CAR and Antibodies Structure-Activity Relationships and molecular architecture

该项目的一个重要重点是通过获得其组成性分子元件（抗体结合部分，铰链，跨膜和刺激结构域）之间相互作用的详细知识及其功能作用的表征，支持汽车（嵌合抗原受体）和抗体的精确自下而上工程。最先进的汽车设计策略依赖于从用作组件的这些模块化结构域构建嵌合体。这是通过一个组合过程，使用昂贵的试错方法来选择最有希望的候选人。在过去的几年里，我的研究重点是分子系统，特别是蛋白质的自缔合决定簇的研究，正如它们在分子组织的几个层次上的结构对称性所揭示的那样：分子组装，蛋白质链，蛋白质结构域和蛋白质超二级结构（原结构域）[Youkharibache 2019]。虽然在大分子组装中对称性很高且很明显，但在蛋白质结构域或受体-配体相互作用的水平上很难检测到。例如，虽然抗体的重链和轻链对称性是众所周知的，但单个免疫球蛋白结构域本身由固有的伪对称原结构域组成[Youkharibache 2019]，这一特性在很大程度上被忽视，可以为抗体工程开辟新的途径，特别是纳米抗体。同时，从T细胞到其靶细胞的许多细胞表面蛋白受体（TCR、CD 4、CD 8、CD 28、CTLA 4、PD 1、PDL 1等），由通过寡聚伪对称排列相互作用的IG结构域组成。我们已经开发了用于其结构分析的方法，以告知并可能设计新的基于Ig的免疫受体。我们使用实验和计算工具来探索这些问题。我们的建模工具的应用帮助我们基于灵活性分析使用可变的CD 8和CD 28铰链组件来表征抗CD 19和抗BCMA汽车[Brudno et al. 2020]，这表明CAR毒性可能与其灵活性有关。我们通过X射线晶体学技术对CAR胞外结构域的探索导致了关于由准对称性介导的CAR scFv自发重排形成的第一份报告，导致了意想不到的VL-VL排列[PDBid：7 JO 8 Cheung et al. 2020]。这些早期有希望的结果支持开发一个管道，用于在不同复杂程度下对汽车进行快速建模，并通过计算技术探索其特性。这些技术基于数据科学方法、大数据存储库挖掘、计算机结构分析新方法和机器学习工具的组合应用[Wang et al. 2020; [Youkharibache et al. 2019，Youkharibache et al. 2020]。观察到的免疫球蛋白结构域交换[Cheung et al. 2020]支持了我们对探索作为数据/知识组织标准的免疫球蛋白相关重排及其在抗体设计中的应用（独立和作为CAR抗原结合部分）日益增长的兴趣。我们希望通过进行稳定性测量来补充现有的数据，从而扩大我们对这些结构域的实验表征。我们计划通过强调超微结构分析和CAR片段分析来改变我们对汽车的实验探索。在有利的情况下，通过薄片TEM研究进行的超微结构分析（汽车如何在膜中组织）可能会导致Cryo-ET研究。使用衍射技术来分析CAR片段可以加速一些缺失结构的发现。这两种方法是互补的，将提供关键的缺失信息，以提高我们对这些复杂系统的理解。

An important focus of this project is to support CARs (Chimeric Antigen Receptors) and antibodies' precise bottom-up engineering by acquiring the detailed knowledge of the interplay between their constitutive molecular elements (Antibody binding moiety, Hinge, Transmembrane, and stimulatory domains) and the characterization of their functional role. State-of-the-art CARs design strategies rely on the buildup of chimeras from these modular domains used as components. This is done through a combinatorial process using an expensive trial and error approach to select the most promising candidates. My research has focused in the last few years on the study of self-association determinants of molecular systems, especially proteins, as revealed by their structural symmetries at several levels of molecular organization: molecular assemblies, protein chains, protein domains, and protein supersecondary structures (protodomains) [Youkharibache 2019]. While symmetry is high and apparent in large molecular assemblies, it is difficult to detect at the level of protein domains or receptor-ligand interactions. For example, while antibodies' heavy and light chain symmetries are well known, the individual Immunoglobulin domains consist themselves of intrinsically pseudo-symmetric protodomains [Youkharibache 2019], a property largely ignored that can open new routes to antibody engineering, especially nanobodies. At the same time, many of the cell surface protein receptors, from T-Cell to their target cells (TCRs, CD4, CD8, CD28, CTLA4, PD1, PDL1, etc.) are composed of Ig domains interacting through oligomeric pseudo-symmetric arrangements. We have developed methods for their structural analysis to inform and possibly design new Ig-based immunoreceptors. We used experimental and computational tools to explore these questions. Our modeling tools' application helped us characterize anti-CD19 and anti-BCMA CARs using variable CD8 and CD28 hinge components based on flexibility analysis [Brudno et al. 2020], suggesting that CAR toxicity can be related to its flexibility. Our exploration of CAR extracellular domains by X-ray crystallography techniques resulted in the first report on the formation of a spontaneous rearrangement of a CAR scFv mediated by quasi-symmetry, resulting in an unexpected VL-VL arrangement [PDBid: 7JO8 Cheung et al. 2020]. These early promising results supported developing a pipeline for the rapid modeling of CARs at different scales of complexity and the exploration of their properties through computational techniques. These techniques are based on the application of a combination of data science methods, mining of big-data repositories, new approaches to in-silico structure analysis, and machine learning tools [Wang et al. 2020; [Youkharibache et al. 2019, Youkharibache et al. 2020]. The Immunoglobulin domain swapping observed [Cheung et al. 2020] gave support to our growing interest in exploring symmetry-related rearrangements as a data/knowledge organizing criteria and its application to antibody design, both stand-alone and as CAR antigen binding moieties. We expect to expand our experimental characterization of these domains by performing stability measurements to complement the available data. We plan to shift our experimental exploration of CARs by emphasizing ultrastructure analysis and CAR fragment analysis. Ultrastructure analysis (how CARs are organized in the membrane) by thin-section TEM studies may lead to Cryo-ET studies in favorable cases. The use of diffraction techniques to analyze CAR fragments may expedite the uncovering of some of the missing structures. The two approaches are complementary and will provide critical missing information to improve our understanding of these complex systems.