Establishment of a mechanism-based in vitro coculture assay for evaluating the efficacy of immune checkpoint inhibitors

Myeong Joon Kim, Kyeong Hee Hong, Bo Ryeong Lee, Sang Jun Ha

Research output: Contribution to journalArticlepeer-review


Cancer immunotherapy, which blocks immune checkpoint molecules, is an effective therapeutic strategy for human cancer patients through restoration of tumor-infiltrating (TI) cell function. However, evaluating the efficacy of immune checkpoint inhibitors (ICIs) is difficult because no standard in vitro assay for ICI efficacy evaluation exists. Additionally, blocking a particular immune checkpoint receptor (ICR) is insufficient to restore T cell functionality, because other ICRs still transduce inhibitory signals. Therefore, limiting inhibitory signals transduced via other ICRs is needed to more accurately assess the efficacy of ICIs targeting a particular immune checkpoint. Here, we introduce a newly developed in vitro coculture assay using human peripheral blood mononuclear cells (hPBMCs) and engineered human cancer cell lines. We enriched CD8+ T cells from hPBMCs of healthy donors through low-dose T cell receptor stimulation and cytokine (human IL-2 and IL-7) addition. These enriched CD8+ T cells were functional and expressed multiple ICRs, especially TIM-3 and TIGIT. We also established immune checkpoint ligand (ICL) knockout (KO) cancer cell lines with the CRISPR-Cas9 system. Then, we optimized the in vitro coculture assay conditions to evaluate ICI efficacy. For example, we selected the most effective anti-TIM-3 antibody through coculture of TIM-3+CD8+ T cells with PD-L1-/-PVR-/- cancer cells. In summary, we developed a mechanism-based in vitro coculture assay with hPBMCs and ICL KO cancer cell lines, which could be a useful tool to identify promising ICIs by providing reliable ICI efficacy information.

Original languageEnglish
Pages (from-to)2777-2789
Number of pages13
JournalCancer Immunology, Immunotherapy
Issue number11
Publication statusPublished - 2022 Nov

Bibliographical note

Funding Information:
The authors declare no competing financial interests. This study was supported by grants funded by the Ministry of Food and Drug Safety (18182MFDS408) and the Ministry of Science and ICT (MSIT) (2017R1A5A1014560, 2019M3A9B6065221). This study was also supported by Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare (HV20C0144) and Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C1410). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Cancer Research


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