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The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8 + and T-helper 1 CD4 + T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4 + T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion. The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease with limited therapeutic options. Here, the authors identify the DNAJB1-PRKACA protein as a source for immunogenic neoepitopes and a potential target of T cell-based immunotherapy.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies. Epigenetic therapies are known to synergize with immunotherapies through the de-repression of endogenous retroviral element (ERV)-encoded promoters. Here the authors identify treatment-induced neoantigens and validate their ability to induce T cell response and anti-tumor effects in vitro and in patient samples.

T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the implementation of ion mobility separation-based time-of-flight (TOF IMS ) MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. Applying TOF IMS -based immunopeptidomics, a novel extensive benign TOFIMS dataset was generated from 94 primary benign samples of solid tissue and hematological origin, which enabled the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides, the refinement of previously described tumor antigens, as well as the identification of frequently presented self antigens and not yet described tumor antigens comprising low abundant mutation-derived neoepitopes that might serve as targets for future cancer immunotherapy development. MS-based immunopeptidomics provides direct evidence for HLA peptide-antigen presentation, which is indispensable for therapeutic use. Here the authors present an ion mobility MS-based immunopeptidome workflow, largely expand benign reference databases and enables next generation tumor antigen discovery.

Confident identification of human leukocyte antigen (HLA)-presented peptides is crucial for advancing cancer immunotherapy. We present MHCquant2, a scalable and modular Nextflow pipeline integrated into nf-core as a reproducible, portable, and standardized workflow for immunopeptidomics. This integration allows a community-driven, robust solution for high-throughput analyses across operating systems and cloud infrastructures. MHCquant2 integrates open-source tools including OpenMS, DeepLC, and MS 2 PIP, improving peptide identifications by up to 27% across diverse MS platforms, particularly enriching low-abundant peptides. MHCquant2 demonstrates state-of-the-art performance on our novel benign MHCquant2 dataset ( n  = 92) and expands the benign human immunopeptidome by over 160,000 unique naturally presented HLA peptides.

BackgroundVarious cancer immunotherapies rely on the T cell-mediated recognition of peptide antigens presented on human leukocyte antigens (HLA). However, the identification and selection of naturally presented peptide targets for the development of personalized as well as off-the-shelf immunotherapy approaches remain challenging.MethodsOver 10,000 raw mass spectrometry (MS) files from over 3,000 tissue samples were analyzed, summing to approximately seven terabytes of data. The raw MS data were processed using the standardized and open-source nf-core pipelines MHCquant2 and epitopeprediction, providing a uniform procedure for data handling. A global false discovery rate was applied to minimize false-positive identifications.ResultsHere, we introduce the open-access Peptides for Cancer Immunotherapy Database (PCI-DB, https://pci-db.org/), a comprehensive resource of immunopeptidome data originating from various malignant and benign primary tissues that provides the research community with a convenient tool to facilitate the identification of peptide targets for immunotherapy development. The PCI-DB includes >6.6 million HLA class I and >3.4 million HLA class II peptides from over 40 tissue types and cancer entities. First application of the database provided insights into the representation of cancer-testis antigens across malignant and benign tissues, enabling the identification and characterization of cross-tumor entity and entity-specific tumor-associated antigens (TAAs) as well as naturally presented neoepitopes from frequent cancer mutations. Further, we used the PCI-DB to design personalized peptide vaccines for two patients suffering from metastatic cancer. In a retrospective analysis, PCI-DB enabled the composition of both a multi-peptide vaccine comprising non-mutated, highly frequent TAAs matching the immunopeptidome of the individual patient’s tumor and a neoepitope-based vaccine matching the mutational profile of a patient with cancer. Both vaccine approaches induced potent and long-lasting T-cell responses, accompanied by long-term survival of these patients with advanced cancer.ConclusionThe PCI-DB provides a highly versatile tool to broaden the understanding of cancer-related antigen presentation and, ultimately, supports the development of novel immunotherapies.

BackgroundEpigenetic modifying therapies, including hypomethylating agents (HMA) and histone deacetylase (HDAC) inhibitors, have shown promising results in the treatment of acute myeloid leukemia (AML). Recent data point to an immunological mode of action suggesting that HMA-induced gene expression of endogenous retroviral element (ERV)-encoded promoters and various cancer/testis antigens (CTA) might result in the presentation of novel peptides on human leukocyte antigen (HLA) molecules. These treatment-induced antigens could represent targets for immune surveillance in malignant disease and guide the development of novel combinatorial T-cell based immunotherapies. Thus, we here investigated the impact of the DNA methyltransferase inhibitor decitabine (DAC) and the HDAC inhibitor vorinostat (VOR) on the immunopeptidome of primary AML cells.MethodsImplementing label-free quantitation mass spectrometry, we assessed HLA class I and II peptide presentation of (i) AML blasts of patients treated in vivo with DAC (n=3), or (ii) primary AML samples (n=7), (iii) AML cell lines (n=3), and (iv) peripheral blood mononuclear cells (PBMC) of healthy donors (n=4), after in vitro treatment with DAC, VOR, or a combination of both.ResultsFlow cytometry-based quantification showed no loss or downregulation of HLA surface expression on AML blasts under DAC or VOR treatment. In total, 638,209 HLA class I binders and 331,765 class II presented peptides were identified with 75,619 and 58,098 unique identifications, respectively. A comparative analysis of DAC and/or VOR treated samples with their untreated controls revealed 16,251 treatment exclusive HLA class I binders and 14,788 treatment exclusive HLA class II peptides. Overlap analysis of the HLA class I binders with our in-house benign tissue (n=404, >140,000 peptides) and untreated malignant tissue (n=552, >240,000 peptides) databases revealed that 5,271 (32%) treatment-exclusive peptides were never identified on benign or malignant tissues before, confirming that these HLA ligands were induced upon treatment. Focusing on alternated CTA-expression of patients treated in vivo with DAC, HLA ligands from several CTA (ATAD2, KIAA0100, CNOT9, CNTN2, CTNA2) were found to be exclusively presented after the epigenetic modifying therapy. By integrating mass spectrometry-based immunopeptidomics with genome-wide CpG methylation screening, and deep short- and long-read RNA sequencing (RNA-seq) technologies followed by reference guided transcriptome assembly, treatment-induced HLA-ligands from canonical proteins as well as novel open reading frames (ORF) were identified.ConclusionsOur results demonstrate that epigenetic therapy modifies the immunopeptidome of primary AML cells by inducing treatment-exclusive novel HLA ligands. These treatment-associated antigens will be further evaluated for their eligibility as suitable targets for immunotherapeutic combinatorial approaches in AML.Ethics ApprovalThe study was performed according to the guidelines of the Declaration of Helsinki and the local ethics committees in Tübingen (713/2018B02, 406/2019BO2). Participants gave informed consent before taking part.

T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4 + T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469. Here, Heitmann et al. report results from a Phase I/II trial evaluating CoVac-1, a peptide-based T-cell activator, in patients with B-cell deficiency, demonstrating potent induction of SARS-CoV-2-specific T-cell responses along with a favorable safety profile.

