bispecific antibodies that bind to vegf/dll4, pharmaceutical composition and prokaryotic, fungal or

专利摘要:
VEGF/DLL4 BINDING AGENTS AND USES THEREOF. The present invention relates to VEGF binding agents, DLL4 binding agents, VEGFIDLL4 bispecific binding agents and methods of using the agents for treating diseases such as cancer. The present invention provides antibodies that specifically bind to human VEGF, antibodies that specifically bind to human DLL4, and bispecific antibodies that specifically bind to human VEGF and/or human DLL4. The present invention further provides methods of using the agents to inhibit tumor growth. Also described are methods of treating cancer which comprise administering a therapeutically effective amount of an agent or an antibody of the present invention to a patient having a tumor or cancer.
公开号:BR112014007035B1
申请号:R112014007035-0
申请日:2012-09-24
公开日:2021-05-04
发明作者:Austin L. Gurney；Aaron Ken Sato；Christopher John Bond
申请人:Oncomed Pharmaceuticals, Inc；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates generally to antibodies and other agents that bind to VEGF, Dll4 or both VEGF and Dll4, particularly bispecific anti-VEGF/anti-DLL4 antibodies, as well as methods of using the antibodies or of other agents for the treatment of diseases such as cancer. BACKGROUND OF THE INVENTION
[002] Angiogenesis plays an important role in the pathogenesis of a number of disorders, including solid tumors and metastasis. The production of new blood vessels is essential for the supply of oxygen and nutrients for the growth and spread of a tumor and therefore angiogenesis is a good target for therapeutic agents for cancer.
[003] Angiogenesis involves a family of proteins that act as angiogenic activators, including vascular endothelial growth factor (VEGF-A), VEGF-B, VEGF-C, VEGF-E and their respective receptors (VEGFR-1 , VEGFR-2 and VEGFR-3). VEGF-A, also referred to as VEGF or vascular permeability factor (VPF), exists in several isoforms that arise from alternative mRNA splicing of a single VEGF gene, with VEGF165 being the most biologically relevant isoform.
Anti-VEGF antibodies have been shown to suppress tumor cell growth in vitro and in vivo. A humanized anti-VEGF monoclonal antibody, bevacizumab (AVASTIN) has been developed and approved in the United States of America as a therapeutic agent for cancer.
[005] The Notch signaling pathway is a universally conserved signal transduction system. It is involved in determining the fate of cells during development including embryo pattern formation and post-embryonic tissue maintenance. In addition, Notch signaling has been identified as a critical factor in the maintenance of hematopoietic stem cells.
[006] The Notch pathway has been linked to the pathogenesis of both hematological and solid tumors and cancers. Several cellular functions and microenvironmental cues associated with tumorigenesis have been shown to be modulated by the Notch signaling pathway, 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 chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, leukemia acute T-cell lymphoblastic cancer, brain cancer, breast cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, and skin cancer. (Leong et al., 2006, Blood, 107:2223-2233).
[007] Delta 4 type 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 EGF-like tandem repeats within the extracellular domain. It has been reported that DLL4 is induced by VEGF and that DLL4 can act as a negative feedback regulator for vascular proliferation.
Anti-DLL4 antibodies have been shown to increase sprouting and angiogenic branching that lead to non-productive angiogenesis and reduced tumor growth (Noguera-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; US Patent No. 7,750,124).
[009] Although there has been significant progress in the development of monoclonal antibodies for use in cancer treatments, there is still enormous potential for further improvement. One class of antibody molecules with the promise of greater potency and/or reduced side effects (eg, toxicity) is bispecific antibodies.
[010] Early bispecific molecules were mainly generated using chemical crosslinking of two antibodies or were hybrid hybridomas or "quadromas". One hit of the quadroma format is triomabs, which are mouse/rat combinations that demonstrate a species-specific heavy/light chain pairing. More recently, advances in antibody engineering have provided a wide variety of new antibody formats, including, but not limited to, tandem scFv (bi-scFv), diantibodies, tandem diantibodies (tetra-bodies), single-chain diantibodies, and dual variable domain antibodies.
[011] It is one of the objectives of the present invention to provide improved molecules for cancer treatment, particularly bispecific antibodies that specifically bind to human VEGF and human DLL4. SUMMARY OF THE INVENTION
[012] The present invention provides binding agents, such as antibodies, that bind to VEGF, DLL4 or both VEGF and DLL4 (binding agents to VEGF/DLL4), as well as compositions, such pharmaceutical compositions, which comprise the liaison officers. Binding agents that bind to VEGF or Dll4, as well as at least one additional antigen or target and pharmaceutical compositions of such binding agents, are also provided. In certain embodiments, binding agents are novel polypeptides, such as antibodies, antibody fragments, and other polypeptides related to such antibodies. In certain embodiments, the binding agents are antibodies that specifically bind to human VEGF. In some embodiments, the binding agents are antibodies that specifically bind to human Dll4. In some embodiments, the binding agents are bispecific antibodies that specifically bind to human VEGF and human Dll4. The invention further provides methods of inhibiting the growth of a tumor by administering the binding agents to an individual having a tumor. The invention further provides methods of treating cancer by administering the binding agents to a subject in need thereof. In some embodiments, methods of treating cancer or inhibiting tumor growth comprise targeting cancer stem cells with the binding agents. 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 tumor, reducing the tumorigenicity of a tumor, and/or reducing the tumorigenicity of a tumor through reduction in the number or frequency of cancer stem cells in the tumor.
[013] In one aspect, the invention provides a binding agent, such as an antibody, that specifically binds to human VEGF. In some embodiments, the binding agent inhibits the binding of VEGF to at least one VEGF receptor. In some embodiments, the binding agent inhibits the binding of VEGF to VEGFR-1 and/or VEGFR-2. In some embodiments, the binding agent modulates angiogenesis. In certain embodiments, the antibody or other binding agent specifically further binds and/or inhibits human Dll4 in addition to human VEGF.
[014] In some embodiments, the binding agent is an antibody comprising a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18) and a CDR3 heavy chain comprising HYDDKYYPLMDY (SEQ ID NO:19); 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).
[015] In certain embodiments, the binding agent is an antibody comprising a heavy chain variable region that has at least 80% sequence identity to SEQ ID NO:11; and/or a light chain variable region that has at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the linker comprises a heavy chain variable region that has at least 90% sequence identity to SEQ ID NO:11; and/or a light chain variable region that has at least 90% sequence identity to SEQ ID NO:12. In certain embodiments, the linker comprises a heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:11; and/or a light chain variable region that has at least 95% sequence 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:11; and/or a light chain variable region of SEQ ID NO:12.
In some embodiments, the binding agent is the antibody 219R45, 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75 or 219R45-MB-21R83.
[017] In another aspect, the invention provides a binding agent, such as an antibody, that specifically binds to human DLL4. In some embodiments, the binding agent inhibits the binding of Dll4 to at least one Notch receptor. In some embodiments, the binding agent inhibits the binding of DLL4 to Notch1, Notch2, Notch3 and/or Notch4. In some embodiments, the binding agent inhibits Notch signaling. In some modalities, the binding agent promotes non-productive angiogenesis. In certain embodiments, the antibody or other binding agent specifically further binds and/or inhibits human VEGF in addition to human Dll4.
[018] In some embodiments, the binding agent is an antibody that binds to human DLL4 and comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a CDR2 of heavy chain comprising YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), where 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 chain CDR3 heavy comprising 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 embodiments, the antibody comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 that comprises YIANYNRATNYNQKFKG (SEQ ID NO:14) and a CDR3 heavy chain comprising 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).
[019] In certain embodiments, the binding agent is an antibody comprising a heavy chain variable region that has at least 90% or at least 95% sequence identity to SEQ ID NO:10; and/or a light chain variable region that has at least 90% or at least 95% sequence 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:10; and a light chain variable region of SEQ ID NO:12.
[020] In some embodiments, the binding agent is the 21R79 antibody or the 219R45-MB-21R79 antibody.
[021] In some embodiments, the binding agent is an antibody that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 that comprises YIAGYKDATNYNQKFKG ( SEQ ID NO:59) and a heavy chain CDR3 comprising 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).
[022] In certain embodiments, the binding agent is an antibody comprising a heavy chain variable region that has at least 90% or at least 95% sequence identity to SEQ ID NO:58; and/or a light chain variable region that has at least 90% or at least 95% sequence 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:58; and a light chain variable region of SEQ ID NO:12.
[023] In some embodiments, the binding agent is the 21R75 antibody or the 219R45-MB-21R75 antibody.
[024] In some embodiments, the binding agent is an antibody that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 that comprises YISNYNRATNYNQKFKG ( SEQ ID NO:65) and a heavy chain CDR3 comprising 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).
[025] In certain embodiments, the binding agent is an antibody comprising a heavy chain variable region that has at least 90% or at least 95% sequence identity to SEQ ID NO:64; and/or a light chain variable region that has at least 90% or at least 95% sequence 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.
[026] In some embodiments, the binding agent is the 21R83 antibody or the 219R45-MB-21R83 antibody.
[027] In certain embodiments of each of the aforementioned aspects or embodiments, as well as other aspects and/or embodiments described elsewhere herein, the binding agent is a bispecific antibody. In some embodiments, the bispecific antibody specifically binds to human VEGF and a second target. In some embodiments, the bispecific antibody specifically binds to human Dll4 and a second target. In some embodiments, the bispecific antibody specifically binds to both human VEGF and human DLL4. In some embodiments, the bispecific antibody modulates angiogenesis. In certain embodiments, the bispecific antibody inhibits Notch signaling. In some embodiments, the bispecific antibody modulates angiogenesis and inhibits Notch signaling. In some embodiments, the bispecific antibody reduces the frequency number of cancer stem cells. In certain embodiments, the bispecific antibody comprises two identical light chains. In certain embodiments the bispecific antibody is an IgG antibody (eg, IgG2).
[028] In some embodiments, the bispecific antibody comprises: a first antigen-binding site that specifically binds to human VEGF, wherein the first antigen-binding site comprises a heavy chain CDR1 that comprises 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 CDR1 which comprises RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises: a first antigen-binding site that specifically binds to human VEGF, wherein the first antigen-binding site comprises (a) a heavy chain CDR1 that comprises 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 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).
[029] In certain embodiments, the bispecific antibody comprises: a first antigen-binding site that specifically binds to human DLL4, wherein the first antigen-binding site comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprising YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), where X1 is serine or alanine, X2 is serine, asparagine or glycine, X3 is as- paragine or lysine and X4 is glycine, arginine or aspartic acid and a heavy chain CDR3 comprising 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 embodiments, the bispecific antibody comprises: a first antigen-binding site that specifically binds to human Dll4, wherein the first antigen-binding site comprises a heavy chain CDR1 that comprises 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 which comprises RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific antibody further 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 bispecific antibody comprises: a first antigen-binding site that specifically binds to human DLL4, wherein the first antigen-binding site comprises (a) a heavy chain CDR1 that comprises 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 AASNQGS (SEQ ID NO:21) and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
[030] In some embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human DLL4, wherein the 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 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 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 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 bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human DLL4, wherein the first site that binds that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the second antigen-binding site comprises 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 comprise a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human DLL4, wherein the first site that binds that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the second antigen-binding site comprises a heavy chain CDR1 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 comprise a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human DLL4, wherein the first site that binds that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the second antigen-binding site comprises a heavy chain CDR1 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 CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human DLL4, wherein the first site that binds that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO: 19); wherein the second antigen-binding site comprises a heavy chain CDR1 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 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22).
[031] In some embodiments, the bispecific antibody that specifically binds to human VEGF and comprises: a heavy chain variable region that has at least 90% sequence identity to SEQ ID NO:11 and/or a variable region of a light chain that has at least 90% sequence identity to SEQ ID NO:12. In some embodiments, the bispecific antibody specifically binds to human VEGF and comprises: a heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:11 and/or a light chain variable region that has at least 95% sequence identity to SEQ ID NO:12.
In some embodiments, the bispecific antibody specifically binds to human DLL4 and comprises: a heavy chain variable region that has at least 90% 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 that has at least 90% sequence identity to SEQ ID NO:12. In some embodiments, the bispecific antibody specifically binds to human Dll4 and comprises: a heavy chain variable region that has at least 95% 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 that has at least 95% sequence identity to SEQ ID NO:12.
[033] In some embodiments, the bispecific antibody specifically binds to human VEGF and human DLL4 and comprises: (a) a first heavy chain variable region that has at least 90% sequence identity to SEQ ID NO: 11; (b) a second heavy chain variable region that has at least 90% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58 or SEQ ID NO:64; and (c) a first and second heavy chain variable regions that have 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 that has at least 95% sequence identity to SEQ ID NO:11; (b) a second heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:9; and (c) a first and second heavy chain variable regions that have 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 that has at least 95% sequence identity to SEQ ID NO:11; (b) a second heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:10; and (c) a first and second heavy chain variable regions that have 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 that has at least 95% sequence identity to SEQ ID NO:11; (b) a second heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:58; and (c) a first and second heavy chain variable regions that have 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 that has at least 95% sequence identity to SEQ ID NO:11; (b) a second heavy chain variable region that has at least 95% sequence identity to SEQ ID NO:64; and (c) a first and second heavy chain variable regions that have at least 95% sequence identity to SEQ ID NO:12.
[034] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising (a) a first human VEGF binding antigen binding site with a KD between approximately 0.1 nM and approx. approximately 1.0 nM and (b) a second antigen-binding site that specifically binds to human Dll4 with a KD of between approximately 0.1 nM and approximately 20 nM. In certain embodiments, the bispecific antibody comprises two identical light chains.
[035] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody selected from the group consisting of 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75 and 219R45-MB-21R83.
[036] 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 isolated.
[037] In another aspect, the invention provides a polypeptide selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:46, SEQ ID NO :47, SEQ ID NO:48, SEQ ID NO:49, 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 polypeptide is isolated. In certain embodiments, the polypeptide is substantially pure. In certain embodiments, the polypeptide is an antibody or part of an antibody, such as an antibody fragment.
[038] In another aspect, the invention provides isolated polynucleotide molecules comprising a polynucleotide encoding the linkers and/or the polypeptides of each of the aforementioned 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 NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID expression vectors comprising the polynucleotides as well as cells comprising the expression vectors and/or the polynucleotides . In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell.
[039] In other aspects, the invention provides methods of inhibiting the growth of a tumor, which comprise contacting the tumor with an efficient amount of an antibody (or other binding agent) that binds to VEGF, DLL4 or both VEGF and Dll4, including each of such antibodies (or other binding agents) described herein.
[040] In another aspect, the invention provides a method of inhibiting the growth of a tumor in an individual, which comprises administering to the individual a therapeutically effective amount of an antibody (or other binding agent) that binds to VEGF, Dll4, or both VEGF and Dll4, including each of such antibodies (or other binding agents) described herein.
[041] In another aspect, the invention provides a method of modulating angiogenesis in a subject, which comprises administering to the subject a therapeutically effective amount of an antibody (or other binding agent) that binds to VEGF, DLL4 or both VEGF and Dll4, including each of such antibodies (or other binding agents) described herein.
[042] In another aspect, the invention provides a method of reducing the tumorigenicity of a tumor in an individual, which comprises administering to the individual a therapeutically effective amount of an antibody (or other binding agent) that binds to VEGF, Dll4, or both VEGF and Dll4, including each of such antibodies (or other binding agents) described herein.
[043] In another aspect, the invention provides a method of reducing the tumorigenicity of a tumor in an individual by reducing the frequency of cancer stem cells in the tumor, which comprises administering to the individual a therapeutically effective amount of an antibody (or other binding agent) that binds to VEGF, DLL4 or both VEGF and DLL4, including each of such antibodies (or other binding agents) described herein.
[044] In other aspects, the invention provides methods of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody (or other binding agent) that binds to VEGF, DLL4 or both to VEGF and Dll4, including each of such antibodies (or other binding agents) described herein.
[045] Pharmaceutical compositions comprising a binding agent (e.g. antibody) described herein and a pharmaceutically acceptable carrier are additionally provided, as are cell lines that express and/or produce the binding agents. Methods of treating cancer and/or inhibiting tumor growth in a subject (eg, a human) which comprise administering to the subject an effective amount of a composition comprising the binding agents are also provided.
[046] When 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 with the absence of one or more of the group members. The present invention also contemplates the explicit exclusion of one or more of any of the members of the group in the claimed invention. BRIEF DESCRIPTIONS OF THE DRAWINGS
[047] Figure 1. 1A) heavy chain and light chain CDRs of bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21M18, 219R45-MB-21M79, 219R45-MB-21M75 and 219R45-MB-21M83 ; 1B) SEQ ID NOs of the heavy and light chain variable region; 1C) SEQ ID NOs of heavy chain and light chain.
[048] 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 relative fluorescence intensity at 665 nm to relative fluorescence intensity at 620 nm. 219R45-MB-21M18 (-•-); 219R45-MB-21R79 (-■-); 219R45 plus 21M18 (-▲-); 219R45 plus 21R79 (-□-); 219R45 (-▼-); 21M18 (-◊-); 21R79 (-o- ); LZ-1 control antibody (-Δ-).
[049] Figure 3. Inhibition of VEGF-induced proliferation of HUVEC by bispecific anti-VEGF/anti-DLL4 antibodies. Fluorescence intensity is read using an excitation wavelength of 530 nm and an emission wavelength of 590. 219R45-MB-21M18 (-•-); 219R45-MB-21R79 (-▲-); 219R45 (-■-); Medium without VEGF (-◊-).
[050] Figure 4. Inhibition of DLL4-induced Notch signaling by bispecific anti-VEGF/anti-DLL4 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 (-■-); 21M18 (-o-); 21R79 (-□-).
[051] Figure 5. Inhibition of colon tumor growth in vivo by a bispecific anti-VEGF/anti-DLL4 antibody. OMP-C8 colon tumor cells were injected subcutaneously into a human skin graft in NOD/SCID mice. Mice were treated with control antibody (-■-), anti-hDLL4 antibody 21M18 (-▲-), anti-VEGF antibody bevacizumab (-o-) or bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21M18 (-▼-). Data are shown in the form of tumor volume (photons/sec) over the post-treatment days. Antibodies were administered intraperitoneally at a dose of 25mg/kg once a week.
[052] Figure 6. Tumorigenicity of pancreatic tumor cells after treatment with bispecific anti-VEGF/anti-DLL4 antibodies. OMP-PN8 tumor cells from mice treated with control antibody, anti-hDLL4 antibody 21M18, anti-VEGF antibody bevacizumab, or bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21M18 or 219R45-MB-21R79 with or without gemcitabine were processed into isolated 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 without treatment. Data is shown as tumor volume (mm3) at day 55. Tumor frequency is shown as the number of tumors relative to the total number of mice injected in each group.
[053] Figure 7. ELISA with bispecific antibody. Bispecific antibodies 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75 and 219R45-MB-21R83 were diluted in blocking buffer (1x PBS, 0.1% gelatin, 0.1% Polysorbate-20, pH 7.4) containing 2 µg/ml of biotin-DLL4-hFc. Antibodies were serially diluted 3-fold starting from 500 ng/ml to 0.008 ng/ml. Antibody samples were incubated for 2 hours in blocking buffer containing the biotin-DLL4-hFc. After incubation, antibody samples were transferred to a VEGF coated assay plate (100 µl/well) and incubated for 2 hours. Streptavidin-HRP was added to each well and incubated for 1 h. 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 450-650nm and data analyzed using 4-parameter fit within the analysis program Softmax Pro.
[054] Figure 8. Capillary isoelectric focusing with images of bispecific anti-VEGF/anti-DLL4 antibodies.
[055] Figure 9. Inhibition of colon tumor growth by bispecific anti-VEGF/anti-DLL4 antibodies in tumor recurrence model. OMP-C8 colon tumor cells were injected subcutaneously into NOD/SCID mice. Mice were treated with control antibody (-■-), anti-hDLL4 antibody 21M18 (-•-), anti-VEGF antibody bevacizumab (-▲-), a combination of 21M18 and bevacizumab (-▼-), bispecific antibody anti-VEGF/anti-DLL4 219R45-MB-21M18 (-◊-) or bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R79 (-o-), all in combination with irinotecan. Antibodies 21M18 and bevacizumab were administered intraperitoneally at a dose of 7.5 mg/kg once a week, the bispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79 were administered intraperitoneally at a dose of 15 mg/kg once a week and irinotecan was administered for the first 4 weeks at a dose of 45 mg/kg. Data are shown in the form of tumor volume (mm3) over the post-treatment days.
[056] Figure 10. Tumorigenicity of OMP-C3 colon tumor cells after treatment with bispecific anti-VEGF/anti-DLL4 antibodies. Tumors from mice treated with control antibody, anti-hDLL4 antibody 21M18, anti-VEGF antibody bevacizumab, a combination of 21M18 and bevacizumab, or bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21M18 or 219R45-MB-21R79 with or without irinotecan were processed into single cell suspensions and serially transplanted into mice. 150 cells from each treatment group were injected subcutaneously into NOD/SCID mice. Tumors were allowed to grow without treatment. Data are shown as tumor volume (mm3) at day 68.
[057] Figure 11. Inhibition of colon tumor growth in vivo by bispecific anti-VEGF/anti-DLL4 antibodies. OMP-C8 colon tumor cells were injected subcutaneously into NOD/SCID mice. The mice were treated with control antibody (-■-), anti-VEGF antibody bevacizumab (-▲- ) or bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21M18 (-◊-), 219R45-MB-21R75 (-•-), 219R45-MB-21R79 (-o-) or 219R45-MB-21R83 (-▼-). Mice were treated with antibodies in the form of single agents (Fig. 10A) or in combination with irinotecan (Fig. 10B). Antibodies were administered intraperitoneally at a dose of 15 mg/kg once a week and irinotecan at a dose of 7.5 mg/kg once a week. Data are shown in the form of tumor volume (mm3) over the post-treatment days. DETAILED DESCRIPTION OF THE INVENTION
[058] The present invention provides novel binding agents, including but not limited to polypeptides such as antibodies, which bind to 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 producing the VEGF/DLL4 binding agents are also provided. Methods of using novel VEGF/DLL4 binding agents, such as methods of inhibiting tumor growth, methods of treating cancer, methods of reducing the tumorigenicity of a tumor, methods of reducing the frequency of cancer stem cells in a tumor and/or angiogenesis modulation methods are additionally provided.
[059] A monoclonal antibody that specifically binds to human VEGF has been identified, 219R45. This antibody has a binding affinity for human VEGF of approximately 0.67 nM and a binding affinity for mouse VEGF of approximately 23 nM. Several monoclonal antibodies that specifically bind to human DLL4 have been identified, 21R79, 21R75 and 21R83. The 21R79 antibody has a binding affinity for human DLL4 of less than 0.1 nM. Bispecific antibodies that specifically bind to human VEGF and human DLL4 have been produced, 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75 and 219R45-MB-21R83 (CDR sequences in Figure 1). As used herein, the "MB" within an antibody name refers to "monovalent/bispecific". The bispecific antibody 219R45-MB-21M18 has a binding affinity for human VEGF of less than 1.0 nM and a binding affinity for human DLL4 of approximately 16 nM. The 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. The bispecific antibody 219R45-MB-21R75 has a binding affinity for human DLL4 of approximately 5 nM, whereas the bispecific antibody 219R45-MB-21R83 has a binding affinity for human DLL4 of approximately 1 nM. Bispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79 bind mouse VEGF (Example 1, Table 3). Bispecific anti-VEGF/anti-DLL4 antibodies bind human VEGF and human DLL4 simultaneously (Example 2, Figure 2). Bispecific anti-VEGF/anti-DLL4 antibodies inhibit VEGF-induced proliferation of HUVEC cells (Example 3, Figure 3). Bispecific anti-VEGF/anti-DLL4 antibodies inhibit Dll4-induced Notch signaling (Example 4, Figure 4). Bispecific anti-VEGF/anti-DLL4 antibodies inhibit tumor growth (Examples 5, 9, 11 and Figures 5, 9, 11). Bispecific anti-VEGF/anti-DLL4 antibodies inhibit tumorigenicity (Examples 6 and 10 and Figures 6, 10). Bispecific anti-VEGF/anti-DLL4 antibodies bind to both VEGF and DLL4 in a bispecific ELISA (Example 7, Figure 7). Bispecific anti-VEGF/anti-DLL4 antibodies are isolated and purified to a product comprising at least 90% heterodimeric antibody (Example 8, Table 7). I. Definitions
[060] To facilitate an understanding of the present invention, a number of terms and expressions are defined below.
[061] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically recognizes and binds to a target, such as a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or combinations of the above, 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, isolated chain antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), isolated Fv chain antibodies ( scFv), multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins that comprise an antibody antigen-binding site and any other modified immunoglobulin molecule that comprises a site of antigen recognition (ie, antigen-binding site) as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM or subclasses (isotypes) thereof (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on identity of its heavy chain constant domains referred to as alpha, delta, epsilon, gamma and mu, respectively. Different 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.
[062] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single chain antibodies and multispecific antibodies formed starting from antibody fragments. "Antibody fragment" as used herein comprises an antigen-binding site or an epitope-binding site.
[063] The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as "hypervariable regions". The CDRs on each chain are held together in close proximity by the framework regions and, with the CDRs on the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (ie, Kabat et al., 1991, Sequences of Proteins of Immunological 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. Mol. Biol., 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
[064] The term "monoclonal antibody" as used herein refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies which typically include a mixture of different antibodies directed against a variety of different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (eg, Fab, Fab', F(ab')2, Fv), single chain antibodies (scFv), fusion proteins which comprise a portion of the antibody and any other modified immunoglobulin molecule which comprises an antigen recognition site (antigen binding site). Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of techniques, including, but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
[065] The term "humanized antibody" as used herein refers to forms of non-human (eg, 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 from the CDRs are replaced by residues from the CDRs from a non-human species (e.g., mouse, rat, rabbit or hamster) that have the specificity, affinity and/or ability to desired binding (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, Fv framework region residues from a human immunoglobulin are replaced by corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and/or binding capacity. The humanized antibody can be further modified by substituting additional residues in the Fv framework region and/or within the substituted non-human residues to refine and optimize the antibody's specificity, affinity and/or binding capacity. In general, the humanized antibody 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 while all or substantially all of the framework regions are those of a consensus sequence of human immunoglobulin. The humanized antibody may 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, for example, in U.S. Patent 5,225,539.
[066] The term "human antibody" as used herein refers to an antibody produced by a human or an antibody which has an amino acid sequence corresponding to an antibody produced by a human. A human antibody can be produced using any of the techniques known in the art. This definition of a human antibody specifically excludes a humanized antibody that comprises CDRs that are not human.
[067] The term "chimeric antibody" as used herein refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies derived from a mammalian species (e.g. mouse, rat, rabbit etc.) with the desired specificity, affinity and/or binding capacity , while the constant regions correspond to the sequences in antibodies derived from another species (generally human).
[068] The term "affinity-matured antibody" as used herein refers to an antibody with one or more changes in one or more CDRs thereof that result in an improvement in the antibody's affinity for the antigen, compared to a parent antibody that does not has such change(s). The definition also includes changes in non-CDR residues made in association with changes in CDR residues. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., 1992, Bio/Technology 10:779-783, describe affinity maturation by scrambling the VH and VL domains. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., 1994, PNAS, 91:3809-3813; Schier et al., 1995, Gene, 169:147155; 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-directed mutagenesis can also be used to obtain antibodies with matured affinity.
[069] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being specifically recognized and bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed either from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained after protein denaturation, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost after protein denaturation. An epitope typically includes at least 3 and more generally at least 5 or 8-10 amino acids in a unique spatial conformation.
[070] The terms "heteromultimeric molecule" or "heteromultimer" 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 polypeptides or can form higher order tertiary structures in which additional polypeptides are present.
[071] The terms "antagonist" and "antagonistic" as used herein refer to any molecule that blocks, inhibits, reduces or partially or completely neutralizes a biological activity of a target and/or a signaling pathway (eg, the pathway of Notch). The term "antagonist" is used herein to include any molecule that blocks, inhibits, reduces or partially or completely neutralizes the activity of a protein. Suitable antagonist molecules specifically include, but are not limited to, antagonist antibodies or antibody fragments.
[072] The terms "modulation" and "modulate" as used herein refer to a modification or a change in a biological activity. Modulation includes but is not limited to stimulating or inhibiting an activity. Modulation can be an increase or a decrease in activity (for example, a decrease in angiogenesis or an increase in angiogenesis), a modification in binding characteristics or any other modification in the biological, functional or immunological properties associated with the activity of a protein, a pathway or other biological point of interest.
[073] The terms "selectively binds" or "specifically binds" mean that a binding agent or an antibody reacts or associates more frequently, more rapidly, with longer duration, with greater affinity or with some combination than has been described earlier with the epitope, protein or target molecule than with alternative substances, including unrelated proteins. In certain embodiments "specifically binds" means, for example, that an antibody binds to a protein with a KD of approximately 0.1 mM or less, but more generally less than approximately 1 µM. In certain embodiments, "specifically binds" means that an antibody binds to a target at times with a KD of at least approximately 0.1 μM or less, at other times at least approximately 0.01 μM or less, and at other times times of at least approximately 1 nM or less. Due to the sequence identity between homologous proteins in different species, specific binding may include an antibody that recognizes a protein in more than one species (for example, human VEGF and mouse VEGF). Similarly, due to homology within certain regions of polypeptide sequences from different proteins, specific binding may include an antibody (or other polypeptide or binding agent) that recognizes more than one protein (for example, human VEGF-A and VEGF-B human). It is understood that, in certain embodiments, an antibody or binding group that specifically binds to a first target may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require (although it may include) exclusive binding, that is, binding to a single target. Thus, an antibody can, in certain embodiments, specifically bind to more than one target. In certain embodiments, multiple targets can be bound through the same antigen-binding site on the antibody. For example, an antibody may, in certain cases, comprise two identical antigen-binding sites, each of which specifically binds to 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 different specificities. For purposes of non-limiting example, a bispecific antibody may comprise an antigen-binding site that recognizes an epitope on a protein (eg, human VEGF) and further comprise a second, different antigen-binding site that recognizes a different epitope on a second protein (eg, human Dll4). Generally, but not necessarily, reference to binding means specific binding.
[074] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms further encompass an amino acid polymer that has been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, due to the fact that the polypeptides of this invention may be based on antibodies, in certain embodiments, the polypeptides may occur as isolated chains or associated chains.
[075] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, nucleotides or modified bases and/or their analogues or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
[076] "High stringency conditions" can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example, 15 mM sodium chloride/1.5 mM sodium citrate/0, 1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent such as formamide, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 in 5x SSC (0.75 m NaCl, 75 mM sodium citrate) at 42°C; or (3) employ during hybridization 50% formamide in 5x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, salmon sperm DNA treated with ultrasound (50 μg/mL), 0.1% SDS and 10% dextran sulfate 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.
[077] The terms "identical" or percentage of "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 residues of amino acids that are the same when compared and aligned (introducing spaces if necessary) for maximum match, not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using string comparison software or algorithms or through visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments 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% identity of nucleotides or amino acid residues when compared and aligned for maximum match, which is 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 residues in length, or any integral value in between. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least approximately 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.
[078] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been identified in the art, including basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), polar side chains uncharged (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-side chains branched (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). For example, replacing a phenylalanine with a tyrosine is a conservative substitution. Preferably, conservative substitutions in the polypeptide and antibody sequences of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the polypeptide or antibody binds. Methods of identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art.
[079] The term "vector" as used herein means a construct, which is capable of providing, and generally expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, DNA or naked RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA vectors expression of DNA or RNA encapsulated in liposomes.
[080] A polypeptide, an antibody, a polynucleotide, a vector, a cell or a composition that is “isolated” is a polypeptide, an antibody, a polynucleotide, a vector, a cell or a composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to a degree that they are no longer in the form in which they are found in nature. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.
[081] The term "substantially pure" as used herein refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure.
[082] The terms "cancer" and "cancerous" as used herein refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
[083] The terms "tumor" and "neoplasm" as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, benign (non-cancerous) or malignant (cancerous) including precancerous lesions.
[084] The term "metastasis" as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion in a new location. A "metastatic" or "metastatic" cell is one that loses adhesion contacts with neighboring cells and migrates through the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
[085] 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 a tumor that: (1) they have extensive proliferation capacity; 2) are capable of asymmetric cell division to generate one or more types of progeny of differentiated cells in which the differentiated cells have proliferative or reduced developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties give cancer stem cells the ability to form or establish a tumor or cancer after serial transplantation into an immunocompromised host (eg, a mouse) compared to most tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic fashion to form tumors with abnormal cell types that can change over time as mutations occur.
[086] The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from a cancer or a tumor or a precancerous lesion, including both non-tumorigenic cells, which comprise the mass of the cell population tumors or tumorigenic stem cells (cancer stem cells). As used herein, the terms "cancer cell" or "tumor cell" will be changed by the term "non-tumorigenic" when referring only to those cells that do not have the capacity to renew and differentiate to distinguish tumor cells from stem cells cancerous.
[087] The term “tumorigenic” as used herein refers to the functional characteristics 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 origin to differentiated and thus non-tumorigenic tumor cells).
[088] The term "tumorigenicity" as used herein refers to the ability of a random cell sample from the tumor to form palpable tumors after serial transplantation into immunocompromised hosts (eg, mice). This definition further includes enriched and/or isolated cancer stem cell populations that form palpable tumors after serial transplantation into immunocompromised hosts (eg, mice).
[089] The term "individual" refers to any animal (eg, a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which shall be the recipient of a private treatment. Typically, the terms "individual" and "patient" are used interchangeably herein in reference to a human individual.
[090] The term "pharmaceutically acceptable" refers to a product or compound approved (or approved) by a regulatory agency of the Federal Government or a state government or listed on the US Pharmacopeia or other pharmacopoeia generally recognized for use in animals , including humans.
[091] The terms "pharmaceutically acceptable excipient, vehicle or adjuvant" or "pharmaceutically acceptable carrier" refer to an excipient, a vehicle or an adjuvant that can be administered to an individual, together with at least one binding agent (e.g., an antibody) of the present disclosure and which does not destroy the activity of the binding agent. The excipient, vehicle or adjuvant must be non-toxic when administered with a binding agent in doses sufficient to provide a therapeutic effect.
[092] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a binding agent, an antibody, a polypeptide, a polynucleotide, a small organic molecule or other drug efficient to "treat" a disease or disorder in an individual or a mammal. In the case of cancer, the therapeutically effective amount of a drug (eg, an antibody) has a therapeutic effect and as such reduces the number of cancer cells; decreases tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduces the number or frequency of cancer stem cells; reduces tumor size; reduces the population of cancer cells; inhibit and/or stop the infiltration of cancer cells into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibits and/or stops metastasis of tumor or cancer cells; inhibits and/or stops the growth of tumor or cancer cells; relieves to some extent one or more of the symptoms associated with cancer; reduces morbidity and mortality; improves quality of life; or a combination of such effects. To the extent that the agent, for example an antibody, prevents the growth and/or kills existing cancer cells, it may be referred to as cytostatic and/or cytotoxic.
[093] The terms "treating" or "treatment" or "treating" or "relieving" or "relieving" refer either to 1) therapeutic measures that cure, delay, lessen the symptoms of and/or halt the progression of a diagnosed health state or pathological disorder in terms of 2) prophylactic or preventative measures that prevent or delay the development of a targeted health state or pathological disorder. So those in need of treatment include those who already have the disorder; those likely to have the disorder; and those in which the disorder must be prevented. In some embodiments, an individual is successfully "treated" in accordance with the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of metastasis of tumor or cancer cells; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.
[094] As used in the present disclosure and the claims, the singular forms "a", "an", "the" and "a" include the plural forms unless the context clearly dictates otherwise.
[095] It is understood that wherever modalities are described here with the language "comprising(s)" otherwise homologous modalities described in terms of "consisting of" and/or "consisting essentially of" are also provided. It is also understood that wherever modalities are described here with the language "consisting essentially of" otherwise homologous modalities described in terms of "consisting of" are also provided.
[096] The term "and/or" as used in an expression such as "A and/or B" is intended herein to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and/or" as used in an expression such as "A, B and/or C" is intended to encompass each of the following modalities: 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
[097] The present invention provides agents that specifically bind to human VEGF proteins and/or human Dll4 proteins. These agents are referred to herein as "VEGF/DLL4 binding agents". The term "VEGF/DLL4 binding agent" encompasses agents that bind only to VEGF, agents that bind only to DLL4, and bispecific agents that bind to both VEGF and DLL4. In certain embodiments, in addition to specifically binding to VEGF and/or DLL4, the VEGF/DLL4 binding agents specifically additionally bind at least one additional 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 to human VEGF. In certain embodiments, the VEGF/DLL4 binding agent specifically binds to human DLL4. In certain embodiments, the VEGF/DLL4 binding agent is a bispecific antibody. In certain embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that specifically binds to human VEGF and human DLL4. The full-length (aa) amino acid sequences for human VEGF (VEGF-A) and for human DLL4 are known in the art and are provided herein as SEQ ID NO:27 (VEGF) and SEQ ID NO:23 (DLL4).
[098] In certain embodiments, the VEGF/DLL4 binding agent or antibody binds to VEGF and/or DLL4 with a dissociation constant (KD) of approximately 1 µM or less, approximately 100 nM or less, approximately 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds VEGF and/or DLL4 with a KD of approximately 20 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds VEGF and/or DLL4 with a KD of approximately 10 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds VEGF and/or DLL4 with a KD of approximately 1 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds VEGF and/or DLL4 with a KD of approximately 0.1 nM or less. In some embodiments, the VEGF/DLL4 binding agent binds to both human VEGF and mouse VEGF with a KD of approximately 100 nM or less. In some embodiments, the VEGF/DLL4 binding agent binds to both human VEGF and mouse VEGF with a KD of approximately 50 nM or less. In some embodiments, a VEGF/DLL4 binding agent binds to both human and mouse DLL4 with a KD of approximately 100 nM or less. In some embodiments, a VEGF/DLL4 binding agent binds to both human DLL4 and mouse DLL4 with a KD of approximately 50 nM or less. In some embodiments, the dissociation constant of the binding agent (e.g., an antibody) to VEGF is the dissociation constant determined using a VEGF fusion protein that comprises at least a portion of VEGF immobilized 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 that comprises at least a portion of Dll4 immobilized on a Biacore chip.
[099] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first antigen-binding site that specifically binds to VEGF and a second antigen-binding site that specifically binds to DLL4 . In some embodiments, a VEGF/DLL4 binding agent or antibody binds to both VEGF and DLL4 with a KD of approximately 100 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds to both VEGF and DLL4 with a KD of approximately 50 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds to both VEGF and DLL4 with a KD of approximately 20 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds to both VEGF and DLL4 with a KD of approximately 10 nM or less. In some embodiments, a VEGF/DLL4 binding agent or antibody binds to both VEGF and DLL4 with a KD of approximately 1 nM 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 approximately 1 nM and the KD of the second antigen-binding site may be approximately 10 nM. In some embodiments, the difference in affinity between the two antigen binding sites can be approximately 2-fold or more, approximately 3-fold or more, approximately 5-fold or more, approximately 8-fold or greater, approximately 10-fold or greater, approximately 15 times or more, about 20 times or more, about 30 times or more, about 50 times or more, or about 100 times or more. The modulation of the affinities of the two antigen-binding sites can affect the biological activity of the bispecific antibody. For example, decreasing the affinity of the antigen binding site for Dll4 or for VEGF can have a desirable effect, for example, lower toxicity of the binding agent or higher therapeutic index.
[0100] By way of non-limiting example, the bispecific antibody may comprise (a) a first antigen-binding site that binds to human VEGF with a KD between approximately 0.1 nM and approximately 1.0 nM and ( b) a second antigen-binding site that specifically binds to human Dll4 with a KD between approximately 0.1 nM and approximately 20 nM, between approximately 0.5 nM and approximately 20 nM, between approximately 1.0 nM and 10 nM. In certain embodiments, the bispecific antibody comprises two identical light chains.
[0101] In certain embodiments, the VEGF/DLL4 binding agent (for example, an antibody) binds VEGF and/or DLL4 with a mean maximum efficient concentration (EC50) of approximately 1 µM or less, approximately 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In certain embodiments, a VEGF/DLL4 binding agent (e.g., an antibody) binds VEGF and/or DLL4 with a mean maximum efficient concentration (EC50) of approximately 1 µM or less, approximately 100 nM or less, approximately 40 nM or less, approximately 20 nM or less, approximately 10 nM or less, approximately 1 nM or less, or approximately 0.1 nM or less.
[0102] In certain embodiments, the VEGF/DLL4 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 IgG1 antibody. In certain embodiments, the antibody is an IgG2 antibody. In certain embodiments, the antibody is an antibody fragment that comprises an antigen-binding site. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is monovalent, monospecific, bivalent, or multispecific. In some embodiments, the antibody is conjugated to a cytotoxic group. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.
[0103] The VEGF/DLL4 binding agents (e.g., antibodies) of the present invention can be analyzed for specific binding by any method known in the art. Immunological assays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA, assay sandwich immunological assay, immunoprecipitation assay, precipitation reaction, precipitin reaction with gel diffusion, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, time-revealed fluorescence assay homogeneous (HTRF) and protein A immunoassay. Such assays are routine and well known in the art (see, for example, Ausubel et al., Editors, 1994-present, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. , New York, NY).
[0104] For example, the specific binding of an antibody to human VEGF and/or human DLL4 can be determined using ELISA. An ELISA assay comprises preparing the antigen, coating the wells of a 96-well microtiter plate with the antigen, adding the antibody or other binding agent conjugated to a detectable compound such as an enzyme substrate (eg, peroxidase horseradish or alkaline phosphatase) to the well, incubation for a period of time and detection of the presence of the binding agent bound to the antigen. In some embodiments, the binding agent or antibody is not conjugated to a detectable compound, but rather a second antibody that recognizes 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, rather than coating the well with the antigen, the binding agent or antibody can be coated onto the well and a second antibody conjugated to a detectable compound can be added after adding the antigen to the coated well. One skilled in the art would be educated as to parameters that can be modified to increase the detected signal as well as other variations of ELISAs known in the art.
[0105] In another example, the specific binding of an antibody to human VEGF and/or human DLL4 can be determined using FACS. A FACS verification assay may comprise producing a cDNA construct that expresses an antigen as a fusion protein, transfection of the construct into cells, expression of the antigen on the surface of the cells, mixing the agent of binding or antibody with the transfected cells and incubation for a period of time. Cells bound by the binding agent or antibody can be identified through the use of a secondary antibody conjugated to a detectable compound (eg, PE-conjugated anti-Fc antibody) and a flow cytometer. One skilled in the art would be educated regarding parameters that can be modified to optimize the detected signal as well as other FACS variations that can improve verification (e.g., verification against blocking antibodies).
[0106] The binding affinity of an antibody or other binding agent to an antigen (eg, VEGF or DLL4) and the “off-rate” of an antibody-antigen interaction can be determined through competitive binding assays . An example of a competitive binding assay is a radioimmunoassay which comprises the incubation of labeled antigen (eg 3H or 125I) or fragment or variant thereof with the antibody of interest in the presence of increasing amounts of unlabeled antigen followed by detection of antibody bound to labeled antigen. Antibody affinity for antigen and binding def-rates can be determined from the data by Scatchard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the binding and off rates of antibodies or agents that bind to an antigen (eg, VEGF or DLL4). Biacore kinetic analysis comprises the analysis of binding and dissociation of antibodies from chips with immobilized antigen (eg, VEGF or DLL4) on their surface.
[0107] In certain embodiments, the invention provides a VEGF binding agent (for example an antibody) that specifically binds to human VEGF, wherein the VEGF binding agent (for example an antibody) comprises one, two , three, four, five and/or six of the CDRs of the 219R45 antibody (see Table 1). In some embodiments, the VEGF binding agent comprises one or more of the 219R45 CDRs, two or more of the 219R45 CDRs, three or more of the 219R45 CDRs, four or more of the 219R45 CDRs, five or more of the 219R45 CDRs or all six CDRs of 219R45. In some embodiments, the VEGF binding agent binds to human VEGF and mouse VEGF.

[0108] In certain embodiments, the invention provides a VEGF binding agent (e.g., an antibody) that specifically binds to human VEGF, wherein the VEGF binding agent comprises a heavy chain CDR1 that comprises 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 VEGF binding agent further comprises a light chain CDR1 which comprises RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the VEGF binding agent comprises: (a) a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18) and a CDR3 of heavy chain comprising HYDDKYYPLMDY (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 CDR3 of a light chain comprising QQSKEVPWTFGG (SEQ ID NO:22).
[0109] In certain embodiments, the invention provides a VEGF binding agent (e.g., an antibody) that specifically binds to human VEGF, wherein the VEGF binding agent comprises: (a) a heavy chain CDR1 which comprises NYWMH (SEQ ID NO:17) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 which comprises DINPSNGRTSYKEKFKR (SEQ ID NO:18) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 which comprises HYDDKYYPLMDY (SEQ ID NO:19) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20) or a variant thereof comprising 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, amino acid substitutions are conservative substitutions.
[0110] In certain embodiments, the invention provides a VEGF binding agent (e.g., an antibody) that specifically binds to VEGF, wherein the VEGF binding agent comprises a heavy chain variable region having at least approximately 80% sequence identity to SEQ ID NO:11 and a light chain variable region that has at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the VEGF binding agent comprises a heavy chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence with respect to SEQ ID NO:11. In certain embodiments, the VEGF binding agent comprises a light chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:12. In certain embodiments, the VEGF binding agent comprises a heavy chain variable region that has at least about 95% sequence identity to SEQ ID NO:11 and a light chain variable region that has at least about 95% of sequence identity to SEQ ID NO:12. In certain embodiments, the VEGF binding agent 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, the VEGF binding agent comprises a heavy chain variable 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 comprises a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:8. In some embodiments, the antibody that binds to VEGF or another agent comprises a heavy chain comprising SEQ IDNO:7 and a light chain comprising SEQ ID NO:8.
[0111] In some embodiments, the VEGF binding agent binds to VEGF with a KD of approximately 10 nM or less. In some embodiments, the VEGF binding agent binds VEGF with a KD of approximately 1 nM or less. In some embodiments, the VEGF binding agent binds VEGF with a K D of approximately 0.1 nM or less. In some embodiments, the VEGF binding agent binds to VEGF with a K D of approximately 0.01 nM or less. In some embodiments, at least one amino acid residue in at least one CDR of the VEGF binding agent is replaced with a different amino acid such that the affinity of the VEGF binding agent for VEGF is altered. In some embodiments, the affinity of the binding agent for VEGF is increased. In some embodiments, the affinity of the binding agent for VEGF is decreased. In some embodiments, the VEGF binding agent binds to human VEGF. In some embodiments, the VEGF binding agent binds to human VEGF and mouse VEGF.
[0112] In certain embodiments, the VEGF binding agent comprises the heavy chain variable region and the light chain variable region of the 219R45 antibody. In certain embodiments, the VEGF binding agent comprises the heavy chain and the light chain of the 219R45 antibody (with or without the leader sequence). In certain embodiments, a VEGF binding agent is the 219R45 antibody.
[0113] In certain embodiments, a VEGF binding agent comprises, consists essentially of, or consists of, the 219R45 antibody.
[0114] In certain embodiments, a VEGF binding agent (for example, an antibody) binds to the same 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 to an epitope on VEGF that overlaps the epitope on VEGF bound by an antibody of the invention. In certain embodiments, a VEGF binding agent (e.g., an antibody) binds to the same or essentially the same epitope, on VEGF as the 219R45 antibody. In another embodiment, the VEGF binding agent is an antibody that binds to an epitope on VEGF that overlaps the epitope on VEGF bound by the 219R45 antibody.
[0115] In some embodiments, the VEGF binding agent inhibits the binding of VEGF to at least one VEGF receptor. In certain embodiments, the VEGF binding agent inhibits the binding of human VEGF to VEGFR-1 or VEGFR-2. In some embodiments, the VEGF-binding agent specifically binds to VEGF and modulates angiogenesis. In some embodiments, the VEGF-binding agent specifically binds to VEGF and inhibits angiogenesis. In some embodiments, the VEGF binding agent specifically binds to VEGF and inhibits tumor growth.
[0116] In certain embodiments, the invention provides a DLL4 binding agent (for example an antibody) that specifically binds to human DLL4, wherein the DLL4 binding agent (for example an antibody) comprises one, two , three, four, five and/or six of the CDRs of the 21R79 antibody (see Table 2). In some embodiments, the DLL4 binding agent comprises one or more of the 21R79 CDRs, two or more of the 21R79 CDRs, three or more of the 21R79 CDRs, four or more of the 21R79 CDRs, five or more of the 21R79 CDRs or all six CDRs of 21R79. 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 the 21R75 antibody (see Table 2). In some embodiments, the DLL4 binding agent comprises one or more of the 21R75 CDRs, two or more of the 21R75 CDRs, three or more of the 21R75 CDRs, four or more of the 21R75 CDRs, five or more of the 21R75 CDRs or all six CDRs of 21R75. In certain embodiments, the invention provides a DLL4 binding agent (for example an antibody) that specifically binds to human DLL4, wherein the DLL4 binding agent (for example an antibody) comprises one, two, three, four, five and/or six of the CDRs of the 21R83 antibody (see Table 2). In some embodiments, the DLL4 binding agent comprises one or more of the 21R83 CDRs, two or more of the 21R83 CDRs, three or more of the 21R83 CDRs, four or more of the 21R83 CDRs, five or more of the 21R83 CDRs or all six CDRs of 21R83. In some embodiments, the DLL4 binding agent binds to human DLL4 and mouse DLL4.

