Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

Abstract

The present invention relates to a fusion protein comprising IGF-I or an IGF-I variant N-terminally linked to the C-terminus of a propeptide. The invention relates also to a method involving the use of the aforementioned fusion protein in the production of a lysine-PEGylated IGF-I or IGF-I variant. The method comprises the steps of cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding the fusion protein and causing the cell to express the fusion protein, recovering and PEGylating said fusion protein, cleaving said PEGylated fusion protein with IgA protease, and recovering lysine-PEGylated IGF-I or IGF-I variant. The invention relates also to a lysine-PEGylated IGF-I or IGF-I variant produced using the above method. In addition, the invention relates to a method for treating a neurodegenerative disorders like Alzheimer's Disease using the lysine-PEGylated IGF-I or IGF-I variant and a composition comprising the lysine-PEGylated IGF-I or IGF-I variant.

1 . A method for treating a patient suffering from Alzheimer's disease comprising administering to said patient a pharmaceutically-effective amount of a fusion protein comprising insulin-like growth factor I (IGF-I) (SEQ ID NO: 1) or an IGF-I variant N-terminally linked to the C-terminus of a propeptide, said C-terminus of said propeptide comprising the amino acid sequence Y-Pro, wherein Y is selected from the group consisting of: Pro; Pro-Ala; Pro-Gly; Pro-Thr; Ala-Pro; Gly-Pro; Thr-Pro; Arg-Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ ID NO: 12); and Pro-Ala-Pro-Arg-Pro (SEQ ID NO: 13) and said variant being a polypeptide which differs from SEQ ID NO: 1 in that one or two of the lysine residues at positions 27, 65, or 68 thereof is independently substituted by a polar amino acid selected from the group consisting of: cysteine; aspartic acid; glutamic acid; histidine; asparagine; glutamine; arginine; serine; and threonine; and the remainder of the amino acid sequence of said variant is the same as that of SEQ ID NO: 1. 
     
     
         2 . The method of  claim 1 , wherein the insulin-like growth factor I or IGF-I variant is lysine PEG-ylated. 
     
     
         3 . The method of  claim 2  wherein the lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant is administered in an amount of from about 0.1 to about 100 mg/ml.