Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.
A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.
Kettleborough
C A
CA
Medical Research Council Collaborative Centre, London, UK.
Saldanha
J
J
Heath
V J
VJ
Morrison
C J
CJ
Bendig
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D002460:Cell Line; D004277:DNA, Single-Stranded; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D004277:DNA, Single-Stranded; D007127:Immunoglobulin Constant Regions; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins