL4, including ific antibodies that specifically bind both VEGF and DLL4. In some
embodiments, the method for producing an antibody that binds VEGF and/or DLL4 comprises using
hybridoma techniques. In some ments, the method of generating an antibody that binds VEGF or
DLL4 or a bispecific antibody that binds VEGF and DLL4 comprises screening a human phage library.
The t invention further provides methods of identifying an antibody that binds VEGF and/or DLL4.
In some embodiments, the antibody is identified by FACS screening for binding to VEGF or a portion
thereof. In some embodiments, the antibody is identified by FACS screening for binding to DLL4 or a
n thereof. In some ments, the antibody is identified by FACS screening for binding to both
VEGF and DLL4 or a portion thereof. In some embodiments, the antibody is fied by screening
using ELISA for g to VEGF. In some embodiments, the dy is identified by screening using
ELISA for binding to DLL4. In some embodiments, the antibody is identified by screening using ELISA
for binding to VEGF and DLL4. In some embodiments, the antibody is identified by FACS screening for
blocking of binding of human VEGF to a human VEGF receptor. In some ments, the antibody is
identified by FACS screening for blocking of binding of human DLL4 to a human Notch receptor. In
some embodiments, the antibody is identified by screening for inhibition or blocking ofNotch signaling.
In some embodiments, the antibody is identified by screening for inhibition or ng ofVEGF activity
(e.g., induction of HUVEC proliferation). In some embodiments, the antibody is identified by ing
for modulation of angiogenesis.
In some embodiments, a method of generating an antibody to human VEGF comprises
immunizing a mammal with a polypeptide comprising amino acids 27-232 of human VEGF. In some
embodiments, a method of ting an antibody to human VEGF comprises immunizing a mammal
with a polypeptide comprising at least a portion of amino acids 27-232 of human VEGF. In some
embodiments, the method further comprises isolating antibodies or antibody-producing cells from the
mammal. In some ments, a method of generating a monoclonal antibody which binds VEGF
comprises: immunizing a mammal with a ptide comprising at least a n of amino acids 27-232
of human VEGF, and isolating antibody-producing cells from the immunized mammal. In some
embodiments, the method r comprises fusing the antibody-producing cells with cells of a a
cell line to form hybridoma cells. In some embodiments, the method further comprises selecting a
oma cell expressing an dy that binds VEGF. In certain embodiments, the mammal is a
mouse. In some embodiments, the antibody is selected using a polypeptide comprising at least a portion
of amino acids 27-232 of human VEGF.
In some embodiments, a method of generating an antibody to human DLL4 comprises
immunizing a mammal with a polypeptide comprising amino acids 27-529 of human DLL4. In some
embodiments, a method of generating an antibody to human DLL4 comprises immunizing a mammal with
a polypeptide comprising at least a portion of amino acids 2?—529 of human DLL4. In some
embodiments, a method of generating a monoclonal antibody which binds DLL4 comprises: immunizing
a mammal with a polypeptide comprising at least a portion of amino acids 27-529 of human DLL4, and
isolating antibody producing cells from the immunized mammal. In some ments, the method
further comprises fusing the antibody-producing cells with cells of a myeloma cell line to form hybridoma
cells. In some embodiments, the method further comprises selecting a hybridoma cell expressing an
antibody that binds DLL4. In certain embodiments, the mammal is a mouse. In some embodiments, the
antibody is selected using a polypeptide comprising at least a portion of amino acids 27-529 of human
DLL4.
In some embodiments, a method of generating an dy to human VEGF comprises screening
an antibody-expressing library for antibodies that bind human VEGF. In some embodiments, a method of
generating an antibody human DLL4 ses screening an antibody-expressing library for antibodies
that bind human DLL4. In some embodiments, a method of generating an antibody to human VEGF
and/or human DLL4 comprises ing an antibody—expressing library for bispecific antibodies that
bind human VEGF and human DLL4. In some embodiments, the antibody-expressing y is a phage
library. In some embodiments, the screening comprises panning. In some embodiments, the antibody-
expressing library (e.g., a phage library) is screened using at least a portion of amino acids 27-232 of
human VEGF. In some embodiments, antibodies identified in the first screening, are screened again using
at least a portion of amino acids 27-529 of human DLL4 to identify a bispecific antibody that binds VEGF
and DLL4. In some ments, the antibody-expressing library (e. g., a phage library) is ed using
at least a portion of amino acids 27-529 of human DLL4. In some embodiments, antibodies identified in
the first screening, are screened again using at least a portion of amino acids 27-232 of human VEGF to
identify a bispecific antibody that binds VEGF and DLL4. In some embodiments, the antibody identified
in the screening is a VEGF antagonist. In some embodiments, the antibody identified in the screening
ts biological activities induced by VEGF. In some embodiments, the antibody identified in the
screening is a DLL4 antagonist. In some embodiments, the antibody identified in the screening inhibits
Notch ing d by DLL4. In some embodiments, the antibody fied in the screening binds
both human VEGF and mouse VEGF. In some embodiments, the antibody identified in the screening
binds both human DLL4 and mouse DLL4.
In certain ments, the antibodies described herein are ed. In certain embodiments, the
antibodies described herein are substantially pure.
In some embodiments of the present invention, the VEGF/DLL4-binding agents are polypeptides.
The polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, that bind VEGF and/or DLL4. It will be recognized in the
art that some amino acid sequences of the binding agents described herein can be varied without
significant effect on the structure or function of the protein. Thus, the invention further includes
variations of the polypeptides which show substantial activity or which include regions of an antibody, or
fragment thereof, against human VEGF andfor DLL4. In some embodiments, amino acid sequence
variations of VEGF/DLL4-binding polypeptides include deletions, insertions, inversions, repeats, and/or
other types of substitutions.
In some embodiments, the polypeptides described herein are isolated. In some embodiments, the
ptides described herein are ntially pure.
The polypeptides, analogs and variants thereof, can be r modified to contain additional
chemical moieties not normally part of the polypeptide. The derivatized moieties can improve or
ise modulate the solubility, the biological half—life, and/or absorption of the polypeptide. The
es can also reduce or eliminate undesirable side effects of the polypeptides and variants. An
overview for chemical moieties can be found in Remington: The Science and Practice ofPharmacy, 21”
Edition, 2005, University of the Sciences, Philadelphia, PA.
The polypeptides described herein can be produced by any suitable method known in the art.
Such methods range from direct protein synthesis methods to ucting a DNA sequence encoding
polypeptide sequences and sing those sequences in a suitable host. In some embodiments, a DNA
sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence
encoding a wild-type protein of st. Optionally, the sequence can be mutagenized by site-specific
mutagenesis to provide functional analogs thereof. See, e.g., Zoeller et al., 1984, PNAS, 81:5662-5066
and US Patent No. 4,588,585.
In some embodiments, a DNA sequence ng a polypeptide of interest may be constructed by
chemical synthesis using an ucleotide synthesizer. Oligonucleotides can be designed based on the
amino acid sequence of the desired polypeptide and selecting those codons that are d in the host cell
in which the recombinant polypeptide of st will be produced. Standard methods can be applied to
synthesize a polynucleotide sequence encoding an isolated polypeptide of st. For example, a
complete amino acid sequence can be used to construct a back—translated gene. Further, a DNA oligomer
containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For
example, l small Oligonucleotides coding for portions of the d polypeptide can be synthesized
and then ligated. The individual Oligonucleotides typically n 5' or 3' overhangs for complementary
assembly.
Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide
sequences encoding a particular polypeptide of interest can be ed into an expression vector and
operatively linked to an expression control sequence appropriate for expression of the protein in a desired
host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or
expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to
obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to
riptional and translational expression control sequences that are functional in the chosen expression
host.
In certain embodiments, inant expression vectors are used to amplify and express DNA
encoding antibodies, or nts thereof, against human VEGF and/or DLL4. For example, recombinant
sion vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA
fragments encoding a polypeptide chain of a VEGF/DLL4-binding agent, such as an anti-VEGF antibody
or an anti-DLL4 antibody, or fragment thereof, ively linked to suitable transcriptional and/or
translational tory elements derived from mammalian, microbial, viral, or insect genes. A
transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a
regulatory role in gene expression, for example, transcriptional promoters or ers, (2) a structural or
coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate
transcription and translation initiation and termination sequences. Regulatory elements can include an
operator sequence to control transcription. The ability to replicate in a host, usually red by an
origin of replication, and a selection gene to facilitate recognition of transformants can additionally be
incorporated. DNA regions are “operatively linked” when they are functionally related to each other. For
example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it
is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively
linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is
operatively linked to a coding sequence if it is positioned so as to permit ation. In some
embodiments, structural elements intended for use in yeast expression systems include a leader sequence
enabling extracellular secretion of translated protein by a host cell. In other embodiments, in situations
where recombinant protein is sed without a leader or transport sequence, it can include an N-
terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed
recombinant protein to provide a final product.
The choice of an expression control sequence and an expression vector depends upon the choice
of host. A wide y of expression host/vector combinations can be employed. Useful sion
vectors for eukaryotic hosts include, for example, vectors sing expression control sequences from
SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial
hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9,
and their tives, and wider host range ds, such as M13 and other filamentous single-stranded
DNA phages.
The VEGF/DLL4-binding agents (e.g., polypeptides) of the present invention can be sed
from one or more vectors. For example, in some embodiments, one heavy chain polypeptide is expressed
by one vector, a second heavy chain polypeptide is expressed by a second vector and a light chain
polypeptide is expressed by a third vector. In some embodiments, a first heavy chain polypeptide and a
light chain polypeptide is expressed by one vector and a second heavy chain polypeptide is sed by a
second vector. In some embodiments, two heavy chain ptides are expressed by one vector and a
light chain ptide is expressed by a second vector. In some embodiments, three polypeptides are
sed from one vector. Thus, in some embodiments, a first heavy chain polypeptide, a second heavy
chain polypeptide, and a light chain polypeptide are expressed by a single vector.
Suitable host cells for sion of a VEGF/DLL4-binding polypeptide or antibody (or a VEGF
or DLL4 protein to use as an antigen) include prokaryotes, yeast cells, insect cells, or higher eukaryotic
cells under the l of appropriate promoters. Prokaryotes e gram-negative or ositive
organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of
mammalian origin as described below. Cell—free translation systems may also be ed. Appropriate
cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are
described in Pouwels et al., 1985, Cloning Vectors: A Laboratory Manual, ElseVier, New York, NY.
onal information regarding methods of protein production, ing antibody production, can be
found, e.g., in US. Patent Publication No. 2008/0187954; US. Patent Nos. 6,413,746; 6,660,501; and
International Patent Publication No. WO 04f009823.
Various mammalian or insect cell e systems may be used to express recombinant
polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these
proteins are generally correctly folded, appropriately d, and biologically onal. Examples of
le mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929
(murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived),
CHO (Chinese hamster ovary—derived), HeLa (human cervical cancer-derived), BHK (hamster kidney
fibroblast-derived), HEK-293 (human embryonic kidney—derived) cell lines and variants of these cell
lines. Mammalian expression vectors can comprise non—transcribed elements such as an origin of
replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking
non-transcribed sequences, and 5' or 3' non—translated sequences, such as necessary ribosome binding
sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
Expression of recombinant proteins in baculovirus also offers a robust method for producing correctly
folded and biologically functional proteins. Baculovirus s for production of logous proteins
in insect cells are well-known to those of skill in the art (see, e.g., Luckow and Summers, 1988,
Bio/Technology, 6:47).
Thus, the present invention provides cells comprising the VEGF/DLL4-binding agents described
herein. In some embodiments, the cells produce the VEGF/DLL4-binding agents described herein. In
certain embodiments, the cells produce an antibody. In some embodiments, the cells produce a VEGF-
binding agent, such as an anti-VEGF antibody. In some embodiments, the cells produce a bispeciflc
antibody that binds VEGF. In some embodiments, the cells produce a DLL4-binding agent, such as an
anti-DLL4 antibody. In some embodiments, the cells produce a ific antibody that binds DLL4. In
certain embodiments, the cells produce a bispecific VEGF/DLL4—binding agent, such as a bispecific
dy that binds VEGF and DLL4. In certain embodiments, the cells produce antibody 219R45. In
certain embodiments, the cells produce dy 21R79. In certain ments, the cells produce
dy 21R75. In certain embodiments, the cells produce antibody 21R83. In certain embodiments, the
cells produce a bispeciflc antibody which comprises an n-binding site from dy . In
certain ments, the cells produce a bispecific antibody which comprises an antigen-binding site
from antibody 21R79. In certain embodiments, the cells produce a bispecific antibody which ses
an antigen-binding site from antibody 21R75. In certain embodiments, the cells produce a bispecific
antibody which ses an antigen-binding site from antibody 21R83. In certain embodiments, the
cells produce a bispecific antibody which comprises an antigen-binding site from antibody 219R45 and an
antigen-binding site from antibody 21R79. In certain embodiments, the cells produce a bispecific
dy which comprises an antigen-binding site from antibody 219R45 and an antigen-binding site from
dy 21M18. In certain embodiments, the cells produce a ific antibody which comprises an
antigen-binding site from antibody 219R45 and an antigen—binding site from dy 21R75. In certain
embodiments, the cells produce a bispecific antibody which comprises an n-binding site from
antibody 219R45 and an antigen-binding site from antibody 21R83. In certain embodiments, the cells
e the ific antibody 219R45-MB-21M18. In certain embodiments, the cells produce the
bispecific antibody 219R45-MB-21R79. In certain embodiments, the cells produce the bispecific
antibody 219R45-MB-21R75. In n embodiments, the cells produce the bispecific antibody 219R45-
The proteins produced by a transformed host can be purified according to any suitable method.
Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any other standard technique for protein
purification. Affinity tags such as hexa—histidine, maltose g domain, influenza coat sequence, and
glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an
appropriate affinity column. y chromatography used for purifying immunoglobulins can include
n A, n G, and Protein L chromatography. Isolated proteins can be physically characterized
using such techniques as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS),
nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high mance liquid chromatography
(HPLC), and x-ray crystallography. The purity of ed proteins can be determined using techniques
known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel
electrophoresis, IEF, and capillary isoelectric focusing (cIEF).
In some embodiments, supernatants from expression systems which secrete recombinant protein
into culture media can be first concentrated using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ltration unit. Following the concentration step, the
concentrate can be d to a suitable purification matrix. In some embodiments, an anion exchange
resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE)
groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed
in protein ation. In some embodiments, a cation exchange step can be employed. le cation
exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some
embodiments, a hydroxyapatite media can be employed, including but not limited to, ceramic
hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps ing
hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be
employed to further purify a recombinant protein (e.g., a VEGF/DLL4-binding agent). Some or all of the
foregoing purification steps, in various combinations, can be employed to provide a homogeneous
inant protein.
In some ments, heterodimeric proteins such as bispecific antibodies are purified according
the any of the methods described herein. In some embodiments, anti-VEGF/anti-DLL4 bispecific
antibodies are isolated and/or purified using at least one chromatography step. In some embodiments, the
at least one chromatography step comprises affinity chromatography. In some embodiments, the at least
one chromatography step further comprises anion exchange chromatography. In some ments, the
isolated and/or purified antibody product comprises at least 90% dimeric antibody. In some
embodiments, the isolated and/or purified antibody t comprises at least 95%, 96%, 97%, 98% or
99% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product
comprises about 100% heterodimeric antibody.
In some embodiments, recombinant protein produced in bacterial culture can be isolated, for
example, by initial extraction from cell pellets, followed by one or more concentration, salting-out,
aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final
purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by
any convenient method, including -thaw cycling, sonication, mechanical disruption, or use of cell
lysing agents.
Methods known in the art for purifying antibodies and other proteins also e, for example,
those described in U.S. Patent ation Nos. 2008/0312425; 2008/0177048; and 2009/0187005.
In certain embodiments, the VEGF/DLL4—binding agent is a ptide that is not an antibody.
A variety ofmethods for fying and ing non-antibody polypeptides that bind with high affinity
to a protein target are known in the art. See, e.g., Skerra, 2007, Curr. Opin. Biotechnol, 18:295-304;
Hosse et al., 2006, Protein Science, 15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol., 17:653-658;
Nygren, 2008, FEBSJ, 275:2668-76; and Skerra, 2008, FEBSJ, 275:2677-83. In n embodiments,
phage or mammalian cell display technology may be used to produce and/or identify a LL4-
binding polypeptide that is not an antibody. In certain embodiments, the ptide comprises a protein
scaffold of a type ed from the group consisting of n A, protein G, a lipocalin, a fibronectin
domain, an ankyrin consensus repeat , and thioredoxin.
In certain embodiments, the VEGF/DLL4-binding agents or antibodies can be used in any one of
a number of ated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms. In certain
embodiments, the antibodies can be used in a non-conjugated form to harness the subject’s natural defense
mechanisms including ment—dependent cytotoxicity and antibody-dependent cellular toxicity to
eliminate malignant or cancer cells.
In some embodiments, the VEGF/DLL4—binding agent (e.g., an antibody or polypeptide) is
conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent
including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C,
chlorambucil, ubicin or other intercalating agents. In some ments, the cytotoxic agent is an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but
not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain,
ricin A chain, abrin A chain, modeccin A chain, alpha—sarcin, Aleurites fordii proteins, dianthin ns,
Phytolaca americana proteins (PAPI, PAPII, and PAP—S), ica charantia inhibitor, curcin, crotin,
Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or
a radioconjugated antibody. A variety of radionuclides are available for the production of
radioconjugated antibodies including, but not d to, 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 1538m, 67Cu,
67Ga, 166Ho, 177Lu, 186Re, 188Re and 212Bi. Conjugates of an antibody and one or more small le
, such as calicheamicins, maytansinoids, trichothecenes, and CC1065, and the tives of these
toxins that have toxin activity, can also be used. Conjugates of an dy and cytotoxic agent can be
made using a variety of bifunctional protein—coupling agents including, but not limited to, N-
succinimidyl(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional tives of
imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes
(such as eldehyde), ido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)—ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
III. cleotides
In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides
that encode a polypeptide (or a fragment of a polypeptide) that specifically binds VEGF, DLL4, both
VEGF and DLL4. The term “polynucleotides that encode a polypeptide” encompasses a polynucleotide
which includes only coding sequences for the polypeptide, as well as a polynucleotide which includes
additional coding and/or non-coding sequences. For example, in some embodiments, the invention
provides a polynucleotide comprising a polynucleotide sequence that encodes an antibody to human
VEGF or encodes a fragment of such an antibody (e.g., a nt comprising the n-binding site).
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide sequence that
encodes an dy to human DLL4 or encodes a fragment of such an antibody (e. g., a fragment
comprising the antigen-binding site). The polynucleotides of the invention can be in the form ofRNA or
in the form of DNA. DNA includes cDNA, c DNA, and synthetic DNA; and can be double-
stranded or single-stranded, and if single—stranded can be the coding strand or non-coding (anti-sense)
strand.
In certain embodiments, the polynucleotide comprises a cleotide encoding a polypeptide
comprising a sequence selected from the group ting of SEQ ID N021, SEQ ID N022, SEQ ID
N0:3, SEQ ID N024, SEQ ID N025, SEQ ID N0:6, SEQ ID N027, SEQ ID N028, SEQ ID N0:9, SEQ
ID N0:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID
N0:49, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:62, SEQ ID N0:63, and SEQ ID
N0:64. In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide
comprising a sequence selected from the group consisting of SEQ ID N0z5, SEQ ID N026, SEQ ID
N027, SEQ ID N028, SEQ ID N029, SEQ ID N0:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:48,
SEQ ID N0249, SEQ ID N0256, SEQ ID NO:58, SEQ ID N0:62, and SEQ ID NO: 64. In some
embodiments, the polynucleotide comprises a polynucleotide sequence selected from the group ting
of SEQ ID N0229, SEQ ID N0:30, SEQ ID N0:3], SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34,
SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ
ID NO:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID NO:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, SEQ ID
N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID
N0:76, SEQ ID N077, and SEQ ID N0:78.
In certain embodiments, the polynucleotide comprises a polynucleotide haVing a nucleotide
sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least
about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a
cleotide comprising a sequence selected from the group consisting of SEQ ID N0229, SEQ ID
N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:55, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:66, SEQ ID N0:67, SEQ ID
N0:68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ ID N0276, SEQ ID N0:77, and SEQ ID N0:78. In certain embodiments,
the polynucleotide comprises a cleotide having a nucleotide sequence at least about 80% cal,
at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some
embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequence
selected from the group consisting of SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0239, SEQ ID N0240, SEQ ID NO:50, SEQ ID N0:51, SEQ ID N0:54, SEQ ID N0:68, SEQ
ID NO:69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0276, SEQ ID N0277, and SEQ ID N0:78. Also ed is a polynucleotide that
comprises a polynucleotide that hybridizes to SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID
N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0240, SEQ ID NO:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:66, SEQ ID
NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78. In certain
embodiments, the hybridization is under ions of high stringency.
In certain embodiments, the polynucleotides comprise the coding sequence for the mature
polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in sion
and secretion of a polypeptide from a host cell (e.g., a leader sequence which fimctions as a secretory
sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader
sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature
form of the ptide. The polynucleotides can also encode for a proprotein which is the mature protein
plus additional 5 ' amino acid residues. A mature protein having a prosequence is a proprotein and is an
inactive form of the protein. Once the prosequence is d an active mature protein remains.
In certain embodiments, the polynucleotides comprise the coding sequence for the mature
polypeptide fused in the same reading frame to a marker sequence that allows, for example, for
ation of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag
supplied by a pQE-9 vector to e for ation of the mature polypeptide fused to the marker in
the case of a bacterial host, or the marker ce can be a hemagglutinin (HA) tag derived from the
influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some
embodiments, the marker sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:45)
which can be used in conjunction with other affinity tags.
The present invention further relates to variants of the hereinabove described cleotides
encoding, for example, fragments, analogs, and/or derivatives.
In certain embodiments, the present invention provides polynucleotides comprising
polynucleotides having a nucleotide sequence at least about 80% identical, at least about 85% identical, at
least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%,
97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a VEGF/DLL4-
binding agent (e.g., an antibody), or fragment f, described herein.
As used herein, the phrase a polynucleotide having a nucleotide sequence at least, for example,
95% “identical” to a reference nucleotide ce is ed to mean that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include
up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words,
to obtain a polynucleotide having a nucleotide ce at least 95% identical to a reference nucleotide
sequence, up to 5% of the nucleotides in the reference sequence can be deleted or tuted with another
nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be
inserted into the nce sequence. These mutations of the reference sequence can occur at the 5' or 3'
terminal positions of the nce nucleotide sequence or anywhere n those terminal positions,
interspersed either individually among nucleotides in the nce ce or in one or more contiguous
groups within the reference sequence.
The polynucleotide variants can contain tions in the coding regions, non-coding regions, or
both. In some embodiments, a polynucleotide variant contains tions which produce silent
substitutions, additions, or deletions, but does not alter the properties or activities of the encoded
polypeptide. In some embodiments, a polynucleotide variant comprises silent tutions that results in
no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon
expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial
host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent
mutation in a non-coding or a coding region of the sequence.
In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or
expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is
produced to increase expression of the encoded ptide. In some embodiments, a polynucleotide
variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a
polynucleotide t has increased expression of the d polypeptide as compared to a parental
polynucleotide sequence. In some ments, a polynucleotide variant has sed expression of the
encoded polypeptide as compared to a parental polynucleotide sequence.
In some embodiments, at least one cleotide variant is produced (without changing the
amino acid sequence of the encoded polypeptide) to increase production of a heteromultimeric molecule.
In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid
sequence of the encoded polypeptide) to increase production of a bispeciflc antibody.
In certain embodiments, the polynucleotides are ed. In n embodiments, the
polynucleotides are substantially pure.
Vectors and cells comprising the polynucleotides described herein are also provided. In some
embodiments, an expression vector ses a polynucleotide molecule. In some embodiments, a host
cell comprises an expression vector sing the polynucleotide molecule. In some ments, a
host cell ses a polynucleotide molecule.
IV. Methods of use and pharmaceutical compositions
The -binding agents (including polypeptides and antibodies) of the invention that bind (e.g.,
specifically bind) VEGF and/or DLL4 are useful in a variety of applications including, but not d to,
therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are
useful for inhibiting VEGF activity, ting DLL4—induced Notch signaling, inhibiting tumor growth,
reducing tumor volume, reducing the frequency of cancer stem cells in a tumor, reducing the
tumorigenicity of a tumor, modulating angiogenesis, and/or inhibiting enesis. The methods of use
may be in vitro, ex vivo, or in vivo. In certain embodiments, a VEGF/DLL4-binding agent is an antagonist
of human VEGF. In certain embodiments, a VEGF/DLL4—binding agent is an antagonist of human DLL4.
In certain embodiments, a VEGF/DLL4-binding agent is an antagonist of both VEGF and DLL4.
In certain embodiments, the VEGFXDLL4—binding agents are used in the treatment of a disease
associated with angiogenesis, i.e. sed angiogenesis and/or aberrant angiogenesis. In n
embodiments, the disease is a disease dependent upon angiogenesis. In certain embodiments, the
VEGF/DLL4-binding agents are used in the treatment of disorders characterized by increased levels of
stem cells and/or progenitor cells.
The present invention provides methods for inhibiting growth of a tumor using the VEGF/DLL4-
binding agents or antibodies described herein. In certain embodiments, the method of inhibiting growth
of a tumor comprises ting a tumor cell with a VEGF/DLL4-binding agent (e.g., antibody) in vilro.
For example, an immortalized cell line or a cancer cell line is cultured in medium to which is added an
EGF antibody, an anti-DLL4 antibody, or an anti—VEGF/anti-DLL4 bispeciflc antibody to t
tumor cell growth. In some embodiments, tumor cells are ed from a patient sample such as, for
example, a tissue biopsy, pleural effusion, or blood sample and cultured in medium to which is added a
VEGF/DLL4-binding agent to inhibit tumor cell growth.
In some ments, the method of inhibiting growth of a tumor comprises ting a tumor
or tumor cells with a VEGF/DLL4-binding agent (e.g., dy) in vivo. In certain embodiments,
contacting a tumor or tumor cell with a VEGF/DLL4—binding agent is undertaken in an animal model.
For e, an anti-VEGF antibody, an anti—DLL4 antibody, or an EGF/anti-DLL4 bispeciflc
antibody may be administered to an immunocompromised host animal (e.g., NOD/SCID mice) which has
a tumor xenograft. In some embodiments, tumor cells and/or cancer stem cells are isolated from a patient
sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and injected into an
immunocompromised host animal (e.g., NOD/SCID mice) that is then administered a VEGF/DLL4-
binding agent to inhibit tumor cell growth. In some embodiments, the VEGF/DLL4-binding agent is
administered at the same time or shortly after introduction of tumorigenic cells into the animal to t
tumor growth (“preventative ). In some embodiments, the VEGF/DLL4—binding agent is
administered as a therapeutic after tumors have grown to a specified size (“therapeutic model”). In certain
embodiments, the VEGF/DLL4-binding agent is a bispecific antibody that specifically binds human
VEGF and human DLL4.
In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a
subject a therapeutically effective amount of a VEGF/DLL4—binding agent. In certain embodiments, the
subject is a human. In certain ments, the t has a tumor or has had a tumor which was
removed. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the
frequency of cancer stem cells in the tumor is reduced by stration of the VEGF/DLL4-binding
agent. The invention also provides a method of reducing the frequency of cancer stem cells in a tumor,
comprising contacting the tumor with an effective amount of a VEGF/DLL4-binding agent (e. g., an anti-
VEGF/anti-DLL4 bispecific antibody). In some embodiments, a method of reducing the frequency of
cancer stem cells in a tumor in a subject, comprises administering to the subject a therapeutically effective
amount of a LL4-binding agent.
In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor
selected from the group ting of colorectal tumor, colon tumor, pancreatic tumor, lung tumor,
ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma,
cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain ments, the
tumor is a colorectal tumor or a colon tumor. In certain embodiments, the tumor is an ovarian tumor. In
some ments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor.
In certain embodiments, the tumor is a breast tumor.
The present invention further provides methods for treating cancer comprising administering a
therapeutically effective amount of a VEGF/DLL4—binding agent to a subject. In some embodiments, the
VEGF/DLL4-binding agent binds VEGF, and inhibits or s growth of the cancer. In some
embodiments, the VEGF/DLL4-binding agent binds DLL4, and inhibits or s growth of the cancer.
In some embodiments, the VEGF/DLL4-binding agent is a bispecific antibody that binds VEGF and
DLL4, and inhibits or s grth of the cancer. In some embodiments, the VEGF/DLL4-binding
agent binds VEGF, interferes with VEGF/VEGF receptor interactions, and inhibits or reduces growth of
the cancer. In some embodiments, the VEGF/DLL4—binding agent binds DLL4, interferes with
DLL4/Notch interactions, and inhibits or reduces growth of the cancer. In some embodiments, the
VEGF/DLL4-binding agent binds both VEGF and DLL4, eres with VEGF/VEGF receptor
interactions and with otch interactions, and inhibits or reduces growth of the cancer. In some
embodiments, the VEGF/DLL4-binding agent binds DLL4, and reduces the frequency of cancer stem
cells in the cancer.
The t invention provides s of treating cancer comprising administering a
therapeutically effective amount of a VEGF/DLL4—binding agent to a subject (e.g., a subject in need of
treatment). In certain embodiments, the t is a human. In certain embodiments, the subject has a
cancerous tumor. In certain ments, the subject has had a tumor removed.
The subject’s cancer/tumor, may, in some embodiments, be refractory to certain treatment(s). As
a non-limiting example, the subj ect’s cancer (or tumor) may be efractory. In certain embodiments,
the subj ect’s cancer may be resistant to anti—VEGF therapy or anti-DLL4 therapy, or both.
