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Chimeric Antigen Receptor (CAR)-T cell therapy has achieved great success with specificity, efficacy, safety and ingenuity. CAR cell technology is advancing rapidly, with ever increasing capabilities for cancer treatment both with regard to tumor types and the CAR cell used (NK, Macrophage, T). To date, the most successful application remains the treatment of B cell lymphoma using CD19-specific CAR-T cells. Here we describe how to efficiently generate and expand a CD19 CAR-T cell and validate their efficacy in in-vitro and in-vivo studies. HLA-A*02:01+ CD3+ T-cells were transduced to express a clinically tested CD19-specific CAR to >80% efficiency and reproducibly expanded to >68 fold over a 19-day period. After sufficient expansion, cells were validated in in-vitro studies to assess their efficacy, utilizing a HLA-A*02:01+ patient derived xenograft (PDX) CD19+ cell line, alongside human cancer cell lines RAJI (CD19+) and K562 (CD19-). Flow cytometry analysis revealed the potent ability of CD19-CAR-T cells to kill CD19+ targets in a dose-dependent fashion. Highly specific killing at all E:T ratios was observed, with negligible off target killing identified. Supportive supernatant analysis showed highly elevated pro-inflammatory cytokine concentrations, identifying enhanced activation of CAR-T cells in the presence of CD19+ target cells only. After successful in-vitro batch testing for on-target killing CAR-T cells were released for in-vivo survival studies. Mice were inoculated with the same strains of CD19+ cells used during in-vitro validation to produce a tumor burdened model. CAR-T cells or non-transduced cells from the same donor were transfused and survival rates, tumor progression and tolerability assessments were observed over several weeks. Ex-vivo tissue and sera analysis further characterized these highly efficacious human CAR-T cells. These assays represent the capability to produce, expand and study CAR-effector cells both in-vitro and in-vivo. Careful consideration of human target cells enables the efficient translation of in-vitro efficacy data to whole organism mouse models. Citation Format: Lauren Kelsey, Jezrom Self-Fordham, Eva Oswald, Ilona Aylott, Daniel Rocca, Robert Nunan, David Harris, Julia Schueler, Rhiannon Jenkinson. Development of a translatable CD19 CAR-T platform for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2843. |
