Growth Factor Binding Constructs Materials and Methods

Abstract

The present invention provides materials and methods for antagonizing the function of vascular endothelial growth factor receptors, platelet derived growth factor receptors and other receptors. Soluble binding constructs able to bind vascular endothelial growth factors, platelet derived growth factors, and other ligands are provided.

1 . A purified polypeptide comprising an amino acid sequence at least 95% identical to a vascular endothelial growth factor C (VEGF-C) binding construct, wherein the VEGF-C binding construct comprises at least three linked immunoglobulin-like domains from VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 including, in order:
 (i) a VEGFR-3 fragment that includes VEGFR-3 immunoglobulin-like domain I amino acids 47-115 of SEQ ID NO: 6;   (ii) a VEGFR-2 fragment that includes VEGFR-2 immunoglobulin-like domain II amino acids 145-203 of SEQ ID NO: 4; and   (iii) a VEGFR fragment that includes immunoglobulin-like domain III amino acids 248-315 of SEQ ID NO: 2 (VEGFR-1) or amino acids 241-310 of SEQ ID NO: 4 (VEGFR-2);   and wherein the polypeptide binds VEGF-C or VEGF-D.   
     
     
         2 . The polypeptide according to  claim 1 , wherein the VEGFR-3 fragment has an amino terminal amino acid selected from the group consisting of positions of 1 to 47 of SEQ ID NO: 6. 
     
     
         3 . The polypeptide according to  claim 1 , wherein the polypeptide further comprises a heterologous peptide connected to the fragment. 
     
     
         4 . The polypeptide according to  claim 3 , wherein the heterologous peptide comprises an immunoglobulin constant domain fragment. 
     
     
         5 . The polypeptide according to  claim 4 , wherein the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 125 and 128. 
     
     
         6 . The polypeptide according to  claim 1 , wherein the polypeptide further comprises a signal peptide. 
     
     
         7 . The polypeptide according to  claim 6 , wherein the signal peptide directs secretion of the polypeptide from a cell that expresses the polypeptide. 
     
     
         8 . The polypeptide according to  claim 1 , wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS: 125 and 128. 
     
     
         9 . A binding construct comprising the polypeptide according to  claim 1  operatively connected with a second polypeptide that binds at least one growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, and PDGF-D, wherein the second polypeptide is selected from the group consisting of a polypeptide comprising a platelet derived growth factor receptor extracellular domain fragment, and a polypeptide comprising an antigen binding fragment of an antibody that immunoreacts with the at least one of said growth factors. 
     
     
         10 . The polypeptide according to  claim 1 , wherein at least one of the VEGFR fragments further include sequence from the VEGFR adjacent to the VEGFR immunoglobulin-like domain. 
     
     
         11 . The binding construct of  claim 9 , further comprising a linker connecting the first and second polypeptides. 
     
     
         12 . The binding construct of  claim 11 , wherein the linker comprises a peptide that links the first and second polypeptides to form a single polypeptide. 
     
     
         13 . The binding construct of  claim 9 , further comprising a signal peptide operatively linked to the single polypeptide. 
     
     
         14 . A composition comprising a polypeptide according to  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         15 . A purified or isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of  claim 1 . 
     
     
         16 . The polynucleotide according to  claim 15 , wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 124 and 127. 
     
     
         17 . The polynucleotide according to  claim 15 , wherein the polynucleotide further comprises a promoter sequence operatively connected to a nucleotide sequence encoding the polypeptide, wherein the promoter sequence promotes transcription of the sequence that encodes the polypeptide in a host cell. 
     
     
         18 . A polynucleotide according to  claim 15 , wherein the polynucleotide further comprises a polyadenylation sequence operatively connected to the sequence that encodes the polypeptide. 
     
     
         19 . A vector comprising the polynucleotide of  claim 15 . 
     
     
         20 . The vector according to  claim 19 , wherein the vector comprises an expression control sequence operatively connected to the sequence that encodes the polypeptide. 
     
     
         21 . The vector according to  claim 20  wherein said vector is selected from the group consisting of a lentivirus vector, an adeno-associated viral vector, an adenoviral vector, a liposomal vector, and combinations thereof. 
     
     
         22 . The vector according to  claim 20  wherein said vector comprises a replication-deficient adenovirus, said adenovirus comprising the polynucleotide operatively connected to a promoter and flanked by adenoviral polynucleotide sequences. 
     
     
         23 . A composition comprising a polynucleotide according to  claim 15 , or comprising a vector that contains the polynucleotide, and a pharmaceutically acceptable carrier. 
     
     
         24 . A host cell comprising the polynucleotide according to  claim 15 , or comprising a vector that contains the polynucleotide. 
     
     
         25 . The polypeptide according to  claim 1  wherein the amino terminal amino acid of the VEGFR-2 fragment is selected from the group consisting of positions 106-145 of SEQ ID NO: 4, and wherein the carboxy terminal amino acid of the VEGFR-2 fragment is selected from the group consisting of positions 203 to 240 of SEQ ID NO: 4. 
     
     
         26 - 30 . (canceled) 
     
     
         31 . The polypeptide according to  claim 25 , wherein the VEGFR-2 fragment consists of an amino acid sequence selected from the group consisting of residues 118-220, 118-226, and 118-232 of SEQ ID NO: 4. 
     