In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal
cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate
cancer, gastrointestinal cancer, ma, cervical cancer, bladder cancer, glioblastoma, and head and
neck . In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is
colorectal cancer or colon cancer. In n embodiments, the cancer is atic cancer. In n
embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In
certain embodiments, the cancer is lung cancer. In some embodiments, the cancer is a hematologic cancer
such as leukemia or ma. In some embodiments, the leukemia or lymphoma is a B-cell leukemia or
lymphoma. In some embodiments, the leukemia or lymphoma is a T—cell leukemia or lymphoma. In
some embodiments the hematologic cancer is acute myelogenous leukemia, Hodgkin lymphoma, non-
ns’s lymphoma, acute lymphocytic leukemia, hairy cell leukemia, chronic lymphocytic leukemia,
multiple myeloma, ous T-cell lymphoma, or T-cell acute lymphoblastic ia.
The invention also provides methods of treating a disease or disorder in a subject, wherein the
disease or disorder is associated with angiogenesis. In some embodiments, the e or disorder is
associated with aberrant angiogenesis. In some embodiments, the disease or disorder is associated with
increased angiogenesis. Thus, the present invention es methods for modulating angiogenesis in a
subject, sing administering to the subject a therapeutically effective amount of any of the
VEGF/DLL4-binding agents described herein. In some embodiments, the VEGF/DLL4-binding agent is
an antibody that binds human VEGF. In some ments, the VEGF/DLL4-binding agent is an
antibody that binds human DLL4. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
antibody that binds human VEGF. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
antibody that binds human DLL4. In some embodiments, the VEGF/DLL4-binding agent is a bispecific
antibody that binds human VEGF and human DLL4.
Methods of treating a disease or disorder in a subject, wherein the disease or disorder is
terized by an increased level of stem cells and/or progenitor cells are further provided. In some
embodiments, the treatment methods comprise administering a therapeutically effective amount of a
VEGF/DLL4-binding agent, polypeptide, or antibody to the t.
In certain ments of any of the methods described herein, the VEGF/DLL4-binding agent is
a bispecific antibody that specifically binds human VEGF and human DLL4. In some embodiments, the
ific antibody comprises a first antigen—binding site that specifically binds human VEGF and a
second antigen-binding site that specifically binds human DLL4, wherein the first antigen-binding site
comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising
DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY
(SEQ ID NO: 19), and the second antigen—binding site comprises a heavy chain CDRl comprising
TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID
NO: 14), YISSYNGATNYNQKFKG (SEQ ID NO: 15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), or
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the ific antibody ses a first antigen—binding site that specifically binds human
VEGF and a second antigen-binding site that specifically binds human DLL4, wherein the first antigen-
binding site ses a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2
comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain CDR3 comprising
HYDDKYYPLMDY (SEQ ID NO:19), and the second antigen-binding site comprises a heavy chain
CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YIANYNRATNYNQKFKG (SEQ ID , and a heavy chain CDR3 comprising VGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen—binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
ments, the bispecific antibody comprises a first antigen—binding site that specifically binds human
VEGF and a second antigen-binding site that specifically binds human DLL4, wherein the first antigen-
binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO: 17), a heavy chain CDR2
comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 sing
HYDDKYYPLMDY (SEQ ID NO: 19), and the second antigen-binding site comprises a heavy chain
CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YISSYNGATNYNQKFKG (SEQ ID , and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
ments, the bispecific antibody comprises a first antigen-binding site that specifically binds human
VEGF and a second antigen-binding site that specifically binds human DLL4, wherein the first antigen-
binding site comprises a heavy chain CDRl comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2
comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain CDR3 comprising
HYDDKYYPLMDY (SEQ ID NO:19), and second antigen—binding site which comprises a heavy chain
CDRl comprising TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising
YIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second antigen-binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID N021), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In some
embodiments, the bispecific dy comprises a first antigen—binding site that specifically binds human
VEGF and a second antigen-binding site that cally binds human DLL4, wherein the first antigen-
binding site comprises a heavy chain CDRl sing NYWMH (SEQ ID NO: 17), a heavy chain CDR2
comprising DINPSNGRTSYKEKFKR (SEQ ID NO: 18), and a heavy chain CDR3 comprising
HYDDKYYPLMDY (SEQ ID NO: 19), and the second antigen-binding site comprises a heavy chain
CDRl comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising
YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY
(SEQ ID NO: 16); and wherein both the first and second n—binding sites comprise a light chain
CDRl comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS
(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID .
In certain embodiments of any of the methods described herein, the VEGF/DLL4 bispecific
antibody comprises a first heavy chain variable region having at least about 80% sequence identity to SEQ
ID NO:11, a second heavy chain variable region having at least about 80% sequence identity to SEQ ID
N019, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID N0264, and a first and a light chain variable region
having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the VEGF/DLL4
bispecific antibody comprises a first heavy chain variable region having at least about 80% sequence
identity to SEQ ID NO:11, a second heavy chain variable region having at least about 80% sequence
identity to SEQ ID N019, and a first and a second light chain variable region having at least 80%
sequence identity to SEQ ID NO:12. In some embodiments, the VEGF/DLL4 bispecific antibody
comprises a first heavy chain variable region having at least about 80% sequence identity to SEQ ID
NO:11, a second heavy chain variable region having at least about 80% sequence identity to SEQ ID
NO: 10, and a first and a second light chain variable region having at least 80% sequence identity to SEQ
ID NO: 12. In some embodiments, the VEGF/DLL4 bispecific antibody comprises a first heavy chain
le region having at least about 80% sequence ty to SEQ ID NO:11, a second heavy chain
le region having at least about 80% sequence ty to SEQ ID NO:58, and a first and a second
light chain variable region having at least 80% sequence identity to SEQ ID NO:12. In some
embodiments, the VEGF/DLL4 bispecific dy comprises a first heavy chain variable region having
at least about 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region having at
least about 80% sequence identity to SEQ ID NO:64, and a first and a second light chain le region
having at least 80% sequence ty to SEQ ID NO:12.
In some embodiments of any of the methods bed herein, the VEGF/DLL4-binding agent is
an antibody. In some embodiments, the LL4-binding agent is an anti-VEGF antibody. In some
embodiments, the anti-VEGF antibody is antibody . In some embodiments, the VEGF/DLL4-
binding agent is an anti—DLL4 antibody. In some embodiments, the anti-DLL4 antibody is antibody
21R79. In some embodiments, the LL4 antibody is antibody 21R75. In some embodiments, the
anti-DLL4 antibody is antibody 21R83. In some embodiments, the VEGF/DLL4-binding agent is a
bispecific antibody comprising an antigen—binding site from antibody 219R45. In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody comprising an antigen-binding site from dy
21R79. In some embodiments, the VEGF/DLL4—binding agent is a bispecific dy comprising an
antigen-binding site from antibody 21R75. In some embodiments, the VEGF/DLL4-binding agent is a
bispecific antibody comprising an n—binding site from antibody 21R83. In some embodiments, the
VEGF/DLL4-binding agent is a ific antibody comprising a first antigen-binding site from antibody
219R45 and a second antigen-binding site from antibody 21R?9. In some embodiments, the
VEGF/DLL4-binding agent is a bispecific antibody comprising a first antigen—binding site from antibody
219R45 and a second antigen-binding site from dy 21M18. In some embodiments, the
LL4-binding agent is a bispecific antibody comprising a first antigen-binding site from antibody
219R45 and a second antigen-binding site from antibody 21R75. In some ments, the
LL4-binding agent is a bispecific antibody comprising a first antigen-binding site from antibody
219R45 and a second antigen—binding site from antibody 21R83. In some embodiments, the
VEGF/DLL4-binding agent is the bispecific antibody 219R45—MB-21M18. In some embodiments, the
VEGF/DLL4-binding agent is the bispecific antibody 219R45—MB-21R79. In some ments, the
VEGF/DLL4-binding agent is the bispecific antibody 219R45—MB-21R75. In some embodiments, the
VEGF/DLL4-binding agent is the bispecific antibody 219R45-MB-21R83.
The present invention further provides pharmaceutical compositions comprising the binding
agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a
pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in inhibiting tumor
growth and/or treating cancer in a subject (e.g., a human patient).
In certain embodiments, the invention provides pharmaceutical compositions comprising
bispecific antibodies, wherein at least about 90%, at least about 95%, at least about 98%, at least about
99% of the antibodies in the ition are bispecific antibodies or heterodimeric antibodies. In certain
embodiments, the bispecific antibodies are IgG (e.g., IgG2 or IgG1) dies. In certain ments,
less than about 10%, less than about 5%, less than about 2% or less than about 1% of the total antibodies
in the compositions are monospecific antibodies or homodimeric antibodies. In certain embodiments, the
antibodies in the composition are at least about 98% heterodimeric.
In n embodiments, formulations are prepared for storage and use by combining a purified
dy or agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or
excipient). Suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers
such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants ing
ic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride,
hexamethonium de, benzalkonium chloride, benzethonium chloride, , butyl or benzyl alcohol,
alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-
cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as
serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, ine, asparagine, histidine, arginine, or lysine; carbohydrates such as
monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal
complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene
glycol (PEG). (Remington: The Science and Practice ofPharmacy, 21st n, 2005, sity of the
Sciences, Philadelphia, PA).
The pharmaceutical compositions of the present invention can be administered in any number of
ways for either local or systemic ent. Administration can be topical by epidermal or transdermal
patches, nts, s, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary by
inhalation or insufflation of s or aerosols, ing by nebulizer, intratracheal, and intranasal;
oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal,
intramuscular (e. g., injection or on), or intracranial (e.g., intrathecal or intraventricular).
The eutic formulation can be in unit dosage form. Such formulations include s, pills,
capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In
solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier.
Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid,
magnesium stearate, ium phosphate or gums, and diluents (e.g., water). These can be used to form a
solid preformulation composition containing a neous mixture of a compound of the present
invention, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition
is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the
ation or composition can be coated or otherwise compounded to provide a dosage form affording
the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition
covered by an outer component. Furthermore, the two components can be separated by an enteric layer
that serves to resist disintegration and permits the inner component to pass intact through the h or
to be delayed in release. A variety of materials can be used for such enteric layers or gs, such
materials include a number of polymeric acids and mixtures of polymeric acids with such materials as
shellac, cetyl alcohol and cellulose acetate.
The VEGF/DLL4-binding agents or antibodies described herein can also be entrapped in
microcapsules. Such apsules are prepared, for example, by coacervation techniques or by
interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-
(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for e,
liposomes, n microspheres, microemulsions, rticles and nanocapsules) or in macroemulsions
as described in Remington: The Science and Practice ofPharmacy, 21st Edition, 2005, University of the
Sciences in Philadelphia, PA.
In certain embodiments, pharmaceutical formulations include a VEGF/DLL4-binding agent (e.g.,
an antibody) of the present invention complexed with liposomes. Methods to produce liposomes are
known to those of skill in the art. For example, some liposomes can be generated by reverse phase
evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to
yield liposomes with the d diameter.
In certain embodiments, sustained—release preparations can be ed. Suitable examples of
sustained-release ations e semi-permeable matrices of solid hydrophobic polymers containing
a LL4-binding agent (e.g., an antibody), where the es are in the form of shaped articles
(e.g., films or microcapsules). Additional examples of sustained-release matrices include polyesters,
hydrogels such as poly(2—hydroxyethyl—methacrylate) or poly(vinyl alcohol), polylactides, copolymers of
L-glutamic acid and 7 ethyl—L—glutamate, non—degradable ethylene-vinyl acetate, degradable lactic acid-
ic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic
lycolic acid copolymer and leuprolide e), sucrose e isobutyrate, and poly-D-(-)
hydroxybutyric acid.
In certain embodiments, in addition to administering a VEGF/DLL4-binding agent (e.g., an
antibody), the method or treatment further comprises administering at least one additional therapeutic
agent. An additional therapeutic agent can be stered prior to, concurrently with, and/or
subsequently to, administration of the VEGF/DLL4—binding agent. Pharmaceutical compositions
comprising a VEGF/DLL4-binding agent and the additional therapeutic agent(s) are also ed. In
some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional
therapeutic agents.
Combination therapy with at least two therapeutic agents often uses agents that work by different
mechanisms of action, although this is not required. Combination therapy using agents with different
mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a
lower dose of each agent than is used in erapy, thereby reducing toxic side effects and/or
increasing the therapeutic index of at least one of the agents. Combination therapy may decrease the
likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises
a therapeutic agent that primarily affects (e.g., inhibits or kills) morigenic cells and a therapeutic
agent that primarily affects (e.g., inhibits or kills) tumorigenic CSCs.
Useful classes of therapeutic agents include, for e, bulin agents, atins, DNA
minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as
cisplatin, mono(platinum), bis(platinum) and tri—nuclear platinum xes and carboplatin),
cyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins,
etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine
antimetabolites, puromycins, ion sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca
alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an
antimetabolite, an totic, a topoisomerase inhibitor, or an angiogenesis inhibitor. In some
embodiments, the second eutic agent is a platinum x such as carboplatin or cisplatin. In
some embodiments, the additional therapeutic agent is a platinum complex in combination with a .
Therapeutic agents that may be administered in combination with the VEGF/DLL4-binding
agents include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves
the administration of an anti-VEGF-binding agent or antibody of the present invention in ation
with a chemotherapeutic agent or cocktail of multiple different herapeutic agents. In some
embodiments, the method or treatment involves the administration of an anti-DLL4-binding agent or
antibody of the t invention in ation with a herapeutic agent or cocktail of multiple
different chemotherapeutic agents. In some embodiments, the method or treatment involves the
administration of a bispecific antibody of the present invention that binds VEGF and DLL4 in
combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic .
Chemotherapeutic agents useful in the instant invention include, but are not limited to, alkylating
agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as an,
improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; en mustards
such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; ureas such as carmustine, chlorozotocin, fotemustine, lomustine,
nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, ine,
bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins,
dactinomycin, daunorubicin, bicin, 6—diazo—5—oxo—L—norleucine, doxorubicin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, enolic acid, nogalamycin, olivomycins,
ycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, atin, zorubicin; etabolites such as methotrexate and 5—fluorouracil (S-FU); folic
acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as bine,
azacitidine, 6-azauridine, carrnofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,
floxuridine, S-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as
folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; xate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;
gallium nitrate; hydroxyurea; lentinan; mine; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;
ran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
(Ara-C); taxoids, e. g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-
thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum;
ide ); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;
novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPTl l; topoisomerase inhibitor RFS
2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and
pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also
include anti-hormonal agents that act to regulate or t hormone action on tumors such as anti-
estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-
hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene (FARESTON); and
anti-androgens such as flutamide, mide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the
second therapeutic agent is tin. In certain embodiments, the second therapeutic agent is carboplatin.
In n embodiments, the second therapeutic agent is paclitaxel.
In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. omerase
inhibitors are chemotherapeutic agents that ere with the action of a topoisomerase enzyme (e.g.,
topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, bicin HCl,
daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, can HCl, teniposide (VM-26),
and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In
n embodiments, the second eutic agent is irinotecan.
In certain embodiments, the chemotherapeutic agent is an anti-metabolite. An anti-metabolite is a
chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet
different enough to interfere with one or more normal fimctions of cells, such as cell division. Anti-
metabolites include, but are not limited to, gemcitabine, fluorouracil, tabine, methotrexate sodium,
ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, S—azacytidine, aptopu1ine,
azathioprine, 6-thioguanine, tatin, fludarabine phosphate, and cladribine, as well as
pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the
second eutic agent is gemcitabine.
In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not
d to, agents that bind tubulin. In some embodiments, the agent is a . In certain embodiments,
the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel
or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE),
albumin-bound paclitaxel (ABRAXANE), DHA—paclitaxel, or PG-paclitaxel. In certain alternative
embodiments, the antimitotic agent comprises a vinca alkaloid, such as stine, binblastine,
vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives f. In some
embodiments, the antimitotic agent is an inhibitor of kinesin EgS or an inhibitor of a mitotic kinase such
as Aurora A or Plkl. In certain embodiments, where the chemotherapeutic agent administered in
combination with a VEGF/DLL4-binding agent is an anti—mitotic agent, the cancer or tumor being treated
is breast cancer or a breast tumor.
In some embodiments, a second therapeutic agent comprises an agent such as a small le.
For example, treatment can involve the combined stration of a VEGF/DLL4-binding agent (e.g. an
antibody) of the t ion with a small molecule that acts as an inhibitor against additional tumor-
associated ns including, but not limited to, EGFR, ErbB2, HERZ, and/or VEGF. In certain
embodiments, the second therapeutic agent is a small molecule that inhibits a cancer stem cell pathway.
In some embodiments, the second eutic agent is a small molecule inhibitor of the Notch pathway.
In some embodiments, the second therapeutic agent is a small molecule inhibitor of the Wnt pathway. In
some embodiments, the second therapeutic agent is a small molecule inhibitor of the BMP y. In
some embodiments, the second eutic agent is a small molecule that inhibits B-catenin signaling.
In some embodiments, a second therapeutic agent comprises a biological molecule, such as an
antibody. For example, treatment can involve the combined administration of a VEGF/DLL4-binding
agent (e. g. an antibody) of the present invention with other antibodies against additional tumor-associated
proteins including, but not limited to, antibodies that bind EGFR, ErbB2, HERZ, and/or VEGF. In certain
embodiments, the second therapeutic agent is an antibody that is an anti-cancer stem cell marker antibody.
In some embodiments, the second therapeutic agent is an dy that binds a component of the Notch
pathway. In some ments, the second therapeutic agent is an antibody that binds a ent of
the Wnt pathway. In certain embodiments, the second therapeutic agent is an antibody that ts a
cancer stem cell pathway. In some embodiments, the second therapeutic agent is an antibody tor of
the Notch pathway. In some embodiments, the second therapeutic agent is an antibody inhibitor of the
Wnt pathway. In some embodiments, the second therapeutic agent is an antibody inhibitor of the BMP
pathway. In some embodiments, the second therapeutic agent is an antibody that inhibits B-catenin
signaling. In certain embodiments, the second eutic agent is an antibody that is an angiogenesis
inhibitor or modulator (e.g., an anti-VEGF or VEGF receptor antibody). In certain ments, the
second therapeutic agent is bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), panitumumab
(VECTIBIX), or cetuximab (ERBITUX). Combined administration can include co-administration, either
in a single pharmaceutical formulation or using separate formulations, or consecutive stration in
either order but generally within a time period such that all active agents can exert their biological
activities simultaneously.
Furthermore, treatment with a VEGF/DLL4—binding agent described herein can include
combination ent with other biologic molecules, such as one or more cytokines (e.g., lymphokines,
eukins, tumor necrosis factors, and/0r growth factors) or can be accompanied by al removal of
tumors, cancer cells, or any other therapy deemed ary by a ng physician.
In certain embodiments, the treatment involves the administration of a VEGF/DLL4-binding
agent (e. g. an antibody) of the present invention in combination with ion therapy. Treatment with a
VEGF/DLL4-binding agent can occur prior to, rently with, or subsequent to administration of
radiation therapy. Dosing schedules for such radiation therapy can be determined by the d medical
practitioner.
It will be appreciated that the combination of a VEGF/DLL4-binding agent and an additional
therapeutic agent may be administered in any order or rently. Treatment with a VEGF/DLL4-
binding agent (e.g., an antibody) can occur prior to, concurrently with, or subsequent to administration of
chemotherapies. ed administration can include co—administration, either in a single
pharmaceutical formulation or using separate formulations, or consecutive administration in either order
but lly within a time period such that all active agents can exert their biological activities
simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used
according to manufacturers' instructions or as determined empirically by the skilled practitioner.
Preparation and dosing schedules for such chemotherapy are also bed in The Chemotherapy Source
Book, 4’17 Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
In some embodiments, the VEGF/DLL4—binding agent will be administered to patients that have
previously undergone treatment with a second therapeutic agent. In certain other embodiments, the
VEGF/DLL4-binding agent and a second therapeutic agent will be administered substantially
simultaneously or concurrently. For example, a subject may be given a VEGF/DLL4-binding agent (e.g.,
an antibody) while undergoing a course oftreatment with a second therapeutic agent (e.g., chemotherapy).
In certain embodiments, a VEGF/DLL4—binding agent will be administered within 1 year of the treatment
with a second therapeutic agent. In certain alternative embodiments, a LL4-binding agent will be
administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain
other embodiments, a VEGF/DLL4-binding agent will be administered within 4, 3, 2, or 1 weeks of any
treatment with a second therapeutic agent. In some embodiments, a VEGF/DLL4-binding agent will be
administered within 5, 4, 3, 2, or 1 days of any ent with a second therapeutic agent. It will further
be appreciated that the two (or more) agents or treatments may be stered to the subject within a
matter of hours or minutes (i.e., substantially simultaneously).
For the treatment of a disease, the appropriate dosage of an VEGF/DLL4-binding agent (e.g., an
antibody) of the present invention depends on the type of disease to be treated, the severity and course of
the disease, the responsiveness of the disease, whether the LL4-binding agent or antibody is
administered for eutic or preventative purposes, previous therapy, the patient’s clinical history, and
so on, all at the discretion of the treating physician. The VEGF/DLL4-binding agent or antibody can be
administered one time or as a series of treatments spread over several days to several months, or until a
cure is effected or a diminution of the e state is ed (e.g., reduction in tumor size). Optimal
dosing schedules can be calculated from measurements of drug accumulation in the body of the patient
and will vary depending on the relative potency of an individual antibody or agent. The stering
physician can determine optimum dosages, dosing methodologies, and repetition rates. In certain
ments, dosage of a VEGF/DLL4-binding agent or antibody is from about 0.01ug to about
100mg/kg of body weight, from about 0.1ug to about 100mg/kg of body weight, from about lug to about
100mg/kg of body weight, from about 1mg to about 100mg/kg of body weight, about 1mg to about
80mg/kg of body weight from about 10mg to about lOOmg/kg of body weight, from about 10mg to about
75mg/kg of body weight, or from about 10mg to about 50mg/kg ofbody . In n embodiments,
the dosage of the antibody or other VEGF/DLL4—binding agent is from about 0.1mg to about 20mg/kg of
body weight. In certain embodiments, dosage can be given once or more daily, weekly, monthly, or
yearly. In certain embodiments, the antibody or other VEGF/DLL4-binding agent is given once every
week, once every two weeks, once every three weeks, or once every month.
In some embodiments, a LL4-binding agent (e.g., an antibody) may be administered at
an initial higher “loading” dose, ed by one or more lower doses. In some embodiments, the
frequency of administration may also change. In some embodiments, a dosing regimen may comprise
administering an initial dose, followed by additional doses (or enance” doses) once a week, once
every two weeks, once every three weeks, or once every month. For example, a dosing regimen may
comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example,
one-half of the l dose. Or a dosing regimen may comprise administering an initial loading dose,
followed by maintenance doses of, for e one-half of the l dose every other week. Or a dosing
regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of,
for e, the same amount every other week. Or a dosing regimen may comprise administering an
initial dose followed by additional doses every 3 weeks or once a month. The treating physician can
estimate repetition rates for dosing based on measured nce times and concentrations of the drug in
bodily fluids or tissues. The progress of therapy can be monitored by conventional techniques and assays.
As is known to those of skill in the art, administration of any therapeutic agent may lead to side
effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude
stration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must
be tinued, and other agents may be tried. However, many agents in the same therapeutic class often
display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if
possible, suffer from the unpleasant side effects associated with the therapeutic agent.
Side effects from therapeutic agents may include, but are not limited to, hives, skin rashes,
g, nausea, vomiting, decreased appetite, diarrhea, chills, fever, fatigue, muscle aches and pain,
headaches, low blood pressure, high blood pressure, hypokalemia, low blood counts, bleeding, and
cardiac problems.
Thus, one aspect of the present invention is directed to methods of treating cancer in a patient
comprising administering an anti-VEGFfanti—DLL4 bispecific antibody using an intermittent dosing
regimen, which may reduce side effects andfor toxicities associated with administration of the anti-
VEGF/anti-DLL4 bispecific antibody. As used herein, mittent dosing” refers to a dosing regimen
using a dosing interval of more than once a week, e.g., dosing once every 2 weeks, once every 3 weeks,
once every 4 weeks, etc. In some ments, a method for treating cancer in a human patient
comprises stering to the patient an effective dose of an anti-VEGF/anti-DLL4 bispecific antibody
according to an intermittent dosing regimen. In some embodiments, a method for treating cancer in a
human patient comprises administering to the patient an ive dose of an anti-VEGF/anti-DLL4
bispecific antibody according to an intermittent dosing regimen, and increasing the therapeutic index of
the anti-VEGF/anti-DLL4 bispecific antibody. In some embodiments, the intermittent dosing regimen
comprises stering an initial dose of an anti—VEGF/anti—DLL4 ific antibody to the patient, and
administering subsequent doses of the anti-VEGF/anti—DLL4 bispecific antibody about once every 2
weeks. In some embodiments, the intermittent dosing regimen comprises administering an initial dose of
an anti-VEGF/anti-DLL4 bispecific antibody to the t, and stering subsequent doses of the
anti-VEGF/anti-DLL4 bispecific antibody about once every 3 weeks. In some embodiments, the
ittent dosing regimen comprises administering an initial dose of an anti-VEGF/anti-DLL4
if1c antibody to the patient, and administering subsequent doses of the EGF/anti-DLL4
bispeciflc antibody about once every 4 weeks.
In some embodiments, the subsequent doses in an intermittent dosing regimen are about the same
amount or less than the initial dose. In other embodiments, the subsequent doses are a greater amount
than the initial dose. As is known by those of skill in the art, doses used will vary depending on the
clinical goals to be achieved. In some embodiments, the initial dose is about 0.25mg/kg to about
g. In some embodiments, the initial dose is about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20mg/kg. In certain embodiments, the initial dose is about 0.5mg/kg. In certain
embodiments, the initial dose is about 1mg/kg. In certain embodiments, the initial dose is about
2.5mg/kg. In certain embodiments, the initial dose is about 5mg/kg. In n embodiments, the initial
dose is about 7.5mg/kg. In certain embodiments, the initial dose is about g. In certain
embodiments, the l dose is about 12.5mg/kg. In certain embodiments, the initial dose is about
15mg/kg. In certain embodiments, the l dose is about 20mg/kg. In some embodiments, the
subsequent doses are about 0.25mg/kg to about 15mg/kg. In certain embodiments, the subsequent doses
are about 0.5, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15mg/kg. In certain embodiments, the
subsequent doses are about 0.5mg/kg. In certain embodiments, the uent doses are about 1mg/kg.
In certain embodiments, the subsequent doses are about kg. In certain embodiments, the
subsequent doses are about 5mg/kg. In some embodiments, the subsequent doses are about 7.5mg/kg. In
some embodiments, the subsequent doses are about lOmg/kg. In some embodiments, the subsequent
doses are about 12.5mg/kg.
In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient
an initial dose of an anti-VEGF/anti-DLL4 bispecific antibody of about 2.5mg/kg and (b) stering
subsequent doses of about 2.5 mg/kg once every 2 weeks. In some ments, the intermittent dosing
regimen comprises: (a) administering to the patient an l dose of an anti-VEGF/anti-DLL4 bispecific
antibody of about 5mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 2 weeks.
In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an
initial dose of an anti-VEGF/anti—DLL4 ific dy of about 2.5mg/kg and (b) administering
subsequent doses of about 2.5 mg/kg once every 3 weeks. In some embodiments, the intermittent dosing
regimen comprises: (a) administering to the t an initial dose of an EGF/anti-DLL4 bispecifrc
antibody of about 5mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 3 weeks.
In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an
l dose of an anti-VEGF/anti-DLL4 bispecific antibody of about 2.5mg/kg and (b) administering
subsequent doses of about 2.5 mg/kg once every 4 weeks. In some embodiments, the intermittent dosing
regimen comprises: (a) stering to the patient an initial dose of an anti-VEGF/anti-DLL4 bispecifrc
antibody of about 5mg/kg and (b) administering subsequent doses of about 5 mg/kg once every 4 weeks.
In certain embodiments, the initial dose and the maintenance doses are different, for example, the initial
dose is about 5mg/kg and the subsequent doses are about 2.5mg/kg. In certain embodiments, an
intermittent dosing regimen may comprise a g dose, for example, the initial dose is about 20mg/kg
and the subsequent doses are about 2.5mgfkg or about 5mg/kg administered once every 2 weeks, once
every 3 weeks, or once every 4 weeks.
Another aspect of the present invention is directed to methods for reducing toxicity of an anti-
VEGF/anti-DLL4 bispecific antibody in a human patient comprises administering to the t the anti-
nti-DLL4 bispecifrc antibody using an intermittent dosing regimen. Another aspect of the present
invention is directed to methods for reducing side effects of an anti-VEGF/anti-DLL4 bispecific antibody
in a human patient comprises administering to the patient the anti-VEGF/anti-DLL4 bispecific antibody
using an intermittent dosing regimen. Another aspect of the t invention is directed to methods for
increasing the therapeutic index of an EGF/anti—DLL4 bispecifrc antibody in a human patient
comprises administering to the patient the anti—VEGF/anti—DLL4 ific antibody using an intermittent
dosing regimen.
The choice of delivery method for the initial and subsequent doses is made ing to the
ability of the animal or human patient to tolerate introduction of the anti-VEGF/anti-DLL4 bispecific
antibody into the body. Thus, in any ofthe s and/or embodiments described herein, the
administration of the anti-VEGF/anti-DLL4 bispecific antibody may be by intravenous injection or
enously. In some embodiments, the administration is by intravenous infusion. In any of the aspects
and/or embodiments described herein, the administration of the anti—VEGF/anti-DLL4 bispecific antibody
may be by a non-intravenous route.
V. Kits comprising VEGF/DLL4-binding agents
The present invention provides kits that comprise the VEGF/DLL4-binding agents (e.g.,
antibodies) described herein and that can be used to perform the methods described herein. In certain
embodiments, a kit ses at least one purified antibody against VEGF and/or DLL4 in one or more
containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to
perform a detection assay, ing all controls, ions for performing assays, and any necessary
software for analysis and tation of results. One skilled in the art will readily recognize that the
disclosed VEGF/DLL4-binding agents of the present invention can be readily incorporated into one of the
established kit formats which are well known in the art.
Further provided are kits comprising a VEGF/DLL4-binding agent (e.g., an anti-VEGF/anti-
DLL4 bispecific antibody), as well as at least one onal therapeutic agent. In certain embodiments,
the second (or more) therapeutic agent is a chemotherapeutic agent. In certain embodiments, the second
(or more) therapeutic agent is an angiogenesis inhibitor.
