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Introduction: The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets., Materials and Methods: Snap-frozen tumor (N Ligand/RNA =40), healthy mucosa (N RNA =6), and healthy tonsils (N Ligand =5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (N Ab =27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins)., Results: 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels., Discussion: This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy., Competing Interests: SL: Advisory Boards: Merck Sharp & Dohme MSD, Bristol Myers Squibb BMS, Sanofi Genzyme, Astra Zeneca AZ. Honoraria: MSD, BMS. Travel reimbursement: Merck Serono, Astra Zeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Abou Kors, Meier, Mühlenbruch, Betzler, Oliveri, Bens, Thomas, Kraus, Doescher, von Witzleben, Hofmann, Ezic, Huber, Benckendorff, Barth, Greve, Schuler, Brunner, Blackburn, Hoffmann, Ottensmeier, Kestler, Rammensee, Walz and Laban.)

Rationale: Immune checkpoint inhibitor-related pneumonitis is a serious autoimmune event affecting up to 20% of patients with non-small cell lung cancer, yet the factors underpinning its development in some patients and not others are poorly understood., Objectives: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis., Methods: The study cohort consisted of non-small cell lung cancer patients who gave blood samples before and during immune checkpoint inhibitor treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with non-small cell lung cancer and patients with melanoma., Measurements and Main Results: Across both cohorts, patients who developed pneumonitis had higher pre-treatment levels of immunoglobulin G autoantibodies targeting surfactant protein-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4 + interferon-gamma-positive surfactant protein B-specific T cells, and expanding T cell clonotypes recognizing this protein, accompanied by a pro-inflammatory serum proteomic profile., Conclusions: Our data suggest that the co-occurrence of surfactant protein-B-specific immunoglobulin G autoantibodies and CD4 + T cells is associated with the development of pneumonitis during ICI therapy. Pre-treatment levels of these antibodies may represent a potential biomarker for elevated risk of developing pneumonitis and on-treatment levels may provide a diagnostic aid. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC + format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rβγ stimulation, whereas the L45E mutation optimized IL-15Rβγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC + constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC + constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC + constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Objectives: T cell immunity is key for the control of viral infections including SARS-CoV-2, in particular with regard to immune memory and protection against arising genetic variants., Methods: We recently evaluated a peptide-based SARS-CoV-2 T cell activator termed CoVac-1 in a first-in-human trial in healthy adults. Here, we report on long-term safety and efficacy data of CoVac-1 until month 12., Results: CoVac-1 is well tolerated without long-term immune-related side effects and induces long-lasting anti-viral T cell responses in 100% of study participants, with potent expandability of clusters of differentiation (CD4 + ) and CD8 + T cells targeting multiple different CoVac-1 T cell epitopes. T cell responses were associated with stronger injection site reaction. Beyond induction of T cell immunity, 89% of subjects developed CoVac-1-specific immunoglobulin G antibodies which associated with the intensity of the T cell response, indicating that CoVac-1-specific CD4 + T cells support the induction of B-cell responses. Vaccination with approved COVID-19 vaccines boosted CoVac-1-specific T cell responses. Overall, a low SARS-CoV-2 infection rate (8.3%) was observed., Conclusion: Together, a single application of CoVac-1 elicits long-lived and broad SARS-CoV-2-specific T cell immunity, which further supports the current evaluation of our T cell activator in patients with congenital or acquired B-cell defects., Competing Interests: Declarations of Competing Interest JSH, HRS, H-GR, and JSW are listed as inventors on a patent (EP 20 169 047.6) related to the SARS-CoV-2 T cell epitopes included in CoVac-1. H-GR and K-HW are listed as inventors on a patent related to the adjuvant XS15 included in CoVac-1 (DE102016005550.2). The other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023. The Author(s).)

