Bacterial glutamine synthetase as a selectable marker in mammalian cells

Abstract

The present invention relates to a mammalian expression vector comprising a polynucleotide encoding bacterial glutamine synthetase as a selection marker, as well as a nucleic acid encoding said bacterial glutamine synthetase and a mammalian host cell. In particular, said bacterial glutamine synthetase is from a bacterium of the order Enterobacteriale and the family Morganellaceae, preferably said bacterial glutamine synthetase is a glutamine synthetase from the genus Providencia, optionally further comprising a mutation at position E130 and / or F226 and / or R345A. The present invention further relates to a method for preparing a mammalian cell stably expressing a protein and / or a non-coding RNA of interest, and a method for producing a protein in a mammalian cell using said bacterial glutamine synthetase.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, as well as a nucleic acid encoding said bacterial glutamine synthetase and a mammalian host cell. In particular, the bacterial glutamine synthetase is from a bacterium of the order Enterobacteriale and the family Morganellaceae, preferably said bacterial glutamine synthetase is a glutamine synthetase from the genus Providencia, optionally further comprising mutations at positions E130 and / or F226 and / or R345. The present invention further relates to a method for preparing a mammalian cell stably expressing a protein and / or a non-coding RNA of interest, as well as a method for producing a protein in a mammalian cell using said bacterial glutamine synthetase.

[0002] background Chinese hamster ovary (CHO) cells are one of the most frequently used mammalian cell lines used for the production of therapeutic proteins, such as antibodies. The key aspect is to generate productive and stable cell lines expressing the protein of interest with high product titers and adequate product quality in a short time. To produce a stable and highly productive cell line, a selection phase is required to generate a stable and heterogeneous cell pool consisting of various clones that need to be isolated and screened, and then the final production cell line can be stored in a cell bank. Metabolic selection systems, such as dihydrofolate reductase (DHFR) and glutamine synthetase (GS) selection systems, are commonly used to improve this process and generate stable cell lines more efficiently.

[0003] In cell lines lacking the dihydrofolate reductase gene, such as CHO-DG44 cells, selection is performed in the absence of hypoxanthine and thymidine in the medium. An additional amplification step by addition of increasing concentrations of methotrexate (MTX) may be used. The glutamine synthetase selection system is advantageous because it requires fewer gene copies for survival and therefore selection is more rapid on highly productive cell pools.

[0004] Glutamine synthetase (EC 6.3.1.2, also known as gamma-glutamyl ammonia ligase) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine. Glutamine synthetases are classified into three subgroups: GSI, GSII, and GSIII. CHO GS is a class II enzyme, which is the subclass expressed primarily by eukaryotic cells, while bacterial GS proteins are typically members of the GSI class. Although GSI and GSII catalyze the same reaction, they show no or very little sequence similarity, except for residues that form part of the active site, and are quite different overall. For example, bacterial type I GS is a 12-subunit complex (Eisenberg et al. (2000), Biochim. Biophys. Acta 1477, 122-145), and GSII has been reported to form stacks of two or three pentameric rings (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228). Also, bacterial glutamine synthetases may have very little sequence similarity to each other, for example, Bacillus coagulans (CN 12625930 A) or Mycobacterium tuberculosis (WO 2006 / 000045) or Cornybacterium glutamicum (CN 1884501 A) share about 50% or less amino acid identity with the glutamine synthetase of Providencia vermicola (Zuo et al., Scientific Reports, 2018, 8(1): 1-8 and supplementary material).

[0005] Glutamine synthetase is a ubiquitous enzyme that is essential for nitrogen metabolism. Therefore, glutamine synthetase has been used as a selection marker introduced via mammalian expression constructs. In cell lines that do not express sufficient levels of endogenous glutamine synthetase, removal of glutamine supplementation from the cell culture medium increases the selection pressure on the cells. In cell lines that have insufficient endogenous glutamine synthetase levels, such as mouse myeloma cell lines, culture in the absence of glutamine or without glutamine supplementation provides sufficient selection pressure to isolate stable recombinant cell lines. In cell lines that have sufficient endogenous glutamine synthetase, such as CHO cells, addition of the glutamine synthetase inhibitor methionine sulfoximine (MSX) or creation of glutamine synthetase knockout cells (GS- / - or GS- / +) is required to allow sufficient selection pressure to isolate production cell lines in the absence of glutamine. Selection stringency in CHO GS knockout cells is strongly improved, and expression of the transfected GS gene under the control of a weak promoter has been reported to improve selection stringency, with or without the use of MSX. In addition to selection stringency and productivity, stability of protein production of highly productive clones has been shown to be an important feature.

[0006] Also, the transfected glutamine synthetase selection marker has a considerable effect on the selection process and the phenotypic stability as well as productivity of CHO-based cell lines. For example, the use of attenuated mutants of CHO glutamine synthetase has been shown to further improve stability, selection behavior, and productivity (Lin et al. (2019), mAbs, 11:5, 965-976; WO 2018 / 093331, US 20190352631, WO 2017 / 197098). The majority of the attenuated mutants described had mutations in conserved substrate-binding residues. For example, two attenuated GS mutants containing R324C and R341C mutations were first identified in two unrelated infants with congenital GS deficiency (Lin et al. (2019), mAbs, 11:5, 965-976). Residues reported to be involved in glutamate binding are E134, E136, E196, E203, N248, G249, H253, R299, R319, E338, and R340; residues reported to be involved in ATP binding are W130, A191, G192, P208, N255, S257, R262, R324, and Y336; and residues involved in ammonia binding are D63, S66, D162, and E305 (amino acid numbering refers to human GS) (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228 and WO 2018 / 093331).

[0007] Attenuated GS mutants carrying mutations in the active site showed increased selection stringency, but the duration of the selection process and overall cell growth characteristics could be compromised, negatively impacting bioprocess performance. Attenuation of a selection marker is a fine balance between selection stringency, integrated copy number, and cell culture performance (e.g., growth behavior) suitable to support recombinant protein production. Therefore, there is a need to discover novel attenuated GS mutants to improve mammalian expression vectors in cell line development.

[0008] Although bacterial GS or peptides thereof from various species have been expressed in eukaryotic cells such as yeast for purification, as immunogens, or for other reasons (see, for example, WO 2018 / 144807, WO 2006 / 000045A1), it has been assumed that bacterial GS (GSI) is not active in mammalian cells, since yeast or plant GS has been shown to be active in E. coli, but not vice versa (WO 2004 / 003175A2 and EP 0240792A1). More importantly, to the best of our knowledge, bacterial GS has not been previously tested or used in mammalian cells as a selection marker.

[0009] Summary of the Invention The present invention uses a specific bacterial glutamine synthetase gene, unrelated to the mammalian glutamine synthetase gene, to generate highly productive and stable mammalian host cell pools expressing therapeutic proteins, particularly therapeutic antibodies, which show more stringent selection behavior and beneficial cell culture performance in mammalian cells, such as CHO cells, due to their attenuated activity compared to wild-type glutamine synthetase. This is the first time that a prokaryotic glutamine synthetase mutant has been used to generate a stable and productive mammalian cell pool for cell line generation. Furthermore, point mutations in Providencia vermicola glutamine synthetase have been identified that show increased selection stringency and improved antibody titers compared to wild-type Providencia vermicola glutamine synthetase. Another advantage of bacterial glutamine synthetase selectable markers is that due to their low sequence similarity to mammalian (e.g. Chinese hamster ovary cell) glutamine synthetase, the probability of recombination events that would restore endogenous glutamine synthetase and thus allow survival of cells that do not produce the gene of interest is dramatically reduced.

[0010] In a first aspect, the present invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker, wherein the bacterial glutamine synthetase has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the bacterial glutamine synthetase is derived from a bacterium of the order Enterobacteriale and the family Morganellaceae. In a particular embodiment, the bacterial glutamine synthetase is derived from the genus Providencia and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the bacterial glutamine synthetase is derived from the genus Photorhabdus and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14. In a preferred embodiment, the bacterial glutamine synthetase is Providencia vermicola glutamine synthetase or Photorhabdus luminescens glutamine synthetase.

[0011] The mammalian expression vector may further encode a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. In a particular embodiment, the amino acid sequence has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0012] In certain embodiments, the mammalian expression vector further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest. The protein of interest is preferably a therapeutic protein, more preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

[0013] The present invention further provides a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or non-coding RNA of interest. In a particular embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0014] In yet another aspect, the present invention provides a bacterial glutamine synthetase from Providencia vermicola comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0015] In yet another aspect, the present invention provides a mammalian host cell comprising an expression vector according to the present invention, a nucleic acid sequence according to the present invention, or a nucleic acid sequence encoding a bacterial glutamine synthetase according to the present invention, preferably wherein the mammalian host cell is (a) a rodent cell, more preferably a CHO cell, and / or (b) a GS gene knockout cell. Preferably, the polynucleotide encoding the bacterial glutamine synthetase is co-integrated into the host cell genome together with at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0016] In yet another aspect, the present invention provides a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising: (a) introducing into a mammalian host cell, preferably a CHO cell, an expression vector according to the present invention or a nucleic acid according to the present invention, comprising a polynucleotide encoding bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest; and (b) culturing the mammalian host cell in a medium free of glutamine under conditions for selecting for bacterial glutamine synthetase, wherein the at least one polynucleotide encoding the protein and / or the non-coding RNA of interest is simultaneously integrated into the host cell genome together with the polynucleotide encoding bacterial glutamine synthetase. In a particular embodiment, the method further comprises (c) selecting a single clone for clonal expansion to prepare a monoclonal cell line. In such a case, the method is a method for preparing a monoclonal cell line stably expressing a protein and / or a non-coding RNA of interest.

[0017] The present invention further provides a method of producing a protein of interest comprising the steps of: (a) introducing a mammalian expression vector according to the present invention or a nucleic acid according to the present invention into a mammalian host cell, preferably a CHO cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium free of glutamine under conditions that select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome with the polynucleotide encoding the bacterial glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the mammalian host cell under conditions that produce the protein of interest; and (e) collecting and optionally purifying the protein of interest.

[0018] In yet another aspect, there is provided a method of producing a protein of interest, comprising the steps of: (a) providing a mammalian host cell of the invention, comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions for producing the protein of interest; and (c) harvesting and optionally purifying the protein of interest. Preferably, the mammalian host cell is a rodent cell, more preferably a CHO cell.

