Label-free imaging of CAR T cell metabolism

项目摘要/摘要 这项提议的目标是开发非侵入性的单细胞技术来提高T细胞的效力 治疗癌症的方法。最近批准了首批6种嵌合抗原受体(CAR)T细胞疗法 800种CAR和T细胞疗法正在进行临床试验。然而，在实现持久缓解方面仍然存在障碍。 (&gt；1年)~50%接受CAR T细胞治疗的患者。由于这些疗法的快速发展 和流程优化的迫切需要，我们专注于改善三个平移障碍，以有效的汽车 T细胞治疗：(1)筛选不适合制造CAR T细胞的患者；(2)体外优化 CAR T细胞的产生，以获得更高的效力；以及(3)确定有效的CAR T细胞在体内的代谢特征。 CAR T细胞治疗可以通过在开始时丰富幼稚和干细胞记忆(SCM)T细胞来改进 材料和最终产品。未经治疗的癌症患者中约50%会出现幼稚T细胞和SCM T细胞缺陷， 而用这些来源制造自体CAR T细胞产品一直没有成功。即使SCM T 细胞可以被分离，在CAR掺入后，扩增过程通常会通过T细胞降低效力 疲惫不堪。在输液后，体内记忆样表型的存在与更好的反应相关。至 到目前为止，还没有强大的、无损的技术来监控汽车T细胞制造来优化 在单细胞水平上生产和评估体内的效力。这些问题限制了CAR T细胞疗法的影响。 目前测量T细胞功能的方法是劳动密集型、破坏性的或缺乏单细胞分辨率， 这限制了这些测量的频率或特异性。为CAR T细胞治疗实现其临床应用 潜在的，需要新的方法来监测T细胞在整个制造和生产后的最佳效力 输液。细胞代谢的变化提供了一种有吸引力但未被充分探索的方法来追踪T细胞的潜能。 以前的研究，包括我们自己的研究，表明T细胞经历了剧烈的代谢变化和激活，并且 幼稚、疲惫和记忆样的T细胞具有明显的新陈代谢特征。我们的初步数据显示， NAD(P)H和FAD(光学)的荧光强度和寿命的无创性单细胞成像 代谢成像，或OMI)可以预测汽车T细胞制造条件，产生更多与更少 体内有效的抗肿瘤反应。考虑到CAR T细胞潜能的这些代谢特征，我们建议 确定无标记T细胞自发荧光OMI和多变量模型是否能识别患者T细胞 CAR T细胞的适合性、体外扩增的最佳条件和体内细胞生物标记物 回应。总体而言，这些技术将简化持续有效的T细胞的过程和干预 治疗并增加我们对CAR T细胞体外和体内代谢的了解。

PROJECT SUMMARY / ABSTRACT The goal of this proposal is to develop non-invasive single-cell technologies to improve the potency of T cell therapies against cancer. The first 6 chimeric antigen receptor (CAR) T cell therapies were recently approved and >800 CAR and T cell therapies are in clinical trials. However, barriers remain in achieving durable remissions (>1 year) for ~50% of patients who receive CAR T cell therapy. Due to the rapid development of these therapies and a great need for process optimization, we focus on improving three translational roadblocks to effective CAR T cell therapy: (1) screening patients whose T cells are unfit for CAR T cell manufacturing, (2) optimizing in vitro CAR T cell production for higher potency, and (3) identifying metabolic features of potent CAR T cells in vivo. CAR T cell therapy could be improved by enriching for naïve and stem cell memory (SCM) T cells in starting materials and final products. Deficiencies in naïve and SCM T cells occurs in ~50% of untreated cancer patients, and manufacturing autologous CAR T cell products from these sources has been unsuccessful. Even if SCM T cells can be isolated, after CAR incorporation, the expansion process typically diminishes potency through T cell exhaustion. After infusion, the presence of memory-like phenotypes in vivo correlate with better responses. To date, there are no robust, non-destructive technologies to monitor CAR T cell manufacturing to optimize production and assess potency in vivo at a single-cell level. These issues limit the impact of CAR T cell therapy. Current approaches to measure T cell function are labor-intensive, destructive, or lack single-cell resolution, which limits the frequency or specificity of these measurements. For CAR T cell therapy to realize its clinical potential, new methods are needed to monitor T cells for optimal potency throughout manufacturing and post- infusion. Changes in cell metabolism provide an attractive yet under-explored assay to track T cell potency. Previous studies, including our own, show that T cells undergo drastic metabolic changes with activation, and that naïve, exhausted, and memory-like T cells have distinct metabolic features. Our preliminary data shows that non-invasive single-cell imaging of the fluorescence intensity and lifetime of NAD(P)H and FAD (optical metabolic imaging, or OMI) can predict CAR T cell manufacturing conditions that produce a more vs. less potent anti-tumor response in vivo. Given these metabolic features of CAR T cell potency, we propose to determine whether label-free OMI of T cell autofluorescence and multivariate models can identify patient T cell fitness, optimal in vitro expansion conditions, and in vivo cell biomarkers of potent and persistent CAR T cell response. Overall, these technologies will streamline processes and interventions for consistently potent T cell therapy and increase our knowledge of CAR T cell metabolism in vitro and in vivo.