EP1888648A2 - Procedes de traitement, de diagnostic ou de detection du cancer - Google Patents

Procedes de traitement, de diagnostic ou de detection du cancer

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Publication number
EP1888648A2
EP1888648A2 EP06771814A EP06771814A EP1888648A2 EP 1888648 A2 EP1888648 A2 EP 1888648A2 EP 06771814 A EP06771814 A EP 06771814A EP 06771814 A EP06771814 A EP 06771814A EP 1888648 A2 EP1888648 A2 EP 1888648A2
Authority
EP
European Patent Office
Prior art keywords
ephb3
mer
cancer
modulator
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06771814A
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German (de)
English (en)
Inventor
Mary Jo Janatpour
Deborah Lee Zimmerman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Novartis Vaccines and Diagnostics Inc
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Filing date
Publication date
Application filed by Novartis Vaccines and Diagnostics Inc filed Critical Novartis Vaccines and Diagnostics Inc
Publication of EP1888648A2 publication Critical patent/EP1888648A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • 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.
  • 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.
  • tumor suppressor genes The normal function of tumor suppressor genes is to stop cellular growth.
  • tumor suppressors include p53, p 16, p21, and APC, all of which, when acting normally, stop a cell from dividing and growing uncontrollably.
  • p53 p 16, p21
  • APC APC
  • 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.
  • EphB3 is a receptor in the ephrin receptor tyrosine kinase family.
  • Eph receptors Ephs
  • Ephrin receptors Ephs
  • their ligands the ephrins
  • 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.
  • 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.
  • EFNA ephrin-A
  • EFNB ephrin-B
  • 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.
  • RTK receptor tyrosine kinase
  • 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).
  • EphrinAl can induce angiogenesis in the rat cornea model and antibodies to ephrinAl can inhibit TNF- ⁇ 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).
  • ACE adrenal-cortex derived microvascular endothelial cells
  • EphB2 has been shown to be upregulated in colon and stomach cancer (Kiyokawa et al., 1994).
  • Ephrins may also play a role in metastasis. 293T human epithelial kidney cells transfected with either EphB3 or E ⁇ hB2 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).
  • 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.
  • EphB3 (also known as Hek2, Sek4, Mdk5, Tyro ⁇ , 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.
  • 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.
  • the composition is a sterile injectable.
  • the EphB3 modulator induces one or more of EphB3 phosphorylation, EphB3 oligomerization, EphB3 receptor internalization and EphB3 degradation.
  • 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.
  • the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody.
  • the EphB3 modulator is a monoclonal antibody which binds to EphB3 with an affinity of at least IxIO 8 Ka.
  • the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and modulates one or more EphB3-related biological activities.
  • the monoclonal antibody is a human antibody, a humanized antibody or chimeric antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the EphB3 modulator induces EphB3 degradation.
  • the EphB3 modulator inhibits EphB3 expression by at least 50% as compared to a control.
  • the EphB3 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody.
  • 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.
  • the antibody is labeled.
  • the label is an enzyme, radioisotope, toxin or fluorophore.
  • the antibody has a binding affinity less than about lxlO 5 K a for a polypeptide other than EphB3.
  • the EphB3 modulator is a monoclonal antibody.
  • 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.
  • the cancer is ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.
  • 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.
  • the methods further comprise the administration of a traditional cancer therapeutic to the patient.
  • the methods further comprise the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.
  • 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.
  • the E ⁇ hB3 modulator is a monoclonal antibody which selectively binds EphB3.
  • the patient has or is predisposed to one or more of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.
  • the E ⁇ hB3 modulator is an antibody and is administered to the subject via in vivo therapeutic antibody gene transfer.
  • 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.
  • 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 E ⁇ hB3 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.
  • 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.
  • 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.
  • the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and induces receptor degradation.
  • the EphB3 modulator is a monoclonal antibody which selectively binds EphB3 and inhibits EphB3 expression.
  • 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 E ⁇ hB3 modulator.
  • 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.
  • the cancer cells are selected from the group consisting of ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer cells.
  • 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.
  • 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.
  • the composition comprises an anti-EphB3 antibody conjugated to an imaging agent.
  • the imaging agent is 18 F, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 87 MSr, 86 Y, 90 Y, 99 MTc, 111 In, 123 1, 125 1, 127 Cs, 129 Cs, 131 1, 132 I, 197 Hg, 203 Pb, Or 206 Bi.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 2 depicts the inhibiton of anchorage independent growth of colon cancer cells using EphB3 siRNA.
  • Figure 3 provides a summary of the effect of EphB3 knockdown on various cancer and normal cell lines.
  • Figure 4 depicts differential expression of EphB3 at tihe mRNA level using
  • Figure 5 provdes a summary of EphB3 immunohistochemistry data.
  • Figure 6 provides EphB3 immunohistochemistry data.
  • 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.
  • EphB3 When bound to an E ⁇ hB3 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)).
  • 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 E ⁇ hB3 could be used to treat various types of cancers by modulating angiogenesis, tumor invasion, or metastasis, etc. [00044] Definitions
  • the term "about” refers to +/- 30%, +/- 20%, +/- 10%, or +/- 5% of a value.
  • 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.
  • accession number NM_004443 nucleotide sequence; SEQ DD NO:1
  • NP_004434 amino acid sequence; SEQ TD NO:2
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • angiogenesis refers to the development of blood vessels in a patient.
  • 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.
  • 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.
  • 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.
  • biological molecule includes, but is not limited to, polypeptides, nucleic acids, and saccharides.
  • 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.
  • 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.
  • the term “modulating cell division” refers to affecting the rate, amount or degree of cell division.
  • the methods will completely inhibit cell division.
  • the methods will decrease the amount of cell division.
  • the methods will prevent cell division.
  • N-terminus refers to the first 10 amino acids of a protein.
  • C-terminus refers to the last 10 amino acids of a protein.
  • cell-cell interaction refers to an interaction between two or more cells.
  • 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).
  • domain 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.
  • 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.
  • region refers to a physically contiguous portion of the primary structure of a biomolecule.
  • a region is defined by a contiguous portion of the amino acid sequence of that protein.
  • a "region" is associated with a function of the biomolecule.
  • portion refers to a physically contiguous portion of the primary structure of a biomolecule.
  • 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.
  • 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.
  • portions of biomolecules have a biological activity.
  • 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.
  • the phrase “induce” refers to a stimulation of an activity.
  • the phrase “EphB3 biological activity” refers to a biological, physiological, or biochemical activity of E ⁇ hB3 affected by activation of the receptor.
  • EphB3 may be activated by EphB3 modulators including ligands of EphB3, oligonucleotides, small molecules, mimetics, decoys or antibodies.
  • EphB3 biological activities include without limitation, receptor phosphorylation, receptor oligomerization, receptor internalization, receptor degradation, signaling, EphB3-mediated cell-cell adhesion, and the like.
  • EphB3-related cells/tumors/samples 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.
  • 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.
  • modulator refers to a composition that modulates one or more physiological or biochemical events associated with cancer.
  • an "EphB3 modulator” promotes EphB3 phosphorylation and degradation.
  • the modulator inhibits one or more biological activities associated with cancer.
  • 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.
  • the modulator acts independently of ligand binding, hi some embodiments the modulator does not compete for a ligand binding site.
  • the modulator blocks expression of a gene product involved in cancer.
  • the modulator blocks a physical interaction of two or more biomolecules involved in cancer
  • 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
  • the EphB3 modulator inhibits EphB3 expression.
  • 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.
  • epitope refers to an antigenic determinant of a polypeptide.
  • an epitope may comprise 3 or more amino acids in a spatial conformation which is unique to the epitope.
  • 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
  • oligonucleotide refers to a series of linked nucleotide residues.
  • 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
  • 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.
  • the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician.
  • 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.
  • 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.
  • detecting means to establish, discover, or ascertain evidence of an activity (for example, gene expression) or biomolecule (for example, a polypeptide).
  • homologous nucleotide sequence 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”.
  • homology identity 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.
  • 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%.
  • 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.
  • Homology may also be at the polypeptide level.
  • 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.
  • probe refers to nucleic acid sequences of variable length.
  • probes comprise at least about 10 and as many as about 6,000 nucleotides.
  • 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.
  • ISH in situ hybridization
  • FISH fluorescent in situ hybridization
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • stringent hybridization 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 5 0 C 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.
  • Tm thermal melting point
  • 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 30 0 C 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.
  • 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)).
  • 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.
  • 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.
  • vidence of gene expression refers to any measurable indicia that a gene is expressed.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.
  • 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.
  • EphB3 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.
  • the present invention is also applicable to any tumor cell-type where EphB3 plays a role in cell adhesion, migration or repulsion.
  • the cancer is ovarian, esophageal, colon, prostate, breast, skin cancer, lung, stomach or pancreatic cancer.
  • the cancers amenable to treatment and/or diagnosis according to the present invention are characterized by overexpression of E ⁇ hB3.
  • such cancers exhibit overexpression of EpliB3 by at least about 25%, at least about 50%, at least about
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods of the invention provide methods of imaging and localizing tumors and/or metastases and methods of diagnosis and prognosis.
  • the methods of the invention provide methods to evaluate the appropriateness of EphB3 -related therapy.
  • the present invention provides EphB3 modulators for, inter alia, the treatment, diagnosis, detection or imaging of cancer.
  • 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.
  • 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.
  • the EphB3 modulator causes phosphorylation of R-Ras.
  • the EphB3 modulator inactivates R-Ras or activates Syndecan.
  • 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.
  • the adaptor protein is Grb2, an adaptor protein not having intrinsic enzymatic activity.
  • the adaptor protein is RasGAP, an adaptor protein having enzymatic activity.
  • 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).
  • 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).
  • 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).
  • 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
  • the EphB3 modulator is an antibody.
  • 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.
  • the antibody has a binding affinity less than about IxIO 5 Ka for a polypeptide other than EphB3.
  • the EphB3 modulator is a monoclonal antibody which binds to EphB3 with an affinity of at least IxIO 8 Ka. In some embodiments, the monoclonal antibody does not bind to the ligand binding domain of EphB3.
  • the monoclonal antibody induces E ⁇ hB3 phosphorylation.
  • the EphB3 modulator induces EphB3 oligomerization.
  • the EphB3 modulator induces EphB3 degradation.
  • the EphB3 modulator induces EphB3 oligomerization and induces EphB3 degradation.
  • 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.
  • 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%.
  • 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.
  • 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”).
  • humanized antibodies will include both “humanized” and “veneered” antibodies.
  • 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.
  • Antibodies of the present invention may function through different mechanisms.
  • antibodies trigger antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • antibodies have multiple therapeutic functions, including, for example, antigen-binding, induction of apoptosis, and complement-dependent cellular cytotoxicity (CDC).
  • antibodies of the present invention may act as agonists of the polypeptides of the present invention.
  • the present invention provides antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • antibodies of the present invention bind an epitope disclosed herein, or a portion thereof.
  • binding of the antibody to the receptor induces receptor degradation.
  • binding of the antibody to the receptor induces receptor oligomerization.
  • binding of the antibody to the receptor induces receptor phosphorylation.
  • binding of the antibody to the receptor induces receptor activation.
  • Receptor activation i.e., signaling
  • 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.
  • 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.
  • the EphB3 antibodies stimulate EphB3 binding to intracellular adaptor proteins.
  • the EphB3 antibodies block and/or interfere with the interaction of the cytoplasmic domain of EphB3 with one or more intracellular adaptor proteins.
  • 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.
  • the EphB3 antibodies inactivate R-ras or activate Syndecan.
  • the E ⁇ hB3 antibodies lead to the phosphorylation of R-Ras.
