The Dark Matter of the Immunopetidome: Cryptic Peptides as Tumor Antigens in Melanoma.

恶性黑色素瘤是最致命的皮肤癌，是全世界皮肤癌相关死亡的主要原因。免疫检查点抑制剂（ICI）在晚期黑色素瘤患者中显示出显著的活性。ICI释放并诱导T细胞介导的抗肿瘤免疫，现已广泛用于现代肿瘤学治疗人类恶性肿瘤。ICI的活性与高负荷的体细胞突变有关，从而产生所谓的新抗原。最近，用质谱法（MS）直接在黑色素瘤转移瘤样品中检测到新抗原。然而，这些突变抗原在所有HLA肽（免疫肽穹窿）总数中的频率似乎比计算机预测的要低。相比之下，T细胞对隐肽的反应在转移性黑色素瘤患者中很常见，但通过MS鉴定这些肽具有挑战性，因为它们无法在蛋白质数据库中找到。De novo测序算法可以直接从MS/MS中确定肽序列，而无需使用数据库。通过使用从头测序，我们能够在肿瘤样本的HLA免疫肽穹窿中识别高达20%的隐肽。这些隐肽的部分源于非规范开放阅读框或非编码区（例如5 ' - utr或ncRNAs）的翻译。然而，大多数隐肽的来源仍然未知。我们的初步数据表明，人类免疫肽穹窿的很大一部分是由隐肽组成的。由于癌症中99%的体细胞突变影响基因组的非蛋白质编码区，我们假设隐肽可能为（突变的）新抗原富集。在本项目中，我们希望结合全基因组测序、RNAseq （RiboMinus）、MS和de novo测序，系统地评估黑色素瘤组织中隐性HLA肽的起源和特异性。如果肿瘤组织中存在特异的隐肽，这些抗原可能为设计个性化免疫疗法提供一种独特而有前途的方法。

Malignant melanoma is the deadliest form of skin cancer and contributes the majority of skin cancer-related mortality worldwide. Immunotherapies called immune checkpoint inhibitors (ICI) have shown significant activity in patients with advanced melanoma. ICI unleashes and induces T cell mediated anti-tumor immunity and is now broadly used in modern oncology to treat human malignancies. Activity of ICI is associated with a high load of somatic mutations, which give rise to so-called neoantigens. Recently, neoantigens were detected directly in samples from melanoma metastases by mass spectrometry (MS). However, the frequency of these mutated antigens among the sum of all HLA peptides (the immunopeptidome) seems to be lower than indicated by in silico predictions. In contrast, T cell responses against cryptic peptides are frequent in patients with metastatic melanoma, but identification of these peptides by MS is challenging since they cannot be found in protein databases. De novo sequencing algorithms can determine peptide sequences directly from MS/MS without using databases. By using de novo sequencing, we were able to identify up to 20% of cryptic peptides within the HLA immunopeptidome of tumor samples. Parts of these cryptic peptides originate from translation of non-canonical open reading frames or non-coding regions (e.g. 5‘-UTRs or ncRNAs). The origin of the majority of cryptic peptides, however, remains unknown. Our preliminary data indicate that a substantial fraction of the human immunopeptidome consists of cryptic peptide. Since 99% of all somatic mutations in cancer affect non-protein coding regions of the genome, we hypothesize that cryptic peptides might be enriched for (mutated) neoantigens. In this project, we want to combine whole genome sequencing, RNAseq (RiboMinus), MS and de novo sequencing to systematically assess the origin and specificity of cryptic HLA peptides in melanoma tissue. If there are cryptic peptides exclusively found in tumor tissue, these antigens might offer a unique and promising way to design personalized immunotherapies.