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形式，占全球皮肤癌相关死亡率的大部分。称为免疫检查点抑制剂（ICI）的免疫疗法在晚期黑色素瘤患者中显示出显着的活性。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中治疗人类恶性肿瘤。ICI的活性与高负荷的体细胞突变相关，这产生了所谓的新抗原。最近，通过质谱法（MS）直接在来自黑素瘤转移的样品中检测到新抗原。然而，这些突变抗原在所有HLA肽（免疫肽组）的总和中的频率似乎低于计算机模拟预测的结果。相比之下，T细胞对隐蔽肽的反应在转移性黑素瘤患者中是常见的，但通过MS鉴定这些肽是具有挑战性的，因为它们不能在蛋白质数据库中找到。从头测序算法可以直接从MS/MS确定肽序列，而不使用数据库。通过使用从头测序，我们能够在肿瘤样品的HLA免疫肽组中鉴定高达20%的隐蔽肽。这些隐蔽肽的一部分来源于非规范开放阅读框或非编码区（例如5 '-UTR或ncRNA）的翻译。然而，大多数隐蔽肽的来源仍然未知。我们的初步数据表明，相当大一部分的人免疫肽组由隐蔽肽。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，我们假设隐藏肽可能富集（突变的）新抗原。在本项目中，我们希望结合联合收割机全基因组测序、RNAseq（RiboMinus）、MS和从头测序来系统地评估黑色素瘤组织中隐藏的HLA肽的来源和特异性。如果存在仅在肿瘤组织中发现的隐蔽肽，则这些抗原可能为设计个性化免疫疗法提供独特且有前途的方法。

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.