Modulation of endothelial cell surface receptor activity in the regulation of angiogenesis

Abstract

A method of modulating angiogenesis in a vertebrate subject, the method comprising administering to the vertebrate subject an ECRTP/DEP-1 activity-modulating amount of a composition, whereby an ECRTP/DEP-1 within the vertebrate subject is contacted by the composition; and modulating angiogenesis through the contacting of the ECRTP/DEP-1 with the composition. Optionally, the composition includes a monoclonal antibody which preferentially binds ECRTP/DEP-1. Methods for screening for modulators of ECRTP/DEP-1 are also disclosed.

1 - 14 . (canceled)  
     
     
         15 . A pharmaceutical composition comprising an isolated and purified biologically active ECRTP/DEP-1 polypeptide, or amide, conjugated, cyclized, fragment, chemically modified embodiment thereof, and a pharmaceutically acceptable carrier.  
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the polypeptide further comprises an ectodomain of the ECRTP/DEP-1.  
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein the polypeptide further comprises an eight amino acid epitope of the ectodomain of the ECRTP/DEP-1, the epitope having the sequence QSRDTEVL, or an eight amino acid epitope of the ectodomain of the ECRTP/DEP-1 having an analog of the sequence QSRDTEVL.  
     
     
         18 . The pharmaceutical composition of  claim 15 , further comprising a cell expressing the ECRTP/DEP-1 polypeptide.  
     
     
         19 . A method of screening a candidate substance for an ability to modulate a receptor tyrosine phosphatase, the method comprising: 
 (a) establishing a test sample comprising a receptor tyrosine phosphatase;    (b) administering a candidate substance to the test sample; and    (c) measuring a receptor tyrosine phosphatase biological activity in the test sample;    (d) detecting phosphotyrosine residues on the receptor tyrosine phosphatase; and    (e) determining that the candidate substance modulates the receptor tyrosine phosphatase if the receptor tyrosine phosphatase biological activity measured for the test sample is greater or less than the receptor tyrosine phosphatase biological activity measured for a control sample and if the amount of phosphotyrosine residues on the receptor tyrosine phosphatase is greater or less than an amount of phosphotyrosine residues on a receptor tyrosine phosphate derived from a control sample.    
     
     
         20 . The method of  claim 19 , wherein the test and control samples further comprise a cell, and the receptor tyrosine phosphatase is expressed in the cell.  
     
     
         21 . The method of  claim 20 , wherein the test and control sample comprise cells expressing an ECRTP/DEP-1.  
     
     
         22 . The method of  claim 21 , wherein the ECRTP/DEP-1 activity is selected from the group consisting of modulation of endothelial cell migration and proliferation, modulation of density induced growth arrest, modulation of angiogenesis and combinations thereof.  
     
     
         23 . The method of  claim 19 , wherein the candidate substance further comprises a cell or cell lysate comprising a natural ligand for the receptor tyrosine phosphatase, and the method further comprises isolating the natural ligand for the receptor tyrosine phosphatase.  
     
     
         24 . The method of  claim 23 , wherein the receptor tyrosine phosphatase comprises the ECRTP/DEP-1.  
     
     
         25 . The method of  claim 24 , wherein the ligand is isolated by lysing the cells and passing the cell lysate over a column containing the ECRTP/DEP-1 bound to a solid phase matrix within the column.  
     
     
         26 . The method of  claim 24 , wherein the ligand is isolated by constructing a cDNA library from the cells binding the ligand; transfecting the cDNA library into a cell line that does not exhibit binding of the ligand; screening the cell line for newly acquired specific binding; isolating DNA form cells exhibiting specific binding; and sequencing the isolated DNA to determine the DNA sequence for the ligand.  
     
     
         27 . A recombinant cell line suitable for use in the assay of  claim 19 .  
     
