Design of antibody fragments specific for amyloidogenic aggregates

描述(申请人提供)：生物分子研究中的一个重大挑战是合理地设计与目标抗原高度亲和力和特异性结合的抗体。这项建议的目的是阐明设计抗体片段的互补决定区(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，即单域抗体可以被设计成识别Aβ42寡聚体和纤维，使用与介导与阿尔茨海默病相关的Aβ聚集的相同分子相互作用(Perchiacca等人，PNAS，2012年)。我们发现疏水的Aβ多肽可以被嫁接到单个CDR环中，所得到的接枝的淀粉样基序抗体(GAMABOBY)以纳摩尔亲和力结合到Aβ寡聚体和纤维上。基于这些发现，我们假设可以根据它们的相对淀粉样蛋白原性来预测其他能够介导伽马抗体结合的Aβ肽。我们还假设，伽马体的结合亲和力将在小到中等的CDR长度时最大，这些长度足以显示Aβ自我识别多肽，但不足以由于熵的增加而不利于结合。此外，我们假设我们的设计方法不仅限于Aβ，还可以扩展到其他淀粉样多肽，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，即使是更高亲和力的伽马体，也可以通过将多个淀粉样多肽嫁接到反平行的CDR中来设计，这些CDR的定向方式与纤维生长(模板)端的相应多肽相同。因此，在目标1中，我们建议确定CDR3的长度和序列如何影响两个Aβ配子体(Aβ15-24和Aβ33-42)的结合亲和力和特异性。然后，在目标2中，我们建议评估我们的预测，当嫁接到CDR3中时，额外的Aβ多肽可以介导Gammabody与Aβ聚集体的结合。接下来，在目标3中，我们建议扩展在AIMS 1和2中进行的分析，以评估我们对另外两个淀粉样蛋白形成多肽(α-突触核蛋白和IAPP)的预测，这些多肽在嫁接到CDR3中时，介导伽马抗体与其相应的聚集构象结合。最后，在目标4中，我们建议评估是否可以通过将两个不同的淀粉样肽嫁接到反平行的CDR中来提高在AIMS 1-3中形成的Aβ和IAPP配子体的亲和力，以匹配纤维生长末端的相应多肽的取向。我们研究的一个重要结果将是阐明如何利用自身互补的淀粉样蛋白多肽来介导抗体-抗原识别。我们预计，我们的发现将导致类似的单域和多域抗体的设计规则，这些抗体对不同的淀粉样蛋白具有特异性，包括那些与人类聚集障碍有关的抗体，如亨廷顿氏病和Pron病。

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 Aβ42 oligomers and fibrils using the same molecular interactions mediating Aβ aggregation associated with Alzheimer's disease (Perchiacca et al., PNAS, 2012). We find that hydrophobic Aβ peptides can be grafted into a single CDR loop, and the resulting Grafted AMyloid-Motif AntiBODIES (gammabodies) bind to Aβ oligomers and fibrils with nanomolar affinity. Based on these discoveries, we posit that additional Aβ 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 Aβ 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 Aβ and 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 Aβ gammabodies (Aβ15-24 and Aβ33-42). Then, in Aim 2, we propose to evaluate our predictions of additional Aβ peptides that mediate gammabody binding to Aβ aggregates 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 Aβ and 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.