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Ep 2391711 B1 (19) TZZ ¥____T (11) EP 2 391 711 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 5/00 (2006.01) C12N 5/07 (2010.01) 08.04.2015 Bulletin 2015/15 G01N 33/50 (2006.01) (21) Application number: 10736548.8 (86) International application number: PCT/US2010/022781 (22) Date of filing: 01.02.2010 (87) International publication number: WO 2010/088633 (05.08.2010 Gazette 2010/31) (54) NOVEL CELL LINES AND METHODS NEUE ZELLLINIEN UND VERFAHREN NOUVELLES LIGNÉES CELLULAIRES ET PROCÉDÉS (84) Designated Contracting States: • SAWCHUK, Dennis AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Fanwood, NJ 07023 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL • SHAH, Purvi, Manoj PT RO SE SI SK SM TR North Brunswick, NJ 08902 (US) (30) Priority: 02.02.2009 US 149311 P (74) Representative: Jennings, Tara Romaine 02.02.2009 US 149318 P FRKelly 02.02.2009 US 149321 P 27 Clyde Road 31.07.2009 US 230536 P Ballsbridge 19.08.2009 US 235181 P Dublin 4 (IE) 02.02.2009 US 149324 P (56) References cited: (43) Date of publication of application: WO-A2-2009/102569 US-A1- 2005 032 158 07.12.2011 Bulletin 2011/49 US-A1- 2006 147 937 US-A1- 2008 262 087 (60) Divisional application: • TOYONO ET AL: "CCAAT/Enhancer-binding 15156204.8 protein beta regulates expression of human T1R3 taste receptor gene in the bile duct carcinoma cell (73) Proprietor: Chromocell Corporation line, HuCCT1", BIOCHIMICA ET BIOPHYSICA North Brunswick, NJ 08902 (US) ACTA . GENE STRUCTURE AND EXPRESSION, ELSEVIER, AMSTERDAM, NL, vol. 1769, no. (72) Inventors: 11-12, 1 November 2007 (2007-11-01), pages • SHEKDAR, Kambiz 641-648, XP022349465, ISSN: 0167-4781 New York, NY 10010 (US) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 391 711 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 391 711 B1 Description Field of the Invention 5 [0001] The invention relates to novel cells and cell lines, and methods for isolating them. In particular embodiments, the invention relates to cells and cell lines stably expressing complex targets. Disclosed herein are methods of making and using such cells and cell lines. The cells and cell lines provided herein are useful in identifying modulators of such complex targets. 10 Background of the Invention [0002] Currently, the industry average failure rate for drug discovery programs in pharmaceutical companies is reported to be approximately 98%. Although this includes failures at all stages of the process, the high failure rate points to a dire need for any improvements in the efficiency of the process. 15 [0003] One factor contributing to the high failure rate is the lack of cell lines expressing therapeutic targets for used in cell-based functional assays during drug discovery. Indisputably, research using cell-based assays, especially drug discovery research, would benefit from cells and cell lines for use in cell-based assays. [0004] Consequently, there is a great need for rapid and effective establishment of cell based assays for more rapid discovery of new and improved drugs. Preferably, for more effective drug discovery, the assay system should provide 20 a more physiologically relevant predictor of the effect of a modulator in vivo. [0005] Beyond the need for cell-based assays is a need for improved cells for protein production, cell-based therapy and a variety of other uses. [0006] Accordingly, there is an urgent need for cells and cell lines that express a functional protein or RNA of interest. [0007] In the mouth, taste receptor cells (TRCs) can be found in several specialized zones that include the tongue, 25 part of the palate, epiglottis, larynx and pharynx. On the tongue, TRCs are organized into groups of cells called taste buds. Taste buds consist of a single apical pore where microv illi of TRCs come into contact with tastants present within the oral cavity. On the tongue, taste buds are embedded in three types of specialized epidermal structures. The fungiform papillae are distributed over the anterior two-thirds of the tongue. The foliate papillae, which are well developed at birth but regress with age, are found on the sides of the posterior one-third of the tongue. Seven to nine circumvallate papillae 30 are located far back on the posterior tongue close to the terminal sulcus. In addition to the ’classical’ TRCs organized in taste buds, chemosensory cell clusters or solitary chemosensory cells are found in non-lingual epithelia in the lung and the intestine. [0008] US 2008/262087 recites that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it recites that co-expression of the T1R2 and T1R3 receptors 35 results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners. Also, it refers to the use of hetero-oligomeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, it refers to cell lines that co-express a combination of T1R1 and T1R3; or T1R2 and T1R3. [0009] WO 2009/102569 refers to cells and cell lines, and methods for making and using them, including a method 40 for isolating a cell that expresses a sweet taste receptor. [0010] Toyono et al, "CCAAT/Enhancer-binding protein beta regulates expression of human T1R3 taste receptor gene in the bile duct carcinoma cell line, HuCCT1", Biochemica et Biophysica Acta 1769 (2007) 641 - 648, refers to, inter alia, the determination of whether a population of cells expresses T1R3. [0011] US 2006/147937 refers to the detection of mRNAs as well as other RNAs in living cells, and also refers to 45 methods for identifying and, if desired, separating cells based on their desired characteristics. Sweet Taste Receptor [0012] Sweet perception is mediated by a heteromeric G-protein coupled receptor (GPCR) composed of two subunits 50 TASR2 (T1R2) and TASR3 (T1 R3). The receptor is named the sweet taste receptor. Both subunits of the receptor are members of the class C GPCR subfamily and possess a large N-terminal extracellular domain, often referred to as the Venus flytrap domain. The T1 R subunits can couple to the G proteins alpha transducin or alpha gustducin, through 2+ which they can activate a phospholipase C (PLC) β2-dependent pathway to increase intracellular Ca concentration. They may also activate a cAMP-dependent pathway. 55 [0013] Sweet taste receptors detect a wide variety of sweet chemicals including simple carbohydrates (such as sugars), amino acids, peptides, proteins, and synthetic sweeteners. Sweet taste receptors are sensitive to both natural and artificial sweeteners. Given the wide diversity of chemical structures known to activate the receptors, multiple binding sites in the receptors have been proposed, including a site in the transmembrane region and a site on T1 R3, which 2 EP 2 391 711 B1 serves as a shared subunit with umami taste receptors. [0014] Sweet taste receptors have also been implicated in conditions such as obesity and diabetes, as these receptors appear to play an important role in nutrient detection and sensing. Taste receptors are expressed in nutrient detection regions of the proximal small intestine in humans, where evidence suggests that they play a role in the detection of 5 nutrients in the intestinal lumen. There is a highly coordinated expression of sweet taste receptors and gustducin, a G- protein implicated in intracellular taste signal transduction, in this region and, more specifically, in the endocrine cells of the gut. The function of these sweet taste receptors thus may show similar ligand-mediated control as other G-protein coupled receptors, that is, they will lose their activity and or expression in the presence of high concentrations of their ligands. This would make intestinal ’taste’ signaling responsive to the dynamic metabolic changes in glucose concen- 10 trations in the blood and lumen. Accordingly, sweet taste receptors and their modulation in the gut may have important roles in diet, appetite and in the treatment of various diseases, such as obesity and diabetes. [0015] Activation of intestinal sweet taste receptors by natural sugars and artificial sweeteners also leads to increased expression of the apical glucose transporter, GLUT2, and other glucose transporters. For example, artificial sweeteners are nutritionally active, because they can signal a functional taste reception system to increase sugar absorption during 15 a meal, a finding that may have important implications in nutrition and appetite, and thus in the potential treatment of malnutrition and eating disorders. Consistently elevated apical GLUT2 levels result in increased sugar absorption and are a characteristic of experimental diabetes and of insulin-resistant states induced by fructose and fat. Additionally, sweet taste receptor activation in neuroendocrine cells leads to the release of glucagon like peptide (GLP-1) and perhaps other modulators of digestion. Overall, sweet taste receptors in the intestine play an important role in sensing the 20 nutritional value of luminal content and help coordinate the body’s response via regulated absorption and digestion. These findings suggest that sweet taste receptors could serve as possible targets for modulators useful in treating obesity and diabetes.
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