(M1030-02-12) In Vitro Evaluation of CAR-T Cell Cytotoxicity Using Flow Cytometry for the Development of a Potency Assay

Monday, October 17, 2022

10:30 AM – 11:30 AM ET

Presenting Author(s)

JJ

Jiu Jiang, PhD

Director of Cell and Immunology Assays
Absorption Systems A Pharmaron Company
Exton, Pennsylvania, United States

Main Author(s)

JJ

Jiu Jiang, PhD

Director of Cell and Immunology Assays
Absorption Systems A Pharmaron Company
Exton, Pennsylvania, United States

Purpose: The purpose of this study was to determine the cytotoxicity of CAR-T cells using a flow cytometry-based potency assay.

Methods: CAR-T cells recognizing CD19 molecule on B cells were used as the effector cells and Toledo cells from B cell lymphoma were used as the target cells. Toledo cells were incubated and resuspended in 10 mL of medium, then plated in 100 µL of medium (5,000 cells/well) in a 96-well round-bottom plate. In parallel, a frozen vial containing CAR-T cells was thawed and the cells were washed and resuspended into the medium with rhIL-2. As effectors, 100 µL of the CAR-T cells was added to the wells in appropriate dilutions to obtain the predetermined effector-to-target (E:T) ratios of 1:1, 2:1, 4:1, 8:1, and 16:1. Negative-control wells contained the target cells with non CAR-T cells. The cells were co-cultured in an incubator at 37°C for 2 h, 5 h, and 24 h, then 20 µL of suspension cells was taken from each well for cell count and the rest of cells were stained with anti-CD3, -CD19 antibodies, Annexin V, and 7-AAD. Flow cytometry was performed with a CytoFLEX machine for determining cytotoxicity of CAR-T cells at different times post co-culture. Data was analyzed using FlowJo software version 10. Killing % = [1-(cell count in CAR-T well × live% × CD19⁺%)/(cell count in non CAR-T well × live% × CD19⁺%)] ×100%.

Results: Flow cytometry data showed that the percentage of tumor cells (CD19⁺) were lower when co-cultured with CAR-T cells at each corresponding E:T ratios for all 3 time points, e.g., the percentage of CD19⁺ cells were 52%, 26%, 18%, 11%, and 9.4% when incubated with non CAR-T cells for 2 h, while 42%, 6.7%, 0.8%, 0.02%, and 0.03% were observed when co-cultured with CAR-T cells for 2 h, at E: T ratio of 1:1, 2:1, 4:1, 8:1, and 16:1, respectively. Two hours after co-culture, around 70% of tumor cells were killed when E:T ratio was 1:1, while up to nearly 100% tumor cells were killed when E:T ratio was over 8:1. Similar results were observed at 5 h post-co-culture. Furthermore, 94% of tumor cells were killed at 24 h when E:T ratio was 1:1, and more than 98% of target cells were eliminated by the CAR-T cells when the E:T ratio was ≥ 2:1 at this time point. In addition, most remaining tumor cells were 7-AAD⁺ and/or Annexin V⁺ in CAR-T cell wells, while few target cells were 7-AAD⁺ and/or Annexin V⁺ in non CAR-T cell wells.

Conclusion: This study demonstrated a profound cytotoxic effect of CAR-T cells on the target cells determined by flow cytometry. The dose of effector cells and incubation time of co-culture affect efficacy of tumor cell killing. The tumor cells can be eradicated within 2 h when co-cultured with high numbers of CAR-T cells. Experiments are ongoing to demonstrate use of an activation factor, such as IFNg as an additional marker, in the evaluation of a comprehensive in vitro CAR-T potency assay with orthogonal endpoints.