Chimeric antigen receptor (CAR) T cell therapies have been used to successfully treat a number of blood cancers and hematologic malignancies; however, using them to treat solid tumors has proven to be more challenging. The tumor microenvironment (TME) contains a variety of cells and expresses molecules that actively suppress immune function, limiting the efficacy of local T cells. Many of these suppression mechanisms exploit the signaling pathways that maintain normal T cell homeostasis. As researchers have gained a better understanding of these evasion mechanisms, they are engineering CAR-T cells to better counter TME immune suppression and maintain immune cells’ anti-tumor activity.
With the development of new CAR-T cell types, researchers must characterize and assess the functionality of these cells to better understand how they work. Because engineered cell products are heterogeneous, it is vital to assess biological functions at the single-cell level as bulk analyses average population characteristics and can miss key functional metrics present in individual cells. IsoPlexis’ Single-Cell Secretome solution gives researchers an automated platform to perform single-cell cytokine analysis, empowering them to easily functionally phenotype cells and characterize key biomarkers for potency and persistence.
Assessing Functional Changes in Engineered T Cells
CAR-T cells engineered to overcome TME immune suppression were characterized in a paper recently published in Cancer Immunology Research. The researchers sought to prevent the loss of T cell efficacy in the TME by targeting Fas signaling. Typically, Fas signaling helps to regulate T cell expansion and attenuate immune function as part of a negative feedback loop in response to immune activation. However, solid tumor cells can take advantage of this signaling by secreting Fas ligand (FasL), which binds to Fas receptors on T cells and induces apoptosis, or cell death. FasL expression has been detected in a wide variety of solid tumors and has shown to be associated with disease progression, severity, and worse outcomes.
To protect effector T cells from activation of the Fas signaling pathway, the researchers engineered CAR-T cells that can secrete a Fas decoy when the T cells engage with a specific antigen, thereby preventing Fas interaction with the FasL. To target solid tumors, the CAR-T cells were engineered to target prostate stem cell antigen (PSCA), an antigen expressed by a variety of solid tumors. The researchers verified that PSCA stimulation caused the CAR-T cells to secrete the Fas decoy molecule and that this secretion resulted in protection of CAR-T cells from initiation of Fas signaling.
Next, the researchers wanted to understand how this modification affected cell function. Using IsoPlexis’ Single-Cell Secretome platform, they assessed cell polyfunctionality, or the ability of cells to secrete two or more cytokines. CAR-T cells modified to secrete the Fas decoy (CAR.FD) were stimulated with pancreatic adenocarcinoma cells expressing FasL. When compared to control CAR-T cells, the CAR.FD cells had a higher proportion of polyfunctional cells, with some cells expressing up to 7 different cytokines while control cells only expressed a maximum of 4 cytokines.
Because polyfunctionality has been previously associated with improved potency, efficacy, and clinical outcomes, the increased polyfunctionality observed in the CAR.FD cells is an informative performance metric to guide further studies. Subsequent in vitro and in vivo experiments revealed that the CAR-T cells expressing the Fas decoy were more effective at controlling tumor growth compared to control CAR-T cells.
Using Single-Cell Metrics to Understand Engineered T Cell Function
Single-cell analysis yields unique metrics, such as polyfunctionality, that are predictive of product quality and efficacy. By using IsoPlexis’ single-cell automated analysis platform, researchers can uncover actionable insights into engineered T cell function, getting critical data early in the development process. These data can help to drive development, uncover mechanisms underlying treatment response, and identify key functional attributes. IsoPlexis’ single-cell functional phenotyping platform empowers scientists to easily assess cell function, all on their own benchtop.
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