CKLF (chemokine-like factor)-MARVEL transmembrane domain containing protein 6 (CMTM6) is a novel regulator to maintain the stability of PD-L1. CMTM6 can colocalize and interact with PD-L1 on the recycling endosomes and cell membrane, preventing PD-L1 from lysosome-mediated degradation and proteasome-mediated degradation thus increasing the half-life of PD-L1 on the cell membrane. The difficulties in obtaining stable full-length PD-L1 and CMTM6 proteins hinder the research on their structures, function as well as related drug development. Using lauryl maltose neopentyl glycol (LMNG) as the optimized detergent and a cell membrane mimetic strategy, we assembled a stable membrane-bound full-length CMTM6-PD-L1 complex with amphipol A8-35. When the PD-1/PD-L1-CMTM6 interactions were analyzed, we found that CMTM6 greatly enhanced the binding and delayed the dissociation of PD-1/PD-L1, thus affecting immunosuppressive signaling and anti-apoptotic signaling. We then used the CMTM6-PD-L1 complex as immunogens to generate immune repertoires in camels, and identified a functional anti-CMTM6 nanobody, called 1A5. We demonstrated that the anti-CMTM6 nanobody greatly decreased T-cell immunosuppression and promoted apoptotic susceptibility of tumor cells in vitro, and mainly relied on the cytotoxic effect of CD8⁺ T-cells to exert tumor growth inhibitory effects in CT26 tumor-bearing mice. In conclusion, the stable membrane-bound full-length CMTM6-PD-L1 complex has been successfully used in studying PD-1/PD-L1-CMTM6 interactions and CMTM6-targeting drug development, suggesting CMTM6 as a novel tumor immunotherapy target.

CKLF（趋化因子样因子）-MARVEL跨膜结构域含有蛋白6（CMTM6）是一种维持PD-L1稳定性的新型调节剂。 CMTM6可以在循环内体和细胞膜上与PD-L1共定位并相互作用，防止PD-L1被溶酶体介导的降解和蛋白酶体介导的降解，从而增加PD-L1在细胞膜上的半衰期。稳定的全长PD-L1和CMTM6蛋白的获得困难阻碍了对其结构、功能的研究以及相关药物的开发。使用月桂基麦芽糖新戊二醇 (LMNG) 作为优化的去垢剂和细胞膜模拟策略，我们与 amphipol A8-35 组装了稳定的膜结合全长 CMTM6-PD-L1 复合物。当分析PD-1/PD-L1-CMTM6相互作用时，我们发现CMTM6极大地增强了PD-1/PD-L1的结合并延迟了PD-1/PD-L1的解离，从而影响了免疫抑制信号和抗凋亡信号。然后，我们使用 CMTM6-PD-L1 复合物作为免疫原在骆驼中产生免疫库，并鉴定了一种功能性抗 CMTM6 纳米抗体，称为 1A5。我们证明抗CMTM6纳米抗体在体外可显着降低T细胞免疫抑制并促进肿瘤细胞凋亡易感性，并且主要依靠CD8 ⁺ T细胞的细胞毒作用对CT26荷瘤小鼠发挥肿瘤生长抑制作用。总之，稳定的膜结合全长CMTM6-PD-L1复合物已成功用于研究PD-1/PD-L1-CMTM6相互作用和CMTM6靶向药物开发，表明CMTM6作为一种新型肿瘤免疫治疗靶点。