Technology Expertise Network and Core Facilities Tübingen
© Department of Immunology / University Tübingen
In Tübingen, a GMP unit consisting of 6 clean air rooms for the manufacture of peptides is now available. The Core Facility “Peptide Vaccines” was specifically designed for rapid, small-scale GMP synthesis for personalized peptide vaccination and serve DKTK members as a Core Facility for the design and synthesis of mainly mutated or overexpressed tumor specific peptides identified via NGS and peptidomics. Tübinger peptide vaccines are already in use. Examples are the DKTK studies “iVacALL” or “NOA-16”. Within the EU project GAPVAC cancer patients are immunized with peptides from our Core Facility.
The Core Facility for the manufacturing of peptide vaccines consists of two areas
Area 1: “Wirkstoffpeptidlabor“, University of Tübingen, is located Auf der Morgenstelle 15, 72076 Tübingen, within the campus of the University. Here, peptides are manufactured as Active Pharmaceutical Ingredients (API) or Drug Substances (DS) under a GMP certificate issued by the local authorities, the Regierungspräsidium Tübingen.
Area 2: The GMP Centre (GMPZ) is located in the Center for Clinical Transfusion Medicine at Otfried-Müller-Str. 4/1, 72076 Tübingen, within the campus of the University Clinic. Here, peptide vaccines ready for use in patients are manufactured as Drug Products (DP) under a manufacturing authorization issued by the local authorities, the Regierungspräsidium Tübingen. In both areas, peptides and vaccines are produced by the personnel of “Wirkstoffpeptidlabor” under the guidance of Prof. Dr. Stefan Stevanović, Head of Quality Control, who is responsible for the release of Drug Substances. Dr. Heinz Rotering, Qualified Person, is responsible for the release of Drug Products.
HLA ligandome core facility at the DKTK partner site Tübingen, © Department of Immunology
HLA ligandome analysis for the identification of tumor associated peptides (TUMAPs).
Essentially all human cells express surface molecules (HLA) that perform a kind of “showcase function”. Samples of peptides, representing most cellular proteins, are presented by HLA molecules. This allows T cells to sense the integrity of all cells. Alterations, for example after viral infections or tumor-specific mutations can thus be sensed by T cells. Each individual tumor express its own set of tumor antigens (overexpressed, mutated, derived from tumor viruses, fusion proteins, etc.) some of which are essential for tumor growth, others being passenger alterations of “normal self”. All categories of tumor antigens can give rise to immunogenic peptides recognizable by T cells that can kill the tumor cells.
Immunoprecipitation of HLA molecules is followed by peptide extraction and identification using capillary HPLC and mass spectrometry. Further data analysis is used to identify tumor specific peptides. © Department of Immunology
The HLA ligandomics Core Facility in Tübingen serves DKTK members as a core facility for the analysis and characterization of HLA presented peptides enabling the identification and informed selection of T cell antigens for immunotherapeutic applications. Our longstanding expertise in the field of HLA ligandome analysis led to the development of standardized MHC peptide extraction protocols as well as data processing and quality control pipelines which allow for efficient and reproducible analysis of HLA presented immunopeptidomes (Fig. 1).
The core facility comprises two Orbitrap LC-MS/MS systems ideally suited for the fast and sensitive analysis of HLA derived peptidomes. In addition to a robust LTQ Orbitrap XL system - our laboratory is equipped with the latest Orbitrap Fusion LumosTM Tribrid mass spectrometer (Fig. 2) providing fast and sensitive analysis of HLA ligandomes from minute amounts of starting material. In addition to our flexible data processing pipeline, an in-house database comprising over 100,000 different HLA ligands from different tumor entities and a range of normal tissues may be interrogated for the confident identification of tumor antigens.
TUMAPs can be synthesized under GMP conditions individually for each patient and provided for vaccination studies - “from bench to bedside”.
Technology Expertise Network: Immune Monitoring
The Immunomonitoring group is located at the Department of Immunology of the University of Tübingen. Our long-term focus is to investigate adaptive immune responses in cancer patients. Subsets of T cells are obtained from the blood or tumor tissues of patients and studied at phenotypical and functional levels. We furthermore investigate various other immune cell subsets, which interact with human tumors. Our specific expertise is the assessment of tumor antigen-specific T cells responses, also during immunotherapy, e.g. multipeptide-based personalized vaccination.
We are a founding member and co-organiser of the Cancer Immunotherapy Immunoguiding Program (CIP/CIMT) which was initiated in 2005. The aim of this international working group is to improve the comparability and sensitivity of established techniques applied for immunomonitoring, to promote the development of complementary tools, and to contribute to progress in the field.
Key methods and technologies
- Multiparameter flow cytometry- Elispot
- Bead-based cytokine measurement
- Assessment of antigen-specific cytotoxicity (VITAL, XCelligence, …)
- T cell priming, culture (also TILs), sorting and cloning
- In general, assessment of antigen-specific CD4 and CD8 T cells
Main monitoring activities (in cooperation with various national and international cooperation partners):
- Characterization of T-cell epitopes derived from newly-identified tumor associated antigens
- Effect of standard therapies on the immune system of cancer patients
- Immune monitoring of peptide-based vaccination studies
- Development of new adjuvants
- Assay development: Establish, optimize, standardize and validate immunoassays