Bacterial glutamine synthase as a selection 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 the Invention The present invention relates to a mammalian expression vector containing a polynucleotide encoding a bacterial glutamine synthetase as a selectable marker having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, and to a nucleic acid encoding the bacterial glutamine synthetase and a mammalian host cell. In particular, the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and the family Morganellaceae, preferably the bacterial glutamine synthetase is a glutamine synthetase of the genus Providencia, and optionally further contains mutations at positions E130 and / or F226 and / or R345. The present invention further relates to a method for preparing mammalian cells stably expressing a protein and / or non-coding RNA of interest, and to a method for producing a protein in mammalian cells using the bacterial glutamine synthetase.

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

[0003] In cell lines lacking the dihydrofolate reductase gene, such as CHO-DG44 cells, selection is carried out in the absence of hypoxanthine and thymidine in the culture medium. An amplification step by adding gradually increasing concentrations of methotrexate (MTX) may be added. The glutamine synthase selection system is advantageous because it requires fewer gene copies for survival, and therefore selection is faster in highly productive cell pools.

[0004] Glutamine synthase (EC 6.3.1.2, also known as γ-glutamyl ammonia ligase) catalyzes the ATP-dependent condensation of ammonia and glutamic acid to form glutamine. Glutamine synthases are classified into three subgroups: GSI, GSII, and GSIII. CHO GS is a class II enzyme, a subclass mainly expressed by eukaryotic cells, while bacterial GS proteins are typically members of the GSI class. Although GSI and GSII catalyze the same reaction, they are quite different overall, showing little to no sequence similarity except for residues that form part of the active site. For example, bacterial type I GS is a complex of 12 subunits (Eisenberg et al. (2000), Biochim. Biophys. Acta 1477, 122-145), and GSII has been reported to form a stack of two or three pentameric rings (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228). Furthermore, bacterial glutamine synthases may have only very slight sequence similarities to one another. For example, Bacillus coagulans (CN 12625930 A), Mycobacterium tuberculosis (International Publication No. 2006 / 000045), or Corynebacterium glutamicum (CN 1884501 A) have a low amino acid identity of about 50% or less compared to the glutamine synthase of Providencia vermicola (Zuo et al., Scientific Reports, 2018, 8(1): 1-8 and supplementary materials).

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

[0006] Furthermore, transfected glutamine synthase selection markers have a significant impact on the selection process, phenotypic stability, and productivity of CHO-based cell lines. For example, the use of attenuated glutamine synthase mutants in CHO has been shown to further improve stability, selection behavior, and productivity (Lin et al. (2019), mAbs, 11:5, 965-976; International Publication No. 2018 / 093331, U.S. Patent No. 20190352631, International Publication No. 2017 / 197098). Most 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). The residues reported to be involved in glutamate binding are E134, E136, E196, E203, N248, G249, H253, R299, R319, E338, and R340; the residues reported to be involved in ATP binding are W130, A191, G192, P208, N255, S257, R262, R324, and Y336; and the 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 International Publication No. 2018 / 093331).

[0007] Attenuated GS mutants with mutations in the active site exhibited increased selectivity, but the duration of the selection process and the overall characteristics of cell proliferation may be impaired, negatively impacting the bioprocess outcomes. Attenuation of the selection marker represents a delicate balance between selectivity, the number of incorporated copies, and the cell culture outcomes (e.g., proliferation behavior) suitable for supporting recombinant protein production. Therefore, the discovery of novel attenuated GS mutants is necessary to improve mammalian expression vectors in cell line development.

[0008] While bacterial GS or its peptides from various species have been expressed in eukaryotic cells such as yeast for purification, as immunogens, or for other reasons (see, for example, International Publication No. 2018 / 144807 and International Publication No. 2006 / 000045A1), and while yeast or plant GS has been shown to be active in Escherichia coli (E. coli), the reverse is not true (International Publication No. 2004 / 003175A2 and European Patent No. 0240792A1), bacterial GS (GSI) was assumed to be inactive in mammalian cells. More importantly, to the best of our knowledge, bacterial GS had not previously been tested or used in mammalian cells as a selection marker.

[0009] Summary of the Invention This invention enables the creation of a highly productive and stable mammalian host cell pool expressing therapeutic proteins, particularly therapeutic antibodies, which exhibit reduced activity compared to wild-type glutamine synthase, resulting in more rigorous selection behavior and beneficial cell culture outcomes in mammalian cells such as CHO cells. This is achieved by using a specific bacterial glutamine synthase gene unrelated to mammalian glutamine synthase genes. This is the first time that a stable and productive mammalian cell pool for cell line development has been created using prokaryotic glutamine synthase mutants. Furthermore, point mutations in Providencia vermicola glutamine synthase were identified that exhibit expanded selectivity and improved antibody titers compared to wild-type Providencia vermicola glutamine synthase. Another advantage of bacterial glutamine synthase selection markers is that, due to their low sequence similarity to mammalian glutamine synthase (e.g., Chinese hamster ovary cells), the probability of recombination events that restore endogenous glutamine synthase and thus allow cells that do not produce the gene of interest to survive is dramatically reduced.

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

[0011] A mammalian expression vector may further encode a bacterial glutamine synthase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, and mutations selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at amino acid position E130 is more preferably a substitution with an aromatic amino acid or a hydrophobic amino acid 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 mutations selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at amino acid position E130 is more preferably a substitution with an aromatic amino acid or a hydrophobic amino acid 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 includes an expression cassette comprising at least one polynucleotide encoding the protein of interest and / or non-coding RNA, preferably at least one polynucleotide encoding the protein of interest and / or non-coding RNA. The protein of interest is preferably a therapeutic protein, and more preferably selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds.

[0013] The present invention further provides a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthase operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein of interest and / or non-coding RNA. In a particular embodiment, the bacterial glutamine synthase 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 the mutation at the E130 position of the amino acid is more preferably 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.

[0014] In yet another embodiment, the present invention provides a bacterial glutamine synthase derived from Providencia vermicola, comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of Sequence 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 the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic amino acid or a hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M and I.

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

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

[0017] The present invention further provides a method for producing a protein of interest, comprising: (a) introducing a mammalian expression vector or nucleic acid described in the present invention into a mammalian host cell, preferably a CHO cell (the expression vector comprising a polynucleotide encoding a bacterial glutamine synthase and at least one polynucleotide encoding a protein of interest operably linked to a mammalian promoter); (b) culturing the mammalian host cell in a glutamine-free medium under conditions selected for bacterial glutamine synthase (the at least one polynucleotide encoding the protein of interest being simultaneously incorporated into the host cell genome together with the polynucleotide encoding bacterial glutamine synthase); (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 embodiment, a method for producing a protein of interest is provided, comprising the steps of: (a) preparing a mammalian host cell of the present invention comprising a polynucleotide encoding a bacterial glutamine synthase operably linked to a mammalian promoter, and at least one polynucleotide encoding the protein of interest; (b) culturing the mammalian host cell under conditions that produce the protein of interest; and (c) collecting 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 embodiment, the present invention relates to a kit comprising the expression vector described in the present invention and a cell culture medium that does not contain glutamine.

[0020] In yet another aspect, the present invention provides the use of bacterial glutamine synthase as a selection marker in mammalian cells. [Brief explanation of the drawing]

