EP1888648A2

EP1888648A2 - Methods of treating, diagnosing or detecting cancer

Info

Publication number: EP1888648A2
Application number: EP06771814A
Authority: EP
Inventors: Mary Jo Janatpour; Deborah Lee Zimmerman
Original assignee: Novartis Vaccines and Diagnostics Inc
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 2005-06-03
Filing date: 2006-06-02
Publication date: 2008-02-20
Also published as: AU2006255536A1; JP2008545750A; WO2006132907A3; CA2610685A1; WO2006132907A2

Abstract

The invention provides, inter alia, methods for treating cancer, compositions for treating cancer, and methods and compositions for diagnosing and/or detecting cancer. In particular, the present invention provides compositions and methods for treating, diagnosing and detecting cancers associated with EphB3 overexpression

Description

METHODS OF TREATING, DIAGNOSING OR DETECTING CANCER

[0001] FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of oncology. More particularly, the invention relates to methods for treating cancer, compositions for treating cancer, and methods and compositions for diagnosing and/or detecting cancer.

[0003] BACKGROUND OF THE INVENTION

[0004] Cancer is the second leading cause of death in the United States. Although "cancer" is used to describe many different types of cancer, i.e. breast, prostate, lung, colon, pancreas, each type of cancer differs both at the phenotypic level and the genetic level. The unregulated growth characteristic of cancer occurs when the expression of one or more genes becomes dysregulated due to mutations, and cell growth can no longer be controlled. [0005] Genes are often classified in two classes, oncogenes and tumor suppressor genes. Oncogenes are genes whose normal function is to promote cell growth, but only under specific conditions. When an oncogene gains a mutation and then loses that control, it promotes growth under all conditions. However, it has been found that for cancer to be truly successful the cancer must also acquire mutations in tumor suppressor genes. The normal function of tumor suppressor genes is to stop cellular growth. Examples of tumor suppressors include p53, p 16, p21, and APC, all of which, when acting normally, stop a cell from dividing and growing uncontrollably. When a tumor suppressor is mutated or lost, that brake on cellular growth is also lost, allowing cells to now grow without restraints.

[0006] Several molecules have been shown to be overexpressed in various cancers including breast and prostate cancer. Although EphB3 has been previously shown to be present in certain cancer cells, there are no published reports of a functional role of EphB3 in several types of cancer including ovarian and esophageal cancers.

[0007] EphB3 is a receptor in the ephrin receptor tyrosine kinase family. Presently there are 14 Eph receptors and 8 ephrin ligands known in humans. Ephrin receptors (Ephs) and their ligands, the ephrins, mediate numerous developmental processes, particularly in the nervous system and vascular systems. Ephrins are also known to play a role in tumor development, angiogenesis, metastatic growth and cell survival. Based on their structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. The Eph family of receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. Eph receptors make up the largest subgroup of the receptor tyrosine kinase (RTK) family.

[0008] Eph receptors have been implicated in cancer. N1H3T3 cells transfected with EphAl and transplanted into nude mice produce 10 mm tumors in 5-6 weeks, while vector controls did not produce any tumors during the same time period (Maru et al., Oncogene. 1990 Mar;5(3):445-7). EphB2 is expressed at higher levels in cancers of the stomach (12/16), colon (3/11), esophagus (3/6), ovarian (1/7), kidney (1/2)- and lung (1/1) when compared to normal tissues (Kiyokawa et al., Cancer Res 1994 JuI 15;54(14):3645-50). EphB6 expression correlates with low grade neuroblastomas. The kinase domain of EphB6 is not active, therefore this receptor has been proposed to act as a naturally occurring dominant negative (Tang et al., Clin Cancer Res 1999a Jun;5(6):1491-6).

[0009] There is an increasing volume of evidence that implicates the involvement of ephrins in angiogenesis. EphrinAl, ephrinBl, ephrinB2, EphB2, EphB3 and EphB4 have been reported to be expressed in blood vessels. EphrinAl can induce angiogenesis in the rat cornea model and antibodies to ephrinAl can inhibit TNF-α induced angiogenesis in this same model (Pandey et al., Science 1995 Apr 28;268(5210):567-9). Clustered ephrinBl induces cell attachment and capillary-like assembly in P 19, a teratocarcinoma-derived murine cell line, and in human renal microvascular endothelial cells (HRMEC) (Stein et al., Genes Dev 1998 Mar l;12(5):667-78). Clustered ephrinBl and ephrinB2 can also induce sprouting of adrenal-cortex derived microvascular endothelial cells (ACE) (Adams et al., Genes Dev 1999 Feb l;13(3):295-306). RNA injection of a dominant negative EphB4 receptor in Xenopus embryos causes intersomitic veins to project abnormally into adjacent somites (Helbling et al., Development 2000 Jan;127(2):269-78). EphB2/EρhB3 double knockout, EphB4 and ephrinB2 knockout mice all have vascular remodeling defects (Adams et al., 1999; Wang et al., Cell 1998 May 29;93(5):741-53; PCT WO 00/30673). EphB2 has been shown to be upregulated in colon and stomach cancer (Kiyokawa et al., 1994).

[00010] Ephrins may also play a role in metastasis. 293T human epithelial kidney cells transfected with either EphB3 or EρhB2 exhibit reduced cell adhesion to fibronectin or collagen coated surfaces in vitro. Failure of 293 cells to adhere was mediated by EphB2 phosphorylation of R-ras followed by integrin de-activation (Zou et al., Proc Natl Acad Sci U S A 1999 Nov 23 ;96(24): 13813-8). [00011] Ephrins appear to function by signaling upon activation. Ephrin binding induces

Eph receptor oligomerization causing phosphorylation of juxtamembrane residues of Ephs.

Activated Ephs have multiple phosphorylated tyrosines that act as docking sites for signaling proteins (RasGaps, Src, LMW-PTP, FAK, cdc42/Rac, PLCg, PI3-kinase, Grb2, Rho and PDZ containing proteins).

[00012] Overexpression of Eph receptors (EphAl, EphA2, EphB2) cause transformation in the absence of receptor phosphorylation. EphB receptors negatively regulate Ras-MAP- kinase pathway and FAK signaling, impairing cell growth.

[00013] - EphB3 (also known as Hek2, Sek4, Mdk5, Tyroό, CeklO and QeklO) is a receptor for ephrin-B family members (ephrin-Bl, ephrin-B2 and ephrin-B3), and is known to be expressed in normal tissue and in certain tumors and cancer cell lines. To date, however, the role of EphB3 in cancer has not been elucidated.

[00014] There is a need to identify compositions and methods that modulate EphB3 and its role in such cancers. The present invention is directed to these, as well as other, important needs.

[00015] SUMMARY OF THE INVENTION

[00016] In some aspects, the present invention provides compositions comprising an EphB 3 modulator and one or more pharmaceutically acceptable carriers. The EphB3 modulator has one or more of the activities selected from the group consisting of inducing receptor phosphorylation, inducing receptor oligomerization, inducing receptor internalization, inducing receptor degradation, inducing ligand-like EphB3 signaling, inducing EphB3 -mediated cell-cell adhesion, and inhibiting EphB3 expression. In some embodiments the composition is a sterile injectable. In some embodiments the EphB3 modulator induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 receptor internalization and EphB3 degradation. In some embodiments the EphB3 modulator induces EphB3 degradation, hi some embodiments the EphB3 modulator stimulates EphB3 binding to intracellular adaptor proteins. In some embodiments the EphB3 modulator inhibits and/or inactivates one or more of FAK, the Erk/MAPK pathway, the Cdc42/Rac pathway, Abl/Arg, Fyn, Src, LMW-PTP, Mersectin, the Cdc42 pathway, Kalirin or the Rac pathway. In some embodiments the EphB3 modulator activates and/or stimulates R-ras. In some embodiments the EphB3 modulator induces phosphorylation of R-ras.

[00017] In some embodiments the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody. In some embodiments the EphB3 modulator is a monoclonal antibody which binds to EphB3 with an affinity of at least IxIO⁸Ka. In some embodiments the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and modulates one or more EphB3-related biological activities. In some embodiments the monoclonal antibody is a human antibody, a humanized antibody or chimeric antibody. In some embodiments the monoclonal antibody binds to an epitope of EphB3, said epitope selected from the group consisting of SEQ ID NOS: 14-424. In some embodiments the monoclonal antibody binds to an epitope of EphB3. In some embodiments the domain is selected from the group consisting of the ligand binding domain, the TNFR domain, the 1^st fibronectin domain, and the 2^nd fibronectin domain. In some embodiments the monoclonal antibody binds to an epitope of the ligand binding domain of EphB3, the epitope selected from the group consisting of SEQ ID NOS:14-148. In some embodiments the monoclonal antibody binds to an epitope of the TNFR domain of EphB3, the epitope selected from the group consisting of SEQ ID NOS: 164-262. In some embodiments the monoclonal antibody binds to an epitope of the 1^st fibronectin domain of EphB3, the epitope selected from the group consisting of SEQ ID NOS:263-304. In some embodiments the monoclonal antibody binds to an epitope of the 2^nd fibronectin domain of EphB3, the epitope selected from the group consisting of SEQ TD NOS :383-424. In some embodiments the monoclonal antibody does not bind to the ligand binding domain of EphB3. In some embodiments the monoclonal antibody does not cross-react with EphB2 or EphB4. In some embodiments the monoclonal antibody induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 internalization and EphB3 degradation.

[00018] In some embodiments the EphB3 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ED NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO: 425. [00019] In some aspects the present invention provides methods of treating cancer or a cancer symptom in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an EphB3 modulator. In some embodiments the EphB3 modulator induces EphB3 degradation. In some embodiments the EphB3 modulator inhibits EphB3 expression by at least 50% as compared to a control. In some embodiments the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody.

[00020] In some embodiments the EphB3 modulator is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single- chain antibody, or a Fab fragment. In some embodiments the antibody is labeled. In some embodiments the label is an enzyme, radioisotope, toxin or fluorophore. In some embodiments the antibody has a binding affinity less than about lxlO⁵K_a for a polypeptide other than EphB3. In some embodiments the EphB3 modulator is a monoclonal antibody. [00021] In some embodiments the EphB3 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ TD NO: 425.In some embodiments of the methods, the cancer is ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.

[00022] In some embodiments cancer symptoms are selected from the group consisting of pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, and difficulty swallowing. [00023] In some embodiments the methods further comprise the administration of a traditional cancer therapeutic to the patient. In some embodiments the methods further comprise the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.

[00024] In some aspects the present inventions provide methods of modulating an EphB3- related biological activity in a patient. The methods comprise administering to the patient an amount of the EphB3 modulator of claim 1 effective to modulate the EphB3 biological activity. In some embodiments the EρhB3 modulator is a monoclonal antibody which selectively binds EphB3. In some embodiments the patient has or is predisposed to one or more of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer. In some embodiments the EρhB3 modulator is an antibody and is administered to the subject via in vivo therapeutic antibody gene transfer.

[00025] In some further aspects, the present invention provides methods of identifying a patient susceptible to EphB3 therapy. The methods comprise detecting the presence or absence of evidence of EphB3 expression in said sample. In some embodiments the expression of EphB3 is increased by at least 30% compared to a control. The presence of evidence of EphB3 expression in said sample is indicative of a patient who is a candidate for EphB3 therapy and the absence of evidence of EphB3 expression in said sample is indicative of a patient who is not a candidate for EρhB3 therapy. The methods also comprise administering a therapeutically effective amount of an EphB3 modulator to the patient if the patient is a candidate for EphB3 therapy; and administering a traditional cancer therapeutic to the patient if the patient is not a candidate for EphB3 therapy.

[00026] In some embodiments evidence of EphB3 expression is detected by measuring EphB3 RNA. In some embodiments evidence of EphB3 expression is detected by measuring EphB3 expression products. In some embodiments the patient has or is predisposed to one or more of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.

[00027] In some aspects the present invention provides methods of inhibiting cancer cell growth in a patient in need thereof comprising administering - a therapeutically effective amount of an EphB3 modulator to the patient. In some embodiments the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and induces receptor degradation. In some embodiments the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and inhibits EphB3 expression.

[00028] In some further aspects the present invention provides methods of inhibiting a cancer cell phenotype in a patient in need thereof. The methods comprise administering to the patient a therapeutically effective amount of an EρhB3 modulator. Ih some embodiments the cancer cell phenotype is one or more of colony formation in soft agar and tubular network formation in a three dimensional basement membrane or extracellular membrane preparation. In some embodiments the cancer cells are selected from the group consisting of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer cells. [00029] Further aspects of the present invention provide methods for detecting a tumor in a patient comprising administering to the patient a composition comprising an EphB3 modulator linked to an imaging agent and detecting the localization of the imaging agent in the patient. In some embodiments the EphB3 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody. In some embodiments the composition comprises an anti-EphB3 antibody conjugated to an imaging agent. In some embodiments the imaging agent is ¹⁸F, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁷⁷Br, ⁸⁷MSr, ⁸⁶Y, ⁹⁰Y, ⁹⁹MTc, ¹¹¹In, ¹²³1, ¹²⁵1, ¹²⁷Cs, ¹²⁹Cs, ¹³¹1, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb, Or ²⁰⁶Bi. [00030] The present invention provides in some further aspects methods of expressing an anti-EphB3 antibody in a CHO or myeloma cell. The methods comprise expressing a nucleic acid encoding the anti-EphB3 antibody in the CHO or myeloma cell.

[00031] In some aspects the present invention provides methods of identifying a cancer inhibitor, the cancer characterized by overexpression of EphB3 compared to a control. The methods comprise contacting a cell expressing EphB3 with a candidate compound and determining whether an EphB3-related biological activity is induced. In some embodiments the induced EphB3-related biological activity is selected from the group consisting of receptor phosphorylation, receptor oligomerization, receptor degradation, and receptor signaling, wherein induction of the EphB3-related biological activity is indicative of a cancer inhibitor. [00032] In some further aspects the present invention provides methods of identifying a cancer inhibitor, said cancer characterized by overexpression of EphB3. The methods comprise contacting a cell expressing EphB3 with a candidate compound and an EphB3 ligand, and determining whether an EphB3-related biological activity is induced. In some embodiments the induced EphB3-related biological activity is -selected from the group consisting of receptor phosphorylation, receptor oligomerization, receptor degradation, and receptor signaling, wherein induction of the EpliB3-related biological activity is indicative of a cancer inhibitor.

[00033] These and other aspects of the present invention will be elucidated in the following detailed description of the invention.

[00034] BRIEF DESCRIPTION OF THE DRAWINGS

[00035] Figures IA and IB depict expression data for EphB3. Figure IA shows that

EphB3 is upregulated more than five-fold in greater than 30% of tested colon cancer patients.

Figure IB shows that EphB3 is highly expressed in colon cnacer "versus essential normal tissues.

[00036] Figure 2 depicts the inhibiton of anchorage independent growth of colon cancer cells using EphB3 siRNA.

[00037] Figure 3 provides a summary of the effect of EphB3 knockdown on various cancer and normal cell lines.

[00038] Figure 4 depicts differential expression of EphB3 at tihe mRNA level using

Affymetrix chips.

[00039] Figure 5 provdes a summary of EphB3 immunohistochemistry data.

[00040] Figure 6 provides EphB3 immunohistochemistry data.

[00041] DETAILED DESCRIPTION

[00042] The present invention provides methods and compositions for the treatment, diagnosis and imaging of cancer, in particular for the treatment, diagnosis and imaging of EphB3-related cancer. [00043] When bound to an EρhB3 ligand, EphB3 becomes phosphorylated and then subsequently degraded. Methods are known in the art to determine the level of receptor phosphorylation, activity, or expression and can be used to assay candidate EpliB3 modulators to determine their agonistic properties. Examples of such methods are set forth, for example in Cancer Research 62:2840 (2002); and Cancer Research 63: 7907 (2003)). Although not wishing to be bound by theory, EphB3 may act by a mechanism similar to other Ephrin receptors to modulate cell adhesion and motility via integrals leading to increased tumor invasion and metastasis. EphB3 expression may also correlate with the presence of distant metastasis in colon cancer patients. Inhibitors to EρhB3 could be used to treat various types of cancers by modulating angiogenesis, tumor invasion, or metastasis, etc. [00044] Definitions

[00045] Various definitions are used throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art. [00046] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, VoIs. I-IV (D .M. Weir and CC. Blackwell, eds., 1986, Blackwell Scientific Publications); and Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

[00047] As used herein, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a mixture of two or more such antibodies.

[00048] As used herein, the term "about" refers to +/- 30%, +/- 20%, +/- 10%, or +/- 5% of a value.

[00049] As used herein, the term "EphB3" refers to a receptor that binds ephrins. In some embodiments EphB3 refers to a receptor that binds to EFNA and EFNB. In some embodiments EphB3 refers to a receptor that primarily binds to EFNB. In some embodiments EphB3 refers to a receptor that binds to EFNB2 and/or EFNB3. In some embodiments the term "EphB3" refers to the ephrin receptor B3. In GeneCard, EphB3 is also known as Eph receptor B3, ephrin receptor EphB3, or ephrin type-B receptor 3. The sequence of the ephrin receptor B3 is set forth in accession number NM_004443 (nucleotide sequence; SEQ DD NO:1) and NP_004434 (amino acid sequence; SEQ TD NO:2), each of which is incorporated by reference.

[00050] The terms "polypeptide" and "protein", are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with ■ or without N-terminal - methionine residues; immunologically tagged proteins; and the like.

[00051] The terms "individual", "subject", "host" and "patient" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments the subject is a human.

[00052] As used herein, "cancer" refers to primary or metastatic cancers. The term "cancer cells" refers to cells that are transformed. These cells can be isolated from a patient who has cancer, or be cells that are transformed in vitro to become cancerous. Cancer cells can be derived from many types of samples including any tissue or cell culture line. In some embodiments the cancer cells are hyperplasias, tumor cells, or neoplasms. In some embodiments, the cancer cells are isolated from ovarian, colon, esophageal, breast, prostate, leukemia, melanoma, lung, brain, liver, pancreas, and lymphoma cancers. In some embodiments, the cancer cells are taken from established cell lines that are publicly available, hi some embodiments, cancer cells are isolated from pre-existing patient samples or from libraries comprising cancer cells. In some embodiments, cancer cells are isolated and then implanted in a different host, e.g., in a xenograft. In some embodiments cancer cells are transplanted and used in a SCID mouse model, hi some embodiments, the cancer is ovarian, colon or esophageal cancer. In some preferred embodiments the cancer is ovarian or esophageal cancer.

[00053] As used herein, the term "transformed" refers to any alteration in the properties of a cell that is stably inherited by its progeny, hi some preferred embodiments, "transformed" refers to the change of normal cell to a cancerous cell, e.g., one that is capable of causing tumors, hi some embodiments, a transformed cell is immortalized. Transformation can be caused by a number of factors, including overexpression of a receptor in the absence of receptor phosphorylation, viral infection, mutations in oncogenes and/or tumor suppressor genes, and/or any other technique that changes the growth and/or immortalization properties of a cell.

[00054] As used herein, the term "metastasis" refers to a cancer which has spread to a site distant from the origin of the cancer, e.g. from the primary tumor. Sites of metastasis include without limitation, the bone, lymph nodes, lung, liver, and brain.

[00055] As used herein, the term "angiogenesis" refers to the development of blood vessels in a patient.

[00056] As used herein, the term "clinical endpoint" refers to a measurable event indicative of cancer. Clinical endpoints include without limitation, time to first metastasis, time to subsequent metastasis, size and/or number of metastases, size and/or number of tumors, location of tumors, aggressiveness of tumors, quality of life, pain and the like. Those skilled in the art are credited with the ability to determine and measure clinical endpoints. Methods of measuring clinical endpoints are known to those of skill in the art.

[00057] As used herein, the term "sample" refers to biological material from a patient. The sample assayed by the present invention is not limited to any particular type. Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supematants or extracts of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized. [00058] As used herein, the term "biological molecule" includes, but is not limited to, polypeptides, nucleic acids, and saccharides.

[00059] As used herein, the term "modulating" refers to a change in the quality or quantity of a gene, protein, or any molecule that is inside, outside, or on the surface of a cell. The change can be an increase or decrease in expression or level of the molecule. The term "modulates" also includes changing the quality or quantity of a biological function/activity including, without limitation, proliferation, secretion, adhesion, apoptosis, cell-to-cell signaling, and the like. For example, in the context of "modulating cell division", the term refers to affecting the rate, amount or degree of cell division. In some embodiments, the methods will completely inhibit cell division. In other embodiments, the methods will decrease the amount of cell division. In other embodiments, the methods will prevent cell division. [00060] As used herein, the term "N-terminus" refers to the first 10 amino acids of a protein.

[00061] As used herein, the term "C-terminus" refers to the last 10 amino acids of a protein.

[00062] As used herein, the term "cell-cell interaction" refers to an interaction between two or more cells. In some embodiments, the interaction between the cells leads to a cell signal. Cell-cell interaction can be detected via a number of methods known to those of skill in the art, including, without limitation, the observation of membrane exchange between co-cultured, pre-labeled cells, labeled, for example, with different fluorescent membrane stains including PKH26 and PKH67 (Sigma).

[00063] The term "domain" as used herein refers to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive with regions or portions thereof and may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region. [00064] As used herein, the term "ligand binding domain" refers to any portion or region of a receptor retaining at least one qualitative binding activity of a corresponding native sequence EphB3 receptor.

[00065] The term "region" refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. In some embodiments a "region" is associated with a function of the biomolecule.

[00066] The term "portion" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a portion is defined by a contiguous portion of the amino acid sequence of that protein and refers to at least 3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids. In the case of oligonucleotides, a portion is defined by a contiguous portion of the nucleic acid sequence of that oligonucleotide and refers to at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45 nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, portions of biomolecules have a biological activity.

[00067] As used herein, the term "agonist" refers to a molecule which is capable of binding to EphB3 and activating one or more biological activities of EphB3. EphB3 agonists include native EphB3 ligands including ephrin-Bl, ephrin-B2 and ephrin-B3. [00068] As used herein, the phrase "induce" refers to a stimulation of an activity. [00069] As used herein, the phrase "EphB3 biological activity" refers to a biological, physiological, or biochemical activity of EρhB3 affected by activation of the receptor. EphB3 may be activated by EphB3 modulators including ligands of EphB3, oligonucleotides, small molecules, mimetics, decoys or antibodies. Examples of EphB3 biological activities include without limitation, receptor phosphorylation, receptor oligomerization, receptor internalization, receptor degradation, signaling, EphB3-mediated cell-cell adhesion, and the like.

[00070] As used herein, the phrase "EphB3-related cells/tumors/samples" and the like - refers to cells, samples, tumors or other pathologies that are characterized by increased evidence of EpliB3 expression relative to non-cancerous and/or non-metastatic cells, samples, tumors, or other pathologies. In some preferred embodiments, EphB3-related cells, samples, tumors or other pathologies are characterized by increased evidence of EphB3 expression relative to non-metastatic cells, samples, tumors, or other pathologies. [00071] As used herein, the term "modulator" refers to a composition that modulates one or more physiological or biochemical events associated with cancer. As used herein, an "EphB3 modulator" promotes EphB3 phosphorylation and degradation. In some preferred embodiments the modulator inhibits one or more biological activities associated with cancer. In some embodiments the modulator is a small molecule, an antibody, a mimetic, a soluble receptor, a decoy receptor or an oligonucleotide, hi some embodiments the modulator acts by blocking ligand binding or by competing for a ligand-binding site. In some embodiments the modulator acts independently of ligand binding, hi some embodiments the modulator does not compete for a ligand binding site. In some embodiments the modulator blocks expression of a gene product involved in cancer. In some embodiments the modulator blocks a physical interaction of two or more biomolecules involved in cancer, hi some embodiments modulators of the invention induce one or more EphB3 biological activities selected from the group consisting of receptor phosphorylation, receptor oligomerization, receptor internalization, receptor degradation, ligand-like EphB3 signaling, and EphB3-mediated cell- cell adhesion, hi some embodiments the EphB3 modulator inhibits EphB3 expression. [00072] As used herein, the term "antibody" refers to monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)- grafted antibodies, that are specific for the target protein or fragments thereof. The term "antibody" further includes in vivo therapeutic antibody gene transfer. Antibody fragments, including Fab, Fab', F(ab')2, scFv, and Fv are also provided by the invention. Antibodies may, in some preferred embodiments, be monoclonal, humanized, primatized, single chain, or chimeric antibodies.

[00073] As used herein, the term "epitope" refers to an antigenic determinant of a polypeptide. In some embodiments an epitope may comprise 3 or more amino acids in a spatial conformation which is unique to the epitope. In some embodiments epitopes are linear or conformational epitopes. Generally an epitope consists of at least 4 such amino acids, and more usually, consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray

•crystallography and 2-dimensional nuclear magnetic resonance.

[00074] As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues.

[00075] As used herein, the term "decoy receptor" refers to an EphB3 receptor comprising at least a portion of a polypeptide, mimetic, or other macromolecule capable of binding an

EρhB3 ligand.

[00076] As used herein, the term "therapeutically effective amount" is meant to refer to an amount of a medicament which produces a medicinal effect observed as reduction or reverse in one or more clinical endpoints, growth and/or survival of cancer cell, or metastasis of cancer cells in an individual when a therapeutically effective amount of the medicament is administered to the individual. Therapeutically effective amounts are typically determined by the effect they have compared to the effect observed when a composition which includes no active ingredient is administered to a similarly situated individual. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.

However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician.

[00077] As used herein, the phrase "modulating cell-cell adhesion" refers to a change in the adhesion or cell to cell contact of one cell with another. In some embodiments, cell adhesion is inhibited by the modulators of the present invention.

[00078] As used herein, the terms "in combination with" or "in conjunction with" refer to administration of the EphB3 modulators of the invention with other therapeutic regimens.

[00079] As used herein, the term "susceptible" refers to patients for whom EphB3 therapy is an acceptable method of treatment, i.e., patients who are likely to respond positively.

Cancer patients susceptible to EphB3 therapy express high levels of EphB3 relative to those patients not susceptible to EphB3 therapy. Cancer patients who are not good candidates for EphB3 therapy include cancer patients with tumor samples that lack or have lower levels of EphB3 in or on their cancer cells.

[00080] As used herein, the term "detecting" means to establish, discover, or ascertain evidence of an activity (for example, gene expression) or biomolecule (for example, a polypeptide).

[00081] As used herein, the phrase "homologous nucleotide sequence," or "homologous amino acid sequence," or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least a specified percentage and is used interchangeably with "sequence identity". Homologous nucleotide sequences- include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.

[00082] Percent homology or identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some preferred embodiments, homology between the probe and target is between about 50% to about 60%. In some embodiments, nucleic acids have nucleotides that are about 60%, preferably about 70%, more preferably about 80%, more preferably about 85%, more preferably about 90%, more preferably about 92%, more preferably about 94%, more preferably about 95%, more preferably about 97%, more preferably about 98%, more preferably about 99% and most preferably about 100% homologous to SEQ ID NO:1, or a portion thereof. [00083] Homology may also be at the polypeptide level. In some embodiments, polypeptides are about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 94%, about 95%, about 97%, about 98%, about 99% and about 100% homologous to SEQ ID NO:2 or a portion thereof.

[00084] As used herein, the term "probe" refers to nucleic acid sequences of variable length. In some embodiments probes comprise at least about 10 and as many as about 6,000 nucleotides. In some embodiments probes comprise at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 50 or at least 75 consecutive nucleotides. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from natural or recombinant sources, are highly specific to the target sequence, and are much slower to hybridize to the target than are oligomers. Probes may be single- or double-stranded and are designed to have specificity in PCR, hybridization membrane-based, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), or ELISA-like technologies.

[00085] As used herein, the term "mixing" refers to the process of combining- one or more compounds, cells, molecules, and the like together in the same area. This may be performed, for example, in a test tube, petri dish, or any container that allows the one or more compounds, cells, or molecules, to be mixed.

[00086] As used herein the term "isolated" refers to a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the antibody naturally occurs. Methods of isolating cells are well known to those skilled in the art. A polynucleotide, a polypeptide, or an antibody which is isolated is generally substantially purified.

[00087] As used herein, the term "substantially purified" refers to a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that is removed from its natural environment and is at least 60% free, at least 75% free, and at least 90% free from other components with which it is naturally associated.

[00088] As used herein, the term "binding" means the physical or chemical interaction between two or more biomolecules or compounds. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Binding can be either direct or indirect, indirect being through or due to the effects of another biomolecule or compound. Direct binding refers to interactions that do not take place through or due to the effect of another molecule or compound but instead are without other substantial chemical intermediates.

[00089] As used herein, the term "contacting" means bringing together, either directly or indirectly, one molecule into physical proximity to a second molecule. The molecule can be in any number of buffers, salts, solutions, etc. "Contacting" includes, for example, placing a polynucleotide into a beaker, microtiter plate, cell culture flask, or a microarray, or the like, which contains a nucleic acid molecule. Contacting also includes, for example, placing an antibody into a beaker, microtiter plate, cell culture flask, or microarray, or the like, which contains a polypeptide. Contacting may take place in vivo, ex vivo, or in vitro. [00090] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences will hybridize with specificity to their proper complements at higher temperatures. Generally, stringent conditions are selected to be about 5⁰C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are hybridized to their complements at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30⁰C for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

[00091] As used herein, the term "moderate stringency conditions" refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a limited number of other sequences. Moderate conditions are sequence-dependent and will be different in different circumstances. Moderate conditions are well-known to the art skilled and are described in, inter alia, Manitatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; 2nd Edition (December 1989)).

[00092] The nucleic acid compositions described herein can be used, for example, to produce polypeptides, as probes for the detection of mRNA in biological samples (e.g., extracts of human cells) or cDNA produced from such samples, to generate additional copies of the polynucleotides, to generate ribozymes or oligonucleotides (single and double stranded), and as single stranded DNA probes or as triple-strand forming oligonucleotides. The probes described herein can be used to, for example, determine the presence or absence of the polynucleotides provided herein in a sample. The polypeptides can be used to generate antibodies specific for a polypeptide associated with cancer, which antibodies are in turn useful in diagnostic methods, prognostic methods, and the like as discussed in more detail herein. Polypeptides are also useful as targets for therapeutic intervention, as discussed in more detail herein. Antibodies of the present invention may also be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies are useful in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). These and other uses are described in more detail below.

[00093] As used herein the term "imaging agent" refers to a composition linked to an antibody, small molecule, or probe of the invention that can be detected using techniques known to the art-skilled. As used herein, the term "evidence of gene expression" refers to any measurable indicia that a gene is expressed.

[00094] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles.

[00095] When bound to an EphB3 ligand, EphB3 becomes phosphorylated and then subsequently degraded. Accordingly, the present invention is based, in part, on the discovery that EphB3 modulators can inhibit cancer cell proliferation and invasiveness by reducing the levels of EphB3 expression in cancer cells and/or by inducing ligand-like EphB3 signaling, EphB3 phosphorylation, and/or EphB3 degradation. Cancer cell growth and/or migration is therefore decreased.

[00096] Specific examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancers that overexpress EphB3. The present invention is also applicable to any tumor cell-type where EphB3 plays a role in cell adhesion, migration or repulsion. In some embodiments, the cancer is ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer. In some embodiments, the cancers amenable to treatment and/or diagnosis according to the present invention are characterized by overexpression of EρhB3. In some embodiments, such cancers exhibit overexpression of EpliB3 by at least about 25%, at least about 50%, at least about

75%, at least about 100%, at least about 150%, at least about 200%, or at least about about

300% as compared to a control.

[00097] The present invention provides methods and compositions that provide for the treatment, inhibition, prevention and management of diseases and disorders associated with

EphB3 overexpression as well as the treatment, inhibition, prevention and management of symptoms of such diseases and disorders. Some embodiments of the invention relate to methods and compositions comprising compositions that inhibit cancer cell proliferation and invasion.

[00098] The present invention further provides methods and compositions for the treatment, inhibition, prevention or management of cancer or cancer metastases. Further compositions and methods of the invention include other active ingredients in combination with the EphB3 modulators of the present invention. In some embodiments, the methods further comprise administering one or more traditional cancer therapeutics to the patient. In some embodiments the methods of the present invention further comprise treating the patient with one or more of chemotherapy, radiation therapy or surgery.

[00099] The present invention also provides methods and compositions for the treatment, inhibition, prevention and management of cancer or other hyperproliferative cell disorder or disease that has become partially or completely refractory to current or standard cancer treatment, such as surgery, chemotherapy, radiation therapy, hormonal therapy, and biological therapy.

[000100] The invention also provides diagnostic and/or imaging methods using the EphB3 modulators of the invention, particularly EphB3 antibodies, to diagnose cancer and/or predict cancer progression. In some preferred embodiments, the methods of the invention provide methods of imaging and localizing tumors and/or metastases and methods of diagnosis and prognosis. In some embodiments, the methods of the invention provide methods to evaluate the appropriateness of EphB3 -related therapy.

[000101] EphB3 Modulators

[000102] The present invention provides EphB3 modulators for, inter alia, the treatment, diagnosis, detection or imaging of cancer.

[000103] In some embodiments, the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody. In some embodiments, the

EphB3 modulator induces EphB3 phosphorylation. In some embodiments, the EphB3 modulator induces EphB3 oligomerization. In some embodiments, the EphB3 modulator induces EphB3 degradation. In some embodiments, the EphB3 modulator induces EphB3 oligomerization and induces EphB3 degradation. In some embodiments the EphB3 modulator stimulates EphB3 binding to intracellular adaptor proteins. In some embodiments, the EphB3 modulator inhibits and/or inactivates FAK, the Erk/MAPK pathway, the Cdc42/Rac pathway, activates RasGAP, inhibits and/or inactivates Abl/Arg, Fyn, Src, LMW- PTP, Mersectin, the Cdc42 pathway, Kalirin or the Rac pathway. In some embodiments the EphB3 modulator causes phosphorylation of R-Ras. In some embodiments, the EphB3 modulator inactivates R-Ras or activates Syndecan.

[000104] As used herein, the term "intracellular adaptor proteins" refers to a protein that connects different segments of a signaling complex. The adaptor protein may or may not have enzymatic activity. In some embodiments the adaptor protein is Grb2, an adaptor protein not having intrinsic enzymatic activity. In some embodiments the adaptor protein is RasGAP, an adaptor protein having enzymatic activity.

[000105] In some embodiments, the EphB3 modulator increases EphB3 phosphorylation by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods are known in the art to determine the level of receptor phosphorylation, activity, or expression and can be used to assay candidate EphB3 modulators in order to determine their properties. Examples of such methods are set forth, for example, in Cancer Research 62:2840 (2002); and Cancer Research 63: 7907 (2003). [000106] In some embodiments, the EphB3 modulator increases EphB3 oligomerization/degradation/internalization by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods of determining levels of receptor oligomerization/degradation/internalization are known to those of skill in the art. (See, for example, Methods 27 (4): 340, 2002; Cancer Res. 64: 781, 2004; Cancer Res. 63: 7907, 2003).

[000107] In some embodiments, the EphB3 modulator increases EphB3 phosphorylation by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods of determining levels of receptor phosphorylation are known to those of skill in the art. (See, for example, Cancer Res. 62: 2840, 2002; Cancer Res. 63: 7907, 2003).

[000108] In some embodiments, the EphB3 modulator inhibits EphB3 expression. In some embodiments, EphB3 expression is inhibited by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods of determining levels of EphB3 expression are known to those of skill in the art. [000109] Antibodies

[000110] In some embodiments the EphB3 modulator is an antibody. In some embodiments, the EphB3 modulator is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a single-chain antibody, or a Fab fragment. The antibody may be labeled with, for example, an enzyme, radioisotope, or fluorophore. In some embodiments the antibody has a binding affinity less than about IxIO⁵Ka for a polypeptide other than EphB3. In some embodiments, the EphB3 modulator is a monoclonal antibody which binds to EphB3 with an affinity of at least IxIO⁸Ka. In some embodiments, the monoclonal antibody does not bind to the ligand binding domain of EphB3.

[000111] In some embodiments, the monoclonal antibody induces EρhB3 phosphorylation. In some embodiments, the EphB3 modulator induces EphB3 oligomerization. In some embodiments, the EphB3 modulator induces EphB3 degradation. In some embodiments, the EphB3 modulator induces EphB3 oligomerization and induces EphB3 degradation. [000112] The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding using, for example, immunoassays. In some embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. [000113] In some embodiments the antibody is selected from the group consisting of a monoclonal antibody, a humanized antibody, a chimeric antibody, a primatized antibody, a phage-displayed antibody, a single chain antibody, or a fragment of any of the preceding. In some preferred embodiments the antibody is a humanized antibody. Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as "humanizing"), or, alternatively, (2) transplanting the entire non-human variable domains, but "cloaking" them with a human-like surface by replacement of surface residues (a process referred to in the art as "veneering"). In the present invention, humanized antibodies will include both "humanized" and "veneered" antibodies. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al, Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sd, U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan* Molec. Immunol. 31(3): 169-217 (1994); and Kettleborough, CA. et al., Protein Eng. 4(7):773-83 (1991) each of which is incorporated herein by reference.

[000114] Antibodies of the present invention may function through different mechanisms. In some embodiments, antibodies trigger antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells. In some embodiments, antibodies have multiple therapeutic functions, including, for example, antigen-binding, induction of apoptosis, and complement-dependent cellular cytotoxicity (CDC).

[000115] In some embodiments, antibodies of the present invention may act as agonists of the polypeptides of the present invention. For example, in some embodiments the present invention provides antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. In some embodiments antibodies of the present invention bind an epitope disclosed herein, or a portion thereof. In some embodiments, binding of the antibody to the receptor induces receptor degradation. In some embodiments, binding of the antibody to the receptor induces receptor oligomerization. In some embodiments, binding of the antibody to the receptor induces receptor phosphorylation. In some embodiments, binding of the antibody to the receptor induces receptor activation. Receptor activation (i.e., signaling) may be determined by techniques known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by Western blot analysis. In some embodiments, antibodies are provided that modulate ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody. [000116] In some embodiments the EphB3 antibodies stimulate EphB3 binding to intracellular adaptor proteins. In some embodiments, the EphB3 antibodies block and/or interfere with the interaction of the cytoplasmic domain of EphB3 with one or more intracellular adaptor proteins. In some embodiments, EphB3 antibodies inhibit and/or inactivate FAK, the Erk/MAPK pathway., the Cdc42/Rac pathway, activates RasGAP, inhibits and/or inactivates Abl/Arg, Fyn, Src, LMW-PTP, Intersectin, the Cdc42 pathway, Kalirin or the Rac pathway. In some embodiments, the EphB3 antibodies inactivate R-ras or activate Syndecan. In some embodiments, the EρhB3 antibodies lead to the phosphorylation of R-Ras. [000117] As used herein, the term "intracellular adaptor proteins" refers to a protein that connects different segments of a signaling complex. The adaptor may or may not have enzymatic activity. Examples of adaptor proteins are known to those of skill in the art. For example, Grb2 is an adaptor protein that does not have intrinsic enzymatic activity, while- RasGAP is an adaptor protein that has enzymatic activity.

[000118] In some embodiments the present invention provides activating antibodies. In some embodiments the activating antibodies act as receptor agonists, i.e., modulating either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing oligomerization of the receptor. In some embodiments the antibodies may be specified as agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. Antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6): 1981-1988 (1998); Chen et al., Cancer Res. 58(16): 3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15): 3209- 3214 (1998): Yoon et al., J. Immunol. 16O(7):3 170-3179 (1998); Prat et al., J. Cell. Sci. 11 l(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4): 233-241 (1997); Carlson et al., J. Biol. Chem. 272( I 7): 11295-1 1301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9): 1153-1167 (1998); Bartunek et al., Cytokine 8(l):14-2O (1996).

[000119] The antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non- covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.

[000120] The present invention also provides antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include, without limitation, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include

1 OK luciferase, luciferin, and aequorin; and examples of suitable radioactive material include I, ¹³¹I, or "Tc.

[000121] In some embodiments the antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of cytotoxins or cytocidals include one or more of paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechloretharnine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (IT) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). [000122] Antibody conjugates of the present invention can be used for modifying a given biological response. For example, in some embodiments the drug moiety may be a protein or polypeptide, or fragments thereof, possessing a desired biological activity. Such proteins include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF- alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AHvI II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al. Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. [000123] Antibodies of the present invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include without limitation, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[000124] Techniques for conjugating such therapeutic moieties to antibodies are well known to the art skilled, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).

[000125] In some embodiments the antibodies of the present invention can be conjugated to a second antibody to form an antibody heteroconjugate (see, for example U.S. Patent No. 4,676,980). [000126] In some embodiments the present invention provides therapeutic antibodies, with or without a therapeutic moiety conjugated thereto, administered alone or in combination with other agents, including, for example, cytotoxic factor(s) and/or cytoldne(s). [000127] In some embodiments, the antibody disrupts or prevents cell-cell interactions. In some embodiments, the antibody inhibits cell migration or chemotactic properties of a cell expressing EphB3.

[000128] In addition to chimeric and humanized antibodies, fully human antibodies can be derived from transgenic mice having human immunoglobulin genes (see, e.g., U.S. Patent Nos. 6,075,181, 6,091,001, and 6,114,598, all of which are incorporated herein by reference), or from phage display libraries of human immunoglobulin genes (see, e.g. McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991)).

[000129] Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256:495-496, or a modification thereof. Typically, a mouse is immunized with a solution containing an antigen. Immunization can be performed by mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally. Any method of immunization known in the art may be used to obtain the monoclonal antibodies of the invention. After immunization of the animal, the spleen (and optionally, several large lymph nodes) are removed and dissociated into single cells. The spleen cells may be screened by applying a cell suspension to a plate or well coated with the antigen of interest. The B cells expressing membrane bound immunoglobulin specific for the antigen bind to the plate and are not rinsed away. Resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium. The resulting cells are plated by serial or limiting dilution and are assayed for the production of antibodies that specifically bind the antigen of interest (and that do not bind to unrelated antigens). The selected monoclonal antibody (mAb)-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). [000130] As an alternative to the use of hybridomas for expression, antibodies can be produced in a cell line such as a CHO or myeloma cell lines, as disclosed in U.S. Patent Nos. 5,545,403; 5,545,405; and 5,998,144; incorporated herein by reference. Briefly the cell line is transfected with vectors capable of expressing a light chain and a heavy chain, respectively. By transfecting the two proteins on separate vectors, chimeric antibodies can be produced. Immunol. 147:8; Banchereau et al. (1991) Clin. Immunol. Spectrum 3:8; and Banchereau et al. (1991) Science 251 :70; all of which are herein incorporated by reference. [000131] Antibodies of the present invention may also be administered to a subject via in vivo therapeutic antibody gene transfer as discussed by Fang et al. (2005), Nat. Biotechnol. 23, 584-590. For example recombinant vectors can be generated to deliver a multicistronic expression cassette comprising a peptide that mediates enzyme independent, cotranslational self cleavage of polypeptides placed between MAb heavy and light chain encoding sequences. Expression leads to stochiometric amounts of both MAb chains. A preferred example of the peptide that mediates enzyme independent, cotranslational self cleavage is the foot-and- mouth-disease derived 2A peptide.

[000132] Fragments of the antibodies are suitable for use in the methods of the invention so long as they retain the desired affinity of the full-length antibody. Thus, a fragment of an anti-EphB3 antibody will retain the ability to bind to the EphB3 cell-surface antigen expressed on a human cell, particularly to EphB3 on the cell surface of EphB3-expressing cancer cells. Such fragments are characterized by properties similar to the corresponding full-length anti- EphB3 antibody, that is, the fragments will specifically bind a human EphB3 antigen expressed on the surface of a human cell.

[000133] Anti-EphB3 antibodies or antibody fragments thereof may be conjugated prior to use in the methods of the present invention. Methods for producing conjugated antibodies are known in the art. Thus, the anti-EphB3 antibody may be labeled using an indirect labeling or indirect labeling approach. By "indirect labeling" or "indirect labeling approach" is intended that a chelating agent is covalently attached to an antibody and at least one radionuclide is inserted into the chelating agent. See, for example, the chelating agents and radionuclides described in Srivagtava and Mease (1991) Nucl. Med. Bio. 18:589-603, herein incorporated by reference.

[000134] Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorabicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and antliramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). The conjugates of the invention can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, IL-I, IL-2, IL-6, GM-CSF, G-CSF, or other growth factors. [000135] Techniques for conjugating such therapeutic moieties to antibodies are well known. See, for example, WO 2004010957 A2; Arnon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, " in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al. (1987) "Antibodies for Drug Delivery, " in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, " in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316.

[000136] hi some embodiments the antibody is specific to the N-terminus of the EphB3 gene product. In other embodiments, the antibody is specific to the C-terminus of the EphB3 gene product. In some embodiments, the antibody is specific to a region, domain, portion, or segment of the EphB3 gene product that is between the N- and C-termini of the protein. In some embodiments, the antibody is specific to a region that spans both the N-terminus and the region that is between the N- and C-termini. In other embodiments, the antibody is specific for a region that spans both the C-terminus and the region that is in between the N- and C- termini. In some embodiments the antibody binds to an epitope of a polypeptide having an amino acid sequence of SEQ ID NO:2. In some embodiments the antibody binds to an epitope having an amino acid sequence of SEQ ID NOS: 14-424.

[000137] In some embodiments, the monoclonal antibody binds to an epitope of EphB3, wherein the epitope is selected from the group consisting of SEQ ID NOS: 14-424. In some embodiments, the monoclonal antibody binds to an epitope of EphB3, wherein the epitope is in the domain selected from the group consisting of the ligand binding domain, the TNFR domain, the 1st fibronectin domain, and the 2nd fibronectin domain. In some embodiments, the monoclonal antibody binds to an epitope of the ligand binding domain of EphB3, wherein the epitope is selected from the group consisting of SEQ ID NOS: 14- 148. In some embodiments, the monoclonal antibody binds to an epitope of the TNFR domain of EphB3, wherein the epitope is selected from the group consisting of SEQ ID NOS: 164-262. In some embodiments, the monoclonal antibody binds to an epitope of the 1st fibronectin domain of EphB3, wherein the epitope is selected from the group consisting of SEQ ID-NOS:263-304. In some embodiments, the monoclonal antibody binds to an epitope of the 2nd fibronectin domain of EphB3, wherein the epitope is selected from the group consisting of SEQ ID NOS-.383-424.

