Research

Our long-term vision is to routinely produce recombinant proteins as single defined glycoforms without the need for genetic manipulation. This will functionally be the equivalent for glycans as site-directed mutagenesis. We will showcase this by the production of therapeutically relevant proteins with homogenous pre-defined N-linked glycoforms. Our approach is a radically new approach to the manipulation of cell function; we will use the delivery of computationally defined mixtures of enzyme-specific inhibitors to tune the activity of glycosyltransferases and thereby the glycans displayed on secreted proteins such as antibodies, hormones and other therapeutic modalities. The untemplated nature of glycan synthesis leads to inherent heterogeneity in these glycans. This variation can compromise the behaviour of these complex molecules which are increasingly important in modern disease treatments. The complexity of glycan biosynthesis is staggering; in mammals there are up to 200 different glycosyltransferases (GTs) that compete for substrates within the Golgi, the organelle in which most glycans are synthesized. Generation of a given glycan structure depends on the activities and levels of a suite of glycosylation enzymes in each of the Golgi’s sub-compartments (cisternae). In order to manipulate glycans, we will build an understanding of how the arrangement of key biosynthetic enzymes, their activities and their locations specify glycan structures. Our approach, termed Inhibitor-Mediated Programming of Glycoforms (IMProGlyco) will provide an effective strategy to manipulate these enzymes and thereby deliver defined mixtures of glycosylated proteins. This strategy will be adaptable and expandable into other cell types and organisms and thereby enable scalable production of highly specified recombinant biomolecules. Supply of these will enable both production of high performance biotherapeutics and modulation of cell function together with fundamental studies of the consequence of variation in glycosylation patterns in laboratory studies.

Oral health maintenance relies significantly on the delicate balance within the oral microbiome. The probiotic Streptococcus dentisani and Rothia species are integral members of this microbial community, playing pivotal roles in oral ecology and health. Both have been shown to counteract the cariogenic pathogen Streptococcus mutans, thereby contributing to the maintenance of oral homeostasis. S. mutans produces a lipopeptide mutanobactin for which a broad-spectrum growth inhibitory effect on members of the oral biofilm community has been observed. This study delves into the potential inhibitory effects of lipopeptide mutanobactin on specifically health-associated species focusing on S. dentisani, R. aeria, R. denticariosa and R. mucilaginosa. Through a series of planktonic and biofilm growth assays, co-culture systems and multispecies biofilm assays, we elucidate the interaction dynamics between S. mutans and S. dentsani/ Rothia spp. and the role of mutanobactin D in this process. Our findings underscore the nuanced relationship between microbial agents and oral health, offering insights into the development of novel strategies for oral microbiome modulation and disease prevention.

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.