[0117] In certain embodiments, the heavy chain CDR1 of the antibody that binds DLL4 is a minimal HC CDR1 that comprises AYYIH (SEQ ID NO:79).
[0118] In some embodiments, the binding agent is an antibody that binds to human DLL4 and comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a CDR2 of heavy chain comprising YIX1X2YX3X4ATNYNQKFKG (SEQ ID NO:80), where 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 chain CDR3 heavy comprising 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).
[0119] 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 heavy chain CDR1 that comprises 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 CDR1 which comprises RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4 binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14) and a CDR3 of heavy chain comprising RDYDYDVGMDY (SEQ ID NO:16) and (b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (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 CDR1 that comprises TAYYIH (SEQ ID NO:13) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 which comprises YIANYNRATNYNQKFKG (SEQ ID NO:14) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 which comprises RDYDYDVGMDY (SEQ ID NO:16) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20) or a variant thereof comprising 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, amino acid substitutions are conservative substitutions.
[0121] 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 approximately 80% sequence identity to SEQ ID NO:10 and a light chain variable region that has at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the Dll4 binding agent comprises a heavy chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:10. In certain embodiments, the Dll4 binding agent comprises a light chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:12. In certain embodiments, the DLL4 binding agent comprises a heavy chain variable region that has at least about 95% sequence identity to SEQ ID NO:10 and a light chain variable region that has at least about 95% of sequence identity 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 variable 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 variable 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 antibody that binds DLL4 or another agent comprises a heavy chain comprising SEQ ID NO:6 and a light chain comprising SEQ ID NO:8. In some embodiments, the antibody is a bispecific antibody.
[0122] 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 heavy chain CDR1 that comprises 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 CDR1 which comprises RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4 binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59) and a CDR3 of heavy chain comprising RDYDYDVGMDY (SEQ ID NO:16) and (b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of a light chain comprising QQSKEVPWTFGG (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 CDR1 that comprises TAYYIH (SEQ ID NO:13) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 which comprises YIAGYKDATNYNQKFKG (SEQ ID NO:59) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 which comprises RDYDYDVGMDY (SEQ ID NO:16) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20) or a variant thereof comprising 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, amino acid substitutions are conservative substitutions.
[0124] 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 approximately 80% sequence identity to SEQ ID NO:58 and a light chain variable region that has at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the Dll4 binding agent comprises a heavy chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:58. In certain embodiments, the Dll4 binding agent comprises a light chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:12. In certain embodiments, the DLL4 binding agent comprises a heavy chain variable region that has at least about 95% sequence identity to SEQ ID NO:58 and a light chain variable region that has at least about 95% of 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 certain embodiments, the DLL4 binding agent comprises a heavy chain variable region consisting essentially of SEQ ID NO:58 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 NO:8.
[0125] 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 heavy chain CDR1 that comprises 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). In some embodiments, the DLL4 binding agent further comprises a light chain CDR1 which comprises RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4 binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65) and a CDR3 of heavy chain comprising RDYDYDVGMDY (SEQ ID NO:16) and (b) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of a light chain comprising QQSKEVPWTFGG (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 CDR1 that comprises TAYYIH (SEQ ID NO:13) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 which comprises YISNYNRATNYNQKFKG (SEQ ID NO:65) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 which comprises RDYDYDVGMDY (SEQ ID NO:16) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20) or a variant thereof comprising 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 which comprises AASNQGS (SEQ ID NO:21) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22) or a variant thereof which comprises 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, amino acid substitutions are conservative substitutions.
[0127] 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 approximately 80% sequence identity to SEQ ID NO:64 and a light chain variable region that has at least 80% sequence identity to SEQ ID NO:12. In certain embodiments, the Dll4 binding agent comprises a heavy chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence with respect to SEQ ID NO:64. In certain embodiments, the Dll4 binding agent comprises a light chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity of sequence relative to SEQ ID NO:12. In certain embodiments, the DLL4 binding agent comprises a heavy chain variable region that has at least about 95% sequence identity to SEQ ID NO:64 and a light chain variable region that has at least about 95% of 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 embodiments, 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.
[0128] In some embodiments, the DLL4 binding agent is an antibody comprising a heavy chain comprising SEQ ID NO:5 and a light chain comprising SEQ ID NO:8. In some embodiments, the antibody is a bispecific antibody.
[0129] In some embodiments, the DLL4 binding agent binds to DLL4 with a KD of 25 nM or less. In some embodiments, the DLL4 binding agent binds to DLL4 with a KD of 10 nM or less. In some embodiments, the DLL4 binding agent binds DLL4 with a KD of approximately 1 nM or less. In some embodiments, the DLL4 binding agent binds DLL4 with a KD of approximately 0.1 nM or less. In some embodiments, the DLL4 binding agent binds DLL4 with a KD of approximately 0.01 nM or less. In some embodiments, at least one amino acid residue in at least one CDR of the DLL4 binding agent is replaced with a different amino acid such that the affinity of the DLL4 binding agent for the DLL4 is changed. In some embodiments, the affinity of the DLL4 binding agent is increased. In some embodiments, the affinity of the DLL4 binding agent is decreased.
[0130] In certain embodiments, the Dll4 binding agent comprises the heavy chain variable region and the light chain variable region of the 21R79 antibody. In certain embodiments, the Dll4 binding agent comprises the heavy chain and light chain of the 21R79 antibody (with or without the leader sequence). In certain embodiments, the Dll4 binding agent is the 21R79 antibody.
[0131] 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.
[0133] In certain embodiments, a DLL4 binding agent comprises, consists essentially of, or consists of, the antibody 21R75.
[0134] 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 embodiments, 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.
[0135] In certain embodiments, a DLL4 binding agent comprises, consists essentially of, or consists of, the 21R83 antibody.
[0136] In some embodiments, a DLL4 binding agent binds to an N-terminal fragment of human DLL4 (amino acids 1-191 of SEQ ID NO:24). In some embodiments, the Dll4 binding agent binds to an epitope comprising amino acids 40-47 of SEQ ID NO:25. In some embodiments, the Dll4 binding agent binds to an epitope comprising amino acids 113-120 of SEQ ID NO:25. In some embodiments, the DLL4 binding agent binds to an epitope comprising amino acids 40-47 of SEQ ID NO:25 and amino acids 113-120 of SEQ ID NO:25.
[0137] In certain embodiments, a DLL4 binding agent (e.g., an antibody) binds to the same 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 to an epitope on DLL4 that overlaps the epitope on DLL4 bound by an antibody of the invention. In certain embodiments, a Dll4 binding agent (e.g., an antibody) binds to the same or essentially the same epitope, on Dll4 as the 21R79 antibody. In another embodiment, the DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R79 antibody. In certain embodiments, a DLL4 binding agent (e.g., an antibody) binds to the same or essentially the same epitope, on DLL4 as the 21R75 antibody. In another embodiment, the DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R75 antibody. In certain embodiments, a DLL4 binding agent (e.g., an antibody) binds to the same or essentially the same epitope, on DLL4 as the 21R83 antibody. In another embodiment, the DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R83 antibody.
[0138] In some embodiments, the DLL4 binding agent inhibits DLL4 from binding to at least one Notch receptor. In certain modes, the Notch receiver is Notch1, Notch2, Notch3 or Notch4. In some embodiments, the DLL4 binding agent specifically binds to DLL4 and inhibits DLL4 activity. In some embodiments, the DLL4 binding agent specifically binds DLL4 and inhibits 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 inhibits 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.
[0139] In certain embodiments, the invention provides a binding agent for VEGF/DLL4 which is a bispecific antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site that specifically binds to human VEGF. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that binds to a target associated with the tumor. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds to human VEGF, wherein the first antigen-binding site comprises a chain CDR1 heavy chain 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 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 VEGF/DLL4 binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds to human VEGF, wherein the first antigen-binding site 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 comprising HYDDKYYPLMDY (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 QQSKEVPWTFGG (SEQ ID NO:22).
[0140] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:11. In some embodiments, the bispecific antibody further comprises a light chain variable region that has 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 that has at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about about 99% sequence identity to SEQ ID NO:11 and a light chain variable region that has at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about about 99% sequence identity to SEQ ID NO:12.
[0141] In certain embodiments, the invention provides a binding agent for VEGF/DLL4 which is a bispecific antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site that specifically binds to human DLL4. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first antigen-binding site that specifically binds to human DLL4 and a second antigen-binding site that binds to a target associated with the tumor. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds to human DLL4, wherein the first antigen-binding site comprises a chain CDR1. heavy chain 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 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 VEGF/DLL4 binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds to human DLL4, wherein the first antigen-binding site comprises a chain CDR1. heavy which comprises TAYYIH (SEQ ID NO:13), a heavy chain CDR2 which comprises YIANYNRATNYNQKFKG (SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59) or YISNYNRATNYNQK NO:65) and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific antibody comprises a first antigen-binding site that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13), a heavy chain CDR2 that comprises 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 that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13), a heavy chain CDR2 that comprises YISSYNGATNYNQKFKG (SEQ ID NO:15) and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific antibody comprises a first antigen-binding site that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13), a heavy chain CDR2 that comprises YIAGYKDATNYNQKFKG (SEQ ID NO:59) and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, the bispecific antibody comprises a first antigen-binding site that comprises a heavy chain CDR1 that comprises TAYYIH (SEQ ID NO:13), a heavy chain CDR2 that comprises 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 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 VEGF/DLL4 binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds to 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. which comprises AASNQGS (SEQ ID NO:21) and a light chain CDR3 which comprises QQSKEVPWTFGG (SEQ ID NO:22).
[0142] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first heavy chain variable region that has at least approximately 80% sequence 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 that has 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 that has at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 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 that has 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 to human VEGF and human DLL4. In some embodiments, the bispecific antibody comprises: a) a first antigen-binding site that specifically binds to human VEGF and b) a second antigen-binding site that specifically binds to human 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 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 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 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, a bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site that binds to human VEGF that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO: 19) and the second 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); 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22).
[0144] In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to antigen binding 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 comprising HYDDKYYPLMDY (SEQ ID NO:19 ) and the second antigen-binding site comprises 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 comprise a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R79.
[0145] In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to antigen binding 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 comprising HYDDKYYPLMDY (SEQ ID NO:19 ) and the second antigen-binding site comprises a heavy chain CDR1 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 comprise a light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21) and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody is 219R45-MB-21M18.
[0146] In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site that binds binds to antigen comprising 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 the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59) and a heavy chain CDR3 comprising comprises 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R75.
[0147] In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to antigen binding 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 comprising HYDDKYYPLMDY (SEQ ID NO:19 ) and the second antigen-binding site comprises a heavy chain CDR1 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 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R83.
[0148] In some embodiments, the VEGF/DLL4 binding agent (e.g., a bispecific antibody) comprises a first heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:11 , a second heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58 or SEQ ID NO:64, and a first and a second heavy chain variable regions which have 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 that has at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about about 99% sequence identity to SEQ ID NO:11; a second heavy chain variable region that has 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 a first and second heavy chain variable regions that have at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to the SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4 binding agent comprises a first heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:11, a second heavy chain variable region which has at least approximately 95% sequence identity to SEQ ID NO:9 and a first and second heavy chain variable regions which have at least approximately 95% sequence identity to SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4 binding agent comprises a first heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:10 and a first and second heavy chain variable regions which have at least approximately 95% sequence identity to SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4 binding agent comprises a first heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:58 and a first and second heavy chain variable regions which have at least approximately 95% sequence identity to SEQ ID NO:12. In certain embodiments, the bispecific VEGF/DLL4 binding agent comprises a first heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 95% sequence identity to SEQ ID NO:64 and a first and second heavy chain variable regions which have at least approximately 95% sequence identity to 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 variable region comprising SEQ ID NO:9, and a first and a second light chain variable regions 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 variable region comprising SEQ ID NO:10, and a first and a second light chain variable regions 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 variable region comprising SEQ ID NO:58, and a first and a second light chain variable regions 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 variable region comprising SEQ ID NO:64, and a first and a second light chain variable regions 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 regions 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:11, a second heavy chain variable region consisting essentially of SEQ ID NO:10, and a first and a second light chain variable regions 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:11, a second heavy chain variable region consisting essentially of SEQ ID NO:58, and a first and a second light chain variable regions 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:11, a second heavy chain variable region consisting essentially of SEQ ID NO:64, and a first and a second light chain variable regions consisting essentially of SEQ ID NO:12.
[0149] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain variable region from the anti-VEGF antibody 219R45. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain variable region from the 21M18 anti-DLL4 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain variable region from the anti-DLL4 antibody 21R79. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain variable region from the 21R75 anti-DLL4 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain variable region from the anti-DLL4 antibody 21R83. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain variable region from anti-VEGF antibody 219R45, a heavy chain variable region from anti-DLL4 antibody 21R79, and two variable regions from identical light chain. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain variable region from anti-VEGF antibody 219R45, a heavy chain variable region from anti-DLL4 antibody 21M18, and two variable regions from identical light chain. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain variable region from anti-VEGF antibody 219R45, a heavy chain variable region from anti-DLL4 antibody 21R75, and two variable regions from identical light chain. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain variable region from anti-VEGF antibody 219R45, a heavy chain variable region from anti-DLL4 antibody 21R83, and two variable regions from identical light chain.
[0150] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first CH3 domain and a second CH3 domain, each of which is modified to promote the formation of heteromultimers. In some modalities, the first and second CH3 domains are modified using a “knobs-into-holes” technique. In some embodiments, the first and second CH3 domains comprise amino acid changes that result in altered electrostatic interactions. In some embodiments, the first and second CH3 domains comprise amino acid changes that result in altered hydrophobic/hydrophilic interactions.
[0151] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising heavy chain constant regions selected from the group consisting of: (a) a first human IgG1 constant region, wherein the amino acids at positions 253 and 292 are replaced by glutamate or aspartate and a second human IgG1 constant region, where amino acids at positions 240 and 282 are replaced by lysine; (b) a first human IgG2 constant region, in which the amino acids at positions 249 and 288 are replaced by glutamate or aspartate, and a second human IgG2 constant region, where the amino acids at positions 236 and 278 are replaced by lysine; (c) a first human IgG3 constant region, wherein the amino acids at positions 300 and 339 are replaced by glutamate or aspartate, and a second human IgG3 constant region, where the amino acids at positions 287 and 329 are replaced by lysine; and (d) a first human IgG4 constant region, in which the amino acids at positions 250 and 289 are replaced by glutamate or aspartate, and a second human IgG4 constant region, where the amino acids at positions 237 and 279 are replaced by lysine.
[0152] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first human IgG1 constant region with amino acid substitutions at positions 253 and 292, where the amino acids are glutamate or aspartate and a second human IgG1 constant region with amino acid substitutions at positions 240 and 282, where the amino acids are lysine. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first human IgG2 constant region with amino acid substitutions at positions 249 and 288, where the amino acids are glutamate or aspartate and a second constant region of human IgG2 with amino acid substitutions at positions 236 and 278, where the amino acids are lysine. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first human IgG3 constant region with amino acid substitutions at positions 300 and 339, where the amino acids are glutamate or aspartate and a second constant region of human IgG2 with amino acid substitutions at positions 287 and 329, where the amino acids are lysine. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first human IgG4 constant region with amino acid substitutions at positions 250 and 289, where the amino acids are glutamate or aspartate and a second constant region of human IgG4 with amino acid substitutions at positions 237 and 279, where the amino acids are lysine.
[0153] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a first human IgG2 constant region with amino acid substitutions at positions 249 and 288, where the amino acids are glutamate and a second region human IgG2 constant with amino acid substitutions at positions 236 and 278, where the amino acids are lysine. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first human IgG2 constant region with amino acid substitutions at positions 249 and 288, where the amino acids are asparate and a second constant region. of human IgG2 with amino acid substitutions at positions 236 and 278, where the amino acids are lysine.
[0154] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain of SEQ ID NO:7. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain of SEQ ID NO:5. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain of SEQ ID NO:56. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a heavy chain of SEQ ID NO:62. In some embodiments, the bispecific antibody further comprises a light chain of SEQ ID NO:12. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising one heavy chain of SEQ ID NO:7, one heavy chain of SEQ ID NO:5, and two light chains of SEQ ID NO:8. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising a heavy chain of SEQ ID NO:7, a heavy chain of SEQ ID NO:6, and two light chains of SEQ ID NO:8. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising one heavy chain of SEQ ID NO:7, one heavy chain of SEQ ID NO:56, and two light chains of SEQ ID NO:8. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody comprising one heavy chain of SEQ ID NO:7, one heavy chain of SEQ ID NO:62, and two light chains of SEQ ID NO:8.
[0155] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds to VEGF with a KD of approximately 50 nM or less, approximately 25 nM or less, approximately 10 nM or less, approximately 1 nM or less than or approximately 0.1 nM or less. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds DLL4 with a KD of about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less or about 0.1 nM or less. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds to VEGF with a KD of approximately 50 nM or less and binds to DLL4 with a KD of approximately 50 nM or less. In some embodiments, the bispecific antibody binds to VEGF with a KD of approximately 25 nM or less and binds to DLL4 with a KD of approximately 25 nM or less. In some embodiments, the bispecific antibody binds to VEGF with a KD of approximately 10 nM or less and binds to DLL4 with a KD of approximately 10 nM or less. In some embodiments, the bispecific antibody binds to VEGF with a KD of approximately 1 nM or less and binds to DLL4 with a KD of approximately 1 nM or less.
[0156] In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises an antigen-binding site with a binding affinity that is weaker than the binding affinity of the second antigen-binding site. For example, in some embodiments, the bispecific antibody can bind to VEGF with a KD ranging from approximately 0.1 nM to 1 nM and can bind to DLL4 with a KD ranging from approximately 1 nM to 10 nM. Or the bispecific antibody can bind VEGF with a KD ranging from approximately 1 nM to 10 nM and can bind DLL4 with a KD ranging from approximately 0.1 nM to 1 nM. In some embodiments, the bispecific antibody can bind Dll4 with a KD ranging from approximately 0.1 nM to 1 nM and can bind VEGF with a KD ranging from approximately 1 nM to 10 nM. Or the bispecific antibody can bind DLL4 with a KD ranging from approximately 1 nM to 10 nM and can bind VEGF with a KD ranging from approximately 0.1 nM to 1 nM. In some embodiments, the difference in affinity between the two antigen-binding sites can be approximately 2-fold or more, approximately 3-fold or more, approximately 5-fold or more, approximately 8-fold or more, approximately 10-fold or more , about 15 times or more, about 30 times or more, about 50 times or more, or about 100 times or more. In some embodiments, at least one amino acid residue in at least one CDR of the antigen binding site for VEGF is replaced with a different amino acid such 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 replaced with a different amino acid such that the affinity of the Dll4 binding site is changed. In some embodiments, 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 the antigen-binding sites for both VEGF and DLL4 are altered.
[0157] The invention provides polypeptides, including, but not limited to antibodies, that specifically bind to VEGF and/or DLL4. In some embodiments, a polypeptide binds to human VEGF. In some embodiments, a polypeptide binds to human Dll4. In some embodiments, a polypeptide binds to human VEGF and mouse VEGF. In some embodiments, a polypeptide binds to human Dll4 and mouse Dll4.
[0158] In some embodiments, a VEGF binding agent comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:47 and SEQ ID NO:49.
[0159] In some embodiments, a 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 NO:4, SEQ ID NO:5 , SEQ NO ID:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, 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.
[0160] 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 NO:3, SEQ ID NO :4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 , SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, 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.
[0161] 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 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 further comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO: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:4, SEQ ID NO:8 and SEQ ID NO:12.
[0162] In some embodiments, a VEGF/DLL4 binding agent comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO: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 further 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.
[0163] In certain embodiments, a VEGF/DLL4 binding agent (e.g. antibody) competes for specific binding to VEGF with an antibody comprising a heavy chain variable region comprising SEQ ID NO:11 and a variable region of a light chain comprising SEQ ID NO:12. In certain embodiments, a VEGF/DLL4 binding agent competes with the 219R45 antibody for specific binding to human VEGF. In some embodiments, a VEGF/DLL4 binding agent or antibody competes for specific binding to VEGF in an in vitro competitive binding assay. In some embodiments, VEGF is human VEGF. In some embodiments, VEGF is mouse VEGF.
[0164] In certain embodiments, a VEGF-DLL4 binding agent (for example, an antibody) binds to the same 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 to an epitope on VEGF that overlaps the epitope on VEGF bound by an antibody of the invention. In certain embodiments, a VEGF/DLL4 binding agent (e.g., an antibody) binds to the same or essentially the same epitope, on VEGF as the 219R45 antibody. In another embodiment, the VEGF/DLL4 binding agent is an antibody that binds to an epitope on VEGF that overlaps the epitope on VEGF bound by the 219R45 antibody.
[0165] In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of VEGF with the 219R45 antibody (for example, in a competitive binding assay).
[0166] In certain embodiments, a VEGF/DLL4 binding agent (e.g. antibody) competes for specific binding to DLL4 with an antibody comprising a heavy chain variable region comprising SEQ ID NO:9, SEQ ID NO :10, SEQ ID NO:58 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 the 21R79 antibody for specific binding to human DLL4. In certain embodiments, a VEGF/DLL4 binding agent competes with the 21R75 antibody for specific binding to human DLL4. In certain embodiments, a VEGF/DLL4 binding agent competes with the 21R83 antibody for specific binding to human DLL4. In some embodiments, a VEGF/DLL4 binding agent or antibody competes for specific binding to DLL4 in an in vitro competitive binding assay. In some embodiments, DLL4 is human DLL4. In some embodiments, DLL4 is mouse DLL4.
[0167] In certain embodiments, a VEGF/DLL4 binding agent (for example, an antibody) binds to the same or essentially the same epitope, on DLL4 as an antibody of the invention. In another embodiment, a VEGF/DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by an antibody of the invention. In certain embodiments, a VEGF/DLL4 binding agent binds to the same or essentially the same epitope, on DLL4 as the 21R79 antibody. In certain embodiments, a VEGF/DLL4 binding agent binds to the same or essentially the same epitope, on DLL4 as the 21R75 antibody. In certain embodiments, a VEGF/DLL4 binding agent binds to the same or essentially the same epitope, on Dll4 as the 21R83 antibody. In another embodiment, the VEGF/DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R79 antibody. In another embodiment, the VEGF/DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R75 antibody. In another embodiment, the VEGF/DLL4 binding agent is an antibody that binds to an epitope on DLL4 that overlaps the epitope on DLL4 bound by the 21R83 antibody.
[0168] In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of DLL4 with the 21R79 antibody (for example, in a competitive binding assay). In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of DLL4 with the 21R75 antibody (e.g., in a competitive binding assay). In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of DLL4 with the 21R83 antibody (e.g., in a competitive binding assay). In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of DLL4 with the 21M18 antibody (e.g., in a competitive binding assay).
[0169] In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of VEGF and/or DLL4 with the bispecific antibody 219R45-MB-21M18 (for example, in a competitive binding assay) . In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of VEGF and/or DLL4 with the bispecific antibody 219R45-MB-21M79 (e.g., in a competitive binding assay). In certain embodiments, the VEGF/DLL4 binding agent is an agent that competes for specific binding of 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 specific binding of VEGF and/or DLL4 with the bispecific antibody 219R45-MB-21M83 (e.g., in a competitive binding assay).
[0170] In certain embodiments, the VEGF/DLL4 binding agent (eg, 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 angiogenesis. 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.
[0171] In certain embodiments, a VEGF/DLL4 binding agent (eg, an antibody) described herein binds to 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 antagonist and modulates angiogenesis. In some embodiments, a VEGF/DLL4 binding agent is a DLL4 antagonist and promotes aberrant angiogenesis. In some embodiments, a VEGF/DLL4 binding agent is a DLL4 antagonist and inhibits tumor growth.
[0172] In certain embodiments, a VEGF/DLL4 binding agent (e.g., an antibody) described herein is a bispecific antibody that binds to 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 to human DLL4 and modulates DLL4 activity. In certain embodiments, a VEGF/DLL4 binding agent (e.g., an antibody) described herein is a bispecific antibody that binds to human VEGF and human DLL4 and modulates both VEGF and DLL4 activity. In some embodiments, the bispecific antibody is a VEGF antagonist and a Dll4 antagonist and inhibits both VEGF activity and Dll4 activity. In some embodiments, the bispecific antibody is a VEGF antagonist and a Dll4 antagonist and inhibits VEGF activity and Notch activity. In some embodiments, the bispecific antibody is a VEGF antagonist 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.
[0173] In certain embodiments, the VEGF/DLL4 binding agent (for example, an antibody or a bispecific antibody) is a VEGF antagonist. In some embodiments, the VEGF/DLL4 binding agent is a VEGF antagonist and inhibits 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 approximately 90% or approximately 100%. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human VEGF activity is the 219R45 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human VEGF activity is a bispecific antibody that comprises 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.
[0174] In certain embodiments, the VEGF/DLL4 binding agent (e.g., an antibody) is a Dll4 antagonist. In some embodiments, the VEGF/DLL4 binding agent is a DLL4 antagonist and inhibits DLL4 activity. In certain 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 approximately 90% or approximately 100%. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the 21R79 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the 21R75 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the 21R83 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human Dll4 activity is a bispecific antibody that comprises the antigen-binding site of 21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is a bispecific antibody that comprises the antigen-binding site of 21R75. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is a bispecific antibody that comprises the antigen-binding site of 21R83. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the bispecific antibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the bispecific antibody 219R45-MB-21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the bispecific antibody 219R45-MB-21R75. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human DLL4 activity is the bispecific antibody 219R45-MB-21R83.
[0175] In certain embodiments, the VEGF/DLL4 binding agent (eg, antibody) is an antagonist of Notch 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 approximately 90% or approximately 100%. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is the 21R79 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is the 21R75 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is the 21R83 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is a bispecific antibody that comprises the antigen-binding site of 21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is a bispecific antibody that comprises the antigen-binding site of 21R75. In certain embodiments, a VEGF/DLL4 binding agent that inhibits Notch signaling is a bispecific antibody that comprises 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 219R45-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.
[0176] In certain embodiments, the VEGF/DLL4 binding agent (e.g., antibody) inhibits the binding of VEGF to at least one receptor. In some embodiments, the VEGF/DLL4 binding agent inhibits the binding of VEGF to VEGFR-1 or VEGFR-2. In certain embodiments, the VEGF/DLL4 binding agent inhibits VEGF binding 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 minus about 90% or at least about 95%. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human VEGF to at least one VEGF receptor is the 219R45 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human VEGF to at least one VEGF receptor is a bispecific antibody that comprises the antigen-binding site of 219R45. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human VEGF binding to at least one VEGF receptor is the bispecific antibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4 binding agent that inhibits human VEGF binding to at least one VEGF receptor is the bispecific antibody 219R45-MB-21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding 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 human VEGF binding to at least one VEGF receptor is the bispecific antibody 219R45-MB-21R83.
[0177] In certain embodiments, the VEGF/DLL4 binding agent (eg, antibody) inhibits the binding of DLL4 protein to at least one Notch receptor. In some embodiments, the VEGF/DLL4 binding agent inhibits the binding of DLL4 to Notch1, Notch2, Notch3 and/or Notch4. In certain embodiments, the VEGF/DLL4 binding agent inhibits the binding of DLL4 to at least one Notch receptor by at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least minus about 90% or at least about 95%. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is antibody 21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is the 21R75 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is the 21R83 antibody. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is a bispecific antibody that comprises the antigen-binding site of 21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is a bispecific antibody that comprises the antigen-binding site of 21R75. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is a bispecific antibody that comprises the antigen-binding site of 21R83. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the 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 the binding of human DLL4 to at least one Notch receptor is the bispecific antibody 219R45-MB-21R79. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is the bispecific antibody 219R45-MB-21R75. In certain embodiments, a VEGF/DLL4 binding agent that inhibits the binding of human DLL4 to at least one Notch receptor is the bispecific antibody 219R45-MB-21R83.
[0178] In vivo and in vitro assays for determining the fact that a VEGF/DLL4 binding agent (or candidate VEGF/DLL4 binding agent) inhibits VEGF or affects angiogenesis are known in the art. In vitro angiogenesis assays include, but are not limited to, HUVEC proliferation assays, endothelial cell tube formation assays, budding (or budding) assays, HUVEC cell migration assays, and HUVEC cell migration assays. invasion. In some embodiments, cells in the presence of VEGF and in the presence of a VEGF/DLL4 binding agent are compared to cells in the presence of VEGF without the VEGF/DLL4 binding agent present and evaluated for effects on angiogenesis (or biological effects associated with angiogenesis). In vivo angiogenesis assays include, but are not limited to, matrigel block assays, corneal micropocket assays, and chicken chorioallantoic membrane (CAM) assays.
[0179] In vivo and in vitro assays for determining the fact that a VEGF/DLL4 binding agent (or candidate VEGF/DLL4 binding agent) inhibits Notch activation or signaling are known in the art. For example, cell-based luciferase reporter assays using a TCF/Luc reporter vector containing multiple copies of the TCF binding domain upstream of a firefly luciferase reporter gene can be used to measure signaling levels of Notch in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica MA). In some embodiments, a cell-based luciferase reporter assay using a CBF/Luc reporter vector containing multiple copies of the CBF binding domain upstream of a firefly luciferase reporter gene can be used. The level of Notch signaling in the presence of one or more Notch ligands (eg Dll4 expressed on the surface of transfected cells or soluble Dll4-Fc fusion protein) and in the presence of a VEGF/DLL4 binding agent is compared to the level of Notch signaling without the VEGF/DLL4 binding agent present.
[0180] In certain embodiments, VEGF/DLL4 binding agents have one or more of the following effects: inhibit tumor cell proliferation, inhibit tumor growth, reduce tumorigenicity of a tumor, reduce stem cell frequency cells in a tumor, activate cell death of tumor cells, prevent metastasis of tumor cells, decrease tumor cell survival, modulate angiogenesis, inhibit angiogenesis, inhibit productive angiogenesis, or promote aberrant angiogenesis.
[0181] In certain embodiments, VEGF/DLL4 binding agents are capable of inhibiting tumor growth. In certain embodiments, VEGF/DLL4 binding agents are capable of inhibiting tumor growth in vivo (eg, in a xenograft mouse model and/or in a human who has cancer). In certain embodiments, tumor growth is inhibited at least about twice, about three times, about five times, about ten times, about 50 times, about 100 times or about 1000 times when compared to an untreated tumor.
[0182] In certain embodiments, VEGF/DLL4 binding agents are capable of reducing the tumorigenicity of a tumor. In certain embodiments, the binding agent or antibody to VEGF/DLL4 is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse xenograft model. In certain embodiments, the binding agent or antibody to VEGF/DLL4 is capable of reducing the tumorigenicity of a tumor by decreasing the number or frequency 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 approximately twice, approximately three times, approximately five times, approximately ten times, approximately 50 times, approximately 100 times, or approximately 1000 times. 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 guidelines 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, for example, in International Publication Number WO 2008/042236; U.S. Patent Publication No. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.
[0183] In certain modalities, VEGF/DLL4 binding agents are able to modulate angiogenesis. In certain embodiments, VEGF/DLL4 binding agents are able to modulate angiogenesis in vivo (eg, in a xenograft mouse model and/or in a human who has cancer). In certain embodiments, VEGF/DLL4 binding agents are capable of inhibiting angiogenesis. In certain modalities, VEGF/DLL4 binding agents are capable of promoting aberrant angiogenesis. In certain modalities, VEGF/DLL4 binding agents are able to inhibit angiogenesis and/or promote aberrant angiogenesis, leading to non-productive vascularization.
[0184] In certain embodiments, the VEGF/DLL4 binding agents described herein have a circulating half-life in mice, cynomolgus monkeys, or humans of at least approximately 2 hours, at least approximately 5 hours, at least approximately 10 hours, at least at least approximately 24 hours, at least approximately 3 days, at least approximately 1 week, or at least approximately 2 weeks. In certain embodiments, the VEGF/DLL4 binding agent is an IgG antibody (eg, IgG1 or IgG2) 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 antibodies include introducing mutations in the Fc region that increase the binding of the pH-dependent antibody to the neonatal Fc receptor (FcRn) at pH 6.0 (see, by example, US Patent Publication Nos. 2005/0276799, 2007/0148164 and 2007/0122403). Known methods of increasing the circulating half-life of antibody fragments that lack the Fc region include techniques such as PEGylation.
[0185] In some embodiments, VEGF/DLL4 binding agents are antibodies. Polyclonal antibodies can be prepared by any known method. In some embodiments, polyclonal antibodies are produced by immunizing an animal (eg, a rabbit, rat, mouse, goat, donkey) with an antigen of interest (eg, a purified peptide fragment, a full-length recombinant protein or a fusion protein) via various subcutaneous or intraperitoneal injections. The antigen may optionally be conjugated to a vehicle such as keyhole limpet hemocyanin (KLH) or serum albumin. The antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (eg, 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, usually from blood or ascites. Polyclonal antibodies can be purified from serum or ascites according to methods standard in the art including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.
[0186] In some embodiments, VEGF/DLL4 binding agents are monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods known to one skilled in the art (see, for example, Kohler and Milstein, 1975, Nature, 256:495-497). In some embodiments, using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to activate lymphocyte production of antibodies that specifically bind to 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.
[0187] After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized means that are known in the art and lymphocytes and unfused myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies specifically directed against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (eg, flow cytometry, FACS, ELISA and radioimmunoassay). Hybridomas can be propagated in cultures in vitro using standard methods (J.W. Goding, 1996, Monoclonal Antibodies: Principles and Practice, 3rd Edition, Academic Press, San Diego, CA) or in vivo as ascites tumors in an animal. Monoclonal antibodies can be purified from culture medium or ascites fluid according to methods standard in the art including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.
[0188] In certain embodiments, monoclonal antibodies can be produced using recombinant DNA techniques that are known to one of skill in the art. Polynucleotides encoding a monoclonal antibody are isolated starting from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the antibody heavy and light chains and their sequence is determined using standardized techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors that produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin proteins.
[0189] In certain other embodiments, recombinant monoclonal antibodies, or fragments thereof, can be isolated from phage display libraries that express variable domains or CDRs from a desired species (see, e.g., McCafferty et al., 1990, Nature , 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).
[0190] The polynucleotide(s) encoding a monoclonal antibody can be modified, for example, through the use of recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains, for example, of a mouse monoclonal antibody may be replaced by those regions, for example, of a human antibody to generate a chimeric antibody or by a non-immunoglobulin polypeptide for generate a fusion antibody. In some embodiments, constant regions are truncated or removed to generate the desired antibody fragment from a monoclonal antibody. Site-directed or high-density variable region mutagenesis can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
[0191] In some embodiments, a monoclonal antibody against VEGF and/or DLL4 is a humanized antibody. Typically, humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced by residues from a CDR from a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the specificity, affinity and /or the desired binding capacity using methods known to one skilled in the art. In some embodiments, Fv framework region residues from a human immunoglobulin are replaced with corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and/or binding capacity. In some embodiments, a humanized antibody can be further modified by substituting additional residues in the Fv framework region and/or within the substituted non-human residues to refine and optimize the antibody's specificity, affinity, and/or capacity. 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 while 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 immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. In certain embodiments, such humanized antibodies are used therapeutically because they can 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 reduced immunogenicity following known techniques (see, for example, U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; and 5,693,762).
[0192] In certain embodiments, the VEGF/DLL4 binding agent is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. In some embodiments, human antibodies can be generated starting from immortalized human B lymphocytes immunized in vitro or starting from lymphocytes isolated from an immunized individual. In either case, cells that produce an antibody directed against a target antigen can be generated and isolated (see, for example, Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boemer et al., 1991, J. Immunol., 147:86-95; 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, wherein the phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; 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, starting from immunoglobulin variable domain gene repertoires from unimmunized donors. Techniques for generating and using 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 the antibodies are identified, affinity maturation strategies known in the art, including, but not limited to, chain shuffling (Marks et al., 1992, Bio/Technology, 10:779-783) and site-directed mutagenesis , can be used to generate high-affinity human antibodies.
[0193] In some embodiments, human antibodies can be produced in transgenic mice that contain human immunoglobulin loci. After immunization these mice are able to produce the complete 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; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0194] This invention further encompasses bispecific antibodies. Bispecific antibodies are able to recognize and specifically bind to at least two different antigens or epitopes. Different epitopes can be within the same molecule (eg two epitopes on a single protein) or on different molecules (eg an epitope on one protein and an epitope on a second protein). In some embodiments, a bispecific antibody has greater potency compared to an individual antibody or a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity when compared to an individual antibody or a combination of more than one antibody. It is known to those skilled in the art that any binding agent (eg antibody) may have unique pharmacokinetics (PK) (eg circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents where the two individual binding agents have different PK profiles. In some embodiments, a bispecific antibody has the ability to focus the actions of two binding agents (eg, antibodies) in a common area (eg, a tumor and/or a tumor environment). In some embodiments, a bispecific antibody has the ability to focus the actions of two binding agents (eg, antibodies) to a common target (eg, a tumor or a tumor cell). In some embodiments, a bispecific antibody has the ability to direct the actions of two binding agents (eg, antibodies) to more than one biological pathway or function.
[0195] In certain embodiments, the bispecific antibody specifically binds to VEGF and a second target. In certain embodiments, the bispecific antibody specifically binds DLL4 and a second target. In certain embodiments, the bispecific antibody specifically binds to VEGF and DLL4. In some embodiments, the bispecific antibody specifically binds to human VEGF and human Dll4. In some embodiments, the bispecific antibody is a human or humanized monoclonal antibody. In some modalities, the bispecific antibody inhibits angiogenesis and reduces the number or frequency of cancer stem cells. In some embodiments, the bispecific antibody inhibits blood vessel growth and inhibits blood vessel maturation. In some embodiments, the bispecific antibody prevents endothelial hyperproliferation. In some embodiments, the bispecific antibody has less toxicity and/or side effects. In some embodiments, the bispecific antibody has less toxicity and/or side effects when compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, the bispecific antibody has a higher therapeutic index. In some embodiments, the bispecific antibody has a greater therapeutic index when compared to a mixture of the two individual antibodies or antibodies as single agents.
[0196] In some embodiments, the bispecific antibody may specifically recognize and bind to a first antigen target, (eg, DLL4) as well as a second antigen target, such as an effector molecule on a leukocyte (eg, CD2 , CD3, CD28 or B7) or an Fc receptor (eg CD64, CD32 or CD16) in order to centralize cellular defense mechanisms in the cell expressing the first antigen target. In some embodiments, bispecific antibodies can be used to target cytotoxic agents to cells that express a particular target antigen. These antibodies have an antigen-binding site (eg, human Dll4) and a second site that binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA or TETA.
Techniques for producing bispecific antibodies are known to 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. WO 2009/089004; and U.S. Patent Publication No. 2011/0123532. In some embodiments, bispecific antibodies comprise heavy chain constant regions with amino acid modifications that form part of the interface between the two heavy chains. In some embodiments, bispecific antibodies can be generated using a "knobs-into-holes" strategy (see, for example, US Patent No. 5,731,168; Ridgway et al., 1996, Prot. Engin., 9:617 -621). From time to time the terminology “knobs” and “holes” is replaced by the terms “bulges” and “cavities”. In some embodiments, bispecific antibodies may comprise variant hinge regions incapable of forming disulfide bonds between heavy chains (see, for example, WO 2006/028936). In some embodiments, the modifications can comprise amino acid changes that result in altered electrostatic interactions. In some embodiments, the modifications can comprise amino acid changes that result in altered hydrophobic/hydrophilic interactions.
[0198] Bispecific antibodies can be intact antibodies or antibody fragments that comprise antigen-binding sites. Antibodies with more than two valences are also considered. For example, trispecific antibodies can be prepared (Tutt et al., 1991, J. Immunol., 147:60). Thus, in certain embodiments the antibodies to VEGF and/or Dll4 are multispecific.
[0199] In certain embodiments, the antibodies (or other polypeptides) described herein may be monospecific. In certain embodiments, each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binding to) a homologous epitope on different proteins.
[0200] In certain embodiments, the VEGF/DLL4 binding agent is an antibody fragment. Antibody fragments may have different functions or capabilities than intact antibodies; for example, antibody fragments may have greater 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 an F(ab')2 fragment produced by pepsin digestion of an antibody molecule. In some embodiments, antibody fragments include a Fab fragment generated by reducing the disulfide bonds of an F(ab')2 fragment. In other embodiments, antibody fragments include a Fab fragment generated by treating the antibody molecule with papain and a reducing agent. In certain embodiments, antibody fragments are produced recombinantly. In some embodiments, antibody fragments include single-chain Fv or Fv (scFv) fragments. Fab, Fv and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, allowing the production of large quantities of these fragments. In some embodiments, antibody fragments are isolated from antibody phage libraries that are discussed herein. For example, the methods can be used to construct Fab expression libraries (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity by VEGF and/or by 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 VEGF/DLL4 binding agent is an scFv. Various techniques can be used to produce single chain antibodies specific for VEGF or DLL4 (see, for example, U.S. Patent No. 4,946,778).
[0201] It may be additionally desirable, especially in the case of antibody fragments, to modify an antibody for the purpose of altering (eg, increasing or decreasing) its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope within the antibody fragment by mutating the appropriate region in the antibody fragment or by incorporating the epitope within a peptide tag that is then fused to the antibody fragment at either end or in the middle (eg, by DNA or peptide synthesis).
[0202] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune cells to unwanted cells (see, for example, U.S. Patent No. 4,676,980). It is also contemplated that heteroconjugate antibodies can be prepared 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 through the formation of a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
[0203] For the purposes of the present invention, it should be considered that modified antibodies can comprise any type of variable region that allows the association of the antibody with the target (i.e., human VEGF or human DLL4). In this regard, the variable region can 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, monkeys, etc.) or rabbit origin. In some modalities, both the variable and constant regions of the modified immunoglobulins are human. In other embodiments, variable regions of compatible antibodies (generally derived from a non-human source) can be engineered or specifically custom-made to enhance the binding properties or reduce the immunogenicity of the molecule. In this regard, the variable regions useful in the present invention can be humanized or otherwise altered by the inclusion of imported amino acid sequences.
[0204] In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by replacement of the partial framework region and sequence modification and/or alteration . Although CDRs may be derived from an antibody of the same class or even subclass as that of the antibody from which the framework regions are derived, it is considered that CDRs may be derived from an antibody of a different class and often from an antibody of a different species. . It may not be necessary to replace all CDRs with all CDRs in the donor variable region to transfer antigen-binding capacity from one variable domain to another. Instead, it may only be necessary to transfer those residues that are needed to maintain the activity of the antigen-binding site.
[0205] Variable region changes notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full length antibodies or immunoreactive fragments thereof) wherein at least a fraction of one or more of the region domains constant has been deleted or otherwise altered in order to provide desired biochemical characteristics such as increased tumor localization or increased serum half-life when compared to an antibody of approximately the same immunogenicity that comprises a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Constant region modifications compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. The modified antibodies disclosed herein may comprise alterations or modifications in one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or the light chain constant domain (CL). In some embodiments, one or more domains are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the omitted constant region domain is replaced with a short amino acid spacer (eg, 10 amino acid residues) that provides some of the molecular flexibility typically conferred by the absent constant region.
[0206] In some embodiments, the modified antibodies are engineered to fuse the CH3 domain directly to the antibody hinge region. In other embodiments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, constructs can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a spacer of 5-20 amino acids. Such a spacer can be added to ensure that the constant domain regulatory elements remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and trigger an unwanted immune response against the construct. Consequently, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic in order to maintain the desired biological qualities of the modified antibodies.
[0207] In some embodiments, modified antibodies may have only a partial deletion of a constant domain or substitution of some or even a single amino acid. For example, mutation of a single amino acid in selected areas of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, it may be desirable to simply delete the part of one or more constant region domains that control a specific effector function (eg, complement C1q binding) that will be modulated. Such partial constant region deletions can enhance selected antibody features (serum half-life) while leaving other desirable functions associated with the individual constant region domain intact. Furthermore, as hinted at above, the constant regions of the disclosed antibodies can be modified by mutating or substituting one or more amino acids that improve the profile of the resulting construct. In this regard it may be possible to interrupt the activity provided by a conserved binding site (eg Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. In certain embodiments, modified antibodies comprise the addition of one or more amino acids in the constant region to enhance desirable characteristics such as decreasing or increasing effector function or providing more cytotoxin or carbohydrate binding sites.
[0208] It is known in the art that the constant region mediates various effector functions. For example, binding the C1 component of complement to the Fc region of IgG or IgM (antigen-bound) antibodies activates the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind to a cell that expresses an Fc receptor (FcR). There are a number of Fc receptors that are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). Antibody binding to Fc receptors on cell surfaces activates a number of important and distinct biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by cells killer (called antibody-dependent cellular cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.
[0209] In certain embodiments, the modified antibodies provide altered effector functions which, in turn, affect the biological profile of the administered antibody. For example, in some embodiments, deletion or inactivation (through point mutations or other means) of a constant region domain can reduce circulating modified antibody Fc receptor binding thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, modifications to the constant region increase the serum half-life of the antibody. In other embodiments, modifications to the constant region reduce the serum half-life of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide groups. Constant region modifications in accordance with this invention can be readily produced using well known biochemical or molecular engineering techniques known to those skilled in the art.
[0210] In certain embodiments, a VEGF/DLL4 binding agent that is an antibody lacks one or more effector functions. For example, in some embodiments, the antibody has no ADCC activity and/or no complement dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind an Fc receptor and/or complement factors. In certain embodiments, the antibody has no effector function.
