Optimizing ex vivo CAR-T cell–mediated cytotoxicity assay through multimodality imaging

Why it’s important

Chimeric antigen receptor T cell therapy (CAR-T cell therapy) is a groundbreaking treatment for cancer. Cancer patients are treated with their own T cells, which have been engineered to express a receptor that targets the T cells to antigens expressed on the surface of cancer cells. The engineered T cells then trigger lysis of the targeted cancer cells. To date, six CAR-T cell therapeutics have been approved by the FDA and are used to treat relapsed and/or refractory multiple myeloma and B cell malignancies.¹ To improve the efficacy of this therapy and expand the number of treatable cancers, reliable ex vivo assays are required to test the targeting ability and killing efficiency of the engineered T cells. 

Challenges

Ex vivo assays exist for assessing cytotoxicity of CAR-T cells, but each assay has limitations. For example, the chromium release assay, where cells are labeled with a radioactive isotope, is considered the gold standard cytotoxicity assay.² Cells release the isotope upon lysis and the levels of radioactivity detected in the media correlate with the amount of cell death.³ However, this assay requires the use of a radioactive isotope, which introduces disposal and personnel safety concerns. Another commonly used cytotoxicity assay, the impedance-based assay, detects changes in electrical resistance in the co-culture as cells die and release from the cell culture plate. ⁴ However, this assay is limited to the analysis of adherent cell lines, and change in electrical resistance is an indirect readout of cell death.²

What’s the solution?

The ideal cytotoxicity assay should be versatile across many different target and effector cell types, and the readout for cytotoxicity should be sensitive and correlate well with the amount of cell lysis. In addition, cells used in the assay should be physiologically relevant to the cancer under study. 

How we can help

To address the need for a high-throughput, sensitive, and reliable ex vivo assay for CAR-T cell therapy research, we engineered ATCC-authenticated cancer cell lines that naturally express high levels of CAR-T target antigens to express a luciferase reporter gene. Luciferase levels in the reporter lines can be easily measured using a commercially available enzymatic assay and a luminescence plate reader. When the luciferase-expressing cancer cells are co-cultured with T cells engineered to target the cancer cells, luciferase levels decrease with higher killing efficiency as the reporter cancer cells die. 

In addition, we chose which lines to engineer based on their endogenously high expression of popular CAR-T therapy targets (CD19, CD20, or HER2). These cell lines are therefore physiologically relevant and are excellent models for B cell lymphomas or breast ductal carcinoma. The reporter lines have also been validated in live imaging assays, which allow researchers to observe the targeting and lysis of the reporter cells in real time. Overall, using these CAR-T target reporter lines, researchers can combine real-time imaging data with the quantitative sensitivity of the luciferase assay. These features improve the accuracy and ease of the cytotoxicity assay and will help researchers expand CAR-T cell therapy applications and improve CAR-T targeting efficacy.

Table 1: CAR-T Target Luciferase Reporter Cells

Did you know?

To help facilitate immuno-oncology studies and immunotherapy development, ATCC offers a variety of reporter-labeled cell models including checkpoint luciferase reporter cells, CAR-T target luciferase reporter cells, and THP-1 reporter cells.

References

1. Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20(6): 359-371, 2023. PubMed: 37055515
2. Kiesgen S, et al. Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nat Protoc 16(3): 1331-1342, 2021. PubMed: 33589826
3. Brunner KT, et al. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14(2):181-196, 1968. PubMed: 4966657
4. Peper JK, et al. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells. J Immunol Methods 405:192-198, 2014. PubMed: 24486140