Publications by authors named "Hans-Georg Rammensee"

238 Publications

Expression levels of HLA-DR in acute myeloid leukemia: implications for antigenicity and clinical outcome.

Leuk Lymphoma 2021 Mar 2:1-18. Epub 2021 Mar 2.

Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany.

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 ( = 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 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 and HLA-DR patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DR and HLA-DR patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DR AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DR AML patients.
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http://dx.doi.org/10.1080/10428194.2021.1885659DOI Listing
March 2021

Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity.

Nat Commun 2021 02 19;12(1):1152. Epub 2021 Feb 19.

NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.

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.
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http://dx.doi.org/10.1038/s41467-021-20973-3DOI Listing
February 2021

The Peptide Vaccine of the Future.

Mol Cell Proteomics 2021 Feb 8;20:100022. Epub 2021 Feb 8.

Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), University Hospital Tübingen, Tübingen, Germany; Cluster of Excellence iFIT (EXC2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen, Germany. Electronic address:

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.
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http://dx.doi.org/10.1074/mcp.R120.002309DOI Listing
February 2021

Obituary: Dr Zoltan A. Nagy (1941-2020).

Scand J Immunol 2021 Feb 10:e13027. Epub 2021 Feb 10.

Institute for Cell Biology, Dept. Immunology, University of Tübingen, Tübingen, Germany.

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http://dx.doi.org/10.1111/sji.13027DOI Listing
February 2021

The Impact of Biomaterial Cell Contact on the Immunopeptidome.

Front Bioeng Biotechnol 2020 16;8:571294. Epub 2020 Dec 16.

NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.

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 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.
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http://dx.doi.org/10.3389/fbioe.2020.571294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773052PMC
December 2020

An IgG-based bispecific antibody for improved dual targeting in PSMA-positive cancer.

EMBO Mol Med 2021 Feb 29;13(2):e11902. Epub 2020 Dec 29.

Department of Immunology, Institute for Cell Biology, Eberhard Karls University Tuebingen, German Cancer Consortium (DKTK), Partner Site Tuebingen, Tuebingen, Germany.

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.
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http://dx.doi.org/10.15252/emmm.201911902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863392PMC
February 2021

The peptide vaccine of the future.

Mol Cell Proteomics 2020 Dec 7. Epub 2020 Dec 7.

Cluster of Excellence iFIT (EXC2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Germany

The approach of peptide-based anti-cancer 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.
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http://dx.doi.org/10.1074/mcp.R120.002309DOI Listing
December 2020

Vaccination against RhoC induces long-lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial.

J Immunother Cancer 2020 11;8(2)

Department of Immunology, Institute for Cell Biology, University of Tübingen, Tubingen, Germany

Background: Peptide-based vaccination is a rational option for immunotherapy of prostate cancer. In this first-in-man phase I/II study, we assessed the safety, tolerability and immunological impact of a synthetic long peptide vaccine targeting Ras homolog gene family member C (RhoC) in patients with prostate cancer. RhoC is a small GTPase overexpressed in advanced solid cancers, metastases and cancer stem cells.

Methods: Twenty-two patients who had previously undergone radical prostatectomy received subcutaneous injections of 0.1 mg of a single RhoC-derived 20mer peptide emulsified in Montanide ISA-51 every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. The drug safety and vaccine-specific immune responses were assessed during treatment and thereafter within a 13-month follow-up period. Serum level of prostate-specific antigen was measured up to 26 months postvaccination.

Results: Most patients (18 of 21 evaluable) developed a strong CD4 T cell response against the vaccine, which lasted at least 10 months following the last vaccination. Three promiscuouslypresented HLA-class II epitopes were identified. Vaccine-specific CD4 T cells were polyfunctional and effector memory T cells that stably expressed PD-1 (CD279) and OX-40 (CD134), but not LAG-3 (CD223). One CD8 T cell response was detected in addition. The vaccine was well tolerated and no treatment-related adverse events of grade ≥3 were observed.

Conclusion: Targeting of RhoC induced a potent and long-lasting T cell immunity in the majority of the patients. The study demonstrates an excellent safety and tolerability profile. Vaccination against RhoC could potentially delay or prevent tumor recurrence and metastasis formation.

Trial Registration Number: NCT03199872.
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http://dx.doi.org/10.1136/jitc-2020-001157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662471PMC
November 2020

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome.

J Proteome Res 2021 Jan 3;20(1):289-304. Epub 2020 Nov 3.

Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland.

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.
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http://dx.doi.org/10.1021/acs.jproteome.0c00386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786382PMC
January 2021

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

Cell 2020 Nov 21;183(5):1264-1281.e20. Epub 2020 Oct 21.

