FRAMEWORK RESIDUE-71 AND RESIDUE-93 OF THE CHIMERIC B72.3 ANTIBODY ARE MAJOR DETERMINANTS OF THE CONFORMATION OF HEAVY-CHAIN HYPERVARIABLE LOOPS

Structural analysis derived from the crystallographic study of the chi meric B72.3 antibody illustrated that some heavy-chain framework resid ues having atomic interactions with heavy-chain CDR residues may direc tly affect the conformation of CDR loops. For example, an alanine resi due at H71 provides room for packing CDR2/CDR1 and lysine residues at H73 and H93 contribute a salt-bridge to aspartic acid at H55 in CDR2 a nd a hydrogen bond to the carbonyl group at H96 in CDR3, respectively We have analysed the contribution of these framework residues to the T AG72-binding affinity. We altered these framework residues by site-dir ected mutagenesis, and determined the affinity of these mutant chimeri c antibodies for the TAG72 antigen by solid phase radioimmunoassay. We found that a single amino acid substitution of alanine by phenylalani ne at H71 or lysine by isoleucine at H93, significantly reduced the bi nding affinity for the TAG72 antigen by 12 and 20-fold, respectively, whereas the substitution of lysine by alanine at H73 reduced the bindi ng affinity only two-fold. Our results indicate that heavy-chain frame work residues alanine at H71 and lysine at H93 of the chimeric B72.3 a ntibody are the major determinants of the conformation of heavy-chain CDR2/CDR1 and CDR3 loops, whereas the salt-bridge between lysine at H7 3 and aspartic acid at H55 is less important. The hydrogen bond betwee n two framework residues, glutamine at H5 and serine at H25 does not a ffect any CDR conformation. Our results will thus be of importance esp ecially when the humanized B72.3 antibody is constructed by grafting t he CDR loops to a human framework. The important framework region inte ractions must be maintained in the final humanized antibody