Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops.
Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops.
复制标题
嵌合 B72.3 抗体的框架残基 71 和 93 是重链高变环构象的主要决定因素。
DOI：
--
复制
发表时间：
1995
影响因子：
5.6
通讯作者：
L. Delbaere
中科院分区：
文献类型：
--
作者：
J. Xiang;Y. Sha;Z. Jia;L. Prasad;L. Delbaere
嵌合B72.3抗体的晶体学分析表明，一些与重链CDR残基发生原子相互作用的重链框架残基可能直接影响CDR环的构象。例如，H71的丙氨酸残基为CDR2/CDR1提供了包装空间，H73和H93的赖氨酸残基分别为CDR2中H55处的天冬氨酸和CDR3中H96处的羰基提供了一个氢键。我们分析了这些框架残基对tag72结合亲和力的贡献。我们通过位点定向诱变改变了这些框架残基，并通过固相放射免疫测定测定了这些突变嵌合抗体对TAG72抗原的亲和力。我们发现，在H71位点上用苯丙氨酸取代丙氨酸或在H93位点上用异亮氨酸取代赖氨酸可显著降低TAG72抗原的结合亲和力，分别降低12倍和20倍，而在H73位点上用丙氨酸取代赖氨酸仅降低2倍的结合亲和力。我们的研究结果表明，嵌合B72.3抗体的重链框架残基H71处丙氨酸和H93处赖氨酸是CDR2/CDR1和CDR3重链环构象的主要决定因素，而H73处赖氨酸和H55处天冬氨酸之间的盐桥不太重要。H5的谷氨酰胺和H25的丝氨酸这两个框架残基之间的氢键不影响任何CDR构象。因此，我们的结果将是重要的，特别是当人源化的B72.3抗体是通过将CDR环嫁接到人的框架来构建的。在最终的人源化抗体中必须保持重要的框架区相互作用。
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.