WO2023180398A1 - Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère - Google Patents

Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère Download PDF

Info

Publication number
WO2023180398A1
WO2023180398A1 PCT/EP2023/057369 EP2023057369W WO2023180398A1 WO 2023180398 A1 WO2023180398 A1 WO 2023180398A1 EP 2023057369 W EP2023057369 W EP 2023057369W WO 2023180398 A1 WO2023180398 A1 WO 2023180398A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
glutamine synthetase
protein
cell
bacterial
Prior art date
Application number
PCT/EP2023/057369
Other languages
English (en)
Inventor
Moritz Schmidt
Daniel HEINZELMANN
Franziska REUSS
Simon Fischer
Patrick SCHULZ
Original Assignee
Boehringer Ingelheim International Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boehringer Ingelheim International Gmbh filed Critical Boehringer Ingelheim International Gmbh
Publication of WO2023180398A1 publication Critical patent/WO2023180398A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker with a sequence identity of at least 85% with the amino acid sequence of SEQ ID NO: 1 and to a nucleic acid and a mammalian host cell encoding said bacterial glutamine synthetase.
  • the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family of Morganellaceae, preferably the bacterial glutamine synthetase is a Providencia species glutamine synthetase, optionally further comprises a mutation in position E130 and/or F226 and/or R345.
  • the invention further relates to methods for preparing a mammalian cell stably expressing a protein of interest and/or a non-coding RNA and to a method for producing a protein in a mammalian cell using said bacterial glutamine synthetase.
  • Chinese hamster ovary (CHO) cells are one of the most commonly employed mammalian cell lines used for the production of therapeutic proteins, such as antibodies.
  • An important aspect is to generate productive and stable cell lines expressing the protein of interest in a short period of time and at high product titers with suitable product quality.
  • a selection phase is required to generate stable heterogenous cell pools consisting of different clones which need to be isolated and screened, before the final production cell line can be preserved in a cell bank.
  • Metabolic selection systems such as the dihydrofolate reductase (DHFR) and the glutamine synthetase (GS) selection system are commonly used to improve this process and to generate stable cell lines more efficiently.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • DHFR gene In cell lines deficient of the DHFR gene, such as CHO-DG44 cells, selection is performed in the absence of hypoxanthine and thymidine in the medium. Amplification steps by adding increasing concentration of methotrexate (MTX) may be used in addition.
  • MTX methotrexate
  • the GS selection system is advantageous as it requires fewer gene copies for the survival and hence selection is faster for high producing cell pools.
  • Glutamine synthetase (EC 6.3.1 .2, also known as Y-glutamyl:ammonia ligase) catalyzes ATP- dependent condensation of ammonia and glutamate to form glutamine.
  • Glutamine synthetases are classified in three subgroups: GSI, GSII and GSIII.
  • the CHO GS is a class II enzyme, the subclass predominantly expressed by eukaryotes, whereas bacterial GS proteins are typically members of the GSI class. While GSI and GSII catalyze the same reaction, they show no or very little sequence similarity except for the residues forming part of the active site and are overall rather different.
  • bacterial type I GS is a 12-subunit complex (Eisenberg et al. (2000), Biochim. Biophys. Acta 1477, 122-145), and GSII has been reported to form two or three pentameric ring stackings (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228).
  • bacterial GS may have very little sequence similarity with each other, as low as about 50 % amino acid identity or less of e.g., Bacillus coagulans (CN 12625930 A) or Mycobacterium tuberculosis (WO 2006/000045) or Cornybacterium glutamicum (CN 1884501 A) with Providencia vermicola GS (Zuo et al., Scientific Reports, 2018, 8(1): 1-8 and supplementary material).
  • Bacillus coagulans CN 12625930 A
  • Mycobacterium tuberculosis WO 2006/000045
  • Cornybacterium glutamicum CN 1884501 A
  • GS is an ubiquitous enzyme essential for nitrogen metabolism. Thus, GS has been used as selection marker that is introduced via a mammalian expression construct. In cell lines that do not express sufficient levels of endogenous GS, removal of glutamine supplementation from the cell culture media increases the selection pressure on cells. In cell lines having insufficient endogenous GS levels, such as mouse myeloma cell lines, culturing in the absence of glutamine or lack of glutamine supplementation provides sufficient selection pressure to isolate stable recombinant cell lines.
  • the transfected glutamine synthetase selection marker has substantial influence on the selection process and the phenotypic stability as well as productivity of CHO-based cell lines.
  • the use of attenuated variants of the CHO glutamine synthetase was further shown to improve stability, selection behavior and productivity (Lin et al. (2019), mAbs, 11 :5, 965-976; WO 2018/093331 , US20190352631 , WO 2017/197098). Most of the described attenuated variants had mutations in the conserved substrate-binding residues.
  • the residues reported to be involved in binding of glutamate are E134, E136, E196, E203, N248, G249, H253, R299, R319, E338 and R340; the residues reported to be involved in binding of ATP are W130, A191 , G192, P208, N255, S257, R262, R324 and Y336; and the residues to be involved in binding of ammonia are D63, S66, D162 and E305 (amino acid numbering refers to human GS) (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228 and WO 2018/093331).
  • Attenuated GS variants harboring mutations in the active site show an increased selection stringency, the duration of the selection process and the overall cell growth characteristics can be impaired, negatively affecting bioprocess performance.
  • Attenuation of the selection marker is a fine balance between selection stringency, integrated copy numbers and suitable cell culture performance (e.g. growth behavior) to support the manufacturing of recombinant proteins.
  • suitable cell culture performance e.g. growth behavior
  • bacterial GS or peptides thereof from various species have been expressed in eukaryotic cells such as yeast, for purification, as immunogen or other reasons (see e.g., WO 2018/144807, WO 2006/000045 A1) and yeast or plant GS was shown to be active in E.coli but not vice versa (WO 2004/003175 A2 and EP 0 240 792 A1), bacterial GS (GSI) were assumed not to be active in mammalian cells. More importantly, to the best of our knowledge bacterial GS have not been tested or used in mammalian cells as selection marker before.
  • the present invention demonstrates that certain bacterial glutamine synthetase genes, unrelated to mammalian glutamine synthetase genes, can be used to generate highly productive and stable mammalian host cell pools expressing a therapeutic protein, particularly a therapeutic antibody, showing a more stringent selection behavior due to the attenuated activity compared to wildtype glutamine synthetase and beneficial cell culture performance in mammalian cells such as CHO cells. This is the first time that prokaryotic glutamine synthetase variants were used to generate stable and productive mammalian cell pools for cell line development.
  • Providencia vermicola glutamine synthetase were identified that exhibit extended selection stringency and improved antibody titers compared to wildtype Providencia vermicola glutamine synthetase.
  • Another advantage of a bacterial GS selection marker is that due to the low sequence similarity to mammalian (e.g., CHO) GS the probability of a recombination event that restores the endogenous GS gene and hence enables survival of cells not producing the gene of interest is dramatically reduced.
  • the invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker, wherein the bacterial glutamine synthetase has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 .
  • the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family of Morganellaceae.
  • the bacterial glutamine synthetase is derived from a Providencia species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 1.
  • the bacterial glutamine synthetase is derived from a Photorhabdus species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 14.
  • the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase or a Photorhabdus luminescens glutamine synthetase.
  • the mammalian expression vector may further encode the bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the amino acid sequence has 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the mammalian expression vector further comprises at least one polynucleotide encoding a protein of interest and/or a non-coding RNA, preferably an expression cassette comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • the protein of interest is preferably a therapeutic protein, more preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule and an antibody mimetic.
  • the invention further provides a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, optionally further comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the invention provides a bacterial glutamine synthetase from Providencia vermicola comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the invention provides a mammalian host cell comprising the expression vector according to the invention, the nucleic acid sequence according to the invention or a nucleic acid sequence encoding the bacterial glutamine synthetase according to the invention, preferably wherein the mammalian host cell is (a) a rodent cell, more preferably a CHO cell and/or (b) a GS gene knockout cell.
  • the polynucleotide encoding the bacterial glutamine synthetase is cointegrated with at least one polynucleotide encoding a protein of interest and/or a non-coding RNA into the host cell genome.
  • the invention provides a method for preparing a cell stably expressing a protein of interest and/or a non-coding RNA, comprising (a) introducing the expression vector according to the invention or the nucleic acid according to the invention comprising a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein of interest and/or a non-coding RNA into a mammalian host cell, preferably into a CHO cell; and (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest and/or a non-coding RNA is co-integrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome.
  • the method further comprises (c) a step of isolating a single clone for clonal expansion to prepare a monoclonal cell line.
  • the method is a method for preparing a monoclonal cell line stably expressing a protein of interest and/or a non-coding RNA.
  • the invention further provides a method of producing a protein of interest, comprising (a) introducing the mammalian expression vector according to the invention or the nucleic acid according to the invention into a mammalian host cell, preferably into a CHO cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is cointegrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome; (c) optionally isolating a single clone for clonal expansion to prepare a monoclonal cell line;
  • a method of producing a protein of interest comprising (a) providing the mammalian host cell of the invention, comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest.
  • the mammalian host cell is a rodent cell, more preferably a CHO cell.
  • the invention relates to a kit comprising the expression vector according to the invention and a cell culture medium not comprising glutamine.
  • the invention provides a use of a bacterial glutamine synthetase as a selection marker in mammalian cells.
  • FIGURE 1 CHO-K1 -GS cell pools expressing mAb1 and wildtype CHO GS as selection marker.
  • FIGURE 2 Viability and viable cell density (VCD) of CHO-K1-GS expressing mAb1 and wildtype CHO GS or Providencia vermicola GS as selection marker.
  • CHO-K1-GS cell pools stably transfected with a vector encoding the transcription cassette of mAb1 and glutamine synthetase selection marker from Cricetulus griseus or Providencia vermicola were cultured in medium not containing L-glutamine.
  • FIGURE 3 Productivity post selection and shake-flask production run of CHO-K1-GS expressing mAb1 and wildtype CHO GS or Providencia vermicola GS as selection marker.
  • CHO-K1- GS cell pools stably transfected with a vector encoding the transcription cassette of mAb1 and glutamine synthetase selection marker from Cricetulus griseus (CHO WT GS) or Providencia vermicola were cultured in medium not containing L-glutamine.
  • Productivity as titer [mg/L] was determined (A) post-selection at the indicated days post transfection using CHO WT GS or P. vermicola GS as selection marker and (B) in a shake-flask production run at the indicated days post transfection for P. vermicola GS only, because CHO WT GS cells lost their productivity after about 20 days.
  • FIGURE 4 Viability and productivity post transfection of CHO-K1-GS expressing mAb1 and GS from Providencia vermicola, Photorhabdus luminescens and Budvicia aquatica as selection marker.
  • CHO-K1-GS cell pools stably transfected with a vector encoding the transcription cassette of mAb1 and glutamine synthetase selection marker from Providencia vermicola (filled circle), Photorhabdus luminescens (filled square) and Budvicia aquatica (filled triangle) were cultured in medium not containing L-glutamine.
  • B Productivity as titer [mg/L] was determined post-selection at the indicated days post transfection.
  • FIGURE 5 Shake-flask production run of CHO-K1-GS expressing mAb1 and wildtype Providencia vermicola GS or Providencia vermicola GS E130F/E130G mutants as selection marker.
  • CHO-K1-GS cell pools stable transfected with a transcription cassette to express mAb1 and Providencia vermicola GS or Providencia vermicola GS harboring a E130F or E130G mutation as a selection marker were cultured in medium not containing L-glutamine. Titer [mg/L] of mAb1 was determined at the indicated days post transfection.
  • FIGURE 6 Productivity and viable cell density (VCD) in shake-flask production run of CHO-K1- GS expressing mAb1 and Providencia vermicola wildtype GS, CHO wildtype GS or Providencia vermicola F226Y variant as selection marker. After stable pool generation and single cell deposition, selected high producing clones were subjected to shaking flask experiment (14 days production run) head-to-head with CHO WT.
  • VCD viable cell density
  • A Productivity as mAb1 titer [mg/L) using of Providencia vermicola wildtype GS, Providencia vermicola GS F226Y or CHO GS WT as selection marker and
  • B viable cell density [1x10 6 cells/ml] of Providencia vermicola wildtype GS and Providencia vermicola GS F226Y as selection marker were determined at the end of production run.
  • FIGURE 7 Viability and productivity post transfection of CHO-K1-GS expressing mAb1 and Providencia vermicola wildtype GS or Providencia vermicola R345A or R360A variants as selection marker.
  • CHO-K1-GS cell pools stably transfected with a vector encoding the transcription cassette of mAb1 and glutamine synthetase selection marker from Providencia vermicola WT GS (filled circle), and Providencia vermicola R345A (filled square) or R360A variants (filled triangle) were cultured in medium not containing L-glutamine.
  • Viability [%] during selection was determined at the indicated days post transfection for Providencia vermicola wildtype GS and the two variants.
  • Productivity as titer [mg/L] was determined post-selection at the indicated days normalized to > 70% viability for Providencia vermicola wildtype GS and Providencia vermicola R345A variant, because Providencia vermicola R360A variant did not recover during selection.
  • FIGURE 8 Sequence of Providencia vermicola glutamine synthetase (SEQ ID NO: 1)
  • FIGURE 9 Sequence of wild type Cricetulus griseus glutamine synthetase (SEQ ID NO: 2) [030]
  • FIGURE 10 Sequence of Photorhabdus luminescens glutamine synthetase (SEQ ID NO: 14)
  • protein is used interchangeably with “amino acid sequence” or “polypeptide” and refers to polymers of amino acids of any length. These terms also include proteins that are post- translationally modified through reactions that include, but are not limited to, glycosylation, acetylation, phosphorylation, glycation or protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with the same properties.
  • nucleic acid sequence is used interchangeably with “polynucleotide” and refers to DNA or RNA of any length.
  • polynucleotide refers to DNA or RNA of any length.
  • Eukaryotic cell refers to cells that have a nucleus within a nuclear envelop and include animal cells, human cells, plant cells and yeast cells. Eukaryotic cell particularly encompasses mammalian cell, such as Chinese hamster ovary (CHO) cell or HEK293 cell derived cells. Mammalian cells as used herein refer to all cells or cell lines of mammalian origin, such as human or rodent cells. Cells as referred to herein are cells maintained in culture and do not relate to primary cells, but cell lines or cell line derived cells, i.e., to immortalized cells.
  • mammalian cells as used herein refer to all cells or cell lines of mammalian origin, such as human or rodent cells. Cells as referred to herein are cells maintained in culture and do not relate to primary cells, but cell lines or cell line derived cells, i.e., to immortalized cells.
  • selection stringency refers to the duration to reach more than 70 % viability and a doubling time of 48 h or less of the cell culture following transfection. The longer the time period, the more stringent the selection behavior. Typically, an attenuated glutamine synthetase shows a more stringent selection behavior compared to CHO wildtype glutamine synthetase.
  • the present invention demonstrates that certain bacterial glutamine synthetase genes, unrelated to mammalian glutamine synthetase genes, can be used as selection marker to generate highly productive and stable mammalian host cell pools or cell lines expressing a therapeutic protein, such as a therapeutic antibody, and/or a non-coding RNA, such as an siRNA or miRNA.
  • a therapeutic protein such as a therapeutic antibody
  • a non-coding RNA such as an siRNA or miRNA
  • point mutations of Providencia vermicola glutamine synthetase in highly conserved amino acids were identified that exhibit extended selection stringency and improved antibody titers compared to wildtype Providencia vermicola glutamine synthetase. Due to the conservation of these amino acids in at least Photorhabdus luminescens glutamine synthetase, it is expected that these point mutations in Photorhabdus luminescens glutamine synthetase similarly exhibit extended selection stringency and improved antibody titers compare to wildtype Photorhabdus luminescens glutamine synthetase.
  • the invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker, wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 ( Figure 8).
  • the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family of Morganellaceae.
  • the bacterial glutamine synthetase mediates increased selection stringency and/or genetic stability of a stably co-integrated protein of interest and/or non-coding RNA in CHO cells compared to a CHO glutamine synthetase having the amino acid sequence of SEQ ID NO: 2 ( Figure 9).
  • the bacterial glutamine synthetase is derived from a Providencia species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 1 ( Figure 8).
  • the bacterial glutamine synthetase is derived from a Photorhabdus species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 14 ( Figure 10).
  • the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase or a Photorhabdus luminescens glutamine synthetase.
  • the glutamine synthetase is from a Providencia species, more preferably from Providencia vermicola.
  • the glutamine synthetase may be a Providencia vermicola glutamine synthetase, particularly a bacterial glutamine synthetase comprising an 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 .
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90% and preferably at least 95% sequence identity with SEQ ID NO: 1.
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 or the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 14, preferably the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.
  • the bacterial glutamine synthetase may have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14.
  • the bacterial glutamine synthetase may further comprise point mutations that further attenuate the glutamine synthetase (e.g., E130X and/or R345A) or that increase catalytic activity (e.g., F226Y).
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position E130 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 .
  • the point mutation at position E130 may be a substitution with any amino acid (X), i.e., E130 may be mutated to any amino acid except E (SEQ ID NO: 3).
  • the mutation in amino acid position corresponding to E130 is a substitution with an aromatic or hydrophobic amino acid
  • the mutation in 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.
  • substitutions to amino acids not comprising polar, acidic or basic side chains are preferred.
  • the mutation is E130F, E130G or E130A, more preferably E130F or E130G (E130F/G) (SEQ ID NO: 4 or SEQ ID NO: 5, respectively).
  • X refers to any amino acid, wherein the reference to a mutation as E130X specifies that X is any amino acid except for the original amino acid, i.e., E (Glu, glutamic acid).
  • E Glu, glutamic acid
  • substitution of the highly conserved glutamic acid (E) corresponding to amino acid position E130 in SEQ ID NO: 1 results in attenuation of the bacterial glutamine synthetase, wherein attenuation means reduced enzymatic activity in this context.
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position F226 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 .
  • the point mutation corresponding to amino acid position F226 of SEQ ID NO: 1 is a substitution with the amino acid Y (Tyr, tyrosine), also referred to as F226Y.
  • F highly conserved phenylalanine
  • Such a mutation that increases catalytic activity of glutamine synthetase may be advantageously combined with a mutation that further attenuates the glutamine synthetase.
  • the mutation is E130X and/or F226Y, wherein X is any amino acid or as specified in this paragraph above. More preferably the mutation is E130X or E130X and F226Y. In a specific embodiment the mutation is E130F/G or E130F/G and F226Y.
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position R345 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 .
  • the point mutation corresponding to amino acid position R345 of SEQ ID NO: 1 is a substitution with the amino acid A (Ala, alanine), also referred to as R345A or alternatively with another amino acid with aliphatic side chain, such as glycine (G), valine (V), leucine (L) or isoleucine (I), preferably glycine (G).
  • R345A amino acid A
  • another amino acid with aliphatic side chain such as glycine (G), valine (V), leucine (L) or isoleucine (I), preferably glycine (G).
  • R highly conserved arginine
  • the bacterial glutamine synthetase may further comprise the point mutation F226Y.
  • the mutation is E130X and/or R345A (or G, V, L or I) optionally further comprising F226Y, wherein X is any amino acid or as specified in this paragraph above.
  • the mutations is E130F/G and/or R345A, optionally further comprising F226Y, such as E130F/G and R345A, optionally further comprising F226Y, or preferably E130F/G or R345A, optionally further comprising F226Y.
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic and/or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the mutation is E130X and/or F226Y, and wherein E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F/G).
  • the term a “mutation of E130X, F226Y, R345A and combinations thereof’ as used herein 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.
  • the mutation is (i) E130X or (ii) E130X and F226Y or (iii) R345A or (iv) R345A and F226Y, wherein X is as described herein.
  • the bacterial glutamine synthetase 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 (or G, V, L or I) and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic and/or hydrophobic amino acid, more preferably selected from the group consisting ofY, W, F, A, G, V, L, M and I.
  • 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, even more preferably the amino acid sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, further comprising the mutation as disclosed herein, such as exemplified in the amino acid sequence 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 as in 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).
  • the bacterial glutamine synthetase 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic and/or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the mutation in the bacterial glutamine synthetase having at least 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 14 is E130X and/or F226Y, and wherein 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, and wherein E130X is E130F, E130G or E130A, more preferably E130F or E130G (E130F/G).
  • the bacterial glutamine synthetase may comprise a mutation (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.
  • 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.
  • a “mutation E130X and/or F226Y” as used herein means a mutation E130X or F226Y or E130X and F226Y. More preferably the mutation is E130X or E130X and F226Y.
  • the term a mutation “F226Y and/or R345A” as used herein means a mutation F226Y or R345A or F226Y and R345A, preferably R345A or F226Y and R345A.
  • the mammalian expression vector according to the invention is for expression of the heterologous sequence in mammalian cells, i.e., it is adapted for expression in mammalian cells (such as mammalian cells), i.e., expression of the bacterial glutamine synthetase and preferably further a protein of interest and/or a non-coding RNA.
  • mammalian expression vector according to the invention is characterized in that it comprises the polynucleotide encoding the bacterial glutamine synthetase operably linked to a mammalian promoter.
  • the mammalian expression vector comprises an expression cassette comprising the polynucleotide encoding the bacterial glutamine synthetase operably linked to a mammalian promoter (such as a CMV promoter or an SV40 promoter).
  • a mammalian promoter such as a CMV promoter or an SV40 promoter.
  • Mammalian promoters regulate transcription in mammalian cells, particularly mammalian promoter regulate transcription in mammalian cells.
  • mammalian promoters without being limited thereto are simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1a promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK) and chicken p-actin promoter coupled to CMV early enhancer (CAGG).
  • the mammalian expression vector may further comprise bacterial sequences, such as an origin of replication and resistance genes for vector amplification in bacterial cells.
  • the mammalian expression vector typically further comprises at least one polynucleotide encoding a protein of interest and/or a non-coding RNA, preferably an expression cassette comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • the polynucleotide encoding the protein of interest and/or the non-coding RNA is operably linked to a mammalian promoter.
  • the protein of interest and/or the non-coding RNA and the bacterial glutamine synthetase may be encoded by the same (multicistronic) or separate expression cassettes.
  • the protein of interest and/or non-coding RNA and the bacterial glutamine synthetase are encoded by at least two separate expression cassettes or at least two separately encoded mRNAs.
  • the present invention does not encompass fusion proteins of the protein of interest with the bacterial glutamine synthetase or parts thereof.
  • the non-coding RNA is an RNA interference (RNAi) mediating RNA, for example miRNA, siRNA, IncRNA or shRNA.
  • RNAi RNA interference
  • expression cassette is a distinct component of a DNA, particularly vector DNA, consisting of one or more coding polynucleotide sequences and the regulatory sequences controlling their expression in a transfected or transduced cell.
  • An expression cassette comprises at least three components: a promoter sequence, an open reading frame, and termination sequence.
  • the termination sequence is referred to as 3’ untranslated region and usually contains a polyadenylation site.
