Pharmacologic drugging of BCL-2 during manufacturing restores T cell fitness and enhances CART efficacy

Abstract While originally developed to induce cancer cell apoptosis, venetoclax, the most clinically relevant BH3-mimetic targeting the anti-apoptotic BCL-2 protein, has emerged as a promising immunomodulator. Through focusing on the effects of targeting BCL-2 family members in T cells, our group previously found that T cells adapt differently following genetic deletion of Bim, the master BH3-only protein bound to all anti-apoptotic members (Ludwig et al. 2020). Additionally, we found that disruption of BCL-2 protein-protein interactions (PPIs) resulted in reorganization of T cell apoptotic control and alteration of lineage-specific gene expression (Ludwig et al. 2021). To better understand if these genetic changes would affect T cell function, we discovered that treatment with venetoclax phenocopied Bcl2 deletion in regulatory T cells (Tregs) and induced a Th17-like phenotypic plasticity change (Liao et al. 2024), that contributed to enhanced CD8+ T cell-mediated anti-tumor killing in the setting of checkpoint blockade. Based upon these results we wondered if such T cell reprogramming could improve the efficacy of autologous chimeric antigen receptor (CAR) T cell products, which can have limited potency due to T cell insufficiency from patient-specific disease- or treatment-related effects. As proof of concept, focusing on BCL-2 targeting in T cells alone, we used venetoclax during ex vivo production of CD19CART prepared from T cells isolated from healthy donors. We found that venetoclax treatment during CART production had only a minor effect on CART expansion and had no effects on the CD4:CD8 ratio, CAR expression, or the memory phenotype in the final CART product when compared to vehicle-treated cells. However, venetoclax treatment resulted in increased protein expression of BCL-2, BCL-XL, and MCL-1, inflammatory cytokines (e.g. IFNγ), granzyme B, and TRAIL. Venetoclax treatment also resulted in increased potency of CART cells in vitro and in vivo against models of acute lymphoblastic leukemia and diffuse large B cell lymphoma, while exhibiting a less exhausted phenotype. To confirm that venetoclax-enhanced antitumor activity of CART was BCL-2 dependent, CART was made to overexpress genes encoding either BCL-2 (wild type; WT) or three different BCL-2 constructs with mutations within their BH3 binding pocket known to incur various levels of venetoclax resistance, BCL-2(F104L), BCL-2(F104C) or BCL-2(G101V). Tumor cell killing was directly proportional to the relative affinities of venetoclax for BCL-2 with treated CART BCL-2(WT) having the highest and CART BCL-2(G101V)having the lowest anti-tumor efficacy.Additionally, CAR T cells expressing mutant forms of BCL-2 regained an exhaustion phenotype that was similar to vehicle-treated cells following incubation with tumor cells. Transcriptomic and functional analyses found that venetoclax reprograms CAR T cells through modulating key signaling (e.g. IL-2/STAT5, PI3K/AKT, NFκB) and metabolic (e.g. PGC1α, OXPHOS, glycolysis) pathways resulting in enhanced cellular fitness. We next tested weather venetoclax could enhance the efficacy of CART prepared from chemotherapeutically pretreated patient-derived T cells that were isolated at the time of apheresis for commercial CD19CART products. Patient-derived T cells had heterogenous baseline BCL-2 family anti-apoptotic protein levels and, as expected, had increased expression of markers of T cell exhaustion compared to cells from healthy donors, particularly in expression of LAG3, PD-1, TIGIT, and TOX. Despite differences in starting material and post-production CD4:CD8 CAR T cell ratios, venetoclax treatment significantly enhanced the in vitro and in vivo CART efficacy of all patient-derived CART products. These results suggest that venetoclax treatment during ex vivo expansion can reprogram CART and increase their efficacy irrespective of the starting cellular material. In summary, this work further establishes BCL-2 as key regulatory node in T cells and demonstrates that therapeutic disruption of critical BCL-2 PPIs can affect T cell function. These findings warrant further investigation into the use of venetoclax to reprogram patient-derived T cells for use in CART-related and other adoptive T cell treatments.