     
         32 - 54 . (canceled) 
     
     
         55 . A binding construct comprising:
 a) a first amino acid sequence at least 90% identical to a fragment of the VEGFR-3 extracellular domain, wherein said fragment comprises VEGFR-3 immunoglobulin-like domain 1 amino acid sequence;   (b) a second amino acid sequence at least 90% identical to a fragment of the VEGFR-2 extracellular domain, wherein the fragment comprises immunoglobulin-like domain 2 amino acid sequence; and,   (c) a third amino acid sequence at least 90% identical to a fragment of the VEGFR-1 extracellular domain, wherein the fragment comprises immunoglobulin-like domain 3 amino acid sequence;   wherein the first, second, and third amino acid sequences are operatively connected, and   wherein the binding construct binds to at least VEGF-A and VEGF-C.   
     
     
         56 . The binding construct of  claim 55 , wherein the binding construct is a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set out in SEQ ID NO: 128. 
     
     
         57 . The binding construct of  claim 56 , comprising the amino acid sequence of SEQ ID NO: 128. 
     
     
         58 . The binding construct of  claim 56 , further comprising a signal peptide operatively linked to the polypeptide. 
     
     
         59 . A composition comprising a binding construct according to  claim 55 , in a pharmaceutically acceptable carrier. 
     
     
         60 . A purified or isolated polynucleotide comprising a nucleotide sequence encoding the binding construct of  claim 55 . 
     
     
         61 . The polynucleotide according to  claim 60 , wherein the polynucleotide further comprises a promoter sequence operatively connected to the nucleotide sequence encoding the polynucleotide, wherein the promoter sequence promotes transcription of the sequence that encodes the binding construct in a host cell transformed or transfected with the polynucleotide. 
     
     
         62 . A polynucleotide according to  claim 60 , wherein the polynucleotide further comprises a polyadenylation sequence operatively connected to the sequence that encodes the binding construct. 
     
     
         63 . A vector comprising the polynucleotide of  claim 60 . 
     
     
         64 . The vector according to  claim 63  wherein the vector comprises an expression control sequence operatively connected to the sequence that encodes the binding construct. 
     
     
         65 . The vector according to  claim 64 , wherein said vector is selected from the group consisting of a lentivirus vector, an adeno-associated viral vector, an adenoviral vector, a liposomal vector, and combinations thereof. 
     
     
         66 . The vector according to  claim 65  wherein said vector comprises a replication-deficient adenovirus, said adenovirus comprising the polynucleotide operatively connected to a promoter and flanked by adenoviral polynucleotide sequences. 
     
     
         67 . A composition comprising a polynucleotide according to  claim 60 , or a vector comprising the polynucleotide and a pharmaceutically acceptable carrier. 
     
     
         68 . A host cell comprising the polynucleotide according to  claim 60 , or comprising a vector comprising the polynucleotide. 
     
     
         69 - 71 . (canceled) 
     
     
         72 . A method of inhibiting endothelial or smooth muscle cell proliferation in a mammal, comprising administering to a mammal a composition, said composition comprising:
 a polypeptide according to  claim 1 ,   a polynucleotide comprising a nucleotide sequence encoding the polypeptide, or   a vector containing the polynucleotide,   in an amount effective to inhibit endothelial cell proliferation in the mammal.   
     
     
         73 . The method according to  claim 72  wherein said composition further comprises a pharmaceutically acceptable diluent, adjuvant, or carrier medium. 
     
     
         74 . The method according to  claim 73  wherein said mammal is human. 
     
     
         75 . The method according to  claim 72  wherein said mammal has a neoplastic disease characterized by endothelial or smooth muscle cell growth. 
     
     
         76 . The method according to  claim 75  wherein said neoplastic disease is selected from the group consisting of carcinomas, squamous cell carcinomas, lymphomas, melanomas, and sarcomas. 
     
     
         77 . The method of  claim 75 , where the composition is administered in an amount effective to inhibit endothelial cell growth and thereby inhibit progression of the neoplastic disease. 
     
     
         78 . The method according to  claim 75 , wherein the composition is administered in an amount effective to inhibit tumor growth or metastasis. 
     
     
         79 . A method of inhibiting endothelial or smooth muscle cell growth comprising steps of:
 (a) screening a mammal to identify a neoplastic disorder characterized by endothelial cell proliferation; and   (b) administering a composition to the mammal identified according to step (a) as having a neoplastic disorder characterized by endothelial cell proliferation, wherein said composition comprises:   a polypeptide according to  claim 1 ,   a polynucleotide comprising a nucleotide sequence encoding the polypeptide, or   a vector containing the polynucleotide,   in an amount effective to inhibit endothelial or smooth muscle cell proliferation in said mammal.   
     
     
         80 . The method according to  claim 79 , wherein the mammal is human. 
     
     
         81 . The method according to  claim 80 , wherein said composition further comprises a pharmaceutically acceptable diluent, adjuvant, or carrier. 
     
     
         82 . The method according to  claim 79 , wherein the screening step comprises screening the mammal for elevated serum levels of at least one growth factor selected from the group consisting of VEGF-A, VEGF-C, and VEGF-D. 
     
     
         83 . The method according to  claim 79 , wherein the screening step comprises obtaining a tissue sample from the tumor and detecting elevated levels of at least one growth factor selected from the group consisting of VEGF-A, VEGF-C, and VEGF-D, or elevated levels of at least one receptor capable of binding the at least one growth factor. 
     
     
         84 . (canceled)