Label-free imaging of CAR T cell metabolism

项目总结/摘要 该提案的目标是开发非侵入性单细胞技术，以提高T细胞的效力。 治疗癌症。前6种嵌合抗原受体（CAR）T细胞疗法最近获得批准 超过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 （>1年），约50%接受CAR T细胞治疗的患者。由于这些疗法的快速发展， 以及对过程优化的巨大需求，我们专注于改善有效CAR的三个翻译障碍 T细胞治疗：（1）筛选T细胞不适合制造CAR T细胞的患者，（2）体外优化 CAR T细胞生产以获得更高的效力，和（3）鉴定体内有效CAR T细胞的代谢特征。 CAR T细胞治疗可以通过在起始阶段富集幼稚和干细胞记忆（SCM）T细胞来改善。 材料和最终产品。幼稚T细胞和SCM T细胞的缺陷发生在约50%的未经治疗的癌症患者中， 并且从这些来源制造自体CAR-T细胞产品是不成功的。即使SCM T 在CAR掺入后，扩增过程通常通过T细胞扩增而降低效力。 疲惫不堪输注后，体内记忆样表型的存在与更好的反应相关。到 迄今为止，还没有强大的，非破坏性的技术来监测CAR T细胞的生产，以优化 生产并在单细胞水平评估体内效力。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的、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。对于CAR T细胞疗法实现其临床 潜在的，需要新的方法来监测T细胞的最佳效力，在整个生产和后， 输液细胞代谢的变化提供了一种有吸引力但尚未开发的测定来跟踪T细胞效力。 以前的研究，包括我们自己的研究，表明T细胞在活化时会发生剧烈的代谢变化， 幼稚的、疲惫的和记忆样的T细胞具有独特的代谢特征。我们的初步数据显示， NAD（P）H和FAD的荧光强度和寿命的非侵入性单细胞成像（光学 代谢成像或OMI）可以预测CAR T细胞的制造条件， 体内有效的抗肿瘤反应。鉴于CAR T细胞效力的这些代谢特征，我们建议 确定T细胞自发荧光的无标记OMI和多变量模型是否可以识别患者T细胞 有效和持久的CAR T细胞的适应性、最佳体外扩增条件和体内细胞生物标志物 反应总的来说，这些技术将简化流程和干预措施， 治疗和增加我们的知识CAR T细胞代谢在体外和体内。

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.