COMPLEMENTARITY-DETERMINING REGION RESIDUES ASPARTIC-ACID AT H55, SERINE AT H95 AND TYROSINES AT H97 AND L96 PLAY IMPORTANT ROLES IN THE B72.3 ANTIBODY TAG72 ANTIGEN INTERACTION

Structural analysis derived from the crystallographic study of the chi meric B72.3 antibody illustrated some major atomic interactions betwee n complementarity determining region (CDR) residues, For example, hydr ogen bonds are formed between H35/H95, L50/H97, H53/H55 and H96/L96 re spectively, These CDR residues may play important roles in the B72.3-T AG72 (antibody-antigen) interaction either by direct interaction with the TAG72 antigen or by maintaining a CDR loop conformation through at omic interactions between CDR residues, In order to confirm these assu mptions, we altered these CDR residues by site-directed mutagenesis an d determined binding affinities of these mutant chimeric antibodies fo r the TAG72 antigen in a solid-phase radioimmunoassay, We found that H 55, H95, H97 and L96 are important CDR residues for the B72.3-TAG72 in teraction, Single amino acid substitutions of aspartic acid and serine by alanine at H55 of CDR2 and at H95 of CDR3 respectively and of tyro sine by phenylalanine at H97 and L96 of CDR3, significantly reduced th e binding affinity for the TAG72 antigen by 20-, 8-, 16- and 45-fold r espectively, Therefore, this study reveals some of the requirements fo r maintaining the integrity of the B72.3 antibody combining sites.