[0021] [Figure 1] A pool of CHO-K1-GS cells expressing monoclonal antibody 1 and wild-type CHO GS as a selection marker. A pool of CHO-K1-GS cells (n=5), stably transfected with a mammalian expression vector to express monoclonal antibody 1 and CHO wild-type GS as a metabolic selection marker, was cultured in a medium without L-glutamine. The titer [mg / L] of monoclonal antibody 1 was determined on a specified day after transfection. [Figure 2] Viability and viable cell density (VCD) of CHO-K1-GS cells expressing monoclonal antibody 1 and wild-type CHO GS or Providencia vermicola GS as a selection marker. A pool of CHO-K1-GS cells, stably transfected with monoclonal antibody 1 and a vector encoding a transcription cassette of a glutamine synthase selection marker derived from Crisetulus griseus or Providencia vermicola, was cultured in L-glutamine-free medium. A) viability [%] and B) viable cell density during selection were determined on a specified day after transfection. [Figure 3]Productivity after selection and production process in a shaking flask for CHO-K1-GS expressing monoclonal antibody 1 and wild-type CHO GS or Providencia vermicola GS as a selection marker. A pool of CHO-K1-GS cells, stably transfected with monoclonal antibody 1 and a vector encoding a transcription cassette of a glutamine synthase selection marker derived from Chryseturus griceus (CHO WT GS) or Providencia vermicola, was cultured in a medium without L-glutamine. Productivity as titer [mg / L] was determined (A) after selection on a specified day after transfection using CHO WT GS or Providencia vermicola GS as a selection marker, and (B) during the production process in a shaking flask on a specified day after transfection, since CHO WT GS cells lost their productivity after approximately 20 days. [Figure 4] Viability and productivity after transfection of CHO-K1-GS cells expressing monoclonal antibody 1 and glutamine synthase derived from Providencia vermicola, Photorhabdus luminescence, and Budvicia aquatica as selection markers. A CHO-K1-GS cell pool stably transfected with a vector encoding a transcription cassette of monoclonal antibody 1 and glutamine synthase selection markers derived from Providencia vermicola (black circle), Photorhabdus luminescence (black square), and Budvicia aquatica (black triangle) was cultured in L-glutamine-free medium. A) Viability during selection [%] was determined on a specified day after transfection. B) Productivity as titer [mg / L] was determined after selection on a specified day after transfection. [Figure 5]Production process of CHO-K1-GS in a shaking flask, expressing monoclonal antibody 1 and either wild-type Providencia vermicola GS or Providencia vermicola GS E130F / E130G mutant as a selection marker. A CHO-K1-GS cell pool, stably transfected in a transcription cassette to express monoclonal antibody 1 and Providencia vermicola GS or Providencia vermicola GS with the E130F or E130G mutant as a selection marker, was cultured in a medium without L-glutamine. The titer [mg / L] of monoclonal antibody 1 was determined on a specified day after transfection. [Figure 6] Productivity and viable cell density (VCD) of CHO-K1-GS expressing monoclonal antibody 1 and a select marker such as Providencia vermicola wild-type GS, CHO wild-type GS, or Providencia vermicola F226Y mutant were determined in a shaking flask production process. After stable pool preparation and single-cell deposition, selected high-productivity clones were subjected to shaking flask experiments in association with CHO WT (14-day production process). (A) Productivity as the titer [mg / L] of monoclonal antibody 1 using Providencia vermicola wild-type GS, Providencia vermicola GS F226Y, or CHO GS WT as a select marker, and (B) viable cell density [1 × 10⁶ cells / ml] of Providencia vermicola wild-type GS and Providencia vermicola GS F226Y as select markers were determined at the end of the production process. [Figure 7]Survival rate and productivity after transfection of CHO-K1-GS expressing monoclonal antibody 1 and the Providencia vermicola wild-type GS or Providencia vermicola R345A or R360A mutants as selection markers. A pool of CHO-K1-GS cells stably transfected with a vector encoding monoclonal antibody 1 and the transcription cassette of the glutamine synthetase selection marker from Providencia vermicola WT GS (black circles), and Providencia vermicola R345A (black squares) or R360A mutants (black triangles) was cultured in medium without L-glutamine. A) The survival rate [%] during selection was determined on 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 over 70% for Providencia vermicola wild-type GS and the Providencia vermicola R345A mutant. This is because the Providencia vermicola R360A mutant did not recover during selection. [Figure 8-1] Sequence of the glutamine synthetase of Providencia vermicola (SEQ ID NO: 1). [Figure 8-2] Sequence of the glutamine synthetase of Providencia vermicola (SEQ ID NO: 1). [Figure 9] Sequence of the glutamine synthetase of wild-type Chryseobacterium glycerus (SEQ ID NO: 2). [Figure 10] Sequence of the glutamine synthetase of Photorhabdus luminescence (SEQ ID NO: 14).

[0022] Detailed description The terms “contains” or “includes” mean “contains, but not limited to.” The terms are unrestricted and are intended to indicate the presence of any described feature, element, integer, process, or component, but do not exclude the presence or addition of one or more other features, elements, integers, processes, components, or groups thereof. Accordingly, the terms “contains” include the more restrictive terms “consist of” and “substantially consist of.” With respect to sequences, the terms “having the amino acid sequence of” and “containing the amino acids of” are used as synonyms and include the embodiment “consisting of the amino acid sequence of.” Similarly, the terms “coding” or “encode” are unrestricted and allow for the presence or addition of one or more other features, elements, or components. Furthermore, singular and plural forms are not used restrictively. The singular forms “a,” “an,” and “the” as used herein refer to both singular and plural forms unless otherwise specified.

[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 alterations, such as fusion with other proteins, substitution, deletion, or insertion of amino acid sequences, can occur within the structure of a polypeptide, while the molecule maintains its biological and functional activity. For example, substitution of a particular amino acid sequence can occur within the 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 their integration into host cell genomes, those skilled in the art will understand that it refers to a DNA sequence or DNA molecule.

[0025] As used herein, the term “eukaryotic cell” refers to a cell having a nucleus within a nuclear envelope, and includes animal cells, human cells, plant cells, and yeast cells. Eukaryotic cells particularly include mammalian cells, such as Chinese hamster ovary (CHO) cells or HEK293 cell-derived cells. As used herein, mammalian cells refer to all cells or cell lines of mammalian origin, such as human cells or rodent cells. The cells referred to herein are cells maintained in culture and are not related to primary cells, but to cell lines or cell line-derived cells, i.e., immortalized cells.

[0026] In this specification, the term "about" refers to a 10% variation of the stated value; for example, about 50% has a variation of 45-55%.

[0027] As used herein, the term “selection rigor” refers to the period during which viability exceeds 70% and the doubling time within 48 hours of cell culture after transfection. The longer the period, the rigorous the selection behavior. Typically, attenuated glutamine synthase exhibits rigorous selection behavior compared to CHO wild-type glutamine synthase.

[0028] Bacterial glutamine synthase, and mammalian expression vectors or host cells encoding bacterial glutamine synthase. This invention demonstrates that 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, can be created by using specific bacterial glutamine synthase genes, unrelated to mammalian glutamine synthase genes, as selection markers. This is the first time that prokaryotic glutamine synthase mutants have been successfully used as selection markers in mammalian cells. Surprisingly, glutamine synthases from Providencia vermicola and Photorhabdus luminescence exhibited more stringent selection behavior due to attenuated activity compared to CHO wild-type glutamine synthase in CHO cells, and showed higher productivity compared to the use of CHO wild-type glutamine synthase as a selection marker in CHO cells. Having demonstrated the principle that specific bacterial glutamine synthase genes can be used as selection markers in mammalian cells, it is possible to identify other bacterial glutamine synthase genes. Furthermore, point mutations in Providencia vermicola glutamine synthase at highly conserved amino acids were identified, exhibiting expanded selectivity and improved antibody titer compared to wild-type Providencia vermicola glutamine synthase. Due to the conservation of these amino acids in Photorhabdus luminescence glutamine synthase, these point mutations in Photorhabdus luminescence glutamine synthase are expected to exhibit similar expanded selectivity and improved antibody titer compared to wild-type Photorhabdus luminescence glutamine synthase.

[0029] More specifically, in a first embodiment, the present invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthase as a selection marker, wherein the bacterial glutamine synthase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 (Figure 8). In a particular embodiment, the bacterial glutamine synthase is derived from bacteria of the Enterobacteriales and Morganelaceae families. During transfection or transduction of the mammalian expression vector described in the present invention, the bacterial glutamine synthase mediates improved selectivity and / or gene stability of the protein of interest and / or non-coding RNA stably and simultaneously incorporated in CHO cells compared to CHO glutamine synthase having the amino acid sequence of SEQ ID NO: 2 (Figure 9). In a particular embodiment, the bacterial glutamine synthase is derived from the genus Providencia and / or comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1 (Figure 8). In certain embodiments, the bacterial glutamine synthase is derived from the genus Photorhabdus and / or includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 (Figure 10). In preferred embodiments, the bacterial glutamine synthase is glutamine synthase from Providencia vermicola or glutamine synthase from Photorhabdus luminescence. Preferably, the glutamine synthase is derived from the genus Providencia, more preferably from Providencia vermicola. Thus, the glutamine synthase may be a bacterial glutamine synthase containing glutamine synthase from Providencia vermicola, particularly the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the bacterial glutamine synthase includes an amino acid sequence having at least 85%, at least 90%, and preferably at least 95% sequence identity with respect to SEQ ID NO: 1.In another embodiment, the bacterial glutamine synthase 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 with the amino acid sequence of SEQ ID NO: 1, or the bacterial glutamine synthase 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 with the amino acid sequence of SEQ ID NO: 14, preferably the bacterial glutamine synthase 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 with the amino acid sequence of SEQ ID NO: 1. Thus, the bacterial glutamine synthase may have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14.