[0211] The present invention further comprises variants and equivalents that are substantially homologous to the chimeric, humanized and human antibodies or antibody fragments thereof presented herein. These can contain, for example, conservative substitution mutations, i.e. the replacement of one or more amino acids with similar amino acids. For example, conservative substitution refers to the replacement of one amino acid with another amino acid within the same general class such as, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid. by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and is described herein.
[0212] Thus, the present invention provides methods for producing an antibody that binds to VEGF and/or DLL4, including bispecific antibodies that specifically bind to both VEGF and DLL4. In some embodiments, the method for producing an antibody that binds to VEGF and/or DLL4 comprises the use of hybridoma techniques. In some embodiments, the method of producing an antibody that binds to VEGF or DLL4 or a bispecific antibody that binds to VEGF and DLL4 comprises screening a human phage library. The present invention further provides methods of identifying an antibody that binds to VEGF and/or DLL4. In some embodiments, the antibody is identified by scanning through FACS for binding to VEGF or a portion thereof. In some embodiments, the antibody is identified by checking through FACS for binding to DLL4 or a portion thereof. In some embodiments, the antibody is identified by scanning through FACS for either VEGF or DLL4 or a portion thereof. In some embodiments, the antibody is identified by checking using an ELISA for binding to VEGF. In some embodiments, the antibody is identified by checking using an ELISA for binding to DLL4. In some embodiments, the antibody is identified by checking using an ELISA for binding to VEGF and DLL4. In some embodiments, the antibody is identified by scanning by FACS for blocking the binding of human VEGF to a human VEGF receptor. In some embodiments, the antibody is identified by scanning through FACS for blocking the binding of human Dll4 to a human Notch receptor. In some embodiments, the antibody is identified by checking for inhibition or blocking of Notch signaling. In some embodiments, the antibody is identified by screening for inhibition or blocking of VEGF activity (e.g., induction of HUVEC proliferation). In some embodiments, the antibody is identified by screening for modulation of angiogenesis.
[0213] 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 generating an antibody to human VEGF comprises immunizing a mammal with a polypeptide that comprises 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 embodiments, a method of generating a monoclonal antibody that binds to VEGF comprises: immunizing a mammal with a polypeptide comprising at least a portion of amino acids 27232 of human VEGF and isolating antibody-producing cells from the immunized mammal . In some embodiments, the method further comprises fusing the antibody-producing cells with cells of a myeloma cell lineage to form hybridoma cells. In some embodiments, the method further comprises selecting a hybridoma cell that expresses an antibody that binds to VEGF. In certain embodiments, the mammal is a mouse. In some embodiments, the antibody is selected using a polypeptide that comprises at least a portion of amino acids 27-232 of human VEGF.
[0214] 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 that comprises at least a portion of amino acids 27-529 of human Dll4. In some embodiments, a method of generating a monoclonal antibody that binds to Dll4 comprises: immunizing a mammal with a polypeptide comprising at least a portion of amino acids 27-529 of human Dll4 and isolating the antibody-producing cells from the immunized mammal. In some embodiments, the method further comprises fusing the antibody-producing cells with cells of a myeloma cell lineage to form hybridoma cells. In some embodiments, the method further comprises selecting a hybridoma cell that expresses an antibody that binds to DLL4. In certain embodiments, the mammal is a mouse. In some embodiments, the antibody is selected using a polypeptide that comprises at least a portion of amino acids 27-529 of human Dll4.
[0215] In some embodiments, a method of generating an antibody to human VEGF comprises screening an antibody expression library for antibodies that bind to human VEGF. In some embodiments, a method of generating a human DLL4 antibody comprises screening an antibody expression library for antibodies that bind to human DLL4. In some embodiments, a method of generating an antibody to human VEGF and/or human DLL4 comprises screening an antibody expression library for bispecific antibodies that bind to human VEGF and human DLL4. In some embodiments, the antibody expression library is a phage library. In some embodiments, verification comprises separation. In some embodiments, the antibody expression library (e.g., a phage library) is verified using at least a portion of amino acids 27-232 of human VEGF. In some embodiments, the antibodies identified in the first screening are rechecked using at least a portion of amino acids 27-529 of human Dll4 to identify a bispecific antibody that binds to VEGF and Dll4. In some embodiments, the antibody expression library (e.g., a phage library) is verified using at least a portion of amino acids 27-529 of human DLL4. In some embodiments, the antibodies identified in the first screening are rechecked using at least a portion of amino acids 27-232 of human VEGF to identify a bispecific antibody that binds to VEGF and DLL4. In some embodiments, the antibody identified in the discovery is a VEGF antagonist. In some embodiments, the antibody identified in the scan inhibits VEGF-induced biological activities. In some embodiments, the antibody identified in the discovery is a Dll4 antagonist. In some embodiments, the antibody identified in the scan inhibits DLL4-induced Notch signaling. In some embodiments, the antibody identified in the scan binds to both human and mouse VEGF. In some embodiments, the antibody identified in the scan binds to both human and mouse DLL4.
[0216] In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.
[0217] In some embodiments of the present invention, VEGF/DLL4 binding agents are polypeptides. Polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides that comprise an antibody or a fragment thereof that binds to VEGF and/or DLL4. It will be recognized in the art that some amino acid sequences of the linkers described herein can be varied without significant effect on protein structure or function. Thus, the invention further includes variations of polypeptides that exhibit substantial activity, or that include regions of an antibody, or a fragment thereof, against human VEGF and/or Dll4. In some embodiments, amino acid sequence variations of polypeptides that bind VEGF/DLL4 include deletions, insertions, inversions, repeats, and/or other types of substitutions.
[0218] In some embodiments, the polypeptides described here are isolated. In some embodiments, the polypeptides described herein are substantially pure.
[0219] Polypeptides, analogs and variants thereof, can be further modified to contain additional chemical groups that are not normally part of the polypeptide. Derivatized groups can improve or otherwise modulate the solubility, biological half-life and/or absorption of the polypeptide. Clusters can also reduce or eliminate unwanted side effects of polypeptides and variants. An overview of the chemical clusters can be found in Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, University of the Sciences, Philadelphia, PA.
[0220] The polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence that encodes polypeptide sequences and expressing such sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence that encodes a wild-type protein of interest. Optionally, the sequence can be subjected to mutagenesis through site-specific mutagenesis to provide functional analogues of the sequence. See, for example, Zoeller et al., 1984, PNAS, 81:5662-5066 and U.S. Patent No. 4,588,585.
[0221] In some embodiments, a DNA sequence encoding a polypeptide of interest can be constructed through chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting the codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence that encodes an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. In addition, a DNA oligomer containing a nucleotide sequence that encodes the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides that encode portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' hanging ends for complementary assembly.
[0222] Once assembled (through synthesis, site-directed mutagenesis or other method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operably linked with a sequence appropriate expression control 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 operably linked to transcriptional and translational expression control sequences that are functional in the host. chosen expression.
[0223] In certain embodiments, recombinant expression vectors are used to amplify and express the DNA encoding antibodies or fragments thereof, against human VEGF and/or DLL4. For example, recombinant expression vectors can be replicable DNA constructs that contain synthetic DNA fragments or cDNA derivatives that encode a polypeptide chain of a VEGF/DLL4 binding agent, such as an anti-VEGF antibody or an antibody anti-DLL4 or a fragment thereof, operably linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcription unit generally comprises an assembly of (1) a genetic element or elements that have a regulatory function in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated. in protein and (3) appropriate transcription and translation start and stop sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication and a selection gene to facilitate recognition of transformants, can be further incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if the latter is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of translated protein by a host cell. In other embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
[0224] The choice of an expression control sequence and an expression vector depends on the choice of the host. A wide variety of host/expression vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as E. coli plasmids, including pCR1, pBR322, pMB9 and its derivatives, and plasmids with broader host range, such as M13 and other single-stranded DNA phages filamentous.
[0225] The VEGF/DLL4 binding agents (for example, polypeptides) of the present invention can be expressed starting from one or more vectors. For example, in some embodiments, a 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 are expressed by one vector and a second heavy chain polypeptide is expressed by a second vector. In some embodiments, two heavy chain polypeptides are expressed by one vector and one light chain polypeptide is expressed by a second vector. In some embodiments, three polypeptides are expressed starting from a 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.
[0226] Suitable host cells for expression of a polypeptide or an antibody that binds to VEGF/DLL4 (or a VEGF or DLL4 protein for use as an antigen) include prokaryotes, yeast cells, insect cells or eukaryotic cells superiors under the control of appropriate prosecutors. Prokaryotes include gram-negative or gram-positive organisms, for example, E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin which are described below. Cell-free translation systems can also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are described in Pouwels et al., 1985, Cloning Vectors: A Laboratory Manual, Elsevier, New York, NY. Additional information regarding protein production methods, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008/0187954; U.S. Patent Nos. 6,413,746; 6,660,501; and International Patent Publication No. WO 04/009823.
[0227] Various mammalian or insect cell culture systems can be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable 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) cell lines , 3T3 (derived from murine fibroblasts), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from kidney fibroblasts from hamster), HEK-293 (derived from human embryonic kidney) 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' untranslated sequences or 3', such as required ribosome binding sites, a polyadenylation site, processing donor and receptor sites, and transcription termination sequences. The expression of recombinant proteins in baculovirus also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art (see, for example, Luckow and Summers, 1988, Bio/Technology, 6:47).
[0228] Thus, the present invention provides cells that comprise the VEGF/DLL4 binding agents described herein. In some embodiments, the cells produce the VEGF/DLL4 binding agents described herein. In certain embodiments, cells produce an antibody. In some embodiments, the cells produce a VEGF binding agent, such as an anti-VEGF antibody. In some embodiments, cells produce a bispecific antibody that binds to VEGF. In some embodiments, the cells produce a DLL4 binding agent, such as an anti-DLL4 antibody. In some embodiments, cells produce a bispecific antibody that binds to DLL4. In certain embodiments, the cells produce a bispecific binding agent for VEGF/DLL4, such as a bispecific antibody that binds to VEGF and DLL4. In certain embodiments, cells produce the 219R45 antibody. In certain embodiments, cells produce the 21R79 antibody. In certain embodiments, cells produce the 21R75 antibody. In certain embodiments, cells produce the 21R83 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 219R45 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 21R79 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site of the 21R75 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 21R83 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 219R45 antibody and an antigen-binding site from the 21R79 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 219R45 antibody and an antigen-binding site from the 21M18 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 219R45 antibody and an antigen-binding site from the 21R75 antibody. In certain embodiments, the cells produce a bispecific antibody that comprises an antigen-binding site from the 219R45 antibody and an antigen-binding site from the 21R83 antibody. In certain embodiments, the cells produce the bispecific 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 certain embodiments, the cells produce the bispecific antibody 219R45-MB-21R83.
[0229] Proteins produced by a transformed host can be purified according to any suitable method. Standardized methods include chromatography (eg, ion exchange, affinity, and size classification column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be linked to the protein to allow easy purification by passage over an appropriate affinity column. Affinity chromatography used for the purification of immunoglobulins can include Protein A, Protein G and Protein L chromatography. Isolated proteins can be physically characterized using techniques such as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC) and X-ray crystallography. The purity of isolated proteins can be determined using techniques known to those skilled in the art, which include, but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).
[0230] In some embodiments, supernatants from expression systems that secrete recombinant protein into the culture medium can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin can be employed, for example, a matrix or a substrate that has pendant diethylaminoethyl groups (DEAE). The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. In some embodiments, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices that comprise sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite medium may be employed, including, but not limited to, ceramic hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing a hydrophobic RP-HPLC medium, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a recombinant protein (e.g., an agent binding to VEGF/DLL4). Part or all of the above purification steps, in various combinations, can be employed to provide a homogeneous recombinant protein.
[0231] In some embodiments, heterodimeric proteins such as bispecific antibodies are purified according to any of the methods described herein. In some embodiments, bispecific anti-VEGF/anti-DLL4 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 embodiments, the isolated and/or purified antibody product comprises at least 90% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product comprises at least 95%, 96%, 97%, 98% or 99% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product comprises approximately 100% heterodimeric antibody.
[0232] In some modalities, the recombinant protein produced in bacterial culture can be isolated, for example, through the initial extraction starting from the cell pellets, followed by one or more steps of concentration, "saltingout", aqueous ion exchange or chromatography by size exclusion. HPLC can be used for final purification steps. Microbial cells employed in the expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycles, ultrasound, mechanical disruption, or use of cell lysing agents.
[0233] Methods known in the art for purifying antibodies and other proteins further include, for example, those described in U.S. Patent Publication Nos. 2008/0312425; 2008/0177048; and 2009/0187005.
[0234] In certain embodiments, the VEGF/DLL4 binding agent is a polypeptide that is not an antibody. A variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target are known in the art. See, for example, Skerra, 2007, Curr. Opinion Biotechnol., 18:295-304; Hosse et al., 2006, Protein Science, 15:1427; Gill et al., 2006, Curr. Opinion Biotechnol., 17:653-658; Nygren, 2008, FEBS J., 275:2668-76; and Skerra, 2008, FEBS J., 275:2677-83. In certain embodiments, mammalian cell or phage display technology can be used to produce and/or identify a polypeptide that binds to VEGF/DLL4 that is not an antibody. In certain embodiments, the polypeptide comprises a protein structure of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
[0235] In certain embodiments, the binding agents or antibodies to VEGF/DLL4 can be used in any of a number of conjugated (i.e., an immunoconjugate or a radioconjugate) or unconjugated forms. In certain embodiments, the antibodies can be used in an unconjugated form to arm the individual's natural defense mechanisms including complement dependent cytotoxicity and antibody dependent cellular toxicity to eliminate malignant or cancerous cells.
[0236] In some embodiments, the VEGF/DLL4 binding agent (eg, an antibody or a polypeptide) is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent that includes, but is not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomicin C, chlorambucil, daunorubicin, or other intercalating agents. In some embodiments, 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, chain Exotoxin A, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII and PAP-S), inhibitor of Momordica charantia, curcina, crotina, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomicin and trichothecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or radioconjugate antibody. A variety of radionuclides are available for the production of radioconjugated antibodies including, but not limited to, 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re and 212Bi. Conjugates of an antibody and one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothecenes, and CC1065 and derivatives of these toxins that possess toxin activity, can also be used. Conjugates of an antibody and a cytotoxic agent can be produced using a variety of bifunctional protein coupling agents including, but not limited to, N-succinimidyl-3-(2-pyridiidithiol) propionate (SPDP), iminothiolane (IT) , bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis derivatives -diazonium (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) . III. Polynucleotides
[0237] In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide (or a fragment of a polypeptide) that specifically binds to VEGF, DLL4, both VEGF and DLL4. The term "polynucleotides encoding a polypeptide" encompasses a polynucleotide that includes only coding sequences for the polypeptide, as well as a polynucleotide that includes additional coding and/or non-coding sequences. For example, in some embodiments, the invention provides a polynucleotide that comprises a polynucleotide sequence that encodes an antibody to human VEGF or encodes a fragment of such an antibody (e.g., a fragment that comprises the antigen-binding site). In some embodiments, the invention provides a polynucleotide that comprises a polynucleotide sequence that encodes an antibody to human Dll4 or encodes a fragment of such an antibody (e.g., a fragment that comprises the antigen-binding site). Polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA; and it can be double-stranded or single-stranded, and if single-stranded, it can be the encoding filament or non-coding filament (antisense).
[0238] In certain embodiments, the polynucleotide comprises a polynucleotide that encodes a polypeptide that comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, 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 certain embodiments, the polynucleotide comprises a polynucleotide that encodes a polypeptide that comprises a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO :9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62 and SEQ ID NO:64. In some embodiments, the polynucleotide comprises the polynucleotide sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ NO:77 and SEQ ID NO:78.
[0239] In certain embodiments, the polynucleotide comprises a polynucleotide that has a nucleotide sequence at least approximately 80% identical, at least approximately 85% identical, at least approximately 90% identical, at least approximately 95% identical, and in some modalities, at least approximately 96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:33, SEQ ID polynucleotide comprises a polynucleotide having a nucleotide sequence at least approximately 80% identical, at least approximately 85% identical, at least approximately 90% identical, at least approximately 80% identical, at least approximately 95% identical, and in some embodiments, at least approximately 96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO:37, S EQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:54, 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. Further provided is a polynucleotide comprising a polynucleotide that hybridizes to SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ NO:78. In certain embodiments, hybridization occurs under conditions of high stringency.
[0240] In certain embodiments, polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide that helps, for example, in the expression and secretion of a polypeptide from a host cell ( for example, a leader sequence that functions as a secretory sequence to control the transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and may have a leader sequence cleaved by the host cell to form the mature form of the polypeptide. Polynucleotides can also encode a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein that has a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains.
[0241] In certain embodiments, polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, the purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag provided by a pQE-9 vector to allow purification of the mature polypeptide fused to the tag in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag ) derived from the influenza hemagglutinin protein when a mammalian host (eg, COS-7 cells) is used. In some embodiments, the tag sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:45) that can be used in association with other affinity tags.
[0242] The present invention further relates to variants of the polynucleotides described hereinbefore that encode, for example, fragments, analogs and/or derivatives.
[0243] In certain embodiments, the present invention provides polynucleotides comprising polynucleotides that have a nucleotide sequence at least approximately 80% identical, at least approximately 85% identical, at least approximately 90% identical, at least approximately 95% identical, and in in some embodiments, at least approximately 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a VEGF/DLL4 binding agent (e.g., an antibody) or fragment thereof, described on here.
[0244] As used herein, expression of a polynucleotide that has a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence is intended 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 for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide that has a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced by another nucleotide or a number of nucleotides of up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These reference sequence mutations can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between such terminal positions, interspersed individually between the nucleotides in the reference sequence or in one or more contiguous groups within of the reference string.
[0245] Polynucleotide variants may contain alterations in the coding regions, non-coding regions or both. In some embodiments, a polynucleotide variant contains alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that do not result in a change in the amino acid sequence of the polypeptide (due to 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 (ie, alter codons in human mRNA by 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 coding region of the sequence.
[0246] In some embodiments, a polynucleotide variant is produced to modulate or alter the expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has greater expression of the encoded polypeptide when compared to a parent polynucleotide sequence. In some embodiments, a polynucleotide variant has less expression of the encoded polypeptide as compared to a parent polynucleotide sequence.
[0247] 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 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 bispecific antibody.
[0248] In certain embodiments, polynucleotides are isolated. In certain embodiments, polynucleotides are substantially pure.
[0249] Vectors and cells comprising the polynucleotides described here are also provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some embodiments, a host cell comprises an expression vector that comprises the polynucleotide molecule. In some embodiments, a host cell comprises a polynucleotide molecule. IV. Methods of use and pharmaceutical compositions
[0250] Binding agents (including polypeptides and antibodies) of the invention that bind (eg specifically bind) to VEGF and/or DLL4 are useful in a variety of applications including, but not limited to, methods of therapeutic treatment, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting VEGF activity, for inhibiting Dll4-induced Notch signaling, for inhibiting tumor growth, for reducing tumor volume, for reducing cell frequency stem cancer cells in a tumor, for reducing the tumorigenicity of a tumor, for modulating angiogenesis, and/or for inhibiting angiogenesis. Methods of use can be in vitro, ex vivo or in vivo. In certain embodiments, a VEGF/DLL4 binding agent is a human VEGF antagonist. In certain embodiments, a VEGF/DLL4 binding agent is a human DLL4 antagonist. In certain embodiments, a VEGF/DLL4 binding agent is an antagonist of both VEGF and DLL4.
[0251] In certain embodiments, VEGF/DLL4 binding agents are used in the treatment of a disease associated with angiogenesis, ie, increased angiogenesis and/or aberrant angiogenesis. In certain modalities, the disease is an angiogenesis-dependent disease. In certain embodiments, VEGF/DLL4 binding agents are used in the treatment of disorders characterized by higher levels of stem cells and/or progenitor cells.
[0252] The present invention provides methods for inhibiting the growth of a tumor using the binding agents or antibodies to VEGF/DLL4 described herein. In certain embodiments, the method of inhibiting the growth of a tumor comprises contacting a tumor cell with a VEGF/DLL4 binding agent (e.g., antibody) in vitro. For example, an immortalized cell line or a cancer cell line is cultured in a medium to which an anti-VEGF antibody, an anti-DLL4 antibody, or a bispecific anti-VEGF/anti-DLL4 antibody is added to inhibit growth of tumor cells. In some embodiments, tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, a pleural effusion, or a blood sample and are cultured in a medium to which a VEGF binding agent is added. /DLL4 to inhibit tumor cell growth.
[0253] In some embodiments, the method of inhibiting the growth of a tumor comprises contacting a tumor or tumor cells with a VEGF/DLL4 binding agent (eg, antibody) in vivo. In certain embodiments, contacting a tumor or tumor cell with a VEGF/DLL4 binding agent is performed in an animal model. For example, an anti-VEGF antibody, an anti-DLL4 antibody, or a bispecific anti-VEGF/anti-DLL4 antibody can be administered to an immunocompromised host animal (e.g., NOD/SCID mice) that 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, a pleural effusion, or a blood sample and are injected into an immunocompromised host animal (by (eg, NOD/SCID mice) which are 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 as or shortly after the introduction of tumorigenic cells into the animal to prevent tumor growth ("preventive model"). In some embodiments, the VEGF/DLL4 binding agent is administered as a therapeutic agent 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 to human VEGF and human DLL4.
[0254] In certain embodiments, the method of inhibiting the growth of a tumor comprises administering to a subject a therapeutically effective amount of a VEGF/DLL4 binding agent. In certain embodiments, the individual is a human being. In certain embodiments, the individual has a tumor or has had a tumor that has been removed. In certain modalities, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administering the VEGF/DLL4 binding agent. The invention further provides a method of reducing the frequency of cancer stem cells in a tumor, which comprises contacting the tumor with an efficient amount of a VEGF/DLL4 binding agent (e.g., an anti-VEGF/anti-bispecific antibody). DLL4). In some embodiments, a method of reducing the frequency of cancer stem cells in a tumor in an individual comprises administering to the individual a therapeutically effective amount of a VEGF/DLL4 binding agent.
[0255] In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of colorectal tumor, colon tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, tumor gastrointestinal, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a colorectal tumor or a colon tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In certain modalities, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a breast tumor.
[0256] 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 to VEGF and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent binds DLL4 and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds VEGF and DLL4 and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent binds to VEGF, interferes with VEGF/VEGF receptor interactions, and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent binds to DLL4, interferes with DLL4/Notch interactions, and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent binds to both VEGF and DLL4, interferes with VEGF/VEGF receptor interactions and DLL4/Notch interactions, and inhibits or reduces cancer growth. In some embodiments, the VEGF/DLL4 binding agent binds DLL4 and reduces the frequency of cancer stem cells in cancer.
[0257] The present invention provides methods 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 individual is a human being. In certain modalities, the individual has a cancerous tumor. In certain embodiments, the individual has had a tumor removed.
[0258] The individual's cancer/tumor may, in some modalities, be refractory to certain treatment(s). As a non-limiting example, the individual's cancer (or tumor) may be chemorefractory. In certain modalities, the individual's cancer may be resistant to anti-VEGF therapy or anti-DLL4 therapy or both.
[0259] In certain modalities, 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, melanoma , cervical cancer, bladder cancer, glioblastoma and head and neck cancer. In certain modalities the cancer is ovarian cancer. In certain modalities the cancer is colorectal cancer or colon cancer. In certain modalities, cancer is pancreatic cancer. In certain modalities, cancer is breast cancer. In certain modalities the cancer is prostate cancer. In certain modalities, cancer is lung cancer. In some modalities, the cancer is a blood cancer such as leukemia or lymphoma. In some modalities, the leukemia or lymphoma is a leukemia or B-cell lymphoma. In some modalities, the leukemia or lymphoma is a leukemia or T-cell lymphoma. In some modalities the blood cancer is acute myelogenous leukemia, lymphoma Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, multiple myeloma, cutaneous T-cell lymphoma, or T-cell acute lymphoblastic leukemia.
[0260] The invention further provides methods of treating a disease or disorder in an individual, wherein the disease or disorder is associated with angiogenesis. In some modalities, the disease or disorder is associated with aberrant angiogenesis. In some modalities, the disease or disorder is associated with increased angiogenesis. Thus, the present invention provides methods for modulating angiogenesis in a subject, which comprise 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 to human VEGF. In some embodiments, the VEGF/DLL4 binding agent is an antibody that binds to human DLL4. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds to human VEGF. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds to human DLL4. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that binds to human VEGF and human DLL4.
[0261] Methods of treating a disease or disorder in an individual, wherein the disease or disorder is characterized by an increased level of stem cells and/or progenitor cells are additionally provided. In some embodiments, the methods of treatment comprise administering a therapeutically effective amount of a VEGF/DLL4 binding agent, a polypeptide, or an antibody to the subject.
[0262] In certain embodiments of any of the methods described herein, the VEGF/DLL4 binding agent is a bispecific antibody that specifically binds to human VEGF and human DLL4. In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human 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 comprising HYDDKYYPLMDY (SEQ ID NO:19) and the second 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); 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to the antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO: 19) and the second antigen binding site comprises 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 comprises 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to the antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO:19) and the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:15) and a heavy chain CDR3 comprising comprises 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site that binds to human VEGF that binds antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO: 19) and the second antigen binding site comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59) and a heavy chain CDR3 comprising comprises 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecific antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human DLL4, where the first site binds to the antigen 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 comprising HYDDKYYPLMDY (SEQ ID NO:19) and the second antigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65) and a heavy chain CDR3 comprising comprises 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 CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22).
[0263] In certain embodiments of any of the methods described herein, the VEGF/DLL4 bispecific antibody comprises a first heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region which has at least approximately 80% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58 or SEQ ID NO:64 and a first and a variable region of a light chain that has 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 that has at least approximately 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:9 and a first and second heavy chain variable regions that have 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 that has at least approximately 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:10 and a first and second heavy chain variable regions that have 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 that has at least approximately 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:58 and a first and second heavy chain variable regions that have 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 that has at least approximately 80% sequence identity to SEQ ID NO:11, a second heavy chain variable region that has at least approximately 80% sequence identity to SEQ ID NO:64 and a first and second heavy chain variable regions that have at least 80% sequence identity to SEQ ID NO:12.
[0264] In some embodiments of any of the methods described herein, the VEGF/DLL4 binding agent is an antibody. In some embodiments, the VEGF/DLL4 binding agent is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody is the 219R45 antibody. In some embodiments, the VEGF/DLL4 binding agent is an anti-DLL4 antibody. In some embodiments, the anti-DLL4 antibody is the 21R79 antibody. In some embodiments, the anti-DLL4 antibody is the 21R75 antibody. In some embodiments, the anti-DLL4 antibody is the 21R83 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises an antigen-binding site of the 219R45 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises an antigen-binding site of the 21R79 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises an antigen-binding site of the 21R75 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises an antigen-binding site of the 21R83 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site from the 219R45 antibody and a second antigen-binding site from the 21R79 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site from the 219R45 antibody and a second antigen-binding site from the 21M18 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first site that binds the antigen of the 219R45 antibody and a second site that binds the antigen of the 21R75 antibody. In some embodiments, the VEGF/DLL4 binding agent is a bispecific antibody that comprises a first antigen-binding site from the 219R45 antibody and a second antigen-binding site from the 21R83 antibody. 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 embodiments, 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.
[0265] The present invention further provides pharmaceutical compositions comprising the binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. These pharmaceutical compositions find use in inhibiting tumor growth and/or treating cancer in an individual (eg, a human patient).
[0266] In certain embodiments, the invention provides pharmaceutical compositions comprising bispecific antibodies, wherein at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99% of the antibodies in the composition are bispecific antibodies or heterodimeric antibodies. In certain embodiments, bispecific antibodies are IgG antibodies (eg, IgG2 or IgG1). In certain embodiments, 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 approximately 98% heterodimeric.
[0267] In certain embodiments, formulations are prepared for storage and use by combining an antibody or purified agent of the present invention with a pharmaceutically acceptable carrier (eg, a carrier or an excipient). Suitable pharmaceutically acceptable carriers include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (eg, less than approximately 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, 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 indicator 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 of Pharmacy, 21st Edition, 2005, University of the Sciences, Philadelphia, PA).
[0268] The pharmaceutical compositions of the present invention can be administered in any number of ways for local or systemic treatment. Administration can be topical through epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular).
[0269] The therapeutic formulation may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media or suppositories. In solid compositions such as tablets the main active ingredient is mixed with a pharmaceutical carrier. Ingredients for conventional tablets include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous 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 the type described above. Pills, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill can comprise an internal composition covered by an external component. Furthermore, the two components can be separated by an enteric layer which serves to resist disintegration and allows the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.
[0270] The binding agents or antibodies to VEGF/DLL4 described herein may also be trapped within microcapsules. Such microcapsules are prepared, for example, through coacervation techniques or through interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, al-bumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, University of the Sciences in Philadelphia, PA.
[0271] In certain embodiments, pharmaceutical formulations include a VEGF/DLL4 binding agent (e.g., an antibody) of the present invention complexed with liposomes. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be generated through reverse-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and phosphatidylethanolamine derivatized with PEG (PEG-PE). Liposomes can be extruded through filters of defined pore size to produce liposomes of the desired diameter.
[0272] In certain embodiments, controlled release preparations can be produced. Suitable examples of controlled release preparations include semipermeable hues of solid hydrophobic polymers containing a VEGF/DLL4 binding agent (eg an antibody), where the matrices are in the form of molded articles (eg films or microcapsules) . Additional examples of controlled 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-ethylene-vinyl acetate degradable, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-acid hydroxybutyric.
[0273] 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 may be administered prior to, concurrently with and/or subsequent to administration of the VEGF/DLL4 binding agent. Pharmaceutical compositions comprising a VEGF/DLL4 binding agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3 or more additional therapeutic agents.
[0274] Combination therapy with at least two therapeutic agents often uses agents that work through different mechanisms of action, although this is not necessary. Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thus reducing toxic side effects and/or increasing the therapeutic index of at least one of the agents. Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, the combination therapy comprises a therapeutic agent that primarily affects (eg, inhibits or kills) non-tumorigenic cells and a therapeutic agent that primarily affects (eg, inhibits or kills) tumorigenic CSCs.
[0275] Useful classes of therapeutic agents include, for example, antitubulin agents, auristatins, minor DNA groove binding agents, DNA replication inhibitors, alkylating agents (eg, platinum complexes such as cisplatin, mono(platinum) ), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizing agents, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, antimetabolites of purine, puromycins, radiation sensitizing agents, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids or similar. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor. In some embodiments, the second therapeutic agent is a platinum complex such as carboplatin or cisplatin. In some embodiments, the additional therapeutic agent is a platinum complex in combination with a taxane.
[0276] Therapeutic agents that can be administered in combination with VEGF/DLL4 binding agents include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves administering a binding agent or an anti-VEGF antibody of the present invention in combination with a chemotherapeutic agent or a cocktail of several different chemotherapeutic agents. In some embodiments, the method or treatment involves administering a binding agent or an anti-DLL4 antibody of the present invention in combination with a chemotherapeutic agent or a cocktail of several different chemotherapeutic agents. In some embodiments, the method or treatment involves administering a bispecific antibody of the present invention that binds to VEGF and Dll4 in combination with a chemotherapeutic agent or a cocktail of several different chemotherapeutic agents.
[0277] Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomisins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-norleucicin do , epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ziubenimexin, ziubenimex; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as callusterone, 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; edatraxate; defofamin; demecolcine; diaziquone; elformitin; elliptinium acetate; ethoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pyrarubicin; podophylinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxan; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside (Ara-C); taxoids, for example, paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; Esperamycins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents further include anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors such as antiestrogens including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene , keoxifene, LY117018, onapristone and toremifene (FARESTON); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the second therapeutic agent is cisplatin. In certain embodiments, the second therapeutic agent is carboplatin. In certain embodiments, the second therapeutic agent is paclitaxel.
[0278] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of a topoisomerase enzyme (eg, topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26) and irinotecan, as well as pharmaceutically acceptable salts, acids or derivatives of any of these. In certain embodiments, the second therapeutic agent is irinotecan.
[0279] In certain embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical reagent with a structure that is similar to a metabolite needed for normal biochemical reactions, yet different enough to interfere with one or more normal cell functions, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, sodium methotrexate, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentosta fludarabine phosphate and cladribine, as well as pharmaceutically acceptable salts, acids or derivatives of any of these. In certain embodiments, the second therapeutic agent is gemcitabine.
[0280] In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind to tubulin. In some modalities, the agent is a taxane. 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 vincristine, binblastine, vinorelbine or vindesine or pharmaceutically acceptable salts, acids or derivatives thereof. In some embodiments, the antimitotic agent is an Eg5 kinesin inhibitor or an inhibitor of a mitotic kinase such as Aurora A or Plk1. In certain embodiments, when the chemotherapeutic agent administered in combination with a VEGF/DLL4 binding agent is an antimitotic agent, the cancer or tumor being treated is breast cancer or a breast tumor.
[0281] In some embodiments, a second therapeutic agent comprises an agent such as a small molecule. For example, treatment may involve the combined administration of a VEGF/DLL4 binding agent (e.g., an antibody) of the present invention with a small molecule that acts as an inhibitor against additional tumor associated proteins including, but not limited to , EGFR, ErbB2, HER2 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 therapeutic 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 pathway. In some embodiments, the second therapeutic agent is a small molecule that inhibits β-catenin signaling.
[0282] In some embodiments, a second therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment may 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 to EGFR, ErbB2, HER2 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 antibody that binds to a component of the Notch pathway. In some embodiments, the second therapeutic agent is an antibody that binds to a component of the Wnt pathway. In certain embodiments, the second therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the second therapeutic agent is a Notch pathway inhibitory antibody. In some embodiments, the second therapeutic agent is a Wnt pathway inhibitory antibody. In some embodiments, the second therapeutic agent is a BMP pathway inhibitor antibody. In some embodiments, the second therapeutic agent is an antibody that inhibits β-catenin signaling. In certain embodiments, the second therapeutic agent is an antibody that is an inhibitor or a modulator of angiogenesis (e.g., an anti-VEGF antibody or to the VEGF receptor). In certain modalities, the second therapeutic agent is bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), panitumumab (VECTIBIX), or cetuximab (ERBITUX). Combined administration may include co-administration, in a single pharmaceutical formulation or using separate formulations, or consecutive administration in any order, but generally within a period of time such that all active agents can exert their biological activities simultaneously.
[0283] In addition, treatment with a VEGF/DLL4 binding agent described herein may include combination treatment with other biological molecules such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors and/or or growth factors) or may be accompanied by surgical removal of tumors, cancer cells, or any other therapy deemed necessary by a treating physician.
[0284] In certain embodiments, treatment involves administration of a VEGF/DLL4 binding agent (eg, an antibody) of the present invention in combination with radiation therapy. Treatment with a VEGF/DLL4 binding agent can occur prior to, concurrently with or subsequent to the administration of radiation therapy. The dosage schedule for such radiation therapy can be determined by the skilled practitioner.
[0285] 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 concurrently. Treatment with a VEGF/DLL4 binding agent (e.g., an antibody) may occur prior to, concurrently with, or subsequent to administration of chemotherapies. Combined administration may include co-administration, in a single pharmaceutical formulation or using separate formulations, or consecutive administration in any order, but generally within a period of time such that all active agents can exert their biological activities simultaneously. Preparation schedules and dosages for such chemotherapeutic agents can be used in accordance with the manufacturer's instructions or as determined empirically by the skilled person. Preparation schedules and dosages for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
[0286] In some embodiments, the VEGF/DLL4 binding agent will be administered to patients who 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, an individual may receive a VEGF/DLL4 binding agent (e.g., an antibody) while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a VEGF/DLL4 binding agent will be administered within 1 year of treatment with a second therapeutic agent. In certain alternative embodiments, a VEGF/DLL4 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 day of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments can be administered to the individual within a matter of hours or minutes (i.e., substantially simultaneously).
[0287] For the treatment of a disease, the appropriate dosage of a VEGF/DLL4 binding agent (eg, an antibody) of the present invention depends on the type of disease that will be treated, the severity and course of the disease, the disease responsiveness, whether the binding agent or antibody to VEGF/DLL4 is administered for therapeutic or preventative purposes, prior therapy, the patient's medical history, and others, all at the discretion of the treating physician. The binding agent or antibody to VEGF/DLL4 can be administered once or as a series of treatments spread over several days to several months or until a cure is achieved or a decrease in the disease state is achieved (by example, reduction in tumor size). Optimal dosage schedules can be calculated from measures of drug accumulation in the patient's body and will vary depending on the relative potency of an antibody or an individual agent. The administering physician can determine optimal dosages, dosing methodologies, and repeat rates. In certain embodiments, the dosage of a VEGF/DLL4 binding agent or an antibody is from approximately 0.01 µg to approximately 100 mg/kg body weight, from approximately 0.1 µg to approximately 100 mg/kg body weight , from approximately 1 μg to approximately 100 mg/kg of body weight, from approximately 1 mg to approximately 100 mg/kg of body weight, approximately 1 mg to approximately 80 mg/kg of body weight from approximately 10 mg to approximately 100 mg/ kg of body weight, from approximately 10 mg to approximately 75 mg/kg of body weight, or from approximately 10 mg to approximately 50 mg/kg of body weight. In certain embodiments, the dosage of antibody or other VEGF/DLL4 binding agent is from approximately 0.1 mg to approximately 20 mg/kg of body weight. In certain embodiments, dosage may be given once or more per day, week, month or year. In certain embodiments, the antibody or other VEGF/DLL4 binding agent is provided once every week, once every two weeks, once every three weeks, or once every month.
[0288] In some embodiments, a VEGF/DLL4 binding agent (eg, an antibody) may be administered at an initial higher “loading” dose, followed by one or more lower doses. In some modalities the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once each month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, half the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example, half the initial dose every other week. Or a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, 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 repeat rates for dosing based on measured residence times and drug concentrations in body fluids or tissues. The progress of therapy can be monitored using conventional techniques and assays.
[0289] As is known to those skilled in the art, administration of any therapeutic agent can lead to side effects and/or toxicities. In some cases, side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued and other agents may be tested. However, many agents in the same therapeutic class often exhibit similar side effects and/or toxicities, meaning that the patient has to discontinue therapy or, if possible, suffer the unpleasant side effects associated with the therapeutic agent.
[0290] Side effects from therapeutic agents may include, but are not limited to, hives, skin rashes, itching, nausea, vomiting, decreased appetite, diarrhea, chills, fever, fatigue, muscle aches and pain, headache, low blood pressure high blood pressure, hypokalemia, low blood counts, bleeding and heart problems.
[0291] Thus, one aspect of the present invention is directed to methods of treating cancer in a patient comprising administering a bispecific anti-VEGF/anti-DLL4 antibody using an intermittent dosing regimen, which can reduce the effects side effects and/or the toxicities associated with the administration of the bispecific anti-VEGF/anti-DLL4 antibody. As used herein, "intermittent dosing" refers to a dosing regimen utilizing a dosing interval of more than once a week, e.g., dosing once every 2 weeks, once every 3 weeks, once a week. every 4 weeks etc. In some embodiments, a method of treating cancer in a human patient comprises administering to the patient an efficient dose of a bispecific anti-VEGF/anti-DLL4 antibody in accordance with an intermittent dosing regimen. In some embodiments, a method for treating cancer in a human patient comprises administering to the patient an efficient dose of a bispecific anti-VEGF/anti-DLL4 antibody according to an intermittent dosing regimen and increasing the therapeutic index of the bispecific anti-VEGF/anti-DLL4 antibody. In some embodiments, the intermittent dosing regimen comprises administering an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody to the patient and administering subsequent doses of the bispecific anti-VEGF/anti-DLL4 antibody approximately once each 2 weeks. In some embodiments, the intermittent dosing regimen comprises administering an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody to the patient and administering subsequent doses of the bispecific anti-VEGF/anti-DLL4 antibody approximately once each 3 weeks. In some embodiments, the intermittent dosing regimen comprises administering an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody to the patient and administering subsequent doses of the bispecific anti-VEGF/anti-DLL4 antibody approximately once every 4 weeks.
[0292] In some embodiments, subsequent doses in an intermittent dosing regimen are approximately the same amount or less than the initial dose. In other embodiments, subsequent doses are an amount greater than the initial dose. As is known to those skilled in the art, the doses used will vary depending on the clinical goals that are to be achieved. In some embodiments, the starting dose is approximately 0.25 mg/kg to approximately 20 mg/kg. In some embodiments, the starting dose is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg. In certain modalities, the starting dose is approximately 0.5 mg/kg. In certain modalities, the starting dose is approximately 1 mg/kg. In certain embodiments, the starting dose is approximately 2.5 mg/kg. In certain modalities, the starting dose is approximately 5 mg/kg. In certain embodiments, the starting dose is approximately 7.5 mg/kg. In certain modalities, the starting dose is approximately 10 mg/kg. In certain embodiments, the starting dose is approximately 12.5 mg/kg. In certain modalities, the starting dose is approximately 15 mg/kg. In certain modalities, the starting dose is approximately 20 mg/kg. In some embodiments, subsequent doses are from approximately 0.25 mg/kg to approximately 15 mg/kg. In certain embodiments, subsequent doses are approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/kg. In certain embodiments, subsequent doses are approximately 0.5 mg/kg. In certain embodiments, subsequent doses are approximately 1 mg/kg. In certain embodiments, subsequent doses are approximately 2.5 mg/kg. In certain embodiments, subsequent doses are approximately 5 mg/kg. In some embodiments, subsequent doses are approximately 7.5 mg/kg. In some embodiments, subsequent doses are approximately 10 mg/kg. In some embodiments, subsequent doses are approximately 12.5 mg/kg.
[0293] In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 2.5 mg/kg and (b) administration of subsequent doses of approximately 2.5 mg/kg once every 2 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 5 mg/kg and (b) administering subsequent doses approximately 5 mg/kg once every 2 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 2.5 mg/kg and (b) administering subsequent doses approximately 2.5 mg/kg once every 3 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 5 mg/kg and (b) administering subsequent doses approximately 5 mg/kg once every 3 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 2.5 mg/kg and (b) administering subsequent doses approximately 2.5 mg/kg once every 4 weeks. In some embodiments, the intermittent dosing regimen comprises: (a) administering to the patient an initial dose of a bispecific anti-VEGF/anti-DLL4 antibody of approximately 5 mg/kg and (b) administering subsequent doses of approximately 5 mg/kg once every 4 weeks. In certain modalities, the starting dose and maintenance doses are different, for example, the starting dose is approximately 5 mg/kg and subsequent doses are approximately 2.5 mg/kg. In certain embodiments, an intermittent dosing regimen may comprise a loading dose, for example, the initial dose is approximately 20 mg/kg and subsequent doses are approximately 2.5 mg/kg or approximately 5 mg/kg administered at a time. once every 2 weeks, once every 3 weeks or once every 4 weeks.
[0294] Another aspect of the present invention is directed to methods for reducing the toxicity of a bispecific anti-VEGF/anti-DLL4 antibody in a human patient comprising administering to the patient the bispecific anti-VEGF/anti-DLL4 antibody using an intermittent dosing regimen. Another aspect of the present invention is directed to methods for reducing the side effects of a bispecific anti-VEGF/anti-DLL4 antibody in a human patient comprising administering to the patient the bispecific anti-VEGF/anti-DLL4 antibody using a intermittent dosing regimen. Another aspect of the present invention is directed to methods for increasing the therapeutic index of a bispecific anti-VEGF/anti-DLL4 antibody in a human patient which comprise administering to the patient the bispecific anti-VEGF/anti-DLL4 antibody using a regimen of intermittent dosage.
[0295] The choice of delivery method for initial and subsequent doses is made according to the ability of the animal or human patient to tolerate the introduction of the bispecific anti-VEGF/anti-DLL4 antibody into the body. Thus, in any of the aspects and/or embodiments described herein, administration of the bispecific anti-VEGF/anti-DLL4 antibody can occur via intravenous injection or intravenously. In some embodiments, administration is via intravenous infusion. In any of the aspects and/or embodiments described herein, administration of the bispecific anti-VEGF/anti-DLL4 antibody can occur via a route other than intravenous. V. Kits comprising VEGF/DLL4 binding agents
[0296] The present invention provides kits comprising the VEGF/DLL4 binding agents (e.g. antibodies) described herein and which can be used to carry out the methods described herein. In certain embodiments, a kit comprises at least one purified antibody to VEGF and/or DLL4 in one or more containers. In some embodiments, kits contain all the necessary and/or sufficient components to perform a detection assay, including all controls, guidelines for performing the assays, and any software necessary for analyzing and reporting 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 that are well known in the art.
[0297] Kits are further provided which comprise a VEGF/DLL4 binding agent (e.g. an anti-VEGF/anti-DLL4 bispecific antibody) as well as at least one additional therapeutic agent. In certain embodiments, the second (or more) therapeutic agent is a chemotherapeutic agent. In certain modalities, the second (or more) therapeutic agent is an angiogenesis inhibitor.
[0298] The embodiments of the present disclosure may be further defined with reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods for using the antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both in materials and methods, can be practiced without departing from the scope of the present disclosure. EXAMPLES Example 1 Binding Affinities of Anti-VEGF/Anti-DLL4 Antibodies
[0299] The KDs of parental antibodies anti-VEGF 219R45 (IgG format), anti-DLL4 21R79 (IgG format), anti-DLL4 21m18 (IgG format) and bispecific antibodies 219R45-MB-21m18 and 219R45-MB- 21R79 were determined using a Biacore 2000 system from Biacore LifeSciences (GE Healthcare). Recombinant human DLL4-Fc or mouse DLL4-Fc proteins were immobilized onto CM5 carboxyl chips using standard amine-based chemistry (NHS/EDC) and blocked with ethanolamine. Human VEGF165 or recombinant mouse VEGF165 was biotinylated and immobilized on streptavidin chips. Antibodies were serially diluted 2-fold starting at 100 nM to 0.78 nM in HBS-P (0.01 M HEPES pH7.4, 0.15 M NaCl, 0.005% v/v Polysorbate 20). For each antibody, all 8 dilutions were sequentially injected onto a specific chip. Kinetics data was collected over time and was fitted using the simultaneous global fit equation to provide affinity constants (KD values) for each bispecific antibody.

