Abstract 5839: Mimicking the immunosuppressive tumor microenvironment: a complex primary immune cell tumor killing assay to support candidate vaccine or immunomodulator screening

Recent advances in oncology vaccine strategies, from off-the-shelf to personalized approaches offer a promising new weapon in the cancer treatment arsenal. The ability of these vaccines to exploit host immunity by priming and expanding Tumor Associated Antigen (TAA)-specific T cells presents a tractable tool to eradicate cancer whilst limiting systemic toxicity. TAA-specific CD8+ T cells possess a potent capacity for tumor cell killing, and it is widely accepted that patients with higher frequencies infiltrating the tumor exhibit improved clinical outcomes. However, the complex tumor microenvironment (TME), composed of various stromal and regulatory immune cells such as regulatory T cells (Tregs), cytokines and extracellular matrices is often immunosuppressive and can reduce immune surveillance and favor uncontrolled tumor growth. Building preclinical screening platforms that better reflect the complexity of the TME are therefore crucial to enable better interrogation of therapeutic efficacy. To address this, we have developed a primary human TAA-specific tumor killing platform utilizing a tri-culture of antigen specific CD8+ T cells (TAA-CD8), expanded natural T regs (nTregs) and appropriately selected tumor cells. Mart-1 was chosen as the model TAA to generate proof-of-concept data. This platform overcomes the anticipated low precursor cell frequencies by expanding both antigen specific T cells and nTregs. Following expansion, tumor killing assays (TKA) were established with TAA-CD8 and HLA-matched tumor targets with either: (1) endogenous TAA expression, (2) over-expression of target antigen following lentivirus transduction or (3) target peptide loading of MHC class I with exogenous peptide. Tumor killing was assessed in these cultures in the presence or absence of nTreg by time-lapse imaging (IncuCyte S3, Sartorious), with readouts including both tumor growth and apoptosis. Robust killing following antigen-specific expansion of CD8+ T cells was demonstrated and addition of expanded nTreg to these cultures resulted in reduced tumor cytotoxicity. Therapeutics can be tested for their efficacy in overcoming nTreg-mediated immunosuppression. Complex primary immune cell cultures provide an opportunity to test both immunomodulators and vaccines in vitro, and to better predict responses in patients by mimicking the suppressive nature of the TME. Adoption of translationally relevant platforms early in hit-to-lead campaigns for candidate vaccines or immunomodulators can accelerate drugs to the clinic through early screening. Citation Format: Oliver J. Brand, Elizabeth Lieverse, Henry Leonard, Elsenoor Klaver, Lauren Schewitz-Bowers, Louise S. Brackenbury. Mimicking the immunosuppressive tumor microenvironment: a complex primary immune cell tumor killing assay to support candidate vaccine or immunomodulator screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5839.