Igfbp-3 derivatives and uses thereof

Abstract

The present invention provides polypeptide derivatives of IGFBP-3 that are resistant to proteolytic cleavage. These IGFBP-3 derivatives are useful in a variety of therapeutic and diagnostic applications. Also provided are pharmaceutical compositions and kits comprising such IGFBP-3 derivatives and methods for using these derivatives for the treatment of a variety of disorders.

1 . An IGFBP-3 polypeptide derivative comprising an N-terminal domain, an intermediary domain and a C-terminal domain, wherein:
 the N-terminal domain comprises the amino acid sequence of the N-terminal domain of wild-type IGFBP-3, of a biologically active variant thereof or of a biologically active fragment thereof;   the intermediary domain comprises a linker resistant to proteolytic cleavage; and   the C-terminal domain comprises the amino acid sequence of the C-terminal domain of wild-type IGFBP-3, of a biologically active variant thereof or of a biologically active fragment thereof.   
     
     
         2 . The IGFBP-3 polypeptide derivative according to  claim 1 , wherein:
 the intermediary domain of the IGFBP-3 polypeptide derivative comprises the amino acid sequence of the intermediary domain of wild-type IGFBP-3, wherein a portion of said amino acid sequence is replaced by the linker resistant to proteolytic cleavage.   
     
     
         3 . The IGFBP-3 polypeptide derivative according to  claim 1 , wherein IGFBP-3 is human IGFBP-3 and wherein:
 the amino acid sequence of the N-terminal domain of wild-type IGFBP-3 is as set forth in SEQ ID NO: 1;   the amino acid sequence of the intermediary domain of wild-type IGFBP-3 is as set forth in SEQ ID NO: 2; and   the amino acid sequence of the C-terminal domain of wild-type IGFBP-3 is as set forth in SEQ ID NO: 3.   
     
     
         4 . The IGFBP-3 polypeptide derivative according to  claim 1 , wherein the linker has the sequence set forth in SEQ ID NO: 4 or any variant thereof that is resistant to proteolytic cleavage. 
     
     
         5 . The IGFBP-3 polypeptide derivative according to  claim 3 , wherein amino acid residues 43 to 47 in SEQ ID NO: 3 are replaced with AGGSG (SEQ ID NO: 5) or any variant thereof that does not bind ALS (Acid Labile Subunit). 
     
     
         6 . The IGFBP-3 polypeptide derivative according to  claim 1  further comprising, fused thereto, the immunoglobulin IgG1 Fc fragment. 
     
     
         7 . The IGFBP-3 polypeptide derivative according to  claim 1  further comprising the amino acid sequence of IGF-I, wherein the IGF-I is complexed to the IGFBP-3 polypeptide derivative. 
     
     
         8 . The IGFBP-3 polypeptide derivative according to  claim 1  further comprising a BirA enzyme substrate covalently bound to the terminal end of the C-terminal domain of the IGFBP-3 polypeptide derivative. 
     
     
         9 . The IGFBP-3 polypeptide derivative according to  claim 8 , wherein the BirA enzyme substrate has the sequence set forth in SEQ ID NO: 6. 
     
     
         10 . The IGFBP-3 polypeptide derivative according to  claim 8  further comprising biotin covalently bound to the BirA enzyme substrate. 
     
     
         11 . The IGFBP-3 polypeptide derivative according to  claim 1  further comprising, fused thereto, the amino acid sequence of SeAP (secreted alkaline phosphatase). 
     
     
         12 . A method for treating a disorder selected from cancers and proliferative retinopathies in a subject, the method comprising a step of administering to the subject an effective amount of an IGFBP-3 polypeptide derivative according to  claim 1 . 
     
     
         13 . A method for treating a disorder in a subject, the method comprising administering to the subject an effective amount of an IGFBP-3 polypeptide derivative according to  claim 7 , wherein the disorder is selected from the group consisting of growth hormone resistance, IGF-I deficiency, severe burns, HIV wasting, cystic fibrosis, celiac disease, anorexia nervosa, muscle wasting disease, myotonic dystrophy, amyotrophic lateral sclerosis, osteoporosis, severe insulin resistance, type I diabetes, type II diabetes, brain ischemia, heart ischemia, and grafts. 
     
     
         14 . A pharmaceutical composition comprising a therapeutically effective amount of at least one IGFBP-3 polypeptide derivative according to  claim 1  and a pharmaceutically acceptable carrier or excipient. 
     
     
         15 . A method for determining pro-IGF-II concentration in a biological sample, the method comprising steps of:
 contacting the biological sample with an IGFBP-3 polypeptide derivative according to  claim 11  so as to allow formation of a complex between the IGFBP-3 polypeptide derivative and any pro-IGF-II present in the biological sample, wherein pro-IGF-II is a partially processed form of IGF-II; and   determining the concentration of pro-IGF-II in the biological sample by measuring the alkaline activity of SeAP in the complex.   
     
     
         16 . A kit comprising an IGFBP-3 polypeptide derivative according to  claim 11  and at least one reagent to measure alkaline activity. 
     
     
         17 . The IGFBP-3 polypeptide derivative according to  claim 4 , wherein amino acid residues 43 to 47 in SEQ ID NO: 3 are replaced with AGGSG (SEQ ID NO: 5) or any variant thereof that does not bind ALS (Acid Labile Subunit). 
     
     
         18 . The IGFBP-3 polypeptide derivative according to  claim 9  further comprising biotin covalently bound to the BirA enzyme substrate. 
     
     
         19 . A pharmaceutical composition comprising a therapeutically effective amount of at least one IGFBP-3 polypeptide derivative according to  claim 7  and a pharmaceutically acceptable carrier or excipient.