A Private Crosstalk Established by Tumor-Targeted Cytokine Release Rescues CAR-T Activity and Engages Host T Cells against Glioblastoma

Chimeric antigen receptor T cells (CAR-Ts) have shown limited efficacy in solid tumors, due to poor penetration, constrained activity, and early exhaustion into the immunosuppressive tumor microenvironment (TME). While cytokines can counteract immune-suppression, their systemic administration entails risk of toxicities and counter-regulatory responses. Here, we leveraged on a population of tumor associated TIE2 expressing macrophages (TEMs) to release IFN- and/or orthogonal IL-2 (oIL2) at the tumor site. TEM-mediated cytokine delivery rescued CAR-Ts functionality against the clinically relevant antigen B7-H3 in an immunocompetent mouse model of glioblastoma multiforme (GBM) which was refractory to anti-B7-H3 CAR-Ts alone. Immunophenotypic and transcriptomic analyses showed that TEM-mediated cytokine delivery counteracted premature CAR-Ts terminal exhaustion and sustained their effector/memory state which accounts for tumor cell elimination. This was mirrored by delayed tumor growth and prolonged mice survival achieving a synergistic effect in the IFN- + oIL2 group receiving CAR-Ts. Importantly, IFN-, especially when combined with oIL2, also triggered an endogenous T cell response against tumor associated antigens other than B7-H3. Altogether our findings suggest that the combination approach between the two genes and cell therapy strategies presented here, and already under clinical testing as monotherapies, could achieve synergistic therapeutic effect also in GBM patients. CAR-T Single-cell RNA sequencing (scRNAseq) of GBM tumor mice (mixed 1:1 ratio of adoptive and endogenous immune cells) belonging to different treatment groups: Empty, IFN, oIL2, and IFN+oIL2.