Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding

The crystallographic study of chimeric B72.3 antibody illustratedthat there are three FR side-chain interactions with eitherCDR residue's side chain or main chain. For example, hydrogenbonds are formed between the hydroxyl group of threonine atL5 in FR1 and the guanidinal nitrogen group of arginine at L24in CDR1, between the hydroxyl group of tyrosine at L36 in FR2and the amide nitrogen group of glutamine at L89 in CDR3 andbetween the hydroxyl group of tyrosine at L71 in FR3 and thecarbonyl group of isoleucine at L29 as well as the amide nitrogengroup of serine at L31 in CDR1. Elimination of these hydrogenbonds at these FR positions may affect CDR loop conformations.To confirm these assumptions, we altered these FR residues bysite-directed mutagenesis and determined binding affinitiesof these mutant chimeric antibodies for the TAG72 antigen. Wefound that the substitution of tyrosine by phenylalanine atL71, altering main-chain hydrogen bonds, significantly reducedthe binding affinity for the TAG72 antigen by 23-fold, whereasthe substitution of threonine and tyrosine by alanine and phenylalanineat L5 and L36, eliminating hydrogen bonds to side-chain atoms,did not affect the binding affinity for the TAG72 antigen. Ourresults indicate that the light-chain FR residue tyrosine atL71 of chimeric B72.3 antibody plays an important role in influencingthe TAG72 antigen binding. Our results will thus be of importancewhen the humanized B72.3 antibody is constructed, since thisimportant mouse FR residue tyrosine at L71 must be maintained.