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Published in Nature Biotechnology: Improving CAR Binding Strength Increases Functionality and Anti-Tumor Activity

Chimeric antigen receptor (CAR)-based cell therapies have improved treatment outcomes for a number of different types of cancers, but treating solid tumors remains elusive. Researchers have explored targeting different antigens associated with solid tumors with some success, but antigen escape can still reduce the efficacy of these therapies.

Another aspect of CAR binding that may affect the efficacy of the treatment, beyond antigen specificity, is the strength of the binding. The characteristics of the bond that CARs form with antigens, called a synapse, have been explored and can be manipulated to enhance the strength of the bond. “Anchor domains,” parts of proteins that help to stabilize synapses, improve binding. These can potentially be exploited to improve the connection between CARs and their antigens, resulting in stronger downstream signaling and improved efficacy. By using IsoPlexis’ single-cell functional proteomics, researchers were able to characterize cell function and show how the novel modifications affected functional cell secretions.

Characterizing Novel CAR Modifications with Single-Cell Proteomics

In a paper recently published in Nature Biotechnology, researchers investigated how adding a synapse formation binding motif changes CAR-NK cell activity. The researchers exploited a domain from a protein called CRTAM that has previously been shown to play a role in NK cell activity and added it to their CAR constructs on NK cells. The CARs were targeted to two different tumor-associated antigens and tested to determine if binding was enhanced and if that increased the NK cell activity.

The modified cells, called CAR.PDZ, were first shown to bind to target antigens more strongly, which activated downstream signaling pathways more robustly compared to control cells. To see how this affected function, the researchers used IsoPlexis’ IsoCode Single-Cell Secretome platform to functionally phenotype the novel CAR-NK cells. When the percentage of cells that were polyfunctional, or secreting two or more cytokines, was compared, the CAR.PDZ NK cells had the highest percentage of polyfunctional cells, compared to typical CAR-NK cells, inactive CAR-NK cells, and unmodified NK cells. Next, the researchers assessed the polyfunctional strength index (PSI), which combines polyfunctionality with the amount of each individual protein being measured. The CAR.PDZ NK cells had significantly higher PSI compared to the other groups, with effector proteins contributing most to the PSI. The researchers also broke down the secretion frequency of each protein across the groups and found an increase in perforin-secreting and interferon-y-secreting cells in the CAR.PDZ NK cells compared to typical CAR-NK cells. These results were consistent with changes in signaling pathways that the authors previously observed, providing more information about the mechanism underlying improved functionality in CAR-NK cells with enhanced binding.


After fully characterizing the modified NK cells, the authors also explored how improved CAR binding changed the function of T cells. Like the NK cells, CAR.PDZ T cells showed improved function when assessed with single-cell functional phenotyping. The modified CAR also improved cytotoxic activity in in vitro and in vivo studies, improving outcomes and survival in animal models.

Using Single-Cell Functional Phenotyping to Assess Novel Therapies

By using IsoPlexis’ single-cell functional analysis solution, researchers can easily assess how modifications affect immune cell activity to gain unique insight into cell function. Polyfunctionality and PSI have both been shown to be predictive of in vivo activity and clinical outcomes, making them powerful metrics for cell characterization. With IsoPlexis’ single-cell functional phenotyping, researchers can get actionable insights into immune cell function that can guide development of more effective therapies and improve clinical outcomes.

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