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Published on: March 14, 2025
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Protocol for preparing metabolically reprogrammed human CAR T cells and evaluating their in vitro effects.
Yue Hu1, Abhijit Sarkar1, Xiaotong Song1
1Department of Translational Medical Sciences, School of Medicine, Texas A&M University, Houston, TX 77030, USA; Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA.
STAR Protocols
|September 22, 2024
Summary
This study details a protocol for creating advanced CAR T-cells targeting HER2 and GPC3. These engineered cells convert immunosuppressive adenosine to inosine, enhancing T-cell survival in challenging tumor environments.
Area of Science:
- Immunology
- Cancer Biology
- Cell Therapy
Background:
- Chimeric antigen receptor (CAR) T-cell therapy is a promising cancer treatment.
- Developing and testing novel CAR T-cells is vital for therapeutic advancement.
- Tumor microenvironments often exhibit glucose deficiency and immunosuppression.
Purpose of the Study:
- To present a protocol for creating and characterizing metabolic reprogramming (MR)-CAR T-cells.
- To engineer CAR T-cells targeting human epidermal growth factor receptor 2 (HER2) and glypican-3 (GPC3).
- To enhance T-cell survival in glucose-deficient tumor microenvironments by modulating adenosine metabolism.
Main Methods:
- Overexpression of adenosine deaminase 1 (ADA1) and CD26 (dipeptidylpeptidase-4 or DPP4) in CAR T-cells.
- Production of retroviral vectors for gene delivery.
- Generation and characterization of engineered CAR T-cells.
- Assays included ecto-ADA1 activity, cytotoxicity, cell migration, and RNA sequencing.
Main Results:
- The protocol enables the creation of HER2- and GPC3-MR-CAR T-cells.
- Engineered CAR T-cells effectively convert immunosuppressive adenosine into inosine.
- This metabolic reprogramming supports T-cell survival in simulated glucose-deficient tumor conditions.
Conclusions:
- The presented protocol provides a method for developing advanced CAR T-cell therapies.
- Metabolic reprogramming via ADA1 and CD26 overexpression offers a strategy to overcome tumor microenvironment challenges.
- This approach holds potential for improving the efficacy of CAR T-cell treatments for HER2- and GPC3-expressing cancers.