Neuroimmunology and MS Research, Neurology Clinic, University Hospital Zurich, University of Zurich, Zurich 8091, Switzerland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2020.09.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707104PMC
November 2020

[Individualized immunotherapy for malignant tumors using peptide vaccines-maybe it does work after all?]

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2020 Nov 9;63(11):1380-1387. Epub 2020 Oct 9.

Interfakultäres Institut für Zellbiologie, Abteilung Immunologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Deutschland.

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).
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http://dx.doi.org/10.1007/s00103-020-03227-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648007PMC
November 2020

SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

Nat Immunol 2021 01 30;22(1):74-85. Epub 2020 Sep 30.

Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany.

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.
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http://dx.doi.org/10.1038/s41590-020-00808-xDOI Listing
January 2021

HLA Evolutionary Divergence as a Prognostic Marker for AML Patients Undergoing Allogeneic Stem Cell Transplantation.

Cancers (Basel) 2020 Jul 8;12(7). Epub 2020 Jul 8.

Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany.

The diversity of human leukocyte antigens (HLAs) enables the presentation of immense repertoires of peptides, including tumor-associated antigens (TAAs). As a surrogate for immunopeptidome diversity, the HLA evolutionary divergence (HED) between individual HLA alleles might directly define the ability to present TAAs, a prerequisite for graft-versus-leukemia effects. We therefore analyzed the impact of HED on survival within a cohort of 171 acute myeloid leukemia (AML) patients after matched donor allogeneic hematopoietic stem cell transplantation (HSCT). Low HED (<25th percentile) of HLA class I (HED) or HLA-DR antigens (HED) was a strong determinant for adverse overall survival after allogeneic HSCT (OS), with a hazard ratio for death of 1.9 (95% CI 1.2-3.2) and 2.1 (95% CI 1.3-3.4), respectively. Defining a cutoff value for the combined HED (HED and HED), the respective 5 year OS was 29.7% and 64.9% in patients with low and high HED ( < 0.001), respectively. Furthermore, the risk of relapse was significantly higher in patients with low HED (hazard ratio (HR) 2.2, 95% CI 1.3-3.6) and event-free survival (EFS) was significantly reduced (5 year EFS 25.7% versus 54.4%, < 0.001). We here introduce HED, a fundamental metric of immunopeptidome diversity, as a novel prognostic factor for AML patients undergoing allogeneic HSCT.
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http://dx.doi.org/10.3390/cancers12071835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408841PMC
July 2020

Towards new horizons: characterization, classification and implications of the tumour antigenic repertoire.

Nat Rev Clin Oncol 2020 10 22;17(10):595-610. Epub 2020 Jun 22.

Department of Oncology, Haematology, Immuno-Oncology and Rheumatology, University Hospital Bonn, Bonn, Germany.

Immune-checkpoint inhibition provides an unmatched level of durable clinical efficacy in various malignancies. Such therapies promote the activation of antigen-specific T cells, although the precise targets of these T cells remain unknown. Exploiting these targets holds great potential to amplify responses to treatment, such as by combining immune-checkpoint inhibition with therapeutic vaccination or other antigen-directed treatments. In this scenario, the pivotal hurdle remains the definition of valid HLA-restricted tumour antigens, which requires several levels of evidence before targets can be established with sufficient confidence. Suitable antigens might include tumour-specific antigens with alternative or wild-type sequences, tumour-associated antigens and cryptic antigens that exceed exome boundaries. Comprehensive antigen classification is required to enable future clinical development and the definition of innovative treatment strategies. Furthermore, clinical development remains challenging with regard to drug manufacturing and regulation, as well as treatment feasibility. Despite these challenges, treatments based on diligently curated antigens combined with a suitable therapeutic platform have the potential to enable optimal antitumour efficacy in patients, either as monotherapies or in combination with other established immunotherapies. In this Review, we summarize the current state-of-the-art approaches for the identification of candidate tumour antigens and provide a structured terminology based on their underlying characteristics.
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http://dx.doi.org/10.1038/s41571-020-0387-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306938PMC
October 2020

Tocilizumab, but not dexamethasone, prevents CRS without affecting antitumor activity of bispecific antibodies.

