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 7

ITK deficiency attenuates exhaustion and promotes memory phenotype in CD19-CAR-T cells derived from CLL patients.

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ITK deficiency attenuates exhaustion and promotes memory phenotype in CD...
(A) Flow cytometric analyses of IFN-γ, TNF-α, and granzyme B expression in CLL-CAR-T cells. CLL-CAR-T cells were cocultured with MEC1 cells at a 2:1 ratio for 48 hours in A–J. (B) Statistical analysis of percentage of IFN-γ, TNF-α, and granzyme B expression in CAR-T cells shown in A (n = 5). (C) Flow cytometry analyses of CD69 expression in indicated CLL-CAR-T cells after coculture with or without MEC1 cells. (D) Statistical analysis of CD69 expression shown in C (n = 3 for CAR-T-only group, n = 4 for CAR-T + MEC1 group). (E) Flow cytometry analyses of LAG-3, PD-1, and TIM-3 expression in indicated CLL-CAR-T cells. (F) Statistical analysis of percentage of LAG-3+, PD-1+, and TIM-3+ cells shown in E (n = 3 for CAR-T-only group, n = 4 for CAR-T + MEC1 group). (G) Flow cytometry analyses of annexin V and 7-AAD expression in indicated CLL-CAR-T cells at indicated time points. (H) Statistical analysis of annexin V+ CAR-T cells shown in G (n = 4). (I) Flow cytometry analyses of CD69, LAG-3, and PD-1 expression in indicated CAR-T cells cocultured with MEC1 cells with or without PF-06465469 (1 μM) treatment. (J) Statistical analysis of percentage of CD69+, LAG-3+, and PD-1+ cells in CD19-CAR-T cells shown in I (n = 4). (K) Representative flow cytometric plots of CD45RA and CCR7 expression in indicated CAR-T cells 15 days after PF-06465469 treatment. (L) Summary of percentages of CAR-T cells expressing CD45RA and/or CCR7 in K (n = 4). Compiled data from 1 independent experiment for B, D, F, H, J, and L. Statistical differences were determined by 2-tailed unpaired Student’s t test. Data represent results of at least 2 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.