Embodiments of the present disclosure can be further defined by reference to the following non-
limiting examples, which describe in detail preparation of certain antibodies of the t sure and
s for using antibodies of the present disclosure. It will be apparent to those skilled in the art that
many modifications, both to materials and methods, may be practiced without departing from the scope of
the present disclosure.
EXAMPLES
Example 1
g affinities of anti-VEGF/anti-DLL4 antibodies
The KDs of parental antibodies anti—VEGF 219R45 (IgG format), anti-DLL4 21R79 (IgG format),
LL4 21M18 (IgG format) and bispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79
were determined using a Biacore 2000 system from e LifeSciences (GE Healthcare). Recombinant
human DLL4-Fc or mouse DLL4-Fc proteins were immobilized on CMS carboxyl chips using standard
amine-based try (NHS/EDC) and blocked with ethanolamine. Recombinant human VEGF165 or
mouse VEGF165 were biotinylated and immobilized on streptavidin chips. The antibodies were serially
diluted 2-fold from 100nM to 0.78nM in HBS—P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% v/v
Polysorbate 20). For each antibody, all 8 dilutions were sequentially injected over a c chip.
Kinetic data were collected over time and were fit using the simultaneous global fit equation to yield
affinity constants (KD values) for each bispecific antibody.
Table 3
219R45-MB-21M18 0.36 . 16
219R45-MB-21R79 0.68 . .
As shown in Table 3, ific antibody 219R45—MB-21M18 had an affinity constant (KD) for
human VEGF of 0.36nM and a KD for human DLL4 of 16nM. Bispecific dy 219R45-MB-21R79
had a KD for human VEGF of 0.68nM and a KD for human DLL4 of 0.53nM. Both bispecific antibodies
demonstrated weaker binding to mouse VEGF as compared to human VEGF and r antibody bound
mouse DLL4. Thus, both bispecific antibodies trated similar binding affinity to human VEGF and
219R45-MB-21R79 demonstrated approximately 30—fold stronger binding to human DLL4 than 219R45-
MB-21M18. Furthermore, bispecific antibody 219R45-MB—21R79 had a similar g affinity to
human VEGF despite the fact the bispecific antibody is monovalent for VEGF as compared to the bivalent
parental antibody.
Several additional anti-DLL4 antibodies were identified that had g affinities intermediate to
the KDs of 21M] 8 and 21R79. Two of these anti—DLL4 antibodies were used to produce anti-VEGF/anti-
DLL4 ific antibodies 219R45-MB—21R75 and 219R45—MB—21R83. Using the e 2000 system
as described above, the KDs of the bispecific antibodies 219R45—MB—21R75 and 219R45-MB-21R83 to
human DLL4 were determined. A comparison of the binding affinity to human DLL4 of these four anti-
VEGF/anti-DLL4 bispecific antibodies is shown in Table 4.
The CDRs for anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18, 219R45-MB-
21R79, 219R45-MB—21R75, and 219R45—MB—21R83 are shown in Figure 1A. The heavy chain and light
chain variable region SEQ ID NOs are shown in Figure 1B and the heavy chain and light chain SEQ ID
NOs (with and t signal sequence) are shown in Figure 1C.
EGF/anti-DLL4 bispecific antibody 219R45-MB-21M18 comprises a (a) heavy chain
encoded by the DNA comprising SEQ ID NO:75 deposited with American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty
on September 21, 2012 and assigned designation number PTA— a heavy chain encoded by the
, (b)
DNA comprising SEQ ID NO:33 deposited with ATCC under the conditions of the Budapest Treaty on
September 21, 2012 and ed designation number PTA— and (c) a light chain encoded by the
DNA comprising SEQ ID NO:34 deposited with ATCC under the conditions of the st Treaty on
September 21, 2012 and assigned designation number PTA-_ .
Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R79 comprises a (a) heavy chain
encoded by the DNA comprising SEQ ID NO:31 deposited with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned designation number PTA- a heavy chain
, (b)
encoded by the DNA comprising SEQ ID NO:33 ted with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned ation number PTA- and (c) a light
chain encoded by the DNA comprising SEQ ID NO:34 deposited with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned designation number PTA-—
Anti-VEGF/anti-DLL4 bispecific antibody —MB-21R83 comprises (a) a heavy chain
encoded by the DNA comprising SEQ ID NO:72 ted with ATCC under the ions of the
Budapest Treaty on and assigned designation number PTA- a heavy chain encoded by
, (b)
the DNA comprising SEQ ID NO:33 deposited with ATCC under the conditions of the Budapest Treaty
on September 21, 2012 and assigned designation number PTA- and (c) a light chain encoded by the
DNA comprising SEQ ID NO:34 deposited with ATCC under the conditions of the Budapest Treaty on
September 21, 2012 and assigned designation number PTA-_ .
Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R75 comprises (a) a heavy chain
encoded by the DNA sing SEQ ID NO:74 deposited with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned ation number PTA- a heavy chain
, (b)
encoded by the DNA comprising SEQ ID NO:33 deposited with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned designation number PTA- and (c) a light
chain encoded by the DNA comprising SEQ ID NO:34 deposited with ATCC under the conditions of the
Budapest Treaty on September 21, 2012 and assigned designation number PTA-
Table 4
Antibody Heavy chain CDR2 hDLL4 (nM)
Y I S S YNGATNYNQKFKG
219R45-MB-21M18 16.00
SEQ ID NO:15
YIANYNRATNYNQKFKG
219R45-MB-21R79
SEQ ID NO:14
YIAGYKDATNYNQKFKG
YISNYNRATNYNQKFKG
Example 2
HTRF Assay for simultaneous binding of bispecific dies to human VEGF and human DLL4
To characterize the binding capabilities of certain antibodies and/or antibody mixtures to both
VEGF and DLL4, homogeneous time resolved fluorescence (HTRF) assays were performed. Antibodies
tested were anti-VEGF/anti-DLL4 bispecific antibodies -MB-21M18 and -MB-21R79,
parental antibodies 219R45 (anti-VEGF), 21M18 (anti-DLL4), 21R79 (anti-DLL4), a combination of
219R45 and 21M18, or a ation of 219R45 and 21R79. The antibodies or dy mixtures were
serially diluted 2-fold from 3000nM to 2.9nM in binding buffer (1X PBS, 0.1% gelatin, 0.1% Polysorbate
, 400mM potassium fluoride) and placed in a white 96—well plate. An equal volume of on
containing 4ug/ml of d2-labeled hDLL4—F0 and 21.4ng/ml Europium crypate-labeled hVEGF165 was
added to each well for a final volume of 100p] (final concentrations of acceptor and donor fluorophores
were 2ug/ml and 10.7ng/ml, respectively). The assay plates were incubated for 2 hours to overnight and
read on a SpectraMax M5e Microplate reader (Molecular Devices, Sunnyvale CA) at an excitation
wavelength of 314nm.
As shown in Figure 2, anti-VEGF/anti-DLL4 bispeciflc antibodies 219R45-MB-21M18 and
219R45-MB-21R79, were able to bind both hVEGF and hDLL4 simultaneously. antly, neither of
the combinations of the parental dies (i.e., 219R45 and 21M18 or 219R45 and 21R79) was able to
bind VEGF and DLL4 simultaneously. These results clearly demonstrate that the anti-VEGF/anti-DLL4
bispeciflc antibodies 219R45-MB-21M18 and 219R45—MB-21R79 are capable of oning differently
than just a mixture of the two individual antibodies.
Example 3
Inhibition ofHUVEC proliferation by anti—VEGF/anti—DLL4 bispecific antibodies
HUVEC cells were obtained from Lonza (Walkersville MD) and cultured in growth media
(M199, 10% heat-inactivated FBS (HI-PBS), SOug/ml EGS, 1X heparin, 1mM L-glutamine). For the
HUVEC proliferation assay, a 96-well plate was ated with 50p] of l rat tail collagen type I
solution (collagen 1 in 0.02N acetic acid) and incubated at 4°C overnight. After incubation, the plate was
thoroughly aspirated to remove unbound collagen I solution and washed once with 200ul DPBS. The
HUVEC cells were removed from the surface of the growth flasks using an endothelial cell subclone
reagent and centrifuged at 1200 rpm for 5 minutes at 4°C. The cells were resuspended in starvation/assay
medium (M199 and 2% HI-FBS, 1X heparin, 5U/ml n—glutamine) at a density of 105 cells/ml. The
cells were seeded into the collagen-coated assay plate at 5000 cells/well, 50ul/well. The cells were
incubated for 3 hours at 37°C, washed one time, refed with 100ul assay media, and incubated overnight at
37°C. The next day, bispecific antibodies 219R45—MB—21Ml8, 219R45-MB-21R79, parental antibody
219R45, or control antibody LZl were prepared in a mixture with human VEGF (R&D Biosystems,
Minneapolis MN). The antibodies were ly diluted 5—fold from 20”M to 0.25nM in assay buffer in
combination with hVEGF (final concentration Sng/ml). The mixture was cubated at 37°C for 2
hours. The medium was removed from the assay plate, and 100p] of the antibody/hVEGF mixture was
added to each well. After 3-4 days incubation, medium was removed and a fresh aliquot of the
dy/hVEGF mixture was added to each well and allowed to te for another 4 days. On day 7,
20ul of Alamar Blue t (Invitrogen, Carlsbad, CA) was added to each well and incubated at 37°C for
-6 hours. The plate was read with a SpectraMax MSe Microplate reader (Molecular Devices, Sunnyvale
CA) using a excitation wavelength of 539nm and an emission wavelength of 590nm.
As shown in Figure 3, anti—VEGF/anti—DLL4 bispecific antibodies 219R45-MB-21M18 and
219R45-MB-21R79, as well as parental anti—VEGF antibody 219R45 inhibited HUVEC proliferation.
These results demonstrated that the bispecific antibodies were capable of inhibiting VEGF-induced
proliferation of HUVEC cells.
Example 4
Inhibition of nduced Notch signalling by bispecific antibodies
Human PC3 cells were transfected with an expression vector encoding a full-length human
Notch2 receptor and a firefly luciferase reporter vector (8xCBF-luciferase reporter) that is responsive to
Notch signaling. The cells were also ected with a Renilla luciferase reporter (Promega, n
WI) as an internal control for transfection efficiency. Purified human DLL4 protein was coated onto 96-
well plates at lOOng/well and Notch2-expressing PC3—luc cells were added to the wells. Anti-VEGF/anti-
DLL4 bispecific antibodies 219R45-MB-21M18, 2 l 9R45—MB—2 1 R79, parental anti-DLL4 antibodies
21M18, 21R79 or a control antibody LZl were serially diluted 55—fold from 20ug/ml to 0.064ug/ml, added
to the appropriate wells, and ted overnight. Luciferase ty was determined using a dual
luciferase assay kit (Promega, Madison, WI) with firefly luciferase ty normalized to Renilla
luciferase activity.
As shown in Figure 4, anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R79 and
parental LL4 antibodies 21M18 and 21R79 inhibited DLL4-induced Notch signaling. Bispecific
antibody 219R45-MB-21M18 inhibited nduced Notch signaling only at high antibody
concentrations. These results trated that bispecific antibody -MB-21R79, and to a lesser
extent bispecific antibody 219R45—MB—21M18, were capable of inhibiting DLL4-induced Notch
signaling. Thus, in combination with the results presented in Example 3, the anti-VEGF/anti-DLL4
bispecific antibodies 219R45-MB-21R79 and 219R45-MB-2 1M1 8 have demonstrated the ability to
inhibit both VEGF-induced and DLL4-induced signaling and/or proliferation functions.
Example 5
Inhibition of tumor growth in vivo by a bispecific antibody in a human skin graft model
A human skin graft model has been reported which comprises a human skin graft and human
tumor cells. A human skin graft is established and then human tumor cells are implanted into the skin
graft, allowing the tumor cells to grow in an nment with human stroma and vasculature (Tahtis et
al., 2003, Mol. Cancer Ther. 22229-737). Human skin samples were obtained from neonatal foreskin
tissue and grafted onto the lateral flank ofNOD-SCID mice. After establishment of the skin graft,
luciferase-labeled OMP—C8 colon tumor cells (20,000 cells) were injected intradermally into the human
skin. Tumor growth was monitored by bioluminescence imaging using an IVIS imaging system (Caliper
Life Sciences, in View, CA). Tumors were d to grow until they reached 1.2 x 106 photons
per second. Tumor-bearing mice (n = 6 mice/group) were randomized and treated with control Ab, anti-
hDLL4 antibody 21M18, anti-VEGF antibody bevacizumab, or anti-VEGF/anti-DLL4 bispecific dy
219R45-MB-21M18. Animals were treated once a week and antibodies were administered
eritoneally at a dose of 25mg/kg. Tumor growth was monitored by bioluminescence imaging on the
indicated days.
As shown in Figure 5, both anti-hDLL4 antibody 21M18 and anti-VEGF antibody bevacizumab
ted tumor grth in this human skin graft/human tumor model. Furthermore, bispecif1c anti-VEGF/
LL4 ific antibody 219R45-MB-21M18 was more effective than either the anti-DLL4
antibody or the anti-VEGF antibody alone. These data demonstrate the utility of simultaneously targeting
DLL4 and VEGF with a bispecific antibody.
Example 6
Tumorigenicity of 8 atic tumor cells after treatment with anti-VEGF/anti-DLL4 bispeciflc
antibodies
Mice bearing OMP-PN8 pancreatic tumors were treated with control antibody (15 mg/kg), antihDLL4
antibody 21M18 (15 mg/kg), anti-VEGF antibody bevacizumab (15 mg/kg), or anti-VEGF/anti-
DLL4 bispeciflc antibodies 219R45-MB-21M18 or 219R45-MB-21R79 (30 mg/kg) with or without
gemcitabine (70 mg/kg). Following four weeks of treatment, tumors were harvested, processed to single
cell suspensions and the human tumor cells were purified by immunomagnetic depletion of murine cells.
90 human tumor cells from each treatment group were erred to a new cohort ofmice (n = 10
mice/group). Tumors were allowed to grow for 55 days without any treatment and tumor volumes were
measured with electronic calipers.
Figure 6 shows the tumor volume from the individual mice in each group. Cells ed from
mice treated with anti-hDLL4 antibody 21M18 had greatly decreased tumorigenicity, 5 out of 10 mice had
tumors, as compared to cells isolated from mice treated with control antibody where 9 out of 10 mice had
tumors. The reduction in tumor growth frequency indicates a reduction in cancer stem cell frequency. In
st, bevacizumab treatment resulted in no reduction of tumor growth ncy, 10 out of 10 mice
had tumors. Similar to bevacizumab, treatment with gemcitabine as a single agent had no effect on tumor
growth frequency as 10 out of 10 mice had tumors. The anti—VEGF/anti—DLL4 ific antibodies
219R45-MB-21M18 and 219R45-MB-21R79 both reduced tumor growth frequency (5 out of 10 mice had
tumors and 4 out of 10 mice had tumors, respectively). ation treatment with gemcitabine
ed to have no effect on tumor growth frequency. These data indicate that targeting DLL4 reduces
cancer stem cell frequency while targeting VEGF alone does not. Importantly, these data indicate that the
anti-CSC activity of the anti—DLL4 antibody is retained in a ific antibody.
