Dual Inhibition of PI3KDelta/Gamma during Manufacturing Reprograms Metabolism of CAR T Cells to Enhance Expansion and Cytotoxicity Against CLL

Chronic lymphocytic leukemia (CLL) creates an immune-suppressive microenvironment that induces terminal T-cell differentiation and T-cell exhaustion, thereby limiting the efficacy of chimeric antigen receptor (CAR) T cells. Indeed, analysis of CLL patients has shown that a high frequency of senescent T-cells (dashed line, Figure 1A) prior to CAR T-cell manufacturing was associated with disease progression and failure to benefit from CAR T (Fraietta et al. 2018). In a cohort of early stage untreated CLL patients, we show that 55% of patients had a predominant senescent phenotype (Figure 1A). This striking observation highlights the need for strategies to enrich non-senescent T cells for CAR T therapy in CLL patients. To address this need, we studied the effect of adding phosphoinositide 3-kinase inhibitors (PI3Ki) during ex vivo CAR T cell-manufacturing on T cell phenotype, metabolism, in vivo expansion, persistence and anti-CLL cytotoxicity in NOG mice bearing the human OSU-CLL compared with conventional CART.

To investigate the relative influence of PI3Kd-selective versus dual PI3K-d/g inhibition, we first cultured T cells from CLL patients activated with anti-CD3/CD28 beads across logarithmic dose scales of duvelisib or idelalisib. With dual-PI3K-d/g inhibition, CLL donor T cell expansion was 150% of conventionally expanded CART (Figure 1B) with dose-dependent decreases in expression of exhaustion markers TIM-3 and LAG-3 (Figure 1C). PI3K-d/g blockade increased frequencies of CD8+ CAR T cells, thus normalizing the ratio of CD4:CD8 CAR T cells (Figure 1D). To further characterize the effects of dual PI3K-d/g inhibition on T-cell phenotype, mass cytometry time-of-flight (CyTOF) analyzed the phenotype of CAR T cells cultured with or without duvelisib. Unsupervised clustering algorithms showed duvelisib increased frequencies and numbers of T-stem cell memory (T_scm), naïve, and central memory CD8+ CAR T cells (data not shown). Finally, unbiased clustering algorithms identified increases in frequencies of CD27+CD45RO- CD8+ CAR T cells which was confirmed in additional patients using flow cytometry (Figure 1E). In summary, duvelisib enriched populations of less differentiated, more potent CD8+ CAR T cells during manufacturing.

To assess whether these phenotypic changes conferred functional benefit, the cytotoxicity of CAR T cells cultured with or without duvelisib was compared using the OSU-CLL cell line. Duvelisib-cultured CAR T cells (Duv-CAR T cells) had greater cytotoxicity against OSU-CLL cell line (Figure 1F). Subsequently, gene expression profiling of Duv-CAR T cells showed decreased glycolysis pathway scores, down-regulated glucose transporters, and up-regulated glutamine transporters relative to control CAR T cells, leading us to explore T cell metabolism. Elevated protein expression of PGC1-a, a critical regulator of mitochondrial biogenesis and autophagy, was associated with increased staining with NAO, a measure of mitochondrial mass in Duv-CAR T cells (data not shown). To directly measure mitochondrial content of cells, transmission electron microscopy of control CAR T and Duv-CAR T cells generated from CLL patients was performed after 14 days of culture with representative images shown in Figure 1H. A 1.45-fold increase in mitochondrial cross-sectional area was observed (representative images in Figure 1G), which imputes a 175% increase in mitochondrial volume for duvelisib cultured CAR T cells relative to control CAR T cells (p=0.0013; Figure 1G).

Following transfer to NOG mice engrafted with a human CLL cell line, Duv-CAR T cells demonstrated greater in vivo expansion (Figure 1H), faster elimination of CLL (data not shown), and improved mouse survival (Figure 1I) in a model of high disease burden OSU-CLL. In summary, dual PI3K-d/g inhibition during CLL patient-derived CAR T-cell manufacturing increased yields of T_scm, naïve, and central memory CD8+ Duv-CAR T cells with greater mitochondrial mass and enhanced efficacy in eliminating CLL in a mouse model.