Differences in antigen‐binding affinity caused by single amino acid substitution in the variable region of the heavy chain

Abstract

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.