[000138] In some embodiments the binding affinity of the antibodies for EphB3 is at least 1 x 10⁶ Ka. In some embodiments the binding affinity of the antibodies for EphB3 is at least 5 x 10⁶ Ka, at least 1 x 10⁷ Ka, at least 2 x 10⁷ Ka, at least 1 x 10⁸ Ka, or greater. Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. In some embodiments binding affinities include those with a Kd less than 5 x 1(T² M, 10^'2 M, 5 x 10^"3 M, 10-³ M, 5 x 10^"4 M, 10^"4 M, 5 x 10^'5 M, 10^'5 M, 5 x 10^"6 M, 10^"6 M, 5 x 10^"7 M, 10^"7 M, 5 x 1(T⁸ M, 10^"8 M, 5 x 10^"9 M, 1(T⁹ M, 5 x 1(T¹⁰ M, 10^"10 M, 5 x 10^"π M, 10^"π M, 5 x 1(T¹² M, 10^"12 M, 5 x 10^"13 M₅ 1(T¹³ M, 5 x 1(T¹⁴ M, 10^"14 M, 5 x 10^"15 M, or 10^"15 M, or less.

[000139] Suitable antibodies according to the present invention can recognize linear or conformational epitopes, or combinations thereof. In some embodiments the antibody is specific for an epitope of the ligand binding domain (SEQ ID NO:3), TNFR domain (SEQ ID NO:4), 1st fibronectin domain (SEQ ID NO:5), or 2nd fibronectin domain of EphB3 (SEQ ID NO:6). It is to be understood that these peptides do not necessarily precisely map one epitope, but may also contain EρhB3 sequence that is not immunogenic. The following sequences are given by amino acid number (i.e., "AAn") where n is the amino acid number of the amino acid sequence set forth in SEQ ID NO:2. For example, in the context of "AA80-AA90", an epitope is defined from about amino acid 80 of SEQ ID NO:2 to about amino acid 90 of SEQ ID NO:2. In the context of epitopes, the term "about" refers to +/- one or two amino acid residues:

[000140] AA1-AA25; AA1-AA50; AA1-AA84; AA9-AA177; AAl-AAlO; AA5-AA20; AA20-AA25; AA35-AA45; AA48-AA52; AA50-AA100; AA40-AA90; AA45-AA65; AA65- AA75; AA80-AA90; AA88-AA92; AA99-AA120; AA95-AA110; AA105-AA120; AAlOO- AA150; AA132-AA137; AA150-AA200; AA155-AA170, AA190-AA210; AA198-AA202; AA200-AA250; AA220-AA240; AA238-AA234; AA245-AA265; AA250-AA300; AA290- AA330; AA290-305; AA300-AA350; AA310-AA330; AA317-AA322; AA348-AA352; AA350-AA400; AA380-AA395; AA382-AA387; AA405-AA495; AA400-AA450; AA405- AA415; AA415-AA425; AA425-AA435; AA437-AA582; AA450-AA500; AA440-AA460; AA446-AA440; AA460-AA470; AA460-AA464; AA472-AA478; AA475-AA495; AA500- AA550; AA511-AA559; AA515-end (C-terminal). Specific EphB3 epitopes are set forth below in Table 1. •

[000141] Methods of predicting other potential epitopes to which an antibody of the invention can bind are well-known to those of skill in the art and include without limitation, Kyte-Doolittle Analysis (Kyte, J. and Dolittle, R.F., J. MoI. Biol. (1982) 157:105-132), Hopp and Woods Analysis (Hopp, T.P. and Woods, K.R., Proc. Natl. Acad. Sci. USA (1981) 78:3824-3828; Hopp, TJ. and Woods, K.R., MoI. Immunol. (1983) 20:483-489; Hopp, TJ., J. Immunol. Methods (1986) 88:1-18.), Jameson- Wolf Analysis (Jameson, B.A. and Wolf, H., Comput. Appl. Biosci. (1988) 4:181-186.), and Emini Analysis (Emini, E.A., Schlief, W.A., Colonno, RJ. and Wimmer, E., Virology (1985) 140:13-20.).

[000142] The term "specific for," when used to describe antibodies of the present invention, indicates that the variable regions of the antibodies of the invention recognize and bind target polypeptides exclusively by virtue of measurable differences in properties including binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between the target protein and other polypeptides). Those skilled in the art readily understood that such specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art, as discussed in Harlow et al. (Eds.), Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6.

[000143] Antibodies are defined to be "specifically binding" if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with known related polypeptide molecules. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). In some embodiments the antibodies of the present invention bind to their target epitopes or mimetic decoys at least 103, at least 104, at least 105, and at least 106 fold higher than to other known members of the Eph or Eck family.

[000144] In some embodiments, the antibodies of the present invention do not bind to known related polypeptide molecules, for example, if they bind EphB3 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al.). Examples of known related polypeptides include, without limitation, other members of the Ephrin receptor protein family such as EphA5 (Ephrin receptor EphA5), EphB2 (Ephrin receptor EphB2), EphB4 (Ephrin receptor EphB4), and the like. In some embodiments antibodies may be screened against known related polypeptides to isolate an antibody population that specifically binds to EphB3 polypeptides. For example, antibodies specific to human EphB3 receptor polypeptides will flow through a column comprising Ephrin receptor family polypeptides (with the exception of EphB3) adhered to insoluble matrix under appropriate buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art (see, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. [000145] In some embodiments, the antibodies of the present invention have at least about 1000 fold, and at least about 10,000 fold greater affinity for EphB3 than for known related family members. In some embodiments, the binding affinity of an antibody of the present invention is less than about 1 x 10⁵ Ka, less than about 1 x 10⁴ Ka, and preferably less than 1 x 10³ Ka, for a related polypeptide other than EphB3. [000146] Oligonucleotides

[000147] In some embodiments, the EρhB3 modulator is an oligonucleotide. In some embodiments the oligonucleotide is an antisense or RNAi oligonucleotide. In some embodiments the oligonucleotide is complementary to a region, domain, portion, or segment of EphB3. In some embodiments, the oligonucleotide comprises from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 12 to about 35, and from about 18 to about 25 nucleotides. In some embodiments, the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous to a region, portion, domain, or segment of the EphB3 gene. In some embodiments there is substantial sequence homology over at least 15, 20, 25, 30, 35, 40, 50, or 100 consecutive nucleotides of the EρhB3 gene. In some embodiments there is substantial sequence homology over the entire length of the EphB3 gene. In some embodiments, the oligonucleotide binds under moderate or stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:1.

[000148] In some embodiments, the EphB3 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11₅ SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO: 425. [000149] In some embodiments, the EphB3 modulator is a double stranded RNA (dsRNA) molecule and works via RNAi (RNA interference). In some embodiments, one strand of the dsRNA is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous to a region, portion, domain, or segment of the EphB3 gene. In some embodiments there is substantial sequence homology over at least 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, or 1000 consecutive nucleotides of the EphB3 gene. In some embodiments there is substantial sequence homology over the entire length of the EphB3 gene.

[000150] In some embodiments oligonucleotides are used in a polymerase chain reaction (PCR). This sequence may be based on (or designed from) a genomic sequence or cDNA sequence and is used to amplify, confirm, or detect the presence of an identical, similar, or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may also be used to modulate the expression of a gene. Oligonucleotides comprise portions of a DNA sequence and have at least about 10 nucleotides and as many as about 500 nucleotides. In some embodiments oligonucleotides comprise from about 10 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 30 nucleotides, and from about 20 nucleotides to about 25 nucleotides. Oligonucleotides may be chemically synthesized and can also be used as probes. In some embodiments oligonucleotides are single stranded. In some embodiments oligonucleotides comprise at least one portion which is double stranded. In some embodiments the oligonucleotides are antisense oligonucleotides (ASO). In some embodiments the oligonucleotides are RNA interference oligonucleotides (RNAi oligonucleotides). [000151] Small molecules

[000152] In some embodiments, the EphB3 modulator is a small molecule. As used herein, the term "small molecule" refers to an organic or inorganic non-polymer compound that has a molecular weight that is less than about 10 kilodaltons. Examples of small molecules include peptides, oligonucleotides, organic compounds, inorganic compounds, and the like. In some embodiments, the small molecule has a molecular weight that is less than about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 kilodalton. [000153] Mimetics

[000154] In some embodiments^ the EphB3 modulator is a mimetic. As used herein, the term "mimetic" is used to refer to compounds which mimic the activity of a peptide. Mimetics are non-peptides but may comprise amino acids linked by non-peptide bonds. U.S. Patent No. 5,637,677, issued on June 10, 1997, and parent applications thereof, all of which are incorporated herein by reference, contain detailed guidance on the production of mimetics. Briefly, the three-dimensional structure of the peptides which specifically interacts with the three dimensional structure of the EphB3 receptor is duplicated by a molecule that is not a peptide. In some embodiments the EphB3 mimetic is a mimetic of EphB3 receptor or a mimetic of a ligand of EphB3 receptor. [000155] Soluble Receptors

[000156] In some embodiments, the EpliB3 modulator is a soluble receptor. As αised herein, the term "soluble receptor" refers to an Eph receptor, preferably an EphB3 receptor, which is essentially free of either a membrane domain or has a disrupted membrane domain.. [000157] Decoy Receptors

[000158] In some embodiments, the EphB3 modulator is a decoy receptor comprising at least a portion of an EphB3 receptor. In some embodiments the decoy receptor competes with natural EphB3 receptors for EphB3 ligands. In some embodiments, the decoy receptor is labeled to facilitate quantification, qualification, and/or visualization. In other embodiments, the decoy receptor further comprises a moiety to facilitate isolation and/or separation of the decoy receptor and or the decoy receptor-EphB3 complex. In some embodiments, the decoy receptor, upon binding with an EphB3 receptor ligand, causes an increased signal (compared to a native EphB3 receptor) to be effected. In some embodiments, the decoy receptor is a non- signaling molecule which functions by capturing EphB3 ligand and preventing it from interacting with the signaling EphB3 receptor. In some embodiments the decoy receptor comprises at least a portion of an EphB3 receptor fused to an antibody or antibody fragment. [000159] Methods of Treating/Preventing Cancer [000160] The present invention provides methods for treating and/or preventing cancer or symptoms of cancer in a subject comprising administering to the subject a therapeutically effective amount of one or more EphB3 modulators. In some embodiments the cancer is a cancer associated with overexpression of EphB3. In some embodiments, the cancer is colon, ovarian, esophageal or lung cancer or neuroblastoma. In some preferred embodiments the cancer is colon cancer. In some embodiments the subject has been diagnosed as having a cancer or as being predisposed to cancer.

[000161] Symptoms of cancer are well-known to those of skill in the art and include, without limitation, pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, difficulty swallowing, and the like.

[000162] A therapeutically effective amount of the modulating compound can be determined empirically, according to procedures well known to medicinal chemists, and will depend, inter alia, on the age of the patient, severity of the condition, and on the ultimate pharmaceutical formulation desired. Administration of the modulators of the present invention can be carried out, for example, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue, orally, topically, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, intra-arterially, subcutaneously, intraoccularly, intrasynovial, transepithelial, and transdermally. In preferred embodiments, the modulators are administered by lavage, orally or inter-arterially. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow or sustained release polymeric devices. As discussed above, the therapeutic compositions of this invention can also be administered as part of a combinatorial therapy with other known anti-cancer agents or other known anti-bone disease treatment regimen.

[000163] The present invention further provides methods of modulating an EphB3-related biological activity in a patient. The methods comprise administering to the patient an amount of an EphB3 modulator effective to modulate one or more EphB3 biological activities. Suitable assays for measuring EphB3 biological activities are set forth supra and infra. [000164] The present invention also provides methods of inhibiting cancer cell growth in a patient in need thereof comprising administering a therapeutically effective amount of one or more EphB3 modulators to the patient. Suitable assays for measuring EphB3-related cell growth are known to those skilled in the art.

[000165] The present invention further provides methods of inhibiting cancer in a patient in need thereof. The methods comprise determining if the patient is a candidate for EphB3 therapy as described herein and administering a therapeutically effective amount of one or more EphB3 modulators to the patient if the patient is a candidate for EphB3 therapy. If the patient is not a candidate for EphB3 therapy, the patient is treated with conventional cancer treatment.

[000166] The present invention also provides methods for inhibiting the interaction of two or more cells in a patient comprising administering a therapeutically effective amount of an EphB3 modulator to said patient. Suitable assays for measuring EphB3-related cell interaction are known to those skilled in the art.

[000167] The present invention also provides methods of modulating one or more symptoms of cancer in a patient comprising administering to said patient a therapeutically effective amount of the EphB3 compositions described herein.

[000168] The present invention further provides methods for inhibiting anchorage- independent cell growth in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an EphB3 modulator. Suitable assays for measuring EphB3-related anchorage-independent cell growth are set forth in the Examples. [000169] The present invention also provides methods for inhibiting migration of cancer cells in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an EphB3 modulator. Suitable assays for measuring EphB3-related cell migration are known to those skilled in the art.

[000170] The present invention further provides methods for inhibiting adhesion of cancer cells in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an EphB3 modulator. Suitable assays for measuring EphB3-related cell adhesion are known to those skilled in the art.

[000171] The present invention also provides methods to prophylactically treat a patient who is predisposed to develop cancer, a cancer metastasis or who has had a metastasis and is therefore susceptible to a relapse or recurrence. The methods are particularly useful in high- risk individuals who, for example, have a family history of cancer or of metastasizing tumors, or show a genetic predisposition for a cancer metastasis. In some embodiments the tumors are EphB3-related tumors. Additionally, the methods are useful to prevent patients from having recurrences of EphB3 -related tumors who have had EphB3 -related tumors removed by surgical resection or treated with a conventional cancer treatment.

[000172] The present invention also provides methods of inhibiting cancer progression and/or causing cancer regression comprising administering to the patient a therapeutically effective amount of an EphB3 modulator.

[000173] In some embodiments, the patient in need of anti-cancer treatment is treated with the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides in conjunction with chemotherapy and/or radiation therapy. For example, following administration of the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides, the patient may also be treated with a therapeutically effective amount of anti-cancer radiation. In some embodiments chemotherapeutic treatment is provided in combination with the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides. In some embodiments antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides are administered in combination with chemotherapy and radiation therapy.

[000174] Methods of treatment comprise administering single or multiple doses of one or more EphB3 modulators to the patient. In some embodiments the EphB3 modulators are administered as injectable pharmaceutical compositions that are sterile, pyrogen free and comprise the EphB3 modulators in combination with a pharmaceutically acceptable carrier or diluent.

[000175] In some embodiments, the therapeutic regimens of the present invention are used with traditional treatment regimens for cancer including, without limitation, surgery, radiation therapy, hormone ablation and/or chemotherapy. Administration of the EphB3 modulators of the present invention may take place prior to, simultaneously with, or after traditional cancer treatment.

[000176] In some embodiments, two or more different EphB3 modulators are administered to the patient.

