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 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 (VH) and light (VL) chains were aligned with their best human homologs to identify framework residues that differ. Initially, 11 framework residues in VH and six in VL were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled single-chain Fv expression cassette. Six VH and five VL 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 VL residues at the interdomain interfacial position 100 and at the buried position 104 to the human sequence increased the activity 8-fold. Changing VH 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.
Benhar
I
I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Padlan
E A
EA
Jung
S H
SH
Lee
B
B
Pastan
I
I
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D001427:Bacterial Toxins; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005098:Exotoxins / Q000737:chemistry* / Q000633:toxicity; D015870:Gene Expression; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D008252:Macaca fascicularis; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011550:Pseudomonas aeruginosa; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A