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1.
Proteomics ; 18(12): e1700284, 2018 06.
Article in English | MEDLINE | ID: mdl-29505699

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/metabolism , HLA Antigens/metabolism , Immunotherapy , Neoplasms/metabolism , Peptide Fragments/metabolism , Precision Medicine , Proteogenomics/methods , Antigen Presentation/immunology , Antigens, Neoplasm/analysis , Antigens, Neoplasm/immunology , Decision Making , Epitopes/immunology , Epitopes/metabolism , HLA Antigens/analysis , HLA Antigens/immunology , High-Throughput Nucleotide Sequencing , Humans , Mass Spectrometry/methods , Neoplasms/immunology , Peptide Fragments/analysis , Peptide Fragments/immunology , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/immunology
3.
Oncoimmunology ; 5(5): e1065369, 2016 May.
Article in English | MEDLINE | ID: mdl-27467910

ABSTRACT

The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8(+) T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3(+) tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8(+) TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

4.
Cancer Immunol Immunother ; 62(9): 1485-97, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23817722

ABSTRACT

INTRODUCTION: Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach. METHODS: We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors. RESULTS: Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide. CONCLUSIONS: This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.


Subject(s)
Epitopes, T-Lymphocyte/immunology , HLA Antigens/immunology , Lung Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Antigen Presentation , CD8-Positive T-Lymphocytes/immunology , Cyclin D1/immunology , DNA-Activated Protein Kinase/immunology , Dendritic Cells/immunology , Gene Expression , Humans , Immunohistochemistry , Immunotherapy , Ligands , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Molecular Sequence Data , Nuclear Proteins/immunology , Peptides/immunology
5.
Int J Oncol ; 39(6): 1593-600, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21874229

ABSTRACT

Peritoneal carcinomatosis describes cancer metastasis onto the surface of the peritoneum. It is frequently caused by ovarian and colorectal cancer. Once a tumor has penetrated the peritoneum, cancer cells disseminate into the abdominal cavity. Additionally, surgery can account for the spread of free tumor cells. Their subsequent adhesion to mesothelial cells (HMCs) initiates peritoneal carcinomatosis. Therefore, this study analyzed the effect of simvastatin on tumor cell adherence. HMCs were isolated from human greater omentum. Fluorescence-labeled tumor cells (SKOV-3, OvCar-29, OAW42, FraWü; ovarian/HT29; colorectal) were incubated on confluent mesothelial monolayers with 10 µM simvastatin for 48 h. Adhesion was quantified using a fluorescence reader. Expression of the adhesion molecules VCAM-1, ICAM-1 and ß1 integrin chain under the influence of simvastatin 0.1-100 µM for 24-72 h was analyzed using flow cytometry. Simvastatin significantly reduced the adhesion of all ovarian cancer cells and HT29 to HMCs (P≤0.001). Concomitantly simvastatin decreased the expression of VCAM-1 on HMCs. ICAM-1 and ß1 integrin chain expression on ovarian cancer cells was also clearly reduced. By contrast, the expression of the analyzed adhesion molecules on HT29 cells remained unchanged. Simvastatin clearly inhibits tumor cell adhesion to HMCs. In the case of ovarian cancer cell lines it appears to be mediated by decreased expression of both VCAM-1 on HMCs and the integrin α4ß1 on tumor cells. As an example of adhesion molecule down-regulating drugs, simvastatin may provide a novel therapeutic approach to the prevention of peritoneal carcinomatosis.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Adhesion/drug effects , Integrin beta1/metabolism , Peritoneum/cytology , Peritoneum/metabolism , Simvastatin/pharmacology , Vascular Cell Adhesion Molecule-1/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation/drug effects , HT29 Cells , Humans , Integrin beta1/genetics , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Ligands , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Protein Binding , Vascular Cell Adhesion Molecule-1/genetics
6.
Biotechnol Appl Biochem ; 51(Pt 1): 53-61, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18471094

ABSTRACT

We have previously shown the cyanobacterium Anabaena sp. PCC 7120 to be a suitable host for the production of isotopically labelled recombinant proteins using the nitrate-inducible nir expression system. However, the expression of toxic proteins such as oncoproteins proved to be difficult, as expression levels decreased shortly after induction, while growth continued. To overcome this limitation, we have developed a method of auto-induction of the nir promoter in which cells are grown to high cell density in a bioreactor in the presence of ammonium and nitrate. Since ammonium is the preferred nitrogen source and acts as a repressor of the nir promoter, induction occurs only when the ammonium had been depleted. Using this novel auto-induction method, both oncoproteins E6 and gankyrin were expressed at high levels in a folded conformation and were shown to be biologically active after purification. Furthermore, under similar conditions of growth in auto-inducing medium, the use of (15)N- and (13)C-labelled mineral salts yielded isotopic enrichment of these proteins at levels above 95%, making them suitable for NMR-based structural analysis in a cost-effective manner.


Subject(s)
Anabaena , Automation/methods , Gene Expression Regulation, Bacterial , Isotope Labeling/methods , Oncogene Proteins/genetics , Protein Engineering/methods , Anabaena/cytology , Anabaena/genetics , Anabaena/growth & development , Base Sequence , Carbon Isotopes , Humans , Molecular Sequence Data , Nitrates/chemistry , Nitrates/metabolism , Nitrogen/chemistry , Nitrogen/metabolism , Nitrogen Isotopes , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , Promoter Regions, Genetic , Protein Conformation , Protein Folding , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/metabolism
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