[0019] In yet another aspect, the present invention relates to a kit comprising an expression vector according to the present invention and a glutamine-free cell culture medium.

[0020] In yet another aspect, the invention provides the use of bacterial glutamine synthetase as a selectable marker in mammalian cells. [Brief description of the drawings]

[0021] [Figure 1] CHO-K1-GS cell pool expressing monoclonal antibody 1 and wild-type CHO GS as a selection marker. CHO-K1-GS cell pools (n=5) stably transfected with a mammalian expression vector to express monoclonal antibody 1 and CHO wild-type GS as a metabolic selection marker were cultured in medium lacking L-glutamine. The titer [mg / L] of monoclonal antibody 1 was determined on the indicated days after transfection. [Diagram 2] Viability and viable cell density (VCD) of CHO-K1-GS expressing monoclonal antibody 1 and wild-type CHO GS or Providencia vermicola GS as selection markers. CHO-K1-GS cell pools stably transfected with monoclonal antibody 1 and vectors encoding a transcription cassette for the glutamine synthetase selection marker from Cricetulus griseus or Providencia vermicola were cultured in medium lacking L-glutamine. A) Viability [%] and B) viable cell density during selection were determined at the indicated days after transfection. [Diagram 3]Productivity after selection and shake flask production run of CHO-K1-GS expressing monoclonal antibody 1 and wild type CHO GS or Providencia vermicola GS as selection markers. CHO-K1-GS cell pools stably transfected with vectors encoding transcription cassettes for glutamine synthetase selection marker from Crisetulus griseus (CHO WT GS) or Providencia vermicola were cultivated in medium without L-glutamine. Productivity as titer [mg / L] was determined (A) after selection on the indicated days after transfection with CHO WT GS or Providencia vermicola GS as selection markers and (B) during the shake flask production run on the indicated days after transfection for Providencia vermicola GS only, since CHO WT GS cells lost their productivity after about 20 days. [Figure 4] Viability and productivity after transfection of CHO-K1-GS expressing monoclonal antibody 1 and glutamine synthetase from Providencia vermicola, Photorhabdus luminescens and Budvicia aquatica as selection markers. CHO-K1-GS cell pools stably transfected with vectors encoding transcription cassettes for monoclonal antibody 1 and the glutamine synthetase selection marker from Providencia vermicola (closed circle), Photorhabdus luminescens (closed square) and Budvicia aquatica (closed triangle) were cultivated in medium lacking L-glutamine. A) Viability [%] during selection was determined on the indicated days after transfection. (B) Productivity as titer [mg / L] was determined on the indicated days after transfection after selection. [Diagram 5]Shake flask production process of CHO-K1-GS expressing monoclonal antibody 1 and wild-type or E130F / E130G mutants of Providencia vermicola as selection markers. CHO-K1-GS cell pools stably transfected with transcription cassettes to express monoclonal antibody 1 and either E130F or E130G mutants of Providencia vermicola as selection markers were cultured in medium without L-glutamine. The titer of monoclonal antibody 1 [mg / L] was determined on the indicated days after transfection. [Figure 6] Productivity and viable cell density (VCD) during shake flask production run of CHO-K1-GS expressing monoclonal antibody 1 and Providencia vermicola wild-type GS, CHO wild-type GS, or Providencia vermicola F226Y mutant as selection markers. After stable pool generation and single cell deposition, selected highly productive clones were subjected to shake flask experiments in tandem with CHO WT (14-day production run). (A) Productivity as titer [mg / L] of monoclonal antibody 1 with Providencia vermicola wild-type GS, Providencia vermicola GS F226Y, or CHO GS WT as selection markers, and (B) viable cell density [1x106 cells / ml] with Providencia vermicola wild-type GS and Providencia vermicola GS F226Y as selection markers were determined at the end of the production run. [Figure 7]Viability and productivity after transfection of CHO-K1-GS expressing monoclonal antibody 1 and Providencia vermicola wild-type GS or Providencia vermicola R345A or R360A mutants as selection markers. CHO-K1-GS cell pools stably transfected with monoclonal antibody 1 and vectors encoding transcription cassettes of glutamine synthetase selection marker from Providencia vermicola WT GS (closed circle), and Providencia vermicola R345A (closed square) or R360A mutants (closed triangle) were cultivated in medium lacking L-glutamine. A) Viability [%] during selection was determined at the indicated days after transfection for Providencia vermicola wild-type GS and the two mutants. (B) Productivity as titer [mg / L] was determined on the indicated days after selection normalized to a survival rate of >70% for Providencia vermicola wild-type GS and the Providencia vermicola R345A mutant, because the Providencia vermicola R360A mutant did not recover during selection. [Figure 8-1] Sequence of glutamine synthetase from Providencia vermicola (SEQ ID NO:1). [Figure 8-2] Sequence of glutamine synthetase from Providencia vermicola (SEQ ID NO:1). [Figure 9] The sequence of wild-type Chryseturus griseus glutamine synthetase (SEQ ID NO:2). [Figure 10] Sequence of glutamine synthetase from Photorhabdus luminescens (SEQ ID NO: 14).

[0022] Detailed Description The term "comprise" or "comprising" means "including, but not limited to". The term is open-ended and is intended to specify the presence of any stated feature, element, integer, step, or component, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "essentially consisting of". With respect to sequences, the terms "having an amino acid sequence of" and "comprising amino acids of" are used synonymously and include the embodiment "consisting of an amino acid sequence of". Similarly, the term "encoding" or "encoding" is open-ended and allows for the presence or addition of one or more other features, elements, or components. Furthermore, the singular and plural forms are not used restrictively. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

[0023] The term "protein" is used synonymously with "amino acid sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes, such as fusion to other proteins, amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, substitutions of specific amino acid sequences can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with the same properties.

[0024] The term "nucleic acid sequence" is used synonymously with "polynucleotide" and refers to DNA or RNA of any length. In the context of expression vectors, particularly plasmids, and integration into a host cell genome, those skilled in the art will understand it to refer to a DNA sequence or DNA molecule.

[0025] The term "eukaryotic cell" as used herein refers to a cell having a nucleus within a nuclear envelope, including animal cells, human cells, plant cells, and yeast cells. Eukaryotic cells specifically include mammalian cells, such as Chinese Hamster Ovary (CHO) cells or cells derived from HEK293 cells. Mammalian cells as used herein refer to any cell or cell line of mammalian origin, such as human cells or rodent cells. Cells as referred to herein are cells maintained in culture, and do not relate to primary cells, but to cell lines or cell line-derived cells, i.e., immortalized cells.

[0026] As used herein, the term "about" refers to a 10% variation of the specified numerical value, for example, about 50% has a variation of 45-55%.

[0027] As used herein, the term "stringency of selection" refers to the time period to reach more than 70% viability and doubling time within 48 hours of cell culture after transfection. The longer the period, the more stringent the selection behavior. Typically, attenuated glutamine synthetase exhibits a more stringent selection behavior compared to CHO wild-type glutamine synthetase.

[0028] Bacterial glutamine synthetase and mammalian expression vectors or host cells encoding bacterial glutamine synthetase The present invention demonstrates that specific bacterial glutamine synthetase genes, unrelated to mammalian glutamine synthetase genes, can be used as selection markers to generate highly productive and stable mammalian host cell pools and cell lines expressing therapeutic proteins, such as therapeutic antibodies, and / or non-coding RNAs, such as small interfering RNAs or microRNAs. This is the first time that prokaryotic glutamine synthetase mutants have been successfully used as selection markers in mammalian cells, and surprisingly, glutamine synthetases from Providencia vermicola and Photorhabdus luminescens showed a more stringent selection behavior due to their attenuated activity compared to CHO wild-type glutamine synthetase in CHO cells, resulting in higher productivity compared to the use of CHO wild-type glutamine synthetase as a selection marker in CHO cells. Having provided proof of principle that specific bacterial glutamine synthetase genes can be used as selection markers in mammalian cells, further other bacterial glutamine synthetase genes can be identified. Furthermore, point mutations in the glutamine synthetase of Providencia vermicola at highly conserved amino acids were identified that show enhanced selection stringency and improved antibody titers compared to wild-type Providencia vermicola glutamine synthetase. At least due to the conservation of these amino acids in P. luminescens glutamine synthetase, these point mutations in P. luminescens glutamine synthetase are expected to similarly show enhanced selection stringency and improved antibody titers compared to wild-type P. luminescens glutamine synthetase.

[0029] More specifically, in a first aspect, the present invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 (Figure 8). In a particular embodiment, the bacterial glutamine synthetase is derived from a bacterium of the order Enterobacteriale and the family Morganellaceae. Upon transfection or transduction of a mammalian expression vector according to the present invention, the bacterial glutamine synthetase mediates improved selection stringency and / or genetic stability of a stably co-integrated protein of interest and / or non-coding RNA in CHO cells compared to a CHO glutamine synthetase having the amino acid sequence of SEQ ID NO: 2 (Figure 9). In a particular embodiment, the bacterial glutamine synthetase is derived from the genus Providencia and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 (Figure 8). In a particular embodiment, the bacterial glutamine synthetase is from the genus Photorhabdus and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14 (FIG. 10). In a preferred embodiment, the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase or a Photorhabdus luminescens glutamine synthetase. Preferably, the glutamine synthetase is from the genus Providencia, more preferably from Providencia vermicola. Thus, the glutamine synthetase may be a Providencia vermicola glutamine synthetase, in particular a bacterial glutamine synthetase comprising an amino acid sequence of SEQ ID NO: 1 or comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90%, and preferably at least 95% sequence identity to SEQ ID NO: 1.In another embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, or the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, preferably the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. Thus, the bacterial glutamine synthetase may have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14.

