LAUSR

The cancer treatment landscape has transformed following the clinical success of early immune checkpoint inhibitors (ICI) which demonstrate the remarkable power of harnessing the immune system to treat a variety of metastatic cancers. Despite the success of Pembrolizumab, Nivolumab and Atezolizumab, the programmed cell death protein-1 (PD1)-programmed death ligand-1/2 (PD-L1/L2) pathway remains a tractable target for many drug discovery programs, particularly for bi-specific or combinational therapies. The PD1 - PD-L1/L2 pathway is important in protecting the host from uncontrolled immune responses, but can be hijacked by tumors, rendering them resistant to immune attack. Antibodies which antagonize this pathway potentiate T-cell mediated tumor destruction yet do not show efficacy in all patients or guarantee long-term protection, in part due to tumor-associated ICI-resistance mechanisms. There is a distinct need to develop novel ICI therapeutics and robust, complex co-culture platforms to better predict clinical efficacy. Here we outline a series of translationally relevant co-culture assays using Pembrolizumab as a benchmark therapeutic alongside other ICI. PD-1 expression levels are known to be low on resting T cells, as indeed are many checkpoint receptors, and so to enhance the therapeutic window, CD3+T cells from healthy human donors were polyclonally stimulated before addition to SK-OV-3 target cell cultures. The SK-OV-3 tumor line was stably transduced with NucLight Red (NLR) to allow co-localization of the caspase 3/7 signal to tumor cells. Autologous, mature monocyte-derived dendritic cells (moDC), expressing high levels of PD-L1/PD-L2 were added into tri-cultures in a 2D tumor killing assay. Tumor killing kinetics were quantified via live cell imaging using an Incucyte S3. Although Pembrolizumab enhanced immune-mediated direct (pre-stimulated) T cell killing, the therapeutic window increased in the presence of autologous moDC. Using a modified 3D (tumor spheroid) format over a longer time course, Pembrolizumab was also shown to enhance PBMC-mediated killing and the ability of moDC to enhance this effect further was determined. In recent years, the development and implementation of combinatorial approaches has resulted in significant improvements in patient responsiveness and disease outcomes for various cancers. Here, we describe a suite of tumor killing assays which can be adapted to multiple tumor or therapeutic targets, with a focus on immune involvement. ICI are shown to work robustly and efficacy tracks expression of the relevant checkpoint receptors, making this a powerful format in which to test combination therapies or novel ICI. Citation Format: Marie Carkill, Kaitlin Mitchell, Lauren Kelsey, Jezrom Self-Fordham, Nunan Robert, Louise Brackenbury. A translatable tumor-killing platform with robust sensitivity towards immune checkpoint inhibitor targeted therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6377.