The goal of this proposal was to engineer programmed T cells with persistent anti-tumor efficacy. Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells has demonstrated significant anti-tumor effects in patients with refractory B-cell malignancies. The remarkable clinical success of CAR⁺ T cells has spurred the development of this approach for other leukemias and solid tumors. In spite of the clinical potential of ACT, its efficacy remains unpredictable, and newer approaches are required to define the key components of the efficacy of CAR⁺ T cells. Abnormal vascularization of tumors leads to regional microenvironments that demonstrate nutrient starvation. Similar to the cancer cells, the proliferation and effector functionality of T cells are all energetically demanding processes that rely on robust cellular metabolism, and the availability of nutrients is essential to the anti-tumor efficacy of T cells. The incomplete understanding of the role of metabolism in the anti-tumor efficacy of cells has severely limited the biomanufacturing T cells with predictable potency, and this is a fundamental limitation since: (a) it leads to unreliable patient outcomes in the clinic, and (b) does not provide for reliable and scalable biomanufacturing due to the lack of manufacturing specifications.  There is, however, no methodology that can map the metabolism of the T cells with their function and persistence capacity at single-cell resolution, and not surprisingly, the link between metabolism and function in determining the potency of cell populations is not known. We developed a suite of innovative high-throughput single-cell methodologies including real-time metabolic profiling, Timelapse Imaging Microscopy in Nanowell Grids (TIMING), and single-cell RNA-seq. As part of this grant, we have identified: (1) biomarkers of CAR T cells that are associated with clinical responses, (2) that T-cell migration is a selectable biomarker that can identify the fittest T cells.

Intellectual merit. This proposal established TIMING as a transformative methodology for quantifying the functionality of human T-cells and served as an integrated technology that combines single-cell functional profiling (cytotoxicity and cytokine secretion) with dynamic metabolic profiling and systems-level molecular profiling. From a biomanufacturing perspective, the molecular/functional/metabolic properties that need to be engineered/elicited to ensure clinical benefit, i.e. defining the potency of T cells, was accomplished. We have shown that migratory T cells are associated with optimal anti-tumor efficacy in vivo and can be used as a biomarker of manufactured T cells.

Broader Impact. This project performed a global and integrated profiling of the anti-tumor efficacy of T cells. The engineering of more potent T cells can have a broad impact on immunotherapy and can set the stage for the clinical translation of the results. TIMING is a single-cell high-throughput technology that broadly allows the quantification of functional immune cell responses in the context of vaccines, viral and bacterial infections, and can be used for correlative studies. From a scientific standpoint, our work established the heterogeneity and correlation between fundamental T-cell processes like metabolism, function, and phenotype and thus will have a broad impact on T-cell immunology. The educational outreach included student engagement in immunotherapy through research experiences for K-12, undergraduate and graduate students, and through interactive media.

Last Modified: 02/13/2023
Modified by: Navin Varadarajan