POLYETHYLENE GLYCOL-MODIFIED CHIMERIC TOXIN COMPOSED OF TRANSFORMING GROWTH-FACTOR-ALPHA AND PSEUDOMONAS EXOTOXIN

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) h as been shown to prolong circulation time and to reduce immunogenicity . To make a mPEG-modified recombinant toxin that retained cytotoxic ac tivity but had a longer residence time in circulation, we have constru cted an altered form of TGFalpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGFalpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell- binding domain. In the newly designed protein, termed TGFalphaR29-L2-C(H)2-PE38QQDELTA ( TCP), there are no lysine residues in the TGFalpha and PE38 portions. Human IgG4 constant region C(H)2 and a tetradecapeptide linker, L2, ar e inserted between TGFalpha and PE38. Together, L2 and C(H)2 contain 1 3 lysine residues as potential modification sites for mPEG. mPEG conju gates of TCP (PEG-TCP) were generated and the products were resolved b y ion exchange chromatography. Two PEG-TCP species termed B4 and B6 re tained 15 and 4% of cytotoxicity, respectively, and 26% of their recep tor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in a nimals. The mean residence times of B4 and B6 were 37 and 68 min, resp ectively, compared to 7 min for TCP. When administered i.v. to tumor b earing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5 -10 times less than that of TCP. We suggest that the prolonged circula ting time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.