  • the expression cassette directs the cell’s machinery to make RNA and may therefore also be referred to as transcriptional cassette.
  • the RNA may be coding RNA, further processed to mRNA encoding for a protein sequence e.g., glutamine synthetase or the protein of interest, or the RNA may be non-coding RNA, such as RNA interference (RNAi) mediating RNA, for example miRNA, siRNA, IncRNA or shRNA.
  • RNAi RNA interference
  • the protein of interest may be any protein, but is typically a therapeutic protein.
  • therapeutic protein refers to proteins that can be used in medical treatment of humans and/or animals. These include, but are not limited to cytokines, growth factors, hormones, blood coagulation factors, vaccines, interferons, fusion proteins, antibodies, antibody-derived molecules and an antibody mimetics.
  • the therapeutic protein is selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule and an antibody mimetic.
  • the protein of interest is an antibody.
  • the mammalian expression vector comprises a polynucleotide comprising a coding sequence for a variable region of the heavy chain and/or a coding sequence for a variable region of the light chain of the antibody.
  • the mammalian expression vector comprises a polynucleotide comprising a coding sequence for a heavy chain and/or a coding sequence for a light chain of the antibody.
  • the polynucleotide comprising a coding sequence for a variable region of the heavy chain and the polynucleotide comprising a coding sequence of a variable region of the light chain may be expressed on the same mammalian expression vector or on separate mammalian expression vectors.
  • the expression vector may comprise a multicistronic expression cassette, such as a bicistronic expression cassette, and/or multiple expression cassettes.
  • a multicistronic expression cassette comprises more than one open reading frames separated by sequences coding for an RNA element that allows for translation initiation, such as an internal ribosomal entry site (IRES).
  • IRS internal ribosomal entry site
  • the two or more open reading frames are under the control of the same promoter.
  • the polynucleotide encoding at least a variable region of the heavy chain and the polynucleotide encoding at least a variable region of the light chain may therefore be expressed within the same expression cassette (separated e.g., by an IRES sequence) or by two separate expression cassettes.
  • the bacterial glutamine synthetase and the protein of interest and/or the non-coding RNA may be expressed by the same or separate expression cassette(s).
  • the bacterial glutamine synthetase and polynucleotide encoding at least a variable region of the heavy chain and/or the polynucleotide encoding at least a variable region of the light chain may be expressed by the same or separate expression cassettes or a mixture thereof.
  • the mammalian expression vector is for stable integration into the host cell’s genome (such as for stable transfection) and the integrating part of the vector comprises the polynucleotide encoding the bacterial glutamine synthetase and the at least one polynucleotide encoding the protein of interest and/or the non-coding RNA.
  • the mammalian expression vector according to the invention preferably the mammalian expression vector according to the invention, is a plasmid, a Bacterial Artificial Chromosome (BAC) or a viral vector. Said plasmid, a Bacterial Artificial Chromosome (BAC) or viral vector (e.g.
  • a lentiviral vector may be introduced into the mammalian host cell (such as the mammalian host cell) via transfection or transduction, respectively, and is preferably stably integrated into the host cell genome.
  • suitable plasmids BACs or viral vectors and that a plasmid may further comprise transposon recognition sequences upstream and downstream of the polynucleotide encoding a bacterial synthetase as a selection marker and the optional at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • a preferred protein of interest is an antibody, including fragments and derivatives thereof.
  • an antibody is monospecific, but an antibody may also be multispecific.
  • the present invention may be used for the production of mono-specific antibodies, multi-specific antibodies, or fragments thereof, preferably of antibodies (mono-specific), bispecific antibodies, trispecific antibodies or fragments thereof, preferably antigen-binding fragments thereof.
  • Exemplary antibodies within the scope of the present invention include but are not limited to anti-CD2, anti-CD3, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD44v6, anti-CD49d, anti-CD52, anti- EGFR1 (HER1), anti-EGFR2 (HER2), anti-GD3, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2, anti-IL- 5R or anti-lgE antibodies, and are preferably selected from the group consisting of anti-CD20, anti- CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2/neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2 and anti-lgE antibodies.
  • antibody refers to any antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • immunoglobulins There are various classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW.
  • the antibody is an IgG antibody, more preferably an lgG1 or an lgG4 antibody.
  • Antibodies can be of any species and include chimeric and humanized antibodies.
  • Chimeric antibodies are molecules in which antibody domains or regions are derived from different species.
  • the variable region of heavy and light chain can be derived from rat or mouse antibody and the constant regions from a human antibody.
  • humanized antibodies only minimal sequences are derived from a non-human species. Often only the CDR amino acid residues of a human antibody are replaced with the CDR amino acid residues of a non-human species such as mouse, rat, rabbit or llama. Sometimes a few key framework amino acid residues with impact on antigen binding specificity and affinity are also replaced by non-human amino acid residues.
  • antibodies are tetrameric polypeptides composed of two pairs of a heterodimer each formed by a heavy and a light chain. Stabilization of both the heterodimers as well as the tetrameric polypeptide structure occurs via interchain disulfide bridges.
  • Each chain is composed of structural domains called “immunoglobulin domains” or “immunoglobulin regions” whereby the terms “domain” or “region” are used interchangeably.
  • Each domain contains about 70 - 110 amino acids and forms a compact three-dimensional structure.
  • Both heavy and light chain contain at their N-terminal end a “variable domain” or “variable region” with less conserved sequences which is responsible for antigen recognition and binding.
  • the variable region of the light chain is also referred to as “VL” and the variable region of the heavy chain as “VH”.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’) 2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Fab fragments consist of the variable regions of both chains, which are held together by the adjacent constant region. These may be formed by protease digestion, e.g., with papain, from conventional antibodies, but similarly Fab fragments may also be produced by genetic engineering. Further antibody fragments include F(ab‘)2 fragments, which may be prepared by proteolytic cleavage with pepsin.
  • antibody protein of this kind is known as a single-chain-Fv (scFv).
  • scFv-antibody proteins are known to the person skilled in the art.
  • antibody fragments and antigen-binding fragments further include Fv-fragments and particularly scFv.
  • scFv As a multimeric derivative, various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity.
  • scFv were prepared as fusion proteins with multimerisation domains.
  • the multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucine-zipper domains.
  • the interaction between the VHA/L regions of the scFv is used for the multimerisation (e.g. dia-, tri- and pentabodies).
  • diabody By diabody the skilled person means a bivalent homodimeric scFv derivative.
  • Diabodies may additionally be stabilized by the incorporation of disulfide bridges. Examples of diabody-antibody proteins are known from the prior art.
  • minibody means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably lgG1 as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from lgG1) and a linker region.
  • an immunoglobulin preferably IgG
  • lgG1 as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from lgG1) and a linker region.
  • Hinge region e.g. also from lgG1
  • linker region e.g. also from lgG1
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative. ScFv derivatives wherein VH-VL is fused directly without a linker sequence lead to the formation of trimers.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerisation is carried out by di-, tri- or tetrameric coiled coil structures.
  • the gene of interest is encoded for any of those desired polypeptides mentioned above, preferably for a monoclonal antibody, a derivative or fragment thereof.
  • Single-domain antibody also be referred to as nanobody, which is an antibody fragment of a single monomeric variable antibody domain.
  • Single-domain antibodies are typically engineered from heavy chain antibodies found in camelids (VHH fragments) or cartilaginous fishes (VNAR fragments).
  • Fc fragment crystallizable
  • These may be formed by protease digestion, e.g. with papain or pepsin from conventional antibodies but may also be produced by genetic engineering.
  • the N-terminal part of the Fc fragment might vary depending on how many amino acids of the hinge region are still present.
  • Antibodies comprising an antigen-binding fragment and an Fc region may also be referred to as full-length antibody.
  • Full-length antibody may be mono-specific and multispecific antibodies.
  • Multispecific antibodies are antibodies which have at least two different antigen-binding sites each of which bind to different epitopes.
  • a multispecific antibody includes bispecific and trispecific antibodies.
  • a bispecific antibody has two different binding binding sites.
  • Multispecific antibodies also include antibody formats other than full-length antibodies such as antibody-derived molecules.
  • Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, NK cells or macrophages) in one molecule.
  • target cells e.g., malignant B cells
  • effector cells e.g., T cells, NK cells or macrophages
  • Exemplary bispecific antibodies without being limited thereto are diabodies, BiTE (Bi-specific T-cell Engager) formats and DART (Du a I- Affinity Re-Targeting) formats.
  • the diabody format separates cognate variable domains of heavy and light chains of the two antigen binding specificities on two separate polypeptide chains, with the two polypeptide chains being associated non-covalently.
  • the DART format is based on the diabody format, but it provides additional stabilization through a C- terminal disulfide bridge.
  • Trispecific antibodies are monoclonal antibodies which combine three antigen-binding specificities. They may be build on bispecific-antibody technology that reconfigures the antigen-recognition domain of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been generated that target CD38 on cancer cells and CD3 and CD28 on T cells. Multispecific antibodies are particularly difficult to product with high product quality.
  • antibody-derived molecule refers to any molecule comprising at least an antigen-binding moiety that is structurally related to antibodies. It includes modified full-length mono- or bispecific antibodies further modified with an additional antigen binding moiety or smaller antibody formats including the ones described herein.
  • antibody mimetic refers to proteins that bind to specific antigens in a manner similar to antibodies, but that are not structurally related to antibodies.
  • Antibody mimetic include, without being limited thereto an anticalin, an affibody, an adnectin, a monobody, a DARPin, an affimer, and an affitin.
  • a single-domain antibody may also be referred to as nanobody.
  • the protein may comprise more than one antigen-binding domain and hence may be multivalent, preferably bivalent (e.g., a bivalent sdAb or a bivalent anticalin or any other bivalent antibody mimetic).
  • Another preferred therapeutic protein is a fusion protein, such as an Fc-fusion protein.
  • the invention can be advantageously used for production of fusion proteins, such as Fc-fusion proteins.
  • the effector part of the fusion protein can be the complete sequence or any part of the sequence of a natural or modified heterologous protein.
  • the immunoglobulin constant domain sequences may be obtained from any immunoglobulin subtypes, such as lgG1 , lgG2, lgG3, lgG4, lgA1 or lgA2 subtypes or classes such as IgA, IgE, IgD or IgM.
  • Fc-fusion proteins are MCP1-Fc, ICAM-Fc, EPO-Fc and scFv fragments or the like coupled to the CH2 domain of the heavy chain immunoglobulin constant region comprising the N- linked glycosylation site.
  • Fc-fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of the heavy chain immunoglobulin constant region comprising the N- linked glycosylation site into another expression construct comprising for example other immunoglobulin domains, enzymatically active protein portions, or effector domains.
  • an Fc- fusion protein according to the present invention comprises also a single chain Fv fragment linked to the CH2 domain of the heavy chain immunoglobulin constant region comprising, e.g., the N-linked glycosylation site.
  • cytokine refers to small proteins, which are released by cells and act as intercellular mediators, for example influencing the behavior of the cells surrounding the secreting cell.
  • Cytokines may be secreted by immune cells or other cells, such as T-cells, B-cells, NK cells and macrophages. Cytokines may be involved in intercellular signaling events, such as autocrine signaling, paracrine signaling and endocrine signaling. They may 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.
  • growth factor refers to proteins or polypeptides that are capable of stimulating cell growth.
  • the invention further relates to a use of the mammalian expression vector of the invention for expression of a protein of interest and/or a non-coding RNA in a mammalian cell, particularly a rodent cell, such as a CHO cell.
  • the bacterial glutamine synthetase encoded by said mammalian expression vector serves as a selection marker in said mammalian cells.
  • the invention in a second aspect, relates to a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 operably linked to a mammalian promoter, optionally further comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • the nucleic acid sequence may be part of the mammalian expression vector of the first aspect.
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90% or 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 and combinations thereof (such as E130X and/or F226Y), wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the mutation is E130X and/or F226 and E130X is E130F or E130G; or the mutation is F226Y and/or R345A; or the mutation is E130X and/or R345A, optionally further comprising the mutation F226Y.
  • the invention relates to a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • the invention relates to a bacterial glutamine synthetase, such as from Providencia vermicola, comprising 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 and combinations thereof (such as E130X and/or F226Y), wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I; or to a bacterial glutamine synthetase comprising 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 E130X and/or F226Y and/or R345A, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a
  • the mutation is E130X and/or F226 and E130X is E130F or E130G; or the mutation is F226Y and/or R345A; or the mutation is E130X and/or R345A optionally further comprising the mutation F226Y, preferably E130X or R345A optionally further comprising the mutation F226Y.
  • the embodiments and examples specified with regard to the first and second aspect similarly apply to this aspect.
  • the invention in a fourth aspect, relates to a kit comprising the expression vector according to the invention and optionally a cell culture medium not comprising glutamine.
  • the invention relates to a use of a bacterial glutamine synthetase as a selection marker in mammalian cells.
  • the bacterial glutamine synthetase is further specified as disclosed with regard to the first aspect above. More specifically, bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family Monganellaceae, preferably of a Providencia species or a Photorhabdus species, more preferably of Providencia vermicola or Photorhabdus luminescens, even more preferably of Providencia vermicola.
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 .
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 85%, at least 90% and preferably at least 95% sequence identity with SEQ ID NO: 1 .
  • the bacterial glutamine synthetase comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with 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 the amino acid sequence of SEQ ID NO: 14.
  • the bacterial glutamine synthetase Upon transfection or transduction of a mammalian expression vector comprising said bacterial glutamine synthetase, the bacterial glutamine synthetase mediates increased selection stringency and/or genetic stability of a stably co-integrated protein of interest and/or non-coding RNA in CHO cells compared to a CHO glutamine synthetase having the amino acid sequence of SEQ ID NO: 2.
  • the bacterial glutamine synthetase is derived from a Providencia species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 1 ; more preferably the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase.
  • the bacterial glutamine synthetase is derived from a Photorhabdus species and/or comprises an amino acid sequence having at least 95% sequence identity with amino acid sequence of SEQ ID NO: 14; more preferably the bacterial glutamine synthetase is a Photorhabdus luminescens glutamine synthetase.
  • the amino acid sequence of Providencia vermicola glutamine synthetase (SEQ ID NO: 1) and Photorhabdus luminescens glutamine synthetase (SEQ ID NO: 14) have a sequence identity of 87.2% (see Table B).
  • the bacterial glutamine synthetase may further comprise point mutations that further attenuate the glutamine synthetase (e.g., E130X and/or R345A) or that increase catalytic activity (e.g., F226Y).
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position E130 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 .
  • the point mutation at position E130 may be a substitution with any amino acid (X), preferably the mutation in amino acid position corresponding to E130 is a substitution with an aromatic or hydrophobic amino acid, such as 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.
  • the mutation is E130F, E130G or E130A, more preferably E130F or E130G (E130F/G) (SEQ ID NO: 4 or SEQ ID NO: 5, respectively).
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position F226 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 , wherein the point mutation is a substitution with the amino acid Y (Tyr, tyrosine), also referred to as F226Y.
  • the mutation is E130X and/or F226Y, wherein X is any amino acid or as specified in this paragraph above. More preferably the mutation is E130X or E130X and F226Y. In a specific embodiment the mutation is E130F/G or E130F/G and F226Y.
  • the bacterial glutamine synthetase may have a point mutation in a highly conserved residue corresponding to position R345 of Providencia vermicola glutamine synthetase having the amino acid sequence of SEQ ID NO: 1.
