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.
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Han W, Ding P, Xu M, Wang L, Rui M, Shi S, et al. Identification of eight genes encoding chemokine-like factor superfamily members 1–8 (CKLFSF1–8) by in silico cloning and experimental validation. Genomics. 2003;81:609–17.
Jin C, Ding P, Wang Y, Ma D. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8. FEBS Lett. 2005;579:6375–82.
Burr ML, Sparbier CE, Chan YC, Williamson JC, Woods K, Beavis PA, et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature. 2017;549:101–5.
Mezzadra R, Sun C, Jae LT, Gomez-Eerland R, de Vries E, Wu W, et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature. 2017;549:106–10.
Guan X, Zhang C, Zhao J, Sun G, Song Q, Jia W. CMTM6 overexpression is associated with molecular and clinical characteristics of malignancy and predicts poor prognosis in gliomas. EBioMedicine. 2018;35:233–43.
Chen L, Yang QC, Li YC, Yang LL, Liu JF, Li H, et al. Targeting CMTM6 suppresses stem cell-like properties and enhances antitumor immunity in head and neck squamous cell carcinoma. Cancer Immunol Res. 2020;8:179–91.
Martinez-Morilla S, Zugazagoitia J, Wong PF, Kluger HM, Rimm DL. Quantitative analysis of CMTM6 expression in tumor microenvironment in metastatic melanoma and association with outcome on immunotherapy. Oncoimmunology. 2020;10:1864909.
Wu X, Lan X, Hu W, Zhang W, Lai X, Xu S, et al. CMTM6 expression in M2 macrophages is a potential predictor of PD-1/PD-L1 inhibitor response in colorectal cancer. Cancer Immunol Immunother. 2021;70:3235–48.
Li X, Chen L, Gu C, Sun Q, Li J. CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients. PeerJ. 2020;8:e9536.
Zhao Y, Zhang M, Pu H, Guo S, Zhang S, Wang Y. Prognostic Implications of Pan-Cancer CMTM6 Expression and Its Relationship with the Immune Microenvironment. Front Oncol. 2020;10:585961.
Zheng Y, Wang C, Song A, Jiang F, Zhou J, Li G, et al. CMTM6 promotes cell proliferation and invasion in oral squamous cell carcinoma by interacting with NRP1. Am J Cancer Res. 2020;10:1691–709.
Wang H, Fan Y, Chen W, Lv Z, Wu S, Xuan Y, et al. Loss of CMTM6 promotes DNA damage-induced cellular senescence and antitumor immunity. Oncoimmunology. 2022;11:2011673.
Mohapatra P, Shriwas O, Mohanty S, Ghosh A, Smita S, Kaushik SR, et al. CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3beta axis. JCI Insight. 2021;6:e143643.
Pang X, Wang SS, Zhang M, Jiang J, Fan HY, Wu JS, et al. OSCC cell-secreted exosomal CMTM6 induced M2-like macrophages polarization via ERK1/2 signaling pathway. Cancer Immunol Immunother. 2021;70:1015–29.
Huang Y, Zhu Y, Yang J, Pan Q, Zhao J, Song M, et al. CMTM6 inhibits tumor growth and reverses chemoresistance by preventing ubiquitination of p21 in hepatocellular carcinoma. Cell Death Dis. 2022;13:251.
Wang Z, Peng Z, Liu Q, Guo Z, Menatola M, Su J, et al. Co-expression with membrane CMTM6/4 on tumor epithelium enhances the prediction value of PD-L1 on Anti-PD-1/L1 therapeutic efficacy in gastric adenocarcinoma. Cancers (Basel). 2021;13:5175.
Althoff T, Mills DJ, Popot JL, Kühlbrandt W. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J. 2011;30:4652–64.
Kevany BM, Tsybovsky Y, Campuzano ID, Schnier PD, Engel A, Palczewski K. Structural and functional analysis of the native peripherin-ROM1 complex isolated from photoreceptor cells. J Biol Chem. 2013;288:36272–84.
Tsybovsky Y, Orban T, Molday RS, Taylor D, Palczewski K. Molecular organization and ATP-induced conformational changes of ABCA4, the photoreceptor-specific ABC transporter. Structure. 2013;21:854–60.
Yang EY, Shah K. Nanobodies: Next generation of cancer diagnostics and therapeutics. Front Oncol. 2020;10:1182.
Ahuja S, Rougé L, Swem DL, Sudhamsu J, Wu P, Russell SJ, et al. Structural analysis of bacterial ABC transporter inhibition by an antibody fragment. Structure. 2015;23:713–23.
Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen L. B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells. Blood. 2008;111:3635–43.
Lan Y, Zhang D, Xu C, Hance KW, Marelli B, Qi J, et al. Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β. Sci Transl Med. 2018;10:eaan5488.
Miao YR, Zhang Q, Lei Q, Luo M, Xie GY, Wang H, et al. ImmuCellAI: A unique method for comprehensive T-cell subsets abundance prediction and its application in cancer immunotherapy. Adv Sci (Weinh). 2020;7:1902880.
Maute RL, Gordon SR, Mayer AT, McCracken MN, Natarajan A, Ring NG, et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proc Natl Acad Sci USA. 2015;112:E6506–E6514.
Cheng X, Veverka V, Radhakrishnan A, Waters LC, Muskett FW, Morgan SH, et al. Structure and interactions of the human programmed cell death 1 receptor. J Biol Chem. 2013;288:11771–85.
Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027–34.
Hegde PS, Chen DS. Top 10 challenges in cancer immunotherapy. Immunity. 2020;52:17–35.
Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms controlling PD-L1 expression in cancer. Mol Cell. 2019;76:359–70.
Wang H, Yao H, Li C, Shi H, Lan J, Li Z, et al. HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. Nat Chem Biol. 2019;15:42–50.
Ren Y, Qian Y, Ai L, Xie Y, Gao Y, Zhuang Z, et al. TRAPPC4 regulates the intracellular trafficking of PD-L1 and antitumor immunity. Nat Commun. 2021;12:5405.
Chen Y, Liu P, Gao F, Cheng H, Qi J, Gao GF. A dimeric structure of PD-L1: functional units or evolutionary relics? Protein Cell. 2010;1:153–60.
Zak KM, Grudnik P, Magiera K, Dömling A, Dubin G, Holak TA. Structural biology of the immune checkpoint receptor PD-1 and its ligands PD-L1/PD-L2. Structure. 2017;25:1163–74.
Zak KM, Grudnik P, Guzik K, Zieba BJ, Musielak B, Dömling A, et al. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1). Oncotarget. 2016;7:30323–35.
Guo Y, Jin Y, Wang B, Liu B. Molecular mechanism of small-molecule inhibitors in blocking the PD-1/PD-L1 pathway through PD-L1 dimerization. Int J Mol Sci. 2021;22:4766.
Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444:770–4.
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.
The authors declare no competing interests.
About this article
Cite this article
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
- cancer immunotherapy
- CMTM6/PD-L1 complex
- protein interactions
- anti-CMTM6 nanobody