TIMING™ assays applied in research that leads to a novel dual-targeting approach to prevent CAR-NK cell exhaustion and tumor escape
TIMING provides direct functional evaluation of exhaustion and killing by natural killer cells that had ingested tumor antigens by trogocytosis
PUBLICATION ALERT
Nature Medicine has published a paper that evaluates chimeric antigen receptor (CAR)-mediated trogocytosis and its contribution to dysfunction of natural killer (NK) cells. The findings resulted in development of a new method for engineering NK cells with a logic gate powered by a second CAR. In the publication, the researchers applied Time-lapse Imaging In Nanowell Grids (TIMING™) to quantify NK-mediated cytotoxicity, fratricide and exhaustion at single-cell resolution.
In a previous study, the authors had observed that after being treated with investigational CAR-NK therapy, some patients did not respond to therapy, and others eventually relapsed. In this follow-up work, the research team tested whether trogocytosis contributes to a lack of response to CAR-NK therapy. Trogocytosis is a process by which one cell (such as a T cell or NK cell) physically “gnaws on” and consumes material from another cell. Through this process, the surface of CAR-NK cells can eventually begin to display the very same tumor proteins that the CAR-NK cells are designed to recognized as “kill me” signals. Ultimately, engineered CAR-NK cells can begin to kill each other, rendering CAR-NK cell therapy ineffective in patients.
To overcome this limitation, the investigators designed a new dual-CAR strategy intended to activate NK cell killing after recognizing a tumor antigen while simultaneously inhibiting NK cell killing when an NK self-antigen is encountered. The approach was successful in limiting CAR NK cells from killing each other—while maintaining the intended effect of CAR-NK cells killing tumor cells.
TIMING assays provided two key contributions to the research. First, TIMING assays provided a direct functional evaluation of exhaustion, which is particularly useful when evaluating NK cells because the field lacks clear phenotypic markers of NK cell exhaustion. Second, the TIMING platform differentiated killing between NK cells that had undergone trogocytosis and those that had not—at single-cell resolution.
In a Research Briefing published alongside the article, the Nature Medicine Editorial Team praised the work as a “tour de force”. An expert in the field who is unaffiliated with the study, Dr. Nathan Singh of Washington University Medical School in St. Louis, Missouri, USA, added “The authors present very rigorous and thorough data to support the conclusions regarding CAR-NK cell trogocytosis.... I am particularly impressed by the robustness of the findings.”
Example TIMING videos. Supplemental videos show representative nanowells containing one target cell (green) with either one CAR-NK cell (red, positive control, upper left), or one CAR-NK cell after co-culture with autologous NK cells that do not express CD19 (co-culture control, upper right), or with one CAR-NK cell after two days (1st repeat challenge; lower left), four days (2nd repeat challenge; lower middle), or six days (3rd repeat challenge; lower right) of co-culture with autoNKgCD19+ cells. Co-culture with autoNKgCD19+ cells allows transfer of CD19 to the NK cells. The second row shows that after co-culture, the CAR-NK cells become exhausted and fail to kill target cells (TIMING: Time-lapse Imaging Microscopy In Nanowell Grids.) (Click to view the videos.) Citation: Li, Y et al. Nature Medicine 2022 https://doi.org/10.1038/s41591-022-02003-x.
Example TIMING videos are available in the publication supplement on the Nature Medicine website. Supplemental Videos 1 show CAR-NK cell fratricide using single-cell cytotoxicity assays. Supplemental Videos 2 show CAR-NK cell-mediated anti-tumor cytotoxicity against K562 target cells. Supplemental Videos 3 show CAR-NK cell-mediated anti-tumor cytotoxicity against Raji target cells.
The Nature Medicine paper is available at doi.org/10.1038/s41591-022-02003-x ($).
The Research Briefing is available at www.nature.com/articles/s41591-022-02009-5.epdf.
About CellChorus
CellChorus is the leader in applying artificial intelligence to visually evaluate how thousands of individual immune cells, such as T cells and NK cells, perform over time. The company applies Time-lapse Imaging Microscopy in Nanowell Grids (TIMING™) with neural network-based artificial intelligence (AI) to identify cells and evaluate their activity, including how they move, activate, kill and survive. The patent-protected CellChorus platform can link TIMING data and insights with information from other analysis modalities such as single-cell RNA sequencing and flow cytometry to provide a comprehensive understanding of cellular function, state and phenotype for the life sciences industry. Please visit cellchorus.com for more information.
Company Contact:
Daniel Meyer
Chief Executive Officer
CellChorus Inc.
TIMING@cellchorus.com
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