SEQUENCE SHAPE AND SPECIFICITY OF ANTIBODIES

抗体的序列形状和特异性

• 批准号: 2087250
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 31.97万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2087250
• 关键词: X ray crystallography; antibody specificity; antiidiotype antibody; chemical binding; computer assisted sequence analysis; gene expression; gene rearrangement; haptens; immunoglobulin structure; laboratory mouse; protein engineering; protein sequence; site directed mutagenesis

Immune responses in certain inbred mouse strains are dominated by antibodies which share common variable (V) region structures (idiotypes) detected serologically by anti-idiotypic reagents. Such idiotypic determinants characterizing certain antibody specificities are useful structural and genetic markers in studies of antibody diversity and regulation. The predominant cross reactive idiotype (Id-CR) in A/J mice immunized with rho-azophenylarsonate (Ars)-protein conjugates is heritable and encoded by a single combination of germline gene segments ("canonical"). Although Jerne proposed that interactions between Id and anti-Id regulate immune responses through recognition of Id determinants, evidence in the Ars system has accumulated that Id dominance may be due to antigen-driven selection of favorable somatic mutants with higher affinity derived from preferred (more "adaptable") V region germline gene combinations. The Ars system is a model for examining regulation by defining the antibody structural changes involved in enhanced antigen affinity occurring temporally during the immune response ("affinity maturation"). Based upon the high resolution X-ray crystal structure of a somatically mutated Ars-binding Id-CR bearing antibody (36-71) and the germline tertiary structure deduced therefrom, we can now examine the detailed structural correlates of Ars binding and idiotypy, as related to somatic mutation and gene junctional variation. We will capitalize on methodologic advances in protein engineering using site-specific mutagenesis and expression, in concert with predictive computer modelling and the crystal structures to: 1) relate somatic mutation and gene junctional diversity to fine structure of the hapten binding site geometry using comparisons of the tertiary structures of germline and mutated antibodies. 2) Engineer novel mutations designed to increase affinity. 3) Examine sites of hypermutation found in vivo to assess whether or not they relate to affinity enhancement or altered idiotopes. 4) Engineer new binding specificities to Ars homologues as a measure of the differentiative capacity of a canonical V region structure. 5) Map idiotopes by mutagenesis and measurement of reactivity with monoclonal anti-Id reagents, and by determination of the crystal structures of Id-anti-Id. The use of antibody engineering to further understand and thus modify antigen-antibody complementarity is necessary to the design of antibodies for therapeutic use in targeting to drugs, toxins, hormones, and cellular receptors.

某些近交系小鼠品系中的免疫应答主要由 具有共同可变（V）区结构的抗体（独特型） 通过抗独特型试剂进行血清学检测。 如此独特 表征某些抗体特异性的决定簇是有用的 抗体多样性研究中的结构和遗传标记， 调控 A/J小鼠的优势交叉反应独特型（Id-CR） 用对-偶氮苯胂酸盐（Ars）-蛋白质缀合物免疫， 可遗传并由生殖系基因片段的单一组合编码 （“canonical”）。 虽然杰恩提出，身份证和 抗Id通过识别Id决定簇来调节免疫应答， 在Ars系统中积累的证据表明，Id优势可能是由于 抗原驱动的选择具有更高 源自优选（更“适应性”）V区生殖系基因的亲和力 组合。 Ars系统是一个通过以下方式检查监管的模型： 定义增强抗原所涉及的抗体结构变化 在免疫应答期间暂时发生的亲和性（“亲和性 成熟”）。 基于高分辨率的X射线晶体结构， 体细胞突变的Ars-结合Id-CR携带抗体（36-71）和 由此推导出的种系三级结构，我们现在可以检查 Ars结合和独特型的详细结构相关性，如与 体细胞突变和基因连接变异。 我们将利用 蛋白质工程的方法学进展 诱变和表达，与预测性计算机建模相一致 和晶体结构：1）将体细胞突变和基因 半抗原结合位点精细结构的连接多样性 几何学使用种系和 变异抗体2)设计新的突变， 亲和力3)检查体内发现的超突变位点，以评估 无论它们是否与亲和力增强或改变的独特位有关。 4)设计针对Ars同系物的新结合特异性，作为 典型V区结构的微分能力。5)地图 通过诱变和测量与单克隆抗体的反应性 抗Id试剂，并通过测定晶体结构 Id-抗Id。利用抗体工程进一步了解和 因此，修饰抗原-抗体互补性是设计所必需的 用于治疗药物，毒素， 激素和细胞受体。