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Polyethylene Glycol-modified Chimeric Toxin Composed of Transforming Growth Factor and Pseudomonas Exotoxin

Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, NIH, Bethesda, Maryland 20892

² To whom requests for reprints should be addressed, at Laboratory of Molecular Biology, National Cancer Institute, NIH, 9000 Rockville Pike, Bldg. 37, Rm. 4E16, Bethesda, MD 20892.

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 altered form of TGF-PE40, a recombinant toxin composed of human transforming growth factor a (TGF) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell- binding domain. In the newly designed protein, termed TGFR²⁹-L2-C_H2-PE38QQ (TCP), there are no lysine residues in the TGF and PE38 portions. Human IgG4 constant region C_H2 and a tetradecapeptide linker, L2, are inserted between TGF 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.

¹ W. D. received a postdoctoral fellowship from the Medical Research Council of Canada and NIH CRADA. Present address: Laboratory of Molecular Targeting, Research Centre, HDM-UM, 3850 St. Urbain St., Montreal, Quebec H2W 1T8, Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/19/93. Accepted 7/27/93.

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