Use of constitutively active variants of growth factor receptors as selection markers for the generation of stable producer cell lines
The present invention relates to methods for the selection of a stable producer cell line expressing one or more protein(s) of interest (POIs), comprising the stable transfection of cells with a gene encoding a constitutively active variant of a growth factor receptor, respective methods for the expression of one or more POIs in such stable producer cell lines, and uses of a constitutively active variant of a growth factor receptor as selection marker in the cultivation of cells.
1. A method for the selection of a stable producer cell line expressing one or more protein(s) of interest (POIs), comprising the steps of:
(a) providing a cell line,
(b) concurrently stably transfecting said cell line with
(i) a gene encoding a constitutively active variant of a growth factor receptor, wherein the variant of a growth factor receptor is a human CD8-IGF-1R fusion protein, and
(ii) one or more gene(s) of interest (GOIs) encoding said POIs, and
(c) cultivating said cell line in a cell culture medium either not containing the growth factor that is recognized by said growth factor receptor or containing said growth factor at concentrations which are too low to support growth of the cells in the absence of expression of said constitutively active variant of a growth factor receptor.
2. The method of claim 1 , wherein the cell line is selected from the group consisting of CHO cells, HEK293 cells, CAP cells, Per.C6 cells, BHK cells, Vero cells, MDCK cells, hybridoma cells, and fibroblasts.
3. The method of claim 1 , wherein the POIs are selected from the group consisting of extracellular matrix proteins, growth factors, peptide hormones, cytokines, enzymes, antibodies, antibody fragments, blood clotting factors, protease inhibitors, and viral protein products.
4. The method of claim 1 , wherein the POIs are selected from the group consisting of human recombinant alpha-1-antitrypsin (rhAAT); fibrinogen; laminins (LAM); interferons (IFN); interleukins (IL); Immunoglobulin G (IgG); Immunoglobulin M (IgM); bispecific monoclonal antibodies (BsAb); erythropoietin (EPO); Factor VII (FVII); Factor VIII (FVIII); Factor IX (FIX); von-Willebrand-Factor (vWF); Cl esterase inhibitor (Cl-inhibitor; Cl Inh); gag-pol from HIV-1, HIV-2, EIAV, SIV, or other retroviridae; rep protein from Adeno-Associated Virus (REP); and cap protein from Adeno-Associated Virus (CAP).
5. The method of claim 1 , wherein the gene encoding a constitutively active variant of a growth factor receptor and the one or more GOIs are present on the same vector in said cell line.
6. The method of claim 1 , wherein said human CD8-IGF-1R fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 1.
7. The method of claim 1 , wherein said human CD8-IGF-1R fusion protein is encoded by a nucleic acid comprising or consisting of the nucleotide of SEQ ID NO: 2.
8. The method of claim 1 , wherein step (c) is performed for a duration that allows selection to occur.
9. A method for the expression of one or more protein(s) of interest (POIs) in a stable producer cell line, comprising the steps of:
(a) performing the method according to claim 1 ,
(b) expressing said POIs in said stable producer cell line, and
(c) recovering said POIs from the cells or the cell culture medium.
10. A cell comprising a polynucleotide expression construct which encodes a constitutively active variant of a growth factor receptor as a selection marker that is expressed in the cell, wherein the variant of a growth factor receptor is a human CD8-IGF-1R fusion protein, and
wherein the polynucleotide expression construct further comprises a gene of interest that is expressed in the cell.
11. The cell of claim 10 , wherein the cell is selected from the group consisting of CHO cells, HEK293 cells, CAP cells, Per.C6 cells, BHK cells, Vero cells, MDCK cells, hybridoma cells, and fibroblasts.