Research

Success rates in treatment of Osteosarcoma (OS), an aggressive cancer with many fatalities in affected children and adolescents, have not improved over the last 40 years. In view of the sizable number of OS-associated mutations and opportunities provided by innovation-driven personalized T-cell-based biomedicine with ever-increasing curative potential, such stalemate is no longer acceptable. As a therapeutic anchor point, OS-related tumor antigens (TAs; both tumor-associated and tumor-specific neoantigens) are displayed on tumors via MHC molecules as short peptides for surveillance by TA-specific tumor-infiltrating CD4+ helper and CD8+ cytotoxic T cells (TILs, CTLs). If equipped and unless undercut by tumor immune evasion mechanisms with adequate TCRs, antigen-scanning CTLs are poised to kill targets in the presence of even a single antigenic peptide/MHC complex (pMHC). Considering the complexities characterizing interactions between OS and the T-cell compartment yet also the limits on TCR-reactivity set by negative thymic selection, we predict that achieving game-changing breakthroughs mandates a highly concerted multidisciplinary approach. To this end our team will combine its considerable expertise in cancer biology, T-cell and molecular immunology as well as in biotechnology and systems biology. More specifically, we seek to identify personal OS-associated antigens and their matching TA-specific TCRs as well as T-cell and OS-intrinsic regulatory mechanisms underlying immune evasion to ultimately engineer autologous CTLs with enhanced tumor clearing capacity and find entry points for OS-preconditioning in immunotherapy. For this we will express recombinant tumor-enriched “orphan” TCRs isolated from TILs of OS patients to screen yeast-displayed peptide/MHC (pMHC) libraries with vast epitope coverage for nominal TAs. As a complementary approach we will first classify T-cell epitopes based on their relevance for tumor lysis and then determine cognate TCR matches. Following functional validation of TCR-epitope matches in vitro, in tumor sections and in vivo, we will combine computational, biophysical and protein engineering approaches to derive functionally enhanced patient-specific TCRs. In parallel, to inform the design of TME-resilient T-cells and drug-based OS-preconditioning for effective immunotherapies, we seek to delineate through CRISPR-Cas9- and DNA-barcoding-based screening T-cell- and OS-intrinsic mechanisms underlying immune evasion. Will focus on OS-related strategies to interfere with surface expression and TCR-accessibility of MHC class I. We further intend to identify as of yet unknown T-cell- and OS-intrinsic pathways undermining CTL-antigen sensitivity and effector function for improved therapeutic intervention. We expect to lay the foundation and streamline curative OS treatment in Austria and beyond and to pioneer effective and low-toxicity targeting of other solid tumor entities.

Science communication is a rapidly developing field and of great importance, especially for young researchers. In this project, scientists (Postdoc Level) will be trained in different forms of science communcation. The coaching involves workshops coaching sessions in small groups. Visibility will be enhanced by featuring the researchers on BOKU social media channels. In addition, a podcast will be launched to highlight the work of young, inspiring researchers that serve as role models for future scientific generations.

Wider research context Of the five human heme peroxidases, evolved to perform widely divergent functions, thyroid peroxidase (TPO) is the least well studied. The multidomain membrane protein catalyses the biosynthesis of thyroid hormones, which are essential for metabolism, growth and development of the human body. With the use of hydrogen peroxide TPO catalyses the iodination and coupling of tyrosine residues on the surface of thyroglobulin (TG) in the thyroid gland. However, there are open questions about substrate selectivity, biochemical characteristics, the coupling mechanism and the roles of the individual domains. Critically, TPO is at the core of two autoimmune thyroid diseases that combined afflict nearly 150 million people but the only two clinically approved TPO inhibitors are not specific. Objectives This project will elucidate the biochemical and structural characteristics of TPO. The main goals are (I) understanding the kinetics and substrate specificity of TPO, (II) provide structural data of TPO alone and of relevant ligated states and (III) clarify the mode of action of TPO inhibitors. Approach In preliminary work an expression and purification protocol for truncated TPO variants was established. Importantly, it was found that it is possible to reconstitute and link the heme cofactor even after purification, yielding highly pure and enzymatically active recombinant TPO. This allows the first detailed spectroscopic, thermodynamic and structural study of the enzyme. This will include an analysis of the reaction kinetics of TPO using a range of methods, including stopped-flow UV-vis spectroscopy for pre-steady kinetics, analysis of the interaction with small molecules (i.e. inhibitors, ligands) and TG with thermodynamic and mass spectroscopic assays. Finally, this project aims to solve the X-ray crystal structure of TPO alone and in complex with biologically relevant substrates, ligands and inhibitors. Level of originality TPO is crucial in thyroid hormone biosynthesis. However, to date the available biochemical and structural data is scarce and does not allow (I) a clear rationalization of the structure-function relationship of the active site architecture and TPO reactivity, (II) the role of the additional TPO domains and (III) the design of new more specific inhibitors. This project aims to provide a complete enzymatic and structural characterization of TPO to address these issues. Primary researchers involved Vera Pfanzagl graduated from the international PhD program BioToP at BOKU in 2019. She worked primarily on structure-function relationships of heme enzymes and during her postdoc focused on human heme peroxidases. Within this Fellowship she aims to obtain her habilitation to advance her academic career. She will be supported and coached by Kristina Djinovic-Carugo, a highly recognized expert in structural biology and head of EMBLE Grenoble and Chris Oostenbrink (professor at BOKU), an expert in molecular modelling and simulation.