Xiang J, Chen Z
Saskatoon Cancer Centre, University of Saskatchewan, Canada.
Immunology. 1992 Feb;75(2):209-16.
Two amino acids, tyrosine at position 96 and histidine at position 99 in the variable heavy chain (VH) CDR3 region of a mouse/human chimeric anti-TAG72 antibody cB72.3-1-3 were substituted with phenylalanine and asparagine respectively by site-directed mutagenesis technique. The expression vector mpSV2neo-EP1-Vm1-3-C gamma 1 containing mutant VH region fragments (Vm1-3) as well as the immunoglobulin enhancer (E), promoter (P1) and human genomic C gamma 1 region fragments, was transfected into a heavy-chain loss mutant cell line B72.3Mut(k). Mutant chimeric cB72.3m1-3 antibodies were purified from the transfectant supernates and compared based upon their binding affinity for the TAG72 antigen relative to that of the original cB72.3-1-3 antibody. The data show that a single amino acid substitution of histidine with asparagine at position 99 in VH CDR3 region contributes to four times increase in binding affinity for the TAG72 antigen. This suggests that the residue at position 99 in VH CDR3 region may play some role in antibody/antigen (B72.3/TAG72) interaction.
通过定点诱变技术，将小鼠/人嵌合抗TAG72抗体cB72.3-1-3可变重链（VH）CDR3区域中第96位的酪氨酸和第99位的组氨酸这两个氨基酸分别替换为苯丙氨酸和天冬酰胺。将含有突变VH区域片段（Vm1-3）以及免疫球蛋白增强子（E）、启动子（P1）和人基因组Cγ1区域片段的表达载体mpSV2neo-EP1-Vm1-3-Cγ1转染至重链缺失突变细胞系B72.3Mut(k)。从转染细胞的上清液中纯化出突变嵌合cB72.3m1-3抗体，并将其与原始cB72.3-1-3抗体对TAG72抗原的结合亲和力进行比较。数据表明，VH CDR3区域第99位的组氨酸被天冬酰胺单氨基酸取代后，对TAG72抗原的结合亲和力增加了四倍。这表明VH CDR3区域第99位的残基可能在抗体/抗原（B72.3/TAG72）相互作用中发挥一定作用。
