Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.
The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.
Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
Masato
M
Kiyoshi
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ; Laboratory of Medical Proteomics, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Jose M M
JM
Caaveiro
Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
Eri
E
Miura
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Satoru
S
Nagatoishi
Laboratory of Medical Proteomics, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Makoto
M
Nakakido
Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan.
Shinji
S
Soga
Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan.
Hiroki
H
Shirai
Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan.
Shigeki
S
Kawabata
Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan ; Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ; Laboratory of Medical Proteomics, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan ; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Kouhei
K
Tsumoto
D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use*; D000915:Antibody Affinity / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology / Q000378:metabolism*; D000941:Antigens / Q000276:immunology*; D018932:Chemokine CCL2 / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D055672:Static Electricity; D013816:Thermodynamics
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D018932:Chemokine CCL2; D007140:Immunoglobulin Fab Fragments