P-037: LONGITUDINAL SINGLE CELL MULTIOMIC ANALYSIS ALLOW THE IDENTIFICATION OF MECHANISM OF PERSISTENCE AND RESISTANCE TO CAR-T CELLS IN MM

Introduction:
Despite high remission rates observed after BCMA CAR-T cells in R/R Multiple Myeloma (MM) patients, a significant number of patients still relapse, and the molecular mechanisms governing CAR-T cell (dys)function are largely unknown. To shed some light on specific transcriptomic programs activated after CAR-T cell administration and identify potential mechanisms of resistance and persistence we interrogated longitudinal samples of CAR-T cells collected from patients enrolled in CARTBCMA-HCB-01 (NCT04309981).

Methods:
We characterized >60.000 CAR-T cells from 17 samples collected from 3 patients, including infusion products and CAR-T cells isolated from paired bone marrow (BM) and peripheral blood (PB) samples at different times after infusion (month 1 to 18). Single-cell RNA and TCR sequencing was performed using Chromium Single-Cell Immune Profiling solution. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel machine learning method.

Results:
scRNA-seq revealed that CAR-T cells remaining after infusion were mainly non-proliferating CD8⁺ cells, with effector/effector-memory phenotypes. Interestingly, we found that CAR-T cells infiltrating BM presented increased expression of cytotoxic and exhaustion markers compared to their PB counterparts. GRN analysis showed that PRDM1 regulon, already associated with CAR-T cell exhaustion, presented increased activity in the BM. Additionally, transcriptomic profile of CAR-T cells differed among patients. Partial responders presented increased presence of terminally differentiated effector cells with an exhausted signature, while complete responders presented CAR-T cells in transition to central or effector memory phenotype. Importantly, combination of scTCR-seq and scRNA-seq allowed the identification of a hyperexpanded CAR-T clone in the BM of the patient with partial response. Deeper characterization showed that this clone had increased expression of IL10. Additional in vitro studies suggested that activation of endogenous TCR along with CAR-T activation led to IL10 production, and functional validations corroborated that IL10 alters CAR-T cell functionality. Then, to deepen in the mechanism of CAR-T cell persistence, we analyzed post-infusion CAR-T cells of one of the few patients with detectable CAR-T one year after infusion. The analysis showed a prevailing CD8⁺ population expressing key markers related to T-cell memory and survival. However, prior to relapse, cells with increased cytotoxic features expanded in the BM, indicating CAR-T cell reactivation. GRN analysis in this patient identified FOS and NR4A2 regulons as potential drivers of CAR-T reactivation in response to tumor recurrence.

Conclusions:
Overall, our analysis combining scRNA-seq/scTCR-seq with novel machine learning models allowed us to identify several molecular markers associated to CAR-T dysfunction, that represent potential targets to be modulated for the development of improved CAR-T therapies against MM.