Methods for determining cell responses through EphB receptors

Abstract

The present invention provides a method for initiating, promoting and/or directing cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating, promoting and directing cell attachment to a matrix or to another cell. Also provided is a method for promoting angiogenesis, comprising contacting EphB receptor-expressing cells which are associated with angiogenesis with a multimeric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting angiogenesis.

What is claimed is:  
     
         1 . A method for initiating, promoting and/or directing cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating, promoting and/or directing cell attachment to a matrix or to another cell.  
     
     
         2 . The method of  claim 1 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         3 . The method of  claim 1 , wherein the cell is selected from the group consisting of endothelial cells, neuron progenitor cells, fibroblasts, embryonic kidney cells and teratocarcinoma cells.  
     
     
         4 . A method for initiating and/or promoting cell migration, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating and/or promoting cell migration.  
     
     
         5 . The method of  claim 4 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         6 . The method of  claim 4 , wherein the cell is selected from the group consisting of endothelial cells, neuron progenitor cells, fibroblasts, embryonic kidney cells and teratocarcinoma cells.  
     
     
         7 . A method for promoting endothelialization of a prosthesis, comprising contacting the prosthesis with EphB receptor-expressing endothelial cells with a tetrameric EphB receptor-binding ligand whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting endothelialization of the prosthesis.  
     
     
         8 . The method of  claim 7 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         9 . A method for promoting expression of a molecule on the surface of an ephB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting the cell with a tetrameric Eph receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting expression of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.  
     
     
         10 . The method of  claim 9 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         11 . The method of  claim 9 , wherein the molecule is selected from the group consisting of α v β 3 , α 5 β 1 , ICAM-1 and E-selectin.  
     
     
         12 . A method for promoting function of a molecule on the surface of an ephB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting the cell with a tetrameric EphB receptor-binding ligand whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting function of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.  
     
     
         13 . The method of  claim 12 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         14 . The method of  claim 12 , wherein the molecule is selected from the group consisting of α v β 3 , α 5 β 1 , ICAM-1 and E-selectin.  
     
     
         15 . A method for promoting migration, survival and/or targeting of an EphB receptor-expressing neural cell, comprising contacting the cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting migration, survival and/or targeting of the neural cell.  
     
     
         16 . The method of  claim 15 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         17 . A method for targeting and/or promoting endothelial cell incorporation at a site of endothelial cell injury or new blood vessel formation in a subject, comprising contacting an EphB receptor-expressing endothelial cell at the site of endothelial cell injury or new blood vessel formation in the subject with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the receptor, thereby targeting and/or promoting endothelial cell incorporation at the site of endothelial cell injury or new blood vessel formation in the subject.  
     
     
         18 . The method of  claim 17 , wherein the receptor is EphB1 and the ligand is ephrin-B1.  
     
     
         19 . A method for inhibiting cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell, which is stimulated to attach to a matrix or to another cell upon binding a tetrameric Eph receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand to the EphB receptor, thereby inhibiting cell attachment to a matrix or to another cell.  
     
     
         20 . A method for inhibiting cell migration, comprising contacting an EphB receptor-expressing cell, which is stimulated to migrate upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand to the EphB receptor, thereby inhibiting cell migration.  
     
     
         21 . A method for inhibiting expression of a molecule on the surface of an EphB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, comprising contacting an EphB receptor-expressing cell, which is stimulated to express a surface molecule which affects leukocyte or platelet attachment and migration upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting expression of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.  
     
     
         22 . A method for inhibiting function of a molecule on the surface of an EphB receptor-expressing cell, wherein the molecule affects leukocyte or platelet attachment and migration, comprising contacting an EphB receptor-expressing cell, which is stimulated to promote function of a surface molecule which affects leukocyte or platelet attachment and migration upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting function of a molecule on the surface of the cell which affects leukocyte or platelet attachment and migration.  
     
     
         23 . A method for inhibiting migration, survival and/or targeting of a neural cell which expresses an EphB receptor, comprising contacting an EphB receptor-expressing neural cell, which is stimulated to promote migration, survival or targeting of the neural cell upon binding a tetrameric EphB receptor-binding ligand, with a substance which prevents binding of the tetrameric ligand, thereby inhibiting migration, survival and/or targeting of the neural cell.  
     
     
         24 . A composition comprising an isolated tetrameric EphB receptor ligand.  
     
     
         25 . A composition comprising an isolated tetrameric EphB receptor.  
     
     
         26 . A composition comprising an isolated tetrameric EphB receptor ligand/tetrameric EphB receptor complex.  
     
     
         27 . A method for screening an EphB receptor-binding ligand for the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and    b) detecting attachment of cells which have bound multimeric ligand as compared to attachment of cells which have not bound multimeric ligand, whereby attachment of cells which have bound multimeric ligand and no attachment of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form.    
     
     
         28 . A method for screening an EphB receptor-binding ligand for the ability to promote cell migration when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and    b) detecting migration of cells which have bound multimeric ligand as compared to migration of cells which have not bound multimeric ligand, whereby migration of cells which have bound multimeric ligand and no migration of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to promote cell migration when in multimeric form.    
     
     
         29 . A method for screening an EphB receptor-binding ligand for the ability to promote expression of a molecule on the surface of an EphB receptor-expressing cell, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with a cell which expresses an EphB receptor under conditions whereby the ligand can bind the receptor; and    b) detecting expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand as compared to expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand, whereby expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand and no expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand identifies a ligand with the ability to promote the expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells, when the ligand is in multimeric form.    
     
     
         30 . A method for screening an EphB receptor-binding ligand for the ability to promote function of a molecule on the surface of an EphB receptor-expressing cell, wherein the function of the molecule affects leukocyte or platelet attachment and migration, when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing cell under conditions whereby the ligand can bind the receptor; and    b) detecting function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand as compared to function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand, whereby function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have bound multimeric ligand and no function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells which have not bound multimeric ligand identifies a ligand with the ability to promote function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells, when the ligand is in multimeric form.    
     
     
         31 . A method for screening an EphB receptor-binding ligand for the ability to promote migration, survival and/or targeting of a neural cell when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with an EphB receptor-expressing neural cell under conditions whereby the ligand can bind the receptor; and    b) determining migration, survival and/or targeting of a neural cell which has bound multimeric ligand as compared to migration, survival or targeting of a neural cell which has not bound multimeric ligand, whereby migration, survival or targeting of a neural cell which has bound multimeric ligand and no migration, survival, or targeting of a neural cell which has not bound multimeric ligand identifies a ligand with the ability to promote migration, survival or targeting of a neural cell when in multimeric form.    
     
     
         32 . A method for screening an EphB receptor for the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when the receptor is in multimeric form, comprising: 
 a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and    b) detecting attachment of cells with a multimeric EphB receptor as compared to attachment of cells without a multimeric EphB receptor, whereby attachment of cells with a multimeric EphB receptor and no attachment of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to initiate, promote and/or direct cell attachment to a matrix or to another cell when in multimeric form.    
     
     
         33 . A method for screening an EphB receptor for the ability to promote cell migration when the receptor is in multimeric form, comprising: 
 a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and    b) detecting migration of cells with a multimeric EphB receptor as compared to migration of cells without a multimeric EphB receptor, whereby migration of cells with a multimeric EphB receptor and no migration of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote cell migration when in multimeric form.    
     
     
         34 . A method for screening an EphB receptor for the ability to promote expression of a molecule on the surface of an EphB receptor-expressing, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the receptor is in multimeric form, comprising: 
 a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and    b) detecting expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor as compared to expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor, whereby expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor and no expression of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote expression of a molecule on the surface of a cell which expresses an EphB receptor, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the EphB receptor is in multimeric form.    
     
     
         35 . A method for screening an Eph receptor for the ability to promote function of a molecule on the surface of an EphB receptor-expressing cell, wherein the function of the molecule affects leukocyte or platelet attachment and migration, when the receptor is in multimeric form, comprising: 
 a) producing a multimeric EphB receptor on the surface of a cell which expresses an EphB receptor; and    b) detecting function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor as compared to function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor, whereby function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells with a multimeric EphB receptor and no function of a molecule which affects leukocyte or platelet attachment and migration on the surface of cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote function of a molecule on the surface of a cell which expresses an EphB receptor, wherein the expressed molecule affects leukocyte or platelet attachment and migration, when the EphB receptor is in multimeric form.    
     
     
         36 . A method for screening an EphB receptor for the ability to promote migration, survival and/or targeting of a neural cell when the receptor is in multimeric form, comprising: 
 a) producing a multimeric EphB receptor on the surface of a neural cell which expresses an EphB receptor; and    b) detecting migration, survival or targeting of neural cells with a multimeric EphB receptor as compared to migration, survival or targeting of neural cells without a multimeric EphB receptor, whereby migration, survival and/or targeting of neural cells with a multimeric EphB receptor and no migration, survival or targeting of neural cells without a multimeric EphB receptor identifies an EphB receptor with the ability to promote migration, survival and/or targeting of neural cells when in multimeric form.    
     
     
         37 . A method for screening a substance for the ability to inhibit the binding of a multimeric EphB receptor-binding ligand to an EphB receptor comprising: 
 a) contacting the substance with a cell expressing an EphB receptor;    b) contacting the cell of step (a) with a multimeric EphB receptor-binding ligand under conditions whereby the multimeric ligand can bind the receptor; and    c) detecting the binding of the multimeric ligand to the receptor, whereby no binding of the multimeric ligand to the receptor identifies a substance with the ability to inhibit the binding of a multimeric EphB receptor-binding ligand to an EphB receptor.    
     
     
         38 . The method of  claim 38 , wherein the detection of binding of the multimeric EphB receptor-binding ligand to the EphB receptor is by detecting an activity selected from the group consisting of cell attachment to a matrix or to another cell, cell migration, expression of a surface molecule affecting leukocyte or platelet attachment and migration, promotion of function of a surface molecule affecting leukocyte or platelet attachment and migration and promotion of migration, survival or targeting of a neural cell.  
     
     
         39 . A method for promoting angiogenesis, comprising contacting EphB receptor-expressing cells which are associated with angiogenesis with a multimeric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting angiogenesis.  
     
     
         40 . A method for disrupting angiogenesis comprising contacting an EphB receptor-expressing cell which promotes angiogenesis upon binding a multimeric EphB receptor-binding ligand with a substance which prevents formation of a multimeric EphB receptor-binding ligand or inhibits binding of the multimeric EphB receptor-binding ligand to the receptor, thereby disrupting angiogenesis.  
     
     
         41 . A method for treating a disease associated with pathological angiogenesis in a subject, comprising contacting an EphB receptor-expressing cell of the subject which promotes angiogenesis upon binding a tetrameric EphB receptor-binding ligand with a substance which prevents binding of the tetrameric ligand, thereby disrupting angiogenesis and treating a disease associated with pathological angiogenesis.  
     
     
         42 . A method for treating a condition associated with interruption of angiogenic processes in a subject, comprising contacting an EphB receptor-expressing cell of the subject with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby promoting angiogenesis and treating a condition associated with interruption of angiogenic processes.  
     
     
         43 . A method for screening a substance for the ability to inhibit angiogenesis, comprising: 
 a) contacting the substance with a cell expressing an EphB receptor;    b) contacting the cell of step (a) with a multimeric EphB receptor-binding ligand, which promotes angiogenesis, under conditions whereby the multimeric ligand can bind the receptor; and    c) detecting angiogenesis in cells contacted with the substance, as compared to angiogenesis in cells not contacted with the substance, whereby no angiogenesis in cells contacted with the substance and angiogenesis in cells not contacted with the substance identifies a substance having the ability to inhibit angiogenesis.    
     
     
         44 . A method for screening an EphB-receptor binding ligand for the ability to promote angiogenesis when in multimeric form, comprising: 
 a) contacting a multimeric EphB receptor-binding ligand with a cell which expresses an EphB receptor under conditions whereby the ligand can bind the receptor; and    b) detecting angiogenesis of cells which have bound multimeric ligand as compared to angiogenesis of cells which have not bound multimeric ligand, whereby angiogenesis of cells which have bound multimeric ligand and no angiogenesis of cells which have not bound multimeric ligand identifies an EphB receptor-binding ligand with the ability to promote angiogenesis when in multimeric form.