Characterization of the HLA ligandome and the exome of Hepato- and Cholangiocellular carinoma with a view to patient-individual peptide vaccine

Bis heute werden Peptide zur Impfung von Krebs in zahlreichen Studien mit viel versprechenden Ergebnissen angewandt, jedoch mit klinischer Limitation. Es wurden Peptid-Cocktails aus HLA-Liganden verwendet, die aus verschiedenen Tumor-assoziierten Antigenen (TAAs) und verschiedenen HLA-Allotypen stammten um so den Tumorescape-Mechanismus durch dessen Verlust der einzelnen Antigene oder HLA-Allele zu verhindern. Der Einsatz von hoch tumorspezifischen Peptiden würde das Risiko von Autoimmunreaktionen minimieren, da diese Peptide Mutationen beinhalten, die im Tumor aber nicht im Normalgewebe zu finden sind. Das Vorhandensein solcher TSAs (tumor specific antigens) sollte mit dieser Arbeit bestätigt werden. Zur Detektion solcher hochspezifischen Peptide kombinieren wir die Methode zur Charakterisierung des HLA-Ligandoms mit der Methode des Next-Generation Sequencing. Die Schnittmenge beider Methoden sollte solche TSAs enthalten. Die Mutationen, die mit NGS im Exom gefunden wurden, führten mit Hilfe der SYFPEITHI-Epitopvorhersage zur Generierung einer patientenindividuellen Datenbank, die diese Mutationen in Form von AS-Sequenzen enthält. Solche mutierten Liganden und deren Zusammenstellung in einer Datenbank könnten in Zukunft zur personalisierten Behandlung des autologen Tumors dienen. Den Fokus auf Leberzellkrebs, bewerteten wir parallel eine Exomsequenzierung und eine HLA-Ligandomanalyse mit malignem und angrenzendem gutartigem Gewebe von 6 Patienten. Ingesamt konnten wir auf Exomebene im Durchschnitt 582 Sequenzveränderungen detektieren, die jedoch mit den insgesamt 2213 gefundendenen Peptiden nicht bestätigt werden konnten bzw. konnten nicht durch die Verwendung von Einschlusslisten aus den Epitopvorhersagen bestätigt werden. Allerdings war es uns dennoch möglich bereits in Impfstudien verwendete Peptide zu bestätigen (MMP7, APOB, VIM etc.).Till today peptide vaccines for the treatment of cancer have been applied in numerous studies with promising results but still limited clinical success. Peptide cocktails made up of HLA ligands derived from different tumor-associated antigens (TAAs) and presented by different HLA allotypes prevent tumor escape by loss of individual antigens or HLA alleles. The use of highly tumour-specific peptides would minimize the risk of autoimmune reactions, with mutations present in tumor but not normal tissue representing utmost tumor-specificity. The presence of such TSAs (tumor-specific antigens) should be confirmed with this work. For the detection of such highly specific peptides (TSAs), we combine the method of characterisation of the HLA ligandome with the method of next generation sequencing (NGS). The intersection of the results of both methods should contain such TSAs. We employed in silico approaches in order to predict mutated HLA ligands from patient-individual mutations. For this purpose, for each patient an individual database of the mutated proteome had to be created. Such mutated HLA ligands in a database might be used to compose a personalised vaccine for treatment of the autologous tumour in the future. Focusing on hepatocellular cancer, we performed exome sequencing and HLA ligandome analysis in parallel with malignant as well as adjacent benign tissue from 6 patients. Overall, we were able to detect an average of 582 sequence changes in the exome, but with the 2213 peptides we have found in in total on the HLA-ligandome, these changes could not be confirmed. But beside this we could confirm peptides, already in clinical use as peptide vaccines (MMP7, POB, VIM etc.)

Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient

Background & AimsWe 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.MethodsTumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands.ResultsAlthough 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.ConclusionsThis remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma.Lay SummaryMetastatic 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