Xiang J, Prasad L, Delbaere L T, Jia Z
Saskatoon Cancer Center, Departments of Oncology and Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 4H4, Canada.
Protein Eng. 1999 May;12(5):417-21. doi: 10.1093/protein/12.5.417.
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.
嵌合B72.3抗体的晶体学研究表明，FR有三个侧链与CDR残基的侧链或主链相互作用。例如，FR1中L5位苏氨酸的羟基与CDR1中L24位精氨酸的胍基氮之间形成氢键，FR2中L36位酪氨酸的羟基与CDR3中L89位谷氨酰胺的酰胺氮之间形成氢键，FR3中L71位酪氨酸的羟基与CDR1中L29位异亮氨酸的羰基以及L31位丝氨酸的酰胺氮之间形成氢键。消除这些FR位置的氢键可能会影响CDR环的构象。为了证实这些假设，我们通过定点诱变改变了这些FR残基，并测定了这些突变嵌合抗体与TAG72抗原的结合亲和力。我们发现，L71位酪氨酸被苯丙氨酸取代，改变了主链氢键，使与TAG72抗原的结合亲和力显著降低了23倍，而L5和L36位苏氨酸和酪氨酸被丙氨酸和苯丙氨酸取代，消除了与侧链原子的氢键，却不影响与TAG72抗原的结合亲和力。我们的结果表明，嵌合B72.3抗体轻链FR的L71位酪氨酸在影响TAG72抗原结合方面起重要作用。因此，在构建人源化B72.3抗体时，我们的结果将具有重要意义，因为必须保留这个重要的小鼠FR残基L71位酪氨酸。
