Chimeric antigen receptor T cells (CAR-T) induce impressive responses in patients with hematologic malignancies but can also mediate a systemic inflammatory toxicity known as cytokine release syndrome (CRS), marked by elevated levels of pro-inflammatory cytokines and chemokines released from activated CAR-T and innate immune cells. Release of the pro-inflammatory cytokine interferon-gamma (IFNγ) in response to antigen is used as a potency assay for CAR-T cells, but elevated levels have been identified in patients suffering from CAR-T-associated toxicities such as CRS and neurotoxicity. Mutations in IFNγ receptor signaling have been identified as a mechanism of resistance in checkpoint blockade in melanoma and other solid tumors, and we have recently identified that IFNγ receptor signaling also confers resistance to CAR-T cell mediated cytotoxicity in solid tumors, but its biologic role in conferring responses in hematologic malignancies is not established.
CD19-targeted CAR-T were generated using either 4-1BB or CD28 costimulatory domains. CAR-T effector functions in vitro and in vivo were assessed in the presence of absence of IFNγ-blocking antibody. Furthermore, we used CRISPR/Cas9 editing to knock out IFNγ in CD19-directed CAR-T cells. The effects of IFNγ inhibition in CAR-T by pharmacologic and genetic approaches on T cell function, immune checkpoint inhibitor expression, cancer cell lysis and macrophage activation/phenotype were assessed using ELISA, flow cytometry, in vitro/in vivo tumor models and Luminex/fluorescence microscopy/NanoString, respectively. Finally, serum from B cell lymphoma patients treated with the CAR-T products tisagenlecleucel or axicabtagene ciloleucel was collected 3 days post-CAR infusion and added to human macrophages in vitro in the presence of blocking antibodies to IFNγ versus the current clinical agents for managing CRS, including those targeting IL-1Rα and IL-6R. Macrophage phenotype and function was determined using NanoString, ELISA, and immunofluorescence microscopy.
We found that pharmacologic blockade or genetic knockout of IFNγ specifically reduces IFNγ signaling without compromising T cell phenotype or effector function, including production of GM-CSF, IL-2, Granzyme B and TNFα. We also observed reduced expression of the immune checkpoint proteins CTLA-4, PD-1, Lag3 and Tim3, which correlated with enhanced antigen-specific CAR-T proliferation. Cytotoxicity assays and NSG xenograft tumor-bearing mouse models revealed that blocking IFNγ has no effect on therapeutic efficacy of CAR T cells against CD19 + leukemias or lymphomas in vitro or in vivo. Furthermore, pharmacologic blockade or genetic knockout of IFNγ in CD19-directed CAR T cells abrogated macrophage activation in vitro and in hybrid in vitro/in vivo models of CRS, as shown by a reduction of activation markers (CD69, CD86) and pro-inflammatory proteins (IL-6, IP-10, MIP-1β and MCP-1). Further interrogation revealed that these findings were IFNγ-dependent but cell contact-independent. Finally, data herein reveals that blocking IFNγ in both healthy donor CAR-T cultures and lymphoma patient serum results in reduced macrophage activation/function to a similar, if not superior, extent as current clinical approaches targeting IL-1Rα and IL-6R. In addition to reduced macrophage function, NanoString analysis revealed a decreased expression of immune checkpoint inhibitor genes HAVCR2, VSIR and PDCD1LG2 and upregulation of co-stimulatory genes DPP4 and ICOSL.
Collectively, these data show that IFNγ is dispensable for the efficacy of CAR-T against hematologic malignancies and blocking IFNγ could simultaneously mitigate cytokine-related toxicities while enhancing T cell proliferation and persistence via reduced expression of immune checkpoint proteins. Furthermore, direct comparison of IFNγ blockade or knockout in the CAR T cell product with current clinical strategies suggests that targeting IFNγ could mitigate major cytokine-related toxicities to a greater extent than existing approaches.
Frigault: Arcellx: Consultancy; Novartis: Consultancy, Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy; Iovance: Consultancy; Takeda: Consultancy; Editas: Consultancy. Maus: WindMIL: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; Tmunity: Consultancy; Novartis: Consultancy; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Kite Pharma: Consultancy, Research Funding; GSK: Consultancy; Intellia: Consultancy; In8bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; Cabaletta Bio (SAB): Consultancy; BMS: Consultancy; Bayer: Consultancy; Atara: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.