     
         28 . A method of screening a candidate substance for an ability to modulate ECRTP/DEP-1 biological activity, the method comprising: 
 (a) establishing a test sample comprising an ECRTP/DEP-1 polypeptide or fragment thereof;    (b) administering a candidate substance to the test sample; and    (c) measuring an interaction, effect, or combination thereof, of the candidate substance on the test sample to thereby determine the ability of the candidate substance to modulate ECRTP/DEP-1 biological activity.    
     
     
         29 . The method of  claim 28 , wherein the test sample further comprises a cell expressing ECRTP/DEP-1, and wherein the step of measuring an interaction, effect, or combination thereof, of the candidate substance on the test sample further comprises: 
 (i) comparing the interaction, effect, or combination thereof, of the candidate substance on the test sample with the interaction, effect, or combination thereof, of the candidate substance on a cell not expressing ECRTP/DEP-1; and    (ii) determining that candidate compound modulates ECRTP/DEP-1 activity by demonstrate a lack of interaction, effect or combination thereof, of the candidate compound on cells not expressing ECRTP/DEP-1.    
     
     
         30 . The method of  claim 28 , wherein said step of measuring an interaction, effect, or combination thereof, of the candidate substance on the test sample further comprises measuring binding between the candidate substance and the test sample by: 
 (i) contacting the candidate substance with an ECRTP/DEP-1 polypeptide or fragment thereof under conditions favorable to binding the candidate with an ECRTP/DEP-1 polypeptide or fragment thereof to form a complex therebetween; and    (ii) detecting the complex.    
     
     
         31 . The method of  claim 30 , wherein the complex is detected via a label conjugated to the ECRTP/DEP-1 polypeptide or fragment thereof; via a labeled reagent that specifically binds to the complex subsequent to its formation; or via a competition assay with a substance known to bind the ECRTP/DEP-1 polypeptide for fragment thereof.  
     
     
         32 . The method of  claim 30 , wherein the ECRTP/DEP-1 polypeptide or fragment thereof is conjugated with a detectable label.  
     
     
         33 . The method of  claim 32 , wherein the step of detecting the complex further comprises: 
 (i) separating the complex from unbound labeled ECRTP/DEP-1 polypeptide or fragment thereof; and    (ii) detecting the detectable label which is present in the complex or which is unbound.    
     
     
         34 . The method of  claim 30 , wherein the ECRTP/DEP-1 polypeptide fragment is a ECRTP/DEP-1 ectodomain fragment.  
     
     
         35 . The method of  claim 34 , wherein the ECRTP/DEP-1 ectodomain fragment comprises an eight amino acid epitope having the sequence n-QSRDTEVL-c.  
     
     
         36 . The method of  claim 30 , wherein the candidate substance is an antibody, or derivative or fragment thereof.  
     
     
         37 . The method of  claim 36 , wherein the candidate antibody, or derivative or fragment thereof, is derived from a recombinant phage-displayed antibody library.  
     
     
         38 . A kit for use screening a candidate substance for an ability to modulate ECRTP/DEP-1 biological activity, the kit comprising a ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, contained in a first container.  
     
     
         39 . The kit of  claim 38 , wherein the ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, comprises an eight amino acid epitope having the sequence n-QSRDTEVL-c.  
     
     
         40 . The kit of  claim 38 , further comprising a solid phase support.  
     
     
         41 . The kit of  claim 40 , where the ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, is immobilized to the solid phase support.  
     
     
         42 . The kit of  claim 38 , further comprising a detectable label.  
     
     
         43 . The kit of  claim 41 , wherein the detectable label is contained in another container or wherein ECRTP/DEP-1 ectodomain polypeptide, or fragment thereof, comprises the detectable label.  
     
     
         44 . The kit of  claim 43 , wherein the detectable label is a radioactive label or an enzyme.  
     
     
         45 . An isolated antibody, or a fragment or derivative thereof, which specifically binds to an epitope present within amino acids 175-536 of a human ECRTP/DEP-1 polypeptide, wherein the human ECRTP/DEP-1 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 4.  
     
     
         46 . The isolated antibody fragment of  claim 45 , wherein the isolated antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2  fragment, an F(v) fragment, and an single chain fragment variable (scFv) fragment.  
     