J Immunother Cancer 2020 05;8(1)

Department of Immunology, University of Tübingen Interfaculty Institute of Cell Biology, Tubingen, Germany

Bispecific antibodies (bsAb) and chimeric antigen receptor (CAR) T cells allow for antibody guided recruitment of T cells against tumors. Both are successfully used for treatment of CD19 expressing leukemias, but may cause cytokine release syndrome (CRS) as a major dose-limiting side effect. For CRS prevention, steroids are recommended prior to bsAb treatment, despite their well-known lymphotoxic activity. The IL-6 receptor antibody tocilizumab is established for treatment of CRS induced by CAR T cells, but was not considered for CRS prevention in bsAb therapy. We here compared the influence of dexamethasone and tocilizumab on bsAb-mediated T cell proliferation and tumor lysis in vitro and in vivo and found that dexamethasone profoundly inhibited T cell proliferation and antitumor activity as induced by two different bsAb, particularly at low effector:target ratios, whereas tocilizumab did not affect efficacy. When we applied tocilizumab early during treatment of three patients with a newly developed PSMAxCD3 bsAb, significant CRS attenuation despite high IL-6 serum levels was observed. Thus, early IL-6 blockade may reduce the undesired sequelae of CRS upon bsAb therapy without affecting therapeutic activity, allowing in turn for safe application of effective doses.
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http://dx.doi.org/10.1136/jitc-2020-000621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264835PMC
May 2020

Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy.

Genome Med 2020 03 30;12(1):32. Epub 2020 Mar 30.

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.

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.
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http://dx.doi.org/10.1186/s13073-020-00731-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106651PMC
March 2020

Mass spectrometry-based identification of a B-cell maturation antigen-derived T-cell epitope for antigen-specific immunotherapy of multiple myeloma.

Blood Cancer J 2020 02 28;10(2):24. Epub 2020 Feb 28.

University Hospital Tübingen, Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Tübingen, Germany.

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA). Additionally, P(BCMA) was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA) induced multifunctional BCMA-specific cells de novo from naïve CD8 T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA) in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)-specific CD8 T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA) using patient-derived P(BCMA)-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.
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http://dx.doi.org/10.1038/s41408-020-0288-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048774PMC
February 2020

Guidance Document: Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Immunopeptidomics Assay for the Identification of HLA Class I Ligands Suitable for Pharmaceutical Therapies.

Mol Cell Proteomics 2020 03 14;19(3):432-443. Epub 2020 Jan 14.

Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. To support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.The immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.
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http://dx.doi.org/10.1074/mcp.C119.001652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050110PMC
March 2020

Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models.

J Exp Med 2020 03;217(3)

Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany.

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.
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http://dx.doi.org/10.1084/jem.20191164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062530PMC
March 2020

A Non-interventional Clinical Trial Assessing Immune Responses After Radiofrequency Ablation of Liver Metastases From Colorectal Cancer.

Front Immunol 2019 19;10:2526. Epub 2019 Nov 19.

Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany.

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 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. 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 ( = 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 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 ( = 9) or without RFA ( = 7). 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 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 ( = 9) as compared to the surgery only mCRC group ( = 7). 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.
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http://dx.doi.org/10.3389/fimmu.2019.02526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877671PMC
November 2020

A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer.

J Immunother Cancer 2019 11 15;7(1):307. Epub 2019 Nov 15.

Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany.

Background: We previously showed that the bacterial lipopeptide PamCys-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 PamCys-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 T1 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 in the granuloma and 20.5 × 10 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.
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http://dx.doi.org/10.1186/s40425-019-0796-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858783PMC
November 2019

Combining alpha radiation-based brachytherapy with immunomodulators promotes complete tumor regression in mice via tumor-specific long-term immune response.

Cancer Immunol Immunother 2019 Dec 22;68(12):1949-1958. Epub 2019 Oct 22.

Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, P.O. Box 39040, 6997801, Tel Aviv, Israel.

Diffusing alpha-emitters radiation therapy (DaRT) is the only known method for treating solid tumors with highly destructive alpha radiation. More importantly, as a monotherapy, DaRT has been shown to induce a systemic antitumor immune response following tumor ablation. Here, immunomodulatory strategies to boost the antitumor immune response induced by DaRT, and the response specificity, were investigated in the colon cancer CT26 mouse model. Local treatment prior to DaRT, with the TLR3 agonist poly I:C, was sufficient to inhibit tumor growth relative to poly I:C or DaRT alone. DaRT used in combination with the TLR9 agonist CpG, or with the TLR1/2 agonist XS15 retarded tumor growth and increased tumor-rejection rates, compared to DaRT alone, curing 41% and 20% of the mice, respectively. DaRT in combination with CpG, the Treg inhibitor cyclophosphamide, and the MDSC inhibitor sildenafil, cured 51% of the animals, compared to only 6% and 0% cure when immunomodulation or DaRT was used alone, respectively. Challenge and Winn assays revealed that these high cure rates involved a specific immunological memory against CT26 antigens. We suggest that DaRT acts in synergy with immunomodulation to induce a specific and systemic antitumor immune response. This strategy may serve as a safe and efficient method not only for tumor ablation, but also for in situ vaccination of cancer patients.
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http://dx.doi.org/10.1007/s00262-019-02418-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877484PMC
December 2019

MHCquant: Automated and Reproducible Data Analysis for Immunopeptidomics.