[0030] Bacterial glutamine synthases may further contain point mutations that attenuate glutamine synthase (e.g., E130X and / or R345A) or increase catalytic activity (e.g., F226Y). In particular, bacterial glutamine synthases may have point mutations at a highly conserved residue corresponding to the E130 position of glutamine synthase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation at the E130 position 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, and more preferably, the mutation at the 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. Therefore, substitutions with amino acids that do not contain polar, acidic, or basic side chains are preferred. In certain embodiments, 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 the reference to the mutation as E130X herein specifies that X is any amino acid excluding the original amino acid, i.e., E (Glu, glutamic acid). Not bound by theory, a substitution of the highly conserved glutamic acid (E) corresponding to the amino acid E130 position in SEQ ID NO: 1 results in attenuation of bacterial glutamine synthase, where attenuation in this context means reduced enzyme activity. Alternatively or additionally, bacterial glutamine synthase may have a point mutation at the highly conserved residue corresponding to the F226 position of the glutamine synthase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation corresponding to amino acid F226 in SEQ ID NO: 1 is a substitution with amino acid Y (Tyr, tyrosine), and is also called F226Y. Although not strictly theoretical, it is thought that substituting the highly conserved phenylalanine (F) at amino acid F266 in SEQ ID NO: 1 with Y improves the catalytic activity of glutamine synthase.Such mutations that enhance the catalytic activity of glutamine synthase may be advantageously combined with mutations that further attenuate glutamine synthase. In certain embodiments, 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 specific embodiments, the mutation is E130F / G, or E130F / G and F226Y. Alternatively or additionally, bacterial glutamine synthase may have a point mutation at a highly conserved residue corresponding to position R345 of glutamine synthase of Providencia vermicola having the amino acid sequence of Sequence ID No. 1. The point mutation corresponding to amino acid position R345 in Sequence ID No. 1 is a substitution with amino acid A (Ala, alanine), also known as R345A, or alternatively, a substitution with another amino acid having an aliphatic side chain, such as glycine (G), valine (V), leucine (L), or isoleucine (I), preferably glycine (G). While not bound by theory, a substitution of the highly conserved arginine (R) corresponding to amino acid position R345 in Sequence ID No. 1 results in attenuation of bacterial glutamine synthase, where attenuation means reduced enzyme activity in this context. In some cases, bacterial glutamine synthase may further contain the point mutation F226Y. Thus, in certain embodiments, the mutation is E130X and / or R345A (or G, V, L, or I), which may further contain F226Y, where X is any amino acid or as specified in this paragraph above. Preferably, the mutation is E130F / G and / or R345A, which may further contain F226Y, for example, E130F / G and R345A, which may further contain F226Y, or preferably E130F / G or R345A, which may further contain F226Y.

[0031] In certain embodiments, the bacterial glutamine synthase 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, and preferably the mutation at amino acid position E130 is more preferably a substitution with an aromatic and / or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M, and I. In certain embodiments, 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 “mutations and combinations thereof of E130X, F226Y, R345A” means 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 synthase 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, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic and / or hydrophobic amino acid 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 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, and more preferably, the amino acid sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1, further comprising mutations as disclosed herein, as exemplified in 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 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 certain embodiments, the bacterial glutamine synthase comprises an amino acid sequence having at least 95% sequence identity with 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 more preferably a substitution with an aromatic and / or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M, and I. In certain embodiments, the mutation of the bacterial glutamine synthase having at least 95% sequence identity with 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, bacterial glutamine synthase may include (1) E130X or F226Y or R345A; or (2) E130X and F226Y, or R345A and F226Y, or E130X and R345A; or (3) mutations of E130X, 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 “mutation of E130X and / or F226Y” means E130X or F226Y, or a mutation of E130X and F226Y. More preferably, the mutation is E130X, or E130X and F226Y. As used herein, the term “mutation of F226Y and / or R345A” means F226Y or R345A, or a mutation of F226Y and R345A, preferably R345A, or F226Y and R345A.

[0033] The mammalian expression vector described in the present 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., bacterial glutamine synthase, and preferably further for the expression of proteins and / or non-coding RNAs of interest. Accordingly, the mammalian expression vector described in the present invention is characterized in that it comprises a polynucleotide encoding bacterial glutamine synthase operably linked to a mammalian promoter. Typically, the mammalian expression vector comprises an expression cassette containing a polynucleotide encoding bacterial glutamine synthase operably linked to a mammalian promoter (e.g., a cytomegalovirus promoter or a monkey virus 40 promoter). The mammalian promoter regulates transcription in mammalian cells, and in particular, mammalian promoters regulate transcription in mammalian cells. Exemplary mammalian promoters include, but are not limited to, the monkey virus 40 early promoter (SV40), the cytomegalovirus very early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1α promoter (EF1A), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken β-actin promoter (CAGG) conjugated to the cytomegalovirus early enhancer. Mammalian expression vectors may further include bacterial sequences, such as origins of replication and resistance genes, for vector amplification within bacterial cells.

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

[0035] As used herein, the term “expression cassette” refers to a distinct component 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 comprises at least three components: a promoter sequence, an open reading frame, and a transcription termination sequence. In mammalian expression vectors containing polynucleotides encoding a protein of interest, the transcription termination sequence is called the 3' untranslated region and typically includes a polyadenylation site. The expression cassette instructs the cellular mechanism to produce RNA and may therefore also be called a transcription cassette. The RNA may be coding RNA, which is further processed into a protein sequence, such as glutamine synthase or mRNA encoding a protein of interest, or the RNA may be non-coding RNA, such as RNA interference (RNAi)-mediated RNA, such as microRNA, small interfering RNA, long non-coding RNA, or 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 medical treatments for humans and / or animals. These include, but are not limited to, cytokines, growth factors, hormones, blood clotting factors, vaccines, interferons, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds. In certain embodiments, the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds.

[0037] In certain embodiments, the protein of interest is an antibody. When the protein of interest is an antibody, the mammalian expression vector includes a polynucleotide containing the coding sequence of the variable region of the antibody's heavy chain and / or the coding sequence of the variable region of the light chain. In certain embodiments, the mammalian expression vector includes a polynucleotide containing the coding sequence of the antibody's heavy chain and / or the coding sequence of the light chain. Thus, the polynucleotide containing the coding sequence of the variable region of the heavy chain and the polynucleotide containing the coding sequence of the variable region of the light chain may be expressed on the same mammalian expression vector or on a different mammalian expression vector. The expression vector may include a polycistronic expression cassette, e.g., a dicistronic expression cassette, and / or multiple expression cassettes. A polycistronic expression cassette includes one or more open reading frames separated by sequences encoding RNA sequences that enable translation initiation, such as internal ribosome entry sites (IRESs). 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 (separated, for example, by an IRES sequence) or in two separate expression cassettes. Furthermore, the bacterial glutamine synthase and the protein of interest and / or non-coding RNA may be expressed in the same or different expression cassette(s). If the protein of interest is an antibody, the bacterial glutamine synthase 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 in the same or different expression cassette(s) 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 portion of the vector comprises a polynucleotide encoding a bacterial glutamine synthase and at least one polynucleotide encoding a protein of interest and / or non-coding RNA. The mammalian expression vector described in the present invention, preferably the mammalian expression vector described in the present invention, is a plasmid, a bacterial artificial chromosome (BAC), or a viral vector. The plasmid, bacterial artificial chromosome (BAC), or viral vector (e.g., a lentiviral vector) can be introduced into a mammalian host cell (e.g., a mammalian host cell) via transfection or transduction, respectively, and preferably stably integrated into the host cell genome. Those skilled in the art know suitable plasmids, BACs, or viral vectors, and know that the plasmid may further include transposon recognition sequences upstream and downstream of the polynucleotide encoding the bacterial synthase as a selection marker, and at least one optionally selected polynucleotide encoding a protein of interest and / or non-coding RNA.

[0039] Preferred proteins of interest are antibodies, which include their fragments and derivatives. Typically, antibodies are monospecific, but they may also be multispecific. Therefore, the present invention can be used to produce monospecific antibodies, multispecific antibodies, or fragments thereof, preferably monospecific antibody fragments, 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, and anti-VEGF (intravascular) antibodies. Examples include, but are not limited to, anti-corticosteroid (CNFα) antibodies, anti-TNFα (tumor necrosis factor α) antibodies, anti-IL-2 antibodies, anti-IL-5R antibodies, or anti-IgE antibodies. Preferably, the antibodies are selected from the group consisting of anti-CD20 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD52 antibodies, anti-HER2 / neu(erbB2) antibodies, anti-EGFR antibodies, anti-IGF antibodies, anti-VEGF antibodies, anti-TNFα antibodies, anti-IL-2 antibodies, and anti-IgE antibodies.

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

[0041] Antibodies can be from any species and include chimeric antibodies and humanized antibodies. A “chimeric” antibody is a molecule in which the domain or region of the antibody originates from a different species. For example, the variable regions of the heavy and light chains may originate from a rat or mouse antibody, while the constant region may originate from a human antibody. In “humanized” antibodies, only a minimal number of sequences originate from a non-human species. Often, only amino acid residues of the CDR of a human antibody are substituted with amino acid residues of the CDR of a non-human species such as mouse, rat, rabbit, or llama. Occasionally, amino acid residues of several important scaffolds that affect binding specificity and affinity to the antigen are also substituted with non-human amino acid residues.

[0042] Typically, antibodies are tetrameric polypeptides composed of a pair of heterodimers, each consisting of a heavy chain and a light chain. Stabilization of both heterodimeric and tetrameric polypeptide structures occurs via disulfide bridges between the chains. Each chain consists of a structural domain called an "immunoglobulin domain" or "immunoglobulin region," where the terms "domain" and "region" are used synonymously. Each domain contains approximately 70–110 amino acids and forms a dense three-dimensional structure. Both the heavy and light chains contain a "variable domain" or "variable region" at their N-terminus, which has a less conserved sequence involved in antigen recognition and binding. The variable region of the light chain is also called the "VL," and the variable region of the heavy chain is also called the "VH."

[0043] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to an antigen to which an intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; chain antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. A Fab fragment consists of variable regions on both chains, which are linked by adjacent constant regions. These can be formed from conventional antibodies by protease digestion, for example, using papain, but Fab fragments can also be produced by genetic engineering. Another example of an antibody fragment is the F(ab')2 fragment, which can be prepared by pepsin-mediated proteolytic cleavage.

[0044] Using genetic engineering techniques, it is possible to produce shortened antibody fragments consisting only of the variable regions of the heavy chain (VH) and light chain (VL). These are called Fv fragments (fragment variable). Because these Fv fragments lack the covalent bond between the two chains by cysteine ​​in the constant chain, they are often stabilized. It is advantageous that the variable regions of the heavy chain and the light chain are linked by a short peptide fragment of, for example, 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. Therefore, antibody fragments and antigen-binding fragments further include Fv fragments and, in particular, scFv.

[0045] In recent years, various strategies have been developed for preparing scFv as a multimerized derivative. This aims to yield recombinant antibodies with improved pharmacokinetic and in vivo distribution characteristics, as well as enhanced binding affinity. To achieve scFv multimerization, scFv has been prepared as a fusion protein having a multimerization domain. The multimerization domain can be, for example, the CH3 region of IgG or a coiled-coil structure (helix structure), such as a leucine-zipper domain. However, there are also strategies that utilize the interaction between the VH / VL regions of scFv for multimerization (e.g., diabodies, tribodies, and pentabodies). By diabodies, those skilled in the art will understand that a divalent homodimeric scFv derivative is formed by shortening the linker within the scFv molecule to 5-10 amino acids, resulting in interchain VH / VL superposition. Diabodies can further be stabilized by the incorporation of disulfide bridges. Examples of diabodies-antibody proteins are known from the prior art.

[0046] By "minibody," those skilled in the art will understand that it refers to a divalent homodimer scFv derivative. It consists of a fusion protein containing an immunoglobulin, preferably IgG, most preferably the CH3 region of IgG1, as a dimerizing region linked to the scFv via a hinge region (e.g., also derived from IgG1) and a linker region. Examples of minibody-antibody proteins are known from the prior art.

[0047] By "tribody," those skilled in the art will understand that it refers to a trivalent homotrimer scFv derivative. The trimer is formed by the ScFv derivative (where VH-VL are directly fused without a linker sequence).

[0048] Those skilled in the art will also be familiar with so-called mini-antibodies having a bivalent, trivalent, or tetravalent structure and derived from scFv. Multimerization is carried out by a dimeric, trimer, or tetrameric coiled-coil structure. In preferred embodiments of the present invention, the gene of interest encodes one of the above desired polypeptides, preferably a monoclonal antibody, a derivative thereof, or a fragment.

[0049] Furthermore, it includes single-domain antibodies (sdAbs), also called nanobodies, which are antibody fragments of a single monomeric antibody variable domain. Single-domain antibodies are typically found in camelids (V H H fragment) or cartilaginous fish (V NAR The heavy chain antibodies found in the fragments are used for engineering.

[0050] Immunoglobulin fragments, composed of the CH2 and CH3 domains of the antibody heavy chain, are called "Fc fragments," "Fc regions," or "Fc" due to their crystallization properties (Fc = crystallizable fragment). These 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 portion of the Fc fragment can vary depending on how many amino acids remain in the hinge region.

[0051] Antibodies containing an antigen-binding fragment and an Fc region may also be called full-length antibodies. Full-length antibodies can be monospecific or multispecific antibodies. A multispecific antibody is an antibody having at least two different antigen-binding sites, each of which binds to a different epitope. Multispecific antibodies include bispecific and trispecific antibodies. A bispecific antibody has 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 specificity for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, natural killer cells, or macrophages) within a single molecule. Exemplary bispecific antibodies include, but are not limited to, diabody, BiTE (bispecific T cell induction) format, and DART (biaffinity retargeting reagent) format. 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 provides further stabilization through a C-terminal disulfide bridge. Triplespecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. They can be constructed using bispecific antibody technology, which reconstitutes the antigen-recognition domains of two different antibodies into a single bispecific molecule. For example, triplespecific antibodies targeting CD38 on cancer cells and CD3 and CD28 on T cells have been created. Producing multispecific antibodies with high product quality is particularly difficult.

[0053] As used herein, the term “antibody-derived molecule” refers to any molecule that is structurally related to an antibody and contains at least one antigen-binding moiety. This includes modified full-length single-specific or bispecific antibodies, or smaller antibody formats, including those described herein, that are further modified with additional antigen-binding moieties.

[0054] As used herein, the term “antibody mimetic” 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. Examples of antibody mimetic proteins include, but are not limited to, anticalin, afibodies, adonectin, monobodies, DARPin (engineered ankyrin repeat protein), afimers, and afitins.

[0055] Single-domain antibodies (sdAbs) may also be referred to as nanobodies. Those skilled in the art will understand that such proteins may contain more than one antigen-binding domain and are therefore polyvalent, preferably bivalent (e.g., bivalent sdAbs or bivalent anticalin or any other bivalent antibody mimetic).

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

[0057] The term "cytokine" refers to small proteins released by cells that act as intercellular mediators, influencing the behavior of surrounding cells, for example, secretory cells. Cytokines can be secreted by immune cells or other cells, such as T cells, B cells, natural killer cells, and macrophages. Cytokines can be involved in intercellular signaling events, such as 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 may be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

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

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

[0060] In a second aspect, the present invention relates to a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthase, which comprises an amino acid sequence having at least 85% sequence identity with respect to the amino acid sequence of Sequence ID No. 1 operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein of interest and / or non-coding RNA. The nucleic acid sequence may be part of a mammalian expression vector of the first aspect. Accordingly, the embodiments and examples specified with respect to the first aspect are similarly applicable to this aspect. In particular, in certain embodiments, the bacterial glutamine synthase 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, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic amino acid or hydrophobic amino acid 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; or the mutation is F226Y and / or R345A; or the mutation is E130X and / or R345A, and optionally further includes the mutation F226Y.

[0061] In a further third aspect, the present invention relates to a bacterial glutamine synthase comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of Sequence ID No. 1, and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic amino acid or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M and I. In certain embodiments, the present invention relates to a bacterial glutamine synthase derived from Providencia vermicola or the like, which includes 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 the mutation at position E130 of the amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I). The invention relates to bacterial glutamine synthase having 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 the mutation E130X and / or F226Y and / or R345A (wherein X is any amino acid, preferably the mutation at position E130 of the amino acid is more preferably a substitution of an aromatic amino acid or hydrophobic amino acid 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; or the mutation is F226Y and / or R345A; or the mutation is E130X and / or R345A, which may further include the F226Y mutation, preferably E130X or R345A, which may further include the F226Y mutation. The embodiments and examples specified with respect to the first and second embodiments apply similarly to this embodiment.

[0062] In a fourth aspect, the present invention relates to a kit that optionally comprises the expression vector described in the present invention and a cell culture medium that does not contain glutamine.

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

[0064] Bacterial glutamine synthase may further include point mutations that further attenuate glutamine synthase (e.g., E130X and / or R345A) or enhance catalytic activity (e.g., F226Y). In particular, bacterial glutamine synthase may have a point mutation in a highly conserved residue corresponding to the E130 position of glutamine synthase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation at the E130 position is a substitution using any amino acid (X), and preferably the mutation at the amino acid position corresponding to E130 is a substitution with an aromatic or hydrophobic amino acid, for example, an aromatic or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M, and I. In certain embodiments, 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, bacterial glutamine synthase may have a point mutation in a highly conserved residue corresponding to position F226 of glutamine synthase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1; the point mutation here is a substitution of amino acid Y (Tyr, tyrosine), and is also referred to as F226Y. In certain embodiments, 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 specific embodiments, the mutation is E130F / G, or E130F / G and F226Y. Alternatively or additionally, bacterial glutamine synthase may have a point mutation in a highly conserved residue corresponding to position R345 of glutamine synthase of Providencia vermicola having the amino acid sequence of SEQ ID NO: 1. The point mutation corresponding to amino acid position R345 in Sequence ID No. 1 is a substitution with amino acid A (Ala, alanine), also known as R345A, or alternatively, a substitution with another amino acid having an aliphatic side chain, such as glycine (G), valine (V), leucine (L), or isoleucine (I), preferably glycine (G).While not bound by theory, substitution of the highly conserved arginine (R) corresponding to amino acid position R345 in Sequence ID No. 1 attenuates bacterial glutamine synthase, where attenuation means reduced enzyme activity in this context. In some cases, bacterial glutamine synthase may further contain the point mutation F226Y. Thus, in certain embodiments, the mutation is E130X and / or R345A (or G, V, L, or I) (where X is any amino acid or as specified in this paragraph above), which may further contain F226Y. Preferably, the mutation is E130F / G and / or R345A, which may further contain F226Y, for example, E130F / G and R345A, which may further contain F226Y, or preferably E130F / G or R345A, which may further contain F226Y. Preferably, the bacterial glutamine synthase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, further comprising point mutations as described above.

