Framework residues 71 and 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 chimeric B72.3 antibody illustrated that some heavy-chain framework residues having atomic interactions with heavy-chain CDR residues may directly affect the conformation of CDR loops. For example, an alanine residue 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 and a hydrogen bond to the carbonyl group at H96 in CDR3, respectively. We have analysed the contribution of these framework residues to the TAG72-binding affinity. We altered these framework residues by site-directed mutagenesis, and determined the affinity of these mutant chimeric antibodies for the TAG72 antigen by solid phase radioimmunoassay. We found that a single amino acid substitution of alanine by phenylalanine at H71 or lysine by isoleucine at H93, significantly reduced the binding affinity for the TAG72 antigen by 12 and 20-fold, respectively, whereas the substitution of lysine by alanine at H73 reduced the binding affinity only two-fold. Our results indicate that heavy-chain framework residues alanine at H71 and lysine at H93 of the chimeric B72.3 antibody are the major determinants of the conformation of heavy-chain CDR2/CDR1 and CDR3 loops, whereas the salt-bridge between lysine at H73 and aspartic acid at H55 is less important. The hydrogen bond between two framework residues, glutamine at H5 and serine at H25 does not affect any CDR conformation. Our results will thus be of importance especially when the humanized B72.3 antibody is constructed by grafting the CDR loops to a human framework. The important framework region interactions must be maintained in the final humanized antibody.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D005816:Genetic Complementation Test; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins