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

通过重新连接 TGF-β 信号传导设计稳健的过继性 T 细胞癌症治疗

• 批准号: 8648535
• 负责人: ZeNan L Chang
• 金额: $ 3.77万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648535
• 关键词: Address; Angiostatins; Antibodies; Binding; Biological Assay; Candidate Disease Gene; Cell Cycle Arrest; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Clinical; Coculture Techniques; Coupled; Cytolysis; Cytotoxic T-Lymphocytes; DNA; Defense Mechanisms; Deoxyuridine; Disease remission; Endostatins; Endothelial Cells; Engineering; Evaluation; Event; Extracellular Domain; Flow Cytometry; Galactosidase; Gene Expression; Genetic; Genetic Transcription; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Granzyme; Heterodimerization; Human; Immune; Immune system; Immunosuppression; Immunosuppressive Agents; Immunotherapy; In Vitro; Infiltration; Interferons; Interleukin-12; Interleukin-15; Jurkat Cells; Label; Leukocytes; Ligand Binding; Ligands; Link; MADH2 gene; Malignant Neoplasms; Measurement; Measures; Mediating; Microscopy; Molecular Cloning; Monitor; Mus; Nuclear Translocation; Output; Pathway interactions; Patients; Production; Proteins; Recruitment Activity; Refractory Disease; Regulatory T-Lymphocyte; Relapse; Reporter; Reporter Genes; Research; Resistance; Signal Pathway; Signal Transduction; Solid Neoplasm; Staining method; Stains; Surface; System; T cell response; T cell therapy; T-Cell Activation; T-Cell Development; T-Cell Proliferation; T-Lymphocyte; TEV protease; TNF gene; Techniques; Testing; Therapeutic; Time; Transcription Coactivator; Transforming Growth Factor beta; Treatment Efficacy; Up-Regulation; Venus; Western Blotting; Zinc Fingers; base; cancer therapy; cell mediated lymphocytolysis test; cellular engineering; colon cancer cell line; conventional therapy; cytokine; cytotoxicity; density; functional improvement; improved; in vitro Assay; in vitro activity; in vivo; interest; melanoma; migration; monocyte; mouse model; neoplastic cell; perforin; peripheral blood; programs; public health relevance; receptor; receptor expression; response; senescence; success; therapeutic effectiveness; tumor; tumor microenvironment

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.

描述（由申请人提供）：虽然过继性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，特别是在实体瘤的治疗中。本研究的目的是提高 通过解决两个相互关联的挑战来提高过继性T细胞疗法的有效性：对抗实体瘤微环境对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。肿瘤细胞过度产生转化生长因子β（TGF-β）抑制细胞毒性T细胞功能，并促进肿瘤微环境中的免疫抑制调节性T细胞发育和活性。我们假设高局部TGF-β浓度可以作为肿瘤微环境标志物，并且合成的TGF-β响应性转录系统可以被编程以降低TGF-β的内源性免疫抑制作用并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，将构建合成的TGF-β诱导型转录系统，该系统可以同时与内源性免疫抑制信号传导途径竞争，并将TGF-β的存在与可定制的转录输出的产生联系起来。在特定目标2中，多种候选基因将整合到合成转录系统中，以将TGF-β的存在与促进细胞内在T细胞活性偶联，包括加强T细胞活化，改善T细胞增殖和/或增强T细胞细胞毒性。在特定目标3中，合成转录系统将与遗传输出偶联，以募集天然免疫组分并改变肿瘤微环境。将使用等温DNA组装和标准分子克隆技术构建所提出的遗传编码的TGF-β诱导型转录系统。将与功能性遗传输出偶联的合成转录系统整合到已建立的和原代人T细胞中，用于使用定量实时PCR、蛋白质印迹、表面和细胞内抗体染色以及效应细胞/靶细胞共培养测定来体外评价适当表达和抗肿瘤活性，所述测定定量工程化T细胞对靶细胞溶解、细胞周期停滞和迁移的影响。蛋白质印迹和细胞内抗体染色也将用于评估合成转录系统对内源性TGF-β信号传导的抑制作用。具有最有希望的遗传输出和对内源性TGF-β信号传导活性的稳健抑制作用的系统将被整合到pmel-1小鼠模型中用于过继性T细胞治疗，以评估体内抗肿瘤T细胞功能和治疗功效的改善。该研究旨在通过重新构建T细胞治疗工具箱中以前无法使用的多功能遗传构建体来解决T细胞治疗癌症进展的关键障碍，从而产生具有更强大和精确靶向的抗肿瘤活性的T细胞用于癌症免疫治疗。