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

Xiao-min Jia1,2,3, Yi-ru Long2,3, Xiao-lu Yu2,3, Run-qiu Chen, Li-kun Gong2,3,4,5, Yong Geng1,2,3
1 The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
5 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China
Correspondence to: Li-kun Gong:, Yong Geng:,
DOI: 10.1038/s41401-022-01020-3
Received: 12 July 2022
Accepted: 23 October 2022
Advance online: 22 November 2022


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.
Keywords: cancer immunotherapy; CMTM6; CMTM6/PD-L1 complex; protein interactions; anti-CMTM6 nanobody

Article Options

Download Citation

Cited times in Scopus