  • intracellular adaptor proteins refers to a protein that connects different segments of a signaling complex.
  • the adaptor may or may not have enzymatic activity.
  • adaptor proteins are known to those of skill in the art.
  • Grb2 is an adaptor protein that does not have intrinsic enzymatic activity
  • RasGAP is an adaptor protein that has enzymatic activity.
  • the present invention provides activating antibodies.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • bioluminescent materials include
  • radioactive material examples include I, 131 I, or "Tc.
  • 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.
  • 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.
  • Antibody conjugates of the present invention can be used for modifying a given biological response.
  • the drug moiety may be a protein or polypeptide, or fragments thereof, possessing a desired biological activity.
  • proteins include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -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.
  • a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
  • 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.
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Antibodies of the present invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include without limitation, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • 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).
  • 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).
  • the antibody disrupts or prevents cell-cell interactions.
  • the antibody inhibits cell migration or chemotactic properties of a cell expressing EphB3.
  • 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)).
  • Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256:495-496, or a modification thereof.
  • 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.
  • the spleen and optionally, several large lymph nodes
  • 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).
  • mAb monoclonal antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-EphB3 antibody may be labeled using an indirect labeling or indirect labeling approach.
  • 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.
  • an antibody 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
  • 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.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • the binding affinity of the antibodies for EphB3 is at least 1 x 10 6 Ka. In some embodiments the binding affinity of the antibodies for EphB3 is at least 5 x 10 6 Ka, at least 1 x 10 7 Ka, at least 2 x 10 7 Ka, at least 1 x 10 8 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.
  • binding affinities include those with a Kd less than 5 x 1(T 2 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(T 8 M, 10 “8 M, 5 x 10 "9 M, 1(T 9 M, 5 x 1(T 10 M, 10 "10 M, 5 x 10 " ⁇ M, 10 " ⁇ M, 5 x 1(T 12 M, 10 "12 M, 5 x 10 "13 M 5 1(T 13 M, 5 x 1(T 14 M, 10 "14 M, 5 x 10 "15 M, or 10 "15 M, or less.
  • Suitable antibodies according to the present invention can recognize linear or conformational epitopes, or combinations thereof.
  • 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).
  • SEQ ID NO:3 the ligand binding domain
  • SEQ ID NO:4 1st fibronectin domain
  • SEQ ID NO:6 2nd fibronectin domain of EphB3
  • these peptides do not necessarily precisely map one epitope, but may also contain E ⁇ hB3 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.
  • an epitope is defined from about amino acid 80 of SEQ ID NO:2 to about amino acid 90 of SEQ ID NO:2.
  • the term “about” refers to +/- one or two amino acid residues:
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.).
  • 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.
  • EphA5 Ephrin receptor EphA5
  • EphB2 Ephrin receptor EphB2
  • EphB4 Ephrin receptor EphB4
  • antibodies may be screened against known related polypeptides to isolate an antibody population that specifically binds to EphB3 polypeptides.
  • EphB3 receptor 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.
  • EphB3 Ephrin receptor family polypeptides
  • Screening and isolation of specific antibodies is well known in the art (see, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv.
  • 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.
  • the binding affinity of an antibody of the present invention is less than about 1 x 10 5 Ka, less than about 1 x 10 4 Ka, and preferably less than 1 x 10 3 Ka, for a related polypeptide other than EphB3.
  • the E ⁇ hB3 modulator is an oligonucleotide.
  • the oligonucleotide is an antisense or RNAi oligonucleotide.
  • the oligonucleotide is complementary to a region, domain, portion, or segment of EphB3.
  • 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.
  • 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 E ⁇ hB3 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.
  • 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 5 SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO: 425.
  • the EphB3 modulator is a double stranded RNA (dsRNA) molecule and works via RNAi (RNA interference).
  • 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.
  • 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.
  • 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
  • the EphB3 modulator is a small molecule.
  • 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
  • the EphB3 modulator is a mimetic.
  • 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.
  • the EphB3 mimetic is a mimetic of EphB3 receptor or a mimetic of a ligand of EphB3 receptor.
  • the EpliB3 modulator is a soluble receptor.
  • 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.
  • the EphB3 modulator is a decoy receptor comprising at least a portion of an EphB3 receptor.
  • the decoy receptor competes with natural EphB3 receptors for EphB3 ligands.
  • the decoy receptor is labeled to facilitate quantification, qualification, and/or visualization.
  • the decoy receptor further comprises a moiety to facilitate isolation and/or separation of the decoy receptor and or the decoy receptor-EphB3 complex.
  • the decoy receptor upon binding with an EphB3 receptor ligand, causes an increased signal (compared to a native EphB3 receptor) to be effected.
  • 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.
  • 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.
  • the cancer is a cancer associated with overexpression of EphB3.
  • the cancer is colon, ovarian, esophageal or lung cancer or neuroblastoma. In some preferred embodiments the cancer is colon cancer.
  • the subject has been diagnosed as having a cancer or as being predisposed to cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the patient 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.
  • chemotherapeutic treatment is provided in combination with the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides.
  • antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotides are administered in combination with chemotherapy and radiation therapy.
  • Methods of treatment comprise administering single or multiple doses of one or more EphB3 modulators to the patient.
  • 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.
  • 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.
  • two or more different EphB3 modulators are administered to the patient.
  • 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
  • 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.
  • the methods and compositions are useful in treating and/or diagnosing cancer metastasis, including, for example, lung metastases.
  • the present invention also provides pharmaceutical compositions comprising one or more of the EphB3 modulators described herein and a pharmaceutically acceptable carrier.
  • 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.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • the present invention also provides methods for detecting EphB3.
  • the EphB3 is present in a patient or in a patient sample.
  • 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.
  • the EphB3 modulator is linked to an imaging agent or is detectably labeled.
  • 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.
  • the sample is a patient sample.
  • the patient sample comprises cancer cells.
  • the EphB3 modulator is linked to an imaging agent or is detectably labeled.
  • the present invention also provides methods for quantifying the amount of E ⁇ hB3 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.
  • 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.
  • samples believed to include tumor cells are obtained and contacted with labeled antibodies, probes, oligonucleotides, and small molecules.
  • 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.
  • PET positron emission tomography
  • the EphB3 modulator is an anti-EphB3 antibody.
  • the modulator is linked to an imaging agent or is detectably labeled.
  • the imaging agent is 18 F, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 87 MSr, 86 Y, 90 Y, 99 MTc, 111 In, 123 1, 125 1, 127 Cs, 129 Cs, 131 1, 132 1, 197 Hg, 203 Pb, Or 206 Bi.
  • Methods of detection are well known to those of skill in the art.
  • 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.
  • 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.
  • RIA radioimmunoassay
  • TR-FIA time-resolved fluoroimmunoassay
  • ICA immunochromatographic assay
  • polynucleotide expression is detected using PCR methodologies and polypeptide production is detected using ELISA technology.
  • 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.
  • 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.
  • 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.
  • 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.
  • induction of one or more of tyrosine kinase activity, receptor phosphorylation, receptor oligomerization, or receptor degradation is indicative of a cancer inhibitor.
  • inhibition of EpliB3 expression is indicative of a cancer inhibitor.
  • 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.
  • induction of one or more of tyrosine kinase activity, receptor phosphorylation, receptor oligomerization, or receptor degradation is indicative of a cancer inhibitor.
  • inhibition of EphB3 expression is indicative of a cancer inhibitor.
  • 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.
  • kits for imaging and/or detecting a gene or gene product correlated with EphB3 overexpression comprise detectable antibodies, small molecules, oligonucleotides, soluble receptors, decoy receptors, mimetics or probes as well as instructions for performing the methods of the invention.
  • 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.
  • 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 ⁇ l) 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.
  • BCA bicinchoninic acid
  • Example 2 Clustered Ligand-Induced Phosphorylation of EphB3
  • 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 ⁇ g/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 ⁇ g / lane) for Western blot analysis.
