Mechanisms of treatment failure in chimeric antigen receptor T cell therapy

项目摘要/摘要 表达嵌合抗原受体(CAR)的转基因T细胞过继免疫治疗 对B细胞恶性肿瘤非常有希望。然而，只有不到一半的复发非霍奇金患者 淋巴瘤(NHL)在CD19靶向CAR T细胞治疗后实现持久缓解。在一些 免疫原性小鼠导致靶抗原丢失或输注细胞发生排斥反应 成分，但在大多数情况下，治疗耐药或在初步应答后复发的原因仍然存在 人们对此知之甚少。我们提出了一项计划，以阐明最近启动的一项CAR治疗失败的原因 T细胞临床试验，仔细评估肿瘤的生物学特性和肿瘤微环境 并在CAR T细胞治疗后检测患者T细胞表型和输注CAR T细胞表型。 作为我们的I/II期临床试验的一部分，一种完全针对人的第三代CD20特异性CAR在 复发或难治性B细胞性NHL(由单独来源资助)，所有患者都接受强制肿瘤治疗 治疗前后进行活组织检查。这将使我们能够发现可预测的生物特征 对治疗的反应性，并评估肿瘤随时间的适应性变化，以揭示 免疫逃逸导致复发的机制。我们将采用循序渐进的方法，使用 最先进的方法学，包括多色流式细胞术、单细胞RNA测序和基因表达 侧写和多重免疫组织化学。我们已经组建了一支世界级的调查团队， 评估成功治疗的潜在障碍，包括肿瘤进入障碍、肿瘤侵袭 抑制性细胞、CAR T细胞暴露于抑制性配体或分泌的蛋白质，以及CAR T细胞耗竭。 大量临床前数据表明，分化程度较低的T细胞亚群 与分化程度更高的T细胞相比，体内扩增、持久性和抗肿瘤效果更佳 子集。最近一项小型试验的数据表明，这些分化较少的CD8细胞的频率 慢性粒细胞白血病患者制造CAR前后T细胞亚群与临床疗效的相关性 接受CD19靶向CAR T细胞的淋巴细胞性淋巴瘤。这些结果具有重要的意义，但 必须在其他设置中进行验证。我们将在白细胞分离前对分化较少的T细胞亚型进行量化， 以及在注射的CAR T细胞产品中也是如此，并将这些特征与抗肿瘤反应相关 以及体内扩张和持久化。 我们预计，这些相关研究将对CAR T细胞 对于一些NHL患者，治疗是成功的，但对另一些患者则不是。我们希望我们的发现将有助于指导 患者选择和咨询，并为未来通过改进克服这些障碍的战略提供信息 细胞制造技术、汽车载体设计和/或组合辅助疗法，不仅适用于 CD20特异性CAR T细胞，也可用于CAR T细胞治疗的其他靶点。

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.