Differences in antigen-binding affinity caused by a single amino acid substitution in the variable region of the heavy chain.
Site-directed mutagenesis has been used to examine the importance of histidine-99 in the VH CDR3 region of a mouse/human chimeric anti-TAG72 antibody, cB72.3-1-3. The expression vectors mpSV2neo-EP1-V-m4-10C gamma 1, containing seven different mutant VH region fragments (Vm4-10) in association with the immunoglobulin enhancer (E), promoter (P1) and human genomic C gamma 1 region fragments, were transfected into a heavy chain loss mutant cell line B72.3 Mut(K), respectively. Mutant chimeric antibodies cB72.3m4-10 were purified from the transfectant supernates, and their binding affinities for the TAG72 antigen relative to that of the original cB72.3-1-3 antibody were compared. Substitution of histidine-99 by glutamine resulted in a higher affinity antibody (cB72.3m4) whereas substitution by isoleucine resulted in a lower affinity antibody (cB72.3m9). The binding affinity of these mutant antibodies varied nearly eight-fold. It was concluded that the residue at position 99 in the VH CDR3 region is in a 'contact' position in the B72.3/TAG72 antibody-combining site. The polar side-chains of glutamine and asparagine or the ionized side-chains of histidine, arginine or glutamic acid contribute to higher binding affinity, whereas the hydrophobic side-chains of isoleucine, leucine or phenylalanine resulted in a lower binding affinity for the TAG72 antigen.
Xiang
J
J
Saskatoon Cancer Center, Department of Microbiology, University of Saskatchewan, Canada.
Chen
Z
Z
Delbaere
L T
LT
Liu
E
E
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D006639:Histidine