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. Overall design: 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.

嵌合抗原受体T细胞（Chimeric antigen receptor T cells, CAR-T）在实体瘤中的治疗效果有限，这源于其穿透能力不足、抗肿瘤活性受限，且会在免疫抑制性肿瘤微环境（immunosuppressive tumor microenvironment, TME）中发生早期耗竭。细胞因子可抵消免疫抑制作用，但全身给药会带来毒性反应与代偿性应答的风险。本研究利用表达TIE2的肿瘤相关巨噬细胞（tumor associated TIE2 expressing macrophages, TEMs）在肿瘤部位释放干扰素γ（IFN-γ）和/或正交白介素2（orthogonal IL-2, oIL2）。在仅使用抗B7-H3 CAR-T治疗呈现难治性的免疫健全多形性胶质母细胞瘤（glioblastoma multiforme, GBM）小鼠模型中，TEM介导的细胞因子递送恢复了CAR-T针对临床相关抗原B7-H3的抗肿瘤功能。免疫表型与转录组学分析显示，TEM介导的细胞因子递送可抵消CAR-T细胞过早发生的终末耗竭，维持其效应/记忆状态，从而实现肿瘤细胞清除；该效果伴随肿瘤生长延迟与小鼠生存期延长，在联合输注IFN-γ与oIL2的CAR-T治疗组中观察到协同效应。值得注意的是，IFN-γ，尤其是与oIL2联合使用时，还可触发针对B7-H3以外其他肿瘤相关抗原的内源性T细胞应答。综上，本研究提出的双基因递送与细胞治疗联合策略（两种策略目前已作为单药疗法进入临床试验），有望在GBM患者中实现协同治疗效果。实验整体设计：对不同治疗组（空白对照组、IFN-γ组、oIL2组、IFN-γ+oIL2组）的GBM荷瘤小鼠进行单细胞RNA测序（single-cell RNA sequencing, scRNAseq），其肿瘤组织中过继性免疫细胞与内源性免疫细胞的比例为1:1。