IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells
Zheng Fu, … , Qiang Shan, Hongling Peng
Zheng Fu, … , Qiang Shan, Hongling Peng
Published November 26, 2024
Citation Information: J Clin Invest. 2025;135(4):e178558. https://doi.org/10.1172/JCI178558.
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Research Article Hematology Immunology

IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

Abstract

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2–inducible T cell kinase (ITK) is a critical modulator of the strength of T cell receptor signaling, while its role in CAR signaling is unknown. By electroporation of CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing analyses revealed downregulation of exhaustion and upregulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T cell products for clinical use.

Authors

Zheng Fu, Zineng Huang, Hao Xu, Qingbai Liu, Jing Li, Keqing Song, Yating Deng, Yujia Tao, Huifang Zhang, Peilong Wang, Heng Li, Yue Sheng, Aijun Zhou, Lianbin Han, Yan Fu, Chenzhi Wang, Saurav Kumar Choudhary, Kaixiong Ye, Gianluca Veggiani, Zhihong Li, Avery August, Weishan Huang, Qiang Shan, Hongling Peng

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Figure 4

ITK deficiency reduces exhaustion and promotes memory phenotype in CD19-CAR-T cells in vitro.

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ITK deficiency reduces exhaustion and promotes memory phenotype in CD19-...
CAR-T cells were cocultured with MEC1 after the serial killing assay protocol detailed in Methods. (A) Representative flow cytometric plots of CD69 expression in the indicated CAR-T cells in the presence or absence of MEC1 cells. CAR-T cells were analyzed after coculture with MEC1 cells (E/T = 2:1) for 48 hours. (B) Summary of percentages of CD69+ cells in A (n = 3 for each CAR-T-only group, and n = 4 for each CAR-T + MEC1 group) (2-way ANOVA with Šidák’s correction for multiple comparisons). (C) Representative flow cytometric plots of PD-1, TIGIT, TIM-3, and CTLA4 expression in CAR-T cells 15 days after coculture with MEC1 cells at the ratio of E/T = 2:1. (D) Summary of percentages of PD-1+, TIGTI+, TIM-3+, and CTLA4+ cells as shown in C (n = 4). (E) Summary of percentages of LAG-3+, PD-1+, and TIM-3+ cells in the indicated CAR-T cells following the indicated rounds of coculture with MEC1 cells (n = 4). CAR-T cells were cocultured with MEC1 cells at a 2:1 ratio for 48 hours for each round for E and F. (F) Percentages of specific lysis determined by in vitro killing assay of MEC1 cells by the indicated CAR-T cells at indicated rounds of coculture (n = 4). (G) Representative flow cytometric plots of CD45RA and CCR7 expression in the indicated CAR-T cells 15 days after coculture with MEC1 cells (E/T = 2:1). (H) Summary of percentages of CAR-T cells expressing CD45RA and/or CCR7 in G (n = 3). Compiled data from 1 independent experiment for B, D–F, and H. Statistical differences were determined by 2-tailed unpaired Student’s t test in D–F and H. Data represent results of at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.