  • the point mutation corresponding to amino acid position R345 of SEQ ID NO: 1 is a substitution with the amino acid A (Ala, alanine), also referred to as R345A or alternatively with another amino acid with aliphatic side chain, such as glycine (G), valine (V), leucine (L) or isoleucine (I), preferably glycine (G).
  • the bacterial glutamine synthetase may further comprise the point mutation F226Y.
  • the mutation is E130X and/or R345A (or G, V, L or I) optionally further comprising F226Y, wherein X is any amino acid or as specified in this paragraph above.
  • the mutations is E130F/G and/or R345A, optionally further comprising F226Y, such as E130F/G and R345A, optionally further comprising F226Y, or preferably E130F/G or R345A, optionally further comprising F226Y.
  • the bacterial glutamine synthetase comprises an amino acid having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 , further comprising the point mutations as indicated above.
  • the bacterial glutamine synthetase 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 (or G, V, L or I) and combinations thereof (e.g. E130X and/or F226Y), wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic and/or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • 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, even more preferably the amino acid sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, further comprising the mutation as disclosed herein, such as exemplified in the amino acid sequence 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).
  • the mutation is E130X and/or F226Y, and preferably wherein 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) optionally further comprising F226Y, wherein X is any amino acid or as specified in this paragraph above.
  • the mutations is E130F/G and/or R345A, optionally further comprising F226Y, such as E130F/G and R345A, optionally further comprising F226Y, or preferably E130F/G or R345A, optionally further comprising F226Y.
  • the invention relates to a mammalian host cell comprising the mammalian expression vector of the invention (first aspect), the nucleic acid sequence of the invention (second aspect) or a nucleic acid sequence encoding the bacterial glutamine synthetase of the invention (third aspect).
  • the mammalian host cell may be any host cell, provided the host cell is an immortalized cell and not a primary cell.
  • the mammalian host cell is a mouse, a human or a rodent cell, more preferably a rodent cell, even more preferably a CHO cell.
  • the mammalian host cell is preferably a GS gene knockout cell (GS knockout mutant) host cell.
  • GS gene knockout cell refers to a cell in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in GS enzyme function disruption. Such cells may be referred to as GS-/- or GS-/+ cells, depending on whether both or only one allele has been deleted or disrupted. Extracellular glutamine supplementation or a GS gene introduced by an expression vector is essential for cell survival of GS gene knockout cells.
  • the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS-/-) cell.
  • the mammalian host cell is a monoclonal cell line generated by a step of single cell cloning and clonal expansion.
  • the mammalian host cell comprises a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein of interest and/or the non-coding RNA co-integrated into the host cell genome.
  • the invention relates to a method for preparing a cell stably expressing a protein of interest and/or a non-coding RNA, comprising (a) introducing the expression vector according to the invention (first aspect) or the nucleic acid of the invention (second aspect) comprising a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein of interest and/or a non-coding RNA into a mammalian host cell; and (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest and/or a non-coding RNA is co-integrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome.
  • the method may further comprise a step of culturing the resulting cell pool stably expressing a protein of interest and/or a non-coding RNA selected in step (b) and/or isolating a cell pool stably expressing a protein of interest and/or a non-coding RNA.
  • a cell stably expressing a protein of interest and/or a non-coding RNA means that the polynucleotide encoding the protein or interest and/or the non-coding RNA is stable integrated into the genome of the host cell and that the protein of interest and/or the non-coding RNA is stable expressed, i.e., over an extended period of time, such as at least 40 days preferably for months or years.
  • the mammalian expression vector or the nucleic acid may be a plasmid, a Bacterial Artificial Chromosome (BAC) or a viral vector.
  • the mammalian expression vector or the nucleic acid of the invention may be introduced by transfection or transduction, respectively.
  • the mammalian expression vector or the nucleic acid is a plasmid. More preferably the mammalian expression vector or the nucleic acid is introduced by stable transfection.
  • Methods of transducing or transfecting a mammalian expression vector or a nucleic acid into mammalian cells are well known in the art and comprise chemical means, such as calcium phosphate precipitation and lipofection, and physical means, such as electroporation.
  • the polynucleotide encoding the bacterial glutamine synthetase and the polynucleotide encoding the protein of interest and/or the non-coding RNA are operably linked to a mammalian promoter and are therefore adapted for expression in a mammalian host cell.
  • the method of the invention may further comprise a step (c) of isolating a single clone for clonal expansion to prepare a monoclonal cell line.
  • a step (c) of isolating a single clone for clonal expansion to prepare a monoclonal cell line The person skilled in the art would understand that transfection or transduction often requires a large number of cells, resulting in a heterogenous pool of recombinant cells with, e.g., varying integration site populations.
  • the cell pool is diluted or sorted for single cell isolation (monoclonal) and each single clone is subjected to clonal expansion to prepare a monoclonal cell line.
  • cell line refers to a population of cell derived from a single cell clone and can be grown for an unlimited time. It is therefore also referred to as monoclonal cell line.
  • a cell line is genetically stable and hence the characteristics of a cell line should not change over time. Particularly phenotypic characteristics such as production levels (titer) and growth rate and density (VCD and maximal VCD), viability as well as genetic integrity as measured via copy number and DNA-fingerprint assays should be maintained when cultured under comparable conditions.
  • the cell pool or the monoclonal cell line prepared according to the method of the invention may be further used for stably producing a protein of interest or for stably producing a non-coding RNA, such as an RNA mediating RNAi, e.g., an miRNA, an siRNA, IncRNA or an shRNA.
  • RNAi is used for gene silencing and may therefore be used for generating a cell pool or a monoclonal cell line in beneficial properties for, e.g., protein production.
  • a difficult to remove host cell protein may be silenced in the cell pool or monoclonal cell line, or an enzyme such as a fucosyltransferase may be silenced to modify the glycosylation profile of the cell pool or monoclonal cell line.
  • HCP host cell protein
  • CHO cells commonly used for large-scale industrial production are often engineered to improve their characteristics in the production process, or to facilitate selection of recombinant cells.
  • Such engineering includes, but is not limited to increasing apoptosis resistance, reducing autophagy, increasing cell proliferation, altered expression of cell-cycle regulating proteins, chaperone engineering, engineering of the unfolded protein response (UPR), engineering of secretion pathways and metabolic engineering.
  • UTR unfolded protein response
  • Such engineering can potentially be achieved using RNAi in mammalian host cells generated by the methods of the present invention.
  • the invention relates to a method of producing a protein of interest, comprising (a) introducing the mammalian expression vector according the invention or the nucleic acid according to the invention into a mammalian host cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select forthe bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome; (c) optionally isolating a single clone for clonal expansion to prepare a monoclonal cell line; (a) introducing the mammalian expression
  • the integrating part of the vector comprises the polynucleotide encoding the bacterial glutamine synthetase and at least one polynucleotide encoding the protein of interest and/or the non-coding RNA.
  • the integrated part of the mammalian expression vector or the integrated nucleic acid sequence of the invention may further be amplified, e.g., by increasing the concentration of a glutamine synthetase inhibitor, such as methionine sulfoximine (MSX).
  • MSX methionine sulfoximine
  • Amplification is optional and may result in higher productivity due to higher copy numbers of the polynucleotide encoding the protein of interest and/or the non-coding RNA, because these become co-amplified together with the bacterial glutamine synthetase.
  • the methods according to the invention may include the generation of a cell pool or a monoclonal cell line.
  • a mammalian host cell generated according to the methods of the invention or a mammalian host cell according to the invention may further be used for producing a protein of interest and/or a non-coding RNA or in a method for producing a protein of interest.
  • the invention relates to a method of producing a protein of interest, comprising (a) providing the mammalian host cell according to the invention or produced by the methods according to the invention comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest.
  • the mammalian host cell is a GS gene knockout cell.
