Engineering robust adoptive T-cell cancer therapy by rewiring TGF-beta signaling

描述(申请人提供)：虽然采用T细胞疗法在治疗难治性疾病方面取得了巨大的临床成功，但许多患者的反应仍然是部分的，特别是在治疗实体瘤方面。这项研究的目标是提高 通过解决两个相互关联的挑战：对抗实体肿瘤微环境对T细胞的免疫抑制作用，以及通过转移的T细胞支持强大的、持久的、肿瘤局部的抗癌功能，过继T细胞疗法的有效性。肿瘤细胞过度产生转化生长因子β抑制细胞毒性T细胞功能，促进免疫抑制调节性T细胞在肿瘤微环境中的发育和活性。我们假设局部高浓度的转化生长因子β可作为肿瘤微环境标记物，合成的、转化生长因子β反应的转录系统可被编程为既可减少转化生长因子β的内源性免疫抑制效应，又可诱导强大的抗肿瘤反应。这些假设将通过三个具体目标进行检验。在具体目标1中，将构建一个人工合成的可诱导转化生长因子-β的转录系统，该系统可以同时与内源性免疫抑制信号通路竞争，并将转化生长因子-β的存在与可定制转录输出的产生联系起来。在特定的目标2中，各种候选基因将被整合到合成转录系统中，以将转化生长因子β的存在与促进细胞固有的T细胞活性结合起来，包括加强T细胞的激活，促进T细胞的增殖，和/或增强T细胞的细胞毒作用。在特定的目标3中，合成的转录系统将与招募本地免疫成分和改变肿瘤微环境的基因输出相耦合。建议的基因编码的转化生长因子可诱导转录系统将使用等温DNA组装和标准分子克隆技术来构建。与功能性基因输出相结合的合成转录系统将被整合到已建立的和原代人类T细胞中，用于体外评估适当的表达和抗肿瘤活性，方法包括定量实时聚合酶链式反应、蛋白质印迹、细胞表面和细胞内抗体染色以及量化工程T细胞对靶细胞溶解、细胞周期停滞和迁移的影响的效应/靶细胞共培养试验。免疫印迹和细胞内抗体染色将用于评估合成转录系统对内源性转化生长因子-β信号的抑制作用。具有最有前景的遗传输出和对内源性转化生长因子-β信号活性的强大抑制作用的系统将被整合到PMEL-1小鼠模型中进行过继T细胞治疗，以评估体内抗肿瘤T细胞功能和治疗效果的改善。这项研究旨在解决T细胞治疗癌症进展的关键障碍，通过从头构建T细胞治疗工具箱中以前没有的多功能基因结构，从而产生具有更强大和精确靶向的抗肿瘤活性的T细胞，用于癌症的免疫治疗。

DESCRIPTION (provided by applicant): While adoptive T-cell therapy has yielded instances of dramatic clinical success in treating refractory diseases, many patient responses remain partial, particularly in the treatment of solid tumors. The goal of this research is to improve the efficacy of adoptive T-cell therapies by addressing two interrelated challenges: countering the solid-tumor microenvironment's immunosuppressive effects on T cells, and supporting robust, persistent, tumor-localized anti-cancer functions by transferred T cells. Overproduction of transforming growth factor beta (TGF-¿) by tumor cells inhibits cytotoxic T-cell functions and promotes immunosuppressive regulatory T-cell development and activity in the tumor microenvironment. We hypothesize that high local TGF-¿ concentrations can serve as a tumor microenvironment marker and that a synthetic, TGF-¿-responsive transcription system can be programmed to both reduce TGF-¿'s endogenous immunosuppressive effects and induce robust anti- tumor responses. These hypotheses will be tested through three specific aims. In Specific Aim 1, a synthetic TGF-¿-inducible transcription system will be constructed that can simultaneously compete against the endogenous immunosuppressive signaling pathway and link the presence of TGF-¿ to the production of a customizable transcriptional output. In Specific Aim 2, a variety of candidate genes will be integrated into the synthetic transcription system to couple the presence of TGF-¿ to the promotion of cell-intrinsic T-cell activities, including strengthening T-cell activation, improving T-cell proliferation, and/or enhancing T-cell cytotoxicity. In Specific Aim 3, the synthetic transcription system will be coupled to genetic outputs that recruit native immune components and modify the tumor microenvironment. The proposed genetically encoded TGF-¿-inducible transcription systems will be constructed using isothermal DNA assembly and standard molecular cloning techniques. Synthetic transcription systems coupled to functional genetic outputs will be integrated into established and primary human T cells for in vitro evaluation of proper expression and anti-tumor activities using quantitative real-time PCR, western blots, surface and intracellular antibody staining, and effector/target cell co-culture assays that quantify the effects of engineered T cells on target cel lysis, cell-cycle arrest, and migration. Western blots and intracellular antibody staining will als be used to evaluate the synthetic transcription system's inhibitory effects on endogenous TGF-¿ signaling. Systems with the most promising genetic outputs and robust inhibitory effects on endogenous TGF-¿ signaling activities will be integrated into the pmel-1 mouse model for adoptive T-cell therapy to evaluate in vivo anti-tumor T-cell functions and improvements in therapeutic efficacy. This research aims to address a critical barrier to progress in T-cell therap for cancer by pursuing the de novo construction of multi-functional genetic constructs previously unavailable in the T-cell therapy toolbox, thereby generating T cells with more robust and precisely targeted anti-tumor activities for immunotherapy against cancer.