Engineering of computational receptors and gene circuits for T-cell immunotherapy

T 细胞免疫治疗的计算受体和基因电路工程

• 批准号: 8415761
• 负责人: Yvonne Yu-Hsuan Chen
• 金额: $ 30.14万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-25 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415761
• 关键词: Address; Adoptive Transfer; Antibodies; Antigen Receptors; Antigen Targeting; Antigens; Autologous; Biological Assay; Blood Cells; Cancer Patient; Cell-Mediated Cytolysis; Cells; Chromium; Chronic; Chronic Lymphocytic Leukemia; Complex; Coupled; Cues; Cytolysis; DNA; Detection; Disease; Disorder by Site; Effectiveness; Elements; Engineering; Evaluation; Extracellular Domain; Genetic; Genetic Transcription; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Human; Hypoxia; Immune; Immune response; Immune system; Immunosuppressive Agents; Immunotherapy; In Vitro; Indolent; Interleukin-2; Logic; Lymphocyte; Malignant Neoplasms; Metastatic Melanoma; Methods; Mus; Normal Cell; Operative Surgical Procedures; Patients; Performance; Predisposition; Probability; Process; Production; Proteins; Radiation; Radiation therapy; Receptor Gene; Recruitment Activity; Refractory; Refractory Disease; Research; Resistance; Resort; Safety; Signal Transduction; Site; Solid; Specificity; Staining method; Stains; Surface; System; T cell therapy; T-Cell Immunologic Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Toxic effect; Transforming Growth Factor beta; Treatment Efficacy; Tumor Antigens; Western Blotting; Xenograft Model; base; biological systems; cancer immunotherapy; cell killing; chemotherapy; clinical application; clinical efficacy; cytokine; cytotoxic; cytotoxicity; granzyme B; improved; in vitro Assay; in vivo; information processing; killings; neoplastic cell; next generation; novel; novel therapeutics; perforin; prevent; programs; promoter; receptor; receptor expression; response; synthetic biology; tool; treatment strategy; tumor; tumor eradication; tumor specificity; tumor xenograft

DESCRIPTION (provided by applicant): Adoptive T-cell therapy is a promising treatment strategy for cancers resistant to conventional methods including surgery, chemotherapy, and radiation therapy. In particular, the adoptive transfer of T cells genetically modified to express tumor-targeting chimeric antigen receptors (CARs) has shown clinical efficacy by redirecting T-cell specificity toward indolent tumors. However, important challenges remain in the use of CAR-modified T cells, including off-target toxicity toward normal cells and susceptibility to mutational escape by targeted tumors. The goal of this research is to improve the safety and efficacy of adoptive T-cell therapy by engineering more robust and versatile tumor-targeting T cells, which will be achieved through two specific aims. In Specific Aim 1, next-generation CARs capable of logical computation of multiple input signals will be developed. OR-gate CARs that trigger T-cell-mediated cytotoxicity in response to multiple tumor-associated antigens will be developed to lower the probability of mutational escape (i.e., loss of all targeted antigens) by tumor cells. AND- and NOT-gate CARs that trigger cytotoxicity only in the presence of the correct combination of antigens will be constructed to lower off-target toxicity toward normal cells. In Specific Aim 2, inducible transcription systems responsive to tumor-specific environmental cues-including hypoxia and increased local concentrations of the immunosuppressive cytokine transforming growth factor beta (TGF- ¿)-will be constructed to express gene products that enhance anti-tumor immune responses, including increased T-cell proliferation and the recruitment of native immune system components to tumor sites. These inducible transcription systems will be combined with the logic-gate CARs developed in Specific Aim 1 to generate tumor-targeting T cells capable of both executing and recruiting robust anti-tumor responses to diseased targets. The proposed receptors and transcription systems will be constructed using rapid, modular DNA assembly technologies developed in the field of synthetic biology. The novel genetic constructs will be stably integrated into established and primary human T cells via lentiviral transduction to enable performance characterization and system optimization. In vitro assays including western blots, surface and intracellular antibody staining, cytokine production profiling, and chromium release (cell lysis) assays will be performed to ascertain the expression and functional activities of new receptors and transcription systems. Constructs showing robust in vitro performance will be further examined in tumor xenograft models in mice to evaluate their effects on tumor eradication by modified T cells. This research aims to address a critical barrier to progress in T-cell therapy 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. PUBLIC HEALTH RELEVANCE: Adoptive T-cell therapy for cancer, or the use of tumor-targeting T cells derived or modified from cancer patients' own blood cells, has shown effectiveness in treating diseases such as metastatic melanoma and B- cell leukemia that are resistant to conventional therapies7, 8. However, adoptive T-cell therapy has been limited to experimental trials thus far due to imperfections in tumor-targeting specificity and a general lack of control over the fate and function of engineered T cells after introduction into patients. The proposed research aims to improve the safety and efficacy of T-cell therapy by engineering T cells with more precise and robust tumor targeting and eradication capabilities, thus contributing to the advancement of a new therapeutic strategy against currently incurable diseases.