[000177] In some embodiments the amount of EphB3 modulator administered to the patient is effective to inhibit angiogenesis. In some embodiments the amount of EphB3 modulator administered to the patient is effective to induce degradation of EphB3 receptor. In some embodiments the amount of EphB3 modulator administered to the patient is effective to induce oligomerization of two or more EphB3 receptors. In some embodiments the amount of

EphB3 modulator administered to the patient is effective to stimulate phosphorylation of the

EphB3 receptor. In some embodiments the amount of EphB3 modulator administered to the patient is effective to stimulate tyrosine kinase activity. In some embodiments the amount of EphB3 modulator administered to the patient is effective to inhibit cancer progression and/or cause cancer regression. [000178] Clinical Aspects

[000179] In some embodiments, the methods and compositions of the present invention are particularly useful in colon cancer, ovarian cancer, small lung cell cancer, gastroesophageal cancer, stomach cancer, and pancreatic cancer, among others. In some embodiments, the methods and compositions are useful in treating and/or diagnosing cancer metastasis, including, for example, lung metastases. [000180] Pharmaceutical Compositions

[000181] The present invention also provides pharmaceutical compositions comprising one or more of the EphB3 modulators described herein and a pharmaceutically acceptable carrier. [000182] In some embodiments the pharmaceutical compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins. [000183] Methods of Detecting EphB3

[000184] The present invention also provides methods for detecting EphB3. In some embodiments the EphB3 is present in a patient or in a patient sample. In some embodiments the method comprises administering a composition comprising one or more EphB3 modulators to the patient and detecting the localization of the imaging agent in the patient, hi some embodiments the patient sample comprises cancer cells. In some embodiments the EphB3 modulator is linked to an imaging agent or is detectably labeled. In some embodiments, the EphB3 modulator is an anti-EphB3 antibody conjugated to an imaging agent and is administered to a patient to detect one or more tumors or to determine susceptibility of the patient to EphB3 therapy. The labeled antibodies will bind to the high density of receptors on cells and thereby accumulate on the tumor cells. Using standard imaging techniques, the site of the tumors can be detected. [000185] The present invention also provides methods of imaging/detecting cells or tumors expressing or overexpressing EphB3 comprising contacting a composition comprising an EphB3 modulator to a sample and detecting the presence of the EphB3 modulator in the sample. In some embodiments the sample is a patient sample. In some embodiments the patient sample comprises cancer cells. In some embodiments the EphB3 modulator is linked to an imaging agent or is detectably labeled.

[000186] The present invention also provides methods for quantifying the amount of EρhB3 present in a patient, cell or sample. The methods comprise administering one or more of antibodies, probes, or small molecules to a patient or sample and detecting the amount of ■ EphB3 present in the sample. In some embodiments the antibodies, probes, or small molecules are linked to an imaging agent or are detectably labeled. Such information indicates, for example, whether or not a tumor is related to EphB3, and, therefore, whether specific treatments should be used or avoided. In some embodiments, using standard techniques well known to the art-skilled, samples believed to include tumor cells are obtained and contacted with labeled antibodies, probes, oligonucleotides, and small molecules. After removing any unbound, labeled antibodies, probes, oligonucleotides or small molecules, the quantity of labeled antibodies, peptides, oligonucleotides or mimetics bound to the cell, or the quantity of antibodies, peptides, oligonucleotides or mimetics removed as unbound is determined. The information directly relates to the amount of EphB3 present. [000187] Imaging can be performed using procedures well known to those of ordinary skill in the art. Imaging can be performed, for example, by radioscintigraphy, nuclear magnetic resonance imaging (MRT) or computed tomography (CT scan). The most commonly employed radiolabels for imaging agents include radioactive iodine and indium. Imaging by CT scan may employ a heavy metal such as an iron chelate. MRI scanning may employ chelates of gadolinium or manganese. Additionally, positron emission tomography (PET) maybe possible using positron emitters of oxygen, nitrogen, iron, carbon, or gallium. [000188] In some embodiments the EphB3 modulator is an anti-EphB3 antibody. In some embodiments the modulator is linked to an imaging agent or is detectably labeled. In some embodiments the imaging agent is ¹⁸F, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁷⁷Br, ⁸⁷MSr, ⁸⁶Y, ⁹⁰Y, ⁹⁹MTc, ¹¹¹In, ¹²³1, ¹²⁵1, ¹²⁷Cs, ¹²⁹Cs, ¹³¹1, ¹³²1, ¹⁹⁷Hg, ²⁰³Pb, Or²⁰⁶Bi.

[000189] Methods of detection are well known to those of skill in the art. For example, methods of detecting polynucleotides include, but are not limited to PCR, Northern blotting, Southern blotting, RNA protection, and DNA hybridization (including in situ hybridization). Methods of detecting polypeptides include, but are not limited to, Western blotting, ELISA, enzyme activity assays, slot blotting, peptide mass fingerprinting, electrophoresis, immunochemistry and immunohistochemistry. Other examples of detection methods include, but are not limited to, radioimmunoassay (RIA), chemiluminescence immunoassay, fluoroimmunoassay, time-resolved fluoroimmunoassay (TR-FIA), two color fluorescent microscopy, or immunochromatographic assay (ICA), all well known by those of skill in the art. In some preferred embodiments of the present invention, polynucleotide expression is detected using PCR methodologies and polypeptide production is detected using ELISA technology.

[000190] Methods for determining susceptibility to EphB3 therapy [000191] The present invention also provides methods for determining susceptibility of a patient to EphB3 therapy. The methods comprise detecting the presence or absence of evidence of EphB3 expression in a patient or patient sample. The presence of evidence of EphB3 expression in the patient or sample is indicative of a patient who is susceptible to EphB3 therapy. The absence of evidence of EphB3 expression in the patient or patient sample is indicative of a patient who is not a candidate for EphB3 therapy.

[000192] In some embodiments the therapeutic methods comprise first identifying patients susceptible to EphB3 therapy comprising administering to the patient in need thereof a composition comprising an EphB3 antibody, probe, primer, or oligonucleotide linked to an imaging agent and detecting the presence or absence of evidence of the gene or gene product in the patient. The presence of evidence of EphB3 expression, especially EphB3 overexpression, in the patient is indicative of a patient who is a candidate for EphB3 therapy and the absence of evidence of EphB3 expression in the patient is indicative of a patient who is not a candidate for EphB3 therapy. Li some embodiments, the therapeutic methods further comprise administering one or more EphB3 modulators to the patient if the patient is a candidate for EphB3 therapy and treating the patient with conventional cancer treatment if the patient is not a candidate for EphB3 therapy. [000193] Methods for Screening

[000194] The present invention also provides methods of screening for anti-cancer agents. The methods comprise contacting a cell expressing EphB3 with a candidate compound and determining whether an EphB3-related biological activity is modulated. In some embodiments, induction of one or more of tyrosine kinase activity, receptor phosphorylation, receptor oligomerization, or receptor degradation is indicative of a cancer inhibitor. In some embodiments, inhibition of EpliB3 expression is indicative of a cancer inhibitor. [000195] The present invention further provides methods of identifying a cancer inhibitor. The methods comprise contacting a cell expressing EphB3 with a candidate compound and an EpliB3 ligand, and determining whether an EphB3-related biological activity is modulated. In some embodiments, induction of one or more of tyrosine kinase activity, receptor phosphorylation, receptor oligomerization, or receptor degradation is indicative of a cancer inhibitor. In some embodiments, inhibition of EphB3 expression is indicative of a cancer inhibitor.

[000196] In some embodiments, the invention provides methods of screening for anti-cancer agents, particularly anti-metastatic cancer agents, by, for example,- screening putative modulators for an ability to increase receptor phosphorylation and/or induce receptor degradation. [000197] Kits

[000198] In some embodiments, the present invention provides kits for imaging and/or detecting a gene or gene product correlated with EphB3 overexpression. Kits of the invention comprise detectable antibodies, small molecules, oligonucleotides, soluble receptors, decoy receptors, mimetics or probes as well as instructions for performing the methods of the invention. Optionally, kits may also contain one or more of the following: controls (positive and/or negative), containers for controls, photographs or depictions of representative examples of positive and/or negative results.

[000199] Each of the patents, patent applications, GenBank accession numbers and publications described herein is hereby incorporated by reference in its entirety. [000200] Various modifications of the invention, in addition to those described herein, will be apparent to those of skill in the art in view of the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The present invention is further demonstrated in the following examples that are for purposes of illustration and are not intended to limit the scope of the present invention.

[000201] EXAMPLES

[000202] Example 1: Biotinylation of proteins

[000203] Cells were cultured in two 15-cm dishes to approximately 85-90% confluence, and washed once with 10 mL PBS. Just before use, a 0.5 mg/mL biotinylation solution was prepared using PBS (pH 8.0) and EZ-Link Sulfo-NHS-LC-LC-Biotin (Sulfosuccinimidyl-6¹-

(biotinamido)-6-hexanamido hexanoate) (Pierce Biotechnology Inc., Rockford, IL; catalogue # 21338), according to the supplier's directions. Cell surface proteins were then biotinylated using 7-10 mL of the biotinylation solution to coat each plate and incubating at room temperature for 15 minutes. Cells were then washed once with 25mM Tris (pH 8.0) and twice with PBS (pH 8.0). 10 mL of Hanks media was then added to the plates, cells were collected by scraping, and 10 mL of scraped cells were transferred to a 15 mL Falcon tube. Tubes of biotinylated cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cell pellet was washed once with PBS. Cells were then resuspended and lysed in an appropriate volume (400-800 μl) with denaturing or nondenaturing lysis buffer, with incubation on ice for 15-30 minutes. Cells were then centrifuged at 14000 rpm for 10 minutes to removed debris, and the supernatant collected. Protein concentration was determined by bicinchoninic acid (BCA) colorimetric assay (Pierce Biotechnology Inc., Rockford, IL), and extracts were aliquotted in small volumes into Eppendorf tubes (to avoid repeated freeze/thaw cycles) and quick frozen in an ethanol/dry ice bath, and stored at -70°C.

[000204] Example 2: Clustered Ligand-Induced Phosphorylation of EphB3 [000205] An anti-human IgG antibody was used to induce clustering of the ephrinB2-Fc ligand for 10 minutes, before adding clustered ligands to cells. Clustered ligand was added to cells at a concentration of 6.25 μg/mL, and cells were incubated in starvation media for various times to observe results at several timepoints. After incubation, starvation media was removed and the cells were washed once with PBS. Cells were then lysed with a denaturing lysis buffer including protease and phosphatase inhibitors. Lysates were clarified by centrifugation and then quantitated using a protein quantitation kit (Pierce Biotechnology Inc., Rockford, IL). Lysates were used either for immunoprecipitation or run directly on electrophoretic gels (15 μg / lane) for Western blot analysis.

[000206] Example 3: Immunoprecipitation

[000207] Immunoprecipitation (IP) buffer was prepared containing 50 niM Tris-HCl pH 7.5, 150 rnM NaCl, 1% TritonX-100 and 1 protease inhibitor tablet (Roche Diagnostic Corp., Indianapolis, IN) per 10 mL total volume. A rabbit polyclonal antibody (Ab) generated in- house, anti-EphB3, was combined at 1 :100 dilution with IP buffer and added to cell lysate in a screw-top tube, which was allowed to mix on a rocker platform for 1-2 hours at 4°C. For immunoprecipitation, either 40 μl of anti-rabbit IgG-conjugated beads (for EρhB3 clustering experiments) or 120 μl of streptavidin beads (for the analysis of biotinylated surface proteins) were added to each tube and incubation was continued overnight at 4°C on the rocking platform. Subsequently, the tubes were centrifoged at 7000 x g for 2 minutes at 4°C, and the supernatant removed. Bead pellets were washed 4 times with cold wash buffer, and then 30 μl of 2X SDS Tris/Glycine sample buffer containing reducing agent was added to each tube. The beads in sample buffer were then twice boiled at 95°C for 5 minutes each time to release the immunoprecipitate from the beads. The boiled bead solution was then centrifuged at 14,000 x g for 5 minutes at room temperature, and the supernatant then removed and transferred to a new tube. Immunoprecipitates were immediately analyzed by electrophoresis on an SDS- PAGE gel or stored at -2O⁰C. Western blot analysis was performed using standard methods. Electrophoresed immunoprecipitates were transferred from the polyacrylamide gel to membrane and the membrane was then probed for 1 hour at room temperature with gentle rocking using the primary antibody (either anti-phosphotyrosine Ab 4G10 (Upstate Group, LLC, Waltham, MA) at a 1:1000 dilution, or the rabbit anti-EρhB3 polyclonal Ab at 1:1000). After several washes with PBS containing 0.05% TweerώO (PBST), the appropriate species- specific secondary antibody conjugated to horseradish peroxidase (HRP) was added and incubated on a rocker platform for 30 minutes at room temperature. After several washes, reactive bands on the membrane were then visualized using the ECL detection system (Amersham Biosciences UK).

[000208] Example 4: FACS analysis

[000209] Non-permeabilized cells were used for the analysis. FACS buffer was prepared containing (cold) PBS, 1% bovine serum albumin (BSA), 2% fetal bovine serum (FBS) and 0.1% sodium azide. Cells were harvested by detaching adherent cells using dissociation buffer (Invitrogen Corp., Carlsbad, CA). To neutralize the dissociation buffer, an equal volume of growth media was added. Cells were then aliquotted into a 5 mL polystyrene round-bottom tube. For each staining, one million cells were centrifuged at 1000 rpm for 5 minutes at 4°C, and then primary antibody (up to 6 μg in 100 μL FACS buffer) was added to the cell pellet, mixed by vortexing and incubated on ice for 30 minutes to one hour. Cells were then washed twice with 3 mL FACS buffer after pelleting by centrifugation at 1000 rpm for 5 minutes at 4°C. After the second wash and centrifugation, secondary antibody (lμg in 50 μL FACS buffer) was then added to cell pellet, mixed by vortexing and incubated on ice for 30 minutes in the dark. Cells were then washed twice with 3 mL FACS buffer after pelleting by centrifugation at 1000 rpm for 5 minutes at 4°C. After the second wash and centrifugation, cells were resuspended in 500 μg in 50 μL FACS buffer containing propidium iodide (PI). (PI was prepared as a 1 μg/μL stock and used at 1:100). FACS / flow cytometry analysis was performed within an hour.

[000210] Example 5: EphB3 Oligonucleotides Inhibit Soft Agar Growth of SW620 Cells [000211] SW620 cells were treated with antisense (SEQ ID NO.425) or reverse control oligonucleotides to EphB3. The cells were plated in 0.35% soft agar and growth quantitated using Alamar Blue after 7 days in culture.

