Background Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in treating refractory cancers. However, this success has been limited to a small fraction of patients. The suboptimal efficacy of CARTs is often due to patient T cell insufficiency either during manufacturing or after infusion. Therefore, combining certain immunomodulators can enhance the competency of CAR T cell products during manufacturing. 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 promising immunomodulator. Our lab has recently reported that in vivo expansion of murine T cells in setting of BCL-2 blockade by venetoclax results in increased expression of BCL-2 family proteins, cell death resistance and upregulation of genes consistent with an activated phenotype and a signature consistent with less T cell exhaustion.¹ In the current study, we hypothesized that the immunomodulatory effect of venetoclax can be employed to enhance CAR T cell fitness during manufacturing and improve the antitumor efficacy post-infusion.

Methods Human CD19CAR T cells (hCD19CARTs) were prepared and expanded in the presence of vehicle or venetoclax, followed by phenotypic and genetic analysis using flow cytometry and single cell sequencing (scRNA-seq). Ex vivo CART cell cytotoxicity and cytokine production following incubation with human diffuse large B cell lymphoma (DLBCL) was measured along with measurement of their in vivo efficacy in DLBCL xenograft murine models.

Results Expanding hCD19CARTs in the presence of venetoclax resulted in increased expression of anti-apoptotic proteins, upregulated transcription factors involved in T cell signaling and function and enhanced antitumor cytotoxicity with less exhaustion, higher cytokine and granzyme B production in vitro. Furthermore, venetoclax-treated hCD19CARTs resulted in significantly improved tumor control and survival in pre-clinical xenograft models. scRNA-seq of venetoclax-treated hCD19CARTs before antigen stimulation revealed improved effector-like transcriptional profiles in various T cell compartments marked by enriched TNFα and IFNγ signatures and significant PI3K/AKT and STAT5 signaling pathway enrichment and shifts towards oxidative phosphorylation metabolism, suggesting broad involvement of these pathways in the enhanced cytolytic function and survival of venetoclax-treated hCD19CARTs.

Conclusions Our findings provide insight into the molecular basis of venetoclax-mediated immune modulation in T cells and suggest potential manufacturing strategies to improve the efficacy of CAR T cell products. We believe that our results provide proof-of-principle evidence for clinical translation of combining BH3-mimetics with CAR T cell therapies and elucidates novel non-apoptotic mechanisms by which certain clinically relevant BH3-mimetics can potentiate T cell function.

Reference

1. Ludwig LM, Hawley KM, Banks DB, Thomas-Toth AT, Blazar BR, McNerney ME, Leverson JD, LaBelle JL. Venetoclax imparts distinct cell death sensitivity and adaptivity patterns in T cells. Cell Death Dis. 2021;12(11):1–13.

Ethics Approval All animal experiments were approved and performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of the University of Chicago using the approved lab protocol IACUC/ACUP # 72295. All blood samples were collected from volunteers following the approved IRB protocol # IRB14–0221-CR009: Peripheral Blood Collection From Healthy Volunteers for Laboratory Based Research.