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Updated: Jun 3, 2025
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
A multi-kinase inhibitor screen identifies inhibitors preserving stem-cell-like chimeric antigen receptor T cells
Feifei Song1, Ourania Tsahouridis1, Simone Stucchi1
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Abstract:
Chimeric antigen receptor T cells (CAR T cells) with T stem (TSCM) cell-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM cell-like CAR T cells. We identified three KIs, UNC10225387B, UNC10225263A and UNC10112761A, that combined in vitro increased the frequency of CD45RA+CCR7+TCF1hi TSCM cell-like CAR T cells from both healthy donors and patients with cancer. KI-treated CAR T cells showed enhanced antitumor effects both in vitro and in vivo in mouse tumor models. The KI cocktail maintains TSCM cell-like phenotype preferentially in CAR T cells originating from naive T cells and causes transcriptomic changes without arresting T cell activation or modulating the chromatin organization. Specific kinases, ITK, ADCK3, MAP3K4 and CDK13, targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM cell-like CAR T cells. Knockdown of these kinases individually or in combination enriches for TSCM cell-like CAR T cells, but only CAR T cells generated in the presence of the KI cocktail show robust expansion and differentiation on stimulation with tumor cells. Overall, transient pharmacological inhibition of strategically targeted kinases maintains stem-like features in CAR T cells and improves their antitumor activity.
Insights
Researchers identified kinase inhibitors that preserve stem cell-like features in CAR T cells, enhancing their ability to fight cancer. This approach improves CAR T cell therapy efficacy by maintaining key characteristics for sustained antitumor effects.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Chimeric antigen receptor T (CAR T) cells are a promising cancer therapy.
- Maintaining T stem cell memory (TSCM) characteristics in CAR T cells is crucial for sustained antitumor effects.
- Identifying methods to preserve TSCM-like phenotypes in CAR T cells is a key challenge.
Purpose of the Study:
- To identify kinase inhibitors (KIs) that preserve human TSCM-like CAR T cells.
- To evaluate the impact of identified KIs on CAR T cell function and antitumor activity.
- To elucidate the specific kinases targeted by the KIs and their role in TSCM preservation.
Main Methods:
- High-throughput screening of a kinase-focused compound library.
- In vitro assessment of TSCM-like CAR T cell frequency using flow cytometry (CD45RA+, CCR7+, TCF1hi).
- In vitro and in vivo evaluation of antitumor efficacy in mouse models.
- Transcriptomic analysis and kinase knockdown experiments.
Main Results:
- Three KIs (UNC10225387B, UNC10225263A, UNC10112761A) were identified that increase TSCM-like CAR T cells.
- KI-treated CAR T cells demonstrated enhanced in vitro and in vivo antitumor activity.
- The KI cocktail preferentially maintained TSCM phenotype in naive T cell-derived CAR T cells without impairing activation or chromatin organization.
- Targeted kinases (ITK, ADCK3, MAP3K4, CDK13) were dose-dependently associated with TSCM preservation.
Conclusions:
- Transient pharmacological inhibition of specific kinases can maintain stem-like features in CAR T cells.
- This strategy significantly improves CAR T cell antitumor activity.
- The identified KIs offer a novel approach to enhance the efficacy of CAR T cell therapy.