[000212] The effect of EphB3 gene expression upon anchorage-independent cell growth of SW620 cells was measured by colony formation in soft agar. Soft agar assays were performed by first coating a non-tissue culture treated plate with PoIy-HEMA to prevent cells from attaching to the plate. Non-transfected cells were harvested using trypsin and washing twice in media. The cells were counted using a hemacytometer and resuspended to 10⁴ cells per ml in media. Fifty μl aliquots were placed in polyHEMA coated 96-well plates and transfected. For each transfection mixture, a carrier molecule, preferably a lipitoid or cholesteroid, was prepared to a working concentration of 0.5 nM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense or control oligonucleotide was then prepared to a working concentration of 100 μM in sterile Millipore water. The oligonucleotides were further diluted in OptiMEM™ (Gibco/BRL) in a microfuge tube to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, lipitoid or cholesteroid, typically in the amount of about 1.5-2 nmol lipitoid/μg antisense oligonucleotide, was diluted in the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide was immediately added to the diluted lipitoid and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of about 300 nM. Following transfection at 37° C for about 30 minutes, 3% GTG agarose was added to the cells for a final concentration of 0.35% agarose by pipeting up and down. After the cell layer agarose solidified, 100 μl of media was dribbled on top of each well. Colonies formed in about 7 days. For a read-out of growth, 20 μl of Alamar Blue was added to each well and the plate was shaken for about 15 minutes. Fluorescence readings (530 nm excitation/590 run emission) were taken after incubation for 6-24 hours. [000213] Tthe application of EphB3 antisense oligonucleotides to SW620 cells results in inhibition of colony formation and shows that EphB3 plays a role in anchorage-independent cell growth. Those antisense oligonucleotides that result in inhibition of colony formation of SW620 cells indicate that EphB3 plays a role in production or maintenance of the metastatic phenotype. [000214] Example 6: Methods of Detecting EphB3 tumors

[000215] Total RNA from normal tissues from multiple individuals was pooled, reverse transcribed and subjected to quantitative PCR using primers to EphB3. Amplified RNA from LCM dissected tissue from eight cancer and peritumoral normal tissue was reverse transcribed and subjected to quantitative PCR using primers to EphB3. mRNA levels in the cancer were found to be approximately four times as high as the peritumoral levels. EphB3 levels in colon cancer samples appeared to be expressed at significantly greater levels in colon cancer samples than in normal colon samples and many other normal tissue - samples. An exception was normal breast, which expressed comparable levels of EphB3 mRNA as colon cancer.

[000216] Example 7: Regulation of Gene Expression

[000217] The expression of the differentially expressed genes represented by the polynucleotides in the cancerous cells was analyzed using antisense and siRNA knockout technology to confirm the role and function of the gene product in tumorigenesis, e.g., in promoting a metastatic phenotype.

[000218] A number of different antisense and siRNA oligonucleotides were generated and tested for their ability to suppress expression of the gene. Once synthesized and quantitated, the oligomers were screened for efficiency of a transcript knock-out in a panel of cell lines.

The efficiency of the knock-out was determined by analyzing mRNA levels using lightcycler quantification.

[000219] The ability of each oligonucleotide to inhibit gene expression was tested through transfection into MDA-MB-231 or MDA231 ("231"); SW620 colon colorectal carcinoma cells; Colo320DM cells; HCTl 16 cells, and MDA-MB-435 or MDA435 cells.

[000220] For each transfection mixture, a carrier molecule (such as a lipid, lipid derivative, lipid-like molecule, cholesterol, cholesterol derivative, or cholesterol-like molecule) was prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense and siRNA oligonucleotides were then prepared to a working concentration of about 100 μM in sterile

Millipore water. The oligonucleotides were further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. hi a separate microfuge tube, the carrier molecule, typically in the amount of about 1.5-2 rrmol carrier/μg antisense oligonucleotide was diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide is immediately added to the diluted carrier and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of 300 nM (antisense oligonucleotides). siRNAs were added to the cells to a final concentration of about 67nM.

[000221] The level of target mRNA that corresponds to a target gene of interest in the transfected cells was quantitated in the cancer cell lines using the ABI GeneAmp 7000™ realtime PCR machine. Values for the target mRNA were normalized versus an internal control. For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) was placed into a sterile 0.5 or 1-.5 ml microcentrifuge tube, and water added to a total volume of 12.5 μl. To each tube was added 7.5 μl of a buffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μl H₂O, 2.0 μl 1OX reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μl RNAsin® (2Ou) (Ambion, Inc., Hialeah, FL), and 0.5 μl MMLV reverse transcriptase (5Ou) (Ambion, Inc.). The contents were mixed by pipetting up and down, and the reaction mixture was incubated at 42°C for 1 hour. The contents of each tube were centrifuged prior to amplification.

[000222] An amplification mixture was prepared using ABI sybr master mix, plus 0.175 pmol of each oligonucleotide. SYBR® Green (Molecular Probes, Eugene, OR) is a dye which fluoresces when bound to double-stranded DNA. As double stranded PCR product is produced during amplification, the fluorescence from SYBR® Green increases. To each 20 μl aliquot of amplification mixture, 2 μl of template RT was added, and amplification carried out according to standard protocols. The results were expressed as the percent decrease in expression of the corresponding gene product relative to non-transfected cells, vehicle-only transfected (mock-transfected) cells, or cells transfected with reverse control oligonucleotides.

[000223] Example 8: Effect of Expression on Proliferation

[000224] The effect of gene expression on the inhibition of cell proliferation was assessed in several cell lines including MDA-MB-231 or MDA231 ("231")); SW620 colon colorectal carcinoma cells; Colo320DM cells; HCTl 16 cells, and MDA-MB-435 or MDA435 cells using antisense and siRNA methodologies.

[000225] Cells were plated to a density that will be about 80-95% confluent after days in 96- well dishes. Oligonucleotides (antisense or siRNA) were diluted to 2 μM in OptiMEM™.

The oligonucleotide-OptiMEM™ was then added to a delivery vehicle, selected so as to be optimized for the particular cell type to be used in the assay. The oligo/delivery vehicle mixture was then further diluted into medium with serum on the cells. The final concentration of antisense oligonucleotides was about 300 nM and the final concentration of siRNA oligonucleotides was 67-100 nM.

[000226] Oligonucleotides (antisense or siRNA) were prepared as described above. Cells were transfected from about 4 hours to overnight at 37⁰C and the transfection mixture was replaced with fresh medium. Transfection was carried out as described above.

[000227] Those oligonucleotides (antisense or siRNA) that resulted in inhibition of proliferation of SW620 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous colon cells. Those oligonucleotides

(antisense or siRNA) that resulted in inhibition of proliferation of MDA231 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous breast cells.

[000228] Example 9: EphB3 Epitopes

[000229] Linear epitopes of EphB3 for antibody recognition and preparation can be identified by any of numerous methods known in the art. Some example methods include probing antibody-binding ability of peptides derived from the amino acid sequence of the antigen. Binding can be assessed by using BLACORE or ELISA methods. Other techniques include exposing peptide libraries on planar solid support ("chip") to antibodies and detecting binding through any of multiple methods used in solid-phase screening. Additionally, phage display can be used to screen a library of peptides with selection of epitopes after several rounds of biopanning.

[000230] Table 1 below provides regions of EphB3 (SEQ ID NO:2) that have been identified as linear epitopes suitable for recognition by anti EphB3 antibodies.

