Rapid humanization of the Fv of monoclonal antibody B3 by using framework exchange of the recombinant immunotoxin B3(Fv)-PE38

Itai Benhar, Eduardo A. Padlan, Sun Hee Jung, Byungkook Lee, Ira Pastan

Research output: Contribution to journalArticlepeer-review

Abstract

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 and B3 immunotoxins in cancer therapy and diagnosis may be limited by the 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 human homologs to identify framework residues that differ. Initially, 11 framework residues in V(H) and six in V(L) were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled 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 affinity when mutated. This basic design resulted in some 20-fold loss of activity. Changing V(L) residues at the interdomain interfacial position 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 human sequence back to that of the mouse restored the activity 2- to 3-fold to the full binding and cytotoxic activity of the mouse sequence. Humanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.

Original languageEnglish
Pages (from-to)12051-12055
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number25
DOIs
StatePublished - 6 Dec 1994
Externally publishedYes

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