RAPID HUMANIZATION OF THE FV OF MONOCLONAL-ANTIBODY B3 BY USING FRAMEWORK EXCHANGE OF THE RECOMBINANT IMMUNOTOXIN B3(FV)-PE38

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of carcinoma-specific antibody B3 is fused to a truncated form of Pseudomonas exotoxin (PE). The efficacy of monoclonal antibody B3 a nd B3 immunotoxins in cancer therapy and diagnosis may be limited by t he human anti-mouse response. Here we describe the humanization of the Fv of B3(Fv)-PE38 by ''framework exchange.'' The variable domains of the heavy (V-H) and light (V-L) chains were aligned with their best hu man homologs to identify framework residues that differ. Initially, 11 framework residues in V-H and six in V-L were changed by site-specifi c mutagenesis to human residues and introduced simultaneously into a p reassembled single-chain Fv expression cassette. Six V-H and five V-L residues that differ were not changed because they were buried, in the interdomain interface, or previously found to result in decreased aff inity when mutated. This basic design resulted in some 20-fold loss of activity. Changing V-L residues at the interdomain interfacial positi on 100 and at the buried position 104 to the human sequence increased the activity 8-fold. Changing V-H residue at position 82b from the hum an sequence back to that of the mouse restored the activity 2- to 3-fo ld to the full binding and cytotoxic activity of the mouse sequence, H umanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.