Altered insulin-like growth factor binding proteins

Abstract

Altered IGFBPs are able to bind IGF, but the release is inhibited by resistance to protease cleavage and/or reduced binding to extracellular matrix (ECM). Alterations have been made in IGFBP-2 to the linker domain in particular and to two amino acid motifs found to be important for ECM binding. IGF-1 mediated proliferation of cancer cells have been inhibited by use of the altered IGFBPs.

1 . A human IGFBP molecule able to bind IGF-I or IGF-II with high affinity, wherein one or more amino acids of the human IGFBP molecule in one or more extracellular matrix (ECM) binding sites and in one or more protease cleavage sites are substituted deleted or inversed, relative to the amino acid sequence of native human IGFBP, and wherein the IGFBP molecule exhibits inhibited release of IGF upon contact with extracellular matrix and exposure to a protease, relative to native human IGFBP. 
     
     
         2 . The human IGFBP molecule of  claim 1 , wherein the IGFBP is selected from the group consisting of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. 
     
     
         3 . The human IGFBP molecule of  claim 2 , wherein the IGFBP is IGFBP-2. 
     
     
         4 . The human IGFBP molecule of  claim 3 , wherein one or more amino acids in a first ECM binding site corresponding to positions 179-184 of native IGFBP-2 comprising the sequence PKKLRP (SEQ ID NO: 1) are deleted, inversed or substituted, relative to the amino acid sequence of native human IGFBP-2. 
     
     
         5 . The human IGFBP molecule of  claim 4 , wherein the first ECM binding site of the IGFBP molecule comprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. 
     
     
         6 . The human IGFBP molecule of  claim 3 , wherein one or more amino acids in a second ECM binding site corresponding to positions 227-244 of native IGFBP-2 comprising the sequence KHGLYNLKQCKMSLNGQR (SEQ ID NO:2) are substituted, relative to the amino acid sequence of native human IGFBP-2. 
     
     
         7 . The human IGFBP molecule of  claim 6 , wherein the second ECM binding site of the IGFBP molecule comprises a sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. 
     
     
         8 . The human IGFBP molecule of  claim 3 , wherein the IGFBP-2 molecule further comprises one or more amino acid deletions, relative to the amino acid sequence of native human IGFBP-2, that enhances resistance to proteolysis by one or more proteases. 
     
     
         9 . The human IGFBP molecule of  claim 3 , wherein one or more amino acids in the linker domain are deleted, relative to the amino acid sequence of native human IGFBP-2. 
     
     
         10 . The human IGFBP molecule of  claim 3 , wherein substantially all of the linker domain has been deleted, relative to the amino acid sequence of native human IGFBP-2. 
     
     
         11 . The human IGFBP molecule of  claim 2 , wherein the IGFBP molecule is IGFBP-1, and wherein one or more amino acids in the ECM binding site corresponding to the sequence CNKNGFYHSRQCETSMDGEAGLC (SEQ ID NO:6) of native human IGFPB-1 are deleted, inversed or substituted, relative to the amino acid sequence of native human IGFBP-1. 
     
     
         12 . The human IGFBP molecule of  claim 2 , wherein the IGFBP molecule is IGFBP-4, and wherein one or more amino acids in the ECM binding site corresponding to the sequence CDRNGNFHPKQCHPALDGQRGKC (SEQ ID NO:7) of native human IGFBP-4 are deleted, inversed or substituted, relative to the amino acid sequence of native human IGFBP-4. 
     
     
         13 . The human IGFBP molecule of  claim 2 , wherein the IGFBP molecule is IGFBP-6, and wherein one or more amino acids in the ECM binding site corresponding to the sequence CDHRGFYRKRQCRSSQGQRRGPC (SEQ ID NO: 8) of native human IGFBP-6 are deleted, inversed or substituted, relative to the amino acid sequence of native human IGFBP-6. 
     
     
         14 . A nucleic acid encoding a human IGFBP molecule able to bind IGF-I or IGF-II with high affinity, wherein one or more amino acids of the human IGFBP molecule in one or more extracellular matrix (ECM) binding sites and in one or more protease cleavage sites are substituted, deleted or inversed, relative to the amino acid sequence of native human IGFBP, and wherein the IGFBP molecule exhibits inhibited release of IGF upon contact with extracellular matrix and exposure to a protease, relative to native human IGFBP. 
     
     
         15 . A host cell transformed with the nucleic acid of  claim 14 . 
     
     
         16 . A pharmaceutical composition comprising (a) a human IGFBP molecule able to bind IGF-I or IGF-II with high affinity, wherein one or more amino acids of the IGFBP molecule in one or more extracellular matrix (ECM) binding sites and in one or more protease cleavage sites are substituted, deleted or inversed, relative to the amino acid sequence of native human IGFBP, and wherein the IGFBP molecule exhibits inhibited release of IGF upon contact with extracellular matrix and exposure to a protease, relative to native human IGFBP; and (b) a carrier. 
     
     
         17 . A method of treating a patient having cancer comprising administering to said patient a pharmaceutical composition comprising (a) a human IGFBP molecule able to bind IGF-I or IGF-II with high affinity, wherein one or more amino acids of the IGFBP molecule in one or more extracellular matrix (ECM) binding sites and in one or more protease cleavage sites are substituted, deleted or inversed, relative to the amino acid sequence of native human IGFBP, and wherein the IGFBP molecule exhibits inhibited release of IGF upon contact with extracellular matrix and exposure to a protease, relative to native human IGFBP; and (b) a carrier. 
     
     
         18 . The method of  claim 17 , wherein the cancer is selected from the group consisting of prostate cancer, colon cancer and breast cancer. 
     
     
         19 . A method of reducing IGF mediated proliferation of cancerous cells, comprising the step of contacting said cells with a human IGFBP molecule able to bind IGF-I or IGF-H with high affinity, wherein one or more amino acids of the IGFBP molecule in one or more extracellular matrix (ECM) binding sites and in one or more protease cleavage sites are substituted, deleted or inversed, relative to the amino acid sequence of native human IGFBP, and wherein the IGFBP molecule exhibits inhibited release of IGF upon contact with extracellular matrix and exposure to a protease, relative to native human IGFBP. 
     
     
         20 . The method of  claim 19 , wherein the cancerous cells are selected from the group consisting of prostate cancer cells, colon cancer cells and breast cancer cells.