Targeting hepatocytes via the asialoglycoprotein-receptor

The asialoglycoprotein-receptor (ASGP-R) is a C-type lectin predominantly expressed on the sinusoidal surface of mammalian hepatocytes and is responsible for the blood-clearance of desialylated glycoproteins by receptormediated endocytosis. The human receptor consists of two homologous subunits, H1 and H2, whose carbohydrate recognition domains (CRDs) specifically bind non-reducing galactose- and N-acetylgalactosamine-residues. The presence of this specific receptor in high numbers on human hepatocytes attracts scientists allover the world to evaluate its potential as a mediator for the targeting of therapeutic agents and foreign genes to the liver. Although the ASGP-receptor has been intensively investigated, numerous questions remain unanswered; e.g., what are the specific functions of the receptor subunits H1 and H2? What is the mechanism leading to an increased rate of endocytosis upon ligand binding? Is there a difference in specificity of ASGP-receptors on hepatocytes, peritoneal blood macrophages and Kupffercells? Can monovalent high affinity ligands be designed which specifically bind to the hepatic receptor. Can antibodies or antibody fragments be developed as specific drug carriers to the liver? Thesis aims The aims aspired in the scope of this thesis, were the implementation of new tools for the further elucidation of some of the questions above and the development of new targeting moieties to deliver drugs specifically to the liver via the ASGP-R. • In order to generate glycomimetics with improved affinity towards the ASGPR, the availability of suitable assay systems is crucial. To date the only competitive in vitro binding assays for ASGP-R ligands described in literature use radioactive-labeled ligands. Within the scope of this thesis a new cell-free competitive binding assay was developed to confirm the basic approach of new rationally designed ligands and to compare their affinities to the receptor with known galactose-derived compounds. In this assay, the purified human H1-CRD of the ASGP-R expressed in E.coli was coated onto 96-well plates and the competition in binding of galactose-derivates with a biotin-labeled polyacrylamide-carrier bearing N-acetyl-galactosamineresidues to the H1-CRD was measured. The features of the assay were characterized and the IC50 values of galactose-derivates evaluated. In the same assay the binding activities of cysteine mutants of the H1-CRD were tested. • On cellular level, methods for visualization of binding and internalization of synthetic drug carriers and antibodies raised against the receptor and the study of their receptor specificity (hepatic ASGP receptor versus galactosespecific receptors on the surfaces of other cells) were established. Although there is much interest in the use of the ASGP-R for hepatotropic drug delivery, no attempt has been made to address the receptor using an antibody instead of the usual sugar-based delivery systems. However, as carbohydrateprotein interactions are exceptionally weak, higher affinity glycomimetics or antibodies would be of great advantage for targeted therapy. • For proof of principle drug targeting into hepatocytes or as a diagnostic tool for liver diseases in Immunohistochemistry (IHC), The adaptation of high affinity murine anti-human H1-CRD IgG antibodies, previously developed in our institute, to a single chain format (scFv) was attempted. • In order to develop a biopharmaceutical drug, phage display technology was used to select a human scFv antibody with high affinity to the H1-CRD of the ASGP-receptor that could be applied as carrier in the form of an immuno-conjugate for the hepatotropic targeting of drugs and genes (e.g.siRNA).