BackgroundThe immune system targets cancer cells through T cell-mediated recognition of antigenic peptides presented on human leucocyte antigen (HLA) molecules, which is the basis of T cell-based immunotherapies. Their success highly depends on the identification of suitable HLA-presented tumor antigens which are identified by mass spectrometry (MS)-based immunopeptidomics. Substantial efforts have been made to identify naturally presented tumor-associated antigens, including neoepitopes. Only a small fraction of mutations at the DNA level are detected in the immunopeptidome. Similarly, a significant portion of the non-mutated, protein-coding genome remains largely unexplored, as it is either poorly or not detected by MS. These so-called dark spots of the immunopeptidome are either of biological origin or due to technical and methodological limitations of current MS-based approaches.MethodsOur recently published immunopeptidomics database1 containing >9 million HLA class I- and >5 million HLA class II-presented peptides from >5,000 malignant and benign samples was used as a reference for comprehensive mapping of the immunopeptidome dark spot landscape. Alignment analysis of cancer mutations, post translational modifications (PTMs), physiochemical amino acid properties, predicted HLA motifs and presentation processing predictions (pepsickle) were used to uncover the underlying technical, methodological, or biological factors. Moreover, usage of high-sensitive MS technologies combined with hydrophobic desalting steps (restricted access material (RAM), basic reversed-phase fractionation (bRP)) were used.ResultsOnly roughly 30% of the human proteome is covered in recent immunopeptidome data, although a sequence coverage of up to 85-90% is achieved using HLA-binding predictors. Analyses revealed overrepresentation of passenger mutations in dark spots (≈36 mutations per 100 amino acids), highlighting the importance of dark spots for future neoepitope vaccine developments. High-sensitive ion mobility MS could resolve dark spots showing a bias towards peptides rich in acidic amino acids (1143/1959) or highly hydrophobic peptides (575/1959). Combining RAM and bRP, dark spots of 995 highly hydrophobic peptides were resolved. Cell line experiments involving RAM and bRP revealed resolution of 19% of cell line-specific dark spots. Including PTMs such as cysteinylation as variable modifications resolved 258 dark spots, underscoring their significance in immunopeptidomics. Proteasomal cleavage site predictions were performed to investigate potential association with dark spots. These results highlight that hydrophobic peptides or peptides rich in acidic residues as well as PTMs are associated with immunopeptidome dark spots.ConclusionsDistinguishing dark spots as either technical and methodological (non-biological), or biological in origin will enhance the accuracy of (neo)epitope selection for T cell-based immunotherapy.ReferenceLemke S, Dubbelaar ML, Zimmermann P, Bauer J, Nelde A, Hoenisch Gravel N, Scheid J, Wacker M, Jung S, Dengler A, Maringer Y, Rammensee HG, Gouttefangeas C, Fillinger S, Bilich T, Heitmann JS, Nahnsen S, Walz JS. PCI-DB: a novel primary tissue immunopeptidome database to guide next-generation peptide-based immunotherapy development. J Immunother Cancer 2025 Apr 15;13(4):e011366. doi: 10.1136/jitc-2024-011366. PMID: 40234091; PMCID: PMC12001369.

T-cell recognition of HLA-presented antigens is central for the immunological surveillance of malignant disease and key for the development of novel T-cell-based immunotherapy approaches. In recent years, large-scale immunopeptidome studies identified naturally presented tumor-associated antigens for several malignancies. Regarding ovarian carcinoma (OvCa), Mucin-16 (MUC16) and Mesothelin (MSLN) were recently described as the top HLA class I- and HLA class II-presented tumor antigens, respectively. Here, we investigate the role and impact of immunopeptidome-presented tumor antigens on the clinical outcomes of 39 OvCa patients with a follow-up time of up to 50 months after surgery. Patients with a HLA-restricted presentation of high numbers of different MSLN-derived peptides on their tumors exhibited significantly prolonged progression-free (PFS) and overall survival (OS), whereas the presentation of MUC16-derived HLA class I-restricted peptides had no impact. Furthermore, a high HLA-DRB gene expression was associated with increased PFS and OS. In line, in silico prediction revealed that MSLN-derived HLA class II-presented peptides are predominantly presented on HLA-DR allotypes. In conclusion, the correlation of MSLN tumor antigen presentation and HLA-DRB gene expression with prolonged survival indicates a central role of CD4+ T-cell responses for tumor immune surveillance in OvCa, and highlights the importance of immunopeptidome-guided tumor antigen discovery.

Medulloblastoma is the most frequent malignant primary brain tumor in children. Despite recent advances in integrated genomics, the prognosis in children with high-risk medulloblastoma remains devastating, and new tumor-specific therapeutic approaches are needed. Here, we present an atlas of naturally presented T cell antigens in medulloblastoma. We map the human leukocyte antigen (HLA)-presented peptidomes of 28 tumors and perform comparative immunopeptidome profiling against an in-house benign database. Medulloblastoma is shown to be a rich source of tumor-associated antigens, naturally presented on HLA class I and II molecules. Remarkably, most tumor-associated peptides and proteins are subgroup-specific, whereas shared presentation among all subgroups of medulloblastoma (WNT, SHH, Group 3 and Group 4) is rare. Functional testing of top-ranking novel candidate antigens demonstrates the induction of peptide-specific T cell responses, supporting their potential for T cell immunotherapy. This study is an in-depth mapping of naturally presented T cell antigens in medulloblastoma. Integration of immunopeptidomics, transcriptomics, and epigenetic data leads to the identification of a large set of actionable targets that can be further used for the translation into the clinical setting by facilitating the informed design of immunotherapeutic approaches to children with medulloblastoma. Medulloblastoma in children is a difficult cancer to treat and the immune response to this tumour is not fully understood. Here the authors characterise and validate T cell epitopes from these cancers using an immunopeptidomics approach, comparing different molecular subtypes.