Project 3 - Immune analysis of clinical trial samples

项目3：临床试验样本的免疫分析。 摘要 与所见相比，CAR疗法治疗实体肿瘤不成功的原因 在血液系统恶性肿瘤中，它是免疫治疗领域中最重要的问题之一。 可整合临床试验(项目1)、临床前研究(项目2)和深入生物相关的方案 需要对从这些人体试验(本项目)中提取的样本进行研究。如项目1所述，我们的 新的临床试验将使用优化的介体素靶向CAR，M5 huCART-MESO，已经被 构建为包含新的抗间充质蛋白单链抗体片段，该片段完全是人的(选自噬菌体 显示库)。M5 huCART-MESO也比我们之前的SS1-Mesothelin Car有更好的临床前疗效 (见项目1)。我们计划的第二个临床试验将使用一种名为huCART-FAP的CAR，它直接针对肿瘤中的成纤维细胞 靶向成纤维细胞激活蛋白(FAP)的基质。项目3的目标是进行与生物相关的研究 这些临床试验中。我们将重点关注两个主要领域。在目标1中，我们将进行研究，以了解我们的 项目1中的CAR T细胞持续存在并向肿瘤转移。我们将分析我们的血液样本 然而，核心B的患者，我们假设，研究汽车通向肿瘤的能力甚至更多 危急时刻。从肿瘤活检组织中分离的CAR T细胞将对其表型和功能进行鉴定。此外, 将检查肿瘤的组织学变化、靶抗原表达和转录图谱。 在项目1中提议的试验中，每个患者将接受治疗后的活组织检查，以帮助确保 从肿瘤中提取的材料可用于此类研究。在目标2和目标3中，我们将进行旨在 在研究表位扩散方面，这是一个关键的，但研究不足的领域，采用T细胞转移。在目标2中， 我们将评估在我们的临床试验中使用的CAR T细胞可以增强/促进 内源性T细胞对共有肿瘤抗原(Aim 2A)和新抗原(Aim 2B)的免疫。在目标2a中， 来自已知肿瘤抗原的重叠多肽文库以及已定义的多肽将被用于询问 PBMC对共享的肿瘤抗原的反应。在目标2B中，我们将使用下一代测序(NGS) 肿瘤识别候选新表位。串联的微基因结构，以及合成肽 编码这些新表位，将被用来询问外周血(在少数情况下，直到)以寻找新的或 CAR治疗后T细胞免疫功能增强。项目3中的目标2链接到项目2中的目标2，其中 研究人员将使用动物模型来寻找抗原传播的存在和增强这种传播的方法。 效果。最后，在目标3中，我们将检验一个假设，即汽车介导的表位传播促进了 利用NSG方法鉴定肿瘤中抗原特异性T细胞反应的不同谱系 来自血液的共享/新抗原特异性T细胞系的克隆型。这些TCR参考库将是 用于询问CAR治疗前后血液/肿瘤活检样本的特征 CAR介导的表位扩散促进肿瘤内呼吸和T细胞免疫成分。

Project 3. Immune Analysis of Clinical Trial Samples. Abstract The reasons for the lack of therapeutic success of CAR therapy for solid tumors, compared to that seen in hematologic malignancies, represents one of the most important questions in the field of immunotherapy. Programs that can integrate clinical trials (Project 1), preclinical studies (Project 2) and in-depth biocorrelate studies of samples derived from these human trials (this Project) are needed. As described in Project 1, our new clinical trial will use an optimized mesothelin targeted CAR, M5 huCART-meso, that has been constructed to contain a new anti-mesothelin scFv fragment that it is fully human (selected from a phage display library). M5 huCART-meso also had better preclinical efficacy than our previous SS1-mesothelin CAR (see Project 1). Our second planned clinical trial will use a CAR, huCART-FAP, directed to fibroblasts in tumor stroma by targeting fibroblast activation protein (FAP). The goal of Project 3 is to conduct biocorrelate studies of these clinical trials. We will focus on two main areas. In Aim 1, we will conduct studies to learn whether our CAR T cells in Project 1 are persisting and trafficking to tumors. We will analyze blood samples from our patients in Core B, however, we hypothesize that studying the ability of CARs to traffic to tumors is even more critical. CAR T cells isolated from tumor biopsies will be characterized for phenotype and function. In addition, the tumor will be examined for histological changes, target antigen expression, and transcriptionally profiled. Post-treatment biopsies will be expected from each patient in the trials proposed in Project 1 helping to ensure that materials from tumors are available for such studies. In Aims 2 and 3, we will conduct experiments aimed at studying epitope spreading which is a crucial, yet an understudied area of adoptive T cell transfer. In Aim 2, we will evaluate the hypothesis that the CAR T cells used in our clinical trials can enhanced/promote endogenous T cell immunity to shared tumor antigens (Aim 2A) and neoantigens (Aim 2B). In Aim 2A, overlapping peptide libraries from known tumor antigens as well as defined peptides will be used to interrogate PBMC for responses to shared tumor antigens. In Aim 2B, we will use next generation sequencing (NGS) of tumors to identify candidate neoepitopes. Tandem mini-gene constructs, as well as synthetic peptides encoding these neoepitopes, will be used to interrogate peripheral blood (and in limited cases, TIL) for new or enhanced T cell immunity after CAR therapy. Aim 2 in Project 3 is linked to Aim 2 in Project 2, where investigators will use animal models to look for the presence of antigen spreading and ways to augment this effect. Finally, in Aim 3 we will examine the hypothesis that CAR-mediated epitope spreading promotes a diverse repertoire of antigen-specific T cell responses in tumors by utilizing NSG methods to characterize TCR clonotypes in shared/neoantigen-specific T cell lines derived from blood. These TCR reference libraries will be employed to interrogate pre- and post- CAR T therapy in blood/tumor biopsy samples in order to characterize the intra-tumoral breath and composition of T cell immunity promoted by CAR-mediated epitope spreading.