  • the person skilled in the art will understand that this refers to the endogenous GS gene, while the bacterial GS gene is present in the host cell following transfection or transduction of the mammalian expression vector orthe nucleic acid sequence of the invention.
  • the mammalian host cell (transfected or transduced with the mammalian expression vector or the nucleic acid sequence of the invention) is cultured in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase in step (b). This may further comprise the addition of the GS inhibitor methionine sulfoximine (MSX).
  • MSX methionine sulfoximine
  • the cell (cell pool) or monoclonal cell line is generated with increased selection stringency and/or has increased genetic stability and/or has higher productivity compared to a cell or cell line generated with a glutamine synthetase from Cricetulus griseus having the amino acid sequence of SEQ ID NO: 2.
  • Genetic stability may be quantified by measuring copy numbers of the integrated transgenes. Genetic stability is assessed by measuring copy number over an extended cultivation time. In addition to that genomic rearrangements are monitored via e.g. Southern Blot analysis. An increase in selection stringency may be determined by the duration until reaching > 70% viability.
  • the mammalian host cell may be any host cell, provided 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 according to the invention are for in vitro use in cell culture.
  • the term “mammalian cell” as used herein refers to mammalian cell lines suitable for the production of a product of interest, such as a heterologous protein of interest and/or a non-coding RNA and may also be referred to as “host cells” or “mammalian host cell”.
  • the mammalian cells are preferably transformed and/or immortalized cell lines. They are adapted to serial passages in cell culture, preferably serum-free cell culture and/or preferably as suspension culture, and do not include primary non-transformed cells or cells that are part of an organ structure.
  • the mammalian host cell is a mouse, a human or rodent cell, more preferably a rodent cell, even more preferably a CHO cell.
  • Preferred mammalian cells for heterologous protein production are murine cells, rodent cells or human cells.
  • Preferred examples of mammalian cells or mammalian cell lines are CHO cells (such as DG44 and K1), NSO cells, HEK293 cells (such as HEK293 cells and HEK293T cells) and BHK21 cells.
  • the mammalian cells or mammalian cell lines are adapted to growth in suspension.
  • the mammalian cells or mammalian cell line is a CHO cell.
  • the mammalian cell is a HEK293 cell or a CHO cell or a HEK293 cell or a CHO cell derived cell, preferably the mammalian cell is a CHO cell or a CHO derived cell.
  • Suitable rodent cells may be e.g., hamster cells, particularly BHK21 , BHK TK, CHO, CHO-K1 , CHO-DXB11 (also referred to as CHO-DUKX or DuxB11), a CHO-S cell and CHO-DG44 cells or the derivatives/progenies of any of such cell line.
  • CHO cells such as CHO- DG44, CHO-K1 and BHK21 , and even more preferred are CHO-DG44 and CHO-K1 cells.
  • Most preferred are CHO-DG44 cells.
  • Glutamine synthetase (GS)-deficient derivatives of the mammalian cell particularly of the CHO-DG44 and CHO-K1 cell are also encompassed.
  • the mammalian cell is a Chinese hamster ovary (CHO) cell, preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO DXB11 cell, a CHO-S cell, a CHO GS deficient cell or a derivative thereof.
  • Suitable human cells are HEK293 or HEK293T cells.
  • the host cells may also be murine cells such as murine myeloma cells, such as NSO and Sp2/0 cells or the derivatives/progenies of any of such cell line.
  • the mammalian host cell is preferably a GS gene knockout cell (GS knockout mutant).
  • GS gene knockout cell refers to a cell in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in GS enzyme function disruption. Such cells may be referred to as GS-/- or GS-/+ cells, depending on whether both or only one allele has been deleted or disrupted. Extracellular glutamine supplementation or a GS gene introduced by an expression vector is essential for cell survival of GS gene knockout cells.
  • the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS-/-) cell.
  • CHO cells that allow for efficient cell line development processes are metabolically engineered, such as by endogenous glutamine synthetase (GS) knockout to facilitate selection with methionine sulfoximine (MSX).
  • GS glutamine synthetase
  • MSX methionine sulfoximine
  • Non-limiting examples of mammalian cells which can be used in the meaning of this invention are also summarized in Table A.
  • derivatives/progenies of those cells, other mammalian cells, including but not limited to human, mice, rat, monkey, and rodent cell lines can also be used in the present invention, particularly for the production of biopharmaceutical proteins.
  • CAP CEVEC's Amniocyte Production
  • CAP cells are an immortalized cell line based on primary human amniocytes. They were generated by transfection of these primary cells with a vector containing the functions E1 and pIX of adenovirus 5.
  • CAP cells allow for competitive stable production of recombinant proteins with excellent biologic activity and therapeutic efficacy as a result of authentic human posttranslational modification.
  • Cells are most preferred, when being established, adapted, and completely cultivated under serum free conditions, and optionally in media, which are free of any protein/peptide of animal origin.
  • Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO Medium (Sigma), and protein-free CHO Medium (Sigma) are exemplary appropriate nutrient solutions.
  • any of the media may be supplemented as necessary with a variety of compounds, non-limiting examples of which are recombinant hormones and/or other recombinant growth factors (such as insulin, transferrin, epidermal growth factor, insulin like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics and trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • recombinant hormones and/or other recombinant growth factors such as insulin, transferrin, epidermal growth factor, insulin like growth factor
  • salts such as sodium chloride, calcium, magnesium, phosphate
  • buffers such as HEPES
  • nucleosides such as adenosine, thymidine
  • glutamine glucose or other equivalent energy sources
  • antibiotics and trace elements Any other necessary
  • the protein of interest encoded by the mammalian expression vector or produced by the methods of the invention is preferably produced in CHO cells in cell culture. Following expression, the recombinant protein is harvested and further purified.
  • the antibody may be recovered from the culture medium as a secreted protein in the harvested cell culture fluid (HCCF) or from a cell lysate (i.e., the fluid containing the content of a cell lysed by any means, including without being limited thereto enzymatic, chemical, osmotic, mechanical and/or physical disruption of the cell membrane and optionally cell wall) and purified using techniques described herein.
  • the method comprises providing a harvested cell culture fluid comprising a protein of interest, such as an antibody as starting material, wherein the HCCF is from CHO cell culture.
  • a protein of interest such as an antibody as starting material
  • the protein of interest is recovered from the harvested cell culture fluid following cell separation, such as by filtration and/or centrifugation.
  • the harvest includes centrifugation and/or filtration to produce a harvested cell culture fluid.
  • Item 1 provides a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthetase as a selection marker (such as a mammalian expression vector), wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 .
  • Item 2 specifies for the mammalian expression vector of item 1 that the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family of Morganellaceae.
  • Item 3 specifies for the mammalian expression vector of item 1 or 2 that the bacterial glutamine synthetase is derived from a Providencia species or a Photorhabdus species, preferably wherein the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase or a Photorhabdus luminescens glutamine synthetase.
  • Item 4 specifies for the mammalian expression vector of any one of items 1 to 3 that the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 5 specifies for the mammalian expression vector of any one of items 1 to 4 that the bacterial glutamine synthetase 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 6 further specifies the mammalian expression vector of item 4 or 5 in that the mutation is E130X and/or F226Y and/or R345A, and wherein E130X is E130F or E130G.
  • Item 7 further specifies the mammalian expression vector of any one of items 1 to 6 in that the polynucleotide encoding the bacterial glutamine synthetase is operably linked to a mammalian promoter.
  • Item 8 further specifies the mammalian expression vector of any one of items 1 to 7 in that the mammalian expression vector further comprises at least one polynucleotide encoding a protein of interest and/or a non-coding RNA, preferably an expression cassette comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • Item 9 further specifies the mammalian expression vector according to any one of items 1 to 8 in that the protein of interest is a therapeutic protein, preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule and an antibody mimetic.
