S188: car-t cells shape the immunological potential of the tumor microenvironment

Background: Cell-based immunotherapies, particularly chimeric antigen receptor modified (CAR-) T cells, have produced impressive responses in a subset of patients with hematological malignancies. However, the limited durability of responses and the development of treatment resistance present significant challenges. Limited expansion and persistence of CAR-T cells, T-cell exhaustion, and a hostile immune microenvironment are key factors that restrict the efficacy of CAR-T cell therapies. Aims: To enhance the efficacy of CAR-T cell therapy and overcome treatment resistance, a better understanding of their co-evolution with the immune microenvironment and their reciprocal interactions is needed. Therefore, this study aimed to define alterations in cellular states in CAR-T cells and the immune compartment upon BCMA CAR-T cell therapy in relapsed/refractory multiple myeloma (RRMM) patients at single cell resolution. We further aimed to investigate cell-cell interactions and identify novel immunomodulatory pathways for the optimization of T cell-based cancer therapies. Methods: We performed droplet-based single-cell RNA sequencing of CD45+ immune cells from the bone marrow of 4 RRMM patients receiving BCMA-directed CAR-T cell therapy (KarMMa-2 trial; NCT03601078) in combination with immune profiling and VdJ sequencing, before as well as one month and six months after CAR-T infusion. We further performed longitudinal full-length single cell RNA-sequencing of CAR-T cells and CD8+ T cells isolated from the peripheral blood and bone marrow of a total of 24 patients (on KarMMA-2/KarMMa-3 trials; NCT03651128). Results: We defined compositional changes in the bone marrow immune microenvironment upon CAR-T cell therapy and observed a temporary expansion of the myeloid compartment at one month following CAR-T infusion. However, we detected the greatest relative changes in abundance in the CD8 T cell compartment, with an expansion of GZMB+ CD8 effector T cells and a decrease in CD8 naïve and CD8 memory T cell populations over time. We were able to show that CAR-T cell treatment drives bystander CD8+ T lymphocyte differentiation, which initially supports a clonal anti-tumor immune response, but ultimately results in T cell exhaustion, reduced potential for self-renewal and sustained depletion of the tumor-reactive T cell repertoire. We observed an expansion of pre-existing T cell clones six months after CAR-T infusion (P = 0.00018 by paired Wilcoxon test) and found that these undergo terminal differentiation. We predict diminished responses to further immunotherapies, such as response to checkpoint blockade. To investigate how exhaustion is regulated, we analyzed the transcriptional modules active in different cell states and identified TCF7, IKZF1 and PRDM1 among the transcription factors regulating divergent cell states. Inferring cell-cell interactions, we identified crucial interactions of the cell-cell communication network that contribute to T cell exhaustion and regulate immune capacity. Summary/Conclusion: The ability of the CAR-T cells to shape a regulatory immune microenvironment may explain why repeated immunotherapies become increasingly less successful, even when targeting distinct antigens. Our studies therefore have important implications for the timing and sequencing of immunotherapies. Our studies provide a framework for assessing and manipulating the ‘mileage’ of the immune system as a predictive marker and a therapeutic opportunity. Keywords: Myeloma, Cancer immunotherapy, T cell response, CAR-T