Engineering the ER Stress Response to Promote the Survival and Cytotoxic Specificity of CAR T cells

项目总结/摘要 嵌合抗原受体（CAR）T细胞彻底改变了癌症疗法并表现出令人难以置信的效果 在复发性或难治性B细胞癌患者中的反应，但在实体瘤中无效。为了 在实体瘤中工作，CAR T细胞必须能够在免疫抑制性肿瘤微环境中存活 (TME)并区分肿瘤细胞和健康组织上的抗原。这两个障碍 可以通过使用已知的生物电路对T细胞进行巧妙的合成工程来克服。我们有 确定了一种对多种类型白细胞的生存和功能至关重要的途径， 然而，令人惊讶的是，在CD 8 + T细胞的活化过程中，它在肿瘤微环境中不被活化。在这里， 我建议合成激活这一途径，以促进CD 8 + CAR T细胞的细胞毒性和靶向， 肿瘤特异性。具体来说，我将首先探讨这一信号通路的多个方面，以及它的能力， 在肿瘤微环境中的应激下促进CAR T细胞存活和细胞毒性。作为第二 为了提高CAR-T细胞对实体瘤的特异性，我将设计一种结构， CAR响应转录后调节。转录后调节的益处包括： 激活和放大信号。通过使转录后调节因子和靶基因 依赖于结合靶细胞抗原，我将在功能上创建一个“与”逻辑门，需要刺激 从2个单独的抗原，以促进CAR T细胞杀伤。对于这两种策略，我将首先展示它们的 在体外使用模拟TME条件的原代小鼠和人细胞，然后在体内使用 同系移植黑素瘤小鼠模型。总的来说，我假设这个关键的合成激活 T细胞途径将增强CAR-T行为和在固体TME中的控制。

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cells have revolutionized cancer therapies and show incredible responses in patients with relapsed or refractory B cell cancers, yet are ineffective in solid tumors. In order to work in solid tumors, CAR T cells must be able to survive the immunosuppressive tumor microenvironment (TME) and distinguish between antigens presented on tumor cells and healthy tissue. Both of these obstacles can be overcome through clever synthetic engineering of the T cell using known biological circuits. We have identified a pathway critical to the survival and function of multiple types of leukocytes and naturally turned on during activation of CD8+ T cells, however surprisingly, it is not activated in the tumor microenvironment. Here, I propose to synthetically activate this pathway in order to promote CD8+ CAR T cell cytotoxicity and on-target, on-tumor specificity. Specifically, I will first explore multiple aspects of this signaling pathway and its ability to promote CAR T cell survival and cytotoxicity under the stresses in the tumor microenvironment. As a second strategy to improve CAR-T cell specificity for solid tumors, I will engineer a construct that selectively activates a CAR in response to post-transcriptional regulation. Benefits of post-transcriptional regulation include speed of activation, and amplification of signal. By making both the post-transcriptional regulator and the target dependent on binding target cell antigens, I will functionally create an “AND” logic gate, requiring stimulation from 2 separate antigens in order to facilitate CAR T cell killing. For both strategies I will first demonstrate their efficacy in vitro with primary mouse and human cells simulating TME conditions, and then in vivo using a syngeneic transplanted melanoma mouse model. Overall, I hypothesize that synthetic activation of this critical T cell pathway will enhance CAR-T behavior and control in the solid TME.