Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples.
Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified "HV4" and "LV4" in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.
a Laboratory of Molecular Biology , National Cancer Institute , Bethesda , MD , USA.
Yi-Fan
YF
Zhang
a Laboratory of Molecular Biology , National Cancer Institute , Bethesda , MD , USA.
Mitchell
M
Ho
D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis* / Q000737:chemistry*; D001666:Binding Sites, Antibody; D022801:Complementarity Determining Regions / Q000737:chemistry*; D006801:Humans; D000090204:Mesothelin; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits
D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; C000715528:Msln protein, mouse; D000090204:Mesothelin
Humanization; mesothelin; paratope; rabbit monoclonal antibody; recombinant immunotoxin; structure alignment