B3(FAB)-PE38(M) - A RECOMBINANT IMMUNOTOXIN IN WHICH A MUTANT FORM OFPSEUDOMONAS EXOTOXIN IS FUSED TO THE FAB FRAGMENT OF MONOCLONAL-ANTIBODY B3

Recombinant immunotoxins were made by fusing the Fab domain of monoclo nal antibody (MAb) B3 to PE38(M), a truncated mutant form of Pseudomon as 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. MAI,B3 binds to a carbohydrate antigen found on man y kinds of carcinomas. Immunotoxins in which MAb B3 has been chemicall y coupled to recombinant forms of PE have been shown to be very active cytotoxic agents. PE has also been targeted to tumor cells by replaci ng the cell-binding domain of PE (domain I) with a single-chain antibo dy to make a single-chain immunotoxin. In the current study, PE38(M), 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. Alternativel y, the Fd fragment of MAb B3 was fused to PE38(M) and combined with th e light chain. Both types of B3(Fab)-PE38(M) were just as active on ta rget cells as previously described single-chain immunotoxins. Furtherm ore, the B3(Fab)-PE38(M) produced complete remissions of human tumor x enografts growing in nude mice. B3(Fab)-PE38(M) has two advantages ove r single-chain immunotoxins. One is that the yield of recombinant Fab- toxin is very high, with 10-22% of the starting protein recovered as c ytotoxically active immunotoxin after chromatographic purification. Th e second is that the B3(Fab)-PE38(M) has a much longer survival in the circulation of mice with a t(1/2 beta) of similar to 5 h.