Inhibition of PI3Kγ/δ Signaling Promotes an Early Memory State in CAR T Cells and Enhances Their <i>In Vivo</i> Persistence and Efficacy

Introduction CAR T cell therapy has improved outcomes for patients (pts) with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Unfortunately, most pts experience disease relapse after receiving CAR T cells, where survival tends to be poor. Enrichment of CAR T cell products for early memory-like T cell phenotypes, such as stem cell memory T cells (TSCM), correlates with improved clinical outcomes (Westin et al, NEJM 2023). We screened a library of FDA-approved drugs for those capable of enhancing TSCM in CAR T cell products and identified the PI3K gamma/delta (γ/δ) inhibitor, duvelisib (Duv), as the lead compound warranting investigation. Methods Second generation anti-CD19 CAR T cells (4-1BBz and CD28z) were generated through lentiviral transduction and exposed to Duv (150nM) or DMSO (ctrl) during manufacturing. CAR T cells were characterized by flow cytometry-based phenotypic and functional assays. TSCM phenotype was defined by CD45RA+CCR7+ staining. Antigen-directed assays were conducted using human OCI-Ly8 DLBCL cells. For experiments exploring a requirement for FOXO1 activity downstream of Duv, the FOXO1 inhibitor AS1842856 was employed. NSG mice were xenografted with OCI-Ly8 cells, and Duv- vs ctrl-generated CAR T cells were transferred 7 days later, and survival was measured. CAR T cell persistence was measured in mouse peripheral blood at specified timepoints. Results PI3Kγ/δ inhibition with Duv during CAR T cell manufacturing significantly increased TSCM cell proportions, predominantly in CD8+ CAR T cells, when compared to controls among healthy donors (n=7) and DLBCL patient samples (n=9) (p&amp;lt;0.01), consistent with prior observations (Funk et al., Blood 2022). Selective PI3Kδ inhibition with idelalisib similarly increased proportions of TSCM CAR T cells (p&amp;lt;0.01), while selective PI3Kγ inhibition (eganelisib) did not (p=0.48), suggesting the predominant effect of PI3Kγ/δ inhibition with Duv is mediated through PI3Kδ. However, dual PI3Kγ/δ inhibition was superior to PI3Kδ inhibition in enhancing CAR TSCM in several donors. Compared to ctrl CAR T cells, effector cytokine (IFNγ and TNFα) production was higher in Duv CAR T cells following restimulation with OCI-Ly8 cells in vitro (p&amp;lt;0.05), whereas cytolytic capabilities were similar. RNAseq of Duv- and ctrl-generated CAR CD8+ TSCM cells revealed increased expression of memory-associated genes (CCR7, TCF7) in Duv CAR CD8+ TSCM cells, which were also enriched for a stem-cell like gene signature (Gattinoni et al, Nat Med 2011) by GSEA (p&amp;lt;0.01). A FOXO1 regulon score (Doan et al, Nature 2024) was also significantly increased in Duv-treated CD8+ TSCM cells compared to ctrl (p=0.036). Pharmacologic inhibition of FOXO1 with AS1842856 during CAR T cell manufacturing with Duv completely abrogated the effect of PI3Kγ/δ inhibition on increasing CAR TSCM, revealing a mechanistic requirement for FOXO1 transcriptional activity downstream of PI3Kγ/δ inhibition in enhancing CAR TSCM. Finally, Duv-treated CAR T cells exhibited markedly improved persistence (p&amp;lt;0.01 at 10 days) and mediated significantly improved survival in NSG mice xenografted with human OCI-Ly8 DLBCL cells (median survival 43 days vs not reached for ctrl and Duv CAR T cells, respectively; p&amp;lt;0.01). Flow sorted CAR TSCM cells generated with Duv demonstrated significantly increased expansion in vivo compared to ctrl CAR TSCM at 10 days post-infusion (p&amp;lt;0.05), indicating that in addition to increasing their proportions in CAR T cell products, PI3Kγ/δ inhibition also mediated favorable cell-intrinsic effects in TSCM CAR T cells. Conclusion Inhibition of PI3Kγ/δ signaling with Duv delays terminal CAR T cell differentiation, enhancing proportions of TSCM phenotype cells. Duv-generated CAR T cells persist longer and mediate improved survival of lymphoma-bearing mice. PI3Kγ/δ inhibition also promotes early memory transcriptional programs, even among TSCM cells. The enhanced FOXO1 regulon score, as well as the requirement for FOXO1 to mediate the Duv-treatment effect suggests that enhanced FOXO1 nuclear translocation is the major downstream mediator promoting early memory phenotypes of Duv-treated CAR T cells. Pharmacological manipulation of CAR T cells offers a safe, affordable, and rapid approach to improve CAR T cell therapy efficacy. This work provides the rationale for early phase clinical trials of Duv-treated CAR T cells for pts with R/R DLBCL.