B Peter1; B Peng1; A Ghidini2; A Gunnarsson2; J Liu1;
1 Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden, Sweden; 2 Mechanistic and Structural Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden, Sweden
AbstractThe asialoglycoprotein receptor (ASGPR) is a heterotrimeric transmembrane receptor that is primarily expressed on the surface of hepatocytes. The receptor can be exploited for the targeted delivery of therapeutic oligonucleotides to the liver via its natural ligand N-acetyl-D-galactosamine (GalNAc). Developing novel proprietary ASGPR receptor ligands for liver-targeted delivery is therefore an important part of the ongoing AstraZeneca Oligonucleotide Platform Build. In vitro and structural studies of ASGPR have been limited to expression of the monomeric carbohydrate binding domain (CBD). In these studies, the absence of a membrane and the transmembrane domain prevents analysis of the trimeric form of the receptor in in vitro assays. Not only is the trimer more physiologically-relevant but it is also expected to have a much higher affinity for trivalent ligands targeting the CBD in in vitro assays. To tackle this challenge, we designed an artificial trimeric construct in the absence of a membrane and comprising the soluble CBD of ASGPR fused to the NC1 trimerisation domain of collagen. The recombinant protein was expressed and secreted in HEK293 cells and purified using a novel SEC-affinity approach. Analysis by SEC-MALS and iSCAT confirmed that the purified CBD of ASGPR exists almost exclusively as a trimer. A thermal shift assay and SPR also confirmed that the trimers are substrate-binding competent. The approach used in this work represents a novel way of tackling membrane receptor drug discovery, with the benefits of a native-like soluble oligomer gained whilst avoiding the potential challenges associated with membrane protein purification.