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Background Immunotherapy for Head and neck squamous cell carcinoma (HNSCC), such as programmed cell death protein 1 inhibitors (PD-1i), continues to grow in clinical application. 1 PD-1i therapy has improved overall survival in recurrent and metastatic HNSCC,2–4 as well as improved event free survival for neoadjuvant PD-1i in resectable locally advanced HNSCC.5 However, cure rates still remain low. Saponin/MPLA nanoparticles (SMNP) is a novel vaccine adjuvant composed of saponin and the toll like receptor 4 agonist monophosphoryl lipid A (MPLA) that enhances humoral immunity, B cell antigen acquisition in draining lymph nodes, and CD4+ helper T cell and B cell response.6 In this study we investigated the combinatorial treatment of intratumoral SMNP and systemic PD-1i in the PD-1i resistant 4MOSC2 syngeneic, orthotopic, murine model of HNSCC that closely mimics the tobacco-related HNSCC mutational signature.7 Methods C57Bl/6 mice were injected orthotopically with 4MOSC2 cells in the anterior buccal space to establish tumors and subsequently treated with SMNP (0.5 μg MPLA/injection) intratumorally, PD-1i (10 mg/kg) systemically or SMNP and PD-1i simultaneously following 6 days after tumor implantation. Throughout a 62-day observation period, mice were monitored for tumor volume and survival. Primary tumor and draining sentinel nodes were assayed for immune response by flow cytometry.Results Intratumoral SMNP combined with systemic PD-1i treatment significantly reduced the tumor volume with an average reduction of 80% (d29 vs. d6) and a complete response rate of 20% (p= 0.0158), whereas PD-1i (p= 0.6427) and SMNP monotherapy (p= 0.6926) showed no significant effect vs. control ( figure 1A). In contrast, all other groups exhibited increased tumor volumes (control: +315.51%, PD-1i: +159.50%, SMNP: +147.47%) and no complete responses were observed. Combination SMNP and PD-1i significantly improved overall survival (p = 0.0018 vs. control) (figure 1B), with 100% survival by day 62, compared to 50% for PD-1i, 25% for SMNP, and 0% for controls. Flow cytometric analysis 6 days following combinatorial PD-1i and SMNP treatment showed significantly elevated levels of CD11b+ myeloid cells (figure 2A) in the tumor tissues and increased CD8+ T cells in the sentinel lymph nodes (figure 2B).Conclusions Combinatorial treatment with intratumoral SMNP adjuvant and systemic PD-1i significantly reduces tumor growth, prolongs survival, and can overcome PD-1i resistance in an oral cancer 4MOSC2 mouse model, with enhancement of immune response at primary tumor and draining sentinel lymph nodes.Acknowledgements The author acknowledges support from the German Research Foundation through a Walter Benjamin Fellowship, and the Gleiberman Head and Neck Cancer Center (Califano).References Ghosh S, Shah PA, Johnson FM. Novel systemic treatment modalities including immunotherapy and molecular targeted therapy for recurrent and metastatic head and neck squamous cell carcinoma. Int J Mol Sci 2022;23(14). 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KEYNOTE-689: phase 3 study of adjuvant and neoadjuvant pembrolizumab combined with standard of care (SOC) in patients with resectable, locally advanced head and neck squamous cell carcinoma. JCO 2019;37(15_suppl):TPS6090-TPS6090. [https://doi.org/10.1200/JCO.2019.37.15_suppl.TPS6090]Silva M, Kato Y, Melo MB, et al. A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity. Sci Immunol 2021;6(66):eabf1152. [https://doi.org/10.1126/sciimmunol.abf1152][PMID: 34860581]Wang Z, Wu VH, Allevato MM, et al. Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4. Nat Commun 2019;10(1):5546. [https://doi.org/10.1038/s41467-019-13471-0][PMID: 31804466]Ethics Approval All animal studies were approved by the University of California San Diego (UCSD) Institutional Animal Care and Use Committee (IACUC) with protocol S16200.Abstract 666 Figure 1Combinatorial SMNP + PD-1i therapy in the 4MOSC2 buccal tumor mouse model shows synergistic effect on tumor growth and overall survival. (A) Tumor growth curves for control (n= 5) and treatment groups (SMNP i.t. d6 n= 4; PD-1i i.p. d6 n= 4; SMNP d6 + PD-1i d6 n= 5) (mean +/-SEM). (B) Overall survival curves for identical respective groups (statistical analysis: Log-rank (Mantel-Cox) test)Abstract 666 Figure 2Flow cytrometric analysis reveals enhanced immune infiltrates in 4MOSC2 tumors and sentinel lymph nodes 6 days following combinatorial SMNP + PD-1i therapy. (A) CD45+CD3-CD11b+ cells are elevated in tumors of PD-1i + SMNP treated mice (p=0.0192, n=5/group). (B) CD45+CD3+CD8+ cells are elevated in sentinel lymph nodes of SMNP (p=0.0440) and PD-1i + SMNP treated mice (p=0.0052; n=5/group)