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A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.
Darin Bloemberg1, Tina Nguyen1, Susanne MacLean1
1Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.
Molecular Therapy. Methods & Clinical Development
|February 22, 2020
Summary
We developed CAR-J, a rapid and cost-efficient assay for evaluating chimeric antigen receptor (CAR) constructs in T-cells. This method effectively assesses CAR sensitivity and specificity, aiding in the selection of promising CAR candidates for clinical development.
Area of Science:
- Immunology
- Biotechnology
- Cancer Research
Background:
- Chimeric antigen receptor (CAR) development requires extensive empirical testing of antigen-binding domains (ABDs) for clinical suitability.
- Current methods for CAR evaluation can be time-consuming and costly.
Purpose of the Study:
- To present a cost-efficient and rapid method, termed CAR-J, for evaluating CAR constructs in human T-cells.
- To assess the sensitivity and specificity of novel anti-CD22 and anti-EGFRvIII CARs.
Main Methods:
- Utilized a modular CAR plasmid, efficient ABD cloning, plasmid electroporation, and short-term co-culture.
- Employed flow-cytometric detection of CD69 to evaluate CAR activity.
- Assessed 16 anti-CD22 ABDs and 5 anti-EGFRvIII CARs.
Main Results:
- CAR-J assay successfully stratified anti-CD22 CAR constructs based on response magnitude to target cells.
- Identified anti-EGFRvIII CAR candidates with varying tonic and target-specific activation.
- Demonstrated that tonic/auto-activating EGFRvIII-CARs in primary T-cells led to off-target effects and loss of specificity upon re-challenge.
Conclusions:
- CAR-J is a straightforward, high-throughput method for assessing CAR constructs as cell-associated antigen receptors.
- The assay is valuable for generating large specificity datasets and optimizing CARs for downstream applications.
- Findings highlight the importance of evaluating CAR tonic signaling and specificity in preclinical development.
