Design of antibody fragments specific for amyloidogenic aggregates

描述（由申请人提供）：生物分子研究的一个重大挑战是合理设计具有高亲和力和特异性的靶向抗原抗体。本文的目的是阐明设计抗体片段的互补决定区域（cdr）的原则，以介导具有构象和序列特异性的聚集蛋白的识别。我们的建议是基于我们最近的发现，单域抗体可以设计为识别Aß42低聚物和原纤维，使用与阿尔茨海默病相关的a ß聚集相同的分子相互作用（Perchiacca等人，PNAS, 2012）。我们发现疏水的亚酰胺肽可以被接枝到单个CDR环中，并且由此产生的淀粉样蛋白基序抗体（γ体）以纳米摩尔亲和力与亚酰胺寡聚物和原纤维结合。基于这些发现，我们假设能够介导γ体结合的其他α肽可以根据其相对淀粉样变性来预测。我们还假设伽马体的结合亲和力将在小到中间的CDR长度上达到最大，这些长度足以显示asb自我识别肽，但由于熵增加而不足以不利于结合。此外，我们假设我们的设计方法不仅限于a ß，而且可以扩展到其他淀粉样蛋白多肽，包括IAPP（2型糖尿病）和α-突触核蛋白（帕金森病）。最后，我们假设可以通过将多个淀粉样蛋白肽嫁接到反平行cdr中来设计更高亲和力的γ体，这些cdr以与原纤维生长（模板）端相应肽相同的方式定向。因此，在Aim 1中，我们提出确定CDR3的长度和序列如何影响两个ß γ小体（Aß15-24和Aß33-42）的结合亲和力和特异性。然后，在Aim 2中，我们建议评估我们对移植到CDR3中时介导γ体与Aß聚集结合的其他α肽的预测。接下来，在Aims 3中，我们建议扩展Aims 1和Aims 2中进行的分析，以评估我们对其他两种淀粉样蛋白形成多肽（α-突触核蛋白和IAPP）的肽段的预测，这些肽段在移植到CDR3中时介导γ -体与相应聚集构象的结合。最后，在Aims 4中，我们提出评估Aims 1-3中形成的a ß和IAPP γ体的亲和力是否可以通过将两种不同的淀粉样蛋白肽嫁接到反平行cdr中来增加，以匹配原纤维生长端相应肽的取向。我们研究的一个重要结果将是阐明如何自我互补，淀粉样蛋白肽可以用来介导抗体-抗原识别。我们期望我们的发现将为设计具有不同淀粉样蛋白特异性的类似单域和多域抗体提供规则，包括与人类聚集性疾病（如亨廷顿氏病和朊病毒疾病）相关的抗体。

DESCRIPTION (provided by applicant): A grand challenge in biomolecular research is to rationally design antibodies that bind to target antigens with high affinity and specificity. The goal of this proposal is to elucidate principles for designing the complementarity-determining regions (CDRs) of antibody fragments to mediate recognition of aggregated proteins with conformational and sequence specificity. Our proposal is based on our recent discovery that single-domain antibodies can be designed to recognize Aß42 oligomers and fibrils using the same molecular interactions mediating Aßaggregation associated with Alzheimer's disease (Perchiacca et al., PNAS, 2012). We find that hydrophobic Aß peptides can be grafted into a single CDR loop, and the resulting Grafted AMyloid-Motif AntiBODIES (gammabodies) bind to Aß oligomers and fibrils with nanomolar affinity. Based on these discoveries, we posit that additional Aß peptides capable of mediating gammabody binding can be predicted based on their relative amyloidogenicity. We also hypothesize that the binding affinity of gammabodies will be maximal at small to intermediate CDR lengths that are sufficiently long to display Aß self-recognition peptides but not long enough to disfavor binding due to increased entropy. In addition, we posit that our design approach is not limited to Aßand can be extended to other amyloidogenic polypeptides, including IAPP (type 2 diabetes) and α-synuclein (Parkinson's disease). Finally, we hypothesize that even higher-affinity gammabodies can be designed by grafting multiple amyloidogenic peptides into anti-parallel CDRs that are oriented in the same manner as the corresponding peptides at the growing (templating) ends of fibrils. Therefore, in Aim 1, we propose to determine how the length and sequence of CDR3 impacts the binding affinity and specificity for two Aß gammabodies (Aß15-24 and Aß33-42). Then, in Aim 2, we propose to evaluate our predictions of additional Aß peptides that mediate gammabody binding to Aßaggregates when grafted into CDR3. Next, in Aim 3, we propose to extend the analysis performed in Aims 1 and 2 to evaluate our predictions of peptide segments from two other amyloid-forming polypeptides (α-synuclein and IAPP) that mediate gammabody binding to their corresponding aggregated conformers when grafted into CDR3. Finally, in Aim 4, we propose to evaluate whether the affinity of the Aßand IAPP gammabodies developed in Aims 1-3 can be increased by grafting two different amyloidogenic peptides into anti-parallel CDRs to match the orientation of the corresponding peptides at the growing ends of fibrils. A significant outcome of our studies will be the elucidation of how self-complementary, amyloidogenic peptides can be used to mediate antibody-antigen recognition. We expect that our findings will lead to rules for designing of similar single- and multidomain antibodies with specificity for diverse amyloidogenic proteins, including those linked to human aggregation disorders such as Huntington's and prion diseases.