Orthogonal Gene Engineering Enables Novel Synthetic States of Powerful Tumor-rejecting CD8+ T Cells Escaping Canonical Exhaustion. Orthogonal Gene Engineering Enables Novel Synthetic States of Powerful Tumor-rejecting CD8+ T Cells Escaping Canonical Exhaustion

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ TILs away from exhausted effector states remains an elusive goal. Our work provides for the first-time evidence that orthogonal gene-engineering of T cells to secrete an IL-2-variant binding the IL-2R and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host, and led – in the absence of lymphodepletion or exogenous cytokine support – to high levels of engraftment and tumor regression. Our work unlocks the novel opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors. Overall design: CD45+ bulk TILs were independently sorted at at days 5 (T1), 12 (T2) and 26 (T3) post cell transfer Groups: Non-Treated tumor-bearing mice (G5); Mice received ACT using Untransduced OT1 T cells (G1);Mice received ACT using PD1d/IL2V-secreting OT1 (G2); Mice received ACT using PD1d/33-secreting OT1 (G3); Mice received ACT using PD1d/2V/33-secreting OT1 (G4)

迄今为止，尚无任何免疫治疗策略能够完全克服T细胞耗竭（T-cell exhaustion）——这一状态是慢性活化效应T细胞的必然归宿，亦是当前免疫治疗领域的核心挑战之一。探索如何将CD8+肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）重编程以脱离耗竭性效应细胞状态，仍是一个悬而未决的棘手目标。本研究首次提供实验证据：通过正交基因工程技术改造T细胞，使其分泌结合IL-2Rβγ的IL-2变体以及警报素IL-33，可使过继转移的T细胞获得一种全新的合成型细胞状态；该状态脱离了经典的耗竭表型，并展现出更优异的效应细胞功能。此类细胞可成功突破宿主体内的稳态屏障，且无需淋巴细胞清除预处理或外源性细胞因子辅助，即可实现高效细胞植入并促使肿瘤显著消退。本研究为理性设计合成型CD8+ T细胞状态开辟了全新途径，这类细胞可规避耗竭并实现对晚期实体瘤的有效控制。
实验设计概况：于细胞移植后第5天（T1）、第12天（T2）及第26天（T3）分别分选CD45+ 总肿瘤浸润淋巴细胞。
实验分组如下：① 未处理的荷瘤小鼠（G5）；② 接受未转导OT1 T细胞过继细胞治疗（adoptive cell transfer, ACT）的小鼠（G1）；③ 接受分泌PD1d/IL2V的OT1 T细胞过继细胞治疗的小鼠（G2）；④ 接受分泌PD1d/33的OT1 T细胞过继细胞治疗的小鼠（G3）；⑤ 接受分泌PD1d/2V/33的OT1 T细胞过继细胞治疗的小鼠（G4）