Abstract 1113: NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells

Abstract

Abstract Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.