Construction of stable membranal CMTM6-PD-L1 full-length complex to evaluate the PD-1/PD-L1-CMTM6 interaction and develop anti-tumor anti-CMTM6 nanobody

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.