[0065] In a preferred embodiment, the bacterial glutamine synthase 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, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic and / or hydrophobic amino acid 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 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, and more preferably the amino acid sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14 and further includes mutations as disclosed herein, such as those exemplified in the amino acid sequences of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 20, 21, 22, and 29 (based on SEQ ID NO: 1), or SEQ ID NOs: 23, 24, 25, 26, 27, 28, and 30 (based on SEQ ID NO: 14), preferably SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 20, and 21 (based on SEQ ID NO: 1), or SEQ ID NOs: 23, 24, 25, 26, and 28 (based on SEQ ID NO: 14). In certain embodiments, the mutation is E130X and / or F226Y, preferably where E130X is E130F, E130G, or E130A, more preferably E130F or E130G (E130F / G). In certain embodiments, the mutation is F226Y and / or R345A, more preferably R345A, or R345A and F226Y. In certain embodiments, the mutation is E130X and / or R345A (or G, V, L, or I), and 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 which may further contain F226Y, for example, E130F / G and R345A which may further contain F226Y, or preferably E130F / G or R345A which may further contain F226Y.

[0066] In a sixth aspect, the present invention relates to a mammalian host cell comprising the mammalian expression vector of the present invention (first aspect), the nucleic acid sequence of the present invention (second aspect), or the nucleic acid sequence encoding the bacterial glutamine synthase of the present invention (third aspect). The mammalian host cell can be any host cell, provided that the host cell is an immortalized cell and not a primary cell. In certain embodiments, the mammalian host cell is a mouse, human, or rodent cell, more preferably a rodent cell, and even more preferably a CHO cell. Furthermore, the mammalian host cell is preferably a glutamine synthase gene knockout cell (GS knockout mutant) host cell. As used herein, the term “GS gene knockout cell” refers to a cell in which the endogenous GS gene is knocked out, i.e., deleted or destroyed, resulting in the 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 are deleted or destroyed. The GS gene, introduced by extracellular glutamine supplementation or expression vector, is essential for the survival of GS gene knockout cells. In preferred embodiments, the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS(GS- / -) cell. In specific embodiments, the mammalian host cell is a monoclonal cell line produced by the steps of single-cell cloning and clonal proliferation. Preferably, the mammalian host cell contains a polynucleotide encoding bacterial glutamine synthase and at least one polynucleotide encoding a protein of interest and / or non-coding RNA that is simultaneously incorporated into the host cell genome.

[0067] Methods for preparing cell lines or producing proteins In a seventh aspect, the present invention relates to a method for preparing cells stably expressing a protein of interest and / or noncoding RNA, comprising the steps of (a) introducing 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 synthase and at least one polynucleotide encoding a protein of interest and / or noncoding RNA, into a mammalian host cell; and (b) culturing the mammalian host cell in a glutamine-free medium under conditions selected for bacterial glutamine synthase (wherein the at least one polynucleotide encoding the protein of interest and / or noncoding RNA is simultaneously incorporated into the host cell genome together with the polynucleotide encoding the bacterial glutamine synthase). Optionally, the method may further include culturing the cell pool obtained as a result of stably expressing the protein of interest and / or noncoding RNA selected in step (b), and / or isolating the cell pool stably expressing the protein of interest and / or noncoding RNA. Cells that stably express the protein and / or non-coding RNA of interest mean that the polynucleotide encoding the protein and / or non-coding RNA of interest is stably integrated into the host cell's genome, and that the protein and / or non-coding RNA of interest is stably expressed, i.e., expressed over a long period of time, for example, at least 40 days, preferably over several months or several years.

[0068] Mammalian expression vectors or nucleic acids may be plasmids, bacterial artificial chromosomes (BACs), or viral vectors. Therefore, the mammalian expression vectors or nucleic acids of the present invention can 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 transfecting or transfecting mammalian expression vectors or nucleic acids into mammalian cells are well known in the art and include chemical means, e.g., calcium phosphate precipitation and lipofection, and physical means, e.g., electroporation. Polynucleotides encoding bacterial glutamine synthase and polynucleotides encoding the protein of interest and / or non-coding RNA are operably ligated to a mammalian promoter and thus applied for expression in mammalian host cells.

[0069] The method of the present invention may further include step (c) of isolating a single clone for clonal proliferation to prepare a monoclonal cell line. Those skilled in the art will understand that transfection or transduction often requires a large number of cells, resulting in a heterogeneous pool of recombinant cells having, for example, a population of individuals with diverse integration sites. For the preparation of a clonal cell line, the cell pool is diluted or sorted for the isolation of single cells (monoclonality), each single clone is subjected to clonal amplification, and a monoclonal cell line is prepared. As used herein, the term “cell line” refers to a population of cells derived from a single cell clone that can be proliferated for indefinite time. Therefore, it is also called a monoclonal cell line. Thus, cell lines are genetically stable, and therefore, the characteristics of a cell line should not change over time. In particular, phenotypic characteristics, such as production levels (titer) and growth rate and density (live cell density and maximum live cell density), viability, and genetic integrity, as measured by copy number and DNA fingerprint assays, should be maintained when cultured under equivalent conditions.

[0070] Cell pools or monoclonal cell lines prepared according to the method of the present invention may be further used to stably produce proteins of interest, or to stably produce non-coding RNAs, such as RNA interference-mediated RNAs, such as microRNAs, small interfering RNAs, long non-coding RNAs, or small hairpin RNAs. RNA interference can be used for gene silencing and therefore to create cell pools or monoclonal cell lines with beneficial properties, such as protein production. For example, the difficulty in removing host cell proteins (HCPs) can be silenced in cell pools or monoclonal cell lines, or enzymes such as fucosyltransferases can be silenced to modify the glycosylation profile of cell pools or monoclonal cell lines. Furthermore, CHO cells 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 operations include, but are not limited to, improved apoptosis resistance, reduced autophagy, increased cell proliferation, altered expression of cell cycle regulatory proteins, chaperone engineering, engineering of the endoplasmic reticulum stress response (UPR), engineering of secretory pathways, and metabolic engineering. Such engineering operations may be achieved using RNA interference in mammalian host cells produced by the methods of the present invention.

[0071] In a related eighth aspect, the present invention relates to a method for producing a protein of interest, comprising: (a) introducing a mammalian expression vector or nucleic acid described in the present invention into a mammalian host cell (wherein the expression vector comprising a polynucleotide encoding a bacterial glutamine synthase and at least one polynucleotide encoding a protein of interest operably linked to a mammalian promoter); (b) culturing the mammalian host cell in a glutamine-free medium under conditions for the selection of bacterial glutamine synthase (wherein the at least one polynucleotide encoding the protein of interest is simultaneously incorporated into the host cell genome together with the polynucleotide encoding the bacterial glutamine synthase); (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the mammalian host cell under conditions for the 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 present invention can 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, with respect to mammalian expression vectors, typically only a small portion of the vector is integrated into the genome of a host cell. Therefore, the integrated portion of the vector comprises a polynucleotide encoding bacterial glutamine synthase, and at least one polynucleotide encoding the protein of interest and / or non-coding RNA. Furthermore, the integrated portion of a mammalian expression vector, or the integrated nucleic acid sequence of the present invention, can be further amplified by increasing the concentration of a glutamine synthase inhibitor, such as methionine sulfoximine (MSX). Amplification is optional, and higher productivity may be achieved with polynucleotides encoding high copy numbers of the protein of interest and / or non-coding RNA, because these will be amplified simultaneously with bacterial glutamine synthase. Methods of the present invention may include the preparation of a cell pool or monoclonal cell line. Furthermore, mammalian host cells prepared according to the methods of the present invention, or mammalian host cells described in the present invention, can be further used to produce the protein of interest and / or non-coding RNA, or in methods for producing the protein of interest.

[0073] Accordingly, in a further ninth aspect, the present invention relates to a method for producing a protein of interest, comprising: (a) preparing a mammalian host cell produced by the present invention or by the method thereof, comprising a polynucleotide encoding a bacterial glutamine synthase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions that produce the protein of interest; and (c) collecting and optionally purifying the protein of interest.

[0074] In a preferred embodiment of the method of the present invention, the mammalian host cells are GS gene knockout cells. Those skilled in the art will understand that this refers to the endogenous GS gene, while the bacterial GS gene is present in the host cells after transfection or transduction with the mammalian expression vector or nucleic acid sequence of the present invention. The mammalian host cells (transfected or transductioned with the mammalian expression vector or nucleic acid sequence of the present invention) are cultured in a glutamine-free medium under conditions selected for bacterial glutamine synthase in step (b). This may further include the addition of the GS inhibitor methionine sulfoximine (MSX).

