Cell based assay for determining antibody or ligand binding and function

#This application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 25, 2018, is named P32944-US-SequenceListing.txt, and is 2,227 bytes in size.

#This application is a 371 application of International Application No. PCT/EP2016/064238, filed on Jun. 21, 2016, which claims priority to European Application No. 15173929.9 filed on Jun. 25, 2015, the contents of which are incorporated herein in their entirety by reference.

#The present invention relates to a new cell based assay for determining antibody or ligand binding and function.

#FIG. 1. Determination of the transfection efficiency as well as viability measurement of the cells after SNAP-Receptor X transfection and labeling with terbium, a donor fluorescent dye. The terbium signal was measured for 10 000 cells per well at a wavelength of 615 nm. The transfection reagents Lipofectamine 2000, Lipofectamine LTX and Xtreme Gene HP were compared.

#FIG. 2. Determination of the transfection efficiency of Lipofectamine LTX as well as viability measurement of HEK NFκB-Luc-GFP and HeLa NFκB-Luc cells after SNAP-Receptor X transfection and labeling with terbium, a donor fluorescent dye. The terbium signal was measured with 10 000 cells per well at a wavelength of 615 nm.

#FIG. 3. Tag-lite® indirect binding assay with Antibody-I and Antibody-II to Receptor X expressing HEK NFκB-Luc-GFP and HeLa NFκB-Luc cells. The antibodies were diluted ranging from 25 nM to 0.05 nM in a two-fold dilution series. A concentration of 150 nM for the d2-labeled secondary antibody was used. The binding curve was fitted by nonlinear regression with Graph Pad Prism 6.0.

#As used herein, a “reporter gene” means a gene whose expression can be assayed. In one preferred embodiment a “reporter gene” is a gene that encodes a protein the production and detection of which is used as a surrogate to detect indirectly the activity of the antibody or ligand to be tested. The reporter protein is that protein encoded by the reporter gene. Preferably, the reporter gene encodes an enzyme whose catalytic activity can be detected by a simple assay method or a protein with a property such as intrinsic fluorescence so that expression of the reporter gene can be detected in a simple and rapid assay requiring minimal sample preparation. Non-limiting examples of enzymes whose catalytic activity can be detected are Luciferase, beta Galactosidase, Alkaline Phosphatase. Luciferase is a monomeric enzyme with a molecular weight (MW) of 61 kDa. It acts as a catalysator and is able to convert D-luciferin in the presence of Adenosine triphosphate (ATP) and Mg2+ to luciferyl adenylate. In addition, pyrophosphate (PPi) and adenosine monophosphate (AMP) are generated as byproducts. The intermediate luciferyl adenylate is then oxidized to oxyluciferin, carbon dioxide (CO2) and light. Oxyluciferin is a bioluminescent product which can be quantitatively measured in a luminometer by the light released from the reaction. Luciferase reporter assays are commercially available and known in the art, e.g. Luciferase 1000 Assay System and ONE-Glo™ Luciferase Assay System.

#The term “protein with intrinsic fluorescence” refers to a protein capable of forming a highly fluorescent, intrinsic chromophore either through the cyclization and oxidation of internal amino acids within the protein or via the enzymatic addition of a fluorescent co-factor. The term “protein with intrinsic fluorescence” includes wild-type fluorescent proteins and mutants that exhibit altered spectral or physical properties. The term does not include proteins that exhibit weak fluorescence by virtue only of the fluorescence contribution of non-modified tyrosine, tryptophan, histidine and phenylalanine groups within the protein. Proteins with intrinsic fluorescence are known in the art, e.g. green fluorescent protein (GFP), red fluorescent protein (RFP), Blue fluorescent protein (BFP, Heim et al. 1994, 1996), a cyan fluorescent variant known as CFP (Heim et al. 1996; Tsien 1998); a yellow fluorescent variant known as YFP (Oruro et al. 1996; Wachter et al. 1998); a violet-excitable green fluorescent variant known as Sapphire (Tsien 1998; Zapata-Hommer et al. 2003); and a cyan-excitable green fluorescing variant known as enhanced green fluorescent protein or EGFP (Yang et al. 1996) and can be measured e.g. by live cell imaging (e.g. Incucyte) or fluorescent spectrophotometry.

#As used herein, the term “functionality of an antibody or ligand” refers to the biological activity of an antibody or ligand, e.g. the ability of an antibody or ligand to elicit a cellular response. For example through binding to a target antigen, the antibody activates or suppresses a cell signaling pathway, i.e. activates of inhibits the function of the target antigen. For example, the antibody to be tested binds to a receptor activating the NF-κB pathway and through this binding a response element in the cell nucleus is activated. When linking this response element to a reporter gene, the activation can be easily monitored in the assay of the invention. The term “functionality” also includes the effector functions of an antibody, e.g. C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.