肿瘤特异性新表位是癌症免疫治疗中有希望的靶标。然而，功能性肿瘤特异性新表位的鉴定仍然具有挑战性。除了最常见的来源，单核苷酸变异 (SNV) 之外，选择性剪接 (AS) 代表了新表位的另一种丰富来源，可用于低 SNV 的癌症，例如葡萄膜黑色素瘤 (UM)。 UM 是最常见的成人眼部恶性肿瘤，由于缺乏有效的治疗方法，临床结果不佳。最近的研究揭示了利用肿瘤新表位来治疗 UM 的前景。之前的研究主要集中在与剪接因子 3b 亚基 1 (SF3B1) 突变相关的新表位靶标，剪接因子 3b 是一个关键的剪接因子。然而，对于独立于 SF3B1 状态的患者常见的新表位知之甚少。为了鉴定 AS 衍生的新表位，无论 SF3B1 状态如何，我们在此使用了综合的 Nanopore 长读长测序方法来阐明 UM 中 AS 和新亚型的情况。我们还进行了高分辨率质谱分析以进一步验证候选新表位的存在，并使用 AlphaFold2 算法分析了它们的结构。我们通过实验评估了这些新表位的抗肿瘤作用，发现它们通过刺激干扰素（IFN的产生和激活基于T细胞的UM肿瘤杀伤来诱导强大的免疫反应）。这些结果为独立于SF3B1和SF3B1的UM特异性新表位提供了新的见解。通过针对这些可操作的新表位来开发治疗方法奠定基础。

Tumor-specific neoepitopes are promising targets in cancer immunotherapy. However, identification of functional tumor-specific neoepitopes remains challenging. In addition to the most common source, single-nucleotide variants (SNVs), alternative splicing (AS) represents another rich source of neoepitopes and can be utilized in cancers with low SNVs such as uveal melanoma (UM). UM, the most prevalent adult ocular malignancy, has poor clinical outcomes due to a lack of effective therapies. Recent studies have revealed the promise of harnessing tumor neoepitopes to treat UM. Previous studies have focused on neoepitope targets associated with mutations in splicing factor 3b subunit 1 (SF3B1), a key splicing factor; however, little is known about the neoepitopes that are commonly shared by patients independent of SF3B1 status. To identify the AS-derived neoepitopes regardless SF3B1 status, we herein used a comprehensive Nanopore long-read-sequencing approach to elucidate the landscape of AS and novel isoforms in UM. We also performed high-resolution mass-spectrometry to further validate the presence of neoepitope candidates and analyzed their structures using AlphaFold2 algorithm. We experimentally evaluated the anti-tumor effects of these neoepitopes and found they induced robust immune responses by stimulating interferon (IFN production and activating T cell-based UM tumor killing. These results provide novel insights into UM-specific neoepitopes independent of SF3B1 and lay the foundation for developing therapies by targeting these actionable neoepitopes.