Image-guided irreversible electroporation directed CAR T-cell delivery to solid tumors

项目摘要 肿瘤浸润淋巴细胞（TIL）可以显著增加免疫治疗的成功率。实体瘤 对TIL的渗透和功能存在障碍。增加TIL及其功能的策略受到挑战 由于无法直接将T细胞输送到实体瘤中，并且缺乏预测其吸收的技术。 通过这一建议，我们将开发使用图像引导的不可逆电穿孔（IRE）作为一种新的 在实体瘤中促进TIL的方法。IRE在临床上用于治疗实体瘤， 微秒长的电脉冲通过细胞膜的快速透化作用杀死细胞。一个独特 IRE的特点是，在杀死细胞的同时，它在治疗过程中保留了细胞外基质和血管 区域，这是刺激局部炎症和促进TILs的理想选择。我们将调查我们的建议 在转化小鼠模型和恶性胸膜间皮瘤（MPM）患者中， 已经显示，其中细胞毒性TIL与免疫抑制细胞的比率较高的实体瘤， 提高患者生存率。在我们的实验中，我们将使用嵌合抗原受体（CAR）T细胞， 经遗传修饰以识别间皮素，一种在间皮瘤上过表达的细胞表面抗原 癌细胞基于PI团队先前的工作，我们合理化了IRE引起的细胞死亡：（i）可以成像 （ii）可以产生促进TIL的肿瘤内趋化因子梯度;和 (iii)因此，FDG-PET引导的IRE可以作为映射和驱动肿瘤靶向CAR T细胞的替代物， 交付.此外，我们的IRE引导的CAR T细胞浸润的策略可以使用电脉冲重复。 这些参数被设计为癌细胞靶向/TIL保留，从而增强免疫功效。 我们工作的长期目标是将图像引导的IRE开发为可预测的、可再现的和可扩展的平台。 CAR T细胞和其他细胞抗癌疗法向实体瘤的可控递送。在目标1中，我们 结合FDG-PET成像、计算机和生物信息学技术， 模拟和定量多重免疫荧光组织学，以开发一种工具，将确定T- 用IRE处理的实体瘤中的细胞定位。然后，iTIM将用于制定临床相关的 本发明涉及一种方案，其与静脉内施用T细胞的组合将用于增加TIL。在目标2中， 将使用PET（癌细胞）和生物发光成像（T细胞）的双重成像来验证IRE脉冲 参数来选择性地富集和增加MPM内的功能性CAR T细胞。在目标3中，我们将执行 在切除术前使用PET引导下经胸IRE对MPM进行初步临床试验，以验证iTIM的概念 及其对TILs的影响。用图像引导的IRE对肿瘤微环境进行调制， 定位T细胞递送，促进CAR T细胞功能，iTIMM的翻译验证提供了创新的 合理组合临床使用的技术，是一个雄心勃勃的尝试，以解决一个关键的挑战， 实体瘤免疫治疗

PROJECT ABSTRACT Tumor infiltrating lymphocytes (TILs) can significantly increase the success of immunotherapy. Solid tumors present barriers to TIL penetration and function. Strategies to increase TILs and their function are challenged by the inability to direct the delivery of T cells into solid tumors and a lack of techniques to predict their uptake. Through this proposal, we will develop the use of image-guided irreversible electroporation (IRE) as a novel approach to promote TILs in solid tumors. IRE is clinically used for the treatment of solid tumors by applying microsecond long electric pulses that kill cells through rapid permeabilization of the cell membrane. A unique feature of IRE is that, while killing cells, it spares the extracellular matrix and blood vessels within the treatment region, which is ideal for stimulating local inflammation and promoting TILs. We will investigate our proposed strategy in translational mouse models and in patients with malignant pleural mesothelioma (MPM), which is a solid tumor where a higher ratio of cytotoxic TIL to immunosuppressor cells has already been shown to increase patient survival. In our experiments, we will use chimeric antigen receptor (CAR) T cells that are genetically modified to recognize mesothelin, a cell surface antigen that is overexpressed on mesothelioma cancer cells. Based on prior work by the PIs' team, we rationalize that cell death from IRE: (i) can be imaged using intra-procedural FDG-PET; (ii) can generate an intratumoral chemokine gradient that promotes TILs; and (iii) therefore, FDG-PET guided IRE can serve as a surrogate to both map and drive tumor-targeted CAR T-cell delivery. Furthermore, our strategy of IRE-directed CAR T-cell infiltration can be repeated using electric pulse parameters designed to be cancer cell-targeted/TIL-sparing, thereby augmenting immunotherapeutic efficacy. The long-term goal of our work is to develop image-guided IRE as a platform for predictable, reproducible, and controllable delivery of CAR T cells and other cellular anticancer therapies to solid tumors. In Aim 1, we will create an integrated tumor immune microenvironment map (iTIMM) combining FDG-PET imaging, computer simulations, and quantitative multiplex immunofluorescence histology to develop a tool that will determine T- cell localization in solid tumors treated with IRE. iTIMM will then be used to develop a clinically relevant protocol that, in combination of intravenous administration of T cells, will be used to increase TILs. In Aim 2, we will use dual imaging with PET (cancer cells) and bioluminescence imaging (T cells) to validate IRE pulse parameters to selectively enrich and increase functional CAR T cells within MPM. In Aim 3, we will perform a pilot clinical trial using PET-guided transthoracic IRE of MPM prior to resection to validate the concept of iTIMM and its effect on TILs. The modulation of the tumor microenvironment with image-guided IRE to map and localize T-cell delivery, promoting CAR T cell function, and translational validation of iTIMM presents innovative rational combinations of clinically used techniques and is an ambitious attempt to address a key challenge to solid tumor immunotherapy.