Enhancing α-CD19 CAR T Antitumor Efficacy Using the BH3 Mimetic, Venetoclax

Abstract

Introduction Clinical outcomes of anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR T therapy can expect a cure rate of 41% and median survival of just 6.3 months if unresponsive. Poor clinical responses to CAR T therapy and a small window of survival highlights the critical need for improvement of CAR T therapy in r/r DLBCL. Our lab previously revealed synergy between the Bcl-2 inhibitor, Venetoclax, and α-CD19 CAR Ts. Though the role of Bcl-2 inhibition within B cell lymphomas is well characterized, further investigation is needed to understand its impact within effector cell populations. Our lab previously established a role for Bcl-2 inhibition in promoting the differentiation of bulk CD8+ CAR T into CD8+ CAR TStem Cell Memory (TSCM) subsets, decreasing the frequency of CD4+ CAR TRegulatory subsets, and increasing cytotoxicity of CAR T when co-cultured with rituximab/chemoresistant malignant B cells, Raji4RH. Here, our lab further elucidates the molecular mechanisms driving these improvements in CAR T quality as a consequence of Bcl-2 inhibition. MethodsTransduction was performed on healthy donor T cells with lentiviral vectors encoding for 2nd generation α-CD19 CAR constructs containing CD28 (28ζ) or 4-1BB (BBζ) costimulatory domain, 24 hours after activation with αCD3/CD28 TransACT (Miltenyi). Cytotoxicity assays were performed by labeling target cells with a lipophilic dye (Sigma-Aldrich), then incubated with GFP+ CAR T at various effector:target ratios and Venetoclax concentrations for 24 hours in 96 well U-bottom plates. Sample wells were stained with viability dye and acquired on BD LSR II cytometer. Mitochondrial membrane potential (ΨM) was assessed using a cell-permeant fluorescent probe, TMRM (Thermofisher), which was added to immunophenotype panels. CAR T immunophenotype was identified using markers for viability, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, CD95, and TMRM. Frequencies of T cell memory subsets were calculated alongside changes in ΨM. B-cell marker expression was assessed for several ABC and GCB DLBCL lines. Antibodies against CD10, CD20, CD22, CD138, and CD19 were used to establish B-lineage epitope density within our target populations. Proliferation tracking was performed using cell-permeant dye, CellTrace Blue (Thermofisher). Bulk untransduced, CD28, or 4-1BB CAR T were stained with tracking dye, activated for 24 hours by co-culture with CD19+ cells, then isolated using CD19+ Microbead positive selection (Miltenyi Biotec). Then, the untransduced control and CAR T fractions were cultured in Venetoclax for up to 96 hours. Samples were then collected and stained with naïve and memory T cell markers and a viability dye. Results Untransduced control, 28ζ, and BBζ CAR T were exposed to Venetoclax during co-culture with DLBCL lines expressing varying densities of CD19 epitope. A significant concentration-dependent decrease in ΨM was observed for each CAR construct. CAR viability did not decrease more than 5% among each concentration and cell line tested. As Venetoclax concentration increases, larger frequencies of CD8+ TNaive and TSCM are observed. Therefore, we conclude an inverse relationship between ΨM and differentiation into CD8+ TSCM cells. CD19+ epitope density does not significantly impact CAR memory phenotype, ΨM, or cytolytic capacity. To uncover the progenitor cell type populating Venetoclax-exposed CD8+ CAR TSCM cells, we tracked dye loss between subsets TNaive, TSCM, and TTotal. After 96 hours of culture, it was apparent that the TSCM cohort was not significantly proliferating, however the TNaive subset experienced the most dye loss, and accounted for observed dye loss within the TTotal cohort. We conclude that Venetoclax encourages differentiation from TNaive cell to TSCM cell. Conclusion We reveal intrinsic changes within the CAR T as a result of Bcl-2 inhibition; and propose that these changes are due to manipulation of mitochondria, allowing for differentiation into a more quiescent, stem-like cell type. Low ΨM has been linked to memory T cell phenotype and increased anti-tumor function in vivo. We seek to uncover the metabolic mechanisms which regulate the observed changes within CAR Ts as a result of Bcl-2 inhibition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal