B3(Fab)-PE38M: a recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the Fab fragment of monoclonal antibody B3.
Recombinant immunotoxins were made by fusing the Fab domain of monoclonal antibody (MAb) B3 to PE38M, a truncated mutant form of Pseudomonas exotoxin (PE). The recombinant toxins were made in Escherichia coli by fusing genes encoding the antibody domains to a gene encoding the mutant form of PE. MAb B3 binds to a carbohydrate antigen found on many kinds of carcinomas. Immunotoxins in which MAb B3 has been chemically coupled to recombinant forms of PE have been shown to be very active cytotoxic agents. PE has also been targeted to tumor cells by replacing the cell-binding domain of PE (domain I) with a single-chain antibody to make a single-chain immunotoxin. In the current study, PE38M, a mutant form of PE, with a deletion of the cell-binding domain (amino acids 1-252) as well as mutations in domain III and some nonessential sequences in domain Ib (amino acids 365-380), was fused to the light chain of MAb B3. This protein was renatured in the presence of the Fd fragment of MAb B3 to produce a Fab-toxin fusion protein. Alternatively, the Fd fragment of MAb B3 was fused to PE38M and combined with the light chain. Both types of B3(Fab)-PE38M were just as active on target cells as previously described single-chain immunotoxins. Furthermore, the B3(Fab)-PE38M produced complete remissions of human tumor xenografts growing in nude mice. B3(Fab)-PE38M has two advantages over single-chain immunotoxins. One is that the yield of recombinant Fab-toxin is very high, with 10-22% of the starting protein recovered as cytotoxically active immunotoxin after chromatographic purification. The second is that the B3(Fab)-PE38M has a much longer survival in the circulation of mice with a t1/2 beta of approximately 5 h.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
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D036002:ADP Ribose Transferases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001427:Bacterial Toxins; D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics; D006207:Half-Life; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007140:Immunoglobulin Fab Fragments; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A