Example 7
Bispeciflc Antibody ELISA
VEGF (ATGEN, South Korea) was coated onto Nunc maxisorb plates at 2ug/ml (100ul/well) and
incubated overnight at 2-8°C. Bispeciflc antibodies 219R45—MB-21M18, 2 l 9R45-MB-2 1 R79, 219R45-
75, and 219R45-MB-21R83 were diluted in blocking buffer (1x PBS, 0. 1% gelatin, 0.1%
Polysorbate-20, pH 7.4) containing 2ug/ml biotin—DLL4—hFc. The antibodies were serially diluted 3-fold
from 500ng/ml to g/ml. The antibody samples were incubated for 2 hours in blocking buffer
containing the -DLL4-hFc. After incubation, the antibody samples were transferred to the VEGF-
coated assay plate (100 ul/well) and incubated for 2 hours. Streptavidin—HRP (Jackson lmmunoResearch,
West Grove, PA) was added to each well and incubated for 1 hr. TMB substrate was added to the wells
with a 10 minute color development and the reaction was stopped with 2M sulfuric acid. Absorbance was
read at 450—65Onm and the data analyzed using the 4—parameter fit within the Softrnax Pro analysis
program (Molecular Devices, Sunnyvale, CA).
Figure 7 shows the titration curves of bispecific antibodies 219R45—MB-21M18 (open circles),
219R45-MB-21R79 (open squares), 219R45—MB—21R75 (open triangles), and 219R45-MB-21R83 (open
ds) in comparison to a reference anti-VEGF/anti-DLL4 bispecific antibody (solid circles).
ve potencies for the bispecific antibodies as compared to the reference bispecific antibody are
shown in Table 5.
Table 5
Antibody Relatlvgfl)Potency
—MB—21M18
219R45—MB-21R79 501
-MB—21R75 422
-MB—21R83 222
Bispecific dy 219R45-MB—21R79 was the most potent, about 7-fold more potent than
219R45-MB-21M18, which reflected the higher affinity of the 21R79 antigen-binding site.
Example 8
Bispecific Antibody Production
Bispecific antibodies were produced using a GS-CHO cell line. CHOK] SV cells (Lonza
Biologics) were transfected via electroporation with the gene(s) of interest coupled with glutamine
synthetase (GS) as the able marker. Transfectants and subclones were screened for antibody
productivity and the high ers were selected for scaled—up production. Cells were grown using a
fed-batch process and fed-batch bioreactors. Accumulated antibody in harvested cell culture fluid
(HCCF) was isolated and purified using chromatography techniques.
Bispecific antibody cell lines 219R45—MB—21M18.010.017 and 219R45-MB-21R79.017.003 were
cultured in SL stirred tank ctors for 14 days. Cell line -MB-21M18.010.017 produced a
final antibody titer of 3.0g/L and cell line 219R45—MB—21R79.017.003 ed a final antibody titer of
0.8g/L. Cell lines -MB-21R75.101 and 219R45—MB—21R83.1 13 were cultured in 25L WAVE
bioreactor systems (GE Healthcare) using a fed-batch process that achieved final antibody titers of .
Bispecific antibody cell lines 219R45-MB-21M18AG.138.007, 219R45-MB-21M18AG.038.009,
219R45-MB-21M18AG.142.002, 219R45-MB—21R79AG.072.014 and -MB-21R83AG.129.003
were cultured in 5 L stirred tank bioreactors for 14 — 15 days. Cell line 219R45-MB-21M18AG. 138.007
produced a final antibody titer of 1.0 g/L after 14 days. Cell line 219R45-MB-21M18AG.038.009
produced a final antibody titer of 1.6 g/L after 14 days. Cell line 219R45-MB-21M18AG. 142.002
produced a final antibody titer of2.6 g/L after 14 days. Cell line 219R45-MB-21R79AG.072.014
produced a final antibody titer of2.1 g/L after 15 days. Cell line 219R45-MB-21Ml8AG.038.009
produced a final antibody titer of 2.4 g/L after 15 days. Culture fluid was harvested by filtration from
each of these four cell lines and subjected to Protein A affinity chromatography. The Protein A column
was washed with a series of buffers and the antibodies were eluted using a low pH elution buffer. l
characterization of the purity of the bispecific antibodies was performed using size exclusion
chromatography (SEC-HPLC) and isoelectric focusing (IEF).
Size exclusion chromatography (SEC) was used to determine the purity of the antibody product.
SEC is a well known chromatographic method in which molecules (e. g., antibodies) in solution are
separated by their size. SEC may be used to distinguish an antibody product from aggregate and/or
impurities, and to determine the percentage of the antibody product as ed to the total mixture. As
used herein, SEC does not distinguish between a homomeric antibody and a heterodimeric bispecif1c
antibody.
lmaged capillary isoelectric ng (icIEF) was used to determine identity and purity of the
bispecific antibody heterodimers. Using iclEF, the charge isoforms of an antibody are separated
according to their pl and the result is a print” of the antibody’s charge distribution. The iclEF
method can also serve as a determination of purity by separating the bispecific antibody heterodimers by
their distinct p1 from any homodimer products or impurities.
Bispecific antibody samples were analyzed by icIEF on a nSimple ICE280 instrument
(ProteinSimple, Santa Clara, CA). For this analysis, a protein mixture is introduced into a capillary, high
voltage is applied across the capillary and ampholytes establish a linear pH gradient along the length of
the capillary. Under the influence of the electric field, the p] markers and the protein e both
migrate the length of the capillary until a pH value is reached where the net charge is zero. Once focused,
the ICE280 instrument uses whole—column imaging detection with a 280-nm UV camera to monitor the
n of n isoforms within the capillary. The resulting opherogram is calibrated using
internal pl markers and integrated to establish the respective percentage areas of the different charged
isoforms of the protein mixture. The charge profiles from several anti-VEGF/anti-DLL4 bispecific
antibodies are shown in Figure 8. For this experiment, Protein A s were diluted with MilliQ water
to a concentration of ml. A total of 18p] ofthe sample was mixed with 100uL of 8M urea, 70ul of
0.5% cellulose, 8”L of 3-10 Pharmalyte, 2”] of high pl marker and 2ul of low pl marker to a final
volume of 200ul. Table 6 shows the percentage of antibody product from cell lines 219R45-MB-
21M18.010.017, 219R45-MB-21R79.017.002, 219R45-MB-21R75.101, 219R45-MB-21R83.113,
219R45-MB-21M18. 138.007, 219R45-MB—21M18AG.038.009, 219R45-MB-21M18AG. 142.002,
219R45-MB-21R79AG.072.014, and 219R45—MB—2 . 129.003 after Protein A affinity
chromatography as determined by SEC-HPLC. Table 6 also shows the percentage of heterodimeric
antibodies from cell lines 219R45-MB-21M18.010.017, 219R45—MB-21R79.017.002, 219R45-MB-
21R75.101, 219R45-MB-21R83.113, —MB—21M18.138.007, 219R45-MB-21Ml8AG.038.009,
219R45-MB-21M18AG.142.002, 219R45—MB—21R79AG.072.014, and 219R45-MB-21R83AG.129.003
after Protein A affinity chromatography as analyzed by iclEF.
Table 6
. dy Purity by SEC Purity by IEF
CC” Lme
Titer (g/L) (% heterodimer)
The purity of the bispecific antibody product can be increased fiarther by additional
chromatography steps. After Protein A affinity chromatography, the eluate fraction was held at a low pH
for no less than 60 minutes at room temperature for viral inactivation. The antibody solution (Protein A
column , pH adjusted) was loaded onto a strong anion-exchange . Product— and process-
related impurities bound to the anion exchange chromatography resin and the flow-through fraction
(antibody product) was collected. In some cases, purity was further ed by use of a multi-modal
chromatography resin such as ceramic hydroxyapatite. In some cases, buffer exchange of the antibody
product was undertaken using ultrafiltration and ration techniques, after which excipients were
added. The formulated antibody was sterile filtered into sterile containers and stored refrigerated or
. Purity of the bispecific antibodies was re—assessed using SEC-HPLC and IEF.
Table 7
Purity by SEC Purity by IEF
Cell Line
(%) (% heterodimer)
2l9R45-\/lB-21Ml8.010.017 98.9 98.5
2l9R45-\/lB-21R79.0l7.002 95.1 99.3
2l9R45-\/lB-21R75.101 97.2 98.2
2l9R45-\/lB-21R83.ll3 95.3 91.4
2l9R45-\/lB-21Ml8.l38.007 98.1 100
2l9R45-VlB-21M18AG. 142.002 100
2l9R45-VlB-21R79AG.072.014 98.2 100
219R45-VIB-21R83AG. 129.003 998.6 100
As shown in Table 7, the purification of the anti-VEGF/anti-DLL4 bispecific antibodieswith
additional chromatography steps after Protein A resulted in isolation of antibody ts that were 95%
to about 99% pure as analyzed by SEC. Analysis by IEF ined that purified EGF/anti-DLL4
bispecific antibody from cell line 219R45—MB—21M18.010.0l7 was 98.5% heterodimeric, anti-
VEGF/anti-DLL4 bispecific antibody from cell line 219R45—MB-21R79.017.002 was 99.3%
heterodimeric, anti-VEGF/anti-DLL4 ific antibody from cell line 219R45-MB-21R75.101 was
98.2% heterodimeric, anti-VEGF/anti-DLL4 bispecific antibody from cell line 219R45-MB-21R83. 1 l3
was 91.4% heterodimeric, anti-VEGF/anti-DLL4 ific antibody from cell line -MB-
21M18. 7 was 100% heterodimeric, anti—VEGF/anti-DLL4 bispecific antibody from cell line
219R45-MB-21M18AG. 142.002 was 100% heterodimeric, anti-VEGF/anti-DLL4 bispecific antibody
from cell line 219R45-MB-21R79AG.072.014 was 100% heterodimeric, and EGF/anti-DLL4
bispecific antibody from cell line -MB-21R83AG. 129.003 was 100% heterodimeric. These
results demonstrated that the anion-exchange chromatography step greatly increased the percentage of
heterodimeric antibodies as ed to purification with Protein A chromatography alone. The addition
of a multi-modal chromatography step such as c hydroxyapatite can also improve monomeric
purity (as determined by SEP-HPLC).
Example 9
Inhibition of OMP-C8 colon tumor growth in viva tumor recurrence model
Single cell suspensions of OMP—C8 colon tumor xenografts (20,000 cells) were injected
subcutaneously into the flanks of 6-8 week old ID mice. Tumors were allowed to grow for 33
days until they reached an average volume of 240mm}. The mice were randomized (n = 10 per group)
and treated with anti-hDLL4 antibody 21M18, anti—VEGF antibody bevacizumab, a combination of
antibodies 21M18 and bevacizumab, anti-VEGF/anti—DLL4 bispecific antibody 219R45-MB-21M18,
anti-VEGF/anti-DLL4 bispecific dy 219R45-MB—21R79, or control dy, all in combination
with irinotecan. Antibodies and irinotecan were dosed weekly by injection into the intraperitoneal cavity.
Antibodies 21M18 and bevacizumab were dosed at 7.5mg/kg, bispecific antibodies 219R45-MB-21M18
and 219R45-MB-21R79 were dosed at 15mg/kg, and irinotecan was dosed at 45mg/kg. Irinotecan was
dosed for four weeks, at which time, it was discontinued and the administration of the dies
continued. Tumor growth was monitored and tumor volumes were measured with electronic calipers at
the indicated time points. Data are sed as mean i S.E.M.
As shown in Figure 9, anti-hDLL4 antibody 21M18 continued to inhibit tumor growth after
treatment with irinotecan was stopped. In contrast, anti—VEGF antibody bevacizumab was not able to
t regrowth of the tumor after irinotecan had been stopped. The combination of anti-DLL4 antibody
21M18 and anti-VEGF antibody bevacizumab resulted in greater inhibition of tumor regrowth than either
agent alone. Furthermore, the anti-VEGF/anti-DLL4 bispecific dy 219R45-MB-21M18 was more
effective at inhibiting tumor regrowth than the mixture of the two antibodies.
Example 10
Reduction in tumorigenicity of OMP—C8 colon tumors
Single cell sions of OMP—C8 colon tumor xenografts (20,000 cells) were injected
subcutaneously into the flanks of 6-8 week old NOD/SCID mice. Tumors were allowed to grow for 33
days until they d an average volume of . The mice were randomized (n = 5 per group) and
treated with anti-DLL4 antibody 21M18, anti—VEGF antibody bevacizumab, a combination of antibodies
21M18 and zumab, anti-VEGF/anti—DLL4 bispecific antibody 2l9R45-MB-21M18, anti-
VEGF/anti-DLL4 bispecific antibody 219R45—MB—21R79, or control antibody, either in combination with
irinotecan or without irinotecan. Antibodies and irinotecan were dosed weekly by injection into the
intraperitoneal cavity. dies 21Ml 8 and bevacizumab were dosed at 7.5mg/kg, bispecific antibodies
219R45-MB-21M18 and 219R45-MB-21R79 were dosed at 15mg/kg, and irinotecan was dosed at
45mg/kg. Tumors were harvested after 4 weeks, processed into single cell suspensions, and the human
tumor cells were isolated. 150 tumor cells from each experimental group were injected subcutaneously
into a new cohort of mice (n = 10 per group) and tumors were allowed to grow without treatment. Tumor
growth was monitored and tumor s were measured with electronic calipers.
Individual tumor volumes at day 68 are shown in Figure 10. Anti-DLL4 antibody 21M18, the
combination of 21M18 with anti—VEGF antibody bevacizumab, bispecific dies 219R45-MB-21M18
and 219R45-MB-21R79, and irinotecan all reduced tumor growth frequency as single agents. In st,
anti-VEGF bevacizumab as a single agent had no effect on tumor growth frequency as compared to the
control dy. In the groups treated with a combination of irinotecan and antibodies, the bispecific
dy 219R45-MB-21M18 had the greatest effect in reducing tumor growth frequency.
Example 11
Inhibition of OMP-C8 colon tumor growth in vivo
Single cell suspensions of OMP-C8 colon tumor xenografts (50,000 cells) were injected
subcutaneously into the flanks of 6-8 week old NOD/SCID mice. Tumors were allowed to grow for 21
days until they reached an average volume of 80mm3. The mice were randomized (n = 8 per group) and
treated with anti-DLL4 antibody 21M18, anti—VEGF antibody bevacizumab, anti-VEGF/anti-DLL4
bispecific dies 219R45-MB-21M18, 219R45—MB—21R?5, 2 l 9R45-MB-2 1 R79, 2 l 9R45-MB-
21R83, or control dy, either alone or in combination with ecan. Antibodies and irinotecan
were dosed weekly by injection into the intraperitoneal cavity. Bevacizumab and bispecific antibodies
219R45-MB-21M18, 219R45-MB-21R75, 219R45—MB—21R79, and 219R45—MB—21R83 were dosed at
15mg/kg, and irinotecan was dosed at 7.5mg/kg. Tumor growth was monitored and tumor volumes were
measured with electronic calipers at the indicated time points. Data are expressed as mean :I: S.E.M.
As single agents, all four anti-VEGF/anti-DLL4 ific antibodies showed enhanced anti-
tumor activity relative to anti—VEGF antibody bevacizumab. In combination with irinotecan, treatment
with anti-VEGF/anti-DLL4 bispecific antibodies 219R45—MB—21M1 8 and 219R45-MB-21R83 resulted in
the greatest tion of tumor growth (Figure 1 1).
Following the treatment phase, tumor sections were prepared and analyzed by xylin and
eosin (H&E) staining. The tumors treated with —MB-21M18 and 219R45-MB-21R83 in
combination with irinotecan showed dark pink ng regions providing evidence of extensive
calcification. This is characteristic of highly necrotic tumor tissue.
Example 12
Non-GLP toxicity study of bispecific antibodies in cynomolgus monkeys
A non-GLP ty study in cynomolgus monkeys was initiated to evaluate and e the toxicity
profile of some of the bispecific antibodies. The animals were dosed with 0 mg/kg (control), 5 mg/kg
(low dose), or 30 mg/kg (high dose) of anti-DLL4/anti-VEGF bispecific antibody 5-MB-21M18,
219R45-MB-21R83, or 219R45—MB—21R79) every 2 weeks Via IV infusion. 3 males and 3 females were
dosed in each group. After 15 weeks, mean body weights were lower in animals receiving the high dose
of 219R45-MB-21R79 than in animals that received the high dose of either 219R45-MB-21R18 or
219R45-MB-21R83. In addition, mean serum albumin levels were lower in animals that received
219R45-MB-21R79 than in those that ed either 219R45—MB-21R18 or 219R45-MB-21R83.
Although preliminary in nature, these early data suggest that 219R45-MB-21R18 and 219R45-MB-21R83
may have a superior toxicity profile compared to 219R45—MB-21R79.
It is understood that the examples and embodiments described herein are for illustrative es
only and that various modifications or changes in light thereof will be suggested to persons skilled in the
art and are to be included within the spirit and purview of this application.
All ations, s, patent applications, intemet sites, and accession numbers/database
ces including both polynucleotide and polypeptide sequences cited herein are hereby incorporated
by reference herein in their entirety for all purposes to the same extent as if each individual publication,
patent, patent application, intemet site, or accession number/database sequence was specifically and
individually indicated to be so incorporated by reference.
SEQUENCES
21M] 8 Heavy chain with signal sequence (underlined) (SEQ ID NO:1)
MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAP
GQG.flW GY SSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYD
YDVGWDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGA_4TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
21R79 Heavy chain with signal sequence (underlined) (SEQ ID N022)
MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAP
GQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYD
YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
219R45 Heavy chain with signal sequence lined) (SEQ ID NO: 3)
E {HIWFFIuIVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAP
MGD NPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCT__HYD
U {YYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS
2 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS-TKVDKTVE?
NCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF WYVDG
<EVI AKTKPR.flQhNSThRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP.|. flKT SKT<G
QPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENVY(TTPPMLKSD
GSFFLYSKLTVD(SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain With signal sequence (underlined) (SEQ ID NO:4)
MVLQTQVFSIUIW SGAYGDIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWF
QQKPGQPP<LH'YAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPW
TFGGGTKVfl KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
21M18 Heavy chain without predicted signal sequence (SEQ ID NO:5)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNV
HEALHNHYTQKSLSLSPGK
21R79 Heavy chain without ted signal sequence (SEQ ID NO:6)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYTANYNRATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMTSRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVViQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRflflMTKNQ
VSLTCLVLGEYPSDHAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNV
FSCSVMHHAHHNHYTQKSLSLSPGK
219R45 Heavy chain without predicted signal sequence (SEQ ID NO:7)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSY
KEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVS
SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQG
VMHEALHNHYTQKSLSLSPGK
Light chain without predicted signal sequence (SEQ ID NO:8)
DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGS
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLT;SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
21M18 Heavy chain variable region (SEQ ID NO:9)
QVQLVQSGAEVKKPGASVK:SCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNY
NQKFKGRVTFTT3TSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS
21R79 Heavy chain variable region (SEQ ID NO:10)
QVQLVQSGAEVK{PGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIANYNRATNY
NQKFKGRVTFTT3TSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS
219R45 Heavy chain variable region (SEQ ID NO:1 l)
QVQLVQSGA.EVK{PGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGD:NPSNGRTSY
KEKFKRRVT_JSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSS
Light chain variable region (SEQ ID NO: 12)
QSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGS
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK
21R75, 21R79 21R83 and 21Ml8 Heavy chain CDRl (SEQ ID NO:13)
TAYY..H
Alternative 21R75, 21R79, 21R83, and 21M18 Heavy chain CDRl (SEQ ID NO:79)
AYYIH
21R79 Heavy chain CDR2 (SEQ ID NO:14)
YIANYNRATNYNQKFKG
21M18 Heavy chain CDR2 (SEQ ID NO:15)
YISSYNGATNYNQKFKG
21R75, 21R79, 21R83, and 21M18 Heavy chain CDR3 (SEQ ID NO:16)
RDYDYDVGMDY
219R45 Heavy chain CDRl (SEQ ID NO:17)
NYWMH
219R45 Heavy chain CDR2 (SEQ ID NO:18)
DL PSNGRTSYK_3KFKR
219R45 Heavy chain CDR3 (SEQ ID NO: 19)
HYDDKYYPLMDY
Light chain CDRl (SEQ ID NO:20)
RASESVDNYGISFMK
Light chain CDR2 (SEQ ID NO:21)
AASNQGS
Light chain CDR3 (SEQ ID NO:22)
QQSKEVPWTFGG
Human DLL4 with signal sequence (underlined) (SEQ ID N0223)
MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRV
CLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLILE
AWHAPGDDLRPEALPPDALISKTAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVLCSD-YY
GDNCSQLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL
CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCK-GATC
S TCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGL{CE
HSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCA-GGQCLN N
GPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVQTS
LDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVG
Human DLL4 without predicted signal sequence (SEQ ID NO:24)
SGVFQIQIQ4b SGQPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT
NSFAVQDDSSGGGRNPLQIPF FTWPGTFSLIIEAWHAPGDDLRPEALPPDAL S< A Q
GSLAVGQ WITDTQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNL
SCLPGWTGTYCQQPC ISGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCST
PWQCTCDHGWGGIFCDQ HSPCKNGATCSNSGQRSYTCTCRPGYTGVDCflLflLS
ECDSNPC? GGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANY
ACECPP FTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELdVSDCAR
PCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTPVC
CPYGFVGSRCEFPVG
Human DLL4 inal Region (SEQ ID NO:25)
SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT
NSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQ
GSLAVGQW
Human DLL4 DSL Domain (SEQ ID NO:26)
WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTG
Human VEGF-A with signal sequence (underlined)(SEQ ID N022?)
MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD
IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHLGEM
SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPG
PHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR
Human VEGF-A without predicted signal sequence (SEQ ID NO:28)
APMAEGGGQNH{EVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGC
CNDflGHflCVPT.flSNTTMQTMRTKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRG.|.
KGKGQ(RKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSC
KNTDSRCKARQLELNERTCRCDKPRR
21M18 Heavy chain nucleotide sequence (13B Version 1) (SEQ ID NO:29)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGGTCAAGCAGGCCCCT
GGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGCGCCACCAACTACAAC
CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACCGCCTACATG
CGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
GTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCCTCC
ACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCTGAGTCTACC
GCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAAC
TCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTG
TACTCCCTGTCTAGCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACC
TGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGC
TGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTG
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAG
GTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCT
AACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
TCTCCTGGCAAGTAG
21R79 Heavy chain nucleotide sequence (13B Version 1) (SEQ ID NO:30)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTGAAACAGGCACCA
GGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGGGCCACCAACTACAAC
CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACAGCCTACATG
GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
GTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCCTCC
ACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCTGAGTCTACC
GCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAAC
TCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTG
TACTCCCTGTCTAGCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACC
GTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGC
TGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTG
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAG
GTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCT
AACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
TCTCCTGGCAAGTAG
21R79 Heavy chain nucleotide sequence (13B Version 2) (SEQ ID NO:31)
ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCGCTAATTATAATAGAGCTACAAACTATAAC
CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
AAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
AATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
TCTCCTGGCAAGTAG
219R45 Heavy chain nucleotide sequence (13A Version 1) (SEQ ID NO:32)
ATGAAGCATCTGTGGTTTTTCCTGTTGCTCGTGGCGGCACCCAGATGGGTGTTGTCCCAA
GTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCAAGCGTAAAAGTATCG
TGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGGTGCGGCAGGCTCCG
GGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGCAGAACATCATATAAG
GAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCTCGACTGCGTACATG
GAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGCACCATCCATTATGAT
GACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGGTCACCGTCTCCAGC
GCGTCGACGAAAGGTCCCTCGGTATTTCCCCTCGCCCCCTGCTCGAGGTCGACATCCGAA
TCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAGAGCCGGTAACGGTGTCG
TGGAACTCGGGAGCGCTTACGTCCGGAGTCCACACATTTCCGGCGGTACTGCAATCCTCG
GGACTGTATTCGTTGTCGTCAGTGGTGACTGTCCCGTCCTCCAATTTCGGGACTCAGACC
TATACGTGCAACGTCGACCACAAACCCTCAAACACCAAGGTGGATAAGACAGTGGAGCGC
AAGTGCTGCGTGGAGTGTCCCCCGTGTCCGGCACCCCCTGTCGCCGGACCCTCAGTCTTT
CCGCCGAAGCCCAAAGATACACTCATGATCTCAAGAACGCCCGAGGTAACATGC
GTGGTGGTCGATGTAAGCCACGAGGATCCAGAAGTACAATTCAATTGGTATGTAGACGGG
GTCGAGGTCCATAACGCAAAGACGAAACCGAGGGAAGAGCAGTTCAATTCGACTTTCCGG
GTGGTGTCGGTGCTTACAGTCGTACATCAGGACTGGTTGAACGGGAAGGAGTACAAGTGT
AAAGTATCGAATAAGGGCCTTCCAGCGCCGATTGAAAAGACCATCTCCAAGACCAAAGGA
CAGCCACGAGAGCCGCAAGTCTATACGCTTCCTCCCAGCCGAGAAAAGATGACTAAAAAC
CAGGTATCGCTTACGTGTCTCGTCAAGGGTTTCTACCCTTCGGACATCGCGGTGGAATGG
GAGAGCAATGGACAACCGGAAAACAACTACAAGACGACACCGCCTATGTTGAAAAGCGAT
GGATCGTTTTTCCTCTATTCGAAACTCACGGTCGATAAGTCACGGTGGCAGCAGGGGAAT
GTGTTCTCCTGTTCAGTGATGCACGAGGCGCTCCACAATCACTATACCCAGAAAAGCCTG
TCACTTTCCCCGGGAAAATGA
219R45 Heavy chain nucleotide sequence (13A Version 2) (SEQ ID NO:33)
ATGAAGCACCTCTGGTTCTTCCTGCTCCTCGTGGCTGCTCCTCGGTGGGTCCTCTCCCAA
GTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCTTCCGTCAAAGTCTCC
TGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGGTGCGGCAGGCTCCT
GGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGCAGAACCTCCTACAAG
TTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCTCCACAGCTTACATG
GAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCACCATCCACTACGAC
GACAAGTACTACCCTCTGATGGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCCAGC
ACAAAAGGACCCTCCGTCTTTCCCCTCGCCCCCTGCTCCCGGTCCACATCCGAA
TCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAGAGCCTGTCACAGTGTCC
TGGAACTCCGGAGCTCTCACATCCGGAGTCCACACATTTCCTGCTGTGCTCCAATCCTCC
GGACTGTATTCCCTCTCCTCCGTGGTGACAGTGCCTTCCTCCAATTTCGGGACACAGACC
TATACATGCAACGTGGACCACAAACCCTCCAACACCAAAGTCGATAAGACAGTGGAGCGC
TGCGTGGAGTGTCCCCCTTGTCCTGCTCCCCCTGTGGCTGGACCTTCCGTCTTT
CTGTTTCCTCCTAAACCTAAAGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGC
GTCGATGTGAGCCACGAGGACCCCGAAGTCCAATTTAATTGGTATGTGGACGGG
GTGGAGGTCCATAACGCTAAGACCAAACCTAGGGAAGAGCAGTTCAATTCCACTTTCCGG
GTGGTGTCCGTGCTGACCGTCGTTCATCAGGACTGGCTCAACGGGAAAGAATACAAATGC
AAAGTCTCTAATAAGGGCCTCCCTGCTCCTATTGAAAAAACAATTTCCAAAACAAAAGGA
CAACCTCGGGAGCCTCAAGTCTACACACTGCCACCTTCCCGGGAAAAAATGACAAAAAAT
CAAGTCTCCCTCACATGTCTCGTCAAGGGATTCTACCCTTCCGACATTGCTGTGGAATGG
GAATCCAATGGACAACCTGAAAACAACTACAAGACAACACCTCCTATGCTCAAAAGCGAT
GGGTCCTTTTTCCTCTATTCCAAACTCACAGTCGATAAGTCTCGGTGGCAGCAGGGGAAT
GTGTTCTCCTGTTCCGTGATGCACGAGGCTCTCCACAATCACTATACCCAGAAAAGCCTG
TCCCTCTCCCCTGGAAAATGA
Light chain nucleotide sequence (SEQ ID NO:34)
ATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGC
GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGAGAGCGGGCCACC
ATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCATGAAGTGGTTC
CAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCCAACCAGGGATCT
CCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACCCTGACCATCAGC
TCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAAGAGGTGCCCTGG
ACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTC
ATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCCTCCGTCGTGTGCCTGCTG
AACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCC
GGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCC
TCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTG
ACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGTTAG
21M18 Heavy chain variable region nucleotide sequence (SEQ ID NO:35)
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATC
TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGGTCAAGCAGGCC
CCTGGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGCGCCACCAACTAC
AACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACCGCCTAC
ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTAC
GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT
21R79 Heavy chain variable region nucleotide sequence (13B) (SEQ ID NO:36)
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATC
TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTGAAACAGGCA
CCAGGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGGGCCACCAACTAC
AACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACAGCCTAC
ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTAC
GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC
21R79 Heavy chain variable region nucleotide sequence (13B Version 2 ) (SEQ ID NO:37)
CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATA
AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCA
CAGGGACTTGAATGGATCGGATATATCGCTAATTATAATAGAGCTACAAACTAT
AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATAC
ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTAT
GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT
219R45 Heavy chain variable region nucleotide sequence (13A n 1) (SEQ ID NO:38)
CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCAAGCGTAAAAGTA
TCGTGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGGTGCGGCAGGCT
CCGGGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGCAGAACATCATAT
AAGGAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCTCGACTGCGTAC
ATGGAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGCACCATCCATTAT
GATGACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGGTCACCGTCTCC
219R45 Heavy chain variable region nucleotide sequence (13A Version 2) (SEQ ID NO:39)
CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCTTCCGTCAAAGTC
TCCTGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGGTGCGGCAGGCT
CCTGGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGCAGAACCTCCTAC
AAGGAAAAATTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCTCCACAGCTTAC
ATGGAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCACCATCCACTAC
GACGACAAGTACTACCCTCTGATGGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCC
Light chain variable region nucleotide ce (SEQ ID NO:40)
GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGAGAGCGGGCCACC
ATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCATGAAGTGGTTC
CAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCCAACCAGGGATCT
GGCGTGCCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACCCTGACCATCAGC
TCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAAGAGGTGCCCTGG
ACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG
Human IgGl Heavy chain constant region (SEQ ID NO:41)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Heavy chain nt region (SEQ ID NO:42)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR
VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN
LVKGFYPSD:AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK
—1o4—
Human IgG3 Heavy chain nt region (SEQ ID NO:43)
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
G.YSUSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC
DTPPPCPQCPEPKSC3TPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDT
U SRTPfiVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLH
QDWL‘GKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE
LHNRFTQKSLSLSPGK
Human IgG4 Heavy chain constant region (SEQ ID NO:44)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
VMHEALHNHYTQKSLSLSLGK
FLAG peptide (SEQ ID NO:45)
Parental 21R79 Heavy chain With signal sequence underlined unmodified chain (SEQ ID NO:46)
(HLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQA?
