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2026 Inhibition of PI3Kγ/δ Signaling Promotes an Early Memory State in CAR T Cells and Enhances Their In Vivo Persistence and Efficacy

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research, Lymphomas, Non-Hodgkin lymphoma, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diseases, Treatment Considerations, Biological therapies, Immunology, Lymphoid Malignancies, Biological Processes
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Alexandra E. Rojek, MD1, Lishi Xie, PhD2*, Alan J. Cooper3*, Xiufen Chen, PhD1*, Peter A. Riedell, MD1,2, Michael R Bishop, MD1,2 and Justin Kline, MD1,4

1Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL
2David and Etta Jonas Center for Cellular Therapy, University of Chicago, Chicago, IL
3Yale School of Medicine, New Haven, CT
4University of Chicago, Chicago, IL

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<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<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<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<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<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<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<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.

Disclosures: Riedell: Calibr: Research Funding; Intellia Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Research Funding; Fate Therapeutics: Research Funding; Genmab: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cargo Therapeutics: Research Funding; Tessa Therapeutics: Research Funding; Adaptive Biotechnologies: Honoraria; ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Research Funding; CVS Caremark: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sana Biotechnology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; Nektar Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bishop: GenMab: Honoraria, Speakers Bureau; Incyte: Honoraria; ADC Therapeutics: Honoraria, Speakers Bureau; Arcellx, Autolus, Bristol-Myers Squibb, CRISPR Therapeutics, Lyell, Gilead Sciences and Novartis: Research Funding; AbbVie, ADC Therapeutics, Bristol-Myers Squibb, Gilead Sciences, Incyte, Novartis, Sanofi and Servier: Honoraria, Speakers Bureau; Achieve Clinics, Arcellx, Autolus, BMS, Chimeric Therapeutics, CRISPR Therapeutics, In8Bio, Iovance Biotherapeutics, Kite-Gilead, Optum Health, Novartis, Sana Biotechnology: Consultancy; Achieve Clinics, In8Bio: Current holder of stock options in a privately-held company; Sana Biotechnology: Consultancy, Honoraria; Chimeric Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyer-Squibb: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; In8bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite/Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Speakers Bureau; Iovance Biotherapeutics: Consultancy; Galapagos: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kline: Abbvie: Consultancy; BeiGene: Consultancy; BMS: Consultancy; Gilead Sciences: Consultancy; Merck: Research Funding; Curio Science: Honoraria; Genmab: Consultancy; Seagen: Consultancy; Targeted Oncology: Honoraria.

*signifies non-member of ASH