PI3K Inhibition with Idelalisib for Optimized Production of Chimeric Antigen Receptor T Cells

Introduction: Genetically modified T cells expressing chimeric antigen receptors (CARTs) are currently among the most encouraging treatment approaches in cancer immunotherapy. Efficacy and persistence of adoptively transferred CARTs are linked to the proportion of naïve-like T cells (T_N) and stem cell memory-like (T_SCM) T cells defined by stem cell-like properties such as a high proliferative and self-renewal capacity. Optimization of CART production is important to shift the balance towards these subsets. The PI3K/mTOR/AKT pathway is one of the main pathways involved in T cell differentiation. In the current study we evaluated the inhibition of PI3K by the clinically approved small molecule idelalisib during culturing of CARTs in order to increase less differentiated T cell subpopulations in the final cell product.

Methods: Human peripheral blood mononuclear cells (PBMCs) of ten healthy donors were transduced with a 3rd generation retroviral CD19-specific CART vector containing CD28 and 4-1BB as costimulatory domains (kindly provided by Malcolm Brenner, Baylor College of Medicine, Houston). PBMCs were activated with anti-CD3/anti-CD28 and stimulated with IL-7/IL-15. Cultivation was performed with or without 1µM idelalisib. T_N was defined as CD45RA+CCR7+, central memory-like (T_CM) as CD45RA-CCR7+, effector memory-like (T_EM) as CD45RA-CCR7- and effector-like (T_Eff) as CD45RA+CCR7- T cells. Detailed analysis of T cell subpopulations, cytokine secretion and expression of exhaustion and homing markers was performed using flow cytometry over 17 days.

Results: Viability (94±3% vs 87±9%; p=0.006) and transduction efficiency (65±13% vs 60±12%; p=0.001) of CARTs were significantly higher in presence of idelalisib at day 14. Both groups showed similar evolution of CD3+/CD4+ cells (48%±13% vs 46±13%, p=0.1) and CD3+/CD8+ cells (50%±13% vs 51±13%, p=0.2) at day 14. In addition, the T_N subset of CD3+ (31±11% vs 23±9%, p<0.001), CD3+/CD8+ (36±15% vs 27±12%, p<0.001) and CD3+/CD4+ (25±11% vs 19±10%, p=0.003) CARTs was significantly increased with idelalisib. Cultivation with the inhibitor reduced the expression of the exhaustion marker Tim-3 (66±10% vs 82±5%, p=0.003) and raised CD62L expression (84±9% vs 70±9%, p<0.001) of CD3+ CARTs at day 14. Both groups showed low PD-1 expression (4±3% vs 6±3%; p=0.003) after 14 days of cultivation. At day 10 of culture CARTs propagated with idelalisib showed significantly lower CD4+ T helper 1 cell (20±10% vs 23±8%; p=0.05) and higher CD4+ T helper 2 cell (72±9% vs 67±7%; p=0.004) counts. Intracellular cytokine staining at day 15 after co-culture with the CD19+ Daudi cell line showed significantly lower TNF-α (47±7% vs 58±6%; p<0.001) and IFN-γ production (33±13% vs 40±13%; p<0.001) in presence of idelalisib at CD3+ CARTs. This effect was reversible and similar cytokine production for TNF-α (64±7%) and IFN-γ (40±13%) could be achieved when CARTs were rested for one day without idelalisib.

Conclusions: PI3K inhibition during CART production shifted the T cells into subpopulations that allow higher persistence, long-term engraftment and sustained antitumor activity. CARTs showed a less exhausted phenotype and a higher capacity to migrate into lymph nodes. Evaluation of the effect of idelalisib for CART cultivation in patient-derived donor cells is currently ongoing. The identification of the optimal culturing condition of CARTs will further increase the benefit of this promising treatment approach in cancer immunotherapy.