     
         47 . The isolated antibody, or the fragment or derivative thereof, of  claim 45 , wherein the isolated antibody comprises a monoclonal antibody, or a fragment or derivative thereof.  
     
     
         48 . The isolated antibody, or the fragment or derivative thereof, of  claim 47 , wherein the isolated antibody is humanized.  
     
     
         49 . The isolated antibody, or the fragment or derivative thereof, of  claim 45 , which binds an eight amino acid epitope consisting of the sequence QSRDTEVL (SEQ ID NO: 1).  
     
     
         50 . The isolated antibody, or the fragment or derivative thereof, of  claim 49 , wherein the isolated antibody, or the fragment or derivative thereof, is a monoclonal antibody, or a fragment or derivative thereof.  
     
     
         51 . The isolated antibody, or the fragment or derivative thereof, of  claim 50 , wherein the antibody, or the fragment or derivative thereof, is human or humanized.  
     
     
         52 . The isolated antibody, or the fragment or derivative thereof, of  claim 45 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         53 . The isolated antibody, or the fragment or derivative thereof, of  claim 52 , wherein the pharmaceutically acceptable diluent or excipient is pharmaceutically acceptable for use in humans.  
     
     
         54 . An isolated antibody, or a fragment or derivative thereof, which specifically binds to an epitope of an ECRTP/DEP-1 polypeptide extracellular domain, wherein the ECRTP/DEP-1 polypeptide extracellular domain comprises amino acids 175-536 of SEQ ID NO: 4 and the epitope comprises SEQ ID NO: 1.  
     
     
         55 . The isolated antibody fragment of  claim 54 , wherein the isolated antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2  fragment, an F(v) fragment, and an single chain fragment variable (scFv) fragment.  
     
     
         56 . The isolated antibody, or the fragment or derivative thereof, of  claim 54 , wherein the isolated antibody is humanized.  
     
     
         57 . The isolated antibody, or the fragment or derivative thereof, of  claim 54 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         58 . The isolated antibody, or the fragment or derivative thereof, of  claim 57 , wherein the pharmaceutically acceptable diluent or excipient is pharmaceutically acceptable for use in humans.  
     
     
         59 . An isolated antibody, or a fragment or derivative thereof, which specifically binds an extracellular domain of an ECRTP/DEP-1 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4 and wherein the antibody, fragment, or derivative thereof has activity in modulating angiogenesis.  
     
     
         60 . The isolated antibody, or the fragment or derivative thereof, of  claim 59 , or a fragment or derivative thereof, wherein the isolated antibody, fragment, or derivative thereof has activity in modulating angiogenesis in an assay selected from the group consisting of a planar endothelial migration assay, an in situ transfection assay for migration, a cornea pocket angiogenesis assay, a chick chorioallantoic membrane assay, a proliferation assay, and an endothelial wound closure assay.  
     
     
         61 . The antibody fragment, or the fragment or derivative thereof, of  claim 59 , wherein the antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2  fragment, an F(v) fragment, and an single chain fragment variable (scFv) fragment.  
     
     
         62 . The isolated antibody, or the fragment or derivative thereof, of  claim 59 , wherein the isolated antibody is a monoclonal antibody, or a fragment or derivative thereof.  
     
     
         63 . The isolated antibody, or the fragment or derivative thereof, of  claim 62 , wherein the isolated antibody is humanized.  
     
     
         64 . The isolated antibody, or the fragment or derivative thereof, of  claim 59 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         65 . The isolated antibody, or the fragment or derivative thereof, of  claim 64 , wherein the pharmaceutically acceptable diluent or excipient is pharmaceutically acceptable for use in humans.  
     
     
         66 . The isolated antibody, or the fragment or derivative thereof, of  claim 59 , wherein the isolated antibody has a binding specificity of a monoclonal antibody produced by a hybridoma cell line having American Type Culture Collection (ATCC) accession number HB12570.  
     
