Quantitative single-cell biomarkers of T-cells to optimize tumor immunotherapy

描述(申请人提供)：免疫疗法注入抗体、疫苗和细胞是治疗人类恶性肿瘤的有效方法。有效的抗肿瘤作用是通过利用免疫系统的特异性和相关效应功能来识别和消除癌细胞来实现的。临床试验已经揭示了基于免疫的疗法的优势，包括(I)明确的作用机制，(Ii)明确的特异性和减少有害的靶外效应，(Iii)比传统方法更低的毒性。过继细胞治疗(ACT)是基于过继转移转基因T细胞以增强嵌合抗原受体(CAR)的表达，在治疗所有其他治疗方法难治的肿瘤的临床试验中显示出相当大的前景。特别是，CD19特异性CAR T细胞的使用在CD19慢性淋巴细胞白血病(CLL)患者中显示了显著的抗肿瘤效果，这些患者对传统疗法无效。这些试验注入了不同种类的转基因T细胞，这些T细胞已经繁殖到临床足够的数量。虽然输注T细胞的治疗潜力取决于它们的持久性，但目前评估输注T细胞存活率的免疫相关研究仅限于(I)描述整个T细胞接种群体的免疫表型和功能，以及(Ii)对输注后恢复的T细胞的免疫表型分析。这项拨款申请旨在调整单细胞成像和检索，通过开发一个统一的平台来评估从免疫治疗接受者直接获得的少数T细胞(和肿瘤细胞)的表型和功能，从而了解临床级CD19特异性CAR T细胞的效力。我们的目标是使用我们开发的高通量单细胞纳米孔筛选(SNS)来对输注前CAR T细胞进行深入的定量功能表征(多路细胞因子分泌、表型、细胞毒性、调节性T细胞的作用，Treg)，并将这些数据与输注后从患者身上提取的T细胞进行比较。这些数据将被用来(I)量化输注的T细胞的功能和抗肿瘤效果的可能性，以及(Ii)改善T细胞的生成，以便在下一代临床试验中获得更好的疗效。我们将在特定目标1中验证我们的方法，在那里我们将定义临床级别CD19特异性CAR T细胞的整合功能和分子图谱。在具体目标2中，我们将实施我们的方法学来量化过继转移细胞在体内的持久性。这将检验SNS可以量化临床级T细胞的治疗潜力和形成下一代临床试验的假设。 与公共卫生相关：转基因T细胞正在进行针对白血病和淋巴瘤的临床试验。这项拨款申请验证了用于研究输注的T细胞靶向肿瘤细胞的能力的工具，并应用这种方法在临床试验中提高了我们对其治疗益处的理解。这将使研究人员能够在下一代临床试验中开发出产生更有效的T细胞来针对血液恶性肿瘤的方法。

DESCRIPTION (provided by applicant): Immunotherapy infusing antibodies, vaccines, and cells is an effective treatment approach for human malignancies. Potent anti-tumor effects are realized by harnessing the specificity and associated effector functions of the immune system to recognize and eliminate cancer cells. Clinical trials have revealed the advantages of immune-based therapies, including (i) defined mechanisms of action, (ii) defined specificity and reduced deleterious off-target effects, (iii) lower toxicities than conventional approaches. Adoptive cell therapy (ACT), based on the adoptive transfer of T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR), has shown considerable promise in clinical trials treating tumors refractory to all other treatment methods. In particular, the use of CAR+ T cells rendered specific for CD19 demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) refractory to conventional therapies. These trials infuse a heterogeneous population of genetically modified T cells which have been propagated to clinically-sufficient numbers. While the therapeutic potential of infused T cells depends on their persistence, immunocorrelative studies evaluating survival of infused T cells are currently limited to (i) describing the immunophenotypes and function of whole populations of the T-cell inoculum and (ii) immunophenotypic analyses on T cells recovered after infusion. This grant application seeks to adapt single- cell imaging and retrieval to inform on the potency of clinical-grade CD19-specific CAR+ T cells by developing a unified platform for assessing both phenotype and function on the few T cells (and tumor cells) directly obtained (without in vitro manipulation) from recipients of immunotherapy. Our objective is to use high throughput single-cell nanowell screening (SNS) that we have developed to undertake an in-depth quantitative functional characterization (multiplexed cytokine secretion, phenotype, cytotoxicity, effect of regulatory T cells, Treg) of pre-infusion CAR+ T cells and compare these data to T cells recovered from the patient, post-infusion. These data will be used to (i) quantify the functionalit of infused T cells and the potential for anti-tumor effects and (ii) improve the generation of T cells for greater efficacy in next-generation clinical trials. We will validate our approach in Specific Aim 1 where we will define the integrated functional and molecular profiles of clinical- grade CD19-specific CAR+ T cells. In Specific Aim 2 we will implement our methodology to quantify the in vivo persistence of adoptively transferred cells. This will test the hypothesis tha SNS can quantify the therapeutic potential of clinical-grade T cells and in the formation of next-generation clinical trials. PUBLIC HEALTH RELEVANCE: Genetically modified T cells are being infused in clinical trials to target leukemia and lymphomas. This grant application validates tools for investigating the ability of the infused T cells to target tumor cells and applies this approach to improve our understanding of their therapeutic benefit in clinical trials. This will enable investigators to develop approaches to generating even more effective T cells to target hematologic malignancies in next-generation clinical trials.