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Development of Selective Carbohydrate Immunomodulators Targeting C-type Lectin Receptors on Antigen Presenting Cells

Periodic Reporting for period 2 - IMMUNOSHAPE (Development of Selective Carbohydrate Immunomodulators Targeting C-type Lectin Receptors on Antigen Presenting Cells)

Reporting period: 2017-01-01 to 2018-12-31

Glycoscience, which focuses on the study of the structural and functional roles of carbohydrates in their biological context, has an increasing impact on many areas of our economy but particularly in biotechnology, health, food and energy, key focus areas also of the Horizon 2020 program. Illustrative examples for the importance of glycoscience are the effects of protein glycosylation on efficacy, immunogenicity and circulatory half-life of recombinant therapeutic proteins, the enormous untapped potential of glycan biomarkers in personalized medicine and the therapeutic use of carbohydrate antigens in the development of synthetic vaccines and for the immunotherapy of cancer, allergy and autoimmune disease. The interaction of antigens with Toll like receptors (TLRs) or C-type lectin receptors (CLRs) on antigen presenting cells (APCs) like dendritic cells (DCs) or macrophages is the initiating step in the adaptive immune response. This primary interaction is followed by the uptake, processing and presentation of antigen by the APC. Binding to certain C-type lectin receptors can lead to activation of DC signaling events accompanied by the secretion of cytokines and the expression of co-stimulatory compounds, required for T-cell activation. Depending on the CLR involved and the cytokines secreted, the immune response can be shaped towards macrophage or B-cell activation or towards a pro-inflammatory or allergic response. An early intervention in the immune response as the targeting of specific C-type lectins can be a powerful strategy to shape the immune response towards a therapeutically desired effect and has wide potential applications from cancer immunotherapy to the treatment of allergy and autoimmune disease and in vaccine development.
IMMUNOSHAPE aims at training a new generation of scientists that will be capable of combining state of the art synthesis and screening technology to develop new lead structures for highly selective glycan based multivalent immunotherapeutics for the treatment of cancer, autoimmune diseases and allergy. To this end, we have set up a training program in a unique academic-industrial environment that will educate young researchers in scientific and practical biomedical glycoscience with the final aim to produce new talent and innovation in the field and improve their career perspectives in both academic and non-academic sectors. The unique combination of 10 academic groups with expertise in automated solid-phase carbohydrate synthesis, microarray based highthroughput screening technology, tumour immunology, structural glycobiology, multivalent systems and medicinal chemistry along with 4 industrial partners active in nanomedicine, immunotherapy, medicinal device development and the fabrication of scientific instrumentation will provide a multidisciplinary and multisectorial training to 15 fellows in biomedical glycoscience and its industrial applications.
Research in Immunoshape is organized in 7 work packages that group together tasks of the same development stage in the quest for novel multivalent glycan based immune therapeutics. At first the research is focused on improving our knowledge on the CLR glycan binding specificities. This basic knowledge is important as we are using natural glycans as leads for the development of glycomimetics with increased selectivity and enhanced affinity. At this stage we have completed existing glycan libraries with novel structures from parasite, fungal and bacterial glycomes. At the same time dedicated laboratories developed stable expression systems for a variety of novel human and murine C-type lectins, the molecular receptors that have been targeted with our later constructs. Murine receptors were included in our study as the repertoire of immune receptors can present subtle differences between species which are important to take into account when employing animals as model systems for human immune studies. High-throughput profiling of carbohydrate-protein interactions was carried out on a microarray format, in which hundreds of printed synthetic glycan structures are profiled against various C-type lectins at the same time with only minimal expense of the valuable reagents. Our primary screens gave interesting new lead structures which were employed for the development of novel glycomimetics. The development of glycomimetics targeted towards DC-SIGN, a more thoroughly studied CLR, was started in parallel to the glycan synthesis based on previous structural knowledge on DC-SIGN glycan binding and has received further input from our ongoing glycan screening studies. A platform for the enzymatic on-chip derivatisation of existing glycan libraries with non-natural appendices was developed to rapidly explore the chemical space around privileged binding structures and elements. The platform permits the analysis of on-chip chemical reactions by surface mass spectrometry and the subsequent screening with CLRs by fluorescence. For a better visualization of the often weak carbohydrate-protein interactions on the chip-based assays we have explored both immobilizing glycans or CLRs and incubating with fluorescently labeled CLRs or multivalent glycan display systems like gold nanoparticles, dendrimers and BSA based neoglycoproteins, respectively. To this end, several groups have designed and prepared multivalent display systems with precise control on number and spatial presentation of glycan ligands. Studies have been undertaken for the evaluation of these multivalent glycomimetic presentations as targeting probes or inhibitors in in vitro immune assays.
Immunoshape has pushed the boundaries for research in carbohydrate related immunology in many ways. Only through the joint effort of a group of experienced chemists and biologists specialized in glycoscience we were able to create novel reagent libraries exemplified by our collection of recombinant murine and human C-type lectins and our dedicated arrays of glycan antigens and derived glycomimetics. Furthermore, the close interaction between groups from immunology and chemistry has allowed us to prepare novel multivalent glycan display systems and evaluate them as selective probes for dendritic cell targeting. The project has already delivered new technology for the on-chip synthesis and analysis of glycomimetics, new flexible display systems for presenting glycan antigens with precise ligand control and novel insights into the binding specificities of a number of CLR employed in the screening of glycan libraries. Several high impact publications and an important number of conference presentations are testimony of the high quality and productivity of the project. Most technological innovations developed during the project are under validation, further experimentation will be required before a clear exploitation plan in collaboration with the participating companies can be drawn up. As a first step, the patentability of selected inventions will be assessed and then, possible applications and market opportunities will be evaluated to decide if a commercial exploitation via network companies can be realized or if external partners are required.
Summary of the IMMUNOSHAPE Work Packages