  • 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.
  • 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 -2O 0 C. 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-E ⁇ hB3 polyclonal Ab at 1:1000).
  • the primary antibody either anti-phosphotyrosine Ab 4G10 (Upstate Group, LLC, Waltham, MA) at a 1:1000 dilution, or the rabbit anti-E ⁇ hB3 polyclonal Ab at 1:1000.
  • PBST PBS containing 0.05% Tweer ⁇ O
  • HRP horseradish peroxidase
  • 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.
  • BSA bovine serum albumin
  • FBS fetal bovine serum
  • 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.
  • Example 5 EphB3 Oligonucleotides Inhibit Soft Agar Growth of SW620 Cells
  • 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.
  • a carrier molecule preferably a lipitoid or cholesteroid
  • a carrier molecule preferably a lipitoid or cholesteroid
  • the antisense or control oligonucleotide was then prepared to a working concentration of 100 ⁇ M in sterile Millipore water.
  • the oligonucleotides were further diluted in OptiMEMTM (Gibco/BRL) in a microfuge tube to 2 ⁇ M, or approximately 20 ⁇ g oligo/ml of OptiMEMTM.
  • lipitoid or cholesteroid typically in the amount of about 1.5-2 nmol lipitoid/ ⁇ g antisense oligonucleotide, was diluted in the same volume of OptiMEMTM 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.
  • 3% GTG agarose was added to the cells for a final concentration of 0.35% agarose by pipeting up and down.
  • 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.
  • the efficiency of the knock-out was determined by analyzing mRNA levels using lightcycler quantification.
  • a carrier molecule such as a lipid, lipid derivative, lipid-like molecule, cholesterol, cholesterol derivative, or cholesterol-like molecule
  • a carrier molecule such as a lipid, lipid derivative, lipid-like molecule, cholesterol, cholesterol derivative, or cholesterol-like molecule
  • the antisense and siRNA oligonucleotides were then prepared to a working concentration of about 100 ⁇ M in sterile
  • oligonucleotides were further diluted in OptiMEMTM (Gibco/BRL), in a microfuge tube, to 2 ⁇ M, or approximately 20 ⁇ g oligo/ml of OptiMEMTM.
  • the carrier molecule typically in the amount of about 1.5-2 rrmol carrier/ ⁇ g antisense oligonucleotide was diluted into the same volume of OptiMEMTM 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.
  • 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 7000TM realtime PCR machine. Values for the target mRNA were normalized versus an internal control. For each 20 ⁇ l reaction, extracted RNA (generally 0.2-1 ⁇ g total) was placed into a sterile 0.5 or 1-.5 ml microcentrifuge tube, and water added to a total volume of 12.5 ⁇ l.
  • a buffer/enzyme mixture prepared by mixing (in the order listed) 2.5 ⁇ l H 2 O, 2.0 ⁇ l 1OX reaction buffer, 10 ⁇ l oligo dT (20 pmol), 1.0 ⁇ l dNTP mix (10 mM each), 0.5 ⁇ l RNAsin® (2Ou) (Ambion, Inc., Hialeah, FL), and 0.5 ⁇ l 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.
  • 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.
  • MDA-MB-231 or MDA231 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.
  • the oligonucleotide-OptiMEMTM 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.
  • Oligonucleotides (antisense or siRNA) were prepared as described above. Cells were transfected from about 4 hours to overnight at 37 0 C and the transfection mixture was replaced with fresh medium. Transfection was carried out as described above.
  • Those oligonucleotides 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 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.
  • 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.
  • 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.
  • 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.
  • 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, Arlington, 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 ⁇ g/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.
  • the cells with the siRNA were incubated from 4 h to overnight at 37 0 C and replaced with complete media. Cells were harvested cells at 24-72 hours to monitor RNA/ protein levels.
