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

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.