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3411 IL-2-Inducible T Cell Kinase Deficiency Enables Development of Sustainable Chimeric Antigen Receptor T Cell Therapy

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Zineng Huang1*, Hao Xu2*, Qingbai Liu3*, Jing Li4*, Keqing Song5*, Yating Deng1*, Yujia Tao1*, Huifang Zhang1*, Peilong Wang1*, Heng Li, MD1*, Yue Sheng, PhD1, Aijun Zhou3*, Lianbin Han6*, Yan Fu6*, Chenzhi Wang6*, Saurav Kumar Choudhary7*, Kaixiong Ye7*, Gianluca Veggiani8*, Avery August9*, Zhihong Li10*, Weishan Huang8*, Qiang Shan2*, Hongling Peng, MD1* and Zheng Fu6*

1Department of Hematology, The Second XIANGYA Hospital, Changsha, China
2National Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China
3Lianshui People's Hospital of Kangda College Affiliated to Nanjing Medical University, Huai’an, China
4Key Laboratory of Cluster Science, Ministry of Education of China, School of Chemistry and Chemical Engineering, Beijing, China
5Tianjin Mogenetics Biotech Co., Ltd, Tianjin, China
6MegaRobo Technologies Co., Ltd, Suzhou, China
7Institute of Bioinformatics, University of Georgia, Athens
8Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge
9Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca
10Department of Orthopedics, The Second XIANGYA Hospital, Changsha, China

Aim:

The chimeric antigen receptor (CAR) T cell therapy has shown promising clinical efficiency in acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). Nevertheless, CD19 CAR-T therapy achieved an unsatisfactory efficiency in chronic lymphocytic leukemia (CLL) partially owing to the acquired dysfunction of T cells in CLL patients. Ibrutinib, a Bruton’s tyrosine kinase inhibitor (BTKi), has been shown to improve the quality of T cells and response rate to CAR-T therapy in CLL patients possibly via an off-target inhibitory effect on IL-2-inducible T cell kinase (ITK) in T cells. However, it is not clear whether ITK inhibitors had a directly beneficial effect on CAR-T cells, and this study aims to illustrate the intrinsic role of ITK in CAR-T cells against tumors.

Methods:

The lentiviral transduction was used to generate anti-CD19 CAR- T cells and ITK was knocked out using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing system: electroporation of Cas9-sgRNA ribonucleoprotein (RNP) complex. In vitro killing assay and flow cytometry were used to explore the effect of ITK knockout on the cytotoxicity, proliferation, apoptosis, memory, and exhaustion of the CAR-T cells. Bulk and single-cell RNA sequencing were performed to reveal the gene signatures and cell differentiation after ITK knockout. NOD-PrkdcscidIl2rγnull (NPG) mice were used for MEC1 cell and Raji cell-derived xenograft models, and flow cytometry analysis of mice peripheral blood as well as in vivo imaging were used to evaluate the performance of ITK-KO CAR-T cells in vivo.

Results:

The ITK gene editing efficiency in CAR-T cells was as high as 86.6% at the DNA level with no obvious off-target effect and was validated at the protein level. Compared to the control group (electroporated with an RNP complex containing a non-targeting sgRNA), ITK deficiency reduces the expression of several exhaustion makers including LAG-3, PD-1, and TIM-3, promotes the long-term expansion of CAR-T cells, while showing no obvious changes in cytokines such as IFN-γ, TNF-α, and Granzyme B. In vitro killing assay indicated that ITK deficiency attenuates the immediate cytotoxicity of CAR-T cells, while reaching comparable levels to the control group at repeated co-culture with tumor cells. Bulk and single-cell RNA sequencing revealed genes involved in T cell exhaustion (such as PDCD1 and LAG3) were downregulated in the ITK-KO CAR T cells, while several genes associated with naïve or progenitor-memory T cells,including IL7R, TCF7, KLF2,were significantly increased in the ITK-KO group. ITK deficiency reduces exhaustion and enhances T cell memory fate in CD19-CAR-T cells. Cell-derived xenograft models indicated ITK deficiency significantly enhances the expansion and long-term persistence of CAR-T cells with comparable tumor control ability in the MEC1 cell-derived xenograft model compared with the control group. More importantly, ITK-KO CD19 CAR-T cells showed better control of tumor relapse in the Raji cell-derived xenograft model.

Conclusion:

In conclusion, the study demonstrates that ITK deficiency enhances the expansion, with reduction of exhaustion, and improvement of the long-term therapeutic effects of CAR-T cells. These findings offer promising insights into the development of more effective and sustainable CAR-T cell therapies for CLL and other cancers.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH