Pun, Suzie HYazdani, Omeed2025-08-012025-08-012025-08-012025Yazdani_washington_0250O_28640.pdfhttps://hdl.handle.net/1773/53387Thesis (Master's)--University of Washington, 2025Peptide cancer vaccines have had limited clinical success despite their safety, characterization, and production advantages. We hypothesize that the poor immunogenicity of peptides can be surmounted by delivery vehicles that overcome the systemic and cellular drug delivery barriers faced by peptides. We introduce polySTING and NPSTING, copolymerized, mannosylated variants of the STING-3 agonist known to activate the cGAS-STING signaling pathway, promoting the release of type-1 interferons and pro-inflammatory cytokines leading to effective tumor immunogenicity. The STING-3 agonist is a non-nucleotide molecule that successfully activates the STING pathway, but it has poor solubility, which limits its usage in vivo. The developed poly-STING platform improves the drug's solubility and provides enzyme-triggered drug release upon delivery, which has been shown to induce improved therapeutic efficacy compared to the free STING-3 agonist. The Pun lab seeks to investigate modalities for optimization of the cGAS-STING pathway activation through investigating effects caused by mannosylation of the polymer. Dendritic cells (DCs) are crucial for initiating cytotoxic T-cell responses through antigen presentation via the MHC class-I pathway. The cGAS-STING signaling pathway enhances DC maturation and antigen cross-presentation but may interfere with pH-dependent antigen release systems such as the VIPER nanocarrier. This thesis investigates whether STING agonist variants alter DC endosomal pH, potentially affecting VIPER-mediated antigen escape. A ratiometric assay using FITC and Alexa Fluor 647-conjugated dextrans was developed and optimized for flow cytometry-based endosomal pH measurement. Protocol refinements—such as transitioning to a 96-well plate format and implementing valinomycin/nigericin permeabilization—enabled robust, high-throughput analysis with improved cell viability. Results confirmed biologically relevant endosomal acidification profiles over time in DCs. Experimentation showed that co-treatment with STING variants does not drastically alter the acidification trend when compared to PBS, but rather there is a slight delayed acidification. This implies that STING activation effects on endosomal pH might require time to become apparent. A 30-minute pretreatment experiment showed minor delayed acidification in experimental groups compared to PBS, with PolySTING being the most drastic. This assay lays the groundwork for evaluating how variant STING agonists, particularly polySTING and NPSTING, modulate endosomal pH and thereby influence antigen release and immune activation, guiding the design of more effective cancer vaccine delivery platforms.application/pdfen-USCC BYBioengineeringBiochemistryBioengineeringThesis