A chemistry-driven approach to Senolytic Bispecific T-Cell Engagers

细胞衰老涉及先前具有复制能力的细胞的应激诱导的、持久的细胞周期停滞，并且与过多的年龄相关的病理学有关，例如肝和肺纤维化、动脉粥样硬化、糖尿病和骨关节炎。由于大多数具有衰老清除活性的小分子缺乏效力并且产生大量副作用，人们对开发选择性消除衰老细胞而不是健康细胞的衰老清除剂越来越感兴趣。本项目将通过建立一种新的化学驱动的方法来解决这个问题，该方法用于组装具有衰老清除活性的独特的双特异性T细胞衔接子（BiTEs），这将通过设计两个针对半胱氨酸的正交反应来实现（其中一种专门靶向N-末端Cys，因此允许不对称连接），用于从单个Cys标记的单克隆抗体（mAb）组装双特异性抗体（bsAb）文库，并且避免了对每个bsAb逐一克隆、表达、纯化和测试的需要。然后，该方法将包括表达两组Cys标记的抗体，一组靶向造血细胞和免疫细胞上的细胞表面抗原，另一组靶向衰老细胞，其将通过设计的接头彼此连接成对衰老和T细胞抗原特异性的bsAb，因此能够触发针对衰老细胞的免疫细胞毒性应答。为了在体外分析该文库，将从衰老细胞和外周血单核细胞的共培养物的细胞裂解和活力测定推断衰老清除BiTE的免疫调节性质。在此之后，将使用组学和免疫学工具阐明具有衰老清除活性的所选bsAb的免疫调节作用。开发的技术将不仅限于bsAbs，而且将允许扩展到任何类型的蛋白质的连接，使未来在基于蛋白质的药物发现中的应用成为可能。

Cellular senescence involves the stress-induced, durable cell-cycle arrest of previously replication-competent cells and has been associated with a plethora of age-related pathologies, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.Since most small molecules with senolytic activity lack potency and produce substantial side effects, there is growing interest in the development of senolytic agents that selectively eliminate senescent over healthy cells. The present project will tackle this problem by establishing a new, chemistry-driven approach for the assembly of unique Bispecific T-Cell Engagers (BiTEs) with senolytic activity.This will be achieved by designing two orthogonal reactions that are specific to cysteine (one of them exclusively targeting N-terminal Cys and therefore allowing asymmetric ligation), for the assembly of a library of bispecific antibodies (bsAbs) from individual Cys-tagged monoclonal antibodies (mAbs) and avoiding the need to clone, express, purify and test each bsAbs one by one. The approach will then consist on expressing two sets of Cys-tagged antibodies, one targeting cell-surface antigens on hematopoietic and immune cells, and the other targeting senescent cells, which will be ligated to each other through designed linkers into bsAbs specific to senescent and T-cells antigens, and therefore capable to trigger an immune cytotoxic response towards senescent cells. To profile this library in vitro, the immunomodulatory properties of the senolytic BiTEs will be inferred from cell lysis and viability assays from cocultures of senescent cells and peripheral blood mononuclear cells. Following this, the immunomodulatory effects of selected bsAbs with senolytic activity will be elucidated using -omics and immunological tools. The technology developed will not be limited to bsAbs but will allow being extended to the ligation of any type of proteins, enabling future applications in protein-based drug discovery.