     
         67 . The isolated antibody, or the fragment or derivative thereof, of  claim 59 , or a fragment or derivative thereof, wherein the activity in modulating angiogenesis is inhibition of angiogenesis.  
     
     
         68 . An isolated antibody, or a fragment or derivative thereof, which specifically binds an epitope present within amino acids 175-536 of a human ECRTP/DEP-1 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4, wherein the isolated antibody, or the fragment or derivative thereof, has activity in modulating angiogenesis.  
     
     
         69 . The isolated antibody, or the fragment or derivative thereof, of  claim 68 , wherein the isolated antibody, or the fragment or derivative thereof, has activity in modulating angiogenesis in an assay selected from the group consisting of a planar endothelial migration assay, an in situ transfection assay for migration, a cornea pocket angiogenesis assay, a chick chorioallantoic membrane assay, a proliferation assay, and an endothelial wound closure assay.  
     
     
         70 . The isolated antibody fragment of  claim 68 , wherein the isolated antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2  fragment, an F(v) fragment, and an single chain fragment variable (scFv) fragment.  
     
     
         71 . The isolated antibody, or the fragment or derivative thereof, of  claim 68 , wherein the isolated antibody is a monoclonal antibody, or a fragment or derivative thereof.  
     
     
         72 . The isolated antibody, or the fragment or derivative thereof, of  claim 71 , wherein the isolated antibody is humanized.  
     
     
         73 . The isolated antibody, or the fragment or derivative thereof, of  claim 68 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         74 . The isolated antibody, or the fragment or derivative thereof, of  claim 73 , wherein the pharmaceutically acceptable diluent or excipient is pharmaceutically acceptable for use in humans.  
     
     
         75 . The isolated antibody, or the fragment or derivative thereof, of  claim 68 , or a fragment or derivative thereof, wherein the activity in modulating angiogenesis is inhibition of angiogenesis.  
     
     
         76 . An isolated antibody, or a fragment or derivative thereof, which specifically binds to an epitope of an ECRTP/DEP-1 polypeptide extracellular domain, the epitope comprising SEQ ID NO: 1, wherein the antibody, fragment, or derivative thereof has activity in modulating angiogenesis.  
     
     
         77 . The isolated antibody, or the fragment or derivative thereof, of  claim 76 , wherein the antibody, fragment, or derivative thereof has activity in modulating angiogenesis in an assay selected from the group consisting of a planar endothelial migration assay, an in situ transfection assay for migration, a cornea pocket angiogenesis assay, a chick chorioallantoic membrane assay, a proliferation assay, and an endothelial wound closure assay.  
     
     
         78 . The antibody fragment of  claim 77 , wherein the antibody fragment is selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′) 2  fragment, an F(v) fragment, and an single chain fragment variable (scFv) fragment.  
     
     
         79 . The isolated antibody, or the fragment or derivative thereof, of  claim 76 , wherein the isolated antibody is a monoclonal antibody or a fragment or derivative thereof.  
     
     
         80 . The isolated antibody, or the fragment or derivative thereof, of  claim 79 , wherein the antibody is humanized.  
     
     
         81 . The isolated antibody, or the fragment or derivative thereof, of  claim 76 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         82 . The isolated antibody, or the fragment or derivative thereof, of  claim 81 , wherein the pharmaceutically acceptable diluent or excipient is pharmaceutically acceptable for use in humans.  
     
     
         83 . The isolated antibody, or the fragment or derivative thereof, of  claim 76 , or a fragment or derivative thereof, wherein the activity in modulating angiogenesis is inhibition of angiogenesis.  
     
     
         84 . A composition for modulating angiogenesis, the composition comprising: 
 (a) a therapeutically effective amount of an isolated antibody, or a fragment or derivative thereof, which binds to an eight amino acid epitope consisting of SEQ ID NO: 1 present within a human ECRTP/DEP-1 density enhanced phosphatase-1 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4; and    (b) a diluent or excipient pharmaceutically acceptable in humans.    
     
     
         85 . The composition of  claim 84 , wherein the antibody, or the fragment or derivative thereof, is human or humanized.