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 cells have revolutionized the treatment of B lymphomas. Unfortunately, recurrent disease after therapy remains a clinical problem with up to 50% early relapses cells. Therefore, there is critical unmet need identify biomarkers associated durable responses guide optimization therapies. Methods: We developed novel multicolor enzyme-linked immunosorbent spot (ELISpot) assay for functional assessment Cell products on single-cell level combining numerical their characterization. Results: first used standard interferon (IFN)γ ELISpot measure CD19 protein-coated beads or coated an irrelevant target. Beads anti-CD3/anti-CD28 antibodies were as positive control. then simultaneously measuring secretion combinations different cytokines single-CAR level. identified IFNγ/TNFα/Granzyme most relevant combination we our functionally numerically characterize two clinical-grade products. Conclusions: multicolor, quantitative The value will be assessed in studies correlating pre-infusion patients’ outcome. Supported by Kehlert Foundation (to D. A) Maryland Department Health's Cigarette Restitution Fund Program National Cancer Institute - Support Grant (CCSG) P30CA134274.