Design of conformation-specific antibodies against unfolded and misfolded proteins

1159943/ Tessier蛋白质工程界的一个重大的、未解决的挑战是设计以高亲和力和特异性结合靶抗原的抗体。该提案的目的是阐明设计针对未折叠和错误折叠（聚集）蛋白质的抗体的一般原则。重要的是，有没有以前报道的抗体特异性不同的未折叠的蛋白质，很少有报道的构象特异性抗体对错误折叠的蛋白质，识别线性序列表位，并没有报道的策略，设计抗体对任何蛋白质构象。这一提议是基于PI实验室最近的两项发现。首先，他们发现单个抗原结合环内富含酪氨酸的基序介导抗体与未折叠蛋白质的结合（但不与折叠蛋白质结合）。其次，他们发现，来自淀粉样β肽的淀粉样蛋白生成基序被移植到单个抗体环中，介导了聚集形式的淀粉样β蛋白的构象特异性抗体识别知识价值：基于这些发现，PI旨在测试与构象特异性抗体设计相关的以下两个假设：i）展示芳香族肽基序的抗体（包括酪氨酸以外的芳香残基）选择性地结合未折叠的蛋白质，并且这种结合通过涉及未折叠蛋白质内溶剂暴露的芳香残基的π堆积相互作用介导（其在折叠的蛋白质内被溶剂屏蔽）;和ii）展示来自不同蛋白质的淀粉样蛋白基序的抗体选择性地识别淀粉样蛋白原纤维抗体识别是通过淀粉样蛋白基序之间的同型相互作用介导的。更广泛的影响：这项工作对于设计针对不同生物分子（包括几种疾病相关蛋白质）的抗体以及理解和操纵生物分子识别具有广泛的意义。研究人员致力于通过两项努力向代表性不足的少数民族和其他弱势群体进行强有力的宣传：i）在一所小学开展四年级科学宣传计划，其中很大一部分是低价午餐（约80%）和非洲裔美国人（约40%）学生，他们使用动画片和动手活动关注分子，以使这些学生在早期对科学感兴趣;以及ii）针对来自纽约首都区的农村城镇的缺乏高级科学课程的不同学生的12年级外展计划，该计划使这些学生参与发现和开发治疗性抗体的过程，以鼓励他们在本科和研究生教育期间追求科学和工程的生物分子方面。

1159943/ Tessier A grand, unmet challenge in the protein engineering community is to design antibodies that bind to target antigens with high affinity and specificity. The goal of this proposal is elucidate general principles for designing antibodies against unfolded and misfolded (aggregated) proteins. Importantly, there are no previously reported antibodies specific for diverse unfolded proteins, few reported conformation-specific antibodies against misfolded proteins that recognize linear sequence epitopes, and no reported strategies for designing antibodies against either protein conformation. This proposal is based on two recent discoveries from the PIs laboratory. First, they have discovered that a tyrosine-rich motif within a single antigen-binding loop mediates antibody binding to unfolded proteins (but not to folded proteins). Second, they have discovered that amyloidogenic motifs from the amyloid beta peptide grafted into a single antibody loop mediate conformation-specific antibody recognition of aggregated forms of amyloid beta (but not monomeric amyloid beta).Intellectual Merit: Based on these discoveries, the PI aims to test the following two hypotheses related to the design of conformation-specific antibodies: i) antibodies displaying aromatic peptide motifs (including aromatic residues other than tyrosine) bind selectively to unfolded proteins, and such binding is mediated via pi-stacking interactions involving solvent-exposed aromatic residues within unfolded proteins (which are solvent-shielded within folded proteins); and ii) antibodies displaying amyloid motifs from diverse proteins selectively recognize amyloid fibrils (but not monomers) containing the cognate motifs, and such antibody recognition is mediated via homotypic interactions between amyloid motifs.Broader Impacts: The work has broad implications for designing antibodies against diverse biomolecules (including several disease-linked proteins), as well as for understanding and manipulating biomolecular recognition. The investigator is committed to strong outreach to underrepresented minorities and other disadvantaged peoples through two efforts: i) a 4th grade science outreach program in an elementary school with a significant fraction of reduced-cost lunch (~80%) and African American (~40%) students focused on molecules using animated cartoons and hands-on activities to interest these students in science at an early age; and ii) a 12th grade outreach program to diverse students from rural towns in New York's Capital District lacking advanced science courses that involves these students in the process of discovery and development of a therapeutic antibody to encourage them to pursue biomolecular aspects of science and engineering during their undergraduate and graduate education.