Paula Harrison

Development and Automation of a 384-Well Cell Fusion Assay to Identify Inhibitors of CCR5/CD4-Mediated HIV Virus Entry:

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/β-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of β-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturisation to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen ~650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z′ consistently above 0.6, which compares favourably with less complex biochemical assays.

Protein engineering and preclinical development of a GM‐CSF receptor antibody for the treatment of rheumatoid arthritis

For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target‐mediated clearance or antigen ‘sink’ effects. The purpose of this work was to engineer an antibody to the GM‐CSF receptor α (GM‐CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients.

A Novel Method for Determination of the Affinity of Protein: Protein Interactions in Homogeneous Assays

Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the KD of the protein-protein interaction. Accurate measurement of KD during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the KD of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50= (([Ki]/KD) × [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured IC50 versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/KD). KD is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF®) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured KD values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed. (Journal of Biomolecular Screening 2008:674-682)

Monoclonal antibody targeting of fibroblast growth factor receptor 1c ameliorates obesity and glucose intolerance via central mechanisms.

We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation.

Feasibility of Implementing Cell-Based Pathway Reporter Assays in Early High- Throughput Screening Assay Cascades for Antibody Drug Discovery

Implementing functional cell-based screens in early antibody discovery has become increasingly important to select antibodies with the desired profile. However, this is limited by assay tolerance to crude antibody preparations and assay sensitivity. The current study aims to address this challenge and identify routes forward. Two common types of high-throughput screening (HTS) antibody sample, derived from either phage display or hybridoma techniques, have been screened across a wide range of CellSensor beta-lactamase reporter assays in a variety of cell backgrounds to more extensively characterize assay tolerance. Pathway-, sample-, and cell background–specific effects were observed. Reporter assays for agonism were less affected by crude antibody preparations, with 8 of 21 sample tolerant, and the potential to implement an additional 8 assays by choosing the best-tolerated sample type. Antagonist mode assays exhibited more complexity, with potentiating as well as inhibitory effects. However, 5 of 24 antagonist assays were fully tolerant, with the potential to implement an additional 11 assays. Different subsets of assays were affected in agonist versus antagonist mode, and hybridoma sample sets were better tolerated overall. The study clearly demonstrates the potential to use cell-based reporter assays in biologics HTS, particularly if the method of antibody production is considered in the context of the required assay mode (agonist/antagonist).

Development of a Homogeneous High-Throughput Screening Assay for Biological Inhibitors of Human Rhinovirus Infection

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule–1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti–ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.