IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells
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
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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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 5

ITK deficiency enhances expansion and long-term persistence of CD19-CAR-T cells in vivo.

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ITK deficiency enhances expansion and long-term persistence of CD19-CAR...
(A) Experimental design of CAR-T cell therapy against intraperitoneally injected MEC1 cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation before intravenous injection into mice. PB, peripheral blood. (B) Representative flow cytometric plots of CAR-GFP and CD3 in PB samples collected from nt-KO or ITK-KO CD19-CAR-T cell recipients at the indicated time points. (C) Summary of percentages of CAR-T cells (CD3+GFP+) shown in B (n = 3 for day 77 ITK-KO group and n = 4 for the rest). (D) Representative flow cytometric plots of expression of the indicated molecules by CAR-T cells in PB samples collected from nt-KO or ITK-KO-CAR T cell recipients 28 days after CAR-T cell infusion. (E) Summary of percentages of CAR-T cells that are LAG-3+, PD-1+, TIM-3+, TIGIT+, or CTLA4+, as shown in D (n = 4). (F and G) Summary of percentages of CAR-T cells that are Ki-67+ (F) and annexin V+ (G) as shown in Supplemental Figure 5, C and D (n = 5). (H) Statistical analysis of different populations of cells shown in Supplemental Figure 5E (n = 4). (I) Experimental design of CAR-T cell therapy against subcutaneously injected MEC1 cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation before intravenous injection into mice. (J) Representative flow cytometric plots of CAR-GFP and CD3 in PB samples collected from nt-KO or ITK-KO CD19-CAR recipients at the indicated time points. (K) Summary of percentages of CAR-T cells (CD3+GFP+) shown in J (n = 4, mean ± SEM). Compiled data from 1 independent experiment for C, E–H, and K. Statistical differences were determined by 2-tailed unpaired Student’s t test. Data represent results of at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.