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
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.
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