Multi-peptide-based vaccines for personalized cancer therapy : Analytical fundamentals translated into clinical applications

Genomics (Oligonukleotid Mikroarray-basierte Expressionsanalyse) und Analyse des MHC Klasse I-Ligandoms (Massenspektrometrie) wurden zur Charakterisierung von primären Nierenzellkarzinomen kombiniert, um Impfstoffe für die Immuntherapie bei Tumorerkrankungen entwickeln zu können. Es konnte gezeigt werden, dass das Verfahren schnell genug ist, um nach chirurgischer Entfernung des Tumors in angemessener Zeit eine Impfung des Patienten zu ermöglichen. An sieben Beispielen wurde dargestellt, dass es möglich ist, aus dem entnommenen Tumor die Informationen zu erhalten, welche für das Design eines Peptid-basierten Multi-Epitop-Impfstoffes benötigt werden. Der Impfstoff ist auf den einzelnen Patienten zugeschnitten und berücksichtigt dessen individuelle Target-Moleküle sowie alle seine exprimierten HLA-Allele. In-vitro T-Zell-Analysen, welche exemplarisch für zwei der neu entdeckten Liganden durchgeführt wurden, bestätigten die Anwendbarkeit des neuen Verfahrens. Es konnte gezeigt werden, dass es sich bei den Liganden erstens um T-Zell-Epitope handelt und zweitens, dass T-Zellen, welche diese Epitope erkennen, Tumorzellen auslöschen können und gleichzeitig gesunde Kontrollzellen verschonen. Als wesentlicher Bestandteil des gesamten Verfahrens wurde eine auf Oligonukleotid Mikroarrays basierende Expressionsdatenbank erstellt, welche es erlaubt, die Expression eines beliebigen sich auf dem Array befindlichen Genes (insgesamt 22.000 Gene) in zwanzig verschiedenen gesunden Geweben und Organen abzurufen. Eine klinische Studie zur Erprobung des Verfahrens wurde begonnen und die ersten vier Patienten bereits rekrutiert und analysiert. Der zweite Teil der Dissertation beschreibt Ergebnisse auf dem Gebiet der Antigenprozessierung. Zum einen wurde eine neue Aminopeptidase, die sich im Lumen des Endoplasmatischen Retikulums befindet und in das Trimming von Epitopen involviert ist, untersucht. Diese wurde inzwischen als ERAP1 identifiziert. Weiter konnte gezeigt werden, dass Tripeptidylpeptidase eine wesentliche Rolle bei der Generierung eines MHC Klasse I-Epitopes spielt.Genomics (oligonucleotide microarray based expression profiling) and MHC class I-ligandome analysis (mass spectrometry) were combined to analyse tumors of renal cell carcinoma patients. This can be performed in an appropriate timeframe after surgical intervention to allow rapid subsequent vaccination. Thus, it was demonstrated in seven cases that it is possible to extract information from a single excised tumor specimen that leads to an optimized design of a multi-epitope, peptide-based vaccine directed against the tumor of an individual patient and considering all expressed HLA-alleles. New tumor associated antigens together with MHC class I-ligands encoded by them can be identified using this approach. In vitro T cell analyses carried out for two of the newly discovered ligands have proven that, first, they were T cell epitopes, and, second, T cells specific for these epitopes mediated lysis of tumor cells but not control cells. As part of the complete approach, a database based on oligonucleotide microarrays was built covering 22.000 gene expression values of twenty one human healthy tissues and organs. Finally, a clinical study using this approach was started and four RCC patients were already analyzed and three patients vaccinated. The results of this thesis presented in part two provided new insights into the antigen processing mechanisms. First, there was evidence for a novel luminal endoplasmic reticulum aminopeptidase involved in epitope trimming events, which in the meantime has been identified as being ERAP1. Second, it could be shown that tripeptidyl peptidase plays an essential role in the generation of an MHC class I epitope

Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02+ glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8+ T cells in vitro. In vivo, glioblastoma-specific CD8+ T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastom

Moderate degradation does not preclude microarray analysis of small amounts of RNA

Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples and that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully

Antigenic expression and spontaneous immune responses support the use of a selected peptide set from the IMA950 glioblastoma vaccine for immunotherapy of grade II and III glioma.

Gliomas are lethal brain tumors that resist standard therapeutic approaches. Immunotherapy is a promising alternative strategy mostly developed in the context of glioblastoma. However, there is a need for implementing immunotherapy for grade II/III gliomas, as these are the most common CNS tumors in young adults with a high propensity for recurrence, making them lethal despite current treatments. We recently identified HLA-A2-restricted tumor-associated antigens by peptide elution from glioblastoma and formulated a multipeptide vaccine (IMA950) evaluated in phase I/II clinical trials with promising results. Here, we investigated expression of the IMA950 antigens in patients with grade II/III astrocytoma, oligodendroglioma or ependymoma, at the mRNA, protein and peptide levels. We report that the BCAN, CSPG4, IGF2BP3, PTPRZ1 and TNC proteins are significantly over-expressed at the mRNA (n = 159) and protein (n = 36) levels in grade II/III glioma patients as compared to non-tumor samples (IGF2BP3 being absent from oligodendroglioma). Most importantly, we detected spontaneous antigen-specific T cell responses to one or more of the IMA950 antigens in 100% and 71% of grade II and grade III patients, respectively (27 patients tested). These patients displayed T cell responses of better quality (higher frequency, broader epitope targeting) than patients with glioblastoma. Detection of spontaneous T cell responses to the IMA950 antigens shows that these antigens are relevant for tumor targeting, which will be best achieved by combination with CD4 epitopes such as the IDH1R132H peptide. Altogether, we provide the rationale for using a selective set of IMA950 peptides for vaccination of patients with grade II/III glioma