Therapy-resistant leukemia stem and progenitor cells (LSC) are a main cause of acute myeloid leukemia (AML) relapse. LSC-targeting therapies may thus improve outcome of patients with AML. Here we demonstrate that LSCs present HLA-restricted antigens that induce T-cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LSCs and identified AML- and AML/LSC-associated HLA-presented antigens absent from normal tissues comprising nonmutated peptides, cryptic neoepitopes, and neoepitopes of common AML driver mutations of NPM1 and IDH2. Functional relevance of shared AML/LSC antigens is illustrated by presence of their cognizant memory T cells in patients. Antigen-specific T-cell recognition and HLA class II immunopeptidome diversity correlated with clinical outcome. Together, these antigens shared among AML and LSCs represent prime targets for T cell-based therapies with potential of eliminating residual LSCs in patients with AML., Significance: The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 430 . This article is featured in Selected Articles from This Issue, p. 419., (©2023 The Authors; Published by the American Association for Cancer Research.)

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., (© 2023. Springer Nature Limited.)

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., (© 2023. Springer Nature Limited.)

Meningiomas are the most common primary intracranial tumors. Although most symptomatic cases can be managed by surgery and/or radiotherapy, a relevant number of patients experience an unfavorable clinical course and additional treatment options are needed. As meningiomas are often perfused by dural branches of the external carotid artery, which is located outside the blood-brain barrier, they might be an accessible target for immunotherapy. However, the landscape of naturally presented tumor antigens in meningioma is unknown. We here provide a T-cell antigen atlas for meningioma by in-depth profiling of the naturally presented immunopeptidome using LC-MS/MS. Candidate target antigens were selected based on a comparative approach using an extensive immunopeptidome data set of normal tissues. Meningioma-exclusive antigens for HLA class I and II are described here for the first time. Top-ranking targets were further functionally characterized by showing their immunogenicity through in vitro T-cell priming assays. Thus, we provide an atlas of meningioma T-cell antigens which will be publicly available for further research. In addition, we have identified novel actionable targets that warrant further investigation as an immunotherapy option for meningioma., (© 2023. The Author(s).)

Introduction: Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data., Methods: In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples., Results: Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it., Discussion: Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wacker, Bauer, Wessling, Dubbelaar, Nelde, Rammensee and Walz.)

Molecular imaging has experienced enormous advancements in the areas of imaging technology, imaging probe and contrast development, and data quality, as well as machine learning-based data analysis. Positron emission tomography (PET) and its combination with computed tomography (CT) or magnetic resonance imaging (MRI) as a multimodality PET-CT or PET-MRI system offer a wealth of molecular, functional and morphological data with a single patient scan. Despite the recent technical advances and the availability of dozens of disease-specific contrast and imaging probes, only a few parameters, such as tumour size or the mean tracer uptake, are used for the evaluation of images in clinical practice. Multiparametric in vivo imaging data not only are highly quantitative but also can provide invaluable information about pathophysiology, receptor expression, metabolism, or morphological and functional features of tumours, such as pH, oxygenation or tissue density, as well as pharmacodynamic properties of drugs, to measure drug response with a contrast agent. It can further quantitatively map and spatially resolve the intertumoural and intratumoural heterogeneity, providing insights into tumour vulnerabilities for target-specific therapeutic interventions. Failure to exploit and integrate the full potential of such powerful imaging data may lead to a lost opportunity in which patients do not receive the best possible care. With the desire to implement personalized medicine in the cancer clinic, the full comprehensive diagnostic power of multiplexed imaging should be utilized., (© 2023. Springer Nature Limited.)

Cytotoxic T lymphocytes are key for controlling viral infection. Unravelling CD8 + T cell-mediated immunity to distinct influenza virus strains and subtypes across prominent HLA types is relevant for combating seasonal infections and emerging new variants. Using an immunopeptidomics approach, naturally presented influenza A virus-derived ligands restricted to HLA-A*24:02, HLA-A*68:01, HLA-B*07:02, and HLA-B*51:01 molecules were identified. Functional characterization revealed multifunctional memory CD8 + T cell responses for nine out of sixteen peptides. Peptide presentation kinetics was optimal around 12 h post infection and presentation of immunodominant epitopes shortly after infection was not always persistent. Assessment of immunogenic epitopes revealed that they are highly conserved across the major zoonotic reservoirs and may contain a single substitution in the vicinity of the anchor residues. These findings demonstrate how the identified epitopes promote T cell pools, possibly cross-protective in individuals and can be potential targets for vaccination.