As shown in Table 3, the bispecific antibody 219R45-MB-21m18 had an affinity constant (KD) for human VEGF of 0.36 nM and a KD for human DLL4 of 16 nM. The bispecific antibody 219R45-MB-21R79 had a KD for human VEGF of 0.68 nM and a KD for human DLL4 of 0.53 nM. Both bispecific antibodies demonstrated weaker binding to mouse VEGF compared to human VEGF and neither antibody bound to mouse DLL4. Thus, both bispecific antibodies demonstrated similar binding affinity to human VEGF and 219R45-MB-21R79 demonstrated approximately 30-fold stronger binding to human Dll4 than 219R45-MB-21m18. Furthermore, the bispecific antibody 219R45-MB-21R79 had a similar binding affinity to human VEGF despite the fact that the bispecific antibody is monovalent to VEGF when compared to the bivalent parent antibody.
[0301] Several additional anti-DLL4 antibodies were identified that had intermediate binding affinities at a KD of 21m18 and 21R79. Two of these anti-DLL4 antibodies were used to produce the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21R75 and 219R45-MB-21R83. Using the Biacore 2000 system as described above, the KDs of the bispecific antibodies 219R45-MB-21R75 and 219R45-MB-21R83 by human DLL4 were determined. A comparison of human DLL4 binding affinity of these four bispecific anti-VEGF/anti-DLL4 antibodies is shown in Table 4.
[0302] The CDRs for the bispecific anti-VEGF/anti-DLL4 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 without signal sequence) are shown in Figure 1C.
[0303] The bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18 comprises a (a) heavy chain encoded by DNA comprising SEQ ID NO:75 deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on September 21, 2012 and received the designation number PTA-13233, (b) a heavy chain encoded by DNA comprising SEQ ID NO:33 deposited with the ATCC under the Budapest Treaty conditions on September 21, 2012 and received the designation number PTA-13236 and (c) a DNA encoded light chain comprising SEQ ID NO:34 deposited with the ATCC under the Budapest Treaty conditions on September 21, 2012 and received the designation number PTA-13235.
[0304] The bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R79 comprises a (a) heavy chain encoded by DNA comprising SEQ ID NO:31 deposited with the ATCC under the conditions of the Budapest Treaty on September 21st of 2012 and received the designation number PTA-13232, (b) a heavy chain encoded by DNA comprising SEQ ID NO:33 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received the number of designation PTA-13236 and (c) a DNA-encoded light chain comprising SEQ ID NO:34 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received designation number PTA-13235.
[0305] The bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R83 comprises (a) a heavy chain encoded by DNA comprising SEQ ID NO:72 deposited with the ATCC under the conditions of the Budapest Treaty on October 24th of 2012 and received the designation number PTA-13278, (b) a heavy chain encoded by DNA comprising SEQ ID NO:33 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received the number of designation PTA-13236 and (c) a DNA-encoded light chain comprising SEQ ID NO:34 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received designation number PTA-13235.
[0306] The bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R75 comprises (a) a heavy chain encoded by DNA comprising SEQ ID NO:74 deposited with the ATCC under the conditions of the Budapest Treaty on September 21st of 2012 and received the designation number PTA-13234, (b) a heavy chain encoded by DNA comprising SEQ ID NO:33 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received the number of designation PTA-13236 and (c) a DNA-encoded light chain comprising SEQ ID NO:34 deposited with the ATCC under the terms of the Budapest Treaty on September 21, 2012 and received designation number PTA-13235.
Example 2 HTRF Assay for Simultaneous Binding of Bispecific Antibodies to Human VEGF and Human Dll4
[0307] To characterize the binding capacities of certain antibodies and/or mixtures of antibodies to both VEGF and DLL4, time homogeneous revealed fluorescence (HTRF) assays were performed. The antibodies tested were the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18 and 219R45-MB-21R79, parental antibodies 219R45 (anti-VEGF), 21m18 (anti-DLL4), 21R79 (anti-DLL4), one combination of 219R45 and 21m18 or a combination of 219R45 and 21R79. Antibodies or antibody mixtures were serially diluted 2-fold starting at 3000 nM to 2.9 nM in binding buffer (1X PBS, 0.1% gelatin, 0.1% Polysorbate 20, 400 mM fluoride). potassium) and placed in a white 96-well plate. An equal volume of solution containing 4 µg/ml of d2-labeled hDLL4-Fc and 21.4 ng/ml of europium cryptate-labeled hVEGF165 was added to each well for a final volume of 100 µL (the final concentrations of receptor fluorophores and donors were 2 μg/mL and 10.7 ng/mL, respectively). Assay plates were incubated for 2 hours until overnight and read on a SpectraMax M5e Microplate reader (Molecular Devices, Sunnyvale CA) at an excitation wavelength of 314 nm.
[0308] As shown in Figure 2, the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18 and 219R45-MB-21R79, were able to bind both hVEGF and hDLL4 simultaneously. Importantly, none of the combinations of the parental antibodies (i.e., 219R45 and 21m18 or 219R45 and 21R79) were able to bind VEGF and Dll4 simultaneously. These results clearly demonstrate that the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18 and 219R45-MB-21R79 are able to function differently than just a mixture of the two individual antibodies. Example 3 Inhibition of HUVEC proliferation by bispecific anti-VEGF/anti-DLL4 antibodies
[0309] HUVEC cells were obtained from Lonza (Walkersville MD) and grown in growth medium (M199, 10% heat inactivated FBS (HI-FBS), 50 μg/ml EGS, 1X heparin, 1 mM L -glutamine). For the HUVEC proliferation assay, a 96-well plate was pre-coated with 50 μL of rat tail type I collagen solution at 10 μg/mL (collagen I in 0.02 N acetic acid) and incubated at 4°C overnight. After incubation, the plate was vigorously aspirated to remove unbound collagen I solution and washed once with 200 µL of DPBS. HUVEC cells were removed from the surface of growth flasks using an endothelial cell subcloning reagent and centrifuged at 1200 rpm for 5 minutes at 4°C. Cells were resuspended in starvation/assay medium (M199 and 2% HI-FBS, 1X heparin, 5 U/ml heparin-glutamine) at a density of 105 cells/ml. Cells were seeded into the collagen coated assay plate at 5000 cells/well, 50 µl/well. Cells were incubated for 3 hours at 37°C, washed once, re-fed with 100 µl of assay medium and incubated overnight at 37°C. The next day, the bispecific antibodies 219R45-MB-21m18, 219R45-MB-21R79, the parental antibody 219R45 or the control antibody LZ1 were prepared in a mixture with human VEGF (R&D Biosystems, Minneapolis MN). Antibodies were serially diluted 5-fold starting from 20 µM to 0.25 nM in assay buffer in combination with hVEGF (final concentration 5 ng/ml). The mixture was pre-incubated at 37°C for 2 hours. The medium was removed from the assay plate and 100 µl of the antibody/hVEGF mixture was added to each well. After an incubation of 3-4 days, the medium was removed and a fresh aliquot of the antibody/hVEGF mixture was added to each well and allowed to be incubated for an additional 4 days. On day 7, 20 µL of Alamar Blue reagent (Invitrogen, Carlsbad, CA) was added to each well and incubated at 37°C for 5-6 hours. The plate was read with a SpectraMax M5e Microplate reader (Molecular Devices, Sunnyvale CA) using an excitation wavelength of 539 nm and an emission wavelength of 590 nm.
[0310] As shown in Figure 3, the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18 and 219R45-MB-21R79, as well as the parent anti-VEGF antibody 219R45 inhibited the proliferation of HUVEC. These results demonstrated that bispecific antibodies were able to inhibit VEGF-induced proliferation of HUVEC cells. Example 4 Inhibition of Dll4-induced Notch signaling by bispecific antibodies
[0311] 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 capable of responding to Notch signaling. Cells were also transfected with a Renilla luciferase reporter (Promega, Madison WI) as an internal control for transfection efficiency. Purified human DLL4 protein was coated onto 96-well plates at 100 ng/well and PC3-luc cells expressing Notch2 were added to the wells. Bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18, 219R45-MB-21R79, parental anti-DLL4 antibodies 21m18, 21R79 or an LZ1 control antibody was serially diluted 5 times starting from 20 μg/ml to 0.064 μg/ml, added to the appropriate wells and incubated overnight. Luciferase activity was determined using a dual luciferase assay kit (Promega, Madison, WI) with firefly luciferase activity normalized to Renilla luciferase activity.
[0312] As shown in Figure 4, the bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R79 and the parental anti-DLL4 antibodies 21m18 and 21R79 inhibited DLL4-induced Notch signaling. The bispecific antibody 219R45-MB-21m18 inhibited Dll4-induced Notch signaling only at high antibody concentrations. These results demonstrated that the bispecific antibody 219R45-MB-21R79, and to a lesser extent the bispecific antibody 219R45-MB-21m18, were able to inhibit Dll4-induced Notch signaling. Thus, in combination with the results presented in Example 3, the bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21R79 and 219R45-MB-21m18 demonstrated the ability to inhibit both VEGF-induced and DLL4-induced signaling and/or the proliferation functions. Example 5 Inhibition of tumor growth in vivo by a bispecific antibody in a human skin graft model
[0313] A human skin graft model comprising a human skin graft and human tumor cells has been reported. A human skin graft is established and then the human tumor cells are implanted into the skin graft, allowing the tumor cells to grow in an environment with human stroma and vasculature (Tahtis et al., 2003, Mol. Cancer Ther. 2:229 -737). Human skin samples were obtained from neonatal foreskin tissue and grafted onto the lateral flank of NOD-SCID mice. After establishment of the skin graft, luciferase-labeled OMP-C8 colon tumor cells (20,000 cells) were injected intradermally into human skin. Tumor growth was monitored by bioluminescence imaging using an IVIS imaging system (Caliper Life Sciences, Mountain View, CA). Tumors were allowed to grow until they reached 1.2 x 106 photons per second. Tumor-bearing mice (n = 6 mice/group) were randomly separated and treated with control Ab, anti-hDLL4 antibody 21m18, anti-VEGF antibody bevacizumab, or bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18 . Animals were treated once a week and antibodies were administered intraperitoneally at a dose of 25 mg/kg. Tumor growth was monitored by bioluminescence imaging on the days indicated.
[0314] As shown in Figure 5, both the anti-hDLL4 antibody 21m18 and the anti-VEGF antibody bevacizumab inhibited tumor growth in this human skin graft/human tumor model. Furthermore, the bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18 was more efficient 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 OMP-PN8 pancreatic tumor cells after treatment with bispecific anti-VEGF/anti-DLL4 antibodies
[0315] Mice carrying OMP-PN8 pancreatic tumors were treated with control antibody (15 mg/kg), anti-hDLL4 antibody 21m18 (15 mg/kg), anti-VEGF antibody bevacizumab (15 mg/kg) or antibodies bispecific anti-VEGF/anti-DLL4 219R45-MB-21m18 or 219R45-MB-21R79 (30 mg/kg) with or without gemcitabine (70 mg/kg). After four weeks of treatment, tumors were harvested, processed into single cell suspensions, and human tumor cells were purified by immunomagnetic elimination of murine cells. 90 human tumor cells from each treatment group were transferred to a new group of mice (n = 10 mice/group). Tumors were allowed to grow for 55 days without any treatment and tumor volumes were measured with electronic gauge.
[0316] Figure 6 shows the tumor volume of individual mice in each group. Cells isolated from mice treated with 21m18 anti-hDLL4 antibody had greatly diminished tumorigenicity, 5 out of 10 mice had tumors, when compared to cells isolated from control antibody treated mice where 9 out of 10 mice had tumors. A reduction in the frequency of tumor growth indicates a reduction in the frequency of cancer stem cells. In contrast, treatment with bevacizumab did not result in a reduction in the frequency of tumor growth, 10 out of 10 mice had tumors. Similar to bevacizumab, treatment with gemcitabine as a single agent had no effect on tumor growth frequency since 10 out of 10 mice had tumors. Both anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21m18 and 219R45-MB-21R79 reduced the frequency of tumor growth (5 of 10 mice had tumors and 4 of 10 mice had tumors, respectively). Combination treatment with gemcitabine appeared to have no effect on the frequency of tumor growth. These data indicate that targeting DLL4 reduces the frequency of cancer stem cells while targeting VEGF alone does not. Importantly, these data indicate that the anti-CSC activity of the anti-DLL4 antibody is maintained in a bispecific antibody. Example 7 Bispecific antibody ELISA
[0317] VEGF (ATGEN, South Korea) was coated onto Nunc maxisorb plates at 2 µg/ml (100 µl/well) and incubated overnight at 2-8°C. Bispecific antibodies 219R45-MB-21m18, 219R45-MB-21R79, 219R45-MB-21R75 and 219R45-MB-21R83 were diluted in blocking buffer (1x PBS, 0.1% gelatin, 0.1% Polysorbate- 20, pH 7.4) containing 2 µg/ml biotin-DLL4-hFc. Antibodies were serially diluted 3-fold starting from 500 ng/ml to 0.008 ng/ml. Antibody samples were incubated for 2 hours in blocking buffer containing the biotin-DLL4-hFc. After incubation, antibody samples were transferred to the VEGF coated assay plate (100 µl/well) and incubated for 2 hours. Streptavidin-HRP (Jackson ImmunoResearch, West Grove, PA) was added to each well and incubated for 1 h. Substrate TMB was added to the wells with a 10 minute color development and the reaction was stopped with 2M sulfuric acid. Absorbance was read at 450-650 nm and data were analyzed using 4-parameter fit within the program Softmax Pro Analysis Tool (Molecular Devices, Sunnyvale, CA).
[0318] 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 diamonds) compared to a reference bispecific anti-VEGF/anti-DLL4 antibody (solid circles). The relative potencies for the bispecific antibodies when compared to the reference bispecific antibody are shown in Table 5.


