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Updated: Jun 15, 2025
Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory
Published on: October 27, 2023
Development of bioassay platforms for biopharmaceuticals using Jurkat-CAR cells by AICD
Dianze Chen1, Song Li1, Yanan Yang1
1Department of R&D, ImmuneOnco Biopharmaceuticals (Shanghai) Inc., Shanghai 201203, China.
Insights
A novel in vitro bioassay platform using Jurkat-chimeric antigen receptor (Jurkat-CAR) cells was developed to assess therapeutic antibodies targeting immune checkpoints. This reproducible platform enables reliable bioactivity analysis for antibody drug screening and release assays.
Area of Science:
- Immunology
- Biotechnology
- Pharmacology
Background:
- Assessing therapeutic antibody bioactivity, especially for immune checkpoint targets, presents significant challenges.
- Existing methods may lack the robustness required for reliable drug release assays.
Purpose of the Study:
- To develop and validate a versatile in vitro bioassay platform for analyzing antibodies targeting immune checkpoints.
- To establish a reproducible system for therapeutic antibody screening and release assays.
Main Methods:
- Engineered Jurkat cells expressing chimeric antigen receptors (Jurkat-CAR) for various immune checkpoints (CD47/SIRPα, VEGF/VEGFR1, PD-1/PD-L1, CD70/CD27).
- Constructed Jurkat-CAR cells, exemplified by Jurkat-CarSIRPα, by fusing receptor components with signaling domains.
- Validated cell response through activation-induced cell death (AICD) and upregulation of markers like CD69, IL-2, and IFN-γ.
Main Results:
- Demonstrated successful development of Jurkat-CAR cell lines for multiple immune checkpoints.
- Confirmed that Jurkat-CarSIRPα cells exhibit AICD and marker upregulation upon CD47 engagement.
- Observed similar functional responses in Jurkat CarVEGFR1, Jurkat CarPD1, and Jurkat CARCD27 cells.
Conclusions:
- The established Jurkat-CAR in vitro assay platform is robust, reproducible, and suitable for bioactivity analysis.
- This platform provides a reliable method for screening and release assays of therapeutic antibody drugs targeting immune checkpoints.
Abstract:
The assessment of bioactivity for therapeutic antibody release assay poses challenges, particularly when targeting immune checkpoints. An in vitro bioassay platform was developed using the chimeric antigen receptor on Jurkat cells (Jurkat-CAR) to analyze antibodies targeting immune checkpoints, such as CD47/SIRPα, VEGF/VEGFR1, PD-1/PD-L1, and CD70/CD27. For CD47/SIRPα, the platform involved a Jurkat-CAR cell line expressing the chimeric SIRPα receptor (CarSIRPα). CarSIRPα was created by sequentially fusing the SIRPα extracellular region with the CD8α hinge region, the transmembrane (TM) and intracellular (IC) domains of CD28, and the intracellular signaling domain of CD3ζ. The resulting Jurkat-CarSIRPα cells can undergo "activation-induced cell death (AICD)" upon incubation with purified or cellular CD47, as evidenced by the upregulation of CD69, IL-2, and IFN-γ. Similar results also appeared in Jurkat CarVEGFR1, Jurkat CarPD1 and Jurkat CARCD27 cells. These cells are perfectly utilized for the bioactivity analysis of therapeutic antibody. Our study indicates that the established in vitro assay platform based on Jurkat-CAR has been confirmed repeatedly and has shown robust reproducibility; thus, this platform can be used for screening or for release assays of given antibody drugs targeting immune checkpoints.
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