Microbial organisms have key roles in numerous physiological processes in the human body and have recently been shown to modify the response to immune checkpoint inhibitors 1,2 . Here we aim to address the role of microbial organisms and their potential role in immune reactivity against glioblastoma. We demonstrate that HLA molecules of both glioblastoma tissues and tumour cell lines present bacteria-specific peptides. This finding prompted us to examine whether tumour-infiltrating lymphocytes (TILs) recognize tumour-derived bacterial peptides. Bacterial peptides eluted from HLA class II molecules are recognized by TILs, albeit very weakly. Using an unbiased antigen discovery approach to probe the specificity of a TIL CD4 + T cell clone, we show that it recognizes a broad spectrum of peptides from pathogenic bacteria, commensal gut microbiota and also glioblastoma-related tumour antigens. These peptides were also strongly stimulatory for bulk TILs and peripheral blood memory cells, which then respond to tumour-derived target peptides. Our data hint at how bacterial pathogens and bacterial gut microbiota can be involved in specific immune recognition of tumour antigens. The unbiased identification of microbial target antigens for TILs holds promise for future personalized tumour vaccination approaches., (© 2023. The Author(s).)

Background: The immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies., Methods: Mapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens., Results: MS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse., Conclusion: Tumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies., (© 2023. The Author(s).)

Despite the successful development of vaccines and neutralizing antibodies to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerging variants prolong the pandemic and emphasize the persistent need to develop effective antiviral treatment regimens. Recombinant antibodies directed to the original SARS-CoV-2 have been successfully used to treat established viral disease. However, emerging viral variants escape the recognition by those antibodies. Here we report the engineering of an optimized ACE2 fusion protein, designated ACE2-M, which comprises a human IgG1 Fc domain with abrogated Fc-receptor binding linked to a catalytically-inactive ACE2 extracellular domain that displays increased apparent affinity to the B.1 spike protein. The affinity and neutralization capacity of ACE2-M is unaffected or even enhanced by mutations present in the spike protein of viral variants. In contrast, a recombinant neutralizing reference antibody, as well as antibodies present in the sera of vaccinated individuals, lose activity against such variants. With its potential to resist viral immune escape ACE2-M appears to be particularly valuable in the context of pandemic preparedness towards newly emerging coronaviruses., Competing Interests: GJ, LZ, HS, MS, NR and MC are listed as inventors on the patent application “Modified ACE2 proteins with improved activity against SARS-CoV-2”, EP22198499, applicant German Cancer Research Center, Heidelberg and University of Tuebingen, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zekri, Ruetalo, Christie, Walker, Manz, Rammensee, Salih, Schindler and Jung.)

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß 2 -integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8 + ) or few hours (CD4 + ) of activation. Here, we present a simple method for detection of activated ß 2 -integrins in combination with established cell surface activation induced markers. We observed that activated ß 2 -integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß 2 -integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4 + and CD8 + T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4 + and CD8 + T cell responses in various physiological situations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schöllhorn, Maia, Kimmerle, Born, Rammensee, Dimitrov and Gouttefangeas.)

Background: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc)., Methods: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay., Results: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1 . Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment., Conclusion: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category., Competing Interests: Competing interests: MWL is an inventor of patents owned by Immatics Biotechnologies and has acted as a speaker and paid consultant in cancer immunology for Boehringer Ingelheim., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8 + and CD4 + cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8 + T cells was dependent on the elongation site of the EP, whereas CD4 + T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8 + and CD4 + T cell reactivities.

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., (© 2022. The Author(s).)

Cancer treatment with immune checkpoint blockade (ICB) often induces immune-related adverse events (irAEs). We hypothesized that proteins coexpressed in tumors and normal cells could be antigenic targets in irAEs and herein described DITAS (discovery of tumor-associated self-antigens) for their identification. DITAS computed transcriptional similarity between lung tumors and healthy lung tissue based on single-sample gene set enrichment analysis. This identified 10 lung tissue-specific genes highly expressed in the lung tumors. Computational analysis was combined with functional T cell assays and single-cell RNA sequencing of the antigen-specific T cells to validate the lung tumor self-antigens. In patients with non-small cell lung cancer (NSCLC) treated with ICB, napsin A was a self-antigen that elicited strong CD8 + T cell responses, with ICB responders harboring higher frequencies of these CD8 + T cells compared with nonresponders. Human leukocyte antigen (HLA) class I ligands derived from napsin A were present in human lung tumors and in nontumor lung tissues, and napsin A tetramers confirmed the presence of napsin A-specific CD8 + T cells in blood and tumors of patients with NSCLC. Napsin A-specific T cell clonotypes were enriched in lung tumors and ICB-induced inflammatory lung lesions and could kill immortalized HLA-matched NSCLC cells ex vivo. Single-cell RNA sequencing revealed that these T cell clonotypes expressed proinflammatory cytokines and cytotoxic markers. Thus, DITAS successfully identified self-antigens, including napsin A, that likely mediate effective antitumor T cell responses in NSCLC and may simultaneously underpin lung irAEs.

Antibodies against the B cell-specific antigens CD20 and CD19 have markedly improved the treatment of B cell-derived lymphoma and autoimmune diseases by depleting malignant and autoreactive B cells. However, since CD20 and CD19 are also expressed on healthy B cells, such antibodies lack disease specificity. Here, we optimize a previously developed concept that uses bispecific antibodies to induce apoptosis selectively in malignant and autoreactive B cells that express the death receptor CD95. We describe the development and characterization of bispecific antibodies with CD95xCD20 and CD95xCD19 specificity in a new IgG-based format. We could show that especially the CD95xCD20 antibody mediated a strong induction of apoptosis in malignant B cells in vitro. In vivo, the antibody was clearly superior to the previously used Fabsc format with identical specificities. In addition, both IgGsc antibodies depleted activated B cells in vitro, leading to a significant reduction in antibody production and cytokine secretion. The killing of resting B cells and hepatocytes that lack CD95 and CD20/CD19, respectively, was marginal. Thus, our results imply that bispecific anti-CD95 antibodies in the IgGsc format are an attractive tool for a more selective and efficient depletion of malignant as well as autoreactive B cells.

Purpose: Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response., Patients and Methods: A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints., Results: The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees., Conclusions: Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I- and class II-restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted., (©2022 American Association for Cancer Research.)

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.

Background: Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood., Methods: We examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs., Results: We provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses., Conclusion: Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides., (© 2022. The Author(s).)

Purpose: The influence of radiotherapy on patient immune cell subsets has been established by several groups. Following a previously published analysis of immune changes during and after curative radiotherapy for prostate cancer, this analysis focused on describing correlations of changes of immune cell subsets with radiation treatment parameters., Patients and Methods: For 13 patients treated in a prospective trial with radiotherapy to the prostate region (primary analysis) and five patients treated with radiotherapy to prostate and pelvic nodal regions (exploratory analysis), already published immune monitoring data were correlated with clinical data as well as radiation planning parameters such as clinical target volume (CTV) and volumes receiving 20 Gy (V20) for newly contoured volumes of pelvic blood vessels and bone marrow., Results: Most significant changes among immune cell subsets were observed at the end of radiotherapy. In contrast, correlations of age and CD8 + subsets (effector and memory cells) were observed early during and 3 months after radiotherapy. Ratios of T cells and T cell proliferation compared to baseline correlated with CTV. Early changes in regulatory T cells (Treg cells) and CD8 + effector T cells correlated with V20 of blood vessels and bone volumes., Conclusions: Patient age as well as radiotherapy planning parameters correlated with immune changes during radiotherapy. Larger irradiated volumes seem to correlate with early suppression of anti-cancer immunity. For immune cell analysis during normofractionated radiotherapy and correlations with treatment planning parameters, different time points should be looked at in future projects., Trial Registration Number: NCT01376674, 20.06.2011., (© 2021. The Author(s).)

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients' samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Understanding the molecular principles that govern the composition of the MHC-I immunopeptidome across different primary tissues is fundamentally important to predict how T cells respond in different contexts in vivo . Here, we performed a global analysis of the MHC-I immunopeptidome from 29 to 19 primary human and mouse tissues, respectively. First, we observed that different HLA-A, HLA-B, and HLA-C allotypes do not contribute evenly to the global composition of the MHC-I immunopeptidome across multiple human tissues. Second, we found that tissue-specific and housekeeping MHC-I peptides share very distinct properties. Third, we discovered that proteins that are evolutionarily hyperconserved represent the primary source of the MHC-I immunopeptidome at the organism-wide scale. Fourth, we uncovered new components of the antigen processing and presentation network, including the carboxypeptidases CPE, CNDP1/2, and CPVL. Together, this study opens up new avenues toward a system-wide understanding of antigen presentation in vivo across mammalian species., Competing Interests: Heiko Schuster is an employee of Immatics Biotechnologies GmbH. Hans-Georg Rammensee is a shareholder of Immatics Biotechnologies GmbH and Curevac AG., (© 2022 The Author(s).)

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins 1,2 , combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4 + and CD8 + T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency., (© 2022. The Author(s).)

Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8 + T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Both B cells and T cells are involved in an effective immune response to SARS-CoV-2, the disease-causing virus of COVID-19. While B cells-with the indispensable help of CD4 + T cells-are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti-SARS-CoV-2 immunity. In this report, we provide insights into the characteristics of individual HLA-A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. We observed that SARS-CoV-2-reactive T-cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re-expression. The SARS-CoV-2-reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS-CoV-2-reactive TCRs conferred powerful T-cell effector function to primary CD8 + T cells as evident by a robust anti-SARS-CoV-2 IFN-γ response and in vitro cytotoxicity. We also provide an example of a long-lasting anti-SARS-CoV-2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti-SARS-CoV-2 T-cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS-CoV-2., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells., Methods: Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM., Results: Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8 + T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors., Conclusions: These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion., Competing Interests: Competing interests: MEy has taken part in pediatric advisory boards of BMS and Atara Biotherapeutics and holds research collaborations with CellSource and Miltenyi Biotec., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Low human leukocyte antigen (HLA)-DR expression might compromise CD4 + T-cell-mediated anti-tumor immunity. Its immunological and clinical significance however remain undefined in non-promyelocytic acute myeloid leukemia (AML). Taking advantage of mass spectrometry-based immunopeptidome analysis of primary AML samples ( n  = 31), we studied the implications of low HLA-DR expression for antigen presentation and analyzed its association with disease characteristics and survival within a cohort of 399 AML patients. Remarkably, overall HLA-DR/DQ immunopeptidome diversity was preserved in AML with low HLA-DR expression (HLA-DR low AML) and was associated with a shift in HLA-DR/DQ allotype abundances (HLA-DQ to HLA-DR/DQ ligand ratio 0.36 vs 0.19 in HLA-DR low and HLA-DR high patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DR low and HLA-DR high patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DR low AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DR low AML patients.

Patients with cancer, in particular patients with hematologic malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4 + and CD8 + T-cell responses in unexposed and SARS-CoV-2-infected patients with cancer to characterize SARS-CoV-2 immunity and to identify immunologic parameters contributing to COVID-19 outcome. Unexposed patients with hematologic malignancies presented with reduced prevalence of preexisting SARS-CoV-2 cross-reactive CD4 + T-cell responses and signs of T-cell exhaustion compared with patients with solid tumors and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between patients with COVID-19 and cancer and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T-cell responses were profoundly reduced in patients with cancer, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T-cell immunity as a potential determinant for dismal outcome of COVID-19 in patients with cancer. SIGNIFICANCE: This first comprehensive analysis of SARS-CoV-2 immune responses in patients with cancer reports on the potential implications of impaired SARS-CoV-2 T-cell responses for understanding pathophysiology and predicting severity of COVID-19, which in turn might allow for the development of therapeutic measures and vaccines for this vulnerable patient population. See related commentary by Salomé and Horowitz, p. 1877 . This article is highlighted in the In This Issue feature, p. 1861 ., (©2021 American Association for Cancer Research.)

Recombinant bispecific antibodies (bsAbs) are increasingly included in regimens for cancer therapy. Strict good manufacturing practice (GMP) compliant quality control measures are required to ensure quality and safety of these innovative biologicals. Gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and size exclusion chromatography (SEC) are the cornerstones of quality control methods. BsAbs are often prone to aggregation or incomplete synthesis due to their artificial nature. In addition, host cell proteins and host cell DNA as well as impurities from the purification process itself constitute potential contaminants. Such impurities may then appear as additional, unexpected bands or peaks on SDS-PAGE gels and SEC, respectively. Here we describe a standardized protocol for rapid analysis of recombinant antibodies by mass spectrometry (MS) after tryptic digestion of bands excised from SDS-PAGE gels. We have used this protocol to characterize unexpected "contaminating bands" that were observed during the clinical development of a novel bsAb with PSMAxCD3 specificity, either during the production of the protein itself or during the development of a surrogate molecule for evaluation in syngeneic mouse models. MS analysis allowed us to precisely determine the origin of these bands, which resulted from artifacts or from incomplete protein synthesis. The combined utilization of SDS-PAGE und MS can therefore substantially support GMP-compliant production of recombinant proteins., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches., Competing Interests: H-GR is shareholder of Immatics Biotechnologies GmbH, Synimmune GmbH, and Curevac AG, and holds a patent application on an adjuvant, XS15. AN, H-GR, and JW are listed as inventors on patents related to peptides described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nelde, Maringer, Bilich, Salih, Roerden, Heitmann, Marcu, Bauer, Neidert, Denzlinger, Illerhaus, Aulitzky, Rammensee and Walz.)

Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8 + and CD4 + T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4 + T cells. The identification of CD4 + T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used in silico prediction to determine promiscuous CD4 + T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4 + T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4 + T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4 + T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Höttler, März, Lübke, Rammensee and Stevanović.)

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans., Competing Interests: The authors have declared that no competing interests exist.

We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual's HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing.

Long-term immunological memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for the development of population-level immunity, which is the aim of vaccination approaches. Reports on rapidly decreasing antibody titers have led to questions regarding the efficacy of humoral immunity alone. The relevance of T cell memory after coronavirus disease 2019 (COVID-19) remains unclear. Here, we investigated SARS-CoV-2 antibody and T cell responses in matched samples of COVID-19 convalescent individuals up to 6 months after infection. Longitudinal analysis revealed decreasing and stable spike- and nucleocapsid-specific antibody responses, respectively. In contrast, functional T cell responses remained robust, and even increased, in both frequency and intensity. Single peptide mapping of T cell diversity over time identified open reading frame-independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses. Identification of these epitopes may be fundamental for COVID-19 vaccine design., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Background: The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level., Methods: Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing., Results: The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference., Conclusion: Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org ., Competing Interests: Competing interests: LB, DJK, ML and LKF are employees of Immatics Biotechnologies. LB, DJK, MWL and SS are inventors of patents owned by Immatics Biotechnologies. MWL has acted as a paid consultant in cancer immunology for Boehringer Ingehlheim Pharma & Co. KG. H-GR is shareholder of Immatics Biotechnologies and Curevac AG., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

We have previously shown that conformational change in the β 2 -integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8 + T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4 + T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β 2 -integrin. The kinetics of β 2 -integrin activation was different on CD4 + and CD8 + T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4 + and CD8 + T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β 2 -integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4 + T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4 + and CD8 + T cell reactivities, with versatile applicability in clinical and vaccination studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schöllhorn, Schuhmacher, Besedovsky, Fendel, Jensen, Stevanović, Lange, Rammensee, Born, Gouttefangeas and Dimitrov.)

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo , after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro , delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Felux, Erbacher, Breckler, Hervé, Lemeiter, Mannherz, Napirei, Rammensee and Decker.)

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.

The prostate-specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such "dual" expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T-cell recruiting bispecific PSMAxCD3 antibodies in Fab- and IgG-based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

The approach of peptide-based anticancer vaccination has proven the ability to induce cancer-specific immune responses in multiple studies for various cancer entities. However, clinical responses remain so far limited to single patients and broad clinical applicability was not achieved. Therefore, further efforts are required to improve peptide vaccination in order to integrate this low-side-effect therapy into the clinical routine of cancer therapy. To design clinically effective peptide vaccines in the future, different issues have to be addressed and optimized comprising antigen target selection as well as choice of optimal adjuvants and vaccination schedules. Furthermore, the combination of peptide-based vaccines with other immuno- and molecular targeted therapies as well as the development of predictive biomarkers could further improve efficacy. In this review, current approaches in the development of peptide-based vaccines and critical implications for optimal vaccine design are discussed., Competing Interests: Conflict of interest A. N. and J. S. W. hold patents on peptides. H.-G. R. holds patents on peptides and adjuvants., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells.