[0319] The bispecific antibody 219R45-MB-21R79 was the most potent, approximately 7 times more potent than 219R45-MB-21m18, which reflected the greater affinity of the binding site to the 21R79 antigen. Example 8 Production of bispecific antibodies
[0320] Bispecific antibodies were produced using a GS-CHO cell line. CHOK1SV cells (Lonza Biologics) were transfected by electroporation with the gene(s) of interest coupled to glutamine synthetase (GS) as the selectable marker. Transfectants and subclones were checked for antibody productivity and high producers were selected for scale-up production. Cells were grown using a fed-batch process and fed-batch bioreactors. Antibody accumulated in the collected cell culture fluid (HCCF) was isolated and purified using chromatography techniques.
[0321] The cell lines of the bispecific antibodies 219R45-MB-21m18.010.017 and 219R45-MB-21R79.017.003 were cultured in 5 L stirred tank bioreactors for 14 days. The 219R45-MB-21m18.010.017 cell line produced a final antibody titer of 3.0 g/L and the 219R45-MB-21R79.017.003 cell line produced a final antibody titer of 0.8 g/L. The 219R45-MB-21R75.101 and 219R45-MB-21R83.113 cell lines were grown in 25 L WAVE bioreactor systems (GE Healthcare) using a fed-batch process that achieved final antibody titers of 0.4 g /L. The 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 219R45-MB-21R83AG.129.003 were cultured in bioreact. - 5 L stirred tank res for 14 - 15 days. The 219R45-MB-21m18AG.138.007 cell line produced a final antibody titer of 1.0 g/L after 14 days. The 219R45-MB-21m18AG.038.009 cell line produced a final antibody titer of 1.6 g/L after 14 days. The 219R45-MB-21m18AG.142.002 cell line produced a final antibody titer of 2.6 g/L after 14 days. The 219R45-MB-21R79AG.072.014 cell line produced a final antibody titer of 2.1 g/L after 15 days. The 219R45-MB-21m18AG.038.009 cell line produced a final antibody titer of 2.4 g/L after 15 days. Culture fluid was collected 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 buffer of low pH elution. Initial characterization of the purity of bispecific antibodies was performed using size exclusion chromatography (SEC-HPLC) and isoelectric focusing (IEF).
[0322] Size exclusion chromatography (SEC) was used to determine the purity of the antibody product. SEC is a well-known chromatographic method in which molecules (eg, antibodies) in solution are separated by their size. SEC can be used to distinguish an antibody product from aggregate and/or impurities and to determine the percentage of antibody product as compared to the total mixture. As used herein, SEC does not distinguish between a homomeric antibody and a bispecific heterodimeric antibody.
[0323] Capillary isoelectric focusing imaging (icIEF) was used to determine the identity and purity of bispecific antibody heterodimers. Using icIEF, an antibody's charge isoforms are separated according to their pI and the result is a “fingerprint” of the antibody's charge distribution. The icIEF method can also serve as a purity determination by separating bispecific antibody heterodimers by their distinct pI from any homodimer or impurity products.
[0324] Bispecific antibody samples were analyzed by icIEF on a ProteinSimple 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, both the pI markers and the protein mixture migrate along the length of the capillary until a pH value at which the net charge is zero is reached. Once focused, the ICE280 instrument uses full-column imaging detection with a 280 nm UV camera to monitor the pattern of protein isoforms within the capillary. The electropherogram is calibrated using internal and integrated pI markers to establish the respective percentage areas of the different charged isoforms of the protein mixture. Loading profiles from various bispecific anti-VEGF/anti-DLL4 antibodies are shown in Figure 8. For this experiment, Protein A eluates were diluted with MilliQ water to a concentration of 6.6 mg/mL. A total of 18 μL of the sample was mixed with 100 μL of 8 M urea, 70 μL of 0.5% methylcellulose, 8 μL of 3-10 Pharmalyte, 2 μL of high pI marker and 2 μL of marker from low pI to a final volume of 200 µL. Table 6 shows the percentage of antibody product from the 219R45-MB-21m18.010.017, 219R45-MB-21R79.017.002, 219R45-MB-21R75.101, 219R45-MB-21R83.113, 219R45-MB- cell lines 21m18.138.007,219R45-MB-21m18AG.038.009, 219R45-MB-21m18AG.142.002, 219R45-MB-21R79AG.072.014 and 219R45-MB-21R83AG.129.003 after affinity chromatography with Protein A which is determined by SEC-HPLC . Table 6 also shows the percentage of heterodimeric antibodies from the 219R45-MB-21m18.010.017, 219R45-MB-21R79.017.002, 219R45-MB-21R75.101, 219R45-MB-21R83.113, 219R45-MB- cell lines 21m18.138.007, 219R45-MB-21m18AG.038.009, 219R45-MB-21m18AG.142.002, 219R45-MB-21R79AG.072.014 and 219R45-MB-21R83AG.129.003 after affinity chromatography with Protein A which is analyzed by icIEF.