  • Cells were seeded at 3000 to 5000 cells/well in 70 ⁇ l on 5 x 96 well flat plates. The cells were incubated at 37 0 C O/N. The cells were transfected with approximately 10OnM siRNA and 5 ⁇ M lipid using Multimek96. The mixture was allowed to incubate for 10 minutes to form the complex. 30 ⁇ l 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.
  • Soft Agar Assay (SW620; Colo320DM; HCT116 and MDA435 cells): [000240] Cells were plated in 70 ⁇ l media/ well (about 500 cells/well) on polyhema coated round bottom 96 well plate. Cells were transfected with 100 nM siRNA and 3.8 ⁇ M lipid by adding 30 ⁇ l of the complex to cells. About 50 ⁇ l/well of 1.05% agarose was added to each well and mixed well to disperse cells. 100 ⁇ l complete media was added on top of agarose and incubated at 37 0 C for 5-7 days.
  • lipid was used per set of cell line. Lipitoid compound(s) were diluted from
  • 96-well microplates were purchased from Nunc. Cell lines were obtained from
  • Chiron Master Culture Collection (Chiron Corporation) and grown at 37 0 C in 5% CO2 incubator in appropriate media supplemented with 10% FBS (Life Technologies, Rockville,
  • 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.
  • the cells attached to the plate were lysed in 200 ⁇ l 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 ⁇ l to a v-bottom plate. After a 5 minute spin at 2000 rpm to remove debris, 100 ⁇ l 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).
  • iLDH intracellular LDH
  • Dye and catalyst were stored at -2O 0 C and thawed before use in a 25 0 C waterbath.
  • the vial containing the catalyst was brought to room temperature, then ImI UF H 2 O was added and allowed to sit for lOmin before using.
  • Unused reagents were stored at 4 0 C.
  • Plates can be stored at 4 0 C 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.
  • the FBS is diluted in half by using a serum free media when used to dilute samples.
  • tLDH rLDH + iLDH
  • the ratio between released LDH and total LDH 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.

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Abstract

La présente invention a trait, entre autres, à des procédés pour le traitement du cancer, à des compositions pour le traitement du cancer, et à des procédés et des compositions pour le diagnostic et/ou la détection du cancer. En particulier, la présente invention a trait à des compositions et des procédés pour le traitement, le diagnostic et la détection des cancers associés à la surexpression de EphB3.
EP06771814A 2005-06-03 2006-06-02 Procedes de traitement, de diagnostic ou de detection du cancer Withdrawn EP1888648A2 (fr)

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JP5406027B2 (ja) * 2006-08-04 2014-02-05 ノバルティス アーゲー ＥｐｈＢ３特異的抗体およびその使用
AU2007329307B2 (en) 2006-12-07 2012-08-02 Novartis Ag Antagonist antibodies against EphB3

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WO2003087841A2 (fr) 2002-04-09 2003-10-23 Oxford Glycosciences (Uk) Ltd Proteine impliquee dans le carcinome

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US20020025536A1 (en) * 2000-06-26 2002-02-28 Jeno Gyuris Methods and reagents for isolating biologically active antibodies
CA2452578A1 (fr) * 2001-07-03 2003-01-16 The Hospital For Sick Children Immunomodulation induite par les recepteurs ephrin et eph
EP1380644A1 (fr) * 2002-07-08 2004-01-14 Kylix B.V. Utilisation de gènes cible spécifiques de TCF pour identifier des medicaments pour le traitement du cancer, en particulier le cancer colorectal, dans lequel TCF/beta-catenin/WNT signalisation joue un rôle central
CA2539651A1 (fr) * 2003-09-22 2005-04-07 Rosetta Inpharmatics Llc Ecran letal synthetique par interference arn
JP2008510008A (ja) * 2004-08-16 2008-04-03 メディミューン，インコーポレーテッド 抗体依存性細胞性細胞傷害活性が増強されたインテグリンアンタゴニスト
EP1662259A1 (fr) * 2004-11-25 2006-05-31 Cellzome Ag Utilisation des inhibiteurs du récepteur Eph pour le traitement des maladies neurodégénératives

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