Inhibition of PI3Kγ/δ signaling promotes an early memory state in CAR T cells and enhances their in vivo persistence and efficacy

Introduction

Most patients (pts) with R/R large B-cell lymphoma (LBCL) experience disease relapse after receiving CAR T cell therapy. Enrichment of CAR T cell products for early memory-like T cell phenotypes, such as stem cell memory T cells (T_SCM), correlates with improved outcomes. We screened FDA-approved drugs for those capable of enhancing T_SCM in CAR T cells and identified the PI3K gamma/delta (γ/δ) inhibitor, duvelisib (Duv), for further investigation.

Methods

Second generation anti-CD19 CAR T cells were generated through lentiviral transduction and exposed to Duv vs control during manufacturing. CAR T cells were characterized by flow cytometry-based phenotypic and functional assays. NSG mice were xenografted with OCI-Ly8 cells, CAR T cells were transferred 7 days later, and survival was measured.

Results

PI3Kγ/δ inhibition with Duv during CAR T cell manufacturing increased T_SCM cell proportions among healthy donors (n=7) and DLBCL pt samples (n=9) (p<0.01), consistent with prior work (Funk et al., Blood 2022). RNAseq of Duv-generated CAR CD8⁺ T_SCM cells revealed increased expression of memory-associated genes in Duv CAR CD8⁺ T_SCM cells, enriched for a stem-cell like gene signature (Gattinoni et al, Nat Med 2011) (p<0.01), as well as an increased FOXO1 regulon score (Doan et al, Nature 2024) (p=0.036). Pharmacologic inhibition of FOXO1 abrogated the effect of PI3Kγ/δ inhibition on increasing CAR T_SCM. Duv-treated CAR T cells exhibited improved persistence (p<0.01) and mediated improved survival in NSG mice xenografted with human OCI-Ly8 DLBCL cells (p<0.01).

Conclusion

Inhibition of PI3Kγ/δ signaling with Duv delays terminal CAR T cell differentiation, mediates improved survival of lymphoma-bearing mice, and promotes early memory transcriptional programs. This work 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.