Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies.

Complex cellular targets such as G protein-coupled receptors (GPCRs), ion channels, and other multi-transmembrane proteins represent a significant challenge for therapeutic antibody discovery, primarily because of poor stability of the target protein upon extraction from cell membranes. To assess whether a limited set of membrane-bound antigen formats could be exploited to identify functional antibodies directed against such targets, we selected a GPCR of therapeutic relevance (CCR1) and identified target binders using an in vitro yeast-based antibody discovery platform (AdimabTM) to expedite hit identification. Initially, we compared two different biotinylated antigen formats overexpressing human CCR1 in a 'scouting' approach using a subset of the antibody library. Binders were isolated using streptavidin-coated beads, expressed as yeast supernatants, and screened using a high-throughput binding assay and flow cytometry on appropriate cell lines. The most suitable antigen was then selected to isolate target binders using the full library diversity. This approach identified a combined total of 183 mAbs with diverse heavy chain sequences. A subset of clones exhibited high potencies in primary cell chemotaxis assays, with IC50 values in the low nM/high pM range. To assess the feasibility of any further affinity enhancement, full-length hCCR1 protein was purified, complementary-determining region diversified libraries were constructed from a high and lower affinity mAb, and improved binders were isolated by fluorescence-activated cell sorting selections. A significant affinity enhancement was observed for the lower affinity parental mAb, but not the high affinity mAb. These data exemplify a methodology to generate potent human mAbs for challenging targets rapidly using whole cells as antigen and define a route to the identification of affinity-matured variants if required.

A Microphysiological System for Studying Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they in vivo or in vitro, do not fully represent key aspects of the human disease state. We have developed a human in vitro co-culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three-dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH-like phenotype. The co-culture microtissues within the MPS display a NASH-like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro-inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild-type and patatin-like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro-inflammatory milieu. Conclusion: The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co-culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.

Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease.

AIM: To develop a human in vitro model of non-alcoholic fatty liver disease (NAFLD), utilising primary hepatocytes cultured in a three-dimensional (3D) perfused platform. METHODS: Fat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids (FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds. RESULTS: Hepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factor-binding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantly reduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known anti-steatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium. CONCLUSION: The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

Copper prevents amyloid-ß(1-42) from forming amyloid fibrils under near-physiological conditions in vitro.

The aggregation and deposition of amyloid-ß((1-42) )(Aß(42)) in the brain is implicated in the aetiology of Alzheimer's disease (AD). While the mechanism underlying its deposition in vivo is unknown its precipitation in vitro is influenced by metal ions. For example, Aß(42) is known to bind copper, Cu(II), in vitro and binding results in aggregation of the peptide. The biophysical properties of Cu(II)-Aß(42) aggregates are of significant importance to their putative involvement in the amyloid cascade hypothesis of AD and are currently the subject of strong debate. In particular the question has been raised if sub- and super-stoichiometric concentrations of Cu(II) act in opposing ways in respectively accelerating and preventing amyloid fibril formation by Aß(42). Herein we have used fluorimetry and transmission electron microscopy to provide unequivocal evidence that under near-physiological conditions both sub- and super-stoichiometric concentrations of Cu(II) prevented the assembly of Aß(42) into ThT-positive ß-sheet rich amyloid fibrils.