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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 Chen1,2,† , Shizhen Qiu1,† , Wentao Li1,8,† , 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 Song5,* , Chenqi Xu3,6,* , Haitao Wu2,* , Haopeng Wang1,7,*

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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