OPTIMIZATION OF A CLINICALLY VALIDATED CART FLOW ASSAY FOR COMMERCIAL ANTI-CD19 CART T-CELL THERAPY AND UNIVERSAL ASSAYS FOR DETECTION OF NOVEL CAR-T CONSTRUCTS

Background & Aim Chimeric antigen receptor T-cell therapy (CAR T) has revolutionized the landscape of immunotherapy in oncology. The ability to readily detect and quantify CAR T-cells in both commercial and research grade CAR T-cell infusion products as well as in patients post infusion often relies on expensive and difficult PCR or individualized flow assays developed by individual research labs and which vary between institutions. Here, we detail our institution's experience developing a highly reproducible, clinically validated anti-CD19 CAR T-cell flow detection assay and our work developing universal assays to detect novel CAR constructs irrespective of target. Methods, Results & Conclusion: Methods The site for flow optimization was the Cell Therapy Lab (CTL). The final clinical grade flow assays were laboratory developed tests validated in the HemePath labs (CAP certified laboratories at The Ohio State University Comprehensive Cancer Center) according to CAP guidelines. Multiparametric flow analysis was used to analyze in-house manufactured CAR T-cell infusion products and post-infusion patient blood samples. Results We used commercially available reagents targeting a recombinant CD19 fusion protein to develop and validate a rigorous clinical grade CAR T-cell flow assay capable of detecting the majority of CD19 directed CAR T-cell products and validated on commercial products. We analyzed blood samples from multiple post-infusion timepoints for >60 patients who received four commercial anti-CD19 CARTs. Results of our assay correlated with reported expansion patterns of each commercial product as analyzed by PCR in initial trials. We had variable success in detecting clinical trial products in post infusion samples for patients treated with multi-targeting products (e.g., CD19/20; CD19/20/22). Universal CAR detection reagents, including protein L, G4S or Whitlow linker detecting antibodies correlated better with our PCR expansion data for an in-house developed tri-specific CAR T-cell than protein binding-based flow assays. We are currently in the process of optimizing universal CAR detection assays to identify a variety of clinical trial CAR products, and further results will be shared. Conclusion Here, we detail our institution's experience developing a clinically validated anti-CD19 CAR T-cell flow assay for reproducible and reliable detection of commercial CAR T-cells as well as our ongoing work utilizing universal detection reagents to detect and analyze a variety of novel CAR constructs. Chimeric antigen receptor T-cell therapy (CAR T) has revolutionized the landscape of immunotherapy in oncology. The ability to readily detect and quantify CAR T-cells in both commercial and research grade CAR T-cell infusion products as well as in patients post infusion often relies on expensive and difficult PCR or individualized flow assays developed by individual research labs and which vary between institutions. Here, we detail our institution's experience developing a highly reproducible, clinically validated anti-CD19 CAR T-cell flow detection assay and our work developing universal assays to detect novel CAR constructs irrespective of target. The site for flow optimization was the Cell Therapy Lab (CTL). The final clinical grade flow assays were laboratory developed tests validated in the HemePath labs (CAP certified laboratories at The Ohio State University Comprehensive Cancer Center) according to CAP guidelines. Multiparametric flow analysis was used to analyze in-house manufactured CAR T-cell infusion products and post-infusion patient blood samples.