Dual-antigen Doggybone DNA vaccine induces potent anti-tumour immunity against immunosuppressive oral cancer

Immunotherapy has demonstrated durable responses in a subset of patients with head and neck cancer, offering potential survival benefits to those with poor prognoses and resistance to first-line treatments. Despite this, only a modest 15-20% are shown to respond. Cancer vaccines have recently demonstrated efficacy in some solid tumours including melanoma, as well as immune-cold pancreatic and liver cancer, inducing a cytotoxic T cell response and eradicating cancer cells. Our previous research identified cancer testis antigens MAGED4B and FJX1 as highly immunogenic and overexpressed in the majority of HPV-independent head-and-neck squamous cell carcinoma (HPVnegHNSCC) patients. In this study, we developed a novel DNA vaccine utilising the non-plasmid doggyboneTM (dbDNATM) platform to deliver these antigens for targeting head and neck cancer. A series of doggyboneTM vaccine prototypes targeting MAGED4B and FJX1 were generated both to optimise the backbone and to present antigens in an immunogenic form. Vaccine immunogenicity and cytotoxicity were accessed using interferon-gamma. Enzyme-Linked Immunospot (ELISpot) assay. Vaccine efficacy was evaluated in the MOC-2 oral cancer model through tumour monitoring, assessing the infiltration of CD4+, CD8+ and regulatory T cells by immunohistochemistry as well as RNAseq analysis. The optimised dual antigen dbDNA DNA vaccine significantly reduced tumour size. Furthermore, combining the vaccine with anti-PD1 therapy significantly delayed tumour growth and metastasis, thereby enhancing survival in tumour-bearing mice. Vaccination resulted in significant recruitment of CD4+ and CD8+ T cells but not regulatory T cells. IHC analysis also confirmed the ability of dbDNA™ vaccine TGL-100 OPT in shifting M2 to M1 polarisation, highlighting their therapeutic promise. Transcriptomic analysis of immune cells in the tumour indicates that vaccination promotes long term anti-tumour responses by upregulating memory T cell genes T cell factor-7 (Tcf7) and lymphoid enhancer binding factor 1 (Lef1) while suppressing PDL-1 and inhibiting anti-inflammatory cytokines such as IL-10 and IL-27. These findings suggest that our cancer vaccine effectively recruits cytotoxic T cells to immune cold tumours, minimising the host’s immune inhibitory mechanisms within the tumour and resulting in long-term protection against head and neck cancer.