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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

Abstract

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.

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Fig. 1: Construction of stable membranal CMTM6-PD-L1 complex and analysis of PD-1/PD-L1-CMTM6 interaction.
Fig. 2: Screening of anti-CMTM6 nanobody and analysis of CMTM6-nanobody interaction.
Fig. 3: CMTM6 nanobody interfered with PD-1/PD-L1 signaling and inhibited tumor growth.
Fig. 4: CMTM6 nanobody relied on CD8+ T-cells to inhibit tumor growth.

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Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 31670743), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA12040326), Science and Technology Commission of Shanghai Municipality (No. 3918JC141540001), Joint Research Fund for Overseas, Hong Kong and Macao Scholars (No. 81628013), Natural Science Foundation of Shanghai (16ZR1442900), National Science Foundation for Young Scholar projects (118180359901) and The grand from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CASIMM0120164013, SIMM1606YZZ-06, SIMM1601KF-06, 55201631121116101, 55201631121108000, 5112345601, 2015123456005, CASIMM0120202003). We would like to thank colleagues from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, who provided advice and technical support.

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YG, LKG, XMJ, and YRL designed the experiments and analyzed the data. XMJ and YRL performed the experiments and prepared the paper. XLY and RQC assisted in performing the experiments and preparing the paper. All authors approved the final draft of the paper.

Corresponding authors

Correspondence to Li-kun Gong or Yong Geng.

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Jia, Xm., Long, Yr., Yu, Xl. et al. 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. Acta Pharmacol Sin (2022). https://doi.org/10.1038/s41401-022-01020-3

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  • DOI: https://doi.org/10.1038/s41401-022-01020-3

Keywords

  • cancer immunotherapy
  • CMTM6
  • CMTM6/PD-L1 complex
  • protein interactions
  • anti-CMTM6 nanobody

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