OAB-018: A BCL2L1 armoured BCMA-targeting CAR T cells to overcome exhaustion and enhance persistence in multiple myeloma

Background Chimeric antigen receptor (CAR) T cells targeting BCMA have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. Methods Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with anti BCMA CAR T cells. Results CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BclxL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD). Conclusions Cognizant of the role BclxL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BclxL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BclxL armored CAR (BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and in vivo studies. While BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCL2L1_CAR viability and cytotoxic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions, where CAR T cells were stimulated every 2 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCL2L1_CAR and BCMA_CAR with respect to the composition of Tem cells (CCR7–CD45RO+CD45RA–) or Tcm cells (CCR7+CD45RO+CD45RA–). However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation and anti-MM cytotoxic activities of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Therefore BCL2L1 blockade of AICD not only enhanced the viability and cytotoxicity of CAR-T cells but surprisingly also reduced their functional exhaustion. Our findings provide a novel approach for CAR-T optimization and overcoming relapse resulting from lack of persistence.