[0075] In certain embodiments of the method of the present invention, cells (cell pools) or monoclonal cell lines are produced with increased selectivity and / or have increased gene stability and / or have higher productivity compared to cells or cell lines produced using glutamine synthase derived from Chryseturus griseus having the amino acid sequence of SEQ ID NO: 2. Gene stability can be quantified by measuring the copy number of the incorporated transgene. Gene stability is evaluated by measuring the copy number over longer culture times. In addition, genomic rearrangement is monitored, for example, via Southern blot analysis. Increased selectivity can be determined by the time it takes to reach a viability of more than 70%.

[0076] Mammalian host cells 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 cells described herein are for in vitro use in cell culture. As used herein, the term “mammalian cell” refers to a mammalian cell line suitable for the production of the product of interest, e.g., a heterologous protein and / or non-coding RNA of interest, and this may also be referred to as “host cell” or “mammalian host cell.” Mammalian cells are preferably transformed and / or immortalized cell lines. They are suitable for serial passage in cell culture, preferably serum-free cell culture and / or preferably suspension culture, and this does not include primary untransformed cells or cells that are part of an organ structure.

[0077] Preferably, the mammalian host cells are mouse, human, or rodent cells, more preferably rodent cells, and even more preferably CHO cells. Preferred mammalian cells for heterologous protein production are mouse cells, rodent cells, 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 for proliferation in suspension. In preferred embodiments, the mammalian cells or mammalian cell lines are CHO cells. In specific embodiments, 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, particularly BHK21 cells, BHK TK cells, CHO cells, CHO-K1 cells, CHO-DXB11 cells (also referred to as CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or derivatives / offspring of any of these 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 synthase (GS)-deficient derivatives of mammalian cells (particularly CHO-DG44 cells and CHO-K1 cells) are also included. In one embodiment of the present invention, the mammalian cells are Chinese hamster ovary (CHO) cells, preferably CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHO-S cells, CHO-GS deficient cells, or derivatives thereof. Suitable human cells are HEK293 cells or HEK293T cells. The host cells may be mouse cells, such as mouse myeloma cells, such as NS0 cells and Sp2 / 0 cells, or derivatives / offspring of any of such cell lines.

[0079] Furthermore, the mammalian host cells are preferably GS gene knockout cells (GS knockout mutants). As used herein, the term “GS gene knockout cells” refers to cells in which the endogenous GS gene is knocked out, i.e., deleted or destroyed, resulting in the 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 destroyed. The GS gene, introduced by extracellular glutamine supplementation or expression vectors, is essential for the survival of GS gene knockout cells. In preferred embodiments, the mammalian host cells are CHO-K1 cells, more preferably CHO-K1-GS(GS- / -) cells.

[0080] Preferably, CHO cells, which enable an efficient cell line development process, are metabolically engineered, for example by knocking out endogenous glutamine synthase (GS), to facilitate selection with methionine sulfoximin (MSX).

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

[0082] [Table 1]

[0083] Cells are most preferably established, adapted, and fully cultured under serum-free conditions and, optionally, in a medium completely free of any animal-derived proteins / peptides. Commercial media, such as Ham's F12 (Sigma-Ace, Deisenhofen, Germany), RPMI-1640 (Sigma-Ace), Dulbecco's Modified Eagle Medium (DMEM; Sigma-Ace), Minimum Essential Medium (MEM; Sigma-Ace), Iskov Modified Dulbecco's Medium (IMDM; Sigma-Ace), CD-CHO (Invitrogen, Carlsbad, CA), Serum-Free CHO Medium (Sigma-Ace), and Protein-Free CHO Medium (Sigma-Ace), are exemplary suitable nutrient solutions. A wide variety of compounds may be added to any of the culture media as needed, and non-limiting examples include 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 auxiliary substances may also be included in appropriate concentrations known to those skilled in the art. Appropriate selectors are added to the culture media for the proliferation and selection of genetically modified cells expressing selected genes.

[0084] The protein of interest encoded by a mammalian expression vector or produced by the method of the present invention is preferably produced in CHO cells in cell culture. After expression, the recombinant protein is collected and further purified. Antibodies may be recovered from the culture medium as secreted proteins in the collected cell culture medium (HCCF) or from the 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 may be purified using the techniques described herein. According to the present invention, the method comprises the step of preparing a collected cell culture medium containing the protein of interest, e.g., an antibody as a starting material, where the HCCF is derived from a CHO cell culture. Preferably, the protein of interest, e.g., an antibody, is recovered from the collected cell culture medium after cell separation, e.g., by filtration and / or centrifugation. Thus, in certain embodiments, collection includes centrifugation and / or filtration to produce a collected cell culture medium.

[0085] From the above perspective, it will also be recognized that the present invention encompasses the following items.

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

[0087] Item 2 specifies the mammalian expression vectors from Item 1 in which bacterial glutamine synthase is derived from bacteria of the Enterobacteriaceae order and Morganellaceae family.

[0088] Item 3 specifies the mammalian expression vector of Item 1 or 2, wherein the bacterial glutamine synthase is derived from the genus Providencia or Photorhabdus, and preferably the bacterial glutamine synthase is the glutamine synthase of Providencia vermicola or the glutamine synthase of Photorhabdus luminescence.

[0089] Item 4 specifies a mammalian expression vector of any one of items 1 to 3, wherein the bacterial glutamine synthase 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 the mutation at the E130 position of the amino acid is more preferably a substitution of an aromatic or hydrophobic amino acid selected from the group consisting of Y, W, F, A, G, V, L, M and I.

[0090] Item 5 specifies a mammalian expression vector of any one of items 1 to 4, wherein the bacterial glutamine synthase 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, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution with an aromatic or hydrophobic amino acid 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 item 4 or 5, in which the mutation is E130X and / or F226Y and / or R345A, where E130X is E130F or E130G.

[0092] Item 7 further specifies a mammalian expression vector of any one of items 1-6, in which a polynucleotide encoding bacterial glutamine synthase is operably ligated to a mammalian promoter.

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

[0094] Item 9 further specifies a mammalian expression vector according to any one of items 1 to 8, wherein the protein of interest is preferably selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds, and is a therapeutic protein.

[0095] Item 10 further specifies a mammalian expression vector as described in Item 9, wherein the protein of interest is an antibody, and preferably the expression vector herein contains a polynucleotide coding sequence for the variable region of the antibody's heavy chain and / or the variable region of its light chain.

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

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

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

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

[0100] Item 15 further specifies the nucleic acid sequence of Item 14, wherein the bacterial glutamine synthase has at least 85% sequence identity to the amino acid sequence of Sequence ID No. 1, and an amino acid sequence having mutations selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution of an aromatic or hydrophobic amino acid 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 synthase has at least 95% sequence identity to the amino acid sequence of Sequence ID No. 1, and has an amino acid sequence having mutations selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution of an aromatic or hydrophobic amino acid 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 synthase has at least 95% sequence identity to the amino acid sequence of Sequence ID No. 14, and an amino acid sequence having mutations selected from the group consisting of E130X, F226Y, R345A, and combinations thereof, where X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably a substitution of an aromatic or hydrophobic amino acid 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 15-17, where the mutation is E130X and / or F226 and / or R345A, where E130X is E130F or E130G.

[0104] Item 19 provides a bacterial glutamine synthase having at least 85% sequence identity to the amino acid sequence of Sequence ID No. 1, and an amino acid sequence having a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably 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.

[0105] Item 20 provides a glutamine synthase derived from Providencia vermicola, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of Sequence 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 the mutation at the E130 position of the amino acid is more preferably 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.

[0106] Item 21 provides a bacterial glutamine synthase derived from Photorhabdus luminescent, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of Sequence ID No. 14, and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid, and preferably the mutation at the E130 position of the amino acid is more preferably 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.

[0107] Item 22 provides a mammalian host cell comprising an expression vector from any one of items 1 to 13, a nucleic acid sequence from any one of items 14 to 18, or a nucleic acid sequence encoding bacterial glutamine synthase from any one of items 19 to 21, wherein the mammalian host cell herein is preferably 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 contains a polynucleotide encoding bacterial glutamine synthase, as well as at least one polynucleotide encoding a protein of interest and / or non-coding RNA that is simultaneously incorporated into the host cell genome.

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

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

[0111] Item 26 provides a method for producing a protein of interest, comprising: (a) introducing a mammalian expression vector described in items 1 to 13 or a nucleic acid described in 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 bacterial glutamine synthase operably linked to a mammalian promoter, and at least one polynucleotide encoding the protein of interest); (b) culturing the mammalian host cell in a glutamine-free medium under conditions selected for bacterial glutamine synthase (wherein the at least one polynucleotide encoding the protein of interest is simultaneously incorporated into the host cell genome together with the polynucleotide encoding bacterial glutamine synthase); (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 by which the mammalian expression vector or nucleic acid is introduced by transfection or transduction, as described in any one of items 24-26.

[0113] Item 28 further specifies the method described in Item 27, in which a mammalian expression vector or nucleic acid is introduced by stable transfection.

