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Despite significant clinical advances in CAR-T cell therapies for cancer, the ability to precisely and reproducibly analyze the potency of these drug products remains a challenge. As new CAR-T products and production methodologies are developed, it is becoming increasingly important to be able to rigorously evaluate the relative potencies of each batch. We have developed two MOA-based bioluminescent assays that overcome challenges in quantitative assessment of T cell therapies. Both assays rely on split NanoBiT luciferase technology. In the HiBiT Target Cell Killing Assay, incubation of CAR-T cells with target cells stably expressing HiBiT results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins bind with high affinity to LgBiT in the detection reagent and form functional NanoBiT luciferase, resulting in production of light in proportion to target cell lysis. Lumit Immunoassays are a simple and homogeneous method for measuring cytokine production from engineered T cells. Monoclonal antibodies are covalently labeled with LgBiT and a low-affinity tag, SmBiT, which have negligible association in solution. When the antibodies are brought together by a common analyte, such as a cytokine, SmBiT and LgBiT can compliment to form NanoBiT luciferase and produce light in proportion to the concentration of analyte in the sample. Lumit Immunoassays can be run directly on cells, removing media transfer, dilution, and wash steps that can be common sources of error. These luminescent assays are homogenous, fast, highly sensitive, and have robust assay windows. They represent a new set of tools for development and characterization of T cell-based immunotherapies. Citation Format: Julia K. Gilden, Pete Stecha, Aileen Paguio, Jim Hartnett, Brock Binkowski, Dan Lazar, Frank Fan, Mei Cong. NanoBiT technologies for CAR-T characterization and potency testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3202. |
