Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays.

Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell-based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa-mediated luciferase reporter activation upon the binding of antibody-coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody-mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ADCC reporter bioassay using engineered ADCC bioassay effector cells Basic Protocol 2: PBMC ADCC bioassay using primary PBMC and engineered HiBiT target cells.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Improved dual-luciferase reporter assays for nuclear receptors.

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neo(R) fusion protein, which provides selection capability (G418 resistance, neo(R)) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.

Luciferase Reporter Assay System for Deciphering GPCR Pathways.

The G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca(2+) mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m(3) muscarinic receptor and endogenous ß(2)-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.