[0030] The bacterial glutamine synthetase may further comprise a point mutation that attenuates the glutamine synthetase (e.g., E130X and / or R345A) or increases the catalytic activity (e.g., F226Y). In particular, the bacterial glutamine synthetase may have a point mutation at a highly conserved residue corresponding to position E130 of the glutamine synthetase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation at position E130 may be a substitution with any amino acid (X), i.e., E130 may be mutated to any amino acid other than E (SEQ ID NO: 3). Preferably, the mutation at the amino acid position corresponding to E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably, the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M and I. Thus, substitution with an amino acid that does not contain a polar, acidic or basic side chain is preferred. In a particular embodiment, the mutation is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G) (SEQ ID NO: 4 or SEQ ID NO: 5, respectively). As used herein, "X" refers to any amino acid, and reference herein to a mutation as E130X specifies that X is any amino acid except the original amino acid, i.e., E (Glu, glutamic acid). Without being bound by theory, substitution of the highly conserved glutamic acid (E) corresponding to amino acid position E130 in SEQ ID NO: 1 results in attenuation of bacterial glutamine synthetase, where attenuation in this context means reduced enzyme activity. Alternatively or additionally, the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position F226 of the glutamine synthetase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation corresponding to amino acid position F226 of SEQ ID NO:1 is a substitution with amino acid Y (Tyr, tyrosine), also referred to as F226Y. Without being bound by theory, it is believed that the substitution of Y for the highly conserved phenylalanine (F) corresponding to amino acid position F266 in SEQ ID NO:1 improves the catalytic activity of glutamine synthetase.Such mutations that enhance the catalytic activity of glutamine synthetase may be advantageously combined with mutations that further attenuate glutamine synthetase. In a particular embodiment, the mutations are E130X and / or F226Y, where X is any amino acid or as specified in this paragraph above. More preferably, the mutations are E130X, or E130X and F226Y. In a specific embodiment, the mutations are E130F / G, or E130F / G and F226Y. Alternatively or additionally, the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position R345 of the glutamine synthetase of Providencia vermicola having the amino acid sequence of SEQ ID NO:1. The point mutation corresponding to amino acid position R345 of SEQ ID NO:1 is a substitution with amino acid A (Ala, alanine), also referred to as R345A, or alternatively with another amino acid having an aliphatic side chain, such as glycine (G), valine (V), leucine (L) or isoleucine (I), preferably glycine (G). Without being bound by theory, the substitution of the highly conserved arginine (R) corresponding to amino acid position R345 in SEQ ID NO:1 results in attenuation of bacterial glutamine synthetase, where attenuation in this context means reduced enzyme activity. Optionally, the bacterial glutamine synthetase may further comprise a point mutation F226Y. Thus, in a particular embodiment, the mutation is E130X and / or R345A (or G, V, L or I), optionally further comprising F226Y, where X is any amino acid or as specified in this paragraph above. Preferably, the mutation is E130F / G and / or R345A, optionally further comprising F226Y, such as E130F / G and R345A, optionally further comprising F226Y, or preferably E130F / G or R345A, optionally further comprising F226Y.

[0031] In a particular embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A (or G, V, L or I) and combinations thereof, where X is any amino acid, preferably the mutation at amino acid position E130 is a substitution with an aromatic and / or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. In a particular embodiment, the mutation is E130X and / or F226Y, where E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G). As used herein, the term "E130X, F226Y, R345A mutations and combinations thereof" refers to at least one mutation of E130X and / or F226Y and / or R345A, i.e. (1) E130X or F226Y or R345A; or (2) E130X and F226Y, or R345A and F226Y, or E130X and R345A; or (3) E130X and F226Y and R345A. Preferably, the mutation is (i) E130X, or (ii) E130X and F226Y, or (iii) R345A, or (iv) R345A and F226Y, where X is as described herein.

[0032] In certain embodiments, the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and a mutation selected from the group consisting of E130X, F226Y, R345A (or G, V, L or I) and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic and / or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. Preferably, the amino acid sequence has at least 96%, preferably at least 98%, preferably at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14, and even more preferably the amino acid sequence has the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 20, 21, 22 and 29 (based on SEQ ID NO:1) or SEQ ID NO:23, 24, 25, 26, 27, 28 and 30 (based on SEQ ID NO:14), preferably the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 further comprising mutations as disclosed herein, as exemplified in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 20 and 21 (based on SEQ ID NO:1) or SEQ ID NO:23, 24, 25, 26 and 28 (based on SEQ ID NO:14). In a particular embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A (or G, V, L or I) and combinations thereof, where X is any amino acid, preferably the mutation at amino acid position E130 is a substitution with an aromatic and / or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. In a particular embodiment, the mutation of the bacterial glutamine synthetase having at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:14 is E130X and / or F226Y, where E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G).In certain embodiments, the mutation is E130X and / or F226Y and / or R345A, where E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G). Thus, the bacterial glutamine synthetase may comprise the mutations (1) E130X or F226Y or R345A; or (2) E130X and F226Y, or R345A and F226Y, or E130X and R345A; or (3) E130X and F226Y and R345A. Preferably, the mutation is E130X or R345A and optionally F226Y, i.e. (i) E130X, or (ii) E130X and F226Y, or (iii) R345A, or (iv) R345A and F226Y. As used herein, the term "E130X and / or F226Y mutation" refers to E130X or F226Y, or E130X and F226Y mutation. More preferably, the mutation is E130X, or E130X and F226Y. As used herein, the term "F226Y and / or R345A" mutation refers to F226Y or R345A, or F226Y and R345A mutation, preferably R345A, or F226Y and R345A.

[0033] The mammalian expression vector according to the invention is for the expression of heterologous sequences in mammalian cells, i.e. it is adapted for expression in mammalian cells (e.g. mammalian cells), i.e. for the expression of bacterial glutamine synthetase and preferably further protein and / or non-coding RNA of interest. Thus, the mammalian expression vector according to the invention is characterized in that it comprises a polynucleotide encoding bacterial glutamine synthetase operably linked to a mammalian promoter. Typically, the mammalian expression vector comprises an expression cassette comprising a polynucleotide encoding bacterial glutamine synthetase operably linked to a mammalian promoter (e.g. cytomegalovirus promoter or simian virus 40 promoter). The mammalian promoter regulates transcription in mammalian cells, in particular the mammalian promoter regulates transcription in mammalian cells. Exemplary mammalian promoters include, but are not limited to, the simian virus 40 early promoter (SV40), the cytomegalovirus immediate early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF1A), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken β-actin promoter linked to the cytomegalovirus early enhancer (CAGG). Mammalian expression vectors may further contain bacterial sequences, such as origins of replication and resistance genes, for amplification of the vector in bacterial cells.

[0034] The mammalian expression vector typically further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest. The polynucleotide encoding the protein and / or the non-coding RNA of interest is operably linked to a mammalian promoter. The protein and / or the non-coding RNA of interest and the bacterial glutamine synthetase may be encoded by the same expression cassette (polycistronic) or by separate expression cassettes. Preferably, the protein and / or the non-coding RNA of interest and the bacterial glutamine synthetase are encoded by at least two separate expression cassettes or at least two separate encoded mRNAs. The present invention does not encompass fusion proteins of the protein of interest with bacterial glutamine synthetase or a part thereof. In certain embodiments, the non-coding RNA is an RNA that mediates RNA interference (RNAi), such as a microRNA, a small interfering RNA, a long non-coding RNA, or a small hairpin RNA.

[0035] The term "expression cassette" as used herein refers to a separate piece of DNA, particularly vector DNA, consisting of one or more coding polynucleotide sequences and regulatory sequences that control their expression in transfected or transduced cells. An expression cassette contains at least three components: a promoter sequence, an open reading frame, and a transcription termination sequence. In mammalian expression vectors containing a polynucleotide encoding a protein of interest, the transcription termination sequence is called the 3' untranslated region and usually contains a polyadenylation site. An expression cassette directs the cell's machinery to make RNA, and therefore may also be referred to as a transcription cassette. The RNA may be a coding RNA that is further processed into an mRNA that encodes a protein sequence, such as glutamine synthetase or a protein of interest, or it may be a non-coding RNA, such as an RNA that mediates RNA interference (RNAi), such as a microRNA, a small interfering RNA, a long non-coding RNA, or a small hairpin RNA.

[0036] The protein of interest can be any protein, but is typically a therapeutic protein. As used herein, the term "therapeutic protein" refers to a protein that can be used in human and / or animal medical treatment. These include, but are not limited to, cytokines, growth factors, hormones, blood clotting factors, vaccines, interferons, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics. In certain embodiments, the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics.

[0037] In certain embodiments, the protein of interest is an antibody. When the protein of interest is an antibody, the mammalian expression vector comprises a polynucleotide comprising a coding sequence for the variable region of the heavy chain and / or a coding sequence for the variable region of the light chain of the antibody. In certain embodiments, the mammalian expression vector comprises a polynucleotide comprising a coding sequence for the variable region of the heavy chain and / or a coding sequence for the variable region of the light chain of the antibody. Thus, the polynucleotide comprising the coding sequence for the variable region of the heavy chain and the polynucleotide comprising the coding sequence for the variable region of the light chain may be expressed on the same mammalian expression vector or on separate mammalian expression vectors. The expression vector may comprise a polycistronic expression cassette, e.g., a bicistronic expression cassette, and / or a plurality of expression cassettes. A polycistronic expression cassette comprises more than one open reading frame separated by a sequence encoding an RNA sequence that allows initiation of translation, such as an internal ribosome entry site (IRES). In a polycistronic expression cassette, two or more open reading frames are under the control of the same promoter. The polynucleotide encoding at least one variable region of the heavy chain and the polynucleotide encoding at least one variable region of the light chain may therefore be expressed in the same expression cassette (e.g. separated by an IRES sequence) or by two separate expression cassettes. Furthermore, the bacterial glutamine synthetase and the protein of interest and / or the non-coding RNA may be expressed by the same or different expression cassette(s). If the protein of interest is an antibody, the bacterial glutamine synthetase and the polynucleotide encoding at least one variable region of the heavy chain and / or the polynucleotide encoding at least one variable region of the light chain may be expressed by the same or different expression cassettes or a mixture thereof.

[0038] In a preferred embodiment, the mammalian expression vector is for stable integration into the host cell genome (e.g. for stable transfection), and the integration part of the vector comprises a polynucleotide encoding bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or non-coding RNA of interest. The mammalian expression vector according to the present invention, preferably the mammalian expression vector according to the present invention, is a plasmid, a bacterial artificial chromosome (BAC) or a viral vector. The plasmid, the bacterial artificial chromosome (BAC) or a viral vector (e.g. lentiviral vector) can be introduced into a mammalian host cell (e.g. a mammalian host cell) via transfection or transduction, respectively, and is preferably stably integrated into the host cell genome. The skilled artisan knows suitable plasmids, BACs or viral vectors, and knows that the plasmid may further comprise transposon recognition sequences upstream and downstream of the polynucleotide encoding bacterial synthetase as a selection marker, and optionally at least one polynucleotide encoding a protein and / or non-coding RNA of interest.

