Objectives Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss.
Design We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models.
Results After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector.
Conclusion CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.
- HEPATITIS B
- MOLECULAR BIOLOGY
- ANTIVIRAL THERAPY
- ANTIBODY TARGETED THERAPY
Data availability statement
Data are available upon reasonable request. All data relevant to the study are included in the article or in the online supplemental materials.
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YW, QL, CL and CW contributed equally.
Contributors YW, QL, CL, SW, WY, XC and TL: acquisition of data. CW: mice operations. DY: quantitative detection of HBsAg and HBeAg. KZ: personal discussion and revision of the manuscript. BS: mice operations. ZY: revision of the manuscript and supervision. ST: revision of the manuscript; sharing experimental materials and facilities. MN: revision of the manuscript. Y-MW: revision of the manuscript; fund support. Y-XW: study concept and design; analysis and interpretation of data; drafting and revision of the manuscript; obtained funding; guarantor.
Funding This study was in part supported by the National Natural Science Foundation of China (32170153, 31370195, 81672017 and 32370165 to Y-XW), Outstanding Young Scholar Project (C850005 to Y-XW) of Shanghai Municipal Commission of Health and Family Planning, a fund from Shanghai Medical College and Zhongshan Hospital Immunotherapy Technology Transfer Center (to Y-XW). Y-XW is indebted to the Alexander von Humboldt Foundation for a revisit fellowship that allowed short-term collaboration work at Dr. Michael Nassal's lab. ST acknowledges the financial support from the National Science and Technology Major Project of China (2017ZX10202203-003-002). ZY acknowledges the financial support from CAMS Innovation Fund for Medical Sciences (2019-I2M-5-040).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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