Humanization of a mouse monoclonal antibody neutralizing TNF-alpha by guided selection.
With the advent of phage display antibody libraries, humanization of murine antibodies can be achieved by epitope guided selection. In present study, guided selection was applied to the humanization of the mouse mAb Z8 that is directed to human TNF-alpha and can neutralize the cytotoxicity of TNF-alpha. First, the Z8 Fd gene was paired as a template with a repertoire of human kappa chains, and displayed on the filamentous phage, forming a hybrid phage antibody library. Selected by four rounds of panning against TNF-alpha, hybrid antibody fragments that bound to TNF-alpha and contained human kappa chains were obtained. Meanwhile human Fd genes were selected by pairing the human Fd repertoire with the Z8 kappa chain and performing the same procedure of panning. One of the isolated human Fd genes (huFd2), which showed the strongest reactivity, was chosen to pair with 12 of selected human kappa chains. Two of the resulting human Fabs (huFd2-hukappa1 and huFd2-hukappa2), with same Fd and different kappa chains, bound to TNF-alpha specifically. Their human origin was proved by ELISA and sequencing analysis. The human Fabs competitive ELISA and in vitro TNF-alpha neutralization assay demonstrated that the human Fabs resembled its parental mouse mAb Z8 in that they both recognized the same epitope and neutralized the cytotoxicity of TNF-alpha. These results suggest that guided selection is a promising strategy in murine mAb humanization.
Central Laboratory, Navy General Hospital, Haidian District, 100037, Beijing, PR China. zwang1@partners.org
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology; D012641:Selection, Genetic; D013045:Species Specificity; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha