A Novel Multicolor Enzyme-linked Immunosorbent Spot Assay for the Functional Assessment of CAR T Cell Products

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapies are powerful anti-tumor immunotherapies and CD19-targeted CAR T cells have revolutionized the treatment of B cell lymphomas. Unfortunately, recurrent disease after CAR T cell therapy remains a clinical problem with up to 50% early relapses after CD19-targeted CAR T cells. Therefore, there is a critical and unmet need to identify biomarkers associated with durable responses to guide the optimization of CAR T cell therapies. Methods: We developed a novel multicolor enzyme-linked immunosorbent spot (ELISpot) assay for the functional assessment of CAR T Cell products on a single-cell level combining the numerical assessment of CAR T cell products with their functional characterization. Results: We first used a standard single-cell interferon (IFN)γ ELISpot assay to measure CD19-targeted CAR T cell responses to CD19 protein-coated beads or to beads coated with an irrelevant target. Beads coated with anti-CD3/anti-CD28 antibodies were used as a positive control. We then developed a multicolor ELISpot assay simultaneously measuring the secretion of combinations of different cytokines on a single-CAR T cell level. We identified IFNγ/TNFα/Granzyme B as the most relevant combination of cytokines and we used our novel multicolor ELISpot assay to functionally and numerically characterize two clinical-grade CAR T cell products. Conclusions: We have developed a novel multicolor, single-cell ELISpot for the quantitative and functional assessment of CAR T cell products. The clinical value of our novel assay will be assessed in clinical studies correlating the pre-infusion assessment of CAR T cell products with the patients’ outcome. Supported by the Kehlert Foundation (to D. A) by the Maryland Department of Health's Cigarette Restitution Fund Program and by the National Cancer Institute - Cancer Support Grant (CCSG) P30CA134274.