The combination of pembrolizumab and chemotherapy is currently used as first-line therapy for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) and has demonstrated survival benefit. Despite this advance, the proportion of patients who achieve clinical responses with this standard-of-care (SOC) regimen remains relatively low. Interleukin (IL)-12 is a highly active immunostimulatory cytokine that bridges innate and adaptive immune responses and is essential for effective tumor control. Alum-bound murine IL-12 (mANK-101) has previously shown strong antitumor efficacy in multiple syngeneic tumor systems, accompanied by heightened immune activation and extended local cytokine persistence. This work examines the therapeutic potential of adding mANK-101 to SOC therapy in the MOC1 and MOC2 mouse models of HNSCC.C57BL/6 mice implanted with MOC1 or MOC2 tumors received a single intratumoral administration of mANK-101 in combination with weekly intraperitoneal doses of cisplatin and α-programmed death 1 (PD-1) for a total duration of 3 weeks. Immune responses in the MOC1 model were evaluated using flow cytometry and cytokine profiling assays. Multiplex immunofluorescence analysis was conducted to investigate treatment-induced alterations in tumor immune organization. Gene expression analyses were performed to enable detailed characterization of macrophage subsets and effector immune cell populations. Treatment with the triple combination of mANK-101, cisplatin, and α-PD-1 produced the most pronounced suppression of tumor progression and the greatest frequency of tumor-free survival in both MOC1 and MOC2 models, outperforming either mANK-101 alone or SOC therapy with cisplatin plus α-PD-1. This regimen also conferred resistance to tumor re-establishment following rechallenge and limited the growth of non-injected distal tumors. Enhanced antitumor efficacy was associated with increased infiltration of CD8+ T cells, augmented functional activity of both CD8+ and CD4+ T cells, and reprogramming of tumor-associated macrophages from an M2-dominant to an M1-dominant phenotype. Sustained elevation of interferon-γ levels was identified as a central contributor to the observed antitumor effects. Additionally, mice treated with mANK-101 in combination with cisplatin and α-PD-1 developed tertiary lymphoid structure–like immune assemblies in the peritumoral microenvironment. These findings support the use of alum-anchored IL-12 in combination with cisplatin and α-PD-1 as a promising therapeutic strategy for HNSCC.
