Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin
Wang, Q.C.; Pai, L.H.; Debinski, W.; FitzGerald, D.J.; Pastan, I.
Cancer Research 53(19): 4588-4594
1993
ISSN/ISBN: 0008-5472 PMID: 8402632 Document Number: 419979
Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an alter-ed form of TGF-alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF-alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF-alpha-R-29-L2-C-H2-PE38QQ-DELTA (TCP), there are no lysine residues in the TGF-alpha and PE38 portions. Human IgG4 constant region C-H2 and a tetradecapeptide linker, L2, are inserted between TGF-alpha and PE38. Together, L2 and C-H2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing 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 circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.