S259: sar444245, a non-alpha il2, rescues chronic antigen- and car-driven t-cell dysfunction

Background: T cell dysfunction from chronic antigen stimulation remains a hurdle limiting the efficacy of CAR T cell therapy. SAR444245 is a PEGylated, non-alpha IL2 where the PEG is attached to a novel amino acid such that it prevents binding to the alpha subunit of the IL-2 receptor, avoiding drug toxicities and expansion of regulatory T-cells. SAR444245 retains binding to the beta-gamma receptors that selectively expand tumor-killing T effector cells. Using in vitro and in vivo models of T cell exhaustion, we demonstrate that SAR444245 stimulates T cell proliferation and effector functions and mitigates T cell exhaustion following chronic stimulation. Aims:1)Demonstrate reversal of T cell exhaustion following chronic antigen exposure through treatment with SAR444245 2)Demonstrate enhanced proliferation and improved anti-tumor activity of CAR T cells following treatment with SAR444245 Methods:T-cell assays: CD8 T cells were isolated from healthy donors and repetitively stimulated with antigen over 12 days to establish exhausted CD8 T cells. They were exposed to SAR444245 for an additional 7 days. Proliferation and co-inhibitory marker expression were measured by flow cytometry. Luminex cytokine arrays were used to measure T-cell cytokine production. CAR T-cell assays: Anti-CD19 CAR T cells were engineered from relapsed LBCL patient PBMCs obtained at the time of leukapheresis for CAR T cell therapy. Short term CAR T cell cytotoxicity was assessed by co-culture with 2 LBCL cell lines in the presence or absence of SAR444245 over 48 hours. Chronic CAR T stimulation was modelled in vitro by repeat addition of target tumor cells to CAR T cells every 48 hours over 8 days in the presence or absence of SAR444245. Live T cell and lymphoma cell counts as well as T-cell immunophenotyping were obtained at baseline and days 4 and 8 using flow cytometry. In vivo: Raji tumor cells expressing luciferase were injected into NSG-MHC Class I/II knockout mice. Tumor burden was monitored via in vivo bioluminescence imaging. Mice were either treated with anti-CD19/22 CAR T cells alone or in combination with SAR444245 (weekly treatment for 3 doses). Blood samples were obtained from mice on days 11 and 18 days post-CAR T cell infusion to immunophenotype T cells. Results: Seven days following exposure to SAR444245, exhausted CD8 T cells demonstrated enhanced IFN-γ and TNF-α secretion, decreased co-inhibitory molecule expression, and increased proliferation compared to cells not exposed to SAR444245. CD8 T cells exposed to SAR444245 on the day of initial activation, followed by repetitive antigen exposure, proliferated and maintained IFN-γ similar to acutely stimulated CD8 T cells. Luminex cytokine array analysis of treated CD8 T cells demonstrated robust expression of cytokines and effector molecules, and increased polyfunctionality. In short term assays, SAR444245 induced T cell expansion without significant increase in cytotoxicity. However, in long-term assays mimicking chronic CAR stimulation and exhaustion, addition of SAR444245 to CAR T-cells resulted in enhanced CAR T-cell proliferation through day 8, and enhanced control of tumor cells through repeated additions. In an in vivo Raji-Luc model, the combination of SAR444245 with CD19/CD22 CAR T cells demonstrated enhanced in vivo CAR T expansion and sustained anti-tumor efficacy compared to CAR T cells alone. Summary/Conclusion: SAR444245 treatment of exhausted antigen-specific or CAR T cells restored functionality and alleviated T cell dysfunction in vitro and in vivo. These data suggest that SAR444245 rescue of CAR T cell exhaustion through maintenance of CAR T cell proliferative capacity, cytotoxicity, and polyfunctionality and provide rationale for future clinical study of SAR444245 with CAR T cells. Keywords: Diffuse large B cell lymphoma, CAR-T, Cancer immunotherapy, Interleukin-2