描述（由申请人提供）：过继性 T 细胞疗法是一种很有前景的治疗策略，用于治疗对手术、化疗和放射治疗等传统方法耐药的癌症。特别是，经过基因改造以表达肿瘤靶向嵌合抗原受体（CAR）的T细胞的过继转移通过将T细胞特异性重定向到惰性肿瘤而显示出临床功效。然而，CAR 修饰的 T 细胞的使用仍然存在重要挑战，包括对正常细胞的脱靶毒性以及对靶向肿瘤突变逃逸的敏感性。这项研究的目标是通过设计更强大和多功能的肿瘤靶向 T 细胞来提高过继性 T 细胞疗法的安全性和有效性，这将通过两个具体目标来实现。在具体目标1中，将开发能够对多个输入信号进行逻辑计算的下一代CAR。将开发针对多种肿瘤相关抗原触发 T 细胞介导的细胞毒性的 OR 门 CAR，以降低肿瘤细胞突变逃逸（即丢失所有目标抗原）的可能性。 AND门和NOT门CAR仅在存在正确抗原组合的情况下才触发细胞毒性，将被构建以降低对正常细胞的脱靶毒性。在具体目标2中，将构建对肿瘤特异性环境线索（包括缺氧和免疫抑制性细胞因子转化生长因子β（TGF-¿）局部浓度增加）做出反应的诱导转录系统，以表达增强抗肿瘤免疫反应的基因产物，包括增加T细胞增殖和将天然免疫系统成分招募到肿瘤位点。这些诱导转录系统将与 Specific Aim 1 中开发的逻辑门 CAR 相结合，生成肿瘤靶向 T 细胞，能够对患病靶点执行和招募强大的抗肿瘤反应。所提议的受体和转录系统将使用合成生物学领域开发的快速、模块化 DNA 组装技术来构建。新型基因构建体将通过慢病毒转导稳定整合到已建立的原代人类 T 细胞中，以实现性能表征和系统优化。体外测定，包括蛋白质印迹、表面和细胞内抗体染色， 将进行细胞因子产生分析和铬释放（细胞裂解）测定，以确定新受体和转录系统的表达和功能活性。将在小鼠肿瘤异种移植模型中进一步检查显示出强大体外性能的构建体，以评估其对修饰 T 细胞根除肿瘤的影响。这项研究旨在通过从头开始解决癌症 T 细胞疗法进展的关键障碍。 构建以前在 T 细胞治疗工具箱中无法获得的多功能遗传结构，从而产生具有更强大和更精确的靶向抗肿瘤活性的 T 细胞，用于癌症免疫治疗。 公共健康相关性：癌症过继性 T 细胞疗法，或使用从癌症患者自身血细胞衍生或修饰的肿瘤靶向 T 细胞，已显示出治疗对传统疗法耐药的转移性黑色素瘤和 B 细胞白血病等疾病的有效性7, 8。然而，由于肿瘤靶向特异性的不完善和普遍缺乏，迄今为止过继性 T 细胞疗法仅限于实验性试验。 控制工程化 T 细胞进入患者体内后的命运和功能。拟议的研究旨在通过改造T细胞使其具有更精确和更强的肿瘤靶向和根除能力来提高T细胞治疗的安全性和有效性，从而有助于针对目前无法治愈的疾病开发新的治疗策略。