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Novel Cell Lines and Methods (19) TZZ¥ZZ_¥_T (11) EP 3 009 513 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 20.04.2016 Bulletin 2016/16 C12N 15/85 (2006.01) C12N 15/67 (2006.01) C07K 14/435 (2006.01) (21) Application number: 15180871.4 (22) Date of filing: 02.02.2009 (84) Designated Contracting States: (72) Inventor: SHEKDAR, Kambiz AT BE BG CH CY CZ DE DK EE ES FI FR GB GR New York, NY 10010 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR (74) Representative: Vossius & Partner Patentanwälte Rechtsanwälte mbB (30) Priority: 01.02.2008 US 63219 P Siebertstrasse 3 81675 München (DE) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: Remarks: 09709529.3 / 2 245 171 •Claims filed after the date of filing of the application (Rule 68(4) EPC). (71) Applicant: Chromocell Corporation •This application was filed on 13-08-2015 as a North Brunswick, NJ 08902 (US) divisional application to the application mentioned under INID code 62. (54) NOVEL CELL LINES AND METHODS (57) The invention relates to novel cells and cell lines, and methods for making and using them. EP 3 009 513 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 009 513 A1 Description Field of the Invention 5 [0001] The invention relates to novel cells and cell lines, and methods for making and using them. Background of the Invention [0002] Currently, the industry average failure rate for drug discovery programs in pharmaceutical companies is reported 10 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. [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. 15 [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 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. 20 [0006] Accordingly, there is an urgent need for cells and cell lines that express a function protein or RNA of interest. Summary of the Invention [0007] In some embodiments, the invention provides a cell that expresses a heterodimeric protein of interest from an 25 introduced nucleic acid encoding at least one of the subunits of the heterodimeric protein of interest, said cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein said protein of interest does not comprise a protein tag, or said protein is produced in that form consistently and reproducibly such that the cell has a Z’ factor of at least 0.4 in the functional assay, or said cell is cultured in the absenc e of selective pressure, or any combinations thereof 30 [0008] In some embodiments, the invention provides a cell that expresses a heterodimeric protein of interest, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one of the subunits of the heterodimeric protein of interest, said cell being characterized in that it produces the heterodimeric protein of interest in a form suitable for use in a functional assay, wherein said protein of interest does not comprise a protein tag, or said protein is produced in that form consistently and reproducibly such that the cell has a Z’ factor of at least 0.4 in the 35 functional assay, or said cell is cultured in the absence of selective pressure, or any combinations thereof. [0009] In some embodiments, the invention provides a cell that expresses a heterodimeric protein of interest from an introduced nucleic acid encoding at least one of the subunits of the heterodimeric protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure. 40 [0010] In some embodiments, the invention provides a cell that expresses a heterodimeric protein of interest wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one of the subunits of the heterodimeric protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active, wherein the cell is cultured in the absence of selective pressure. [0011] In some embodiments, the nucleic acid encoding the second subunit of the heterodimeric protein of interest is 45 endogenous. In other embodiments, the nucleic acid encoding the second subunit of the heterodimeric protein of interest is introduced. In yet other embodiments, the protein of interest does not comprise a protein tag. [0012] In some embodiments, the heterodimeric protein of interest is selected from the group consisting of: an ion channel, a G protein coupled receptor (GPCR), tyrosine receptor kinase, cytokine receptor, nuclear steroid hormone receptor and immunological receptor. In some embodiments, the heterodimeric protein of interest is selected from the 50 group consisting of: a sweet taste receptor and an umami taste receptor. In other embodiments, the heterodimeric protein of interest has no known ligand. [0013] In some embodiments, the heterodimeric protein of interest is not expressed in a cell of the same type. In some embodiments the cell is a mammalian cell. [0014] In some embodiments, the cell is further characterized in that it has an additional desired property selected 55 from the group consisting of: a signal to noise ratio greater than 1, being stable over time, growth without selective pressure without losing expression, physiological EC50 values, and physiological IC50 values. In some embodiments, the heterodimeric protein of interest is produced in a form consistently and reproducibly for a period of time selected from: at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three 2 EP 3 009 513 A1 months at least four months, at least five months, at least six months, at least seven months, at least eight months, and at least nine months. In some embodiments, the functional assay is selected from the group consisting of: a cell-based assay, a fluorescent cell-based assay, a high throughput screening assay, a reporter cell-based assay, a G protein mediated cell-based assay, and a calcium flux cell-based assay. In other embodiments, the cell is suitable for utilization 5 in a cell based high throughput screening. [0015] In some embodiments, the selective pressure is an antibiotic. In other embodiments, the cell expresses the heterodimeric protein in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 days, or 150 days. [0016] In some embodiments, the invention provides a cell that expresses a heteromultimeric protein of interest wherein 10 said heteromultimeric protein comprises at least 3 subunits, wherein at least one subunit of the heteromultimeric protein interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the heteromultimeric protein of interest in a form suitable for use in a functional assay, wherein said protein of interest does not comprise a protein tag, or said protein produced in that form consistently and reproducibly such that the ceii has a Z’ factor of at least 0.4 in the functional assay, or said cell is cultured in the absence of selective pressure, or any combinations thereof. 15 [0017] In some embodiments, the invention provides a cell that expresses a heteromultimeric protein of interest wherein said heteromultimeric protein comprises at least 3 subunits, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one of the subunits of the heteromultimeric protein of interest, said cell being characterized in that it produces the heteromultimeric protein of interest in a form suitable for use in a functional assay, wherein said protein of interest does not comprise a protein tag, or said protein produced in that form consistently 20 and reproducibly such that the cell has a Z’ factor of at least 0.4 in the functional assay, or said cell is cultured in the absence of selective pressure, or any combinations thereof. [0018] In some embodiments, the invention provides a cell that expresses a heteromultimeric protein of interest wherein said heteromultimeric protein comprises at least 3 subunits, wherein at least one subunit of the heteromultimeric protein interest is encoded by an introduced nucleic acid, said cell being characterized in that it produces the protein of interest 25 in a form that is or is capable of becoming biologically active. [0019] In some embodiments, the invention provides a cell that expresses a heteromultimeric protein of interest wherein said heteromultimeric protein comprises at least 3 subunits, wherein the cell is engineered to activate transcription of an endogenous nucleic acid encoding at least one of the subunits of the heteromultimeric protein of interest, said cell being characterized in that it produces the protein of interest in a form that is or is capable of becoming biologically active .
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