Table 1

98-115 9-mer DVQRVYVEL 101-109 15 28

98-115 9-mer VQRVYVELK 102-110 16 29

98-115 9-mer QRVYVELKF 103-111 17 30

98-115 9-mer RVYVELKFT 104-112 18 31

98-115 9-mer VYVΞLKFTV 105-113 19 32

98-115 9-rτner YVELKFTVR 106-114 20 33

98-115 9-mer VELKFTVRD 107-115 21 34

98-115 10-mer WRRDVQRVYV 98-107 22 35

98-115 10-mer RRDVQRVYVE 99-108 23 36

98-115 10-mer RDVQRVYVEL 100-109 24 37

98-115 10-mer DVQRVYVΞLK 101-110 25 38

98-115 10-mer VQRVYVΞLKF 102-111 26 39

98-115 10-mer QRVYVELKFT 103-112 27 40

98-115 10-mer RVYVELKFTV 104-113 28 41

98-115 10-mer VYVELKFTVR 105-114 29 42

98-115 10-mer YVELKFTVRD 106-115 30 43

152-194 8-mer HPYVKVDT 152-159 1 44

152-194 8-mer PYVKVDTI 153-160 2 45

152-194 8-mer YVKVDTIA 154-161 3 46

152-194 8-mer VKVDTIAP 155-162 4 47

152-194 8-mer KVDTIAPD 156-163 5 48

152-194 8-mer VDTIAPDE 157-164 6 49

152-194 8-mer DTIAPDES 158-165 7 50

152-194 8-mer TIAPDESF 159-166 8 51

152-194 8-mer IAPDESFS 160-167 9 52

152-194 8-mer APDESFSR 161-168 10 53

152-194 8-mer PDESFSRL 162-169 11 54

152-194 8-mer DESFSRLD 163-170 12 55

152-194 8-mer ESFSRLDA 164-171 13 56

152-194 8-mer SFSRLDAG 165-172 14 57

152-194 8-mer FSRLDAGR 166-173 15 58

152-194 8-mer SRLDAGRV 167-174 16 59

152-194 8-mer RLDAGRVN 168-175 17 60

152-194 8-mer LDAGRVNT 169-176 18 61

152-194 8-mer DAGRVNTK 170-177 19 62

152-194 8-mer AGRVNTKV 171-178 20 63

152-194 8-mer GRVNTKVR 172-179 21 64

152-194 8-mer RVNTKVRS 173-180 22 65

152-194 8-mer VNTKVRSF 174-181 23 66

152-194 8-mer NTKVRSFG 175-182 24 67

152-194 8-mer TKVRSFGP 176-183 25 68

152-194 8-mer KVRSFGPL 177-184 26 69

152-194 8-mer VRSFGPLS 178-185 27 70

152-194 8-mer RSFGPLSK 179-186 28 71

152-194 8-mer SFGPLSKA 180-187 29 72

152-194 8-mer FGPLSKAG 181-188 30 73

152-194 8-mer GPLSKAGF 182-189 31 74

152-194 8-mer PLSKAGFY 183-190 32 75

152-194 8-mer LSKAGFYL 184-191 33 76

152-194 8-mer SKAGFYLA 185-192 34 77

152-194 8-mer KAGFYLAF 186-193 35 78

152-194 8-mer AGFYLAFQ 187-194 36 79

152-194 9-mer NPYVKVDTI 152-160 37 80 152-194 10-mer AGRVNTKVRS 171-180 91 134

152-194 10-mer GRVNTKVRSF 172-181 92 135

152-194 10-mer RVNTKVRSFG 173-182 93 136

152-194 10-mer VNTKVRSFGP 174-183 94 137

152-194 10-mer NTKVRSFGPL 175-184 95 138

152-194 10-mer TKVRS FGPLS 176-185 96 139

152-194 10-mer KVRSPGPLSK 177-186 97 140

152-194 10-mer VRSFGPLSKA 178-187 98 141

152-194 10-mer RSFGPLSKAG 179-188 99 142

152-194 10-mer SFGPL.SKAGF 180-189 100 143

152-194 10-mer FGPLSKAGFY 181-190 101 144

152-194 10-mer GPLSKAGFYL 182-191 102 145

152-194 10-mer PLSKΆGFYLA 183-192 103 146

152-194 10-mer LSKAGFYLAF 184-193 104 147

152-194 10-mer SKAGPYLAFQ 185-194 105 148

244-256 8-mer NAVEVSVP 244-251 1 149

244-256 8-mer AVEVSVPL 245-252 2 150

244-256 8-mer VEVSVPLK 246-253 3 151

244-256 8-mer EVSVPLKL 247-254 4 152

244-256 8-mer VSVPLKLY 248-255 5 153

244-256 8-mer SVPLKLYC 249-256 6 154

244-256 9-mer NAVEVSVPL 244-252 7 155

244-256 9-mer AVEVSVPLK 245-253 8 156

244-256 9-mer VEVSVPLKL 246-254 9 157

244-256 9-mer EVSVPIiKLY 247-255 10 158

244-256 9-mer VSVPL. KLYC 248-256 11 159

244-256 10-mer NAVEVSVPLK 244-253 12 160

244-256 10-mer AVEVSVPLKL 245-254 13 161

244-256 10-mer VEVSVPLKLY 246-255 14 162

244-256 10-mer EVSVPLKLYC 247-256 15 163

274-298 8-mer GHEPAAKE 274-281 1 164

274-298 8-mer HEPAAKES 275-282 2 165

274-298 8-mer EPAAKESQ 276-283 3 166

274-298 8-mer PAAKESQC 277-284 4 167

274-298 8-mer AAKESQCR 278-285 5 168

274-298 8-mer AKESQCRP 279-286 6 169

274-298 8-mer KESQCRPC 280-287 7 170

274-298 8-mer ESQCRPCP 281-288 8 171

274-298 8-mer SQCRPCPP 282-289 9 172

274-298 8-mer QCRPCPPG 283-290 10 173

274-298 8-mer CRPCPPGS 284-291 11 174

274-298 8-mer RPCPPGSY 285-292 12 175

274-298 8-mer PCPPGSYK 286-293 13 176

274-298 8-mer CPPGSYKA 287-294 14 177

274-298 8-mer PPGSYKAK 288-295 15 178

274-298 8-mer PGSYKAKQ 289-296 16 179

274-298 8-mer GSYKAJKQG 290-297 17 180

274-298 8-mer SYKAKQGE 291-298 18 181

274-298 9-mer GHEPAA.KES 274-282 . 19 182

274-298 9-mer HEPAAKESQ 275-283 20 183

274-298 9-mer EPAAKBSQC 276-284 21 184

274-298 9-mer PAAKESQCR 277-285 22 185

274-298 9-mer AAKESQCRP 278-286 23 186 274-298 9-mer AKESQCRPC 279-287 24 187

274-298 9-mer KESQCRPCP 280-288 25 188

274-298 9-mer ESQCRPCPP 281-289 26 189

274-298 9-mer SQCRPCPPG 282-290 27 190

274-298 9-mer QCRPCPPGS 283-291 28 191

274-298 9-mer CRPCPPGSY 284-292 29 192

274-298 9-mer RPCPPGSYK 285-293 30 193

274-298 9-mer PCPPGSYKA 286-294 31 194

274-298 9-mer CPPGSYKAK 287-295 32 195

274-298 9-mer PPGSYKAKQ 288-296 33 196

274-298 9-mer PGSYKAKQG 289-297 34 197

274-298 9-mer GSYKAKQGE 290-298 35 198

274-298 10-mer GHEPAAKESQ 274-283 36 199

274-298 10-mer HEPAAKESQC 275-284 37 200

274-298 10-mer EPAAKESQCR 276-285 38 201

274-298 10-mer PAAKESQCRP 277-286 39 202

274-298 10-mer AAKESQCRPC 278-287 40 203

274-298 10-mer AKESQCRPCP 279-288 41 204

274-298 10-mer KΞSQCRPCPP 280-289 42 205

274-298 10-mer ESQCRPCPPG 281-290 43 206

274-298 10-mer SQCRPCPPGS 282-291 44 207

274-298 10-mer QCRPCPPGSY 283-292 45 208

274-298 10-mer CRPCPPGSYK 284-293 46 209

274-298 10-mer RPCPPGSYKA 285-294 47 210

274-298 10-mer PCPPGSYKAK 286-295 48 211

274-298 10-mer CPPGSYKAKQ 287-296 49 212

274-298 10-mer PPGSYKAKQG 288-297 50 213

274-298 10-mer PGSYKAKQGE 289-298 51 214

313-336 8-mer PAASICTC 313-320 1 215

313-336 8-mer AASICTCH 314-321 2 216

313-336 8-mer AS ICTCHN 315-322 3 217

313-336 8-mer S I CTCHNN 316-323 4 218

313-336 8-mer ICTCHNNF 317-324 5 219

313-336 8-mer CTCHNNFY 318-325 6 220

313-336 8-mer TCHNNFYR 319-326 7 221

313-336 8-mer CHNNFYRA 320-327 8 222

313-336 8-mer HNNFYRAD 321-328 9 223

313-336 8-mer NNFYRADS 322-329 10 224

313-336 8-mer NFYRADSD 323-330 11 225

313-336 8-mer FYRADSDS 324-331 12 226

313-336 8-mer YRADSDSA 325-332 13 227

313-336 8-mer RADSDSAD 326-333 14 228

313-336 8-mer ADSDSADS 327-334 15 229

313-336 8-mer DSDSADSA 328-335 16 230

313-336 8-mer SDSADSAC 329-336 17 231

313-336 9-mer PAASICTCH 313-321 18 232

313-336 9-mer AASICTCHN 314-322 19 233

313-336 9-mer ASICTCHNN 315-323 20 234

313-336 9-mer SICTCHNNF 316-324 21 235

313-336 9-mer ICTCHNNFY 317-325 22 236

313-336 9-mer CTCHNNFYR 318-326 23 237

313-336 9-mer TCHNNFYRA 319-327 24 238

313-336 9-mer CHNNFYRAD 320-328 25 239 313-336 9-mer HNNFYRADS 321-329 26 240

313-336 9-mer NNFYRADSD 322-330 27 241

313-336 9-mer NFYRADSDS 323-331 28 242

313-336 9-mer FYRADSDSA 324-332 29 243

313-336 9-mer YRADSDSAD 325-333 30 244

313-336 9-mer RADSDSADS 326-334 31 245

313-336 9-mer ADSDSADSA 327-335 32 246

313-336 9-mer DSDSADSAC 328-336 33 247

313-336 10-mer PAASICTCHN 313-322 34 248

313-336 10-mer AASICTCHNN 314-323 35 249

313-336 10-mer ASICTCHNNF 315-324 36 250

313-336 10-mer SICTCHNNFY 316-325 37 251

313-336 10-mer ICTCHNNFYR 317-326 38 252

313-336 10-mer CTCHNNFYRA 318-327 39 253

313-336 10-mer TCHNNFYRAD 319-328 40 254

313-336 10-mer CHNNFYRADS 320-329 41 255

313-336 10-mer HNNFYRADSD 321-330 42 256

313-336 10-mer NNFYRADSDS 322-331 43 257

313-336 10-mer NFYRADSDSA 323-332 44 258

313-336 10-mer FYRADSDSAD 324-333 45 259

313-336 10-mer YRADSDSADS 325-334 46 260

313-336 10-mer RADSDSADSA 326-335 47 261

313-336 10-mer ADSDSADSAC 327-336 48 262

362-383 8-mer PRDLGGRD 362-369 1 263

362-383 8-mer RDLGGRDD 363-370 2 264

362-383 8-mer DLGGRDDL 364-371 3 265

362-383 8-mer LGGRDDLL 365-372 4 266

362-383 8-mer GGRDDLLY 366-373 5 267

362-383 8-mer GRDDLLYN 367-374 6 268

362-383 8-mer RDDLLYNV 368-375 7 269

362-383 _L_ 8-mer DDLLYNVI 369-376 8 270

362-383 8-mer DLLYNVIC 370-377 9 271

362-383 8-mer LLYNVICK 371-378 10 272

362-383 8-mer LYNVICKK 372-379 11 273

362-383 8-mer YNVICKKC 373-380 12 274

362-383 8-mer NVICKKCH 374-381 13 275

362-383 8-mer VICKKCHG 375-382 14 276

362-383 8-mer ICKKCHGA 376-383 15 277

362-383 9-mer PRDLGGRDD 362-370 16 278

362-383 9-mer RDLGGRDDL 363-371 17 279

362-383 9-mer DLGGRDDLL 364-372 18 280

362-383 9-mer LGGRDDLLY 365-373 19 281

362-383 9-mer GGRDDLLYN 366-374 20 282

362-383 9-mer GRDDLLYNV 367-375 21 283

362-383 9-mer RDDLLYNVI 368-376 22 284

362-383 9-mer DDLLYNVIC 369-377 23 285

362-383 9-mer DLLYNVICK 370-378 24 286

362-383 9-mer LLYNVICKK 371-379 25 287

362-383 9-mer LYNVICKKC 372-380 26 288

362-383 9-mer YNVICKKCH 373-381 27 289

362-383 9-mer NVICKKCHG 374-382 28 290

362-383 9-mer VICKKCHGA 375-383 29 291

362-383 10-mer PRDLGGRDDL 362-371 30 292 362-383 10-mer RDLGGRDDLL 363-372 31 293

362-383 10-mer DLGGRDDLLY 364-373 32 294

362-383 10-mer LGGRDDLLYN 365-374 33 295

362-383 10-mer GGRDDLLYNV 366-375 34 296

362-383 10-mer GRDDLLYNVI 367-376 35 297

362-383 10-mer RDDLLYNVIC 368-377 36 298

362-383 10-mer DDLLYNVICK 369-378 37 299

362-383 10-mer DLLYNVICKK 370-379 38 300

362-383 10-mer LLYNVICKKC 371-380 39 301

362-383 10-mer LYNVICKKCH 372-381 40 302

362-383 10-mer YNVICKKCHG 373-382 41 303

362-383 10-mer NVICKKCHGA 374-383 42 304

436-469 8-mer PLPPRYAA 436-443 1 305

436-469 8-mer LPPRYAAV 437-444 2 306

436-469 8-mer PPRYAAVN 438-445 3 307

436-469 8-mer PRYAAVNI 439-446 4 308

436-469 8-mer RYAAVNIT 440-447 5 309

436-469 8-mer YAAVNITT 441-448 6 310

436-469 8-mer AAVNITTN 442-449 7 311

436-469 8-mer AVNITTNQ 443-450 8 312

436-469 8-mer VNITTNQA 444-451 9 313

436-469 8-mer NITTNQAA 445-452 10 314

436-469 8-mer ITTNQAAP 446-453 11 315

436-469 8-mer TTNQAAPS 447-454 12 316

436-469 8-mer TNQAAPSE 448-455 13 317

436-469 8-mer NQAAPSEV 449-456 14 318

436-469 8-mer QAAPSEVP 450-457 15 319

436-469 8-mer AAPSEVPT 451-458 16 320

436-469 8-mer APSEVPTL 452-459 17 321

436-469 8-mer PSEVPTLR 453-460 18 322

436-469 8-mer SEVPTLRL 454-461 19 323

436-469 8-mer EVPTLRLH 455-462 20 324

436-469 8-mer VPTLRLHS 456-463 21 325

436-469 8-mer PTLRLHSS 457-464 22 326

436-469 8-mer TLRLHSSS 458-465 23 327

436-469 8-mer LRLHSSSG 459-466 24 328

436-469 8-mer RLHSSSGS 460-467 25 329

436-469 8-mer LHSSSGSS 461-468 26 330

436-469 8-mer HSSSGSSL 462-469 27 331

436-469 9-mer PLPPRYAAV 436-444 28 332

436-469 9-mer LPPRYAAVN 437-445 29 333

436-469 9-mer PPRYAAVNI 438-446 30 334

436-469 9-mer PRYAAVNIT 439-447 31 335

436-469 9-mer RYAAVNITT 440-448 32 336

436-469 9-mer YAAVNITTN 441-449 33 337

436-469 9-mer AAVNITTNQ 442-450 34 338

436-469 9-mer AVNITTNQA 443-451 35 339

436-469 9-mer VNITTNQAA 444-452 36 340

436-469 9-mer NITTNQAAP 445-453 37 341

436-469 9-mer ITTNQAAPS 446-454 38 342

436-469 9-mer TTNQAAPSE 447-455 39 343

436-469 9-mer TNQAAPSEV 448-456 40 344

436-469 9-mer NQAAPSEVP 449-457 41 345 436-469 9-mer QAAPSΞVPT 450-458 42 346

436-469 9-mer AAPSEVPTL 451-459 43 347

436-469 9-mer APSEVPTLR 452-460 44 348

436-469 9-mer PSEVPTLRL 453-461 45 349

436-469 9-mer SEVPTLRLH 454-462 46 350

436-469 9-mer EVPTLRLHS 455-463 47 351

436-469 9-mer VPTLRLHSS 456-464 48 352

436-469 9-mer PTLRLHSSS 457-465 49 353

436-469 9-mer TLRLHSSSG 458-466 50 354

436-469 9-mer LRLHSSSGS 459-467 51 355

436-469 9-mer RLHSSSGSS 460-468 52 356

436-469 9-mer LHSSSGSSL 461-469 53 357

436-469 10-mer PLPPRYAAVW 436-445 54 358

436-469 10-mer LPPRYAAVNI 437-446 55 359

436-469 10-mer PPRYAAVNIT 438-447 56 360

436-469 10-mer PRYAAVNITT 439-448 57 361

436-469 10-mer RYAAVNITTN 440-449 58 362

436-469 10-mer YAAVNITTNQ 441-450 59 363

436-469 10-mer AAVNITTNQA 442-451 60 364

436-469 10-mer AVNITTNQAA 443-452 61 365

436-469 10-mer VNITTNQAAP 444-453 62 366

436-469 10-mer NITTNQAAPS 445-454 63 367

436-469 10-mer ITTNQAAPSE 446-455 64 368

436-469 10-mer TTNQAAPSΞV 447-456 65 369

436-469 10-mer TNQAAPSEVP 448-457 66 370

436-469 10-mer NQAAPSEVPT 449-458 67 371

436-469 10-mer QAAPSEVPTL 450-459 68 372

436-469 10-mer AAPSEVPTLR 451-460 69 373

436-469 10-mer APSEVPTLRL 452-461 70 374

436-469 10-mer PSEVPTLRLH 453-462 71 375

436-469 10-mer SEVPTLRLHS 454-463 72 376

436-469 10-mer EVPTLRLHSS 455-464 73 377

436-469 10-mer VPTLRLHSSS 456-465 74 378

436-469 10-mer PTLRLHSSSG 457-466 75 379

436-469 10-mer TLRLHSSSGS 458-467 76 380

436-469 10-mer LRLHSSSGSS 459-468 77 381

436-469 10-mer RLHSSSGSSL 460-469 78 382

509-530 8-mer QLDGLRPD 509-516 1 383

509-530 8-mer LDGLRPDA 510-517 2 384

509-530 8-mer DGLRPDAR 511-518 3 385

509-530 8-mer GLRPDARY 512-519 4 386

509-530 8-mer LRPDARYV 513-520 5 387

509-530 8-mer RPDARYW 514-521 6 388

509-530 8-mer PDARYWQ 515-522 7 389

509-530 8-mer DARYWQV 516-523 8 390

509-530 8-mer ARYWQVR 517-524 9 391

509-530 8-mer RYWQVRA 518-525 10 392

509-530 8-mer YWQVRAR 519-526 11 393

509-530 8-mer WQVRART 520-527 12 394

509-530 8-mer VQVRARTV 521-528 13 395

509-530 8-mer QVRARTVA 522-529 14 396

509-530 8-mer VRARTVAG 523-530 15 397

509-530 9-mer QLDGLRPDA 509-517 16 398 509-530 9-mer LDGLRPDAR 510-518 17 399

509-530 9-mer DGLRPDARY 511-519 18 400

509-530 9-mer GLRPDARYV 512-520 19 401

509-530 9-mer LRPDARYW 513-521 20 402

509-530 9-mer RPDARYWQ 514-522 21 403

509-530 9-mer PDARYWQV 515-523 22 404

509-530 9-mer DARYWQVR 516-524 23 405

509-530 9-mer ARYWQVRA 517-525 24 406

509-530 9-mer RYWQVRAR 518-526 25 407

509-530 9-mer YWQVRART 519-527 26 408

509-530 9-mer WQVRARTV 520-528 27 409

509-530 9-mer VQVRARTVA 521-529 28 410

509-530 9-mer QVRARTVAG 522-530 29 411

509-530 10-mer QLDGLRPDAR 509-518 30 ^' 412

509-530 10-mer LDGLRPDARY 510-519 31 413

509-530 10-mer DGLRPDARYV 511-520 32 414

509-530 10-mer GLRPDARYW 512-521 33 415

509-530 10-mer LRPDARYWQ 513-522 34 416

509-530 10-mer RPDARYWQV 514-523 35 417

509-530 10-mer PDARYWQVR 515-524 36 418

509-530 10-mer DARYWQVRA 516-525 37 419

509-530 10-mer ARYWQVRAR 517-526 38 420

509-530 10-mer RYWQVRART 518-527 39 421

509-530 10-mer YWQVRARTV 519-528 40 422

509-530 10-mer WQVRARTVA 520-529 41 423

509-530 10-mer VQVRARTVAG 521-530 42 424

[000231] Example 10: Immunohistochemistry

[000232] Commercially available paraffin embedded human tissue microarrays consisting of breast, colon, ovarian, lung and esophageal carcinoma, as well as normal tissue arrays were used to evaluate the expression of EphB3 by means of immunohistochemistry. (Zymed Laboratories Inc, San Francisco CA; Cybrdi, Gaithersburg, MD; Clinomics Biosciences Inc, Cambridge UK). Naive and EphB3-transfected 293T cells were used as controls to validate the expression.

[000233] Tissue sections were deparaffinized and hydrated to water. Antigen retrieval was performed in the Decloaker (Biocare, Walnut Creek, CA) for 5 minutes using Reveal (Biocare) diluted 1:10 at 201b pressure. Immunohistochemistry procedures were performed on the DAKO Autostainer Plus (DAKO, Carpenteria, CA). Endogenous biotin was blocked using Avidin Biotin Blocking solutions (Vector Labs, Burlingame, CA) followed by endogenous peroxidase quenching with DAKO Peroxidase block (DAKO). Endogenous immunoglobulins were blocked using the antibody diluent (Ventana, Tucson, AZ) for 30 minutes followed by a 30-minute incubation in the primary antibodies. A rabbit anti-human EphB3 antibody (Chiron, Emeryville, CA) and rabbit IgG Isotype control (NeoMarker, Fremont, CA) were used at 4ug/ml. A biotinylated AffmiPure F(ab')2 fragment goat anti-rabbit IgG F(ab')2 fragment specific secondary antibody (Jackson ImmunoResearch, West Grove, CA) at 2.5 μg/ml followed by Vectastain ABC Elite (Vector Labs) was used for detection. Chromogenic colorization was performed using Stable DAB (Invitrogen, Carlsbad, CA). Mayer's Hematoxylin was used as a counter stain and sections were dehydrated in graded alcohols, cleared in xylene and coverslipped using a synthetic mounting media.

[000234] Example 11 : Gene Silencing

[000235] SiRNA Knockdown and Cell-Based Assays

[000236] Cells were seeded from 250,000 to 350,000 cells/well in a 6 well-plate in 2 ml of medium the day before transfection. Plates were incubated at 37⁰C O/N. The next day the medium was removed and 1.8 ml of complete medium was added. In an Eppendorf tube, 100 μl of OptiMem was added. Diluted siRNA (20 μM stock) at 10OnM and diluted lipid (0.5mM stock) at 3.75 μM were mixed together to form the complex and added to the cells dropwise.

The cells with the siRNA were incubated from 4 h to overnight at 37⁰C and replaced with complete media. Cells were harvested cells at 24-72 hours to monitor RNA/ protein levels.

[000237] Proliferation assay (SW620; Colo320DM; HCT116; MDA-MB-435 or MDA231; 184B5 and MRC9 cells)

[000238] Cells were seeded at 3000 to 5000 cells/well in 70 μl on 5 x 96 well flat plates. The cells were incubated at 37⁰C O/N. The cells were transfected with approximately 10OnM siRNA and 5 μM lipid using Multimek96. The mixture was allowed to incubate for 10 minutes to form the complex. 30 μl was added to each well. Incubate transfection for 4 — 6 hours and then replace with complete media. Proliferation was monitored for 4 — 5 days by Cell TiterGlo kit. One plate was assayed each day.

