Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells rendered specific for CD19 has demonstrated significant anti-tumor effects in patients with refractory CD19+ B-cell malignancies. There are however no biomarkers that can predict the potency of T-cell infusion products and thus the ability to enrich functionally superior cells is lacking. Existing clinical data supports at least two important functions of the T cells in mediating tumor regression: (a) immediate cytotoxicity to enable tumor cell killing, and (b) long-term persistence to ensure lasting and durable responses. We demonstrate here that migratory capacity of T cells can be used as a biomarker to segregate functionally superior cells that display enhanced polyfunctionality in vitro and superior tumor control in vivo. The simplicity of sorting cells based on motility and the scalability of the approach imply that these can be routinely adapted in standard GMP settings to enrich for functional T cells among any heterogeneous population.
Populations of CD19-specific chimeric antigen receptor (CAR) T cells were biomanufactured using our standard clinical protocol. Timelapse Imaging In Nanowell Grids (TIMING) was used to enable the simultaneous quantification of the interaction between thousands of individual tumor-specific CD8+ T cells and multiple tumor cells. These results demonstrated that IFN-gamma secretion was the most common function elicited upon tumor cell conjugation. We found that CD8+ T cells with killing ability, especially serial killing ability, required shorter durations of target cell conjugation in comparison to IFN-gamma secreting mono-functional cells, indicating rapid synapse termination by T cells capable of killing versus cytokine secretion. Tracking the velocities of these cells by time-lapse imaging revealed that these serial killer T cells (with or without IFN-gamma secretion) had a high out-of-contact basal motility. In order to gain a better understanding of the phenotypic characteristics of the migratory T cells, we performed molecular profiling of T cells identified only by their basal motility (in the absence of tumor cells) using single-cell multiplexed transcriptional profiling, microscopy and flow cytometry, and found that the costimulatory molecules CD2 and CD244, and the chemokine receptor CXCR3 were expressed at higher levels in highly motile T cells compared to the non-motile T cells.
In order to test the hypothesis that migratory capacity can serve as a biomarker T cell polyfunctionality, we next sorted motile cells using a modified migration assay, and confirmed that the migrating cells were enriched in highly motile cells. We characterized these cells phenotypically by flow-cytometry and demonstrated that T-cell differentiation markers including CCR7, CD95 and CD45RA were not differentially expressed between the motile and non-motile cells. Consistently however, the migrated cells displayed superior killing and serial killing in comparison to the non-migrated T cells. This superior in vitro polyfunctionality of migratory T cells was confirmed in a mouse xenograft model of leukemia in which motile CAR+ T cells were superior in controlling the disease in comparison to either their non-motile counterparts or the parent unsorted population.
In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T-cell behavior but also the ability to identify subpopulations of T-cell with enhanced polyfunctionality. Our studies support the use of motility as a surrogate and selective marker of higher CAR+ T cell bioactivity. These results also open up avenues to molecularly engineer T cells for an increased motility that could translate to better in vivo outcomes.
Note: This abstract was not presented at the conference.
Citation Format: Gabrielle Romain, Harjeet Singh, Ivan Liadi, Jay R Adolacion, Laurence J.N. Cooper, Navin Varadarajan. Migratory T cells demonstrate superior persistence and enhanced tumor control [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B082.
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