[0039] A preferred protein of interest is an antibody, including fragments and derivatives thereof. Typically, antibodies are monospecific, although antibodies may be multispecific. Thus, the present invention may be used to generate monospecific antibodies, multispecific antibodies, or fragments thereof, preferably fragments of antibodies (monospecific), bispecific antibodies, trispecific antibodies, or fragments thereof, preferably antigen-binding fragments thereof. Exemplary antibodies within the scope of the present invention include anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD44v6 antibodies, anti-CD49d antibodies, anti-CD52 antibodies, anti-EGFR (epidermal growth factor receptor) 1 (HER1) antibodies, anti-EGFR2 (HER2) antibodies, anti-GD (ganglioside) 3 antibodies, anti-IGF (insulin growth factor) antibodies, anti-VEGF (endovascular Examples of the antibody include, but are not limited to, anti-CD20 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD40 antibody, anti-CD44 antibody, anti-CD52 antibody, anti-HER2 / neu (erbB2) antibody, anti-EGFR antibody, anti-IGF antibody, anti-VEGF antibody, anti-TNFα antibody, anti-IL-2 antibody, and anti-IgE antibody.

[0040] The terms "antibody", "antibodies" or "immunoglobulin(s)" are used herein in the broadest sense to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. There are various immunoglobulin classes: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, the antibody is an IgG antibody, more preferably an IgG1 antibody or an IgG4 antibody.

[0041] The antibody may be of any species, including chimeric and humanized antibodies. A "chimeric" antibody is a molecule in which the domains or regions of the antibody are derived from different species. For example, the variable regions of the heavy and light chains may be derived from a rat or mouse antibody, and the constant region may be derived from a human antibody. In a "humanized" antibody, only minimal sequences are derived from a species other than human. Often, only amino acid residues of the CDRs of a human antibody are replaced with amino acid residues of the CDRs of a species other than human, such as mouse, rat, rabbit, or llama. Sometimes, some important framework amino acid residues that affect the binding specificity and affinity to the antigen are also replaced by non-human amino acid residues.

[0042] Typically, antibodies are tetrameric polypeptides composed of two heterodimeric pairs, each formed by a heavy chain and a light chain. Stabilization of both the heterodimeric as well as the tetrameric polypeptide structures occurs via disulfide bridges between the chains. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", the terms "domain" and "region" being used synonymously herein. Each domain contains approximately 70-110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain at their N-terminus "variable domains" or "variable regions" with less conserved sequences that are involved in antigen recognition and binding. The variable region of the light chain is also referred to as "VL" and the variable region of the heavy chain is also referred to as "VH".

[0043] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Fab fragments consist of the variable regions of both chains, which are held together by adjacent constant regions. They can be formed from conventional antibodies by digestion with proteases, such as with papain, but Fab fragments can also be generated by genetic engineering as well. Still other antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0044] Using genetic engineering techniques, it is possible to generate shortened antibody fragments consisting only of the variable regions of the heavy (VH) and light (VL) chains. These are called Fv fragments (Fragment variable = fragment of the variable part). These Fv fragments are often stabilized, since they lack the covalent bond between the two chains by the cysteines of the constant chains. Advantageously, the variable region of the heavy chain and the variable region of the light chain are linked by a short peptide fragment, for example of 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This type of antibody protein is known as single chain Fv (scFv). Examples of scFv antibody proteins are known to those skilled in the art. Thus, antibody fragments and antigen-binding fragments further include Fv fragments and especially scFv.

[0045] In recent years, various strategies have been developed to prepare scFv as multimeric derivatives. This is aimed in particular at resulting in recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as increased avidity. To achieve multimerization of scFv, scFv have been prepared as fusion proteins with multimerization domains. The multimerization domain can be, for example, the CH3 region of IgG or coiled-coil structures (helical structures), such as leucine-zipper domains. However, there are also strategies in which interactions between the VH / VL regions of scFv are used for multimerization (e.g. diabodies, tribodies and pentabodies). By diabody, the skilled person means a bivalent homodimeric scFv derivative. Homodimers are formed by shortening the linker in the scFv molecule to 5-10 amino acids, leading to the superposition of the interchain VH / VL. Diabodies can be further stabilized by the incorporation of disulfide bridges. Examples of diabody-antibody proteins are known from the prior art.

[0046] By minibody the skilled person means a bivalent homodimeric scFv derivative, which consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably an IgG, most preferably an IgG1, as a dimerization region connected to the scFv via a hinge region (for example also derived from IgG1) and a linker region. Examples of minibody-antibody proteins are known from the prior art.

[0047] By tribody the skilled person means a trivalent homotrimeric scFv derivative, in which the VH-VL are directly fused without a linker sequence to form a trimer.

[0048] The skilled person will also be familiar with the so-called mini-antibodies, which have a bivalent, trivalent or tetravalent structure and are derived from scFv. The multimerization is achieved by dimeric, trimeric or tetrameric coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest encodes any of these above desired polypeptides, preferably a monoclonal antibody, derivative or fragment thereof.

[0049] Additionally, single domain antibodies (sdAbs), also called nanobodies, which are antibody fragments of a single monomeric antibody variable domain, are encompassed. Single domain antibodies are typically derived from camelids (V H H fragment) or cartilaginous fish (V NAR The heavy chains are engineered from antibodies found in the human IgG1 heavy chains (HbA1, HbA2, HbA3, HbA4, HbA5, HbA6, HbA7, HbA8, HbA9, HbA10, HbA11, HbA12, HbA13, HbA14, HbA15, HbA16, HbA17, HbA18, HbA19, Hb

[0050] Immunoglobulin fragments consisting of the CH2 and CH3 domains of the antibody heavy chain are called "Fc fragments", "Fc regions" or "Fc" because of their crystallization properties (Fc = fragment crystallizable). They can be formed from conventional antibodies by digestion with proteases such as papain or pepsin, but can also be produced by genetic engineering. The N-terminal part of the Fc fragment can vary depending on how many amino acids of the hinge region are still present.

[0051] An antibody comprising an antigen-binding fragment and an Fc region may also be referred to as a full-length antibody. Full-length antibodies may be monospecific and multispecific antibodies. Multispecific antibodies are antibodies that have at least two different antigen-binding sites, each of which binds to a different epitope. Multispecific antibodies include bispecific and trispecific antibodies. Bispecific antibodies have two different binding sites. Multispecific antibodies also include antibody formats other than full-length antibodies, such as antibody-derived molecules.

[0052] Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, natural killer cells, or macrophages) in one molecule. Exemplary bispecific antibodies are, but are not limited to, diabodies, BiTE (bispecific T cell targeting) formats, and DART (dual affinity retargeting reagent) formats. The diabody format separates the cognate variable domains of heavy and light chains with two antigen-binding specificities on two separate polypeptide chains, which are non-covalently linked. The DART format is based on the diabody format, but it provides additional stabilization through a C-terminal disulfide bridge. Trispecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. They can be constructed with bispecific antibody technology, which reconstitutes the antigen recognition domains of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been generated that target CD38 on cancer cells and CD3 and CD28 on T cells. Multispecific antibodies are particularly difficult to produce with high production quality.

[0053] As used herein, the term "antibody-derived molecule" refers to any molecule that contains at least one antigen-binding portion that is structurally related to an antibody, including modified full-length monospecific or bispecific antibodies that have been further modified with additional antigen-binding portions, or smaller antibody formats, including those described herein.

[0054] As used herein, the term "antibody mimic" refers to a protein that binds to a specific antigen in a manner similar to an antibody, but is not structurally related to an antibody. Antibody mimics include, but are not limited to, anticalins, affibodies, adnectins, monobodies, DARPins (designed ankyrin repeat proteins), affimers, and affitins.

[0055] Single domain antibodies (sdAbs) may also be referred to as nanobodies. The skilled artisan will understand that the protein may comprise more than one antigen-binding domain and therefore may be multivalent, preferably bivalent (e.g. a bivalent sdAb or a bivalent anticalin or any other bivalent antibody mimetic).

[0056] Another preferred therapeutic protein is a fusion protein, such as an Fc-fusion protein. Thus, the present invention may advantageously be used for the creation of fusion proteins, such as Fc-fusion proteins. The effector portion of the fusion protein may be the complete sequence or any part of the sequence of a native or modified heterologous protein. The immunoglobulin constant domain sequences may be obtained from any immunoglobulin subtype, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtype or class, such as IgA, IgE, IgD or IgM. Preferentially, they are derived from human immunoglobulins, more preferably from human IgG, even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc-fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc and scFv fragments linked to the CH2 domain of the constant region of an immunoglobulin heavy chain, containing an N-linked glycosylation site. Fc-fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of the constant region of an immunoglobulin heavy chain, which contains an N-linked glycosylation site, into another expression construct, which contains, for example, another immunoglobulin domain, an enzymatically active protein part, or an effector domain. Thus, Fc-fusion proteins according to the invention also include single chain Fv fragments linked to the CH2 domain of the constant region of an immunoglobulin heavy chain, which contains, for example, an N-linked glycosylation site.

[0057] The term "cytokine" refers to small proteins released by cells that act as intercellular mediators, e.g., affect the behavior of cells surrounding the secreting cell. Cytokines can be secreted by immune cells or other cells, e.g., T cells, B cells, natural killer cells, and macrophages. Cytokines can be involved in intercellular signaling events, e.g., autocrine signaling, paracrine signaling, and endocrine signaling. They can mediate a range of biological processes, including, but not limited to, immunity, inflammation, and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

[0058] As used herein, "growth factor" refers to a protein or polypeptide that is capable of stimulating cell proliferation.

[0059] In a related aspect, the present invention further relates to the use of a mammalian expression vector of the invention for the expression of a protein and / or a non-coding RNA of interest in mammalian cells, in particular rodent cells, such as CHO cells, in which the bacterial glutamine synthetase encoded by the mammalian expression vector serves as a selection marker.

[0060] In a second aspect, the invention relates to a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest. The nucleic acid sequence may be part of a mammalian expression vector of the first aspect. Thus, the embodiments and examples specified for the first aspect apply to this aspect as well. In particular, in certain embodiments, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof (e.g. E130X and / or F226Y), where X is any amino acid, preferably where the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. Preferably, the mutation is E130X and / or F226, where E130X is E130F or E130G; alternatively, the mutation is F226Y and / or R345A; alternatively, the mutation is E130X and / or R345A, optionally further comprising the mutation F226Y.

[0061] In a further third aspect, the present invention relates to a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. In a particular embodiment, the present invention provides a bacterial glutamine synthetase, such as from Providencia vermicola, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof (e.g., E130X and / or F226Y), where X is any amino acid, preferably where the mutation at amino acid position E130 is more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. or a bacterial glutamine synthetase comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14 and an E130X and / or F226Y and / or R345A mutation, where X is any amino acid, preferably where the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. Preferably, the mutation is E130X and / or F226, where E130X is E130F or E130G; alternatively, the mutation is F226Y and / or R345A; alternatively, the mutation is E130X and / or R345A, optionally further comprising an F226Y mutation, preferably E130X or R345A, optionally further comprising an F226Y mutation. The embodiments and examples specified with respect to the first and second aspects apply to this aspect as well.

[0062] In a fourth aspect, the present invention relates to a kit comprising an expression vector according to the invention and optionally a glutamine-free cell culture medium.

[0063] In a fifth aspect, the present invention relates to the use of bacterial glutamine synthetase as a selectable marker in mammalian cells. The bacterial glutamine synthetase is further specified as disclosed in relation to the first aspect above. More specifically, the bacterial glutamine synthetase is from a bacterium of the order Enterobacteriales and the family Morganellaceae, preferably from the genus Providencia or Photorhabdus, more preferably Providencia vermicola or Photorhabdus luminescens, even more preferably Providencia vermicola. Preferably, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1. In a particular embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90%, preferably at least 95% sequence identity to SEQ ID NO:1. In other embodiments, the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14. Upon transfection or transduction of a mammalian expression vector containing the bacterial glutamine synthetase, the bacterial glutamine synthetase mediates increased selection stringency and / or genetic stability of a protein of interest and / or non-coding RNA stably co-integrated in a CHO cell compared to a glutamine synthetase of a CHO cell having the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, the bacterial glutamine synthetase is derived from the genus Providencia and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1; more preferably, the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase.In another preferred embodiment, the bacterial glutamine synthetase is derived from the genus Photorhabdus and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14; more preferably, the bacterial glutamine synthetase is Photorhabdus luminescens glutamine synthetase. The amino acid sequences of Providencia vermicola glutamine synthetase (SEQ ID NO: 1) and Photorhabdus luminescens glutamine synthetase (SEQ ID NO: 14) have 87.2% sequence identity (see Table B).

[0064] The bacterial glutamine synthetase may further comprise a point mutation that further attenuates the glutamine synthetase (e.g. E130X and / or R345A) or enhances its catalytic activity (e.g. F226Y). In particular, the bacterial glutamine synthetase may have a point mutation within the highly conserved residue corresponding to position E130 of the glutamine synthetase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation at position E130 is a substitution with any amino acid (X), preferably the mutation at the amino acid position corresponding to E130 is a substitution with an aromatic or hydrophobic amino acid, for example a substitution with an aromatic or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M and I. In a particular embodiment, the mutation is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G) (SEQ ID NO: 4 or SEQ ID NO: 5, respectively). Alternatively or additionally, the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position F226 of the Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO:1; wherein the point mutation is a substitution with amino acid Y (Tyr, tyrosine), also referred to as F226Y. In a particular embodiment, the mutation is E130X and / or F226Y, where X is any amino acid or as specified in this paragraph above. More preferably, the mutation is E130X, or E130X and F226Y. In a specific embodiment, the mutation is E130F / G, or E130F / G and F226Y. Alternatively or additionally, the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position R345 of the Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO:1. The point mutation corresponding to amino acid position R345 of SEQ ID NO:1 is a substitution with the amino acid A (Ala, alanine), also referred to as R345A, or alternatively with another amino acid having an aliphatic side chain, such as glycine (G), valine (V), leucine (L) or isoleucine (I), preferably glycine (G).Without being bound by theory, the substitution of the highly conserved arginine (R) corresponding to amino acid position R345 in SEQ ID NO: 1 attenuates bacterial glutamine synthetase, where attenuation in this context means reduced enzymatic activity. Optionally, bacterial glutamine synthetase may further comprise a point mutation F226Y. Thus, in a particular embodiment, the mutation is E130X and / or R345A (or G, V, L or I) (wherein X is any amino acid or as specified in this paragraph above) optionally further comprising F226Y. Preferably, the mutation is E130F / G and / or R345A, optionally further comprising F226Y, such as E130F / G and R345A, optionally further comprising F226Y, or preferably E130F / G or R345A, optionally further comprising F226Y. Preferably, the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, further comprising a point mutation as set forth above.

[0065] In a preferred embodiment, the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and a mutation selected from the group consisting of E130X, F226Y, R345A (or G, V, L or I) and combinations thereof (e.g. E130X and / or F226Y), where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic and / or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I. Preferably, the amino acid sequence has at least 96%, preferably at least 98%, preferably at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14, even more preferably the amino acid sequence has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and further comprises mutations as disclosed herein, e.g. as exemplified in the amino acid sequences of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 20, 21, 22 and 29 (based on SEQ ID NO:1), or SEQ ID NO:23, 24, 25, 26, 27, 28 and 30 (based on SEQ ID NO:14), preferably in the amino acid sequences of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 20 and 21 (based on SEQ ID NO:1), or SEQ ID NO:23, 24, 25, 26 and 28 (based on SEQ ID NO:14). In a particular embodiment, the mutation is E130X and / or F226Y, preferably wherein E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F / G). In a particular embodiment, the mutation is F226Y and / or R345A, more preferably R345A, or R345A and F226Y. In a particular embodiment, the mutation is E130X and / or R345A (or G, V, L or I), optionally further comprising F226Y, where X is any amino acid or as specified in this paragraph above. Preferably, the mutation is F130F / G and / or R345A optionally further comprising F226Y, such as E130F / G and R345A optionally further comprising F226Y, or preferably E130F / G or R345A optionally further comprising F226Y.

[0066] In a sixth aspect, the present invention relates to a mammalian host cell comprising a mammalian expression vector of the invention (first aspect), a nucleic acid sequence of the invention (second aspect), or a nucleic acid sequence encoding a bacterial glutamine synthetase of the invention (third aspect). The mammalian host cell may be any host cell, provided that the host cell is an immortalized cell and not a primary cell. In a particular embodiment, the mammalian host cell is a mouse, human or rodent cell, more preferably a rodent cell, even more preferably a CHO cell. Furthermore, the mammalian host cell is preferably a glutamine synthetase gene knockout cell (GS knockout mutant) host cell. The term "GS gene knockout cell" as used herein refers to a cell in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in disruption of the enzymatic function of GS. Such cells may be referred to as GS- / - cells or GS- / + cells, depending on whether both alleles or only one have been deleted or disrupted. Exogenous glutamine supplementation or the GS gene introduced by an expression vector is essential for cell survival of GS gene knockout cells. In a preferred embodiment, the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS- / -) cell. In a particular embodiment, the mammalian host cell is a monoclonal cell line, generated by a process of single cell cloning and clonal expansion. Preferably, the mammalian host cell comprises a polynucleotide encoding bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or non-coding RNA of interest simultaneously integrated into the host cell genome.

[0067] Methods for preparing cell lines or for producing proteins In a seventh aspect, the present invention relates to a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising the steps of: (a) introducing into a mammalian host cell an expression vector according to the present invention (first aspect) or a nucleic acid according to the present invention (second aspect), comprising a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest; and (b) culturing the mammalian host cell in a medium free of any glutamine under conditions selecting for bacterial glutamine synthetase, wherein the at least one polynucleotide encoding the protein and / or the non-coding RNA of interest is co-integrated into the host cell genome together with the polynucleotide encoding the bacterial glutamine synthetase. Optionally, the method may further comprise the steps of culturing the resulting pool of cells stably expressing the protein and / or the non-coding RNA of interest selected in step (b) and / or isolating the pool of cells stably expressing the protein and / or the non-coding RNA of interest. A cell stably expressing a protein and / or non-coding RNA of interest means that a polynucleotide encoding the protein and / or non-coding RNA of interest is stably integrated into the genome of the host cell and the protein and / or non-coding RNA of interest is stably expressed, i.e. expressed for an extended period of time, e.g. for at least 40 days, preferably for several months or years.

[0068] The mammalian expression vector or nucleic acid may be a plasmid, a bacterial artificial chromosome (BAC) or a viral vector. Thus, the mammalian expression vector or nucleic acid of the present invention may be introduced by transfection or transduction, respectively. Preferably, the mammalian expression vector or nucleic acid is a plasmid. More preferably, the mammalian expression vector or nucleic acid is introduced by stable transfection. Methods for transducing or transfecting mammalian expression vectors or nucleic acids into mammalian cells are well known in the art and include chemical means, such as calcium phosphate precipitation and lipofection, and physical means, such as electroporation. The polynucleotide encoding bacterial glutamine synthetase and the polynucleotide encoding the protein and / or non-coding RNA of interest are operably linked to a mammalian promoter and thus adapted for expression in mammalian host cells.

[0069] The method of the present invention may further comprise a step (c) of isolating a single clone for clonal propagation to prepare a monoclonal cell line. Those skilled in the art will appreciate that transfection or transduction often requires a large number of cells, resulting in a heterogeneous pool of recombinant cells, e.g., with a diverse population of integration sites. For the generation of a clonal cell line, the cell pool is diluted or selected for the isolation of a single cell (monoclonality), and each single clone is subjected to clonal amplification to prepare a monoclonal cell line. The term "cell line" as used herein refers to a cell population derived from a single cell clone and can be propagated for an indefinite period of time. It is therefore also referred to as a monoclonal cell line. Thus, the cell line is genetically stable, and therefore the characteristics of the cell line should not change over time. In particular, phenotypic characteristics such as production levels (titer) and growth rate and density (viable cell density and maximum viable cell density), viability, and genetic integrity as measured via copy number and DNA fingerprinting assays should be maintained when cultured under comparable conditions.

[0070] The cell pool or monoclonal cell line prepared according to the method of the present invention can further be used to stably produce a protein of interest or to stably produce non-coding RNA, such as RNA that mediates RNA interference, such as microRNA, small interfering RNA, long non-coding RNA, or small hairpin RNA. RNA interference can be used to silence genes and therefore to create cell pools or monoclonal cell lines with beneficial properties, such as for the production of proteins. For example, the difficulty of removing host cell proteins (HCPs) can be silenced in the cell pool or monoclonal cell line, or enzymes such as fucosyltransferases can be silenced to modify the glycosylation profile of the cell pool or monoclonal cell line. Furthermore, CHO cells, which are commonly used for large-scale industrial production, are often engineered to improve their characteristics in the production process or to facilitate the selection of recombinant cells. Such engineering includes, but is not limited to, improved resistance to apoptosis, reduced autophagy, increased cell proliferation, altered expression of cell cycle regulatory proteins, chaperone engineering, unfolded protein response (UPR) engineering, secretory pathway engineering, and metabolic engineering. Such engineering may potentially be achieved using RNA interference in mammalian host cells generated by the methods of the invention.

[0071] In a related eighth aspect, the invention relates to a method for producing a protein of interest, comprising the steps of: (a) introducing a mammalian expression vector according to the invention or a nucleic acid according to the invention into a mammalian host cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium free of glutamine under conditions for selection of the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding the protein of interest is co-integrated into the host cell genome with the polynucleotide encoding the bacterial glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the mammalian host cell under conditions for production of the protein of interest; and (e) collecting and optionally purifying the protein of interest. The mammalian expression vector or nucleic acid of the invention may be introduced by transfection or transduction. Preferably, the mammalian expression vector or nucleic acid is introduced by stable transfection.

[0072] Those skilled in the art will understand that for mammalian expression vectors, typically only a small portion of the vector is integrated into the genome of the host cell. Thus, the integrated portion of the vector comprises a polynucleotide encoding bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or non-coding RNA of interest. Furthermore, the integrated portion of the mammalian expression vector or the integrated nucleic acid sequence of the present invention may be further amplified, for example, by increasing the concentration of a glutamine synthetase inhibitor, such as methionine sulfoximine (MSX). Amplification is optional, and may result in higher productivity due to a higher copy number of polynucleotides encoding the protein and / or non-coding RNA of interest, since they become co-amplified together with the bacterial glutamine synthetase. The method according to the present invention may include the generation of cell pools or monoclonal cell lines. Furthermore, the mammalian host cell generated according to the method of the present invention or the mammalian host cell described in the present invention may be further used to produce a protein and / or non-coding RNA of interest or in a method for producing a protein of interest.

[0073] Thus, in a further ninth aspect, the present invention relates to a method for producing a protein of interest comprising the steps of: (a) providing a mammalian host cell according to the present invention or produced by a method according to the present invention, comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions for production of the protein of interest; and (c) harvesting and optionally purifying the protein of interest.

[0074] In a preferred embodiment of the method of the present invention, the mammalian host cell is a GS gene knockout cell. Those skilled in the art will understand that this refers to the endogenous GS gene, whereas the bacterial GS gene is present in the host cell after transfection or transduction of the mammalian expression vector or nucleic acid sequence of the present invention. The mammalian host cell (transfected or transduced with the mammalian expression vector or nucleic acid sequence of the present invention) is cultured in a medium that does not contain any glutamine under conditions that select for bacterial glutamine synthetase in step (b). This may further include the addition of the GS inhibitor methionine sulfoximine (MSX).

[0075] In a particular embodiment of the method of the invention, the cells (cell pools) or monoclonal cell lines are generated with increased selection stringency and / or have increased genetic stability and / or have higher productivity compared to cells or cell lines generated with glutamine synthetase from Chryseturus griseus having the amino acid sequence of SEQ ID NO: 2. Genetic stability can be quantified by measuring the copy number of the integrated transgene. Genetic stability is evaluated by measuring the copy number over a longer period of culture time. In addition, genome rearrangements are monitored, for example via Southern blot analysis. Increased selection stringency can be determined by the time to reach a viability of more than 70%.

[0076] The mammalian host cell can be any host cell, provided that the host cell is an immortalized cell and not a primary cell. The methods described herein are in vitro methods, and the mammalian host cell described in the present invention is for in vitro use in cell culture. The term "mammalian cell" as used herein refers to a mammalian cell line suitable for the production of a product of interest, such as a heterologous protein of interest and / or a non-coding RNA, and may also be referred to as a "host cell" or a "mammalian host cell". The mammalian cells are preferably transformed and / or immortalized cell lines. They are adapted to continuous passage in cell culture, preferably serum-free cell culture and / or preferably suspension culture, and do not include primary untransformed cells or cells that are part of an organ structure.

[0077] Preferably, the mammalian host cell is a mouse, human or rodent cell, more preferably a rodent cell, even more preferably a CHO cell. Preferred mammalian cells for heterologous protein production are mouse, rodent or human cells. Preferred examples of mammalian cells or mammalian cell lines are CHO cells (e.g. DG44 and K1), NS0 cells, HEK293 cells (e.g. HEK293 cells or HEK293T cells) and BHK21 cells. Preferably, the mammalian cells or mammalian cell lines are adapted to grow in suspension. In a preferred embodiment, the mammalian cells or mammalian cell lines are CHO cells. In a particular embodiment, the mammalian cells are HEK293 cells or CHO cells or HEK293 cells or CHO cell derived cells, preferably the mammalian cells are CHO cells or CHO derived cells.

[0078] Suitable rodent cells may be, for example, hamster cells, in particular BHK21 cells, BHK TK cells, CHO cells, CHO-K1 cells, CHO-DXB11 cells (also called CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or derivatives / progenies of any of such cell lines. Particularly preferred are CHO cells, such as CHO-DG44 cells, CHO-K1 cells, and BHK21 cells, and even more preferred are CHO-DG44 cells and CHO-K1 cells. Most preferred are CHO-DG44 cells. Glutamine synthetase (GS)-deficient derivatives of mammalian cells, in particular CHO-DG44 cells and CHO-K1 cells, are also included. In one embodiment of the invention, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO DXB11 cell, a CHO-S cell, a CHO GS-deficient cell, or a derivative thereof. A suitable human cell is a HEK293 cell or a HEK293T cell. The host cell may be a mouse cell, such as a mouse myeloma cell, for example a NS0 cell and a Sp2 / 0 cell, or a derivative / descendant of any of such cell lines.

[0079] Moreover, the mammalian host cell is preferably a GS gene knockout cell (GS knockout mutant). As used herein, the term "GS gene knockout cell" refers to a cell in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in disruption of the enzymatic function of GS. Such cells may be referred to as GS- / - or GS- / + cells, depending on whether both alleles or only one are deleted or disrupted. Exogenous glutamine supplementation or the GS gene introduced by the expression vector is essential for cell survival of the GS gene knockout cell. In a preferred embodiment, the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS- / -) cell.

[0080] Preferably, CHO cells are metabolically engineered, for example by knockout of endogenous glutamine synthetase (GS), to allow for an efficient cell line development process, to facilitate selection with methionine sulfoximine (MSX).

[0081] Non-limiting examples of mammalian cells that may be used in the context of the present invention are also summarized in Table A. However, derivatives / progeny of such cells, other mammalian cells, including but not limited to human, mouse, rat, monkey and rodent cell lines, may also be used in the present invention, particularly for the production of biopharmaceutical proteins.

[0082] [Table 1]

[0083] Most preferably, the cells are established, adapted and cultured completely under serum-free conditions and, optionally, in a medium completely free of any proteins / peptides of animal origin. Commercially available media, such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any of the media may be supplemented with a wide variety of compounds as necessary, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium phosphate, magnesium phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose, or other equivalent energy sources, antibiotics, and trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. For the growth and selection of genetically modified cells expressing the selection gene, an appropriate selection agent is added to the culture medium.

[0084] The protein of interest encoded by a mammalian expression vector or produced by the method of the invention is preferably produced in CHO cells in cell culture. After expression, the recombinant protein is harvested and further purified. The antibody may be recovered from the culture medium as a secreted protein in harvested cell culture fluid (HCCF) or from a cell lysate (i.e., a liquid containing the contents of cells lysed by any means, including but not limited to enzymatic, chemical, osmotic, mechanical, and / or physical disruption of the cell membrane and optionally the cell wall) and purified using the techniques described herein. According to the invention, the method comprises the step of providing a harvested cell culture fluid comprising the protein of interest, e.g., an antibody, as a starting material, wherein the HCCF is derived from a CHO cell culture. Preferably, the protein of interest, e.g., an antibody, is recovered from the harvested cell culture fluid after cell separation, e.g., by filtration and / or centrifugation. Thus, in certain embodiments, harvesting comprises centrifugation and / or filtration to produce a harvested cell culture fluid.

[0085] In view of the above, it will be appreciated that the present invention also encompasses the following:

[0086] Item 1 provides a mammalian expression vector (e.g., a mammalian expression vector) comprising a polynucleotide encoding a bacterial glutamine synthetase as a selectable marker, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0087] Item 2 specifies the mammalian expression vector of item 1, wherein the bacterial glutamine synthetase is derived from a bacterium of the order Enterobacteriale and the family Morganellaceae.

[0088] Item 3 specifies the mammalian expression vector of items 1 or 2, wherein the bacterial glutamine synthetase is from the genus Providencia or Photorhabdus, preferably the bacterial glutamine synthetase is Providencia vermicola glutamine synthetase or Photorhabdus luminescens glutamine synthetase.

[0089] Item 4 specifies the mammalian expression vector of any one of items 1 to 3, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0090] Item 5 specifies the mammalian expression vector of any one of items 1 to 4, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and a mutation selected from the group consisting of E130X, F226Y, R345A, and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, V, L, M and I.

[0091] Item 6 further specifies the mammalian expression vector of items 4 or 5, wherein the mutation is E130X and / or F226Y and / or R345A, where E130X is E130F or E130G.

[0092] Paragraph 7 further specifies the mammalian expression vector of any one of paragraphs 1 to 6, wherein the polynucleotide encoding the bacterial glutamine synthetase is operably linked to a mammalian promoter.

[0093] Item 8 further specifies the mammalian expression vector of any one of items 1 to 7, wherein the mammalian expression vector further comprises an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0094] Item 9 further specifies the mammalian expression vector according to any one of items 1 to 8, wherein the protein of interest is a therapeutic protein, preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

[0095] Item 10 further specifies the mammalian expression vector according to item 9, wherein the protein of interest is an antibody, preferably wherein the expression vector comprises a polynucleotide comprising a coding sequence for the variable region of the heavy chain and / or a coding sequence for the variable region of the light chain of the antibody.

[0096] Item 11 further specifies the mammalian expression vector according to any one of items 8 to 10, wherein the mammalian expression vector comprises a polycistronic expression cassette and / or a plurality of expression cassettes, preferably wherein the expression vector comprises a plurality of expression cassettes.