[000239] Soft Agar Assay (SW620; Colo320DM; HCT116 and MDA435 cells): [000240] Cells were plated in 70 μl media/ well (about 500 cells/well) on polyhema coated round bottom 96 well plate. Cells were transfected with 100 nM siRNA and 3.8 μM lipid by adding 30 μl of the complex to cells. About 50 μl/well of 1.05% agarose was added to each well and mixed well to disperse cells. 100 μl complete media was added on top of agarose and incubated at 37⁰C for 5-7 days.

[000241] Alarnar Blue (20 μl/well) was used to quantify cell growth in the colonies formed. Plates were incubated at 37⁰C and fluorescence was measured. [000242] Spheroid Assay (SW620; HCT116; and MDA-MB231 cells):

[000243] Cell Plating

[000244] Cells were trypsinized using 0.1% STV and counted using a hemacytometer.

Dilutions were calculated so that 8-10,000 cells/per well were seeded in 70 μl media on

Poly(HEMA)-coated round bottom 96 well plates (Costar#3799). PoIy(HEMA) is a known anti-adhesive that prevents cells from attaching to the bottom of the plate.

[000245] Cell transfection

[000246] To transfect cells for spheroid assay, cells were usually transfected the same day the cells were plated cells (before the spheroid is totally formed). The siRNA were tested in triplicate. Wild type (untransfected cells) as well as a positive siRNA control and a negative control (usually QiaRef for Qiagen siRNA)) were used.

[000247] Lipitoid plate preparation:

[000248] One lipid was used per set of cell line. Lipitoid compound(s) were diluted from

0.5mM to a concentration of 0.2mM using sterile water.

[000249] Transfection of Complex

[000250] 4.5 μl of diluted lipid (0.2mM) was added into the 30ul OptiMEM on a mixing plate. 30 μl of diluted siRNA (0.7uM) was added into the mixing plate and mixed. The complex was allowed to form for 10 min at RT. 30 μl of siRNA/lipid complex was added to cells in 70 μl media, and mixed. Cells were incubated at 37⁰C on Standard Waver platform rocker for 4-7 days.

[000251] Example 12: LDH assay

[000252] Harvesting for LDH readout

[000253] Cytotoxicity assays was performed using Cytotoxicity Detection

[000254] 96-well microplates were purchased from Nunc. Cell lines were obtained from

Chiron Master Culture Collection (Chiron Corporation) and grown at 37⁰C in 5% CO2 incubator in appropriate media supplemented with 10% FBS (Life Technologies, Rockville,

MD) and 2mM L-Glutamine (Bio Whittaker, Walkersville, MD). [000255] Cytotoxicity assay was performed using Cytotoxicity Detection Kit (LDH) purchased from Roche (#1644793). Microtiter Plate Reader (Molecular Devices) with 490nm filter was used to monitor LDH activity.

[000256] For each assay 1 plate per day was recovered: at day 0 (before transfection) to assess even plating, then at days 1, 2 and 3 after transfection. For each assay plate both culture supernatant and cell lysate were monitored for lactate dehydrogenase or LDH amounts.

[000257] Culture supernatant LDH.

- [000258] From each well of an assay plate, 100 μl of culture supernatant was collected and transferred to 96well v-bottom containing 100 μl serum-free media and spun (5 minutes at 2000 rpm) to remove any floating dead cells. It was then assayed, by transferring 100 μl to a new 96 well flat-plate, and adding to each well 100 μl of the kit dye and catalyst (45:1 ratio, and ~20min RT incubation in dark) according to manufacturer's instructions, in order to determine the amount of released LDH (rLDH). An increase in amount of dead or plasma- membrane damaged cells results in an increased release of LDH into the culture supernatant. An increase in the LDH activity directly correlates to the amount of a dye (formazan) formed in a limited time period. The formation of this dye was detected at 490nm.

[000259] Lysate LDH

[000260] After removal of the culture supernatant, the cells attached to the plate were lysed in 200 μl media/1% Triton XlOO solution (equal volumes of growth media to 2% Triton XlOO in serum-free media) by mixing 4-5X and transferring the 200 μl to a v-bottom plate. After a 5 minute spin at 2000 rpm to remove debris, 100 μl of lysate for each well was recovered to a flat bottom plate and assayed by adding the kit dye and catalyst in the same conditions as for the culture supernatant. This allows the determination of the amount of intracellular LDH (iLDH).

[000261] Assay Development

[000262] Dye and catalyst were stored at -2O⁰C and thawed before use in a 25⁰C waterbath. The vial containing the catalyst was brought to room temperature, then ImI UF H₂O was added and allowed to sit for lOmin before using. Unused reagents were stored at 4⁰C. [000263] Plates can be stored at 4⁰C wrapped in saran wrap for about 1 week before developing. Column 1 on each plate was used as the assay blank. After reagent was added to plate, the plate was placed in the dark (covered box) and incubated for 20min at room temperature. Normal values for the blank are 0.2-0.3 OD490. Generally the FBS is diluted in half by using a serum free media when used to dilute samples.

[000264] RLDH/tLDH ratio

[000265] The total amount of LDH was calculated by adding released LDH + intracellular LDH (tLDH = rLDH + iLDH). In order to compare the amount of cytotoxicity between antisense and reverse control-treated samples, the ratio between released LDH and total LDH (rLDH/tLDH) was used. This ratio expresses the proportion of dead to live cells and bypasses the problem of having different number of cells in- different wells due to varied cytotoxic effects.

[000266] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the present invention.

Claims

We claim:

1. A composition comprising an EphB3 modulator and one or more pharmaceutically acceptable carriers, wherein said EphB3 modulator has one or more of the activities selected from the group consisting of inducing receptor phosphorylation, inducing receptor oligomerization, inducing receptor internalization, inducing receptor degradation, inducing ligand-like EphB3 signaling, inducing EphB3 -mediated cell-cell adhesion, and inhibiting EphB3 expression.

2. ■ The composition of claim 1 wherein the composition is a sterile injectable-.

3. The composition of claim 1 wherein the EphB3 modulator induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 receptor internalization and EphB3 degradation.

4. The composition of claim 1 wherein said EphB3 modulator induces EphB3 degradation.

5. The composition of claim 1 wherein said EphB3 modulator stimulates EphB3 binding to intracellular adaptor proteins.

6. The composition of claim 1 wherein said EphB3 modulator inhibits and/or inactivates one or more of FAK, the Erk/MAPK pathway, the Cdc42/Rac pathway, AbI/ Arg, Fyn, Src, LMW-PTP, Intersectin, the Cdc42 pathway, Kalirin or the Rac pathway.

7. The composition of claim 1 wherein said EphB3 modulator activates and/or stimulates R-ras.

8. The composition of claim 1 wherein said EphB3 modulator induces phosphorylation of R-ras

9. The composition of claim 1 wherein the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody.

10. The composition of claim 1 wherein the EphB3 modulator is a monoclonal antibody which binds to EphB3 with an affinity of at least IxIO⁸Ka.

11. The composition of claim 1 wherein the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and modulates one or more EphB3-related biological activities.

12. The composition of claim 10 wherein the monoclonal antibody is a human antibody, a humanized antibody or chimeric antibody.

13. The composition of claim 10 wherein the monoclonal antibody binds to an epitope of EphB3, said epitope selected from the group consisting of SEQ ID NOS: 14-424.

14. The composition of claim 10 wherein the monoclonal antibody binds to an epitope of EphB3, said epitope in the domain selected from the group consisting of the ligand binding domain, the TNFR domain, the 1^st fibronectin domain, and the 2^nd fibronectin domain.

15. The composition of claim 14 wherein the monoclonal antibody binds to an epitope of the ligand binding domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS: 14-148.

16. The composition of claim 14 wherein the monoclonal antibody binds to an epitope of the TNFR domain of EphB3, said epitope selected from the group consisting o>f SEQ DD NOS-: 164-262.

17. The composition of claim 14 wherein the monoclonal antibody binds to an epitope of the 1^st fibronectin domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS:263-304.

18. The composition of claim 14 wherein the monoclonal antibody binds to an epitope of the 2^nd fibronectin domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS :383-424.

19. The composition of claim 10 wherein the monoclonal antibody does not bind to the ligand binding domain of EρhB3.

20. The composition of claim 10 wherein the monoclonal antibody does not cross-react with EρhB2 or EρhB4.

21. The composition of claim 10 wherein the monoclonal antibody induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 internalization and EphB3 degradation.

22. The composition of claim 1 wherein the EphB3 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID N^"O:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO.ll, SEQ ID NO.12, SEQ ID NO:13 and SEQ ID NO: 425.

23. A method of treating cancer or a cancer symptom in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the EphB3 modulator of claim 1.

24. The method of claim 23 wherein the EρhB3 modulator induces EphB3 degradation.

25. The method of claim 24 wherein the EphB3 modulator inhibits EphB3 expression by at least 50% as compared to a control.

26. The method of claim 23 wherein the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody.

27. The method of claim 23 wherein the EphB3 modulator is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single- chain antibody, or a Fab fragment.

28. The method of claim 27 wherein the antibody is labeled.

29. The method of claim 28 wherein the label is an enzyme, radioisotope, toxin or fluorophore.

30. The method of 27 wherein the antibody has a binding affinity less than about IxIO⁵K₃ for a polypeptide other than EphB3.

31. ■ The method of claim 23 wherein the EphB3 modulator is a monoclonal antibody.

32. The method of claim 31 wherein the monoclonal antibody binds to EphB3 with an affinity of at least IxIO⁸Ka.

33. The method of claim 31 wherein the monoclonal antibody binds to an epitope of EphB3, said epitope selected from the group consisting of SEQ DD NOS:14-424.

34. The method of claim 31 wherein the monoclonal antibody binds to an epitope of EphB3, said epitope in the domain selected from the group consisting of the ligand binding domain, the TNFR domain, the 1^st fibronectin domain, and the 2^nd fibronectin domain.

35. The method of claim 34 wherein the monoclonal antibody binds to an epitope of the ligand binding domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS: 14-148.

36. The method of claim 34 wherein the monoclonal antibody binds to an epitope of the TNFR domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS: 164-262.

37. The method of claim 34 wherein the monoclonal antibody binds to an epitope of the 1^st fibronectin domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS:263-304.

38. The method of claim 34 wherein the monoclonal antibody binds to an epitope of the 2^nd fibronectin domain of EphB3, said epitope selected from the group consisting of SEQ ID NOS:383-424.

39. The method of claim 31 wherein the monoclonal antibody does not bind to the ligand binding domain of EphB3.

40. The method of claim 31 wherein the monoclonal antibody does not bind to EphB2 or EphB4.

41. The method of claim 31 wherein the monoclonal antibody induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 internalization, and EphB3 degradation.

42. The method of claim 23 wherein the cancer is ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.

43. The method of claim 26 wherein the EphB3 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO-.IO, SEQ ID NO:11, SEQ ID NO.12, SEQ ID NO:13 and SEQ ID NO: 425.

44. The method of claim 23 further comprising the administration of a traditional cancer therapeutic to the patient.

45. The method of claim 23 further comprising the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.

46. The method of claim 23 wherein the cancer symptom is selected from the group consisting of pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, and difficulty swallowing.

47. A method of modulating an EphB3-related biological activity in a patient, the method comprising administering to the patient an amount of the EphB3 modulator of claim 1 effective to modulate the EphB3 biological activity.

48. The method of claim 47 wherein the EphB3 modulator is a monoclonal antibody which selectively binds EphB3.

49. The method of claim 47 wherein the patient has or is predisposed to one or more of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.

50. The method of claim 23 wherein the EphB3 modulator is an antibody and is administered to the subject via in vivo therapeutic antibody gene transfer.

51. A method of identifying a patient susceptible to EphB3 therapy comprising:

(a) detecting the presence or absence of evidence of EphB3 expression in said sample, wherein the presence of evidence of EphB3 expression in said sample is indicative of a patient who is a candidate for EρhB3 therapy and the absence of evidence of EphB3 expression in said sample is indicative of a patient who is not a candidate for EphB3 therapy;

(b) administering a therapeutically effective amount of the composition of claim 1 to the patient if the patient is a candidate for EphB3 therapy; and c) administering a traditional cancer therapeutic to the patient if the patient is not a candidate for EphB3 therapy.

52. The method of claim 51 wherein the expression of EphB3 is increased at least 30% compared to a control.

53. The method of claim 51 wherein evidence of EphB3 expression is detected by measuring EphB3 RNA.

54. The method of claim 51 wherein evidence of EphB3 expression is detected by measuring EphB3 expression products.

55. The method of claim 51 wherein the patient has or is predisposed to one or more of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.

56. The method of claim 51 further comprising the administration of a traditional cancer therapeutic to the patient.

57. A method of inhibiting cancer cell growth in a patient in need thereof comprising administering a therapeutically effective amount of the EphB3 modulator of claim 1 to the patient.

58. The method of claim 57 wherein the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and induces receptor degradation.

59. The method of claim 58 wherein the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and inhibits EphB3 expression.

60. A method of inhibiting a cancer cell phenotype in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of the EphB3 modulator of claim 1.

61. The method of claim 60 wherein the cancer cell phenotype is one or more of colony formation in soft agar and tubular network formation in a three dimensional basement membrane or extracellular membrane preparation.

62. The method of claim 60 further comprising the administration of a traditional cancer therapeutic to the patient.

63. The method of claim 60 further comprising the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.

64. The method of claim 60 wherein the cancer cells are selected from the group consisting of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer cells.

65. A method for detecting a tumor in a patient comprising administering to the patient a composition comprising the EphB3 modulator of claim 1 linked to an imaging agent and detecting the localization of the imaging agent in the patient.

66. The method of claim 65 wherein the EphB3 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody.

67. The method of claim 65 wherein the composition comprises an anti-EpliB3 antibody conjugated to an imaging agent.

68. The method of claim 67 wherein the imaging agent is ¹⁸F, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷-Ga, ⁷⁷Br, ⁸⁷MSr, ⁸⁶Y₅ ⁹⁰Y, ⁹⁹MTc, ¹¹¹In, ¹²³1, ¹²⁵1, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I₅ ¹³²1, ¹⁹⁷Hg, ²⁰³Pb, or ²⁰⁵Bi.

69. A method of expressing an anti-EphB3 antibody in a CHO or myeloma cell, the method comprising expressing a nucleic acid encoding the anti-EphB3 antibody in said CHO or myeloma cell.

70. A method of identifying a cancer inhibitor, the cancer characterized by overexpression of EphB3 compared to a control, said method comprising contacting a cell expressing EphB3 with a candidate compound and determining whether an EphB3 -related biological activity is induced, wherein the induced EphB3 -related biological activity is selected from the group consisting of receptor phosphorylation, receptor oligomerization, receptor degradation, and receptor signaling, wherein induction of the EphB3-related biological activity is indicative of a cancer inhibitor.

71. A method of identifying a cancer inhibitor, said cancer characterized by overexpression of EphB3, said method comprising contacting a cell expressing EphB3 with a candidate compound and an EphB3 ligand, and determining whether an EphB3-related biological activity is induced, wherein the induced EphB3-related biological activity is selected from the group consisting of receptor phosphorylation, receptor oligomerization, receptor degradation, and receptor signaling, wherein induction of the EphB3-related biological activity is indicative of a cancer inhibitor.