开发激活T细胞的核酸融合疫苗以预防转移性葡萄膜黑色素瘤的建立

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.