Simultaneous Targeting of Tumor and Stroma Cells to Enhance Solid Tumor CAR-T Cell Therapy

摘要 使用经工程改造以表达特异性嵌合抗原受体（汽车）的T细胞来治疗癌症， 为多种类型的癌症产生了持久的治疗方法，并导致了第一个FDA批准的CAR-T细胞疗法， 治疗儿童急性淋巴细胞白血病。尽管取得了这一成功，但CAR-T免疫疗法仍被 在针对实体瘤方面效果差得多。这种有限的成功部分源于实体瘤 微环境，其形成免疫细胞浸润的物理屏障，并产生可溶性因子， 下调T细胞活性并加速T细胞耗竭。虽然针对实体瘤的免疫疗法 最初有效，肿瘤微环境对T细胞的抑制阻止了这些治疗产生 持久的反应。在本申请中，我们提出了一种新的CAR-T细胞疗法，旨在改善 通过克服困扰当前CAR-T细胞的缺陷， 治疗为此，我们将设计T细胞以表达多种汽车，使这些细胞能够靶向肿瘤。 细胞和免疫抑制肿瘤微环境中的细胞。具体来说，我们将利用非病毒， Tc Buster DNA转座子系统插入含有多个汽车的大型多顺反子遗传构建体 和选择标记物进入T细胞。利用这个平台，我们将产生具有靶向间皮素的汽车的T细胞， （MSLN，一种由80-85%的胰腺癌肿瘤表达的蛋白质）和成纤维细胞活化蛋白（FAP，一种 肿瘤微环境中的癌症相关成纤维细胞的标志物。然后我们将选择一个纯种群， 在一些实施方案中，本发明涉及表达MSLN和FAP-CAR的T细胞，并在体外确定这些细胞的活性和特异性。我们 预期工程化T细胞将产生特异性和强有力的反应，仅引发细胞毒性功能， 针对表达其靶抗原的细胞。然后，我们将确定工程化T细胞在体内的功效， 使用异种移植物小鼠模型产生MSLN和FAP阳性胰腺癌，随后进行过继性移植， 转移T细胞。我们预期表达FAP-CAR和MSLN的双特异性T细胞的免疫递送。 汽车将引发针对MSLN+/FAP+实体瘤的稳健且持久的T细胞应答，从而导致肿瘤免疫应答。 收缩和增加存活率。此外，我们期望开发一种灵活、高效和可靠的 用单一的非病毒基因递送方法产生双特异性T细胞的过程将促进 新的疗法，以克服许多问题，今天面临的工程T细胞治疗，包括抗原逃逸， 和目标特异性。

Abstract The use of T cells engineered to express specific chimeric antigen receptors (CARs) to treat cancer has generated durable cures for many types of cancer and resulted in the first FDA approved CAR-T cell therapy to treat childhood acute lymphoblastic leukemia in 2017. Despite this success, CAR-T immunotherapies have been much less effective at targeting solid tumors. Part of this limited success stems from the solid tumor microenvironment, which forms a physical barrier to immune cell infiltration and produces soluble factors that downregulate T cell activity and accelerate T cell exhaustion. While immunotherapies targeting solid tumors are initially effective, the tumor microenvironment’s inhibition of T cells prevents these treatments from producing durable responses. In this application, we propose a novel CAR-T cell therapy aimed to improve outcomes for patients with advanced stage pancreatic cancer by overcoming the deficiencies that plague current CAR-T cell therapies. To this end, we will engineer T cells to express multiple CARs, enabling these cells to target tumor cells and cells in the immune-suppressive tumor microenvironment. Specifically, we will leverage the non-viral, Tc Buster DNA transposon system to insert a large multicistronic genetic construct containing multiple CARs and a selection marker into T cells. Using this platform, we will generate T cells with CARs targeting mesothelin (MSLN), a protein expressed by 80-85% of pancreatic cancer tumors, and fibroblast activation protein (FAP), a marker of cancer associated fibroblasts in the tumor microenvironment. We will then select a pure population of T cells expressing MSLN- and FAP-CARs and determine the activity and specificity of these cells in vitro. We expect that engineered T cells will generate a specific and robust response, eliciting cytotoxic functions only against cells expressing their target antigen. We will then determine the efficacy of engineered T cells in vivo using a xenograft mouse model to generate MSLN and FAP positive pancreatic carcinomas followed by adoptive transfer of T cells. We expect immunotherapeutic delivery of bispecific T cells expressing FAP-CARs and MSLN- CARs will elicit a robust and long-lasting T cell response against MSLN+/FAP+ solid tumors resulting in tumor shrinkage and increased survival. Furthermore, we expect the development of a flexible, efficient, and reliable process to generate bispecific T cells with a single, non-viral gene delivery approach will facilitate the emergence of novel therapies to overcome many issues facing engineered T cell therapy today, including antigen escape and target specificity.