Development of T cell activating nucleic acid fusion vaccine to prevent metastatic uveal melanoma establishment

Uveal melanoma (UM) is the most common intraocular malignancy in adults. Primary tumor treatment with brachytherapy achieves approximately 80% five-year survival rate, however, later in life nearly half of patients develop predominant liver metastases, resulting in a five-year survival rate of 16% and a median overall survival (OS) of only 3.9 months. Systemic therapy with a new T cell engager Tebentafusp (Kimmtrak) increases median OS to 21.7 months, an objective response rate remains low at about 9%. Given that UM may metastasize to the liver early and remain dormant, we hypothesize that vaccine-based activation of T cell immunity in primary UM patients could prevent the establishment of metastatic disease. Our initial data showed that GNAQ/GNA11 epitopes harboring Q209L tumor driver mutation with mutated leucine in a P9 and anchoring modification at P2 positions of the nonamer activates epitope-specific T cells response. To develop a nucleic acid vaccine, we engineered a plasmid backbone containing 5′ and 3′ untranslated regions, VP22, PADRE, Tetanus toxoid P2 epitopes, and an MHC class I trafficking domain. DNA encoding either a single anchor-modified mutant GNAQ epitope or a fusion with MART-1, Tyrosinase, and MC1R epitopes generated two vaccine constructs that activated mtGNAQ-specific T cells ex vivo . Activation was confirmed by IFN-gamma secretion and cytotoxicity assays using mtGNAQ peptides and UM cells as targets. Solid-phase enrichment of UM-reactive T cells on mtGNAQ peptides further enhanced cytolytic activity. To improve vaccine efficacy, we identified additional immunogenic UM-associated epitopes. Comparative analysis of UM-specific gene expression revealed approximately 50 differentially expressed genes, with four-PMEL17, TYRP1, PRAME, and SLC45A2-showing markedly higher expression in UM compared to cutaneous melanoma or melanocytes. Regions within these antigens enriched for HLA-A*01, -A*02, and -A*03 putative binders were incorporated into the established construct to produce a multiepitope vaccine. cDNA coding for CCL21 chemokine was transcriptionally linked to the open reading frame via a P2A ribosomal skipping peptide to enhance vaccine immunogenicity. T cells activated by the vaccine ex vivo exhibited a fourfold increase in UM cell targeting compared to two other constructs. In vivo testing in HLA-A2/Kb transgenic mice confirmed that vaccinated animals generated T cells capable of recognizing UM-derived peptides and exerting cytolytic activity against melanocytic cells mimicking human UM. Overall, these findings indicate that the engineered multiepitope vaccine effectively activates UM-specific T cells. Future work will focus on identifying the most immunogenic epitopes and evaluating whether vaccination can prevent metastatic UM development and progression in relevant animal models of MUM micrometastases.