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Updated: Sep 16, 2025
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
Screening of a pooled library of chimeric antigen receptor T cells based on secretory function
Abstract:
Chimeric antigen receptor (CAR) T cell therapies have shown promise in treating hematologic malignancies, but challenges remain due to immune suppression, antigen heterogeneity, and insufficient functional screening platforms. Here, we present a modular nanovial-based platform for high-throughput, single-cell functional screening of pooled CAR T cell libraries. Nanovials, hydrogel microparticles with nanoliter-scale cavities, were functionalized with recombinant HER2 antigen and cytokine-capture antibodies to simulate antigen-presenting cells and capture secreted interferon-γ (IFNγ). This system enabled the selective capture, activation, and functional profiling of CAR T cells based on antigen engagement and cytokine secretion. We screened a 32-variant CAR library with diverse intracellular signaling domains, using nanovials to isolate IFNγ-secreting cells at 3- and 12-hour timepoints. IL15RA-containing CARs, particularly IL15RA-CD28, were preferentially enriched in the sorted T cells after 3 hours of stimulation, consistent with early effector activation profiles. By 12 hours, IL15RA-containing constructs remained enriched while other CD40-containing domains showed delayed but substantial enrichment, suggesting prolonged signaling dynamics. The platform's high-throughput capacity (>2 million cells screened), compatibility with downstream sequencing, and tunable antigen presentation make it ideal of identifying CAR constructs associated with various time-dependent secretion phenotypes.
Insights
This study introduces a novel nanovial platform for high-throughput screening of chimeric antigen receptor (CAR) T cells, enabling efficient identification of potent CAR T cell therapies for cancer treatment.
Area of Science:
- Immunology
- Biotechnology
- Cancer Therapy
Background:
- Chimeric antigen receptor (CAR) T cell therapies show promise for hematologic malignancies.
- Challenges include immune suppression, antigen heterogeneity, and limited functional screening.
- Existing platforms lack high-throughput, single-cell functional profiling capabilities.
Purpose of the Study:
- To develop and validate a modular nanovial-based platform for high-throughput, single-cell functional screening of CAR T cell libraries.
- To identify optimal CAR T cell constructs based on antigen engagement and cytokine secretion.
- To analyze time-dependent signaling dynamics of different CAR variants.
Main Methods:
- Utilized nanovials (hydrogel microparticles with nanoliter cavities) functionalized with HER2 antigen and cytokine-capture antibodies.
- Simulated antigen-presenting cells and captured secreted interferon-γ (IFNγ) for CAR T cell selection.
- Screened a 32-variant CAR library, isolating IFNγ-secreting cells at 3- and 12-hour timepoints.
- Enabled selective capture, activation, and functional profiling of CAR T cells.
Main Results:
- The nanovial platform successfully enabled high-throughput screening (>2 million cells).
- IL15RA-containing CARs, especially IL15RA-CD28, were enriched early (3 hours), indicating rapid effector activation.
- Delayed but substantial enrichment of CD40-containing CARs was observed at 12 hours, suggesting prolonged signaling.
- Identified time-dependent secretion phenotypes associated with specific CAR constructs.
Conclusions:
- The nanovial platform provides a powerful tool for functional screening of CAR T cell libraries.
- It facilitates the identification of CAR constructs with desired effector functions and signaling dynamics.
- This technology can accelerate the development of more effective CAR T cell therapies for cancer.