[0097] Item 12 further specifies the mammalian expression vector of any one of items 8 to 11, wherein the mammalian expression vector is for stable transfection and the integration portion of the vector comprises a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0098] Item 13 further specifies the mammalian expression vector according to any one of items 1 to 12, wherein the mammalian expression vector is a plasmid, a bacterial artificial chromosome (BAC) or a viral vector.

[0099] Item 14 provides a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0100] Item 15 further specifies the nucleic acid sequence of item 14, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0101] Item 16 further specifies the amino acid sequence of item 15, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0102] Item 17 further specifies the nucleic acid sequence of item 15, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a mutation selected from the group consisting of E130X, F226Y, R345A, and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0103] Item 18 further specifies the nucleic acid sequence of any one of items 15 to 17, wherein the mutation is E130X and / or F226 and / or R345A, wherein E130X is E130F or E130G.

[0104] Item 19 provides a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0105] Item 20 provides a glutamine synthetase from Providencia vermicola comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0106] Item 21 provides a bacterial glutamine synthetase from Photorhabdus luminescens comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0107] Item 22 provides a mammalian host cell comprising the expression vector of any one of items 1 to 13, the nucleic acid sequence of any one of items 14 to 18, or the nucleic acid sequence encoding a bacterial glutamine synthetase of any one of items 19 to 21, preferably wherein the mammalian host cell is a rodent cell, more preferably a CHO cell and / or (b) a GS gene knockout cell.

[0108] Item 23 further specifies the mammalian host cell of item 22, which comprises a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest simultaneously integrated into the host cell genome.

[0109] Item 24 provides a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising: (a) introducing into a mammalian host cell, e.g., a CHO cell, an expression vector according to items 1 to 13, or a nucleic acid according to any one of items 14 to 18, comprising a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest; and (b) culturing the mammalian host cell in a medium free of glutamine under conditions selecting for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding the protein and / or the non-coding RNA of interest is simultaneously integrated into the host cell genome together with the polynucleotide encoding the bacterial glutamine synthetase.

[0110] Item 25 specifies the method of item 24, further comprising the step of (c) isolating a single clone for clonal expansion to prepare a monoclonal cell line.

[0111] Item 26 provides a method for producing a protein of interest, comprising: (a) introducing the mammalian expression vector according to items 1 to 13 or the nucleic acid according to any one of items 14 to 18 into a mammalian host cell, preferably a mammalian host cell, such as a CHO cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium free of glutamine under conditions that select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is simultaneously integrated into the host cell genome together with the polynucleotide encoding the bacterial glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the mammalian host cell under conditions that produce the protein of interest; and (e) collecting and optionally purifying the protein of interest.

[0112] Item 27 further specifies the method of any one of items 24 to 26, wherein the mammalian expression vector or nucleic acid is introduced by transfection or transduction.

[0113] Paragraph 28 further specifies the method of paragraph 27, wherein the mammalian expression vector or nucleic acid is introduced by stable transfection.

[0114] Item 29 provides a method for producing a protein of interest, comprising: (a) providing a mammalian host cell of item 22 or 23, the mammalian host cell comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions for producing the protein of interest; and (c) collecting and optionally purifying the protein of interest.

[0115] Item 30 further specifies the method of any one of items 24 to 29, wherein (a) the mammalian host cell is a GS gene knockout cell, and / or (b) the step of culturing the mammalian host cell in a medium that is completely devoid of glutamine under conditions that select for the bacterial glutamine synthetase of step (b) comprises the addition of the GS inhibitor methionine sulfoximine (MSX).

[0116] Item 31 further specifies the method of any one of items 24 to 30, wherein the mammalian host cell is a rodent cell, preferably a CHO cell.

[0117] Item 32 further specifies the method of any one of items 24 to 30, wherein the cell or cell line is produced with increased selection stringency and / or exhibits increased genetic stability and / or has higher productivity compared to a cell or cell line produced using a glutamine synthetase from Chrysetrus griseus having the amino acid sequence of SEQ ID NO:2.

[0118] Item 33 provides a kit comprising the expression vector of any one of Items 1 to 13 and a cell culture medium that does not contain glutamine.

[0119] Item 34 provides the use of bacterial glutamine synthetase as a selectable marker in a mammalian cell, preferably wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0120] Item 35 further specifies the use of item 34, wherein the bacterial glutamine synthetase is derived from a bacterium of the order Enterobacteriale and the family Morganellaceae, more preferably from the genus Providencia or Photorhabdus, even more preferably from Providencia vermicola or Photorhabdus luminescens.

[0121] Item 36 further specifies the use of item 35, wherein the bacterial glutamine synthetase (a) is derived from the genus Providencia and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, more preferably wherein the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase.

[0122] Item 37 further specifies the use of any one of items 34 to 36, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0123] Item 38 further specifies the use of item 34, wherein the bacterial glutamine synthetase is derived from the genus Photorhabdus and / or comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, more preferably wherein the bacterial glutamine synthetase is the glutamine synthetase of Photorhabdus luminescens.

[0124] Item 39 further specifies the use of any one of items 34, 35 and 38, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0125] Item 40 further specifies the use of items 37 or 39, wherein the mutation is E130X and / or F226Y and / or R345A, where E130X is E130F or E130G.

[0126] Working Example Linearization of plasmids for transfection The plasmids used for stable transfection contain a cytomegalovirus-driven antibody transcription cassette, an ampicillin transgene, and a glutamine synthetase transgene as a metabolic selection marker. Plasmids were linearized for transfection using PvuI (cuts once in the ampicillin transgene). Restriction enzyme digestion of 20 μg of plasmid DNA was performed with PvuI (NEB) for 3 hours at 37° C. according to the manufacturer's protocol. The linearized plasmid DNA was purified (Qiagen Plasmid Maxi Kit) according to the manufacturer's protocol. Final DNA concentration was determined via a Nanodrop spectrophotometer.

[0127] Host cell culture Host cells of the CHO-K1-GS knockout cell line (also called CHO-K1-GS KO or simply CHO-K1-GS), which has a genomic knockout of the endogenous glutamine synthetase gene, were cultured in host cell medium supplemented with L-glutamine. The host cell culture was at 3 × 10 cells per mL. 5 A seeding density of 1000 cells was initiated. Growth conditions were set at 36.5°C and 5% carbon dioxide in a shaking incubator at 120 rpm in shake flasks. Cell density and viability determinations were performed with a Sedex HiRes© cell counter.

[0128] Transfection of linearized plasmids into CHO cells One day before transfection, host cells were cultured at 0.8 × 10 6Cells were seeded at a cell density of 10 cells / mL. On the day of transfection, cell density and viability were determined and the amount of cells required for transfection was centrifuged at 750 x G for 7 min. The supernatant was discarded. 10 μg of linearized plasmid DNA per transfection was transfected using the Neon© transfection system (Invitrogen) and electroporation. An electroporation cuvette was filled with 3 mL of electroporation buffer E2 and 89 μl of buffer R was added to the cells (5 x 10 6 The cells were resuspended and mixed with 10 μg of linearized plasmid. Transfection was performed using 1500 volts, 10 ms, and 2 pulses. The transfected cells were transferred to 5 ml of pre-warmed host cell medium in a T25 ml flask and incubated at 8% carbon dioxide and 37° C. for at least 24 hours.

[0129] Selection of stable CHO cell pools 24 hours after transfection, cells were transferred to selection medium (medium without L-glutamine). 10 mL of selection medium was pre-warmed for every pool in a T75 flask. Two stable pools were cultured for each transfection. Cells were centrifuged (750×G for 7 min), resuspended in 20 mL selection medium, and incubated at 37° C. with 8% carbon dioxide. Cells were monitored by microscope during selection. Additional selection medium (5 mL) was added after 7 days.

[0130] Stable pool passaging and production process The selection phase was deemed successful once the cells reached at least 70% viability and a doubling time of 48 h or less. After selection, cells were cultured at 3 x 10 per mL in a total volume of 30 mL of medium (containing no L-glutamine) in 125 mL shake flasks. 5Starting with 1 cell, cells were passaged every 2 to 3 days at 36.5° C. and 5% carbon dioxide with shaking at 120 rpm. Samples for titer determination were taken periodically. If the antibody titer was stable for at least 14 days, indicating phenotypic stability, the production run was started for at least 7 days.

[0131] For the production run, cells were cultured at 7 x 10 per mL in shake flasks. 5 At a density of viable cells per well, the cells were seeded in a total volume of 30 mL of basal medium without glutamine and cultivated at 34.5° C. with 5% carbon dioxide and shaking at 120 rpm. During a production run of at least 7 days, cells were counted, samples were withdrawn once a day for pH, glucose and titer measurements, and pH and glucose feeds were adjusted if necessary. Starting from day 2, a daily feed (glutamine water-in-oil) was supplemented. pH was determined using a RAPIDlab348Ex© and glucose levels were measured via an EKF diagnostic instrument. During and / or at the end of the culture, cell culture medium supernatants were analyzed for antibody titers after centrifugation using a ForteBio Octet© instrument with a Protein A biosensor. Sample dilutions and standard curves were processed during the same production run.

[0132] Example 1: Production of antibodies in CHO cells using CHO wild-type glutamine synthetase as a metabolic selection marker CHO-K1-GS cells, once transfected with a vector carrying the glutamine synthetase derived from Chryseturus griseus (CHO wild-type glutamine synthetase (GS), SEQ ID NO:2) selectable marker, can survive selection under culture in medium not supplemented with L-glutamine if the transgene vector is stably integrated into the genome of the cells.

[0133] CHO-K1-GS cells were transfected with a vector carrying an expression cassette for monoclonal antibody 1 (mAb1) and CHO wild-type glutamine synthetase. After selection, stable CHO pools were passaged as described above and samples were taken periodically for measurement of the titer of monoclonal antibody 1. Under the applied experimental conditions, the stable CHO pools (transfected with CHO wild-type GS) remained stable and productive for about 20 days after transfection, after which the level of productivity (titer) decreased (Figure 1).

[0134] Example 2: Production of antibodies in CHO cells using GS from Providencia vermicola and other bacteria as selection markers To functionally test the novel bacterial glutamine synthetase mutants, the stringency of selection (time to reach >70% viability after transfection), the number of productive passages, and the specific productivity were measured over time. For CHO pools that remained productive after extended passages, a 7-day production run in shake flasks was followed in a controlled process setting to further evaluate cell culture process parameters.

[0135] CHO-K1-GS cells were transfected with vectors carrying expression cassettes for monoclonal antibody 1 and glutamine synthetase from Crisetulus griseus (SEQ ID NO: 2) or Providencia vermicola (SEQ ID NO: 1), and viability, viable cell count (VCD) and productivity were measured over time during the selection period. Surprisingly, CHO cells transfected with bacterial class I glutamine synthetase from Providencia vermicola survived the selection process, suggesting that this prokaryotic metabolic selection marker is functional in CHO cells. Cells transfected with Providencia glutamine synthetase took longer to fully recover from selection (Figure 2A) and cell growth was slower (Figure 2B) compared to CHO glutamine synthetase, suggesting an increased stringency of selection.

[0136] The titers were measured during cell selection and showed that the productivity of cells carrying the CHO GS gene dropped rapidly. Cells transfected with Providencia glutamine synthetase stably expressed monoclonal antibody 1 even after the selection process (Figure 3A). An additional production step of at least 7 days was performed after selection and passaging, suggesting that the phenotypic stability of cells transfected with Providencia glutamine synthetase was significantly higher compared to CHO glutamine synthetase (Figure 3B). Cells transfected with CHO glutamine synthetase were not transferred to the production step due to lack of productivity.

[0137] Overall, CHO cells transfected with Providencia vermicola glutamine synthetase showed increased selection stringency and significantly higher productivity. Furthermore, cell pools showed increased phenotypic stability and remained productive for at least 40 days, whereas cells transfected with CHO glutamine synthetase completely lost their productivity about 20 days after transfection.

[0138] In addition to the glutamine synthetase from Providencia vermicola, a total of 10 prokaryotic glutamine synthetases were tested, 9 of which were from bacteria and 1 from Archaea (Methanocaldococcus janaschii). Six were found to be functional in CHO cells and 4 were found to be non-functional. Functional was defined as surviving the selection process and producing antibody-producing cells. The results are summarized in Table B below, where (+++) indicates excellent performance, superior to wild-type CHO GS, (++) indicates moderate performance, similar to wild-type CHO GS, and (+) indicates poor performance, clearly inferior to wild-type CHO GS. The majority of bacterial GSs that were at least successfully used for selection belong to the order Enterobacteriales and the family Morganellaceae and / or have at least 80% sequence identity to the GS from Providencia vermicola. However, most of these bacterial GSs were found to show moderate performance, clearly inferior to wild-type CHO GS, taking into account the stringency of selection and genetic stability, except for the GS derived from Photorhabdus luminescens, which showed excellent performance (stringency of selection / genetic stability) superior to wild-type CHO GS.

[0139] [Table 2]

[0140] Selection stringency is analyzed by measuring the recovery rate of viability above 70%, and genetic stability is demonstrated by stable productivity over a specific period of time, such as more than 20 days after transfection. Representative data of a selection experiment monitoring the viability of stable CHO pools using GS derived from Providencia vermicola, Photorhabdus luminescens, and Budvisia aquatica as selection markers are shown in Figure 4A, and representative data of the productivity of CHO pools transfected with these bacterial GS are shown in Figure 4B. Although the Budvisia aquatica-derived GS was functional in CHO cells, the selection was not robust. In contrast, the Photorhabdus luminescens-derived GS remained functionally active and showed even stronger attenuated activity compared to the Providencia vermicola-derived GS (Figure 4A). Stable pools transfected with P. luminescens-derived GS recovered (reaching >70% viability) 16 to 17 days after transfection compared to 13 days after transfection for P. luminescens-derived GS. Productivity was comparable for P. luminescens- and P. vermicola-derived GS, but was slightly delayed for P. luminescens-derived GS and strongly decreased for B. aquatica-derived GS (Figure 4B).

[0141] These experiments demonstrate for the first time that bacterial GS, such as that from Providencia vermicola, can be used as a selection marker in mammalian cells and further identify bacterial GS that are closely related to other bacterial GS, such as GS from Photorhabdus luminescens, that have at least 85% sequence identity to the amino acid sequence of GS from the Enterobacteriales and Morganellaceae and / or from Providencia vermicola (SEQ ID NO:1).

[0142] Example 3: Production of antibodies in CHO cells using wild-type GS and E130F or E130G mutants of Providencia vermicola as selection markers Furthermore, two distinct point mutations (E130F and E130G) in GS from Providencia vermicola were tested in selection and production experiments. CHO-K1-GS cells were transfected with monoclonal antibody 1 and a vector carrying an expression cassette for wild-type glutamine synthetase from Providencia vermicola (SEQ ID NO:1) or glutamine synthetase from Providencia vermicola containing the mutations E130F (SEQ ID NO:4) or E130G (SEQ ID NO:5).

[0143] Both mutations appeared to further extend the extent of attenuation, extending the period during which cells could grow out of selection compared to wild-type Providencia vermicola glutamine synthetase, and both demonstrated phenotypic stability. Subsequent production runs yielded significantly improved titers overall (Figure 5).

[0144] Example 4: Antibody production and viable cell density in CHO cells using wild type GS and F226Y mutant of Providencia vermicola as selection markers CHO-K1-GS cells were transfected with vectors carrying expression cassettes for monoclonal antibody 1 and CHO wild type GS (CHO WT GS), Providencia wild type GS (SEQ ID NO: 1) or Providencia GS F226Y mutant (SEQ ID NO: 6). After stable pool generation and single cell deposition, the top clones were subjected to shake flask experiments against CHO WT GS. Both Providencia wild type GS and Providencia GS F226 outperformed CHO wild type glutamine synthetase and showed about 50% higher titers (mg / L) compared to CHO wild type GS clones. Compared to Providencia wild type GS, cell clones carrying Providencia GS F226Y showed an even increased growth behavior and higher viable cell density (Figure 6B). This may provide advantages for biopharmaceutical production, especially for combinations containing the E130 mutation.

[0145] Example 5: Antibody production and viability in CHO cells using wild-type GS and R345A or R360A mutants of Providencia vermicola as selection markers In addition, two additional point mutants of Providencia vermicola glutamine synthetase were tested and evaluated in the generation of stable antibody-producing CHO expression pools. Wild-type glutamine synthetase was tested against mutants with mutations in the catalytic domain of a selectable marker.

[0146] Whereas R360A lost functionality and no pools could be recovered from the selection experiment, R345A remained functionally active, but with a strongly attenuated activity (Figure 7A). Stable pools transfected with R345A recovered 31 days after transfection (achieving >70% viability) compared to the wild-type protein, which recovered in about 15 days. Interestingly, the strongly attenuated mutant R345A (SEQ ID NO: 20) showed a 5-10 fold increase in productivity (Figure 7B).

[0147] Overall, the results of Examples 3, 4 and 5 demonstrate that the GS of bacteria such as those from Providencia vermicola can be further attenuated and thereby improved as a selection marker by introducing one or more point mutations in conserved residues involved in binding to the substrate, such as E130F, E130G, F226Y and / or R345A. Since these residues E130, F226 and R345 are also present in the GS from Photorhabdus luminescens (amino acid sequence of SEQ ID NO: 14), these mutations are expected to similarly further attenuate the GS of this bacterium and thereby improve it as a selection marker. In particular, the F226Y mutation showed a further increased growth behavior and a higher viable cell density (Figure 6B). This may provide advantages for the production of biopharmaceuticals, especially in combination with the E130 mutation or the R345A mutation.

[0148] [Table 3]

Claims

1. 1. A mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker, the bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

1.

2. 2. The mammalian expression vector of claim 1, wherein said bacterial glutamine synthetase is derived from a bacterium of the order Enterobacteriale and the family Morganellaceae, preferably said bacterial glutamine synthetase is derived from the genus Providencia or Photorhabdus, more preferably said bacterial glutamine synthetase is Providencia vermicola glutamine synthetase or Photorhabdus luminescens glutamine synthetase.

3. 3. The mammalian expression vector of claim 1 or 2, wherein said bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

4. 4. The mammalian expression vector of any one of claims 1 to 3, wherein said bacterial glutamine synthetase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:14 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

5. 5. The mammalian expression vector of any one of claims 1 to 4, wherein said mammalian expression vector further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

6. 6. The mammalian expression vector of claim 5, wherein the protein of interest is a therapeutic protein, preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

7. A nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

8. 8. The nucleic acid sequence of claim 7, wherein said bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

9. 1. A bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, preferably wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.

10. A mammalian host cell comprising an expression vector according to any one of claims 1 to 6, a nucleic acid sequence according to claim 7 or 8, or a nucleic acid sequence encoding a bacterial glutamine synthetase according to claim 9, Preferably, said mammalian host cell is (a) a rodent cell, more preferably a Chinese Hamster Ovary (CHO) cell and / or (b) a glutamine synthetase gene knockout cell.

11. (a) introducing an expression vector according to claims 1 to 6 or a nucleic acid according to claims 7 or 8, comprising at least a polynucleotide encoding a bacterial glutamine synthetase and a polynucleotide encoding a protein and / or a non-coding RNA of interest, into a mammalian host cell, preferably a CHO cell; and (b) culturing the mammalian host cell in a medium that is devoid of glutamine under conditions that select for bacterial glutamine synthetase, wherein at least one polynucleotide encoding a protein and / or non-coding RNA of interest is co-integrated into the host cell genome with a polynucleotide encoding bacterial glutamine synthetase; 1. A method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising:

12. 12. The method of claim 11, further comprising the step of: (c) isolating a single clone for clonal expansion to prepare a monoclonal cell line.

13. (a) introducing a mammalian expression vector according to claims 1 to 6, or a nucleic acid according to claim 7 or 8 into a mammalian host cell, preferably a CHO cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, and at least one polynucleotide encoding a protein of interest; (b) culturing mammalian host cells in a medium that is devoid of glutamine under conditions that select for bacterial glutamine synthetase, wherein at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome with a polynucleotide encoding bacterial glutamine synthetase; (c) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (d) culturing the mammalian host cells under conditions to produce the protein of interest; and (e) harvesting and optionally purifying the protein of interest A method for producing a protein of interest comprising:

14. (a) providing a mammalian host cell of claim 10 comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cells under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest A method for producing a protein of interest comprising:

15. 15. The mammalian host cell of claim 10 or the method of any one of claims 11 to 14, wherein said mammalian host cell is a rodent cell, more preferably a CHO cell.

16. A kit comprising the expression vector of any one of claims 1 to 6 and a cell culture medium that does not contain glutamine.

17. Use of bacterial glutamine synthetase as a selectable marker in mammalian cells.

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