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 6

ITK-deficient CAR-T cells enhance control of tumor relapse in vivo.

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ITK-deficient CAR-T cells enhance control of tumor relapse in vivo. (A) ...
(A) Experimental design of CAR-T cell therapy against intraperitoneally injected Raji cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation. (B) Flow cytometric plots of CAR-GFP and CD3 in PB samples collected from indicated recipients at the indicated time points. (C) Summary of CAR-T cell percentages (CD3+GFP+) in B (n = 1 for day 95 nt-KO group, n = 3 for day 95 ITK-KO group, n = 4 for the rest). (D) Representative flow cytometric plots of annexin V and 7-AAD expression by CAR-T cells from PB samples 28 days after CAR-T cell injection. (E) Summary of annexin V+ CAR-T cells percentages in D (n = 4). (F) Flow cytometric plots of LAG-3 and TIGIT expression by CAR-T cells from PB samples 24 days after CAR-T cell infusion. (G) Summary of LAG-3+ or TIGIT+ CAR-T cell pecentages in F (n = 4). (H) Bioluminescence images of NPG mice xenografted with Raji cells as in A. Representative figures from 1 independent experiment. (I) Kaplan-Meier survival of Raji-bearing NPG mice (n = 9) (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons). Compiled data from 2 independent experiments. (J) Statistical analysis of CD45RO and/or CCR7 expression by CAR-T cells in Supplemental Figure 6G (n = 4). (K, L) Statistical analysis of Raji to CAR-T cells ratios (K) and CAR-T cell number fold changes (L) as shown in Supplemental Figure 6H (n = 4). (M) Statistical analysis of IFN-γ, TNF-α, and granzyme B expression in CAR-T cells shown in Supplemental Figure 6I (n = 3). Compiled data from 1 independent experiment for C, E, G, and J–M. Two-tailed unpaired Student’s t test was performed in C, E, G, and J–M. Data represent at least 2 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.