[0114] Item 29 provides a method for producing a protein of interest, comprising the steps of (a) preparing a mammalian host cell of item 22 or 23 containing a polynucleotide encoding a bacterial glutamine synthase operably linked to a mammalian promoter, and at least one polynucleotide encoding the protein of interest; (b) culturing the mammalian host cell under conditions that produce 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 24-29, wherein (a) the mammalian host cells are GS gene knockout cells and / or (b) the mammalian host cells are cultured in a glutamine-free medium under conditions of selecting the bacterial glutamine synthase of step (b), the step of which includes the addition of the GS inhibitor methionine sulfoximine (MSX).

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

[0117] Item 32 further specifies a method of any one of items 24-30, wherein the cells or cell line are produced with increased selectivity and / or exhibit increased genetic stability and / or have higher productivity compared to cells or cell lines produced using glutamine synthase derived from Chryseturus griceus having the amino acid sequence of Sequence ID No. 2.

[0118] Item 33 provides a kit that includes an expression vector from any one of items 1-13 and a cell culture medium that does not contain glutamine.

[0119] Item 34 provides the use of bacterial glutamine synthase as a selection marker in mammalian cells, preferably here comprising an amino acid sequence having at least 85% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1.

[0120] Item 35 further specifies the use of item 34, wherein the bacterial glutamine synthase is derived from bacteria of the order Enterobacteriales and the family Morganellaceae, more preferably from the genera Providencia or Fotorhabdus, and even more preferably from Providencia vermicola or Fotorhabdus luminescence.

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

[0122] Item 37 further specifies the use of any one of items 34-36, wherein the bacterial glutamine synthase 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 here the mutation at amino acid position E130 is more preferably a substitution of an aromatic or hydrophobic amino acid 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 synthase is derived from the genus Photorhabdus and / or comprises an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of Sequence ID No. 1, more preferably herein the bacterial glutamine synthase is glutamine synthase of Photorhabdus luminescence.

[0124] Item 39 further specifies the use of any one of items 34, 35, and 38, wherein the bacterial glutamine synthase 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 here the mutation at amino acid position E130 is more preferably a substitution of an aromatic or hydrophobic amino acid 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, where the mutation is E130X and / or F226Y and / or R345A, and here E130X is E130F or E130G.

[0126] Examples Transfection of plasmids for transfection The plasmid used for stable transfection contains a cytomegalovirus-driven antibody transcription cassette, an ampicillin transgene, and a glutamine synthase transgene as a metabolic selection marker. The plasmid was stratified for transfection using PvuI (single cleavage at the ampicillin transgene). Restriction enzyme digestion of 20 μg of plasmid DNA was performed using PvuI(NEB) at 37°C for 3 hours, according to the manufacturer's protocol. The stratified plasmid DNA was purified according to the manufacturer's protocol (Qiagen Plasmid Maxi Kit). Final DNA concentrations were determined via nanodrop spectroscopy.

[0127] Culture of host cells Host cells of the CHO-K1-GS knockout cell line (also called CHO-K1-GS KO or simply CHO-K1-GS), which have a genomic knockout of the endogenous glutamine synthase gene, were cultured in host cell medium supplemented with L-glutamine. The host cell culture was performed at a rate of 3 × 10⁶ cells per 1 mL. 5 Cell seeding was initiated at a seeding density of 100 cells. Growth conditions were set to 120 rpm in a shaking flask and 36.5°C and 5% carbon dioxide in a shaking incubator. Cell density and viability were determined using a Sedex HiRes (copyright) cell counter.

[0128] Transfection of chain-like plasmids into CHO cells One day before transfection, the host cells were placed in a shaking flask at a rate of 0.8 × 10⁶ 6Cells were seeded at a cell density of 5 × 10¹ cells / mL. On the transfection day, cell density and viability were determined, and the amount of cells required for transfection was centrifuged at 750 × G for 7 minutes. The supernatant was discarded. 10 μg of chained plasmid DNA per transfection was transfected using the Neon (copyright) transfection system (Invitrogen) and electroporation. An electroporation cuvette was filled with 3 mL of electroporation buffer E2, and cells (5 × 10¹⁶) were added to 89 μl of buffer R. 6 The cells were resuspended and mixed with 10 μg of chain-transfected plasmid. Transfection was performed using 1500 volts, 10 milliseconds, and 2 pulses. The transfected cells were transferred to 5 ml of pre-warmed host cell medium in a 25 ml T flask and incubated with 8% carbon dioxide at 37°C for at least 24 hours.

[0129] Selection of a stable CHO cell pool Twenty-four hours after transfection, the cells were transferred to selective medium (L-glutamine-free medium). 10 mL of selective medium was pre-warmed for each pool in the T75 flask. Two stable pools were cultured for each transfection. The cells were centrifuged (750 × G for 7 minutes), resuspended in 20 mL of selective medium, and incubated at 37°C with 8% carbon dioxide. Cells were monitored by microscope during selection. An additional 5 mL of selective medium was added after 7 days.

[0130] Stable pool subdivision and production process The selection phase was considered successful once the cells achieved a viability of at least 70% and a doubling time of 48 hours or less. After selection, the cells were placed in a 125 mL shaking flask in 30 mL of medium (without L-glutamine) at a rate of 3 × 10⁶ cells per mL. 5Cellular cultures were started and subcultured every 2 to 3 days at 36.5°C and 5% carbon dioxide with shaking at 120 rpm. Samples were periodically collected for titer measurement. If the antibody titer was stable for at least 14 days, phenotypic stability was indicated, and a production process of at least 7 days was initiated.

[0131] For the production process, the cells are placed in a shaking flask at a density of 7 × 10 per mL. 5 Cells were seeded at a density of 100 viable cells in 30 mL of glutamine-free basal medium, and cultured at 34.5°C with 5% carbon dioxide and shaking at 120 rpm. During the production process for at least 7 days, cells were counted, and samples were collected once daily for pH, glucose, and titer measurement, and the pH and glucose feeds were adjusted as needed. Starting from day 2, daily feeds (water-in-oil glutamine) were added. pH was determined using RAPIDlab348Ex (copyright), and glucose levels were measured via an EKF diagnostic instrument. During and / or at the end of culture, the cell culture medium supernatant was analyzed for antibody titer after centrifugation using a ForteBio Octet (copyright) instrument with a Protein A biosensor. Sample dilutions and standard curves were processed during the same production process.

[0132] Example 1: Antibody production in CHO cells using CHO wild-type glutamine synthase as a metabolic selection marker. CHO-K1-GS cells, once transfected with a vector containing glutamine synthase derived from the Chryseturus griseus (CHO wild-type glutamine synthase (GS), SEQ ID NO: 2) selection marker, can survive selection under culture conditions in a medium without L-glutamine supplementation, provided that the transgene vector is stably integrated into the cell genome.

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

[0134] Example 2: Antibody production in CHO cells using GS of Providencia vermicola and other bacteria as selective markers. To functionally evaluate novel bacterial glutamine synthase variants, selection rigor (time to achieve over 70% viability after transfection), productive passage count, and specific productivity were measured over time. For CHO pools that remained productive after prolonged passage, a 7-day production process in shaking flasks was followed under controlled process settings to further evaluate cell culture process parameters.

[0135] CHO-K1-GS cells were transfected with monoclonal antibody 1 and a vector containing an expression cassette of glutamine synthase derived from Chryseturus griceus (SEQ ID NO: 2) or Providencia vermicola (SEQ ID NO: 1). Viability, viable cell count (VCD), and productivity were measured over time during the selection period. Surprisingly, CHO cells transfected with class I glutamine synthase from Providencia vermicola survived the selection process, suggesting that this prokaryotic metabolic selection marker is functional in CHO cells. Cells transfected with Providencia glutamine synthase took longer to fully recover from selection (Figure 2A) and exhibited slower cell proliferation (Figure 2B) compared to CHO glutamine synthase, suggesting increased selection rigor.

[0136] Titer measurements during cell selection showed a rapid decline in productivity of cells possessing the CHO GS gene. Cells transfected with Providencia glutamine synthase stably expressed monoclonal antibody 1 even after the selection process (Figure 3A). An additional production process of at least 7 days was performed after selection and passage, suggesting that the phenotypic stability of cells transfected with Providencia glutamine synthase was significantly higher compared to those transfected with CHO glutamine synthase (Figure 3B). Cells transfected with CHO glutamine synthase did not proceed to the production process due to a loss of productivity.

[0137] Overall, CHO cells transfected with Providencia vermicola glutamine synthase showed increased selection rigor and significantly higher productivity. Furthermore, the cell pool exhibited increased phenotypic stability, remaining productive for at least 40 days, while cells transfected with CHO glutamine synthase completely lost their productivity approximately 20 days after transfection.

