SITE-SPECIFIC ATTACHMENT TO RECOMBINANT ANTIBODIES VIA INTRODUCED SURFACE CYSTEINE RESIDUES
SITE-SPECIFIC ATTACHMENT TO RECOMBINANT ANTIBODIES VIA INTRODUCED SURFACE CYSTEINE RESIDUES
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DOI：
10.1093/protein/3.8.703
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发表时间：
1990-08-01
期刊：
影响因子：
--
通讯作者：
ADAIR, JR
中科院分区：
文献类型：
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作者：
LYONS, A;KING, DJ;ADAIR, JR
单克隆抗体的许多诊断和治疗应用需要效应子或报告分子与免疫球蛋白多肽的共价连接。现有方法通常涉及氨基酸侧链的非选择性修饰，产生一个或多个随机分布的附着位点。这导致抗体分子的异质标记，并且由于靠近抗原结合位点的残基的修饰而常常导致抗原结合的减少。我们报告了一种通过表面半胱氨酸残基对抗体进行位点特异性标记的新策略。分子结构检查用于鉴定 IgG 重链 CH1 结构域的氨基酸，这些氨基酸可被溶剂接触，但不能被较大分子接触。使用定点诱变来取代肿瘤结合单克隆抗体 B72.3 的小鼠/人嵌合版本重链中这些位置的半胱氨酸残基。修饰后的抗体基因在哺乳动物细胞中的表达产生了正确组装的蛋白质，该蛋白质在预定位置具有硫醇基团，并且没有表现出抗原结合活性的损失。其中一种突变体用于证明放射性碘配体与嵌合 B72.3 抗体的位点特异性连接。
Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.