Focused ultrasound control of intratumoral NKG2DL BTE production by CART cells to potentiate epitope spread

项目摘要 嵌合抗原受体（CAR）T细胞疗法正在改变血液恶性肿瘤的临床护理; 然而，它们在治疗实体瘤方面显示出有限的临床成功。目前正在作出各种努力， 扩大CAR T细胞在不同癌症类型中的应用，但它们在治疗实体瘤方面的有效性 由于实体瘤是异质性的，因此受到限制;通常使用CAR T细胞靶向单一抗原， 导致肿瘤抗原逃逸，导致疾病复发。虽然系统性地提供 免疫调节剂可增强抗肿瘤反应，但也可导致非免疫性疾病的衰弱性不良事件。 靶向、肿瘤外毒性、细胞因子释放综合征，以及在某些情况下，危及生命的自身免疫。通过 相比之下，免疫调节剂的肿瘤内给药已显示可降低全身暴露， 提高系统治疗的功效。针对肿瘤抗原的免疫力，由肿瘤抗原表位扩散引起， 肿瘤细胞杀伤，导致表位特异性从最初的表位多样化-启动内源性肿瘤细胞。 免疫系统对更大的目标池。该提案寻求使用热敏CAR T细胞， 在肿瘤内运输和浸润，以在肿瘤内产生双特异性T细胞增殖物（BTE）并诱导 表位通过内源性抗肿瘤T细胞的DC引发而扩散，以产生强大的免疫力。聚焦超声 (FUS)将用于在异质性肿瘤内空间存款热量并激活工程化的CAR T细胞 具有热基因开关（TS），其允许热触发控制鼠T细胞中的转基因表达。 TS构建体在体温（37°C）下是转录沉默的，但在暴露于短时间的热刺激后，TS构建体在体温（37°C）下是转录沉默的。 加热（40-42 ℃，10分钟），表达转基因至高于基础水平200倍水平。这些研究 将专注于BTE靶向自然杀伤组2，成员D配体（NKG 2DL），这是压力诱导的 在肿瘤细胞以及免疫抑制细胞上高度表达的抗原。临床前评价将 在完全免疫活性小鼠的乳腺癌鼠模型中进行。预计这一做法将 显著增强局部CAR T细胞对异源性肿瘤的活性，同时增强内源性CAR T细胞对异源性肿瘤的活性， 抗肿瘤免疫为了评估这一点，将通过DC介导的内源性T细胞的引发进行表位扩散。 研究了成功完成这项研究的目的，预计将阐明机制， CAR T细胞的BTE分泌可以引发内源性T细胞免疫。

Project Summary Chimeric antigen receptor (CAR) T cell therapies are transforming clinical care of hematological malignancies; however, they have shown limited clinical success in treating solid tumors. Numerous efforts are underway to expand the use of CAR T cells for different cancer types, but their effectiveness in treating solid tumors have been limited by the fact that solid tumors are heterogenous; targeting a single antigen using CAR T cells typically results in tumor antigen escape, leading to relapse of the disease. Although systemic delivery of immunomodulators can potentiate anti-tumor responses, it can also lead to debilitating adverse events from on- target, off-tumor toxicity, cytokine release syndrome, and in some cases, life-threatening autoimmunity. By contrast, intratumoral administration of immunomodulators has been shown to reduce systemic exposure while improving efficacy of systemic therapies. Immunity against tumor antigens, resultant of epitope spreading after tumor cell killing, leads to a diversification of epitope specificity from the initial epitope – priming the endogenous immune system against larger pool of targets. This proposal seeks to use thermal sensitive CAR T cells, which traffic and infiltrate deep within tumors, to intratumorally produce bispecific T cell engagers (BTEs) and induce epitope spread by DC priming of endogenous anti-tumor T cells to develop robust immunity. Focused ultrasound (FUS) will be used to spatially deposit heat within heterogenous tumors and activate CAR T cells engineered with a thermal gene switch (TS) which allows for heat-triggered control of transgene expression in murine T cells. TS constructs are transcriptionally silent at body temperature (37°C) but after exposure to a short duration of heat (10 min. at 40–42°C), express transgenes to levels greater than 200-fold above basal levels. These studies will focus on BTEs targeting natural killer group 2, member D ligands (NKG2DL), which are stress-induced antigens highly expressed on tumor cells as well as on immunosuppressive cells. Preclinical evaluation will be performed in murine models of breast cancer in fully immunocompetent mice. This approach is expected to significantly enhance local CAR T cell activity against heterogenous tumors, while potentiating endogenous antitumor immunity. To assess this, epitope spread by DC mediated priming of the endogenous T cells will be investigated. Successful completion of the aims of this study is expected to elucidate mechanisms by which local BTE secretion by CAR T cells can prime endogenous T cell immunity.