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 Article has an altmetric score of 4

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 8

ITK-deficient CAR-T cells derived from CLL patients improve control of tumor relapse in vivo.

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ITK-deficient CAR-T cells derived from CLL patients improve control of t...
(A) Experimental design of CAR-T cell therapy against intravenously injected MEC1 cells expressing luciferase in NPG mice via the lateral tail vein. CAR-T cells were expanded for 12 days following electroporation before intravenous injection into mice. (B) Representative flow cytometric plots of CAR-GFP and CD3 in PBMCs collected from CLL-CAR-T cell recipients at the indicated time points. (C) Summary of percentages of CAR-T cells (CD3+GFP+) as shown in B (mean ± SEM; n = 5 for the nt-KO groups on days 26, 33, and 40, as well as the ITK-KO groups on days 40 and 52; n = 4 for nt-KO day 52 group; n = 6 for the rest). (D) Representative flow cytometric plots of TIM-3 expression on CAR-T cells in PBMCs collected from nt-KO or ITK-KO CLL-CAR-T recipients 26 days after infusion. (E) Summary of percentages of CAR-T cells that are TIM-3+ as shown in D (n = 5). (F) Representative flow cytometric plots of CD62L and CD45RA expression in the indicated CAR-T cells collected. PBMCs were collected on day 33 after CAR-T cell injection. (G) Statistical analysis of different cell populations as shown in F (n = 5). (H) Representative bioluminescence images of NPG mice xenografted with MEC1 cells as designed in A. (I) Survival of MEC1-bearing NPG mice treated with PBS or nt-KO CLL-CAR-T or ITK-KO CLL-CAR-T cells (n = 6) (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons). Compiled data from 2 independent experiments in C, E, G, and I. Statistical differences were determined by 2-tailed unpaired Student’s t test in C, E, and G. Data represent results of 2 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.