Molecular and Structural Studies of Antibody Diversity Mechanisms

摘要 抗体作为免疫反应的主要媒介是极其重要的 感染剂和疫苗，但也作为重组分子用于治疗， 诊断学和研究试剂。因此，了解抗体产生的机制 形成、结合和中和它们的靶标在许多生物医学领域都是非常重要的。多数 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曲目，其中 互补决定区(CDR)形成相对平坦的相互作用结合面 带着抗原。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，其子集 可长达70多个氨基酸，形成带状“柄”和二硫键“旋钮”。 远离典型抗体表面的突起的小颗粒。值得注意的是，来自 这种能力可以有效和广泛地中和艾滋病毒，而正常的人或小鼠 抗体谱系不能。因此，它们具有独特的结合和中和能力 尤其是具有挑战性的抗原。在这里，我们建议进行研究，以了解分子细节 形成这些抗体的遗传机制，即茎的结合特性 和旋钮微域，以及这些抗体的独特潜力，以二价结合和 双管齐下。这些研究将为深入了解病毒中和的机制提供帮助。 一般说来，特别是艾滋病毒的中和。此外，考虑到这些潜在的 为了结合隐含的表位，我们将产生额外的抗体，特别是 挑战抗原，如酶活性部位和离子通道。我们将采用诱变技术， 结合，结构方法，以及细胞分析，以评估独特的性质 这些抗体。对这类新抗体的详细了解可以提供 有效的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，我们独一无二的 在这项提议中发现的抗体和特征最终可能成为治疗性抗体 候选人本人。

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.