Cell-based functional assays

GLP-1 R IgG clones were tested for their potential effects on GLP-1 R signaling by performing cAMP assays obtained from Eurofins DiscoverX. The technology involved in detecting cAMP levels is a no wash gain-of-signal competitive immunoassay based on Enzyme Fragment Complementation technology. Experiments were designed to test for either agonist or antagonist activity of the IgG clones. To test for agonist activity of the IgGs, cells were stimulated with IgG incubating for 30 min at 37°C (titrations 1:3 starting from 100 nM and diluting down to 0.046 nM with PBS) or with the known agonist GLP-1 7–36 peptide (MedChemExpress, Cat. #HY-P005), titrated 1:3 starting from 12.5 nM with 12 points serial dilution with PBS. To test for antagonist activity, GLP1R-expressing cells (DiscoverX, Cat. #95-0062E2) are seeded at 15,000 cells per well. After overnight incubation, the cells were treated with titrated antibodies or antagonistic control Exendin 9–39 (AnaSpec Inc. AS-24467) for 1 hour at 37°C, followed by agonist stimulation of GLP-1 or Exendin-4 for 30 minutes at 37°C. cAMP level is evaluated one day after adding detection reagent.

β-arrestin recruitment assay was obtained from Eurofins DiscoverX (Cat #93-0300E2) that used untagged GLP-1 R-overexpressing CHO-K1 cells. This experiment tests whether TB01-3 has an effect on GLP-1 7–36 agonist-induced β-arrestin recruitment upon GLP-1 R activation. GLP1R-expressing cells (DiscoverX, Cat. #93-0300E2) are seeded at 10000 cells per well. After overnight incubation, the cells were treated with titrated antibodies for 1 hour at 37°C, followed by agonist stimulation of GLP-1 for 1 hour at 37°C. The level of β-arrestin is evaluated 3 hours after adding detection reagent by a Chemiluminescence plate reader (Molecular Devices SpectraMax M5) and output relative light unites were analyzed using GraphPad Prism.