J Proteome Res 2019 11 22;18(11):3876-3884. Epub 2019 Oct 22.

Biomolecular Interactions , Max Planck Institute for Developmental Biology , Tübingen 72076 , Germany.

Personalized multipeptide vaccines are currently being discussed intensively for tumor immunotherapy. In order to identify epitopes-short, immunogenic peptides-suitable for eliciting a tumor-specific immune response, human leukocyte antigen-presented peptides are isolated by immunoaffinity purification from cancer tissue samples and analyzed by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Here, we present MHCquant, a fully automated, portable computational pipeline able to process LC-MS/MS data automatically and generate annotated, false discovery rate-controlled lists of (neo-)epitopes with associated relative quantification information. We could show that MHCquant achieves higher sensitivity than established methods. While obtaining the highest number of unique peptides, the rate of predicted MHC binders remains still comparable to other tools. Reprocessing of the data from a previously published study resulted in the identification of several neoepitopes not detected by previously applied methods. MHCquant integrates tailor-made pipeline components with existing open-source software into a coherent processing workflow. Container-based virtualization permits execution of this workflow without complex software installation, execution on cluster/cloud infrastructures, and full reproducibility of the results. Integration with the data analysis workbench KNIME enables easy mining of large-scale immunopeptidomics data sets. MHCquant is available as open-source software along with accompanying documentation on our website at https://www.openms.de/mhcquant/ .
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http://dx.doi.org/10.1021/acs.jproteome.9b00313DOI Listing
November 2019

Targeting tumor-resident mast cells for effective anti-melanoma immune responses.

JCI Insight 2019 10 3;4(19). Epub 2019 Oct 3.

Department of Dermatology and Allergy, School of Medicine, Technical University of Munich, Munich, Germany.

Immune checkpoint blockade has revolutionized cancer treatment. Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LPS signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LPS-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival. These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.
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http://dx.doi.org/10.1172/jci.insight.125057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795496PMC
October 2019

Human CCR5high effector memory cells perform CNS parenchymal immune surveillance via GZMK-mediated transendothelial diapedesis.

Brain 2019 11;142(11):3411-3427

Department of Neurology with Institute of Translational Neurology, University Hospital Münster, University of Münster, Münster, Germany.

Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity.
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http://dx.doi.org/10.1093/brain/awz301DOI Listing
November 2019

PD-1 and LAG-3 Dominate Checkpoint Receptor-Mediated T-cell Inhibition in Renal Cell Carcinoma.

Cancer Immunol Res 2019 Nov 4;7(11):1891-1899. Epub 2019 Sep 4.

Department of Immunology, University of Tübingen, Tübingen, Germany.

Drugs targeting the programmed cell death protein 1 (PD-1) pathway are approved as therapies for an increasing number of cancer entities, including renal cell carcinoma. Despite a significant increase in overall survival, most treated patients do not show durable clinical responses. A combination of checkpoint inhibitors could provide a promising improvement. The aim of the study was to determine the most promising checkpoint blockade combination for renal cell carcinoma patients. Tumor-infiltrating lymphocytes (TIL) and autologous peripheral blood mononuclear cells (PBMC) were isolated from patients undergoing surgery for primary tumors. Cells were stained for multicolor flow cytometry to determine the (co)expression of five inhibitory receptors (iR), PD-1, LAG-3, Tim-3, BTLA, and CTLA-4, on T-cell populations. The function of these TILs was assessed by intracellular cytokine staining after stimulation in the presence or absence of PD-1 ± LAG-3 or Tim-3-specific antibodies. Although the percentage of iR T cells was low in PBMCs, both CD4 and CD8 T cells showed increased frequencies of PD-1, LAG-3, and Tim-3 cells on TILs. The most frequent iR combination was PD-1 and LAG-3 on both CD4 and CD8 TILs. Blockade of PD-1 resulted in significant LAG-3, but not Tim-3, upregulation. The dual blockade of PD-1 and LAG-3, but not PD-1 and Tim-3, led to increased IFNγ release upon stimulation. Together, these data suggest that dual blockade of PD-1 and LAG-3 is a promising checkpoint blockade combination for renal cell carcinoma.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0146DOI Listing
November 2019

High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules.

Sci Immunol 2019 07;4(37)

Immatics Biotechnologies GmbH, Tübingen, Germany.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8 T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.
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http://dx.doi.org/10.1126/sciimmunol.aav0860DOI Listing
July 2019

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Mol Cell Proteomics 2019 06;18(6):1255-1268

From the ‡Department of Biology, Technion, Israel Institute of Technology, Haifa 32000, Israel;

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.
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http://dx.doi.org/10.1074/mcp.RA119.001524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553928PMC
June 2019