IL-2 mutein-mediated improvement of CAR T function in Relapsed/Refractory diffuse B cell lymphoma

Abstract

Abstract Although chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment landscape of B-cell malignancies, many patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) experience disease relapse driven by poor CAR-T cell persistence and functional impairment within the immunosuppressive tumor microenvironment. Interleukin-2 (IL-2) has potential to enhance T-cell activity, but its clinical application is hindered by undesirable effects, particularly the stimulation of regulatory T cells (Tregs) and toxicities. To selectively enhance effector T-cell activation while limiting Treg expansion and toxicities, our team engineered a novel IL-2 variant (called IL2-mutein, or IL2m) incorporating four point mutations that disrupt its binding to the IL-2 receptor alpha chain (CD25), which is predominantly expressed on Tregs. The safety and efficacy of this IL2m is being investigated as a monotherapy for patients with solid tumor malignancies (clinical trial #RPCEC00000234). We evaluated whether IL2m can improve CAR-T cell efficacy by enhancing CAR-T cell persistence and decreasing dysfunction. We investigated the effects of IL2m on the function of CD19 CAR-T cells in an in vitro model of DLBCL using Eµ-ALL tumor cells. We showed that IL2m promoted antigen-driven expansion of CD19 CAR-T cells, enhancing CD8+CAR-T proliferation (80.3 vs 35.6 % CD8+ cells of lymphocytes) and increasing granzyme B (99.6 vs 47.8 % GzmB+of CD8+ lymphocytes) and TNFα secretion (31.2 vs 24.9 pg/mL, p < 0.0001) upon co-culture with tumor cells, as assessed by real-time imaging and flow cytometry. Moreover, IL2m-treated CAR-T cells exhibited reduced PD-1 expression (3.7 vs 20.2 % PD-1+cells of CD8+ lymphocytes), suggesting improved functional fitness. To evaluate the translational relevance of this compound, we evaluated IL2m in a syngeneic r/r DLBCL mouse model in which CD19 CAR-T cells alone show limited efficacy with a median overall survival of only 24 days post-CAR infusion (n = 16-18 mice per group). IL2m treatment significantly improved overall median survival in mice to 46 days and reduced tumor burden (total flux of 1.02e+09 vs 7.44e+09 p/s 17 days post-CAR infusion, p < 0.0001) compared to those injected with CAR-T cells alone, as measured by bioluminescent imaging. Notably, a substantial proportion of mice responded to the treatment (8/18 mice), with enhanced efficacy observed using a twice-daily low-dose regimen. Indeed, because of its short half-life, administering IL2m in two daily doses of 600 IU resulted in better responses than a single daily dose of 1200 IU. Mechanistically, IL2m enhanced the proliferation and activation of endogenous CD8+T cells (10.6 vs 5.3 % CD8+CD44+Ki67+ cells of lymphocytes, p = 0.0004), increased the frequency of circulating CAR-T cells (0.3 vs 0.1 cells per µL of blood, p = 0.0223), and boosted granzyme B+CD8+populations (2.9 vs 0.5 cells per µL of blood, p = 0.0099), without promoting immunosuppressive Treg expansion – resulting in improved tumor cell killing (12.5 vs 614.0 Eµ cells per µL of blood, p = 0.0191), as evaluated by flow cytometry analyses on the peripheral blood of the animals. Altogether, our findings support IL2m as a promising immunomodulatory adjunct to improve the efficacy and durability of CAR-T cell therapy, leading to the development of a phase I trial combining IL2m with CAR-T cell therapy in r/r DLBCL.