EAGER: Biomanufacturing: Large-Scale Isolation of T Cells with High-Performance Phenotype for Enhancing Adoptive T-Cell Therapy

164506-chent细胞被设计成表达肿瘤靶向受体，在治疗对手术、化疗和放射等传统疗法耐药的癌症方面显示出显著的临床疗效。然而，这种新的治疗策略的广泛应用需要一个有效和一致的生产过程，为每个患者产生高质量的T细胞。目前，这种生产过程的发展受到以下因素的阻碍：(1)缺乏对哪些可选择的特征与高性能T细胞最密切相关的了解，以及(2)缺乏能够精确分离显示所需特征的T细胞的细胞分选技术。该项目旨在通过(1)系统地研究具有高和低肿瘤杀伤力的T细胞表面蛋白质表达的差异来应对这两个挑战，以及(2)开发一种基于使用微流控设备的磁分离的高通量和经济的细胞分离技术。这项拟议研究的成功完成将提高肿瘤靶向T细胞的治疗效果，并使这一有希望的癌症治疗方案更广泛地适用于需要过继T细胞治疗的患者。过继T细胞疗法在治疗难治性癌症，特别是B细胞恶性肿瘤方面显示出显著的临床疗效，然而，在这种新的治疗策略作为一线治疗方案广泛应用之前，必须建立一个强大的生产平台，支持一致、可扩展和经济的高质量T细胞的制造。这种生产平台的开发需要两项基础知识和技术：(I)必须确定区分高性能和低性能T细胞的表型，以便制造过程可以丰富高功能T细胞；(Ii)必须存在高通量和经济的细胞分离方法，以精确分离表现出与高治疗能力相关的表型的细胞。在这里，该项目将系统地比较高性能和低性能CAR-T细胞的转录组，识别与强大的抗肿瘤能力相关的表面标记，并优化基于磁棘轮的细胞分离平台，以实现一致、快速和大规模分离表现出与高治疗效果相关的表型的CAR-T细胞。目的1将分析功能与功能障碍的CAR-T细胞的转录组；将识别编码在功能与功能障碍的CAR-T细胞中差异表达的表面定位蛋白的基因，并将经验性地验证这些蛋白标记物与抗原刺激下T细胞效应功能的相关性。这一目标的成功完成将有助于我们理解与强大的抗肿瘤能力相关的表型标记，从而有助于在T细胞制造过程中识别高性能细胞。目的2旨在优化棘轮细胞术分离CAR-T细胞，从而提供一种新的细胞分离技术，该技术结合了荧光激活细胞分选的定量特异性和基于磁珠的细胞分离的温和、高通量的性质。在目标3中，CAR-T细胞将通过棘轮细胞术进行分选，以选择具有与强大的T细胞效应功能强烈相关的蛋白质标记的细胞，并将在小鼠肿瘤异种移植模型中评估这些CAR-T细胞的体内功能。该项目的成功完成将导致一种可扩展的制造工艺，为下一代癌症免疫疗法生产具有始终如一的强大抗肿瘤功能的治疗性T细胞。

164506 - ChenT cells engineered to express tumor-targeting receptors have shown remarkable clinical efficacy in treating cancers that are resistant to conventional therapies such as surgery, chemotherapy, and radiation. However, broad application of this novel treatment strategy requires an efficient and consistent production process to generate high-quality T cells for each individual patient. The development of such a production process is currently hampered by (1) lack of understanding on what selectable features are most strongly associated with high-performing T cells, and (2) the absence of a cell-sorting technology that would enable precise isolation of T cells that display the desirable features. This project aims to address both of these challenges by (1) systematically studying differences in protein expression on the surface of T cells with confirmed high vs. low tumor-killing capabilities, and (2) developing a high-throughput and economical cell isolation technique based on magnetic separation using microfluidic devices. Successful completion of the proposed research would increase the therapeutic efficacy of tumor-targeting T cells and make this promising cancer treatment option more widely available to patients in need of adoptive T-cell therapy. Adoptive T-cell therapy has shown remarkable clinical efficacy in treating refractory cancers, particularly B-cell malignancies, However, before this novel therapeutic strategy can be made broadly available as a front-line treatment option, a robust production platform that supports consistent, scalable, and economical manufacturing of high-quality T cells must be established. The development of such a production platform requires two pieces of foundational knowledge and technology: (i) the phenotypes that distinguish high-performing vs. low-performing T cells must be identified, such that the manufacturing process could enrich for highly functional T cells; and (ii) a high-throughput and economical cell-separation method must exist to enable precise isolation of cells exhibiting phenotypes associated with high therapeutic capacity. Here, the project will systematically compare the transcriptome of high- vs. low-performing CAR-T cells, identify surface markers that correlate with robust anti-tumor capabilities, and optimize a magnetic-ratcheting-based cell separation platform to achieve consistent, rapid, and large-scale isolation of CAR-T cells exhibiting phenotypes that correlate with high treatment efficacy. Aim 1 will analyze the transcriptome of functional vs. dysfunctional CAR-T cells; genes encoding surface-localized proteins that are differentially expressed in functional vs. dysfunctional CAR-T cells will be identified, and these protein markers will be empirically validated for correlation to T-cell effector function upon antigen stimulation. Successful completion of this aim will contribute to our understanding of phenotypic markers that correlate with robust anti-tumor capabilities, and can thus serve to identify high-performing cells during T-cell manufacturing. Aim 2 seeks to optimize ratcheting cytometry for the isolation of CAR-T cells, thus providing a novel cell-separation technique that combines the quantitative specificity of fluorescence-activated cell sorting with the gentle, high throughput nature of magnetic bead-based cell separation. In Aim 3, CAR-T cells will be sorted by ratcheting cytometry to select cells with protein markers that strongly correlate with robust T-cell effector functions, and the in vivo function of these CAR-T cells will be evaluated in mouse tumor xenograft models. Successful completion of the project will result in a scalable manufacturing process that yields therapeutic T cells with consistently robust anti-tumor functions for next-generation cancer immunotherapy.