The Tyrosine Residue at Position 97 in the V H CDR3 Region of a Mouse/Human Chimeric Anti-Colorectal Carcinoma Antibody Contributes Hydrogen Bonding to the TAG72 Antigen

One amino acid, tyrosine at position 96 and 97 in the V_H CDR3 region of a mouse/human chimeric anti-TAG72 antibody cB72.3m4 was substituted by the phenylalanine residue and by a number of amino acids from different amino acid groups by the site-directed mutagenesis technique. The expression vector mpSV2neo-EPl-V_mll-16C₁ containing mutant V_H region fragments (V_mll-16) as well as the immunoglobulin enhances (E), promoter (P1) and the human genomic C₁ region fragments, were transfected into a heavy-chain-loss mutant cell line B72.3Mut(K), respectively. Mutant chimeric cB72.3mll-16 antibodies were purified from the transfectant supernates and compared based upon their binding affinity for the TAG72 antigen relative to that of the original cB72.3m4 antibody. The data showed that a single amino acid substitution of tyrosine by phenylalanine and a number of amino acids including serine, asparagine, histidine and arginine at position 97 in the V_H CDR3 region all resulted in approximate 18-fold lower binding affinity, whereas the substitution of tyrosine by phenylalanine at position 96 in the V_H CDR3 region did not affect the binding affinity of the cB72.3m4 antibody. This suggests that the tyrosine residue at position 97 in the V_H CDR3 region is in a contact position in the B72.3/TAG72 antibody/antigen interaction, and that the terminal hydroxyl group of the position 97 tyrosine side-chain contributes hydrogen bonding to the TAG72 antigen, whereas the position 96 tyrosine side-chain does not.