Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

Xinyi Xu; Haotian Chen; Zhengxu Ren; Xiaomin Xu; Wei Wu; Haochen Yang; JinJiao Wang; Yumeng Zhang; Qiuping Zhou; Hua Li; Shaoqing Zhang; Haopeng Wang; Chenqi Xu

doi:10.1016/j.immuni.2024.11.005

Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

Immunity. 2024 Nov 25:S1074-7613(24)00520-X. doi: 10.1016/j.immuni.2024.11.005. Online ahead of print.

Authors

Xinyi Xu¹, Haotian Chen², Zhengxu Ren¹, Xiaomin Xu³, Wei Wu⁴, Haochen Yang¹, JinJiao Wang², Yumeng Zhang², Qiuping Zhou³, Hua Li¹, Shaoqing Zhang¹, Haopeng Wang⁵, Chenqi Xu⁶

Affiliations

¹ Key Laboratory of Multi-cell Systems, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
² School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
³ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.
⁴ Cancer Center, The First Hospital of Jilin University, Changchun, China.
⁵ School of Life Science and Technology, ShanghaiTech University, Shanghai, China. Electronic address: wanghp@shanghaitech.edu.cn.
⁶ Key Laboratory of Multi-cell Systems, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China; School of Life Science and Technology, ShanghaiTech University, Shanghai, China; Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China. Electronic address: cqxu@sibcb.ac.cn.

PMID: 39609126
DOI: 10.1016/j.immuni.2024.11.005

Abstract

Major challenges of chimeric antigen receptor (CAR)-T cell therapy include poor antigen sensitivity and cell persistence. Here, we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered T cell receptor CD3ε motif, E_B6I, into the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. E_B6I CAR formed a mature immunological synapse with the CD2 corolla to transduce efficient antigen and costimulatory signaling, although its tonic signaling remained low. Functionally, E_B6I CAR-T cells exhibited improved signaling and cytotoxicity against low-antigen tumor cells and persistent tumor-killing function. In multiple primary and relapsed murine tumor models, E_B6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

Keywords: CAR-T cell therapy; CD2 costimulation; antigen sensitivity; chimeric antigen receptor; exhaustion; immunological synapse; persistence; phase separation; tonic signaling; trogocytosis.