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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).)

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 ( COL6A3 ) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

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.)

Knowledge about the peptide repertoire presented by human leukocyte antigens (HLA) holds the key to unlock target-specific cancer immunotherapies such as adoptive cell therapies or bispecific T cell engaging receptors. Therefore, comprehensive and accurate characterization of HLA peptidomes by mass spectrometry (immunopeptidomics) across tissues and disease states is essential. With growing numbers of immunopeptidomics datasets and the scope of peptide identification strategies reaching beyond the canonical proteome, the likelihood for erroneous peptide identification as well as false annotation of HLA-independent peptides as HLA ligands is increasing. Such "fake ligands" can lead to selection of nonexistent targets for immunotherapeutic development and need to be recognized as such as early as possible in the preclinical pipeline. Here we present computational and experimental methods that enable the identification of "fake ligands" that might be introduced at different steps of the immunopeptidomics workflow. The statistics presented herein allow discrimination of true HLA ligands from coisolated HLA-independent proteolytic fragments. In addition, we describe necessary steps to ensure system suitability of the chromatographic system. Furthermore, we illustrate an algorithm for detection of source fragmentation events that are introduced by electrospray ionization during mass spectrometry. For confirmation of peptide sequences, we present an experimental pipeline that enables high-throughput sequence verification through similarity of fragmentation pattern and coelution of synthetic isotope-labeled internal standards. Based on these methods, we show the overall high quality of existing datasets but point out limitations and pitfalls critical for individual peptides and how they can be uncovered in order to identify true ligands., Competing Interests: Conflict of interest All authors are employees and shareholders of Immatics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Background: Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy., Methods: We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n = 452). DNA methylation (TCGA KIRC, n = 273), somatic mutations (TCGA KIRC, n = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n = 30) and immune-oncological markers (cohort 1, n = 37) were analyzed to characterize regulatory and functional involvements. CD8 + T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture., Results: A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8 + T cells, including peptides derived from EGLN3. Functional analysis of EGLN3 revealed possible tumor-promoting functions., Conclusions: Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC.

In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identifying the repertoire of T cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. Here, we present a novel approach that employs HCMV deletion mutant viruses lacking HLA class I immunoevasins and allows direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. We identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. Multiple HCMV-directed specificities in the memory T cell pool of single individuals indicate that physiologic anti-HCMV T cell responses are directed against a broad range of antigens. Thus, the unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T cell specificities in seropositive individuals., Competing Interests: Disclosures: M. Lübke reported a patent to HCMV epitopes (pending). S. Spalt reported a patent to HCMV epitopes (pending) and is currently an employee of Immatics Biotechnologies GmbH. D.J. Kowalewski reported a patent to HCMV epitopes (pending) and is currently an employee of Immatics Biotechnologies. C. Zimmermann reported a patent to HCMV epitopes (pending). L. Bauersfeld reported a patent to HCMV epitopes (pending). A. Nelde reported a patent to HCMV epitopes (pending). V.T.K. Le-Trilling reported a patent to HCMV epitopes (pending). H. Hengel reported a patent to HCMV epitopes (pending). H.-G. Rammensee reported "other" from Immatics GmbH and "other" from Curevac AG during the conduct of the study; in addition, H.-G. Rammensee had a patent to HCMV epitopes (pending). A. Halenius reported a patent to HCMV epitopes (pending). No other disclosures were reported., (© 2019 Lübke et al.)

Background: Radiofrequency ablation (RFA) is an established treatment option for malignancies located in the liver. RFA-induced irreversible coagulation necrosis leads to the release of danger signals and cellular content. Hence, RFA may constitute an endogenous in situ tumor vaccination, stimulating innate and adaptive immune responses, including tumor-antigen specific T cells. This may explain a phenomenon termed abscopal effect, namely tumor regression in untreated lesions evidenced after distant thermal ablation or irradiation. In this study, we therefore assessed systemic and local immune responses in individual patients treated with RFA. Methods: For this prospective clinical trial, patients with liver metastasis from colorectal carcinoma (mCRC) receiving RFA and undergoing metachronous liver surgery for another lesion were recruited ( n = 9) during a 5-year period. Tumor and non-malignant liver tissue samples from six patients were investigated by whole transcriptome sequencing and tandem-mass spectrometry, characterizing naturally presented HLA ligands. Tumor antigen-derived HLA-restricted peptides were selected by different predefined approaches. Further, candidate HLA ligands were manually curated. Peripheral blood mononuclear cells were stimulated in vitro with epitope candidate peptides, and functional T cell responses were assessed by intracellular cytokine staining. Immunohistochemical markers were additionally investigated in surgically resected mCRC from patients treated with ( n = 9) or without RFA ( n = 7). Results: In all six investigated patients, either induced immune responses and/or pre-existing T cell immunity against the selected targets were observed. Multi-cytokine responses were inter alia directed against known tumor antigens such as cyclin D1 but also against a (predicted) mutation contained in ERBB3. Immunohistochemistry did not show a relevant influx of immune cells into distant malignant lesions after RFA treatment ( n = 9) as compared to the surgery only mCRC group ( n = 7). Conclusions: Using an individualized approach for target selection, RFA induced and/or boosted T cell responses specific for individual tumor antigens were more frequently detectable as compared to previously published observations with well-characterized tumor antigens. However, the witnessed modest RFA-induced immunological effects alone may not be sufficient for the rejection of established tumors. Therefore, these findings warrant further clinical investigation including the assessment of RFA combination therapies e.g., with immune stimulatory agents, cancer vaccination, and/or immune checkpoint inhibitors., (Copyright © 2019 Löffler, Nussbaum, Jäger, Jurmeister, Budczies, Pereira, Clasen, Kowalewski, Mühlenbruch, Königsrainer, Beckert, Ladurner, Wagner, Bullinger, Gross, Schroeder, Sipos, Königsrainer, Stevanović, Denkert, Rammensee, Gouttefangeas and Haen.)

Background: We previously showed that the bacterial lipopeptide Pam 3 Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8 + T cells in mice, when covalently coupled to a synthetic peptide., Case Presentation: We now designed a new water-soluble synthetic Pam 3 Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8 + T and NK cells by 6-sulfo LacNAc + monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging. XS15 induced strong ex vivo CD8 + and T H 1 CD4 + responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4 + and CD8 + effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 10 5 in the granuloma and 20.5 × 10 6 in peripheral blood., Conclusion: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.

Background: Although mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs., Methods: In-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data., Results: The approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations., Conclusions: This study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.

MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8 + T cell responses. Despite the appreciation of CD8 + T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8 + T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8 + T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8 + T-cell-based immunotherapies.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients., (© 2019 by The American Society of Hematology.)

The large-scale and in-depth identification of MHC class I- and II-presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as for developing innovative immunotherapeutic approaches against cancer and other diseases. In this chapter we briefly review the existing strategies for the isolation of MHC-restricted peptides and provide a detailed protocol for the immunoaffinity purification of MHC class I- and II-presented peptides from primary tissues or cells.

Most T cell-based immunotherapies of cancer depend on intact antigen presentation by HLA class I molecules (HLA-I). However, defects in the antigen-processing machinery can cause downregulation of HLA-I, rendering tumor cells resistant to CD8 + T cells. Previously, we demonstrated that a unique category of cancer antigens is selectively presented by tumor cells deficient for the peptide transporter TAP, enabling a specific attack of such tumors without causing immunopathology in mouse models. With a novel combinatorial screening approach, we now identify 16 antigens of this category in humans. These HLA-A*02:01 presented peptides do not derive from the mutanome of cancers, but are of "self" origin and therefore constitute universal neoantigens. Indeed, CD8 + T cells specific for the leader peptide of the ubiquitously expressed LRPAP1 protein recognized TAP-deficient, HLA-I low lymphomas, melanomas, and renal and colon carcinomas, but not healthy counterparts. In contrast to personalized mutanome-targeted therapies, these conserved neoantigens and their cognate receptors can be exploited for immune-escaped cancers across diverse histological origins., (© 2018 Marijt et al.)

Immune cell infiltrates have proven highly relevant for colorectal carcinoma prognosis, making colorectal cancer a promising candidate for immunotherapy. Because tumors interact with the immune system via HLA-presented peptide ligands, exact knowledge of the peptidome constitution is fundamental for understanding this relationship. Here, we comprehensively describe the naturally presented HLA ligandome of colorectal carcinoma and corresponding nonmalignant colon (NMC) tissue. Mass spectrometry identified 35,367 and 28,132 HLA class I ligands on colorectal carcinoma and NMC, attributable to 7,684 and 6,312 distinct source proteins, respectively. Cancer-exclusive peptides were assessed on source protein level using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein analysis through evolutionary relationships (PANTHER), revealing pathognomonic colorectal carcinoma-associated pathways, including Wnt, TGFβ, PI3K, p53, and RTK-RAS. Relative quantitation of peptide presentation on paired colorectal carcinoma and NMC tissue further identified source proteins from cancer- and infection-associated pathways to be overrepresented merely within the colorectal carcinoma ligandome. From the pool of tumor-exclusive peptides, a selected HLA-ligand subset was assessed for immunogenicity, with the majority exhibiting an existing T-cell repertoire. Overall, these data show that the HLA ligandome reflects cancer-associated pathways implicated in colorectal carcinoma oncogenesis, suggesting that alterations in tumor cell metabolism could result in cancer-specific, albeit not mutation-derived, tumor antigens. Hence, a defined pool of unique tumor peptides, attributable to complex cellular alterations that are exclusive to malignant cells, might comprise promising candidates for immunotherapeutic applications. Significance: Cancer-associated pathways are reflected in the antigenic landscape of colorectal cancer, suggesting that tumor-specific antigens do not necessarily have to be mutation-derived but may also originate from other alterations in cancer cells. Cancer Res; 78(16); 4627-41. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The course of chronic lymphocytic leukemia (CLL), inducing an immunosuppressed state that also affects T cells as central components of adaptive immunity, predisposes patients to develop second malignancies with skin cancer being the most common. Recently, we found that prevalence of memory T cells with specificity for CLL-associated antigens defined by mass spectrometry-based immunopeptidome analysis correlated with a significant survival benefit. Here, we analyzed our CLL patient cohort for second skin (pre)malignancies and found a significantly lower incidence of skin cancer in the patients showing immune responses to CLL-associated antigens. Surprisingly, CLL-associated antigen-specific immune responses did not associate with clinical characteristics including leukocyte, neutrophil, and thrombocyte count, hemoglobin, immunoglobulin levels, or CD8 + and CD4 + T-cell immune status. Our data indicate that the CLL-specific immune signature of a given patient, defined by antigen-specific T-cell responses, might represent an independent marker to identify CLL patients susceptible for the development of skin malignancies.

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.

Immunotherapy is revolutionizing cancer treatment and has shown success in particular for tumors with a high mutational load. These effects have been linked to neoantigens derived from patient-specific mutations. To expand efficacious immunotherapy approaches to the vast majority of tumor types and patient populations carrying only a few mutations and maybe not a single presented neoepitope, it is necessary to expand the target space to non-mutated cancer-associated antigens. Mass spectrometry enables the direct and unbiased discovery and selection of tumor-specific human leukocyte antigen (HLA) peptides that can be used to define targets for immunotherapy. Combining these targets into a warehouse allows for multi-target therapy and accelerated clinical application. For precise personalization aimed at optimally ensuring treatment efficacy and safety, it is necessary to assess the presence of the target on each individual patient's tumor. Here we show how LC-MS paired with gene expression data was used to define mRNA biomarkers currently being used as diagnostic test IMADETECT™ for patient inclusion and personalized target selection within two clinical trials (NCT02876510, NCT03247309). Thus, we present a way how to translate HLA peptide presentation into gene expression thresholds for companion diagnostics in immunotherapy considering the peptide-specific correlation to its encoding mRNA., (© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translation initiation. We experimentally validated translation using major histocompatibility complex class I (MHC I) peptidomics and observed that sORF-derived peptides efficiently enter the MHC I presentation pathway and thus constitute a substantial fraction of the antigen repertoire.

Despite multidisciplinary local and systemic therapeutic approaches, the prognosis for most patients with brain metastases is still dismal. The role of adaptive and innate anti-tumor response including the Human Leukocyte Antigen (HLA) machinery of antigen presentation is still unclear. We present data on the HLA class II-chaperone molecule CD74 in brain metastases and its impact on the HLA peptidome complexity.We analyzed CD74 and HLA class II expression on tumor cells in a subset of 236 human brain metastases, primary tumors and peripheral metastases of different entities in association with clinical data including overall survival. Additionally, we assessed whole DNA methylome profiles including CD74 promoter methylation and differential methylation in 21 brain metastases. We analyzed the effects of a siRNA mediated CD74 knockdown on HLA-expression and HLA peptidome composition in a brain metastatic melanoma cell line.We observed that CD74 expression on tumor cells is a strong positive prognostic marker in brain metastasis patients and positively associated with tumor-infiltrating T-lymphocytes (TILs). Whole DNA methylome analysis suggested that CD74 tumor cell expression might be regulated epigenetically via CD74 promoter methylation. CD74 high and TIL high tumors displayed a differential DNA methylation pattern with highest enrichment scores for antigen processing and presentation. Furthermore, CD74 knockdown in vitro lead to a reduction of HLA class II peptidome complexity, while HLA class I peptidome remained unaffected.In summary, our results demonstrate that a functional HLA class II processing machinery in brain metastatic tumor cells, reflected by a high expression of CD74 and a complex tumor cell HLA peptidome, seems to be crucial for better patient prognosis.

Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro . We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies.

Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation., Competing Interests: Conflict of interest statement: H.-G.R. is a shareholder of Immatics Biotechnologies, Tübingen, and CureVac GmbH, Tübingen. H.S. and D.J.K. are employees of Immatics Biotechnologies GmbH. The authors declare that Immatics did not provide neither financial nor scientific support in any direct relation to this manuscript or the underlying studies, and was not involved in data collection, analysis, or decision to publish., (Copyright © 2017 the Author(s). Published by PNAS.)

Hematological malignancies (HM) are highly amenable targets for immunotherapeutic intervention and may be effectively treated by antigen-specific T-cell based treatment. Recent studies demonstrate that physiologically occurring anti-cancer T-cell responses in certain HM entities target broadly presented non-mutated epitopes. HLA ligands are thus implied as prime targets for broadly applicable and antigen-specific off-the-shelf compounds. With the aim of assessing the presence of common targets shared among different HM which may enable addressing a larger patient collective we conducted a meta-analysis of 83 mass spectrometry-based HLA peptidome datasets (comprising 40,361 unique peptide identifications) across four major HM (19 AML, 16 CML, 35 CLL, and 13 MM/MCL samples) and investigated similarities and differences within the HLA presented antigenic landscape. We found the cancer HLA peptidome datasets to cluster specifically along entity and lineage lines, suggesting that the immunopeptidome directly reflects the differences in the underlying (tumor-)biology. In line with these findings, we only detected a small set of entity-spanning antigens, which were predominantly characterized by low presentation frequencies within the different patient cohorts. These findings suggest that design of T-cell immunotherapies for the treatment of HM should ideally be conducted in an entity-specific fashion.

Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8 + T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88 , Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88 L265P -derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88 L265P -containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88 L265P -derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88 L265P mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88 L265P+ NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling.

There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients., Competing Interests: The commercial affiliations Immatics, Biotechnologies GmbH, Biomolecular Mass Spectrometry and Proteomics do not alter our adherence to PLOS ONE policies on sharing data and materials.

Background & Aims: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing., Methods: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands., Results: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued., Conclusions: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma., Lay Summary: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Despite recent advances in immunotherapy of renal cell carcinoma (RCC), peptide vaccination strategies still lack an approach for personalized peptide vaccination that takes intra- and inter-tumoral heterogeneity and biological characteristics into account. In this study, we use an immunoprecipitation and mass spectrometry-based approach supplemented by network analysis of HLA ligands to target this goal. By analyzing HLA-presented peptides for HLA class I and II of 11 malignant and 6 non-malignant kidney tissue samples, more than 2,700 peptides and 1,600 corresponding source proteins were identified. A high overlap with HLA ligands derived from peripheral blood mononuclear cells (PBMCs) was detected most likely due to tumor-infiltrating inflammatory cells and therefore excluded from network analysis. Subsequent biological function analysis of HLA ligands by the GeneMANIA online platform showed enrichment for well established, but also novel, pathways and biological processes involved in carcinogenesis of RCC almost exclusively in malignant tissue samples. By exploring source proteins involved in these overrepresented pathways, we verified various known tumor-associated antigens (TAAs) for RCC (e.g., CA9, EGLN3, IGFBP3, MMP7, PAX2, VEGFA, or EGFR) but could also detect novel TAAs for RCC (e.g., PLOD2, LOX, ENPEP, or TGFBI). Some of these new TAAs had already been shown to elicit a T cell response in cancer patients. Thus, network analysis of HLA ligands provided a new platform for implementing personalized, multipeptide vaccines with potentially synergistic antitumor effects.

Recent studies suggest that multiple myeloma is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell immunotherapy. As standard of care in myeloma includes proteasome inhibitor therapy, it is of great importance to characterize the effects of this treatment on HLA-restricted antigen presentation and implement only robustly presented targets for immunotherapeutic intervention. Here, we present a study that longitudinally and semi-quantitatively maps the effects of the proteasome inhibitor carfilzomib on HLA-restricted antigen presentation. The relative presentation levels of 4780 different HLA ligands were quantified in an in vitro model employing carfilzomib treatment of MM.1S and U266 myeloma cells, which revealed significant modulation of a substantial fraction of the HLA-presented peptidome. Strikingly, we detected selective down-modulation of HLA ligands with aromatic C-terminal anchor amino acids. This particularly manifested as a marked reduction in the presentation of HLA ligands through the HLA allotypes A*23:01 and A*24:02 on MM.1S cells. These findings implicate that carfilzomib mediates a direct, peptide motif-specific inhibitory effect on HLA ligand processing and presentation. As a substantial proportion of HLA allotypes present peptides with aromatic C-termini, our results may have broad implications for the implementation of antigen-specific treatment approaches in patients undergoing carfilzomib treatment.

The myriad of peptides presented at the cell surface by class I and class II major histocompatibility complex (MHC) molecules are referred to as the immunopeptidome and are of great importance for basic and translational science. For basic science, the immunopeptidome is a critical component for understanding the immune system; for translational science, exact knowledge of the immunopeptidome can directly fuel and guide the development of next-generation vaccines and immunotherapies against autoimmunity, infectious diseases, and cancers. In this mini-review, we summarize established isolation techniques as well as emerging mass spectrometry-based platforms (i.e. SWATH-MS) to identify and quantify MHC-associated peptides. We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Direct analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, nonmutant antigenome of multiple myeloma (MM). Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigens on the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD8(+) T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell-based immunotherapy of MM., (© 2015 by The American Society of Hematology.)

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

Effective antigen-specific cancer immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens. Although the current paradigm views mutation-derived neoantigens as the most promising targets, we have recently demonstrated that leukemia-specific T-cell responses associated with survival benefit in CLL patients target a panel of non-mutated tumor-associated antigens.

Identification of physiologically relevant peptide vaccine targets calls for the direct analysis of the entirety of naturally presented human leukocyte antigen (HLA) ligands, termed the HLA ligandome. In this study, we implemented this direct approach using immunoprecipitation and mass spectrometry to define acute myeloid leukemia (AML)-associated peptide vaccine targets. Mapping the HLA class I ligandomes of 15 AML patients and 35 healthy controls, more than 25 000 different naturally presented HLA ligands were identified. Target prioritization based on AML exclusivity and high presentation frequency in the AML cohort identified a panel of 132 LiTAAs (ligandome-derived tumor-associated antigens), and 341 corresponding HLA ligands (LiTAPs (ligandome-derived tumor-associated peptides)) represented subset independently in >20% of AML patients. Functional characterization of LiTAPs by interferon-γ ELISPOT (Enzyme-Linked ImmunoSpot) and intracellular cytokine staining confirmed AML-specific CD8(+) T-cell recognition. Of note, our platform identified HLA ligands representing several established AML-associated antigens (e.g. NPM1, MAGED1, PRTN3, MPO, WT1), but found 80% of them to be also represented in healthy control samples. Mapping of HLA class II ligandomes provided additional CD4(+) T-cell epitopes and potentially synergistic embedded HLA ligands, allowing for complementation of a multipeptide vaccine for the immunotherapy of AML.

The breakthrough development of clinically effective immune checkpoint inhibitors illustrates the potential of T-cell-based immunotherapy to effectively treat malignancies. A remaining challenge is to increase and guide the specificities of anticancer immune responses, e.g., by therapeutic vaccination or by adoptive T-cell transfer. By analyzing the landscape of naturally presented HLA class I and II ligands of primary chronic lymphocytic leukemia (CLL), we delineated a novel category of tumor-associated T-cell antigens based on their exclusive and frequent representation in the HLA ligandome of leukemic cells. These antigens were validated across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. We demonstrate specific immune recognition of these antigens exclusively in CLL patients, with the frequencies of representation in CLL ligandomes correlating with the frequencies of immune recognition by patient T cells. Moreover, retrospective survival analysis revealed survival benefits for patients displaying immune responses to these antigens. These results directly imply these nonmutant self-peptides as pathophysiologically relevant tumor antigens and encourages their implementation for cancer immunotherapy.

To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8(+) T-cell responses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The large-scale identification of MHC class I presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as it is an invaluable tool in identifying potential targets for the immunotherapy of disease. In this chapter we briefly review the existing strategies for the analysis of MHC ligandomes and provide an in-depth protocol for the immunoaffinity purification of MHC class I presented peptides from primary tissues or cells.