The Dual PI3Kdg Inhibitor Duvelisib Potently Inhibits Cytokine Release Syndrome (CRS) While Maintaining CAR-T Function

Clinical efficacy of CAR-T cell therapy is limited by toxicities such as Cytokine Release Syndrome (CRS) in over 30% of patients. Using a protein kinase inhibitor library, we identified duvelisib, a novel and selective dual PI3Kδγ inhibitor as a potent inhibitor of CRS in vitro and in vivo without attenuating CAR-T function. To study the mechanisms of inhibition of CRS, CART19 (19-28BBζ), CD19+ Ramos CBR GFP (CG), and immature dendritic cells (IDC) were co-cultured with duvelisib and ruxolitinib (JAK1/2 inhibitor; a less potent inhibitor of CRS also identified in screen) and submitted for scRNA-seq. Across 6 experimental conditions, we identified 38,330 CART19 cells, 8,602 IDCs and 2,537 Ramos CG cells (Fig 1A). The transcriptional landscape encompassed five distinct populations of IDCs and three subsets of CART19. In experiments lacking RamosCG or IDCs, CART19 cells were largely in G1 and exhibited interferon signaling response (IFN) by the Effector /Effector Memory population (Fig 1B). Persistent IFN signaling and pro-inflammatory cytokines expressed by CART19 were dampened by both inhibitors. With both IDC and Ramos CG, CART19 cells shifted to a proliferative (G2/M) state while IDCs shifted from an immature (MMP12+) to mature (CCL22+CCL17+) state. Of note, the proportion of immature (MMP12+) iDCs was expanded in conditions with duvelisib, but not ruxolitinib. By evaluating cell-cell signaling and transcriptional pathways between IDC and CART19 cells we show that each drug is able to inhibit CRS by different mechanisms. This single-cell analyses using an in vitro model of CART-mediated CRS has allowed for improved understanding of pathways of CRS in response to CART19 therapy and vulnerabilities for pharmacologic interventions.

Jayasinghe:Multiple Myeloma Research Foundation: Consultancy; Wugen: Consultancy. Pachter:Verastem Oncology: Current Employment. DiPersio:WUGEN: Current equity holder in private company, Research Funding; Incyte: Consultancy, Research Funding; Macrogenics: Research Funding; BioLineRx, Ltd.: Research Funding; Amphivena Therapeutics: Research Funding; NeoImmune Tech: Research Funding; RiverVest Venture Partners: Consultancy, Membership on an entity's Board of Directors or advisory committees; CAR-T cell Product with Washington University and WUGEN: Patents & Royalties; VLA-4 Inhibitor with Washington University and Magenta Therapeutics: Patents & Royalties; hC Bioscience, Inc.: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.