Molecular and Structural Studies of Antibody Diversity Mechanisms

抗体多样性机制的分子和结构研究

• 批准号: 9982985
• 负责人: Vaughn Vasil Smider
• 金额: $ 39.94万
• 依托单位: APPLIED BIOMEDICAL SCIENCE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982985
• 关键词: Active Sites; Amino Acids; Antibodies; Antibody Diversity; Antibody Formation; Antibody Repertoire; Antigen Targeting; Antigens; Area; Binding; Biochemical; Biological; Biological Response Modifiers; Cattle; Cells; Cellular Assay; Color; Complementarity Determining Regions; Complex; Cysteine; Data; Diagnostic; Diagnostics Research; Disulfides; Electron Microscopy; Enzyme Activation; Enzymes; Epitopes; Event; Fab Immunoglobulins; Family; Flow Cytometry; Gene Mutation; Genes; Genetic; Genetic Structures; HIV; HIV Antibodies; HIV-1; Human; Immune response; Immunization; Immunize; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; In Vitro; Infectious Agent; Investigational Therapies; Ion Channel; Label; Left; Length; Light; Maps; Matrix Metalloproteinases; Mediating; Medicine; Metalloproteases; Methods; Modeling; Molecular; Molecular Genetics; Molecular Structure; Mus; Mutagenesis; Mutate; Mutation; Pathway interactions; Pattern; Play; Polysaccharides; Process; Property; Proteins; Reagent; Recombinants; Role; Site-Directed Mutagenesis; Structure; Surface; System; Tertiary Protein Structure; Testing; Therapeutic; Time; V(D)J Recombination; Vaccine Design; Vaccines; Vertebrates; Viral; Virus; activation-induced cytidine deaminase; antibody engineering; antigen binding; antigen challenge; base; combinatorial; disulfide bond; electron crystallography; in vivo; insight; knob protein; neutralizing antibody; novel; response; therapeutic candidate; vaccine response

Abstract Antibodies are extremely important as the primary mediators of the immune response to infectious agents and vaccines, but also as recombinant molecules used as therapeutics, diagnostics, and research reagents. Thus, understanding the mechanisms by which antibodies form, bind, and neutralize their targets is very important in multiple biomedical areas. Most vertebrate antibody repertoires form their diversity through V(D)J recombination, where combinatorial rearrangement of V, D, and J genes form a vast repertoire where the complementarity determining regions (CDRs) form a relatively flat binding surface for interaction with antigen. Remarkably, cows appear to have a different mechanism for generating diversity and binding antigen; cow antibodies have particularly long CDR H3 regions, a subset of which can be over 70 amino acids long, which form -ribbon “stalk” and disulfide-bonded “knob” minidomains that protrude far from the typical antibody surface. Remarkably, antibodies from this repertoire can potently and broadly neutralize HIV, whereas normal human or mouse antibody repertoires cannot. Therefore, they have unique abilities to bind and neutralize particularly challenging antigens. Here we propose studies to understand in molecular detail the genetic mechanisms underlying formation of these antibodies, the binding properties of the stalk and knob minidomains, and the unique potential of these antibodes to bind bivalently and bispecifically. These studies will provide insight into mechanisms of viral neutralization generally, and particularly HIV neutralization. Additionally, given the potential of these antibodies to bind recessed epitopes, we will generate additional antibodies against particularly challenging antigens like enzymatic active sites and ion channels. We will employ mutagenesis, binding, and structural methods, as well as cellular assays to evaluate the unique properties of these antibodies. Understanding this novel class of antibodies in detail could provide fundamental insights needed for effective vaccine design as well as discovery and engineering of antibodies against some of the most challenging targets in biomedicine. Similarly, our unique antibodies discovered and characterized in this proposal could eventually become therapeutic candidates themselves.

摘要 抗体作为免疫应答的主要介质是极其重要的， 感染剂和疫苗，而且作为重组分子用作治疗剂， 诊断试剂和研究试剂。因此，了解抗体 形成、结合和中和它们的靶标在多个生物医学领域中非常重要。最 脊椎动物抗体库通过V（D）J重组形成其多样性，其中 V、D和J基因的组合重排形成了一个庞大的库， 互补决定区（CDR）形成用于相互作用的相对平坦的结合表面 抗原。值得注意的是，奶牛似乎有一种不同的产生多样性的机制， 和结合抗原;牛抗体具有特别长的CDR H3区，其中一个亚组 可以超过70个氨基酸长，形成带状的“茎”和二硫键键合的“节”， 突出远离典型抗体表面的微小区。值得注意的是， 这套系统可以有效和广泛地中和HIV，而正常的人或小鼠， 抗体库不能。因此，它们具有独特的能力， 特别是挑战性抗原。在这里，我们提出研究，以了解分子的细节， 这些抗体形成的遗传机制，茎的结合特性， 以及这些抗体结合生物素的独特潜力， 双特异性抗体。这些研究将提供深入了解病毒中和的机制 一般来说，特别是HIV中和。此外，鉴于这些潜力 抗体结合凹陷表位，我们将产生额外的抗体，特别是针对 挑战抗原如酶活性位点和离子通道。我们将采用诱变， 结合和结构方法，以及细胞测定，以评估的独特性质， 这些抗体。详细了解这类新型抗体可以提供 有效疫苗设计以及发现和工程所需的基本见解 针对生物医学中一些最具挑战性的目标的抗体。同样，我们独特的 在这项提议中发现和鉴定的抗体最终可能成为治疗性的 候选人自己。