QGUfiW GY ANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYD
DVG DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS_TKVDKTV_3
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
Vi A<TKPRflfiQh STERVVSVLTVVHQDWLNGKEYKCKVSNKGLPA?IEKTTSKT{GQP
R.EPQVYTPPPSRflflMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
F "11LYSKLTV){SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Parental 219R45 Heavy chain with signal sequence underlined (SEQ ID NO:47)
M<iIWFFIIIVAAPQWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAP
GQGUflWMGD NPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCT__HYD
D{YYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS
W GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV.ER
KCCVZCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVH AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ?ENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Parental 21R79 Heavy chain without predicted signal sequence (SEQ ID NO:48)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIANYNRATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK
Parental 219R45 Heavy chain t signal sequence (SEQ ID NO:49)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSY
KLKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVS
SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV
FLFPP<PKDTL ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
Parental 21R79 Heavy chain variable region nucleotide sequence (SEQ ID NO:50)
CAAGTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGCATCGGTCAAAATC
AAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGGTAAAGCAGGCG
CCAGGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGCGCGACGAACTAT
AACCAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACATCGACGGCCTAC
ATGGAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGCGCAAGAGATTAC
GACTATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAGTCTCCTCC
Parental 219R45 Heavy chain variable region nucleotide sequence (SEQ ID NO:51)
CAGGTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGCGTCGGTAAAGGTC
AGCTGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGGTCAGACAGGCC
CCTGGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGACGGACTAGCTAT
AAGGAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCCTCAACGGCCTAC
TTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTACTATCCACTAT
GACGATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTGGTAACCGTGAGC
Parental 21R79 Heavy chain nucleotide sequence with signal sequence (SEQ ID NO:52)
ATGAAACACTTGTGGTTTTTCCTCTTGCTCGTGGCAGCTCCTCGGTGGGTACTTTCACAA
GTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGCATCGGTCAAAATCTCG
TGTAAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGGTAAAGCAGGCGCCA
GGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGCGCGACGAACTATAAC
CAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACATCGACGGCCTACATG
GAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGCGCAAGAGATTACGAC
TATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAGTCTCCTCCGCCAGC
ACCAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAGAGCACA
GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC
TCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGT
TGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTG
GTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTC
AGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC
AATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCC
CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG
TCTCCGGGTAAA
Parental 219R45 Heavy chain nucleotide sequence with signal ce (SEQ ID NO:53)
ATGAAACACCTCTGGTTCTTTTTGCTCCTGGTGGCAGCTCCCCGATGGGTGCTTAGCCAG
GTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGCGTCGGTAAAGGTCAGC
TGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGGTCAGACAGGCCCCT
GGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGACGGACTAGCTATAAG
GAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCCTCAACGGCCTACATG
GAGTTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTACTATCCACTATGAC
GATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTGGTAACCGTGAGCAGC
GCGTCCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAG
GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACC
TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGC
AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC
GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGC
GTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT
GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGG
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAA
Parental 21R79 and 219R45 light chain variable region nucleotide sequence (SEQ ID NO:54)
GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACC
ATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTC
CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCC
GGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGC
AGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGG
ACATTCGGAGGAGGGACCAAGGTGGAAATCAAA
Parental 21R79 and 219R45 light chain nucleotide sequence (SEQ ID NO:55)
ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCGGAGCCTACGGG
GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACC
ATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTC
CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCC
GGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGC
AGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGG
ACATTCGGAGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCCCCCTCCGTCTTC
CCCCCCAGCGATGAGCAGCTGAAAAGCGGCACTGCCAGCGTGGTGTGCCTGCTG
AATAACTTCTATCCCCGGGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAAAGC
GGCAACTCCCAGGAGAGCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACCCTGAGCAAAGCCGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC
ACCCATCAGGGCCTGAGCAGCCCCGTCACAAAGAGCTTCAACAGGGGCGAGTGTTGA
21R75 Heavy chain without predicted signal sequence (SEQ ID NO:56)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIAGYKDATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSA
VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMISRTPLVTCVVVDVSHEDBEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL?PSREEMTKNQ
VSLTCLVEGEYPSD-AVLWESNGQBLNNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK
21R75 Heavy chain with predicted signal sequence (underlined) (SEQ ID NO:57)
MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYTHWVKQAP
GQGHflW GY AGYKDATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYD
YDVG DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVTQSSGLYSTSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
VH A(TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
21R75 Heavy chain le region (SEQ ID NO:58)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIAGYKDATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS
21R75 Heavy chain CDR2 (SEQ ID NO:59)
Y-AGYKDATNYNQKFKG
21R75 Heavy chain nucleotide sequence with signal sequence (13B Version 1) (SEQ ID NO: 60)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCT
GGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTACAAC
TTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTACATG
GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTTCC
ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACC
GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAAC
TCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTG
TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACC
TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGC
TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTC
CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG
GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAA
GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCC
CCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC
AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCA
TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
AGCCCCGGCAAG
21R75 Heavy chain nucleotide sequence with signal sequence (13B Version 1T) (SEQ ID NO:77)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCTCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCT
GGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTACAAC
CAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTACATG
GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTTCC
ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACC
GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAAC
GCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTG
TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACC
TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGC
TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTC
AAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG
GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAA
GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCC
CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC
AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCA
TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
AGCCCCGGCAAG
21R75 Heavy chain nucleotide sequence with signal sequence (13B n Sl-2) (SEQ ID NO:61)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAAC
TTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
CGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
TCTCCTGGCAAG
21R83 Heavy chain without predicted signal sequence (SEQ ID NO:62)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISNYNRATNY
NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK
21R83 Heavy chain with predicted signal sequence (underlined) (SEQ ID NO:63)
MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQA?
GQGLEWIGYISNYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYD
YDVG DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW-
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
Vi AKTKPRflflQhNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFHYS?LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
21R83 Heavy chain le region (SEQ ID NO:64)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISNYNRATNY
RVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS
21R83 Heavy chain CDR2 (SEQ ID NO:65)
YISNYNRATNYNQKFKG
21R83 Heavy chain nucleotide sequence with signal sequence underlined (13B n 1) (SEQ ID
NO:66)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCT
GGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTACAAC
CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTACATG
GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGCGCTTCC
ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACC
GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAAC
TCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTG
CTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACC
TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGC
TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTC
CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG
GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAA
GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG
AAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCC
CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC
AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCA
TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
AGCCCCGGCAAG
21R83 Heavy chain nucleotide sequence with signal sequence underlined (13B Version 1T) (SEQ ID
NO:78)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCTCAG
GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCC
TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCT
GGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTACAAC
CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTACATG
GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGAC
TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGCGCTTCC
ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACC
GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAAC
TCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTG
TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACC
TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGC
GAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTC
CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG
GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAA
GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTG
TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG
AAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCC
CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC
AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCA
TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG
AGCCCCGGCAAG
21R75 Heavy chain nucleotide sequence with signal sequence underlined (13B Version S1-2) (SEQ ID
NO:67)
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAAC
CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
GAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
TCTCCTGGCAAG
21R75 Heavy chain variable region nucleotide sequence (13B Version 1) (SEQ ID NO:68)
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATC
TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCC
CCTGGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTAC
AAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTAC
ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTAC
GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT
21R75 Heavy chain variable region nucleotide sequence (13B Version 2) (SEQ ID NO:69)
CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATA
AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCA
CCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTAT
AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATAC
ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTAT
-1ll-
GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT
21R83 Heavy chain variable region tide sequence (133 Version 1) (SEQ ID NO:70)
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATC
TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCC
CCTGGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTAC
AACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTAC
ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTAC
GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGC
21R75 Heavy chain variable region nucleotide ce (13B n 2) (SEQ ID NO:71)
CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATA
AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCA
CCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTAT
AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATAC
ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTAT
GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT
21R83 Heavy chain nucleotide sequence with signal sequence underlined (13B Version 2) (SEQ ID
NO:72)
ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAGCTACAAACTATAAC
CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
GTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
AAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
AAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
TCTCCTGGCAAGTAG
21R83 Heavy chain variable region nucleotide sequence (13B Version 2) (SEQ ID NO:73)
CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATA
AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCA
CCAGGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAGCTACAAACTAT
AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATAC
ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTAT
GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT
-ll2-
21R75 Heavy chain nucleotide sequence with signal sequence underlined (13B Version 2) (SEQ ID
NO:74)
ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAAC
CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
TCTCCTGGCAAGTAG
21M18 Heavy chain nucleotide sequence on 2) (SEQ ID NO:75)
ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAG
GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGT
TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCA
GGACAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTACAAACTATAAC
CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATG
GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGAT
TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCC
ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACT
GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAAC
TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTA
TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACC
TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGC
GAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTC
CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTT
GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAG
AATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTT
TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTG
TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCT
CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTG
TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCT
AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCC
TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTA
GGCAAGTAG
21M] 8 Heavy chain variable region (version 2) (SEQ ID N0276)
CAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGC
AAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGA
CAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTACAAACTATAACCAA
AAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAA
TTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTAT
GATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT
LL4 heavy chain CDR2 consensus sequence (SEQ ID NO:80):
YIX1X2YX3X4ATNYNQKFKG, where X1 is serine or alanine, X2 is , asparagine, or glycine, X3 is
asparagine or lysine, and X4 is glysine, arginine, or aspartic acid
Claims (17)
1. A bispecific antibody comprising: a) a first antigen-binding site that specifically binds human vascular endothelial growth factor (VEGF), and b) a second antigen-binding site that specifically binds human delta-like 4 ligand (DLL4), wherein the first antigen-binding site comprises a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 sing HYDDKYYPLMDY (SEQ ID NO:19); n the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), wherein X1 is serine or alanine, X2 is serine, asparagine, or glycine, X3 is asparagine or lysine, and X4 is glycine, arginine, or aspartic acid , and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and second antigen-binding sites comprise a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 sing AASNQGS (SEQ ID NO:21), and a light chain CDR3 sing QQSKEVPWTFGG (SEQ ID NO:22).
2. The bispecific antibody of claim 1, wherein the second antigen-binding site ses a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising RATNYNQKFKG (SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16).
3. The ific antibody of claim 2, n the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 11283960_1 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16).
4. The bispecific antibody of claim 1 or claim 2, which comprises: (a) a first heavy chain variable region having at least 90% ce identity to SEQ ID NO:11; (b) a second heavy chain variable region having at least 90% sequence ty to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64; and (c) a first and a second light chain variable region having at least 90% sequence identity to SEQ ID NO:12.
5. The bispecific antibody of claim 1 or claim 2, which comprises: (a) a first heavy chain variable region sing SEQ ID NO:11; (b) a second heavy chain variable region comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64; and (c) a first and a second light chain variable region comprising SEQ ID NO:12.
6. The bispecific antibody of claim 5, which comprises: (a) a first heavy chain variable region comprising SEQ ID NO:11; (b) a second heavy chain variable region comprising SEQ ID NO:64; and (c) a first and a second light chain variable region comprising SEQ ID NO:12.
7. The bispecific antibody of any one of claims 1-6, which is a monoclonal antibody, a recombinant antibody, a ic antibody, a humanized antibody, a human antibody, an IgG1 antibody, or an IgG2 antibody.
8. The bispecific antibody of any one of claims 1-6, which comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:42, wherein the amino acids are ed with glutamate or aspartate, and a second human IgG2 constant region with amino acid tutions at 11283960_1 positions corresponding to positions 236 and 278 of SEQ ID NO:42, wherein the amino acids are replaced with lysine.
9. The bispecific antibody of any one of claims 1-8, which: (i) inhibits binding of VEGF to at least one VEGF receptor; (ii) inhibits binding of DLL4 to at least one Notch receptor; (iii) inhibits Notch signaling; and/or (iv) modulates angiogenesis.
10. A bispecific dy that specifically binds human VEGF and human DLL4, which comprises: (a) a heavy chain of SEQ ID NO:7; (b) a heavy chain of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:56, or SEQ ID NO:62; and (c) two light chains of SEQ ID NO:8.
11. A bispecific antibody of claim 10, which comprises: (a) a heavy chain of SEQ ID NO:7; (b) a heavy chain of SEQ ID NO:62; and (c) two light chains of SEQ ID NO:8.
12. A bispecific antibody that specifically binds human VEGF and human DLL4, which ses: (a) a heavy chain encoded by the DNA sing SEQ ID NO:75, a heavy chain encoded by the DNA comprising SEQ ID NO:33, and a light chain d by the DNA comprising SEQ ID NO:34; (b) a heavy chain encoded by the DNA comprising SEQ ID NO:31, a heavy chain encoded by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA comprising SEQ ID NO:34; 11283960_1 (c) a heavy chain encoded by the DNA comprising SEQ ID NO:72, a heavy chain d by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA comprising SEQ ID NO:34; or (d) a heavy chain encoded by the DNA comprising SEQ ID NO:74, a heavy chain encoded by the DNA comprising SEQ ID NO:33, and a light chain encoded by the DNA comprising SEQ ID NO:34.
13. An isolated antibody that specifically binds human VEGF, which comprises: (a) a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 sing YPLMDY (SEQ ID NO:19); and (b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising PWTFGG (SEQ ID NO:22).
14. The antibody of claim 13, which comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:11; and (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:12.
15. The antibody of claim 13, which comprises: (a) a heavy chain variable region sing SEQ ID NO:11; and (b) a light chain variable region comprising SEQ ID NO:12.
16. The antibody of claim 13, which comprises: (a) a heavy chain comprising SEQ ID NO:49 or SEQ ID NO:7; and (b) a light chain comprising SEQ ID NO:8.
17. The antibody of any one of claims 13-16, which inhibits binding of VEGF to at least one VEGF receptor. 11283960
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161538454P | 2011-09-23 | 2011-09-23 | |
US61/538,454 | 2011-09-23 | ||
US201261597409P | 2012-02-10 | 2012-02-10 | |
US61/597,409 | 2012-02-10 | ||
US201261692978P | 2012-08-24 | 2012-08-24 | |
US61/692,978 | 2012-08-24 | ||
PCT/US2012/056886 WO2013044215A1 (en) | 2011-09-23 | 2012-09-24 | Vegf/dll4 binding agents and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ623724A NZ623724A (en) | 2016-09-30 |
NZ623724B2 true NZ623724B2 (en) | 2017-01-05 |
Family
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