[0325] The purity of the bispecific antibody product can be further increased through additional chromatography steps. After Protein A affinity chromatography, the eluate fraction was kept at a low pH for not less than 60 minutes at room temperature for viral inactivation. Antibody solution (eluate from Protein A column, pH adjusted) was loaded onto a strong anion exchange column. Product- and process-related impurities bound to the anion exchange chromatography resin and the eluted fraction (antibody product) were collected. In some cases, purity has been further improved through the use of a multimodal chromatography resin such as ceramic hydroxyapatite. In some cases, buffer exchange of the antibody product was performed using ultrafiltration and diafiltration techniques, after which excipients were added. The formulated antibody was filter sterilized into sterile containers and stored refrigerated or frozen. The purity of the bispecific antibodies was re-evaluated using SEC-HPLC and IEF. Table 7

[0326] As shown in Table 7, purification of bispecific anti-VEGF/anti-DLL4 antibodies with additional chromatography steps after Protein A resulted in isolation of antibody product that was 95% to approximately 99% pure when analyzed by SEC. IEF analysis determined that the purified bispecific anti-VEGF/anti-DLL4 antibody from the 219R45-MB-21m18.010.017 cell line was 98.5% heterodimeric, the bispecific anti-VEGF/anti-DLL4 antibody from the cell line 219R45-MB-21R79.017.002 cell was 99.3% heterodimeric, the bispecific anti-VEGF/anti-DLL4 antibody from the 219R45-MB-21R75.101 cell line was 98.2% heterodimeric, the bispecific anti-VEGF antibody /anti-DLL4 from the 219R45-MB-21R83.113 cell line was 91.4% heterodimeric, the bispecific anti-VEGF/anti-DLL4 antibody from the 219R45-MB-21m18.138.007 cell line was 100% heterodi- meric, the bispecific anti-VEGF/anti-DLL4 antibody from the 219R45-MB-21m18AG.142.002 cell line was 100% heterodimeric, the bispecific anti-VEGF/anti-DLL4 antibody from the 219R45-MB- cell line 21R79AG.072.014 was 100% heterodimeric and the bispecific anti-VEGF/anti-DLL4 antibody came from The patient of the 219R45-MB-21R83AG.129.003 cell line was 100% heterodimeric. These results demonstrated that the anion exchange chromatography step greatly increased the percentage of heterodimeric antibodies when compared to purification by chromatography with Protein A alone. The addition of a multimodal chromatography step such as ceramic hydroxyapatite can also improve monomeric purity (which is determined by SEP-HPLC). Example 9 Inhibition of OMP-C8 colon tumor growth in the in vivo tumor recurrence model
[0327] Isolated cell suspensions from 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 reached an average volume of 240 mm3. Mice were randomly separated (n = 10 per group) and treated with anti-hDLL4 antibody 21m18, anti-VEGF antibody bevacizumab, a combination of 21m18 and bevacizumab antibodies, bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18, bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R79 or control antibody, all in combination with irinotecan. Antibodies and irinotecan were weekly dosed by injection into the intraperitoneal cavity. Antibodies 21m18 and bevacizumab were dosed at 7.5 mg/kg, bispecific antibodies 219R45-MB-21m18 and 219R45-MB-21R79 were dosed at 15 mg/kg and irinotecan was dosed at 45 mg/kg. Irinotecan was dosed for four weeks, during which time it was discontinued and the administration of antibodies continued. Tumor growth was monitored and tumor volumes were measured with electronic gauges at the indicated time points. Data are expressed as mean ± S.E.M.
[0328] As shown in Figure 9, the 21m18 anti-hDLL4 antibody continued to inhibit tumor growth after treatment with irinotecan was stopped. In contrast, the anti-VEGF antibody bevacizumab was not able to inhibit tumor regrowth after irinotecan was 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 bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18 was more efficient in inhibiting tumor regrowth than the mixture of the two antibodies. Example 10 Reduction in tumorigenicity of OMP-C8 colon tumors
[0329] Isolated cell suspensions from 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 reached an average volume of 300 mm3. Mice were randomly separated (n = 5 per group) and treated with anti-DLL4 antibody 21m18, anti-VEGF antibody bevacizumab, a combination of 21m18 and bevacizumab, bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21m18, bispecific anti-VEGF/anti-DLL4 antibody 219R45-MB-21R79 or control antibody, each in combination with irinotecan or without irinotecan. Antibodies and irinotecan were weekly dosed by injection into the intraperitoneal cavity. Antibodies 21m18 and bevacizumab were dosed at 7.5 mg/kg, bispecific antibodies 219R45-MB-21m18 and 219R45-MB-21R79 were dosed at 15 mg/kg and irinotecan was dosed at 45 mg/kg. Tumors were collected after 4 weeks, processed into single cell suspensions and human tumor cells were isolated. 150 tumor cells from each experimental group were injected subcutaneously into a new group of mice (n = 10 per group) and the tumors were allowed to grow without treatment. Tumor growth was monitored and tumor volumes were measured with electronic gauges.
[0330] Individual tumor volumes at day 68 are shown in Figure 10. The anti-DLL4 antibody 21m18, the combination of 21m18 with the anti-VEGF antibody bevacizumab, the bispecific antibodies 219R45-MB-21m18 and 219R45-MB - 21R79 and irinotecan all reduced the frequency of tumor growth as single agents. In contrast, the anti-VEGF bevacizumab as an isolated agent had no effect on the frequency of tumor growth when compared to the control antibody. In groups treated with a combination of irinotecan and antibodies, the bispecific antibody 219R45-MB-21m18 had the greatest effect in reducing the frequency of tumor growth. Example 11 Inhibition of OMP-C8 colon tumor growth in vivo
[0331] Isolated cell suspensions from 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 80 mm3. The mice were randomly separated (n = 8 per group) and treated with anti-DLL4 antibody 21m18, anti-VEGF antibody bevacizumab, bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18, 219R45-MB-21R75, 219R45-MB-21R79, 219R45-MB-21R83 or control antibody, alone or in combination with irinotecan. Antibodies and irinotecan were weekly dosed by injection into the intraperitoneal cavity. Bevacizumab and the bispecific antibodies 219R45-MB-21m18, 219R45-MB-21R75, 219R45-MB-21R79 and 219R45-MB-21R83 were dosed at 15 mg/kg and irinotecan was dosed at 7.5 mg/kg . Tumor growth was monitored and tumor volumes were measured with electronic gauges at the indicated time points. Data are expressed as mean ± S.E.M.
[0332] In the form of isolated agents, all four bispecific anti-VEGF/anti-DLL4 antibodies exhibited greater antitumor activity compared to the anti-VEGF antibody bevacizumab. In combination with irinotecan, treatment with bispecific anti-VEGF/anti-DLL4 antibodies 219R45-MB-21m18 and 219R45-MB-21R83 resulted in the greatest inhibition of tumor growth (Figure 11).
[0333] After the treatment phase, tumor sections were prepared and analyzed by staining with hematoxylin and eosin (H&E). Tumors treated with 219R45-MB-21m18 and 219R45-MB-21R83 in combination with irinotecan exhibited regions of dark pink color 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 toxicity study in cynomolgus monkeys was initiated to evaluate and to compare 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 bispecific anti-DLL4/anti-VEGF antibody (219R45-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 that received the high dose 219R45-MB-21R79 than in animals that received the high dose 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 received 219R45-MB-21R18 or 219R45-MB-21R83. Although preliminary in nature, these initial data suggest that 219R45-MB-21R18 and 219R45-MB-21R83 may have a superior toxicity profile compared to 219R45-MB-21R79.
[0334] It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested by those skilled in the art and should be included within the spirit and purview of this patent application .
[0335] All publications, patents, patent applications, internet sites and database accession/sequence numbers including both polynucleotide and polypeptide sequences cited herein are incorporated herein by reference in their entirety for all the purposes to the same extent as if each individual publication, patent, patent application, website or accession number/database sequence were specifically and individually indicated as being incorporated by reference. SEQUENCE heavy chain with 21m18 signal sequence (underlined) (SEQ ID NO: 1) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKISC KAS GYS FTA YYIHWVKQAP GQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTA VYYCARDYD YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGS FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain with 21R79 signal sequence (underlined) ( SEQ ID NO: 2) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKISC KAS GYS FTA YYIHWVKQAP GQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTA VYYCARDYD YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP IE KTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGS FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain with 219R45 signal sequence (underlined) (SEQ ID NO: 3) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKVSC KAS GYTFTN YWMHWVRQAP GQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDT AVYFCTIHYD DKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLKSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK light chain with signal sequence (underlined) (SEQ ID NO: 4) MVLQTQVFISLLLWISGAYG DIVMTQSPDS LAVS LGE RAT IS C RAS ESVDNY GISFMKWF QQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY CQQSKEVPW TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC chain bp exit without predicted signal sequence 21m18 (SEQ ID NO: 5) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI SSYNGATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQ VSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVD KSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK no sequence heavy chain signal expected 21R79 (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI ANYNRATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRV VSVLTVVHQDWNTPAPKTIKQCKPSNKVPL SELTVD KSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK no sequence heavy chain signal expected 219R45 (SEQ ID NO: 7) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWM GDINPSNGRTSY KEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYW GQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREKMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLT VDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK no sequence light chain signal provided (SEQ ID NO: 8) DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLI YAASNQGS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK RTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 21m18 Heavy Chain Variable Region (SEQ ID NO:9) QVQLVQSGAEVKKNYPGISSQVFTAKSTYSCTG TAVYYCARDYDYDVGMDYW GQGTLVTVSS region heavy chain variable 21R79 (SEQ ID NO: 10) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI ANYNRATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSS region heavy chain variable 219R45 (SEQ ID NO: 11) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWM GDINPSNGRTSY KEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYW GQGTLVTVSS light chain variable region (SEQ ID NO: 12) DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLI YAASNQGS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK 21R75 heavy chain CDR1, 21R79, 21R83 and 21RY83 heavy chain 21R, 21R79H 79H, alternative heavy chain 21R, SEQ ID:21R79H79H SEQ ID 21YR:21R79H79HDI (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 CDR1 (SEQ ID NO:14) ID NO:17) NYWMH heavy chain CDR2 of 219R45 (SEQ ID NO:18) DINPSNGRTSYKEKFKR 219R45 Heavy Chain CDR3 (SEQ ID NO:19) HYDDKYYPLMDY Light Chain CDR1 (SEQ ID NO:20) RASESVDNYGISFMK Light Chain CDR2 (SEQ ID NO:21) AASNQGS Chain CDR3 light (SEQ ID nO: 22) QQSKEVPWTFGG human DLL4 with signal sequence (underlined) (SEQ ID nO: 23) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQE FINE RGVLAS CPRM EPGCRTFFRV CLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFT WPGTFSLIIE AWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYR VICSDNYY GDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQ NGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYC THHSPCKNGATC SNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCL CPPGYYGLHCE HSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSN PCANGGQCLNR GPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAG FSGRRCEVRTS IDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVG Dll4 human without signal sequence provided (SEQ ID NO:24) SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTF GTVSTPVLGT NSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWH APGDDLRPEALPPD ALISKIAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFG HYVCQPDGNL SCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIP HNGCRHGTCST PWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRP GYTGVDCELELS ECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGG SCRERNQGANY ACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGT YCELHVSDCAR NPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCY TDLSTDTFVC NCPYGFVGSRCEFPVG the N-terminal Human DLL4 (SEQ ID NO: 25) SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTF GTVSTPVLGT NSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPD ALISKIAIQ GSLAVGQN DSL domain from human DLL4 (SEQ ID NO: 26) WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDG NLSCLPGWTG EYC VEGF -A with human signal sequence (underlined) (SEQ ID NO: 27) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQ RSYCHPIETLVD IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPH QGQHIGEM SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGA RCCLMPWSLPG PHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDK PRR VEGF-A human without signal sequence provided (SEQ ID NO: 28) APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSC VPLMRCGGC CNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRA RQEKKSVRG KGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHL FVQDPQTCKCSC KNTDSRCKARQLELNERTCRCDKPRR sequence heavy chain nucleotide 21m18 (13B version 1) (SEQ ID NO: 29) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGATCTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGGTCA AGCAGGCCCCT GGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGCGCC ACCAACTACAAC CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCC ACCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGC CAGAGACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGT GTCCTCTGCCTCC ACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACC TCTGAGTCTACC GCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACC GTGTCCTGGAAC TCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCTAGCGTGGTGAC CGTGCCTTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTG GAGCGGAAGTGC TGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTG GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGCGTGGAG GTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTG GAGTGGGAGTCT AACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG TCTCCTGGCAAGTAG heavy chain sequence of nucleotide 21R79 (13B version 1) (SEQ ID NO: 30) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGAT CTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTGA AACAGGCACCA GGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGGGCC ACCAACTACAAC CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCC ACAGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGC CAGAGACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGT GTCCTCCGCCTCC ACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACC TCTGAGTCTACC GCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACC GTGTCCTGGAAC TCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCTAGCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTG GAGCGGAAGTGC TGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTG GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGCGTGGAG GTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGC GAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTG GAGTGGGAGTCT AACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG TCTCCTGGCAAGTAG stranded nucleotide sequence heavy 21R79 (13B Version 2) (SEQ ID NO: 31) ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGG TGTTATCGCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCGCTAATTATAATAGAGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTA GAGATTATGAT TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTG CCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAGTAG heavy chain sequence of nucleotide 219R45 (13A version 1) (SEQ ID NO: 32) ATGAAGCATCTGTGGTTTTTCCTGTTGCTCGTGGCGGCACCCAGATGGG TGTTGTCCCAA GTGCAGCT GGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCAAG CGTAAAAGTATCG TGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGGTGC GGCAGGCTCCG GGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGCAGA ACATCATATAAG GAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCTCG ACTGCGTACATG GAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGCACC ATCCATTATGAT GACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGGTCA CCGTCTCCAGC GCGTCGACGAAAGGTCCCTCGGTATTTCCCCTCGCCCCCTGCTCGAGG TCGACATCCGAA TCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAGAGCCG GTAACGGTGTCG TGGAACTCGGGAGCGCTTACGTCCGGAGTCCACACATTTCCGGCGGTA CTGCAATCCTCG GGACTGTATTCGTTGTCGTCAGTGGTGACTGTCCCGTCCTCCAATTTCG GGACTCAGACC TATACGTGCAACGTCGACCACAAACCCTCAAACACCAAGGTGGATAAGA CAGTGGAGCGC AAGTGCTGCGTGGAGTGTCCCCCGTGTCCGGCACCCCCTGTCGCCGG ACCCTCAGTCTTT TTGTTTCCGCCGAAGCCCAAAGATACACTCATGATCTCAAGAACGCCCG AGGTAACATGC GTGGTGGTCGATGTAAGCCACGAGGATCCAGAAGTACAATTCAATTGGT ATGTAGACGGG GTCGAGGTCCATAACGCAAAGACGAAACCGAGGGAAGAGCAGTTCAAT TCGACTTTCCGG GTGGTGTCGGTGCTTACAGTCGTACATCAGGACTGGTTGAACGGGAAG GAGTACAAGTGT AAAGTATCGAATAAGG GCCTTCCAGCGCCGATTGAAAAGACCATCTCCA AGACCAAAGGA CAGCCACGAGAGCCGCAAGTCTATACGCTTCCTCCCAGCCGAGAAAAG ATGACTAAAAAC CAGGTATCGCTTACGTGTCTCGTCAAGGGTTTCTACCCTTCGGACATCG CGGTGGAATGG GAGAGCAATGGACAACCGGAAAACAACTACAAGACGACACCGCCTATG TTGAAAAGCGAT GGATCGTTTTTCCTCTATTCGAAACTCACGGTCGATAAGTCACGGTGGC AGCAGGGGAAT GTGTTCTCCTGTTCAGTGATGCACGAGGCGCTCCACAATCACTATACCC AGAAAAGCCTG TCACTTTCCCCGGGAAAATGA stranded nucleotide sequence heavy 219R45 (13A Version 2) (SEQ ID NO: 33) ATGAAGCACCTCTGGTTCTTCCTGCTCCTCGTGGCTGCTCCTCGGTGG GTCCTCTCCCAA GTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCTTC CGTCAAAGTCTCC TGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGGTGC GGCAGGCTCCT GGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGCAGA ACCTCCTACAAG GAAAAATTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCTCCA CAGCTTACATG GAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCACCA TCCACTACGAC GACAAGTACTACCCTCTGATGGACTACTGGGGCCAGGGAACCCTGGTC ACCGTGTCCAGC GCTTCCACAAAAGGACCCTCCGTCTTTCCCCTCGCCCCCTGCTCCCGG TCCACATCCGAA TCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAG AGCCT GTCACAGTGTCC TGGAACTCCGGAGCTCTCACATCCGGAGTCCACACATTTCCTGCTGTGC TCCAATCCTCC GGACTGTATTCCCTCTCCTCCGTGGTGACAGTGCCTTCCTCCAATTTCG GGACACAGACC TATACATGCAACGTGGACCACAAACCCTCCAACACCAAAGTCGATAAGA CAGTGGAGCGC AAGTGCTGCGTGGAGTGTCCCCCTTGTCCTGCTCCCCCTGTGGCTGGA CCTTCCGTCTTT CTGTTTCCTCCTAAACCTAAAGACACCCTCATGATCTCCCGGACCCCCG AGGTCACATGC GTGGTCGTCGATGTGAGCCACGAGGACCCCGAAGTCCAATTTAATTGG TATGTGGACGGG GTGGAGGTCCATAACGCTAAGACCAAACCTAGGGAAGAGCAGTTCAATT CCACTTTCCGG GTGGTGTCCGTGCTGACCGTCGTTCATCAGGACTGGCTCAACGGGAAA GAATACAAATGC AAAGTCTCTAATAAGGGCCTCCCTGCTCCTATTGAAAAAACAATTTCCAA AACAAAAGGA CAACCTCGGGAGCCTCAAGTCTACACACTGCCACCTTCCCGGGAAAAA ATGACAAAAAAT CAAGTCTCCCTCACATGTCTCGTCAAGGGATTCTACCCTTCCGACATTG CTGTGGAATGG GAATCCAATGGACAACCTGAAAACAACTACAAGACAACACCTCCTATGC TCAAAAGCGAT GGGTCCTTTTTCCTCTATTCCAAACTCACAGTCGATAAGTCTCGGTGGC AGCAGGGGAAT GTGTTCTCCTGTTCCGTGATGCACGAGGCTCTCCACAATCACTATACCC AGAAAAGCCTG TCCCTCTCCCCTGGAAAATGA light chain nucleotide sequence (SEQ ID NO: 34) ATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCT GCTGTGGATCTCC GGCGCCTACGGC GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGA GAGCGGGCCACC ATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCA TGAAGTGGTTC CAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCC AACCAGGGATCT GGCGTGCCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACC CTGACCATCAGC TCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAA GAGGTGCCCTGG ACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGC TCCCTCCGTGTTC ATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCCTCCGTC GTGTGCCTGCTG AACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAAC GCCCTGCAGTCC GGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACC TACTCCCTGTCC TCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC GCCTGCGAAGTG ACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGTTAG chain variable region nucleotide sequence 21m18 heavy (SEQ ID NO: 35) CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGC CTCCGTGAAGATC TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGG TCAAGCAGGCC CCTGGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGC GCCACCAACTAC AACCAGAAATTCAAGGGCCGCG TGACCTTCACCACCGACACCTCCACC TCCACCGCCTAC ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGC GCCAGAGACTAC GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCAC CGTGTCCTCT chain variable region nucleotide sequence heavy 21R79 (13B) (SEQ ID NO: 36) CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGC CTCCGTGAAGATC TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGG TGAAACAGGCA CCAGGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGG GCCACCAACTAC AACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACC TCCACAGCCTAC ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGC GCCAGAGACTAC GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGAC AGTGTCCTCC nucleotide sequence of the heavy chain variable region 21R79 (13B Version 2 ) (SEQ ID NO:37) CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCAT CCGTGAAAATA AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGG TTAAACAGGCA CCAGGACAGGGACTTGAATGGATCAATCGATATCAGTAACCTACCTGACCTCGTCGTGAATGATCAGCATACCTTCGATCAGT ACACTGCCGTTTACTATTGCG CTAGAGATTAT GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACA GTGTCTTCT chain variable region nucleotide sequence heavy 219R45 (13A version 1) (SEQ ID NO: 38) CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGC AAGCGTAAAAGTA TCGTGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGG TGCGGCAGGCT CCGGGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGC AGAACATCATAT AAGGAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCT CGACTGCGTAC ATGGAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGC ACCATCCATTAT GATGACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGG TCACCGTCTCC AGC chain variable region nucleotide sequence heavy 219R45 (13A Version 2) (SEQ ID NO: 39) CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGC TTCCGTCAAAGTC TCCTGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGG TGCGGCAGGCT CCTGGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGC AGAACCTCCTAC AAGGAAAAATTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCT CCACAGCTTAC ATGGAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCA CCATCCACTAC GACGACAAGTACTACCC TCTGATGGACTACTGGGGCCAGGGAACCCTG GTCACCGTGTCC AGC light chain variable region nucleotide sequence (SEQ ID NO: 40) GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGA GAGCGGGCCACC ATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCA TGAAGTGGTTC CAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCC AACCAGGGATCT GGCGTGCCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACC CTGACCATCAGC TCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAA GAGGTGCCCTGG ACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG heavy chain constant region of human IgG1 (SEQ ID NO: 41) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain constant region of human IgG2 (SEQ ID NO: 42) G ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSS LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP APPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK region heavy chain constant human IgG3 (SEQ ID NO: 43) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTC PRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSV FLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTF RVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHE ALHNRFTQKSLSLSPGK Region chain constant heavy human IgG4 (SEQ ID NO: 44) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPA PEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQVQVQFVQVQTVFTVNTKTVQVQVVVDVSQVDKRVESKYGPPCPSCPA PEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQVQVQVQFVQTVLFTVQTVFTV VSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK Peptide FLAG (SEQ ID NO: 45) DYKDDDDK heavy chain with chain unmodified underlined in 21R79 parent signal sequence (SEQ ID NO: 46) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKISC KAS GYS FTA YYIHWVKQAP GQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTA VYYCARDYD YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain signal sequence underlined with parental 219R45 (SEQ ID NO: 47) MKHLWFFLLLVAAPRWVLSQVQ LVQ SGAEVKKPGASVKVSC KAS GYTFTN YWMHWVRQAP GQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDT AVYFCTIHYD DKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain without sequence predicted signal 21R79 Parental (SEQ ID NO: 48) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI ANYNRATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK heavy chain without sequence 219R45 parental signal (SEQ ID NO:49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWM GDINPSNGRTSY KEKFKRRVTLSVDK SSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYW GQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK nucleotide sequence of the heavy chain variable region 21R79 Parental (SEQ ID NO: 50) CAAGTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGC ATCGGTCAAAATC TCGTGTAAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGG TAAAGCAGGCG CCAGGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGC GCGACGAACTAT AACCAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACAT CGACGGCCTAC ATGGAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGC GCAAGAGATTAC GACTATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAG TCTCCTCC sequence of the variable region nucleotides of parental 219R45 heavy chain (SEQ ID NO:51) CAGGTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGC GTCGGT AAAGGTC AGCTGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGG TCAGACAGGCC CCTGGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGA CGGACTAGCTAT AAGGAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCC TCAACGGCCTAC ATGGAGTTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTA CTATCCACTAT GACGATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTG GTAACCGTGAGC AGC stranded nucleotide sequence weighed 21R79 parent signal sequence (SEQ ID NO: 52) ATGAAACACTTGTGGTTTTTCCTCTTGCTCGTGGCAGCTCCTCGGTGGG TACTTTCACAA GTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGCATC GGTCAAAATCTCG TGTAAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGGTAA AGCAGGCGCCA GGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGCGCG ACGAACTATAAC CAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACATCGA CGGCCTACATG GAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGCGCA AGAGATTACGAC TATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAGTCT CCTCCGCCAGC ACCAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACC AGCGAGAGCACA GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAAC TCAGGCGCTCTGACCAGCGGCGTG CACACCTTCCCAGCTGTCCTACAG TCCTCAGGACTC TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACC CAGACCTACACC TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTT GAGCGCAAATGT TGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCA GTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACGTGCGTGGTG GTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG GACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACG TTCCGTGTGGTC AGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTC TCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCA AAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACC AAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTG GAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGAC TCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTC TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTG TCTCCGGGTAAA heavy chain sequence of nucleotides with 219R45 parent signal sequence (SEQ ID NO: 53) ATGAAACACCTCTGGTTCTTTTTGCTCCTGGTGGCAGCTCCCCGAT GGG TGCTTAGCCAG GTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGCGTC GGTAAAGGTCAGC TGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGGTCA GACAGGCCCCT GGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGACGG ACTAGCTATAAG GAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCCTCAA CGGCCTACATG GAGTTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTACTA TCCACTATGAC GATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTGGTAA CCGTGAGCAGC GCGTCCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGG AGCACCAGCGAG AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCG TGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTC CTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTC GGCACCCAGACC TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGC AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACGTGC GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGACGGC GTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGT GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTA CAAGTGC AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCA AAACCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC GCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATG CTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTC TCCCTGTCTCCGGGTAAA nucleotide sequence of the light chain variable region 21R79 and 219R45 parental (SEQ ID NO: 54) GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGC GAGAGGGCCACC ATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTAT GAAGTGGTTC CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCA ACCAAGGGTCC GGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACT CTCACCATCAGC AGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAG GAGGTGCCTTGG ACATTCGGAGGAGGGACCAAGGTGGAAATCAAA Nucleotide Sequence parental 21R79 and 219R45 light chain (SEQ ID NO:55) ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCG GAGCCTACGGG GACATCGT GATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGC GAGAGGGCCACC ATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTAT GAAGTGGTTC CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCA ACCAAGGGTCC GGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACT CTCACCATCAGC AGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAG GAGGTGCCTTGG ACATTCGGAGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCC CCCTCCGTCTTC ATCTTCCCCCCCAGCGATGAGCAGCTGAAAAGCGGCACTGCCAGCGTG GTGTGCCTGCTG AATAACTTCTATCCCCGGGAGGCCAAAGTGCAGTGGAAGGTGGATAAC GCCCTCCAAAGC GGCAACTCCCAGGAGAGCGTCACAGAGCAGGACAGCAAGGACAGCAC CTACAGCCTCAGC AGCACCCTGACCCTGAGCAAAGCCGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTC ACCCATCAGGGCCTGAGCAGCCCCGTCACAAAGAGCTTCAACAGGGGC GAGTGTTGA heavy chain without signal sequence predicted 21R75 (SEQ ID NO: 56) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI AGYKDATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFL FPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQ VSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVD KSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK heavy chain with predicted signal sequence 21R75 (underlined) (SEQ ID NO: 57) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKISC KAS GYS FTA YYIHWVKQAP GQGLEWIGYIAGYKDATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTA VYYCARDYD YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGS FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Region chain variable 21R75 heavy (SEQ ID NO:58) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYYIHWVKQAPGQGLEWIGYI AGYKDATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSS heavy chain NO:21R75 (SEQ ID NO:58) NQKFKG heavy chain sequence of nucleotides with 21R75 signal sequence (13B version 1) (SEQ ID NO: 60) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGATCTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCA AGCAGGCCCCT GGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCC ACCAACTACAAC CAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTA CCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGC CAGAGACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGT GTCCTCTGCTTCC ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCT CCGAGTCTACC GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACA GTGTCTTGGAAC TCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTG GAACGGAAGTGC TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGC GTGTTCCTGTTC CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCC CGAGGTGCAGTTCAATTGGTACGTG GACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCA AGGGACAGCCC CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGTCC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGAC TCCGACGGCTCA TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG AGCCCCGGCAAG heavy chain sequence of nucleotides with 21R75 signal sequence (13B 1Q version) (SEQ ID NO: 77) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCTCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGATCTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCA AGCAGGCCCCT GGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCC ACCAACTACAAC CAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTA CCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTAC TACTGCGC CAGAGACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGT GTCCTCTGCTTCC ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCT CCGAGTCTACC GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACA GTGTCTTGGAAC TCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTG GAACGGAAGTGC TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGC GTGTTCCTGTTC CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTG GACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCA AGGGACAGCCC CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGTCC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGAC TCCGACGGCTCA TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCA L GGCAACGTGTTC TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG AGCCCCGGCAAG heavy chain sequence of nucleotides with 21R75 signal sequence (13B Version S1-2) (SEQ ID NO: 61) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTA GAGATTATGAT TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAG without following heavy chain signal expected 21R83 (SEQ ID NO: 62) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYI SNYNRATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQ VSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVD KSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK heavy chain with predicted signal sequence 21R83 (underlined) (SEQ ID NO: 63) MKHLWFFLLLVAAPRWVLS QVQ LVQ SGAEVKKPGASVKISC KAS GYS FTA YYIHWVKQAP GQGLEWIGYISNYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTA VYYCARDYD YDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGS FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK region heavy chain variable of 21R83 (SEQ ID NO:64) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYYIHWVKQAPGQGLEWIGYI SNYNRATNY NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW GQGTLVTVSS Nucleus KNYQN83 heavy chain Sequence IDNQG 21R72 (heavy chain Sequence IDQNATG) 21R85 adeia weighed sublinha- signal sequence of 21R83 (13B version 1) (SEQ ID NO: 66) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGATCTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCA AGCAGGCCCCT GGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCC ACCAATTACAAC CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTA CCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGC CAGAGACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGT GTCTAGCGCTTCC ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCT CCGAGTCTACC GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACA GTGTCCTGGAAC TCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTG GAACGGAAGTGC TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGC GTGTTCCTGTTC CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTG GTGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGT L GACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCA AGGGACAGCCC CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGTCC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGAC TCCGACGGCTCA TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG AGCCCCGGCAAG heavy chain sequence of nucleotides with underlined signal sequence 21R83 (13B 1Q version) (SEQ ID NO: 78) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCTCAG GTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCC GTGAAGATCTCC TGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCA AGCAGGCCCCT GGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCC ACCAATTACAAC CAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTA CCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGC CAGA GACTACGAC TACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGT GTCTAGCGCTTCC ACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCT CCGAGTCTACC GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACA GTGTCCTGGAAC TCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAG TCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCC AGACCTACACC TGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTG GAACGGAAGTGC TGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGC GTGTTCCTGTTC CCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTG GACGGCGTGGAA GTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACC TTCCGGGTGGTG TCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCA AGGGACAGCCC CGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGTCC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGAC TCCGACGGCTCA TTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTT C TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTG AGCCCCGGCAAG chain nucleotide sequence with heavy underlined sequence signal 21R75 (S1-2 Version 13B) (SEQ ID NO: 67) ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGG GTGCTGTCCCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTA GAGATTATGAT TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAG nucleotide sequence of the heavy chain variable region 21R75 (13B version 1) (SEQ ID NO: 68) CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGC CTCCGTGAAGATC TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGG TCAAGCAGGCC CCTGGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGAC GCCACCAACTAC AACCAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCT CTACCGCCTAC ATGGAACTGCGGTCCCCTGC GGAGCGACGACACCGCCGTGTACTACTGC GCCAGAGACTAC GACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGAC CGTGTCCTCT chain variable region nucleotide sequence heavy 21R75 (13B Version 2) (SEQ ID NO: 69) CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCAT CCGTGAAAATA AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGG TTAAACAGGCA CCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATG CTACAAACTAT AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCT CGACAGCATAC ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCG CTAGAGATTAT GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACA GTGTCTTCT nucleotide sequence of the heavy chain variable region 21R83 (13B version 1) (SEQ ID NO: 70) CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGC CTCCGTGAAGATC TCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGG TCAAGCAGGCC CCTGGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGG GCCACCAATTAC AACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCT CTACCGCCTAC ATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGC GCCAGAGACTAC GACTACGACGTGGGCAT GGACTACTGGGGCCAGGGCACACTCGTGAC CGTGTCTAGC chain variable region nucleotide sequence heavy 21R75 (13B Version 2) (SEQ ID NO: 71) CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCAT CCGTGAAAATA AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGG TTAAACAGGCA CCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATG CTACAAACTAT AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCT CGACAGCATAC ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCG CTAGAGATTAT GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACA GTGTCTTCT heavy chain sequence of nucleotides with the signal sequence underlined in 21R83 ( 13B Version 2) (SEQ ID NO: 72) ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGG TGTTATCGCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTA TAT GAGATTATGAT GATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGT AATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAGTAG chain variable region nucleotide sequence heavy 21R83 (13B Version 2) (SEQ ID NO: 73) CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCAT CCGTGAAAATA AGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGG TTAAACAGGCA CCAGGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAG CTACAAACTAT AACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCT CGACAGCATAC ATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCG CTAGAGATTAT GATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACA GTGTCTTCT heavy chain sequence of nucleotides with underlined signal sequence 21R75 (13B Version 2) (SEQ ID NO: 74) ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGG TGTTATCGCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACT ATTGCGCTA GAGATTATGAT TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAGTAG stranded nucleotide sequence heavy 21m18 (version 2) (SEQ ID NO: 75) ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGG TGTTATCGCAG GTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCC GTGAAAATAAGT TGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTA AACAGGCACCA GGACAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTA CAAACTATAAC CAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGA CAGCATACATG GAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTA GAGATTATGAT TATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTG TCTTCTGCATCC ACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCT CTGAATCGACT GCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACC GTTTCCTGGAAC TCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAG TCTTCTGGCCTA TACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCA AACTTACACC TGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTG AGCGAAAGTGC TGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCT GTGTTCCTGTTC CCTCCAAAACCTAAGGACACTCTA ATGATCTCTCGGACTCCTGAGGTGA CTTGCGTGGTT GTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTG GACGGAGTCGAG GTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACC TTCCGGGTGGTT TCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTG TCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACC AAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACC AAGAACCAGGTG TCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTT GAGTGGGAGTCT AACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGAC TCCGACGGCTCC TTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAG GGCAACGTGTTC TCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGT CCCTATCCTTA TCTCCTGGCAAGTAG heavy chain variable region 21m18 (version 2) (SEQ ID NO: 76) CAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTG AAAATAAGTTGC AAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAAC AGGCACCAGGA CAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTACAA ACTATAACCAA AAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAG CATACATGGAA TTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAG ATTATGATTAT GATGTTGGAA TGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCT TCT Anti-DLL4 heavy chain CDR2 consensus sequence (SEQ ID NO:80): YIX1X2YX3X4ATNYNQKFKG, where X1 is serine or alanine, X2 is serine, as-paragine or glycine, X4 is glycine and asparagine , arginine or aspartic acid.

权利要求:
Claims (25)
[0001]
1. Bispecific antibody CHARACTERIZED by the fact that it comprises: a) a first antigen-binding site that specifically binds to human vascular endothelial growth factor (VEGF), and b) a second antigen-binding site that specifically binds to the ligand human delta 4 type (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 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the second antigen binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO: 13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO: 14) or 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 CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21), and a CDR3 of light chain comprising QQSKEVPWTFGG (SEQ ID NO:22).
[0002]
2. Bispecific antibody according to claim 1, CHARACTERIZED by the fact that the second antigen-binding site comprises 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).
[0003]
3. Bispecific antibody according to claim 1, CHARACTERIZED by the fact that the second antigen-binding site comprises a heavy chain CDR1 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).
[0004]
4. Bispecific antibody according to claim 1, CHARACTERIZED by the fact that it comprises: (a) a first heavy chain variable region having the sequence of SEQ ID NO: 11; (b) a second heavy chain variable region having the sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 58; and (c) a first and a second light chain variable region having the sequence of SEQ ID NO:12.
[0005]
5. Bispecific antibody, according to any one of claims 1 to 4, CHARACTERIZED by the fact that it comprises a first CH3 domain and a second CH3 domain, each of which is modified to promote the formation of heteromultimers.
[0006]
6. Bispecific antibody according to claim 5, CHARACTERIZED by the fact that the first and second CH3 domains are modified based on electrostatic effects.
[0007]
7. Bispecific antibody according to any one of claims 1 to 6, CHARACTERIZED by the fact that it is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, an IgG1 antibody or an antibody of IgG2.
[0008]
8. Bispecific antibody, according to any one of claims 1 to 7, CHARACTERIZED by the fact that it comprises a first human IgG2 constant region with amino acid substitutions in positions corresponding to positions 249 and 288 of SEQ ID NO: 42, in that the amino acids are replaced by glutamate or aspartate, and a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO: 42, where the amino acids are replaced by lysine.
[0009]
9. Bispecific antibody, according to any one of claims 1 to 8, CHARACTERIZED by the fact that: (i) it inhibits the binding of VEGF to at least one VEGF receptor; (ii) inhibits DLL4 binding to at least one Notch receptor; (iii) inhibits Notch signaling; and/or (iv) modulates angiogenesis.
[0010]
10. A bispecific antibody that specifically binds to human VEGF and human DLL4, CHARACTERIZED in that it comprises: (a) a heavy chain of SEQ ID NO:7; (b) a heavy chain of SEQ ID NO: 5 or SEQ ID NO: 6; and (c) two light chains of SEQ ID NO: 8.
[0011]
11. Bispecific antibody CHARACTERIZED by the fact that it is selected from the group consisting of 219R45-MB-21M18 and 219R45-MB-21R79.
[0012]
12. Pharmaceutical composition CHARACTERIZED by the fact that it comprises the antibody, as defined in any one of claims 1 to 11, and a pharmaceutically acceptable carrier.
[0013]
13. Prokaryotic, fungal or yeast cell, CHARACTERIZED by the fact that it comprises or produces the antibody, as defined in any one of claims 1 to 11.
[0014]
14. A polynucleotide molecule CHARACTERIZED in that it comprises: (i) a heavy chain sequence of SEQ ID NO: 32 or 33, (ii) a heavy chain sequence of SEQ ID NO: 30 or 31, and (iii) two light chain sequences of SEQ ID NO: 34, operably linked to an expression control sequence.
[0015]
15. A polynucleotide molecule CHARACTERIZED in that it comprises: (i) a heavy chain variable region sequence of SEQ ID NO: 38 or 39, (ii) a heavy chain variable region sequence of SEQ ID NO: 36, or 37, and (iii) two light chain variable region sequences of SEQ ID NO: 40, operably linked to an expression control sequence.
[0016]
16. A polynucleotide molecule CHARACTERIZED in that it comprises: (i) a heavy chain sequence of SEQ ID NO: 32 or 33, (ii) a heavy chain sequence of SEQ ID NO: 29 or 75, and (iii) two light chain sequences of SEQ ID NO: 34, operably linked to an expression control sequence.
[0017]
17. A polynucleotide molecule CHARACTERIZED in that it comprises: (i) a heavy chain variable region sequence of SEQ ID NO: 38 or 39, (ii) a heavy chain variable region sequence of SEQ ID NO: 35, or 76, and (iii) two light chain variable region sequences of SEQ ID NO: 40, operably linked to an expression control sequence.
[0018]
18. Vector CHARACTERIZED by the fact that it comprises the polynucleotide, as defined in any one of claims 14 to 17, operably linked to a heterologous promoter and termination sequences.
[0019]
19. Prokaryotic, fungal or yeast cell, CHARACTERIZED by the fact that it comprises the vector, as defined in claim 18.
[0020]
20. Use of the antibody as defined in any one of claims 1 to 11, CHARACTERIZED by the fact that it is for the manufacture of a drug for the treatment of cancer.
[0021]
21. Use of the antibody according to claim 20, CHARACTERIZED by the fact that the cancer is selected from the group consisting of colorectal cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer breast, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphoma and leukemia.
[0022]
22. Use of the antibody, as defined in any one of claims 1 to 11, CHARACTERIZED by the fact that it is for the manufacture of a drug for inhibiting the growth of a tumor.
[0023]
23. Use of the antibody, according to claim 22, CHARACTERIZED by the fact that the tumor is selected from the group consisting of colorectal tumor, colon tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, tumor of breast, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
[0024]
24. Use of the antibody, according to any one of claims 20 to 23, CHARACTERIZED by the fact that the use comprises the antibody and at least one additional chemotherapeutic agent.
[0025]
25. Method for the production of an antibody, CHARACTERIZED by the fact that it comprises expressing at least one polynucleotide, as defined in any one of claims 14 to 17, in a cell.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US3773919A|1969-10-23|1973-11-20|Du Pont|Polylactide-drug mixtures|

---

US4485045A|1981-07-06|1984-11-27|Research Corporation|Synthetic phosphatidyl cholines useful in forming liposomes|

---

CA1176659A|1981-09-14|1984-10-23|Gerhard Degischer|Process for producing n-substituted-n-acetyl-2,6-dialkylanilines|

---

US4588585A|1982-10-19|1986-05-13|Cetus Corporation|Human recombinant cysteine depleted interferon-β muteins|

---

US4816567A|1983-04-08|1989-03-28|Genentech, Inc.|Recombinant immunoglobin preparations|

---

US4544545A|1983-06-20|1985-10-01|Trustees University Of Massachusetts|Liposomes containing modified cholesterol for organ targeting|

---

US4676980A|1985-09-23|1987-06-30|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Target specific cross-linked heteroantibodies|

---

US5681718A|1986-03-14|1997-10-28|Celltech Limited|Methods for enhanced production of tissue plasminogen activator in cell culture using alkanoic acids or salts thereof|

---

US5225539A|1986-03-27|1993-07-06|Medical Research Council|Recombinant altered antibodies and methods of making altered antibodies|

---

US5567610A|1986-09-04|1996-10-22|Bioinvent International Ab|Method of producing human monoclonal antibodies and kit therefor|

---

US4946778A|1987-09-21|1990-08-07|Genex Corporation|Single polypeptide chain binding molecules|

---

GB8823869D0|1988-10-12|1988-11-16|Medical Res Council|Production of antibodies|

---

US5530101A|1988-12-28|1996-06-25|Protein Design Labs, Inc.|Humanized immunoglobulins|

---

US5013556A|1989-10-20|1991-05-07|Liposome Technology, Inc.|Liposomes with enhanced circulation time|

---

US5229275A|1990-04-26|1993-07-20|Akzo N.V.|In-vitro method for producing antigen-specific human monoclonal antibodies|

---

US5219837A|1990-06-21|1993-06-15|Trustees Of The University Of Pennsylvania|Method of stimulating myelination of cells|

---

GB9015198D0|1990-07-10|1990-08-29|Brien Caroline J O|Binding substance|

---

US6172197B1|1991-07-10|2001-01-09|Medical Research Council|Methods for producing members of specific binding pairs|

---

US5661016A|1990-08-29|1997-08-26|Genpharm International Inc.|Transgenic non-human animals capable of producing heterologous antibodies of various isotypes|

---

CA2089661C|1990-08-29|2007-04-03|Nils Lonberg|Transgenic non-human animals capable of producing heterologous antibodies|

---

US5545806A|1990-08-29|1996-08-13|Genpharm International, Inc.|Ransgenic non-human animals for producing heterologous antibodies|

---

US5633425A|1990-08-29|1997-05-27|Genpharm International, Inc.|Transgenic non-human animals capable of producing heterologous antibodies|

---

US5625126A|1990-08-29|1997-04-29|Genpharm International, Inc.|Transgenic non-human animals for producing heterologous antibodies|

---

WO1992009690A2|1990-12-03|1992-06-11|Genentech, Inc.|Enrichment method for variant proteins with altered binding properties|

---

US6582959B2|1991-03-29|2003-06-24|Genentech, Inc.|Antibodies to vascular endothelial cell growth factor|

---

IL101728A|1991-05-03|2007-08-19|Univ Yale|Human notch and delta, binding domains in toporythmic proteins, and methods based thereon|

---

PT1024191E|1991-12-02|2008-12-22|Medical Res Council|Production of anti-self antibodies from antibody segment repertoires and displayed on phage|

---

US5786158A|1992-04-30|1998-07-28|Yale University|Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids|

---

US20050112121A1|1992-04-30|2005-05-26|Yale University|Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids|

---

US5840299A|1994-01-25|1998-11-24|Athena Neurosciences, Inc.|Humanized antibodies against leukocyte adhesion molecule VLA-4|

---

US5731168A|1995-03-01|1998-03-24|Genentech, Inc.|Method for making heteromultimeric polypeptides|

---

US5641870A|1995-04-20|1997-06-24|Genentech, Inc.|Low pH hydrophobic interaction chromatography for antibody purification|

---

EP0861261B9|1995-06-28|2010-02-24|Imperial Cancer Research Technology Limited|Nucleotide and protein sequences of vertebrate delta genes and methods based thereon|

---

US6828422B1|1995-08-18|2004-12-07|Morphosys Ag|Protein/peptide libraries|

---

DK1143006T3|1995-08-18|2008-07-14|Morphosys Ip Gmbh|Vectors / DNA sequences from human combinatorial antibody libraries|

---

US5730977A|1995-08-21|1998-03-24|Mitsui Toatsu Chemicals, Inc.|Anti-VEGF human monoclonal antibody|

---

JPH09124697A|1995-11-01|1997-05-13|Toagosei Co Ltd|Peptide and monoclonal antibody|

---

US6337387B1|1995-11-17|2002-01-08|Asahi Kasei Kabushiki Kaisha|Differentiation-suppressive polypeptide|

---

US20020137890A1|1997-03-31|2002-09-26|Genentech, Inc.|Secreted and transmembrane polypeptides and nucleic acids encoding the same|

