T 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.
