Abstract 5962: NUC-7738 causes reduction of soluble and exosome-associated PD-L1 in melanoma cell lines and patients
Authors: Elshani Mustafa , Zhang Ying , Kaghazchi Boback , Dickson Alison L. , Blagden Sarah P. , Symeonides Stefan , Plummer Ruth , Harrison David J. 
Published: April 2023

Background: Immune checkpoint inhibitors (ICIs), including PD-(L)1 pathway inhibitors, are now standard of care for a variety of cancers. Characterization of PD-L1 protein has revealed multiple secreted forms; soluble (sPD-L1) and exosomal (ExoPD-L1) variants. In the tumor microenvironment these interfere with T-cell activation, facilitating tumor immune evasion. High circulating levels of sPD-L1 are correlated with advanced disease, a worse prognosis and/or poor response to ICIs. High levels of ExoPD-L1 are reported to contribute to T-cell dysfunction. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), currently in Phase 1/2 in patients with solid tumors (NuTide:701 NCT03829254), has shown encouraging efficacy signals in several tumor types, including melanoma. This study investigates the dynamic between NUC-7738 and secreted forms of PD-L1 in a melanoma cell line and patients.

Material and Methods: A375 malignant melanoma cells were treated with NUC-7738 (10 μM) or DMSO for 6 - 72 hours. Cell supernatant was collected for sPD-L1 and ExoPD-L1 analysis and cells were measured for mRNA and metabolites. sPD-L1 mRNA and protein expression were measured in supernatant using RT-qPCR and sandwich ELISA, respectively. NUC-7738 and 3’-dATP were measured by LC-MS. ExoPD-L1 protein levels were assessed by Jess Western analysis (normalized to CD81) and PD-L1 cell surface expression by flow cytometry. Findings were validated in patient plasma samples taken before and 24 hours after C1D1 treatment with NUC-7738 and prior to C2D1of a 14-day cycle.

Results: NUC-7738 was converted into anti-cancer metabolite 3’-dATP within 6 hours of treatment with an average concentration of 80 pmoles/106 cells, which was maintained for at least 24 hours and decreased by approximately 50% by 72 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. NUC-7738 treatment reduced sPD-L1 mRNA expression by up to 40% and caused a time dependent decrease in sPD-L1 protein in the media supernatant by up to 3-fold. NUC-7738 also reduced ExoPD-L1 protein by 50%. NUC-7738 did not alter cell surface PD-L1 expression. Preliminary studies in serum from 4 study patients treated with NUC-7738 showed reductions in Exo-PD-L1 of ≤50% compared to pre-treatment levels.

Conclusion: NUC-7738 reduces secreted forms of PD-L1 whilst having no effect on cell surface protein levels. These in vitro results (melanoma cell line) were validated in the clinical setting, whereby ExoPD-L1 was reduced in plasma samples from patients treated with NUC-7738. These findings indicate that, by reducing sPD-L1 levels, NUC-7738 may have the potential to act as an immune sensitizer. We propose that NUC-7738 given in combination with PD-(L)1 pathway inhibitors may offer a promising treatment option, and are currently exploring this in patients who have experienced therapeutic resistance to ICI treatment.

Citation Format: Mustafa Elshani, Ying Zhang, Boback Kaghazchi, Alison L. Dickson, Sarah P. Blagden, Stefan Symeonides, Ruth Plummer, David J. Harrison. NUC-7738 causes reduction of soluble and exosome-associated PD-L1 in melanoma cell lines and patients [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 5962.