Project 2 - Preclinical studies: Overcoming tumor heterogeneity

项目2.临床前试验 摘要 非小细胞肺癌(NSCLC)和恶性胸膜间皮瘤(MPM)是高度致命的癌症， 次优疗法。本计划项目和项目2的目标是成功实施 表达嵌合抗原受体(CARS)的T细胞，这一方法在 白血病。我们的团队是首批启动CAR T细胞试验的人之一，通过靶向 肿瘤相关抗原--间硫蛋白。然而，尽管显示出安全性和可行性，但治疗效果 是有限的。我们推测，限制实体肿瘤治疗成功的重要问题之一是肿瘤抗原。 异质性。即使在基线水平，也可能不是所有的肿瘤细胞都会表达靶向抗原。这个 该项目的主要重点将是探索克服实体瘤T细胞的异质性的方法 细胞疗法。我们的第一个方法将是攻击肿瘤间质。我们之前展示了CAR T细胞 靶向小鼠成纤维细胞激活蛋白(FAP)对肿瘤成纤维细胞的作用可减少肿瘤基质， 增强内源性肿瘤内T细胞，以最小的毒性减缓肿瘤生长。在目标1中，我们建议 研究一种可用于我们的临床试验的靶向人类FAP的汽车。在目标1A中，我们将评估 人CAF与人CAF结合的新小鼠模型对抗人CAR T细胞免疫效果的影响 免疫缺陷小鼠的肿瘤。在目标1B中，我们将评估抗人FAP CAR T细胞在体外的安全性 原代人类细胞系。在AIM 1C中，我们将评估使用基质靶向治疗的联合疗法 (FAP-CAR)和肿瘤细胞靶向CAR(Mesothelin-CAR)。在目标2中，我们将探讨以下关键问题 间甲肾上腺素和FAP汽车是否能够产生旁观者效应。在目标2a中，我们将研究CAR- 注射不同比例表达或不表达肿瘤细胞的小鼠诱导旁观者效应 并将旁观者效应定义为非靶向肿瘤细胞仍然允许 消除肿瘤。建议进行更多的研究来确定旁观者效应的机制 发生。在目标2B中，将通过测定中和的能力来研究表位的扩散过程 FAP-CARS诱导或刺激针对表达异种抗原的肿瘤的内源性T细胞 病毒抗原和一种新的抗原。我们将定义存在的基准表位扩散的量 四聚体实验和T细胞交叉提呈增殖实验。在目标3中，我们将探索以下方法 免疫激活剂可增强CAR诱导的抗原扩散，如抗PD1和 CTLA-4或IDO抑制剂(AIM 3A)，并通过遗传改变CAR T细胞来产生FMS样酪氨酸 激酶3配体(Flt3L)，一种强大的树突状细胞诱导剂(Aim 3B)。成功完成这些任务 研究将为实体瘤的T细胞过继转移领域提供关键信息(特别是在 关于克服肿瘤抗原异质性这一研究很少的问题)，并提供重要数据 为项目1中的临床试验设计提供信息。 1

Project 2. Preclinical Trials Abstract Non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) are highly lethal cancers with suboptimal therapies. The goal of this Program Project and of Project 2 is to successfully implement the use of T cells expressing chimeric antigen receptors (CARs), an approach that has been highly successful in leukemias. Our group was among the first to inititate CAR T cell trials focused on solid tumors by targeting the tumor-associated antigen mesothelin. However, despite showing safety and feasibility, therapeutic efficacy was limited. We hypothesize that one of the important issues limiting success in solid tumors is tumor antigen heterogeneity. Even at baseline, it is likely that not all tumor cells will express the targeted antigen. The primary focus of this Project will be to explore ways to overcome heterogeneity in solid tumor CAR T cell therapy. Our first approach will be to attack the tumor stroma. We previously showed that CAR T cells targeted to mouse fibroblast activation protein (FAP) on tumor fibroblasts can decrease the tumor matrix, augment endogenous intratumoral T cells, and slow tumor growth with minimal toxicity. In Aim 1, we propose to study a CAR targeted to human FAP that can be used in our clinical trial. In Aim 1A, we will assess the efficacy of the anti-human-CAR T cells using a new mouse model which combines human CAFs and human tumors in immunodeficient mice. In Aim 1B, we will evaluate the safety of anti-human FAP CAR T cells on primary human cell lines. In Aim 1C, we will evaluate combination therapies using the stromal-targeted CAR (FAP-CAR) with a tumor-cell targeted CAR (mesothelin-CAR). In Aim 2, we will explore the key issue of whether mesothelin and FAP CARs are able to induce bystander effects. In Aim 2A, we will study CAR- induced bystander effects by injecting mice with varying ratio's of tumor cells expressing or not expressing mesothelin and defining bystander effect as the minimal percentage of non-targeted tumor cells that still allow tumor elimination. Additional studies are proposed to determine the mechanisms by which the bystander effect occurs. In Aim 2B, the process of epitope spreading will be studied by determining the ability of Meso- and FAP-CARs to induce or stimulate endogenous T cells directed against tumors expressing a xenoantigen, a viral antigen, and a neoantigen. We will define the amount of baseline epitope spreading that is present using tetramer assays and a T cell cross presentation proliferation assay. In Aim 3, we will explore ways in which CAR-induced antigen spreading can be augmented by immune activators such as antibodies to PD1 and CTLA-4 or an IDO inhibitor (Aim 3A) and by genetically altering CAR T cells to produce FMS-like tyrosine kinase 3 ligand (FLT3L), a powerful inducer of dendritic cells (Aim 3B). Successful completion of these studies will provide key information for the field of adoptive transfer of T cells for solid tumors (especially with regard to the very understudied issue of overcoming tumor antigen heterogeneity) and provide important data to inform the designs of the clinical trials in Project 1. 1