Znf740 in the regulation of CD8+T cell exhaustion

摘要： CAR-T细胞疗法是一种新兴的癌症治疗选择，但其疗效有限，特别是在固体 肿瘤，因为效应CD 8 +T细胞在肿瘤微环境中变得功能失调并耗尽 （TME）。然而，定义效应器与耗尽状态之间微妙平衡的关键途径， CD 8 +T细胞仍不清楚。我们的初步数据表明，Znf 740，一个新的锌指成员， 转录因子家族，对于效应CD 8 +T细胞至关重要。Znf 740与SUMO化T-bet结合 并促进CD 8+肿瘤浸润淋巴细胞（TIL）的效应子功能和抗肿瘤活性。 相反，在耗尽的PD 1 + Tim 3 + CD 8 +T细胞中，Znf 740表达下调，这破坏了细胞的免疫功能。 Znf 740：T-bet络合物。重要的是，Znf 740表达的重建拯救了耗尽的CD 8 + TIL， 恢复其效应器功能。此外，Znf 740在CD 8 +T细胞中的转基因表达导致肿瘤细胞减少。 生长，这与TIL产生的IFN-γ升高有关。这些关键发现让我们假设 Znf 740：T-bet复合物对于CD 8 +T细胞的效应子功能是至关重要的， PD 1 + Tim 3+细胞中的这种复合物促进了可作为治疗靶向的CD 8 + TIL的耗尽。 在Aim 1中，我们将研究Znf 740如何促进效应CD 8 +T细胞功能和抗肿瘤反应。使用 新产生的Znf 740-/-和T-bet-K208 R基因敲入小鼠，我们将描述Znf 740 通过其SUMO相互作用基序（SIM）与T-bet结合，形成Znf 740：T-bet复合物，并反式激活 效应CD 8 + TIL中的IFN-γ启动子。在目标2中，我们将靶向Znf 740以克服T细胞耗竭并促进T细胞的增殖。 肿瘤消退我们将研究在晚期肿瘤中Znf 740：T-bet复合物的破坏如何促进肿瘤细胞的增殖。 PD 1 + Tim 3+耗尽的TIL的替代转录谱。使用新产生的T细胞特异性Znf 740 在转基因小鼠中，我们将测试在CAR-T细胞中过表达Znf 740对癌胚抗原的影响。 在MC 38结肠癌模型中，最后，过度表达Znf 740在糖尿病中的治疗潜力也被证实。 CAR-T细胞将在结肠癌患者来源的异种移植物（PDX）模型中进行测试。 这些研究的完成将导致建立1）一种新的Znf 740：T-bet复合物，其对于 效应CD 8 + T细胞功能，2）确定Znf 740表达减少如何破坏该复合物，导致 在晚期肿瘤中耗尽的CD 8 + TIL中的替代转录谱，和3）评估靶向靶向的手段， Znf 740克服了目前CAR-T细胞治疗实体瘤的局限性。这可能导致临床 使用“抗耗竭CAR-T细胞”改善晚期肿瘤患者的结局。

ABSTRACT: CAR-T cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors, because the effector CD8+T cells become dysfunctional and exhausted in the tumor microenvironment (TME). However, the key pathways that define the delicate balance between the effector vs. exhausted state of CD8+T cells remain unclear. Our preliminary data demonstrate that Znf740, a novel member of the zinc finger family of transcription factors, is critically essential for effector CD8+T cells. Znf740 binds to SUMOylated T-bet and promotes the effector function and anti-tumor activity of CD8+ tumor-infiltrating lymphocytes (TILs). Conversely, in exhausted PD1+Tim3+CD8+T cells, Znf740 expression is downregulated, which disrupts the Znf740:T-bet complex. Importantly, reconstitution of Znf740 expression rescues exhausted CD8+TILs and restores their effector function. Further, transgenic expression of Znf740 in CD8+T cells resulted in reduced tumor growth which was associated with elevated IFN- production by TILs. These key findings led us to hypothesize that the Znf740:T-bet complex is critically essential for the effector function of CD8+T cells, and the disruption of this complex in PD1+Tim3+ cells promotes exhaustion of CD8+TILs which can be therapeutically targeted. In Aim1, we will investigate how Znf740 promotes effector CD8+T cell function and anti-tumor response. Using newly generated Znf740-/- and T-bet-K208R knock-in mice, we will delineate the mechanism by which Znf740 binds to T-bet through its SUMO-interacting motif (SIM) to form the Znf740:T-bet complex and transactivates the IFN- promoter in effector CD8+TILs. In Aim 2, we will target Znf740 to overcome T cell exhaustion and promote tumor regression. We will investigate how disruption of the Znf740:T-bet complex in advanced tumors promotes an alternate transcription profile of PD1+Tim3+ exhausted TILs. Using newly generated T cell-specific Znf740 transgenic mice, we will test the effect of overexpressing Znf740 in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 colon cancer model. Finally, the therapeutic potential of overexpressing Znf740 in CAR-T cells will be tested in colon cancer patient-derived xenograft (PDX) models. Completion of these studies will result in the establishment of 1) a novel Znf740:T-bet complex that is critical for the effector CD8+ T cell function, 2) determine how reduced Znf740 expression disrupts this complex leading to alternate transcription profile in exhausted CD8+ TILs in advanced tumors, and 3) evaluate the means to target the Znf740 to overcome the current limitations of CAR-T cell therapy for solid tumors. This could lead to clinical trials using "Exhaustion Resistant CAR-T cells" for improved outcomes in patients with advanced tumors.