---

US20060122373A1|1997-04-04|2006-06-08|Millennium Pharmaceuticals, Inc.|Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 and integrin alpha subunit protein and nucleic acid molecules and uses thereof|

---

US20030180784A1|1997-04-04|2003-09-25|Millennium Pharmaceuticals, Inc.|Novel human Delta3 compositions and therapeutic and diagnostic uses therefor|

---

AU6952498A|1997-04-04|1998-10-30|Millenium Pharmaceuticals, Inc.|Novel human delta3 compositions and therapeutic and diagnostic uses therefor|

---

US6121045A|1997-04-04|2000-09-19|Millennium Biotherapeutics, Inc.|Human Delta3 nucleic acid molecules|

---

EP1325932B9|1997-04-07|2006-07-19|Genentech, Inc.|Anti-vegf antibodies|

---

US20070059302A1|1997-04-07|2007-03-15|Genentech, Inc.|Anti-vegf antibodies|

---

CA2288600C|1997-05-02|2010-06-01|Genentech, Inc.|A method for making multispecific antibodies having heteromultimeric and common components|

---

US20020062010A1|1997-05-02|2002-05-23|Genentech, Inc.|Method for making multispecific antibodies having heteromultimeric and common components|

---

US7951917B1|1997-05-02|2011-05-31|Genentech, Inc.|Method for making multispecific antibodies having heteromultimeric and common components|

---

EP1004669B1|1997-05-14|2007-04-18|Asahi Kasei Kabushiki Kaisha|Novel differentiation inhibitor|

---

AU8162898A|1997-06-18|1999-01-04|Trustees Of Columbia University In The City Of New York, The|Angiogenic modulation by notch signal transduction|

---

US20020032315A1|1997-08-06|2002-03-14|Manuel Baca|Anti-vegf antibodies|

---

US6004528A|1997-09-18|1999-12-21|Bergstein; Ivan|Methods of cancer diagnosis and therapy targeted against the cancer stemline|

---

AU5099499A|1998-07-13|2000-02-01|Yale University|Delta cleavage products and methods based thereon|

---

EP1100899A2|1998-07-27|2001-05-23|Amgen Inc.|Delta-related polypeptides|

---

CA2491610A1|1999-12-01|2001-06-07|Kevin P. Baker|Secreted and transmembrane polypeptides and nucleic acids encoding the same|

---

CA2372053C|1999-04-28|2008-09-02|Board Of Regents, The University Of Texas System|Compositions and methods for cancer treatment by selectively inhibiting vegf|

---

JP4794789B2|1999-07-02|2011-10-19|モルフォシス・アクチェンゲゼルシャフト|Generation of specific binding partners for genomic DNA fragments or peptides encoded by ESTs|

---

US6703221B1|1999-08-19|2004-03-09|Chiron Corporation|Notch receptor ligands and uses thereof|

---

US20110131679A2|2000-04-19|2011-06-02|Thomas La Rosa|Rice Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement|

---

US20020028488A1|2000-06-19|2002-03-07|Sujay Singh|Transgenic avian species for making human and chimeric antibodies|

---

US8044259B2|2000-08-03|2011-10-25|The Regents Of The University Of Michigan|Determining the capability of a test compound to affect solid tumor stem cells|

---

US6984522B2|2000-08-03|2006-01-10|Regents Of The University Of Michigan|Isolation and use of solid tumor stem cells|

---

US6689744B2|2000-09-22|2004-02-10|Genentech, Inc.|Notch receptor agonists and uses|

---

ES2727425T3|2000-12-12|2019-10-16|Medimmune Llc|Molecules with prolonged half-lives, compositions and uses thereof|

---

US7667004B2|2001-04-17|2010-02-23|Abmaxis, Inc.|Humanized antibodies against vascular endothelial growth factor|

---

EP1446424A2|2001-11-14|2004-08-18|Lorantis Limited|Composition comprising inhibitors of the notch signalling pathway for the modulation of the immune system|

---

EP1409005A1|2001-07-25|2004-04-21|Lorantis Limited|Modulators of notch signalling for use in immunotherapy|

---

US7820178B2|2001-08-01|2010-10-26|University of Brisol|VEGF isoforms and their use as anti-angiogenic, anti-vasodilatory, anti-permeability and anti-proliferative agents|

---

US6833441B2|2001-08-01|2004-12-21|Abmaxis, Inc.|Compositions and methods for generating chimeric heteromultimers|

---

US20050137130A1|2001-11-14|2005-06-23|Bodmer Mark W.|Medical treatment|

---

JP2005526701A|2001-11-14|2005-09-08|ロランティスリミテッド|Internal medicine|

---

WO2004024764A1|2002-09-10|2004-03-25|Lorantis Limited|Pharmaceutical composition and medical treatments comprising notch ligand proteins|

---

EP1461023A4|2001-12-07|2005-08-31|Univ Michigan|Prospective identification and characterization of breast cancer stem cells|

---

US8034904B2|2002-06-14|2011-10-11|Immunogen Inc.|Anti-IGF-I receptor antibody|

---

DK2314629T3|2002-07-18|2014-01-20|Merus B V|Recombinant preparation of mixtures of antibodies|

---

GB0216648D0|2002-07-18|2002-08-28|Lonza Biologics Plc|Method of expressing recombinant protein in CHO cells|

---

GB0218879D0|2002-08-14|2002-09-25|Lorantis Ltd|Medical treatment|

---

CA2498008C|2002-09-11|2014-02-04|Genentech, Inc.|Novel composition and methods for the treatment of immune related diseases|

---

US7365168B2|2002-10-15|2008-04-29|Pdl Biopharma, Inc.|Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis|

---

CN1511850A|2002-12-31|2004-07-14|王小宁|Process for preparing MHC-peptide antibody polymer|

---

US7354578B2|2003-06-06|2008-04-08|Regeneron Pharmaceuticals, Inc.|Method of tumor regression with VEGF inhibitors|

---

US20050106667A1|2003-08-01|2005-05-19|Genentech, Inc|Binding polypeptides with restricted diversity sequences|

---

US7758859B2|2003-08-01|2010-07-20|Genentech, Inc.|Anti-VEGF antibodies|

---

EP1651663B1|2003-08-08|2017-05-17|Immunomedics, Inc.|Bispecific antibodies for inducing apoptosis of tumor and diseased cells|

---

FR2859725B1|2003-09-16|2006-03-10|Neovacs|HIGH EFFICIENCY PROCESS FOR OBTAINING HUMAN ANTIBODIES THAT NEUTRALIZE THE BIOLOGICAL ACTIVITY OF A HUMAN CYTOKINE|

---

WO2005047327A2|2003-11-12|2005-05-26|Biogen Idec Ma Inc.|NEONATAL Fc RECEPTOR -BINDING POLYPEPTIDE VARIANTS, DIMERIC Fc BINDING PROTEINS AND METHODS RELATED THERETO|

---

EP1699818A2|2003-12-05|2006-09-13|Angiogenetics Sweden AB|Angiogenesis affecting polypeptides, proteins, and compositions, and methods of use|

---

EP1718767B1|2004-02-03|2012-04-11|The Regents Of The University Of Michigan|Compositions for treating breast and pancreatic cancer|

---

EP1725585A2|2004-03-10|2006-11-29|Lonza Ltd|Method for producing antibodies|

---

WO2005123104A2|2004-06-10|2005-12-29|Regeneron Pharmaceuticals, Inc.|Use of vegf inhibitors for the treatment of human cancer|

---

CA2567738C|2004-06-15|2010-12-21|F. Hoffmann-La Roche Ag|The use of cardiac hormones for diagnosing the risk of suffering from a cardiovascular complication as a consequence of cardiotoxic medication|

---

AU2005265071A1|2004-06-18|2006-01-26|Memorial Sloan-Kettering Cancer Center|VEGF inhibitors for the treatment of malignant pleural effusion|

---

EP1615036B1|2004-07-07|2007-09-19|F.Hoffmann-La Roche Ag|Multimarker panel for diabetes type 1 and 2|

---

AU2005279347A1|2004-08-30|2006-03-09|Lonza Biologics Plc.|Affinity- plus ion exchange- chromatography for purifying antibodies|

---

WO2006028936A2|2004-09-02|2006-03-16|Genentech, Inc.|Heteromultimeric molecules|

---

EP1786906A2|2004-09-09|2007-05-23|Exonhit Therapeutics SA|Tumor specific genes and variant rnas and uses thereof as targets for cancer therapy and diagnosis|

---

WO2006033386A1|2004-09-22|2006-03-30|Kirin Beer Kabushiki Kaisha|STABILIZED HUMAN IgG4 ANTIBODIES|

---

US20060134121A1|2004-10-29|2006-06-22|Gavin Thurston|DII4 antagonists, assays, and therapeutic methods thereof|

---

US8048418B2|2004-10-29|2011-11-01|Regeneron Pharmaceuticals, Inc.|Therapeutic methods for inhibiting tumor growth with combination of Dll4 antagonists and VEGF antagonists|

---

BRPI0517564A|2004-11-10|2008-10-14|Hubrecht Lab|methods for modifying the fate of an adenoma and / or adenocarcinoma cell, to induce the formation of a post-mitotic cell from an adenoma and / or adenocarcinoma cell, and to at least partially shrink an adenoma and / or Intestinal adenocarcinoma present in an animal, use of a notch path inhibitor, and pharmaceutical composition|

---

WO2006083355A2|2004-11-19|2006-08-10|Cornell Research Foundation, Inc.|Use of vascular endothelial growth factor receptor 1+ cells in treating and monitoring cancer and in screening for chemotherapeutics|

---

US7432107B2|2005-01-24|2008-10-07|Roche Diagnostics Operations, Inc.|Cardiac hormones for assessing cardiovascular risk|

---

EP1986012B1|2005-01-24|2012-06-06|F. Hoffmann-La Roche AG|The use of Bnp-type peptidesfor assessing the risk of suffering from heart failure as a consequence of volume overload|

---

CA2597247A1|2005-02-11|2006-08-17|Regeneron Pharmaceuticals, Inc.|Therapeutic combination of a vegf antagonist and an anti-hypertensive agent|

---

WO2006106905A1|2005-03-31|2006-10-12|Chugai Seiyaku Kabushiki Kaisha|Process for production of polypeptide by regulation of assembly|

---

US9296816B2|2005-04-15|2016-03-29|Macrogenics, Inc.|Covalent diabodies and uses thereof|

---

EP3530736A3|2005-05-09|2019-11-06|ONO Pharmaceutical Co., Ltd.|Human monoclonal antibodies to programmed death 1 and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics|

---

EP1891107B1|2005-05-12|2011-07-06|ZymoGenetics, Inc.|Compositions and methods for modulating immune responses|

---

KR20080026562A|2005-06-02|2008-03-25|갤럭시 바이오테크, 엘엘씨|Methods of treating brain tumors with antibodies|

---

EP1917024A2|2005-08-12|2008-05-07|Regeneron Pharmaceuticals, Inc.|Methods of treating diseases with a vegf antagonist|

---

US7774903B2|2005-08-16|2010-08-17|Lummus Corporation|Roller gin apparatus, method and system|

---

US20070213266A1|2005-09-01|2007-09-13|Vasgene Therapeutics, Inc.|Methods for using and identifying modulators of Delta-like 4|

---

US7906116B2|2005-09-01|2011-03-15|Parkash Gill|Methods for using and identifying modulators of Delta-like 4|

---

TW200732350A|2005-10-21|2007-09-01|Amgen Inc|Methods for generating monovalent IgG|

---

EP1945754B1|2005-10-31|2014-07-23|The Regents Of The University Of Michigan|Compositions and methods for treating and diagnosing cancer|

---

PL1962895T3|2005-12-16|2013-06-28|Regeneron Pharma|THERAPEUTIC USE OF A Dll4 ANTAGONIST AND A VEGF INHIBITOR FOR INHIBITING TUMOR GROWTH|

---

WO2007098387A2|2006-02-17|2007-08-30|Gilead Colorado, Inc.|Antihypertensive therapy|

---

US8133857B2|2006-03-07|2012-03-13|The Brigham and Women's FHospital, Inc.|NOTCH inhibition in the treatment of atherosclerosis|

---

US7354582B2|2006-03-10|2008-04-08|Regeneron Pharmaceuticals, Inc.|Use of VEGF antagonists for the treatment of malignant gliomas|

---

JP5474531B2|2006-03-24|2014-04-16|メルクパテントゲゼルシャフトミットベシュレンクテルハフツング|Engineered heterodimeric protein domains|

---

KR20090027227A|2006-06-06|2009-03-16|제넨테크, 인크.|Anti-dll4 antibodies and methods using same|

---

RU2008152435A|2006-06-06|2010-07-20|Дженентек, Инк. |COMPOSITIONS AND METHODS OF REGULATING VESSEL DEVELOPMENT|

---

WO2007145840A2|2006-06-13|2007-12-21|Oncomed Pharmaceuticals, Inc.|Compositions and methods for diagnosing and treating cancer|

---

WO2007147901A1|2006-06-22|2007-12-27|Novo Nordisk A/S|Production of bispecific antibodies|

---

US20080181893A1|2006-08-07|2008-07-31|Lobov Ivan B|Therapeutic methods for treating vascular eye disorders with DII4 antagonists|

---

EP2471816A1|2006-08-30|2012-07-04|Genentech, Inc.|Multispecific antibodies|

---

NZ576404A|2006-09-29|2012-02-24|Oncomed Pharm Inc|Compositions and methods for diagnosing and treating cancer involving dll4|

---

CN101583624B|2006-09-29|2012-11-21|昂考梅德药品有限公司|Compositions and methods for diagnosing and treating cancer|

---

MX2009004027A|2006-10-20|2009-09-28|Schering Corp|Fully human anti-vegf antibodies and methods of using.|

---

WO2008070350A2|2006-10-27|2008-06-12|The Board Of Regents Of The University Of Texas System|Methods and compositions related to wrapping of dehydrons|

---

US20100166746A1|2006-12-04|2010-07-01|Medlmmune Way|High potency recombinant antibodies, methods for producing them and use in cancer therapy|

---

RU2482877C2|2006-12-11|2013-05-27|Дженентек, Инк.|Compositions and methods for treating disease|

---

RU2448979C2|2006-12-14|2012-04-27|Ридженерон Фармасьютикалз, Инк.|Human antibodies to delta-like human ligand-4|

---

EP2101807B1|2006-12-19|2016-05-25|Genentech, Inc.|Vegf-specific antagonists for adjuvant and neoadjuvant therapy and the treatment of early stage tumors|

---

EP2120996A2|2006-12-20|2009-11-25|Vasgene Therapeutics, Inc.|Methods for using and identifying modulators of delta-like 4|

---

US7691980B2|2007-01-09|2010-04-06|Bio-Rad Laboratories, Inc.|Enhanced capacity and purification of antibodies by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers|

---

EP2125013A4|2007-01-26|2010-04-07|Bioinvent Int Ab|Dll4 signaling inhibitors and uses thereof|

---

US20120040025A9|2007-05-04|2012-02-16|Currie Mark G|Compositions and Methods for Treating Disorders Associated with Salt or Fluid Retention|

---

GB0709333D0|2007-05-15|2007-06-20|Smart Targeting Ltd|Binding protein|

---

US8216571B2|2007-10-22|2012-07-10|Schering Corporation|Fully human anti-VEGF antibodies and methods of using|

---

AU2008315414A1|2007-10-25|2009-04-30|Philogene, Inc.|Antibodies specific to pro-angiogenic isoforms of vascular endothelial growth factor |

---

AR069501A1|2007-11-30|2010-01-27|Genentech Inc|ANTI-VEGF ANTIBODIES |

---

WO2009075565A1|2007-12-12|2009-06-18|Erasmus University Medical Center Rotterdam|Methods for controlling vasculogenesis|

---

US9266967B2|2007-12-21|2016-02-23|Hoffmann-La Roche, Inc.|Bivalent, bispecific antibodies|

---

ES2563027T3|2008-01-07|2016-03-10|Amgen Inc.|Method for manufacturing antibody Fc heterodimer molecules using electrostatic conduction effects|

---

US8093364B2|2008-01-18|2012-01-10|Bio-Rad Laboratories, Inc.|Enhanced purification of antibodies and antibody fragments by apatite chromatography|

---

EP2260058A2|2008-04-07|2010-12-15|Ablynx N.V.|Single variable domains against the notch pathways|

---

MX364200B|2008-04-09|2019-04-16|Genentech Inc|Novel compositions and methods for the treatment of immune related diseases.|

---

AU2009241589B2|2008-04-29|2013-10-10|Abbvie Inc.|Dual variable domain immunoglobulins and uses thereof|

---

US20100260668A1|2008-04-29|2010-10-14|Abbott Laboratories|Dual Variable Domain Immunoglobulins and Uses Thereof|

---

ES2535614T3|2008-07-08|2015-05-13|Oncomed Pharmaceuticals, Inc.|Notch binding agents and antagonists and methods of use thereof|

---

EP2321651B1|2008-07-23|2017-08-23|F.Hoffmann-La Roche Ag|Identification of subjects being susceptible to anti-angiogenesis therapy|

---

EP2324358A1|2008-07-23|2011-05-25|F. Hoffmann-La Roche AG|Monitoring anti-angiogenesis therapy|

---

EP2927244A1|2008-09-19|2015-10-07|MedImmune, LLC|Antibodies directed to DLL4 and uses thereof|

---

DK2356270T3|2008-11-07|2016-12-12|Fabrus Llc|Combinatorial antibody libraries and uses thereof|

---

MY150984A|2008-12-12|2014-03-31|Boehringer Ingelheim Int|Anti-agf antibodies|

---

JP2012524083A|2009-04-20|2012-10-11|ジェネンテック，インコーポレイテッド|Adjuvant cancer treatment|

---

WO2010124009A2|2009-04-21|2010-10-28|Schering Corporation|Fully human anti-vegf antibodies and methods of using|

---

AU2010245011B2|2009-04-27|2015-09-03|Oncomed Pharmaceuticals, Inc.|Method for making heteromultimeric molecules|

---

TWI513465B|2009-06-25|2015-12-21|Regeneron Pharma|Method of treating cancer with dll4 antagonist and chemotherapeutic agent|

---

JP2012532608A|2009-07-08|2012-12-20|アムジェンインコーポレイテッド|Design of stable and non-aggregating antibody Fc molecules through CH3 domain interface manipulation|

---

MX2012002605A|2009-08-29|2012-04-02|Abbott Lab|Therapeutic dll4 binding proteins.|

---

TW201119673A|2009-09-01|2011-06-16|Abbott Lab|Dual variable domain immunoglobulins and uses thereof|

---

US20110195494A1|2009-10-02|2011-08-11|Boehringer Ingelheim International Gmbh|Dll4-binging molecules|

---

UY32920A|2009-10-02|2011-04-29|Boehringer Ingelheim Int|BISPECIFIC UNION MOLECULES FOR ANTI-ANGIOGENESIS THERAPY|

---

EP3485908B1|2009-10-16|2021-08-18|Mereo BioPharma 5, Inc.|Therapeutic combination and use of dll4 antagonist antibodies and anti-hypertensive agents|

---

JP2013508308A|2009-10-23|2013-03-07|ガーバンインスティテュートオブメディカルリサーチ|Modified variable domain molecules and methods for their production and use|

---

WO2011053822A2|2009-11-01|2011-05-05|The Brigham And Women's Hospital, Inc.|Notch inhibition in the treatment and prevention of obesity and metabolic syndrome|

---

US20110165162A1|2009-12-01|2011-07-07|Oncomed Pharmaceuticals, Inc.|Methods for Treating Cancers Comprising K-ras Mutations|

---

JO3183B1|2010-01-29|2018-03-08|Regeneron Pharma|Methods of treating autoimmune diseases with dll4 antagonists|

---

KR20120117931A|2010-02-12|2012-10-24|온코메드 파마슈티칼스, 인크.|Methods for identifying and isolating cells expressing a polypeptide|

---

TWI619509B|2010-02-23|2018-04-01|建南德克公司|Combination of carboplatin,paclitaxel and anti-vegf antibody for the treatment of previously untreated,stage iii or stage ovarian,fallopian tube,or primary peritoneal cancer|

---

CN102906113B|2010-03-02|2015-08-12|Abbvie公司|Therapeutic DLL4 associated proteins|

---

UY33702A|2010-11-02|2012-05-31|Abbott Lab|IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME|

---

US20130323265A1|2010-11-15|2013-12-05|Oncomed Pharmaceuticals, Inc.|Methods for treating cancer with dll4 antagonists|

---

WO2012092539A2|2010-12-31|2012-07-05|Takeda Pharmaceutical Company Limited|Antibodies to dll4 and uses thereof|

---

TWI530489B|2011-03-22|2016-04-21|必治妥美雅史谷比公司|Bis-1,4-benzodiazepinone compounds|

---

WO2012174394A1|2011-06-17|2012-12-20|Harris Adrian L|Methods of enhancing the response to radiation in tumor therapy using anti-dll4 antibodies|

---

DK2758073T3|2011-09-23|2019-01-14|Oncomed Pharm Inc|VEGF / DLL4 BINDING AGENTS AND APPLICATIONS THEREOF|

---

EP2855522A1|2012-02-03|2015-04-08|MedImmune Limited|Process for reducing antibody aggregate levels and antibodies produced thereby|

---

SG11201404417PA|2012-03-13|2014-10-30|Hoffmann La Roche|Combination therapy for the treatment of ovarian cancer|

---

JP6448533B2|2012-05-15|2019-01-09|ブリストル−マイヤーズ スクイブ カンパニーＢｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ Ｃｏｍｐａｎｙ|Cancer immunotherapy by disrupting PD-1 / PD-L1 signaling|

---

JP2015520172A|2012-05-31|2015-07-16|ソレント・セラピューティクス・インコーポレイテッドＳｏｒｒｅｎｔｏ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｉｎｃ．|Antigen binding protein that binds to DLL-4|

---

AR092325A1|2012-05-31|2015-04-15|Regeneron Pharma|STABILIZED FORMULATIONS CONTAINING ANTI-DLL4 ANTIBODIES AND KIT|

---

KR101535341B1|2012-07-02|2015-07-13|한화케미칼 주식회사|Novel monoclonal antibody that specifically binds to DLL4 and use thereof|

---

RS62509B1|2012-07-13|2021-11-30|Roche Glycart Ag|Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases|

---

EP2900260A1|2012-09-28|2015-08-05|Boehringer Ingelheim International GmbH|Pharmaceutical combinations comprising dual angiopoietin-2 / dll4 binders and anti-vegf agents|

---

CA2889638A1|2012-10-31|2014-05-08|Oncomed Pharmaceuticals, Inc.|Methods and monitoring of treatment with a dll4 antagonist|

---

TW202014439A|2012-11-01|2020-04-16|美商艾伯維有限公司|Anti-vegf/dll4 dual variable domain immunoglobulins and uses thereof|

---

AR093445A1|2012-11-14|2015-06-10|Regeneron Pharma|METHODS TO TREAT OVARY CANCER WITH DLL4 ANTAGONISTS|

---

US20140154255A1|2012-11-30|2014-06-05|Abbvie Biotherapeutics Inc.|Anti-vegf antibodies and their uses|

---

US9873740B2|2013-07-16|2018-01-23|Genentech, Inc.|Methods of treating cancer using PD-1 axis binding antagonists and TIGIT inhibitors|

---

WO2015130751A1|2014-02-26|2015-09-03|Medimmune, Llc|Methods of treatment with dll4 antagonists|

---

MX2017000363A|2014-07-11|2017-04-27|Genentech Inc|Notch pathway inhibition.|

---

CN107073108A|2014-07-16|2017-08-18|豪夫迈·罗氏有限公司|Use TIGIT inhibitor and the method for anti-cancer agent therapy cancer|

---

JOP20200192A1|2014-08-19|2017-06-16|Merck Sharp & Dohme|Anti-tigit antibodies|

---

PE20171244A1|2014-12-23|2017-08-24|Bristol-Myers Squibb Company|ANTIBODIES AGAINST THE T-CELL IMMUNORECEPTOR WITH IMMUNOGLOBULIN DOMAINS AND INHIBITOR PORTION BASED ON IMMUNORRECEPTOR |

---

CN113956358A|2015-09-25|2022-01-21|豪夫迈·罗氏有限公司|anti-TIGIT antibodies and methods of use|NZ576404A|2006-09-29|2012-02-24|Oncomed Pharm Inc|Compositions and methods for diagnosing and treating cancer involving dll4|

---

AU2010245011B2|2009-04-27|2015-09-03|Oncomed Pharmaceuticals, Inc.|Method for making heteromultimeric molecules|

---

MX2012002605A|2009-08-29|2012-04-02|Abbott Lab|Therapeutic dll4 binding proteins.|

---

EP3485908B1|2009-10-16|2021-08-18|Mereo BioPharma 5, Inc.|Therapeutic combination and use of dll4 antagonist antibodies and anti-hypertensive agents|

---

CN102906113B|2010-03-02|2015-08-12|Abbvie公司|Therapeutic DLL4 associated proteins|

---

US8735546B2|2010-08-03|2014-05-27|Abbvie Inc.|Dual variable domain immunoglobulins and uses thereof|

---

US8551479B2|2010-09-10|2013-10-08|Oncomed Pharmaceuticals, Inc.|Methods for treating melanoma|

---

US9527925B2|2011-04-01|2016-12-27|Boehringer Ingelheim International Gmbh|Bispecific binding molecules binding to VEGF and ANG2|

---

DK2758073T3|2011-09-23|2019-01-14|Oncomed Pharm Inc|VEGF / DLL4 BINDING AGENTS AND APPLICATIONS THEREOF|

---

US9550830B2|2012-02-15|2017-01-24|Novo Nordisk A/S|Antibodies that bind and block triggering receptor expressed on myeloid cells-1 |

---

KR102134088B1|2012-08-24|2020-07-14|더 리젠츠 오브 더 유니버시티 오브 캘리포니아|Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis|

---

US20150368329A1|2012-10-15|2015-12-24|Oncomed Pharmaceuticals, Inc.|Methods of Treating Ocular Diseases|

---

CA2889638A1|2012-10-31|2014-05-08|Oncomed Pharmaceuticals, Inc.|Methods and monitoring of treatment with a dll4 antagonist|

---

TW202014439A|2012-11-01|2020-04-16|美商艾伯維有限公司|Anti-vegf/dll4 dual variable domain immunoglobulins and uses thereof|

---

AU2014287984B2|2013-07-09|2016-09-22|Ablbio|Novel dual-targeted protein specifically binding to DLL4 and VEGF, and use thereof|

---

WO2015130751A1|2014-02-26|2015-09-03|Medimmune, Llc|Methods of treatment with dll4 antagonists|

---

EP3125937A4|2014-04-04|2017-11-01|Oncomed Pharmaceuticals, Inc.|Treatment of gastric cancer|

---

TWI593967B|2014-05-01|2017-08-01|高雄醫學大學|Secondary antibody detected epitope and use thereof|

---

MX2017000363A|2014-07-11|2017-04-27|Genentech Inc|Notch pathway inhibition.|

---

JP6738316B2|2014-07-17|2020-08-12|ノヴォ ノルディスク アクティーゼルスカブ|Site-directed mutagenesis of TREM-1 antibody to reduce viscosity|

---

PT3212233T|2014-10-31|2020-07-16|Oncomed Pharm Inc|Combination therapy for treatment of disease|

---

WO2016094881A2|2014-12-11|2016-06-16|Abbvie Inc.|Lrp-8 binding proteins|

---

TW201710286A|2015-06-15|2017-03-16|艾伯維有限公司|Binding proteins against VEGF, PDGF, and/or their receptors|

---

JP6967003B2|2015-09-23|2021-11-17|メレオ バイオファーマ ５ インコーポレイテッド|Methods and compositions for the treatment of cancer|

---

EP3440190A1|2016-04-05|2019-02-13|Pfizer Inc|Cell culture process|

---

CN105669871A|2016-04-19|2016-06-15|中国药科大学|Fusion protein of thymulin alpha1|

---

EP3474854A4|2016-06-27|2020-02-19|The Regents of The University of California|Cancer treatment combinations|

---

BR112019021182A2|2017-04-24|2020-06-02|Memorial Sloan Kettering Cancer Center|ANTI-CD33 ANTIBODY AGENTS|

---

AU2019248547A1|2018-04-02|2020-09-10|Bristol-Myers Squibb Company|Anti-TREM-1 antibodies and uses thereof|

---

EP3880697A2|2018-11-15|2021-09-22|OncoMed Pharmaceuticals, Inc.|Methods and monitoring of treatment with vegf/dll4 binding agent|

---

TW202132573A|2019-11-07|2021-09-01|美商昂克賽納醫療公司|Classification of tumor microenvironments|

---

CN111234003B|2020-02-07|2022-02-01|中国人民解放军第四军医大学|Short peptide for cancer targeted therapy, ultrasound response nano drug-loaded microbubble based on short peptide and application of short peptide|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-01-28| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-09-15| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-04| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/09/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161538454P| true| 2011-09-23|2011-09-23|

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US61/538,454|2011-09-23|

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US201261597409P| true| 2012-02-10|2012-02-10|

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US61/597,409|2012-02-10|

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US201261692978P| true| 2012-08-24|2012-08-24|

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US61/692,978|2012-08-24|

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PCT/US2012/056886|WO2013044215A1|2011-09-23|2012-09-24|Vegf/dll4 binding agents and uses thereof|

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