508 A novel in vitro assay to evaluate the potency of anti-PD-1 and anti-PD-L1 drugs in NSCLC

<h3>Background</h3> Programmed cell death protein 1 (PD-1), expressed by activated T cells, modulates T cell function upon binding to its ligands, PD-L1 and PD-L2, found on tumor cells or antigen-presenting cells. The goal of immunotherapy is to restore the ability of immune cells, particularly T cells, to recognize and eliminate cancer. Antibodies targeting PD-1 or PD-L1, aim to block their inhibitory signaling, have demonstrated effective <i>in vivo</i> efficacy in certain tumor models. This study aims to establish an <i>in vitro</i> assay to assess the effects of PD-1/PD-L1 blockade in co-cultures of tumor cells or organoids with immune cells. <h3>Methods</h3> PBMCs from healthy donors were activated with stimuli, T cells were isolated and PD-1 expression was detected by flow cytometry. PD-L1 expressing non-small cell lung cancer (NSCLC) cells, NCI-H2228, NCI-H1975 or LU5178E-lucg were seeded in microplates and treated with PBMCs or T cells at an E:T ratio of 1:1 in the presence of anti-PD-1 pembrolizumab or anti-PD-L1 atezolizumab. Tumor cell viability was analyzed with live cell imaging (IncuCyte) or luminescence. After 72h or 120h, the supernatant was collected and IFNγ concentration was measured by ELISA. <h3>Results</h3> After three days of PBMC activation, the major cell populations were T and NK cells, with significantly higher PD-1 expression. CellTracker red-labeled PD-L1⁺ cells (HLA-A*02:01, 03:01) were co-cultured with T cells (HLA-A*02:01, 26:01) in the presence of anti-PD-1 or isotype. A change in viable tumor cells, quantified via IncuCyte, demonstrated enhanced tumor cell killing by anti-PD-1, in a dose-dependent manner (30% killing with the highest dose of 10 µg/mL). A luciferase readout offered similar results for anti-PD-1 and indicated that anti-PD-L1 could also enhance tumor cell killing, albeit not as strongly as anti-PD-1. Cytokine analysis showed that increased IFNγ secretion was also dose-dependent with anti-PD1 treatment (2.48 fold with highest dose 10 µg/mL anti-PD-1). An additional PD-L1⁺ cell line (HLA-A*01:01, 01:01) and PD-L1⁺ organoid model (HLA-A*02:01, 02:01) were tested with activated PBMCs (HLA-A*23:17, 30:01), with luminescence readout measured to estimate tumor cell viability. The results showed enhanced tumor cell killing of ~10%-30% by anti-PD-1. <h3>Conclusions</h3> We have established an <i>in vitro</i> assay in NSCLC to evaluate the potency of anti-PD-1 or anti-PD-L1 drugs. By co-culturing tumor cells or organoids with activated T cells or PBMCs, the direct tumor cell killing effect can be evaluated via live cell imaging or luminescence reading. This assay could be used to evaluate future biologic molecules targeting the PD-1/PD-L1 pathway.