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 illustrated that ther e are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of argin ine at L21 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 an d the amide nitrogen group of glutamine at L89 in CDR3 and between the hydr oxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine a t L29 as well as the amide nitrogen group of serine at L31 in CDR1, Elimina tion of these hydrogen bonds at these FR positions may affect CDR loop conf ormations. To confirm these assumptions, we altered these FR residues by si te-directed mutagenesis and determined binding affinities of these mutant c himeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, sign ificantly reduced the binding affinity for the TAG72 antigen by 23-fold, wh ereas the substitution of threonine and tyrosine by alanine and phenylalani ne at LS and L36, eliminating hydrogen bonds to side-chain atoms, did not a ffect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody play s an important role in influencing the TAG72 antigen binding. Our results w ill thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.