The efficacy of CAR-T therapy can be hindered by T cell exhaustion, leading to reduced effector function. In a recent study, researchers utilized a FluoroSpot assay to evaluate the potency of CAR-T cells. The assay detects various cytokine combinations at the single-cell level, enabling an indirect evaluation of T cell exhaustion levels within a specific CAR-T cell product. Learn about the potential of FluoroSpot assays in providing a clearer picture of CAR-T cell performance.
The persistence of relapse post-CAR-T cell therapy poses a significant challenge for these therapies. Therefore, there's a pressing need to better understand the mechanisms contributing to the partial (in)efficacy of CAR-T treatments, pinpoint biomarkers or secretory profiles associated with durable responses, and optimize the potency of CAR-T products accordingly. Apart from considering the phenotype and quantity of CAR-T cells infused, evaluating the functional (secretory) attributes of these cells at the time of infusion is crucial.
Atanackovic et al. utilized FluoroSpot to assess the functional dynamics of CAR-T cell products at a single-cell level, thus merging numerical assessment with their functional characterization. Among the tested cytokine combinations, IFN-γ/Granzyme B/TNF-α emerged as the most pertinent for characterizing clinical-grade CD19-targeted CAR-T cell products. Conversely, Th2 cytokines such as IL-4, IL-5, and IL-10 were notably scarce across the assays.
CD19-coated magnetic beads were used to stimulate CAR-T cell products to assess the optimal analyte secretory profiles using triple-colored FluoroSpot.
CD19-targeted CAR-T cells were exposed to CD19-coated beads as a relevant target, beads coated with recombinant BCMA as an irrelevant target or no beads. Anti-CD3/anti-CD28 antibody-coated beads were used as a positive control. A FluoroSpot assay measuring secretion of IFN-gamma, Granzyme B, and TNF-alpha was used as a read-out.
IFN-γ has conventionally been used in ELISpot assays for assessing tumor-specific T cell responses, owing to its correlation with anti-tumor cytotoxicity results. It is not surprising, but great to see evidence in this paper, that the cytotoxic Granzyme B and the pro-inflammatory TNF-α can be combined with IFN-γ for the evaluation of CAR-T cell functionality.
The authors outlined four methodological advantages of using FluoroSpot as a potency assay for CAR-T product assessment:
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The antigen-specific T cells are quantified based on the secretion of cytokines in response to exposure to the target antigen, rather than their expression of the CAR.
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The approach allows for the identification of functionally relevant CAR-T cells at a single-cell level.
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The clinical relevance of T cells detected using FluoroSpot is further increased by the fact that they are detected by active secretion of effector cytokines, rather than mere production, as seen in intracellular cytokine staining and flow cytometry.
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The FluoroSpot assay is cost-effective, easy to perform, robust, and can be performed overnight, allowing for fast delivery of the product to the patient. Additionally, the assay requires relatively minimal maintenance and training.
We’d like to add that, unlike ELISpot, FluoroSpot enables the assessment of polyfunctionality, indicating cells that simultaneously secrete multiple analytes. Such polyfunctional cells have repeatedly been shown to correlate with clinical responses in various settings (Seder et. al., 2008). In the present study, CAR-T cells were indeed shown to secrete multiple analytes, showing polyfunctionality, but a clinical correlation has yet to be made. Thus, the clinical value of the FluoroSpot assay will, according to the authors, be assessed in prospective studies to correlate the pre-infusion CAR-T product assessments with patient outcomes.
Learn more about why ELISpot and FluoroSpot are key immunoassays to use in cell and gene therapy evaluation and get inspired!
The researchers used the following Mabtech kits in the study: