Functional single-cell screening of chimeric antigen receptor (CAR)-T cells based on multiple cytokines using Nanovial Technology

Abstract

Abstract CAR-T therapies, while potent against some cancers, face challenges including toxicities and tumor relapse, particularly in solid tumors with hostile TMEs and sub-optimal CAR designs. A major obstacle is the lack of efficient functional screening methods for CAR-T cells, crucial for T-cell-based immunotherapy progress. Our study introduces a novel functional screening approach using hydrogel microparticles (Nanovials) to analyze millions of individual CAR-T cells based on antigen specificity and functional cytokine secretion. We modified Nanovials with Mesothelin (MSLN) antigen, enriching and binding specific CAR-T cells. Additionally, Nanovials were adapted with antibodies to detect key cytokines (IFN-gamma, GranzymeB) from activated CAR-T cells, identified through fluorescent markers. This setup allows for analyzing and sorting CAR-T cells with unique functional traits using standard flow cytometers. Using the Nanovial assay we distinguished MSLN CAR-T variants by cytokine profiles, revealing minor CAR engineering differences and enabling early functional assessment. This technique identified distinct CAR-T cell subpopulations within patient samples, surpassing traditional bulk analysis methods like ELISA and cell killing assays. In summary, our Nanovial-based method offers a scalable, efficient solution for CAR-T cell functional screening, enhancing CAR design and improving therapeutic manufacturing quality control.