Abstract：
Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VH domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.
摘要：
双特异性和多特异性药物已越来越多地用于癌症治疗和免疫疗法。然而，他们复杂的设计参数对开发成功的疗法提出了挑战。只有当两个相对细胞上的受体交联的双特异性细胞在空间上紧密接近时，才能提供受体的特异性激活。例如肿瘤中的免疫细胞和癌细胞。这些特工,包括T细胞激活双特异性，通过利用肿瘤靶标聚集T细胞受体以获得选择性共刺激信号，可以通过仅在肿瘤微环境中激活来避免肿瘤外毒性。这里,我们研究了一组PD-1/CD137靶向HumabodyVH结构域，以确定T细胞活化的关键因素，比如亲和力，价,表达水平，域取向,和表位位置。靶表达是决定T细胞活化的特异性和效力的主要因素。给定内在表达水平，可以调节亲和力以调节活化水平和IC50并实现低和高表达水平之间的特异性。改变表位位置和接头长度显示在低表达水平下对活化的微小改善,但是增加目标的效价会降低所有表达水平的激活。通过组合靶标的非重叠表位，我们在低表达水平下实现了更高的受体激活。动力学模型能够捕捉到这些趋势，为机械解释提供支持。这项工作提供了通过细胞交联双特异性试剂量化T细胞活化因子的框架和新试剂设计的指导原则。
