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 there 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 arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.
Xiang
J
J
Saskatoon Cancer Center, Departments of Oncology and Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 4H4, Canada.
Prasad
L
L
Delbaere
L T
LT
Jia
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D002417:Cattle; D006023:Glycoproteins / Q000276:immunology*; D006860:Hydrogen Bonding; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014443:Tyrosine / Q000276:immunology
D000941:Antigens; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009077:Mucins; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72; D014443:Tyrosine; D010649:Phenylalanine