[0138] In addition to glutamine synthase derived from Providencia vermicola, glutamine synthase from a total of 10 prokaryotic cells was tested, nine of which were derived from bacteria and one from an archaea (Methanocaldococcus janaschii). In CHO cells, six were found to be functional and four were found to be non-functional. Functionality 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 GS cells used for selection, at least, belong to the Enterobacteriaceae and Morganelaceae families and / or have at least 80% sequence identity with GS cells derived from Providencia vermicola. However, most of these bacterial GS strains, with the exception of the *Photorhabdus luminescent*-derived GS strain which showed outstanding performance (selection rigor / genetic stability) compared to wild-type CHO GS, were found to exhibit moderate performance, clearly inferior to wild-type CHO GS when considering selection rigor and genetic stability.

[0139] [Table 2]

[0140] The rigor of selection was analyzed by measuring the recovery rate of viability exceeding 70%, and genetic stability was demonstrated by stable productivity over specific periods, such as more than 20 days after transfection. Representative data from selection experiments monitoring the viability of a stable CHO pool using GS derived from Providencia vermicola, Photorhabdus luminescence, and Budovicia aquatica as selection markers are shown in Figure 4A, and representative data on the productivity of CHO pools transfected with GS from these bacteria are shown in Figure 4B. While GS from Budovicia aquatica was functional in CHO cells, the selection was not robust. In contrast, GS from Photorhabdus luminescence remained functionally active and showed even more strongly attenuated activity compared to GS from Providencia vermicola (Figure 4A). Stable pools transfected with GS derived from Photolabus luminescence recovered 16 to 17 days after transfection (reaching a viability of over 70%), compared to 13 days after transfection with GS derived from Providencia vermicola. Productivity was comparable for GS derived from Photolabus luminescence and Providencia vermicola, but slightly delayed for GS derived from Photolabus luminescence and sharply decreased for GS derived from Budovicia aquatica (Figure 4B).

[0141] These experiments demonstrate for the first time that bacterial GSs, such as those from Providencia vermicola, can be used as selection markers in mammalian cells, and furthermore, that bacterial GSs closely related to other bacterial GSs, such as those from Photorhabdus luminescence, can be identified, having at least 85% sequence identity to the amino acid sequence (SEQ ID NO: 1) of GSs from Enterobacteriaceae and Morganelaceae and / or Providencia vermicola.

[0142] Example 3: Antibody production 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 Providencia vermicola GS were tested in selection and production experiments. CHO-K1-GS cells were transfected with a vector containing an expression cassette for monoclonal antibody 1 and Providencia vermicola-derived glutamine synthase (SEQ ID NO: 1), or mutant E130F (SEQ ID NO: 4) or E130G (SEQ ID NO: 5).

[0143] Both mutations appeared to further amplify the attenuation, extending the time during which cells escape selection and proliferate compared to the glutamine synthase of wild-type Providencia vermicola, and both demonstrated phenotypic stability. Subsequent production processes 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 mutants of Providencia vermicola as select markers. CHO-K1-GS cells were transfected with a vector containing monoclonal antibody 1 and expression cassettes for 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 pooling and single-cell deposition, the top clones were compared with CHO WT GS and subjected to shaking flask experiments. Both Providencia wild-type GS and Providencia GS F226 were superior to CHO wild-type glutamine synthase, showing approximately 50% higher titers (mg / L) compared to CHO wild-type GS clones. Compared to Providencia wild-type GS, cell clones with Providencia GS F226Y showed even greater proliferation and higher viable cell density (Figure 6B). This may offer advantages in the production of biopharmaceuticals, particularly combinations including 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 select markers. Furthermore, two additional point mutants of Providencia vermicola glutamine synthase were tested and evaluated in the creation of a stable antibody-producing CHO expression pool. Wild-type glutamine synthase was compared against mutants with mutations in the catalytic domain of the selection marker.

[0146] R360A lost its functionality, and the pool could not be recovered from the selection experiment, while R345A remained functionally active but with significantly reduced activity (Figure 7A). Stable pools transfected with R345A recovered 31 days after transfection compared to wild-type protein (achieving a viability rate of over 70%), while wild-type protein recovered in approximately 15 days. Interestingly, the strongly attenuated mutant R345A (SEQ ID NO: 20) showed a 5- to 10-fold increase in productivity (Figure 7B).

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

[0148] [Table 3]

Claims

1. A mammalian expression vector comprising a polynucleotide encoding bacterial glutamine synthase as a selection marker, wherein the bacterial glutamine synthase comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

14.

2. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase is derived from bacteria of the Enterobacteriaceae order and the Morganellaceae family.

3. The mammalian expression vector according to claim 2, wherein the bacterial glutamine synthase is derived from the genus Providencia or Photorhabdus.

4. The mammalian expression vector according to claim 3, wherein the bacterial glutamine synthase is Providencia vermicola glutamine synthase or Photorhabdus luminescens glutamine synthase.

5. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase comprises an amino acid sequence having a sequence identity of at least 90% to the amino acid sequence of SEQ ID NO: 1 or at least 95% 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.

6. The mammalian expression vector according to claim 5, wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid.

7. The mammalian expression vector according to claim 6, wherein the aromatic amino acid or hydrophobic amino acid is selected from the group consisting of Y, W, F, A, G, V, L, M, and I.

8. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase comprises an amino acid sequence having at least 95% sequence identity with 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.

9. The mammalian expression vector according to claim 1, further comprising at least one polynucleotide encoding a protein of interest and / or a non-coding RNA.

10. The mammalian expression vector according to claim 1, further comprising an expression cassette containing at least one polynucleotide encoding a protein of interest and / or a non-coding RNA.

11. The mammalian expression vector according to claim 9, wherein the protein of interest is a therapeutic protein.

12. The mammalian expression vector according to claim 11, wherein the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds.

13. A nucleic acid comprising a polynucleotide encoding bacterial glutamine synthase, the amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14, operably linked to a mammalian promoter.

14. The nucleic acid according to claim 13, further comprising at least one polynucleotide encoding a protein of interest and / or a non-coding RNA.

15. The nucleic acid according to claim 13, wherein the bacterial glutamine synthase comprises an amino acid sequence having a sequence identity of at least 90% with respect to the amino acid sequence of SEQ ID NO: 1 or at least 95% with respect 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.

16. A bacterial glutamine synthase comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with 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.

17. A mammalian host cell comprising an expression vector according to any one of claims 1 to 12, a nucleic acid according to any one of claims 13 to 15, or a nucleic acid encoding bacterial glutamine synthase according to claim 16.

18. The mammalian host cell according to claim 17, wherein the mammalian host cell is (a) a rodent cell and / or (b) a glutamine synthase gene knockout cell.

19. The mammalian host cell according to claim 18, wherein the rodent cell is a Chinese hamster ovary (CHO) cell.

20. (a) Introducing into a mammalian host cell an expression vector according to any one of claims 1 to 12 or a nucleic acid according to any one of claims 13 to 15, which comprises a polynucleotide encoding bacterial glutamine synthase and at least one polynucleotide encoding a protein of interest and / or non-coding RNA; and (b) A step of culturing mammalian host cells in a glutamine-free medium under conditions selected for bacterial glutamine synthase (wherein at least one polynucleotide encoding the protein and / or non-coding RNA of interest is simultaneously incorporated into the host cell genome together with the polynucleotide encoding bacterial glutamine synthase). A method for preparing cells that stably express a protein of interest and / or non-coding RNA, including the above.

21. (c) The method according to claim 20, further comprising the step of isolating a single clone for clonal proliferation to prepare a monoclonal cell line.

22. (a) The step of introducing a mammalian expression vector according to any one of claims 1 to 12, or a nucleic acid according to any one of claims 13 to 15, into a mammalian host cell (the expression vector herein comprises a polynucleotide encoding bacterial glutamine synthase operably linked to a mammalian promoter, and at least one polynucleotide encoding a protein of interest); (b) A step of culturing mammalian host cells in a glutamine-free medium under conditions of selection for bacterial glutamine synthase (where at least one polynucleotide encoding the protein of interest is simultaneously incorporated into the host cell genome along with the polynucleotide encoding bacterial glutamine synthase); (c) Depending on the case, the step of isolating a single clone for clonal proliferation and preparing a monoclonal cell line; (d) A step of culturing mammalian host cells under conditions that produce the protein of interest; and (e) The process of collecting the protein of interest and, if applicable, purifying it. A method for producing a protein of interest, including [specific example].

23. (a) A step of preparing a mammalian host cell according to claim 17, comprising a polynucleotide encoding a bacterial glutamine synthase operably linked to a mammalian promoter, and at least one polynucleotide encoding a protein of interest; (b) A step of culturing mammalian host cells under conditions that produce the protein of interest; and (c) The process of collecting the protein of interest and, if necessary, purifying it. A method for producing a protein of interest, including [specific example].

24. The mammalian host cell according to claim 17, wherein the mammalian host cell is a rodent cell.

25. The method according to claim 20, wherein the mammalian host cell is a rodent cell.

26. A kit comprising an expression vector according to any one of claims 1 to 12 and a cell culture medium that does not contain glutamine.

27. Use of bacterial glutamine synthase according to claim 16 as a selection marker in mammalian cells.

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