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.

Purified IgG from patients with obstetric but not IgG from non-obstetric antiphospholipid syndrome inhibit trophoblast invasion.

PROBLEM: Some patients with antiphospholipid syndrome (APS) suffer pregnancy morbidity (PM) but not vascular thrombosis (VT), whilst others suffer VT only. Therefore, we compared the effects of IgG from VT+/PM- and VT-/PM+ subjects on human first-trimester trophoblast (HTR8) cells. METHOD OF STUDY: HTR-8 cells were incubated with APS VT+/PM-, APS VT-/PM+ or healthy control (HC) IgG. We measured trophoblast invasion by cell invasion assay; mRNA expression of TLR4 and adaptor proteins; phosphorylation of p38 MAPK, NFκB and ERK; and expression of interleukin (IL)-8 and IL-6. RESULTS: VT-/PM+ IgG, but not VT+/PM- IgG significantly reduced HTR-8 invasion. The effects on invasion were blocked by TLR-4 inhibition. Neither VT+/PM- nor VT-/PM+ IgG altered MyD88 mRNA expression, phosphorylation of signalling molecules or cytokine expression. CONCLUSIONS: VT-/PM+ IgG exert functionally relevant effects on human trophoblast cells but VT+/PM- IgG do not.

Immunomodulatory and antioxidant function of albumin stabilises the endothelium and improves survival in a rodent model of chronic liver failure.

BACKGROUND & AIMS: Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. METHODS: In vivo: systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague-Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro: human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. RESULTS: Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. CONCLUSIONS: These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction.

Changes in regulation of human monocyte proteins in response to IgG from patients with antiphospholipid syndrome.

The effects of immunoglobulin G (IgG) from patients with the antiphospholipid syndrome (APS) upon monocyte activation have not been fully characterized. We carried out a comprehensive proteomic analysis of human monocytes treated with IgG from patients with different manifestations of the APS. Using 2-dimensional differential gel electrophoresis (2D DiGE), 4 of the most significantly regulated proteins (vimentin [VIM], zinc finger CCH domain-containing protein 18, CAP Gly domain-containing linker protein 2, and myeloperoxidase) were differentially regulated in monocytes treated with thrombotic or obstetric APS IgG, compared with healthy control (HC) IgG. These findings were confirmed by comparing monocytes isolated from APS patients and HC. Anti-VIM antibodies (AVAs) were significantly increased in 11 of 27 patients (40.7%) with APS. VIM expression on HC monocytes was stimulated more strongly by APS IgG from patients with higher-avidity serum AVA. We further characterized the proteome of thrombotic APS IgG-treated monocytes using a label-free proteomics technique. Of 12 proteins identified with the most confidence, 2 overlapped with 2D DiGE and many possessed immune response, cytoskeletal, coagulation, and signal transduction functions which are all relevant to APS and may therefore provide potential new therapeutic targets of this disease.

Examining how antiphospholipid antibodies activate intracellular signaling pathways: a systematic review.

OBJECTIVES: Diverse experimental evidence exists implicating the activation of various different cell surface receptors and intracellular pathways by antiphospholipid antibodies (aPL). This evidence has been generated using a number of different cell types with varying numbers of aPL from different sources and disease subtypes. This experimental variability complicates the comparison of results from different studies. We therefore undertook a systematic review of the literature to provide a critical analysis of the strength of the evidence that specific receptors and signaling pathways are important in the pathogenesis of antiphospholipid syndrome. METHODS: We searched PubMed and EMBASE for studies in which the effects of aPL on cell surface receptors or intracellular signaling pathways were measured in vitro or in vivo. Each publication was systematically examined to note the following points: antibody type and source, outcome measures, use of receptor/signaling pathway inhibitors, and cell type and origin. RESULTS: We identified 10 original studies on toll-like receptors (TLR), 14 on protein kinases, and 13 on nuclear factor kappa B (NFκB). There was considerable heterogeneity between studies. Nevertheless, convincing evidence from multiple approaches implicates TLR4, p38 mitogen-activated protein kinase (MAPK), and NFκB in mediating pathogenic effects of antiphospholipid antibodies. CONCLUSIONS: TLR4, p38 MAPK and NFκB are involved in mediating pathogenic effects of aPL on different cell types and may be potential therapeutic targets in antiphospholipid syndrome.

Interactions of human monoclonal and polyclonal antiphospholipid antibodies with serine proteases involved in hemostasis.

OBJECTIVE: To characterize the interaction between procoagulant and/or anticoagulant serine proteases and human monoclonal IgG antiphospholipid antibodies (aPL) and polyclonal IgG derived from patients with the antiphospholipid syndrome (APS). METHODS: Five human monoclonal IgG with small differences in their sequences were tested for binding to protein C, activated protein C, plasmin, factor VIIa (FVIIa), FIX, FIXa, and FXII. Serum levels of antithrombin and anti-activated protein C were compared in 32 patients with APS, 29 patients with systemic lupus erythematosus (SLE), and 22 healthy controls. Purified polyclonal IgG derived from APS patients with elevated levels of serum antithrombin antibodies was also tested for its functional effects on thrombin and antithrombin activity. RESULTS: Studies of monoclonal antibodies showed that sequence changes in human aPL are important in determining their ability to bind procoagulant and anticoagulant/fibrinolytic serine proteases. Mean IgG antithrombin levels were significantly elevated in patients with APS and in SLE patients with aPL but no APS (SLE/aPL+) compared to healthy controls, but anti-activated protein C levels were not increased in these patients. Moreover, IgG purified from patients with APS displayed higher avidity for thrombin and significantly inhibited antithrombin inactivation of thrombin compared with IgG from SLE/aPL+ patients. CONCLUSION: High-avidity antithrombin antibodies, which prevent antithrombin inactivation of thrombin, distinguish patients with APS from SLE/aPL+ patients, and thus may contribute to the pathogenesis of vascular thrombosis in APS.