QUALIFIABLE POTENCY ASSAY FOR CAR-T CELLS USING TWO SCALABLE PLATFORMS

• Published in: Cytotherapy (Oxford, England) Vol. 26; no. 6; pp. S178 - S179
• Main Authors: Kandell, J., Sylakowski, K., Lakshmipathy, U.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-06-2024
• Subjects: CAR-T; Cell Therapy; Potency Assays; CAR-T; Potency Assays; Cell Therapy
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.353

Assessment of Chimeric Antigen Receptor (CAR) T-Cell function can be achieved using direct methods via cytotoxicity assay or via indirect surrogate method by measuring the release of critical cytokines. In both cases, effector CAR-T cells are co-cultured with cells expressing the target antigen and engineered with reporters such as GFP and/or Luciferase. Additionally, different instrument platforms can be used to perform these experiments. To establish scalable and qualifiable methods that can be easily tech-transferred to decentralized manufacturing facilities, methods that are robust and portable between different instruments offer a unique advantage.
Here, we examined cell killing by CAR-T cells using NALM-6 cells, a CD19 expressing acute lymphoblastic leukemia cell line engineered with GFP and Luciferase with two different platforms, live cell image-based Incucyte S3 cell-by-cell analyzer and Varioskan LUX standard plate reader. CD-19 CAR-T cells were generated with either viral or non-viral methods (CAR % range: 7.5% to 25.9%) and further phenotyped to account for donor variability. In addition to the measurement of reporter GFP and Luciferase, Lactate Dehydrogenase (LDH) was used as an additional readout. Various effector CAR-T cells to target ratios were examined and cell killing was measured either through the decrease in GFP and Luciferase signal or an increase in LDH. For each instrument platform and assay, sensitivity ranges of the measurement were established. GFP detection by the image-based Incucyte platform demonstrated a higher sensitivity and broader range of quantification than the Varioskan LUX plate reader platform. A dynamic range of effector-to-target cell ratios includes 20:1 to 0.5:1 E:T which produced cytotoxicity from 0.5% – 95% depending on CAR-T cell potency. Based on assay output, cell killing by GFP was consistent across both platforms for effector-to-target cell ratios of 5:1, 2:1, and 1:1 E:T. This suggests that GFP assay measurement would be the most translatable across broader instrument and analysis platforms. Additionally, luciferase-based reporter analysis was comparable to GFP measurement on the plate reader platform and could serve as a suitable orthogonal method. The established CAR-T potency assays across platforms demonstrate utility in a variety of measurement systems for smooth integration in manufacturing applications.