Mechanisms of treatment failure in chimeric antigen receptor T cell therapy

项目总结/摘要 使用表达嵌合抗原受体（CAR）的遗传修饰的T细胞的连续性免疫疗法是 对B细胞恶性肿瘤很有希望然而，不到一半的复发性非霍奇金淋巴瘤患者 淋巴瘤（NHL）在用靶向CD 19的CAR T细胞治疗后实现持久缓解。在一些 在某些情况下，这是由于免疫原性鼠CAR引起的输注细胞的靶抗原丢失或排斥所致 但在大多数情况下，治疗耐药性或初始反应后复发的原因仍然存在 不太了解。我们提出了一个计划，以阐明在最近启动的CAR治疗失败的原因 T细胞临床试验前仔细评估肿瘤的生物学特性和肿瘤微环境 以及患者T细胞和输注的CAR T细胞的表型。 作为我们的I/II期临床试验的一部分，在患有以下疾病的患者中进行了完全人第三代CD 20特异性CAR： 复发性或难治性B细胞NHL（由单独来源资助），所有患者均接受强制性肿瘤治疗 治疗前后的活检。这将使我们能够发现生物学特征， 对治疗的反应性，并评估肿瘤随时间的适应性变化，以揭示 免疫逃逸机制导致复发。我们将采用逐步的方法，使用状态- 最先进的方法，包括流式细胞术，单细胞RNA测序和基因表达 分析和多重免疫组织化学。我们已经组建了一个世界级的调查小组， 评估成功治疗的潜在障碍，包括肿瘤进入障碍，肿瘤浸润， 抑制性细胞、CAR T细胞暴露于抑制性配体或分泌蛋白以及CAR T细胞耗竭。 有大量的临床前数据表明，分化程度较低的T细胞亚群赋予了 与更分化的T细胞相比，具有上级体内扩增、持久性和抗肿瘤功效 子集最近一项小型试验的数据表明，这些分化程度较低的CD 8+细胞的频率， CAR T细胞制造前后的亚群与慢性乙型肝炎患者的临床反应相关 接受CD 19靶向CAR T细胞的淋巴细胞性淋巴瘤。这些结果具有重要意义，但 必须在其他设置中验证。我们将在白细胞分离术之前定量低分化T细胞亚型， 以及输注的CAR T细胞产物，并将这些特征与抗肿瘤反应相关联 以及体内扩增和持久性。 我们预计这些相关研究将对CAR-T细胞为什么能够在体内发挥作用的原因产生重要的见解。 治疗对于一些NHL患者是成功的，但对于其他患者不是。我们希望我们的发现将有助于指导 病人的选择和咨询，并告知未来的战略，以克服这些障碍，通过改善 细胞制造技术、CAR载体设计和/或组合辅助疗法，不仅用于 CD 20特异性CAR T细胞，但也用于其他靶点的CAR T细胞治疗。

PROJECT SUMMARY / ABSTRACT Adoptive immunotherapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is highly promising for B-cell malignancies. However, fewer than half of patients with relapsed non-Hodgkin lymphoma (NHL) achieve durable remissions following treatment with CD19-targeted CAR T cells. In some cases this results from target antigen loss or rejection of the infused cells due to immunogenic murine CAR components, but in most cases the causes of treatment resistance or relapse after an initial response remain poorly understood. We propose a plan to elucidate the reasons for treatment failure in a recently initiated CAR T cell clinical trial by carefully evaluating biological features of the tumor and tumor microenvironment before and after CAR T cell therapy as well as phenotypes of patient T cells and infused CAR T cells. As part of our phase I/II clinical trial of a fully human 3rd generation CD20-specific CAR in patients with relapsed or refractory B-cell NHL (funded by a separate source), all patients undergo mandatory tumor biopsies before and after treatment. This will allow us to discover biological characteristics predictive of responsiveness to treatment, and to evaluate adaptive changes in the tumor over time to reveal the mechanisms of immune escape leading to relapse. We will employ a step-wise approach using state-of- the-art methodologies, including multicolor flow cytometry, single-cell RNA sequencing and gene expression profiling, and multiplex immunohistochemistry. We have assembled a world class team of investigators that will evaluate the potential obstacles to successful therapy, including tumor entry barriers, tumor infiltration by suppressive cells, CAR T-cell exposure to inhibitory ligands or secreted proteins, and CAR T-cell exhaustion. There is a robust body of preclinical data demonstrating that less-differentiated T cell subsets impart superior in vivo expansion, persistence, and anti-tumor efficacy, compared with more differentiated T cell subsets. Recent data from a small trial suggests that the frequency of these less-differentiated CD8+ cell subsets before and after CAR T cell manufacturing correlates with clinical responses in patients with chronic lymphocytic lymphoma receiving CD19-targeted CAR T cells. These results have important implications, but must be validated in other settings. We will quantify less-differentiated T cell subtypes prior to leukapheresis, as well as in the infused CAR T cell products, and correlate these characteristics with anti-tumor responses and in vivo expansion and persistence. We anticipate that these correlative studies will yield critical insights into the reasons why CAR T cell therapy is successful for some NHL patients but not others. We are hopeful that our findings will help to guide patient selection and counseling, and inform future strategies to overcome these obstacles through improved cell manufacturing technologies, CAR vector design, and/or combinatorial adjuvant therapies, not only for CD20-specific CAR T cells, but also for CAR T cell therapy for other targets.