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Updated: Jul 11, 2025
Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory
Published on: October 27, 2023
Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging
Siddharth Subham1, John D Jeppson2, Benjamin K Gibbs3
1Department of Radiation Oncology, University of Kansas Cancer Center; Department of Cancer Biology, University of Kansas Cancer Center; BioEngineering Program, University of Kansas.
Abstract:
Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.
Insights
This study introduces a rapid platform for evaluating hinge-optimized chimeric antigen receptor (CAR) T cell constructs. The new method uses Jurkat cells to quickly assess CAR designs before testing in primary T cells.
Area of Science:
- Immunology
- Cellular Engineering
- Oncology
Background:
- Chimeric antigen receptor (CAR) T cells represent a significant advancement in cancer therapy.
- CAR construct design, particularly modifications to the intra-chain linker and hinge domains, critically impacts CAR-mediated cytotoxicity.
- The extensive permutations of CAR designs necessitate efficient evaluation methods due to the time and resource intensity of CAR T cell production.
Purpose of the Study:
- To develop and present a novel platform for the rapid evaluation of hinge-optimized CAR constructs.
- To enable faster screening of various CAR designs before extensive testing.
- To optimize CAR T cell therapy development through efficient construct assessment.
Main Methods:
- Utilized Jurkat cells (CAR-J), an immortalized T cell line amenable to efficient lentiviral transduction.
- Developed a protocol for rapid assessment of CAR-J using a fluorescent imager.
- Confirmed cytolytic activity in peripheral blood mononuclear cell (PBMC)-derived T cells following initial screening.
Main Results:
- Established a high-throughput platform for evaluating CAR construct variations.
- Demonstrated efficient CAR transduction and assessment in Jurkat cells.
- Validated the platform's ability to identify effective CAR designs for subsequent testing in primary T cells.
Conclusions:
- The described platform significantly accelerates the evaluation of hinge-optimized CAR constructs.
- This approach reduces the time and material investment required for CAR T cell development.
- The CAR-J platform facilitates rapid screening, paving the way for faster optimization of CAR T cell therapies.
