Volume 33, No 5, May 2023

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 33 Issue 5, May 2023: 341-354   |  Open Access

ORIGINAL ARTICLES

Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness

Jian Chen^1,2,† , Shizhen Qiu^1,† , Wentao Li^1,8,† , Kun Wang¹ , Yu Zhang³ , Han Yang¹ , Baichuan Liu¹ , Guangfei Li² , Li Li¹ , Min Chen² , Junjie Lan⁴ , Jiahua Niu⁵ , Peijie He² , Lei Cheng² , Gaofeng Fan¹ , Xin Liu³ , Xianmin Song^5,* , Chenqi Xu^3,6,* , Haitao Wu^2,* , Haopeng Wang^1,7,*

¹School of Life Science and Technology, ShanghaiTech University, Shanghai, China
²ENT institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai, China
³State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
⁴Xiamen Shuangshi Middle School of Fujian, Xiamen, Fujian, China
⁵Department of Hematology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
⁶School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, Zhejiang, China
⁷Shanghai Clinical Research and Trial Center, Shanghai, China
⁸Present address: CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
^†These authors contributed equally: Jian Chen, Shizhen Qiu, Wentao Li
Correspondence: Xianmin Song(shongxm@sjtu.edu.cn)Chenqi Xu(cqxu@sibcb.ac.cn)Haitao Wu(eentwuhaitao@163.com)Haopeng Wang(wanghp@shanghaitech.edu.cn)

Tonic signaling of chimeric antigen receptor (CAR), i.e., the spontaneous CAR activation in the absence of tumor antigen stimulation, is considered to be a pivotal event controlling CAR-T efficacy. However, the molecular mechanism underlying the spontaneous CAR signals remains elusive. Here, we unveil that positively charged patches (PCPs) on the surface of the CAR antigen-binding domain mediate CAR clustering and result in CAR tonic signaling. For CARs with high tonic signaling (e.g., GD2.CAR and CSPG4.CAR), reducing PCPs on CARs or boosting ionic strength in the culture medium during ex vivo CAR-T cell expansion minimizes spontaneous CAR activation and alleviates CAR-T cell exhaustion. In contrast, introducing PCPs into the CAR with weak tonic signaling, such as CD19.CAR, results in improved in vivo persistence and superior antitumor function. These results demonstrate that CAR tonic signaling is induced and maintained by PCP-mediated CAR clustering. Notably, the mutations we generated to alter the PCPs maintain the antigen-binding affinity and specificity of the CAR. Therefore, our findings suggest that the rational tuning of PCPs to optimize tonic signaling and in vivo fitness of CAR-T cells is a promising design strategy for the next-generation CAR.

https://doi.org/10.1038/s41422-023-00789-0

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