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).