Biomaterials play an increasing role in clinical applications and regenerative medicine. A perfectly designed biomaterial should restore the function of damaged tissue without triggering an undesirable immune response, initiate self-regeneration of the surrounding tissue and gradually degrade after implantation. The immune system is well recognized to play a major role in influencing the biocompatibility of implanted medical devices. To obtain a better understanding of the effects of biomaterials on the immune response, we have developed a highly sensitive novel test system capable of examining changes in the immune system by biomaterial. Here, we evaluated for the first time the immunopeptidome, a highly sensitive system that reflects cancer transformation, virus or drug influences and passes these cellular changes directly to T cells, as a test system to examine the effects of contact with materials. Since monocytes are one of the first immune cells reacting to biomaterials, we have tested the influence of different materials on the immunopeptidome of the monocytic THP-1 cell line. The tested materials included stainless steel, aluminum, zinc, high-density polyethylene, polyurethane films containing zinc diethyldithiocarbamate, copper, and zinc sulfate. The incubation with all material types resulted in significantly modulated peptides in the immunopeptidome, which were material-associated. The magnitude of induced changes in the immunopeptidome after the stimulation appeared comparable to that of bacterial lipopolysaccharides (LPS). The source proteins of many detected peptides are associated with cytotoxicity, fibrosis, autoimmunity, inflammation, and cellular stress. Considering all tested materials, it was found that the LPS-induced cytotoxicity-, inflammation- and cellular stress-associated HLA class I peptides were mainly induced by aluminum, whereas HLA class II peptides were mainly induced by stainless steel. These findings provide the first insights into the effects of biomaterials on the immunopeptidome. A more thorough understanding of these effects may enable the design of more biocompatible implant materials using in vitro models in future. Such efforts will provide a deeper understanding of possible immune responses induced by biomaterials such as fibrosis, inflammation, cytotoxicity, and autoimmune reactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Ghosh, Hartmann, Jakobi, März, Bichmann, Freudenmann, Mühlenbruch, Segan, Rammensee, Schneiderhan-Marra, Shipp, Stevanović and Joos.)

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4 + T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4 + T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4 + T cells in MS., Competing Interests: Declaration of Interests R.M. received unrestricted grants (Biogen and Novartis) and compensation for advice/lecturing (Biogen, Novartis, Sanofi Genzyme, Merck, Hoffmann La Roche, Neuway, CellProtect, and Third Rock Ventures). R.M., M.S., and A.L. are listed as inventor on several NIH- and University of Zurich-held patents and are co-founders and co-owners of Cellerys. I.J. received compensation for advice by Sanofi Genzyme, none of which has affected this work. R.M. and M.S. are listed as co-inventors on a patent on “Immunodominant proteins and fragments in autoimmune diseases,” including RAGSRP2., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The physician and scientist Paul Ehrlich put forward the thesis that the immune system not only fights infections but can also fight cancer. The possible positive effects of a simultaneous infection on the course of cancer were reported in ancient Egypt around 2600 BC. However, it was not until the 1960s that it became apparent that the immune system could specifically fight cancer cells, and it was not until the 1990s that researchers slowly clarified how this happens.Against this background, the efforts over the last 30 years to develop therapeutic vaccines against cancers are briefly summarized, and their lack of success to date is highlighted. In addition, potentially promising future developments in this context are discussed. The available scientific literature as well as our own results are taken into account.Central questions arise, such as the following: How do cancer cells differ from normal cells? How can the immune system recognize these differences? What are tumor-specific antigens? Why do they need to be selected and applied in an individualized fashion? How can an efficient immune response be induced? Which pharmaceutical formulations, adjuvants, and vaccination routes are effective?Finally, we explain why it may still be worth pursuing peptide vaccination, which has so far been completely unsuccessful (when measured in terms of already approved therapeutics).