  • the protein of interest is a therapeutic protein, preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule and an antibody mimetic.
  • Item 10 further specifies the mammalian expression vector according to item 9 in that the protein of interest is an antibody, preferably wherein expression vector comprises a polynucleotide comprising a coding sequence for a variable region of the heavy chain and/or a coding sequence for a variable region of the light chain of the antibody.
  • Item 1 1 further specifies the mammalian expression vector according to any one of items 8 to 10 in that the mammalian expression vector comprises a multicistronic expression cassette and/or multiple expression cassettes, preferably wherein the expression vector comprises multiple expression cassettes.
  • Item 12 further specifies the mammalian expression vector of any one of items 8 to 11 in that the mammalian expression vector is for stable transfection and the integrating part of the vector comprises the polynucleotide encoding the bacterial glutamine synthetase and at least one polynucleotide encoding the protein of interest and/or the non-coding RNA.
  • Item 13 further specifies the mammalian expression vector according to any one of items 1 to 12 in that the mammalian expression vector is a plasmid, a Bacterial Artificial Chromosome (BAC) or a viral vector.
  • the mammalian expression vector is a plasmid, a Bacterial Artificial Chromosome (BAC) or a viral vector.
  • BAC Bacterial Artificial Chromosome
  • Item 14 provides a nucleic acid sequence comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter, optionally further comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  • Item 15 further specifies the nucleic acid sequence of item 14 in that the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 16 further specifies the nucleic acid sequence of item 15 in that the bacterial glutamine synthetase 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 and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 17 further specifies the nucleic acid sequence of item 15 in that the bacterial glutamine synthetase comprises an amino acid sequence having 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 18 further specifies the nucleic acid sequence of any one of items 15 to 17 in that the mutation is E130X and/or F226 and/or R345A and wherein E130X is E130F or E130G
  • Item 19 provides a bacterial glutamine synthetase comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 20 provides a bacterial glutamine synthetase from Providencia vermicola comprising 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 and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 21 provides a bacterial glutamine synthetase from Photorhabdus luminescens comprising an amino acid sequence having 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 22 provides a mammalian host cell comprising the expression vector of any one of items 1 to 13, the nucleic acid sequence of any one of items 14 to 18, or a nucleic acid sequence encoding the bacterial glutamine synthetase of any one of items 19 to 21 , preferably wherein the mammalian host cell is a rodent cell, more preferably a CHO cell and/or (b) a GS gene knockout cell.
  • Item 23 further specifies the mammalian host cell of item 22 in that it comprises a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein of interest and/or the non-coding RNA co-integrated into the host cell genome.
  • Item 24 provides a method for preparing a cell stably expressing a protein of interest and/or a non-coding RNA, comprising (a) introducing the expression vector according to items 1 to 13 or the nucleic acid of any one of items 14 to 18 comprising a polynucleotide encoding a bacterial glutamine synthetase and at least one polynucleotide encoding a protein of interest and/or a non-coding RNA into a mammalian host cell, such as a CHO cell; and (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest and/or the non-coding RNA is co-integrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome.
  • Item 25 further specifies the method of item 24, to comprise (c) a step of isolating a single clone for clonal expansion to prepare a monoclonal cell line.
  • Item 26 provides a method of producing a protein of interest, comprising (a) introducing the mammalian expression vector according to items 1 to 13 or the nucleic acid of any one of items 14 to 18 into a mammalian host cell, preferably into a mammalian host cell, such as a CHO cell, wherein the expression vector comprises a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated with the polynucleotide encoding the bacterial glutamine synthetase into the host cell genome; (c) optionally isolating a single clone
  • Item 27 further specifies the method of any one of items 24 to 26 in that the mammalian expression vector or the nucleic acid is introduced by transfection or transduction.
  • Item 28 further specifies the method of item 27 in that the mammalian expression vector or the nucleic acid is introduced by stable transfection.
  • Item 29 provides a method of producing a protein of interest, comprising (a) providing the mammalian host cell of item 22 or 23, comprising a polynucleotide encoding a bacterial glutamine synthetase operably linked to a mammalian promoter and at least one polynucleotide encoding a protein of interest; (b) culturing the mammalian host cell under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest.
  • Item 30 further specifies the method of any one of items 24 to 29, wherein (a) the mammalian host cell is a GS gene knockout cell; and/or (b) culturing the mammalian host cell in a medium comprising no glutamine under conditions to select for the bacterial glutamine synthetase in step (b) comprises the addition of the GS inhibitor methionine sulfoximine (MSX).
  • MSX methionine sulfoximine
  • Item 31 further specifies the method of any one of items 24 to 30 in that the mammalian host cell is a rodent cell, preferably a CHO cell.
  • Item 32 further specifies the method of any one of items 24 to 30 in that the cell or cell line is generated with increased selection stringency and/or has increased genetic stability and/or has higher productivity compared to a cell or cell line generated with a glutamine synthetase from Cricetulus griseus having the amino acid sequence of SEQ ID NO: 2.
  • Item 33 provides a kit comprising the expression vector of any one of items 1 to 13 and a cell culture medium not comprising glutamine.
  • Item 34 provides a use of a bacterial glutamine synthetase as a selection marker in a mammalian cell, preferably wherein the bacterial glutamine synthetase comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 .
  • Item 35 further specifies the use of item 34 in that the bacterial glutamine synthetase is derived from bacteria of the order Enterobacterales and of the family Monganellaceae, more preferably of a Providencia species or a Photorhabdus species, even more preferably of Providencia vermicola or Photorhabdus luminescens.
  • Item 36 further specifies the use of item 35 in that the bacterial glutamine synthetase is (a) derived from a Providencia species 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 wherein the bacterial glutamine synthetase is a Providencia vermicola glutamine synthetase.
  • Item 37 further specifies the use of any one of items 34 to 36 in that the bacterial glutamine synthetase 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 and combinations thereof, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 38 further specifies the use of item 34 in that the bacterial glutamine synthetase is derived from a Photorhabdus species 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 wherein the bacterial glutamine synthetase is a Photorhabdus luminescens glutamine synthetase.
  • Item 39 further specifies the use of any one of items 34, 35 and 38 in that the bacterial glutamine synthetase comprises an amino acid sequence having 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, wherein X is any amino acid, preferably wherein the mutation in amino acid position E130 is a substitution with an aromatic or hydrophobic amino acid, more preferably selected from the group consisting of Y, W, F, A, G, V, L, M and I.
  • Item 40 further specifies the use of item 37 or 39, wherein the mutation is E130X and/or F226Y and/or R345A, and wherein E130X is E130F or E130G.
  • the plasmids used for stable transfection comprise an CMV driven antibody transcription cassette, an ampicillin transgene and a glutamine synthetase transgene as metabolic selection marker.
  • the plasmids were linearized for transfection using Pvul (single cut in the ampicillin transgene).
  • the restriction digest of 20 pg plasmid DNA was conducted at 37°C for 3 h with Pvul (NEB) according to the manufacturers protocol.
  • the linearized plasmid DNA was purified (Qiagen Plasmid Maxi Kit) according to the manufacturers protocol. The final DNA concentration was determined via Nanodrop spectral photometer.
  • CHO-K1-GS knock out cell line host cell also referred to as CHO-K1-GS KO or simply CHO- K1-GS
  • CHO-K1-GS KO or simply CHO- K1-GS
  • the cultivation of the host cell was started with a seeding density of 3x10E05 cells per mL.
  • the growth conditions were set to 36.5°C and 5% CO2 in a shaking incubator with 120rpm in shake flasks. Determination of cell density and viability took place in the Cedex HiRes ⁇ cell count analyzer.
  • the transfection was performed using 1500 Volt, 10 mS and a pulse of 2.
  • Transfected cells were transferred in 5 ml prewarmed host cell medium in T25ml flasks and incubated with 8 % CO2 and 37°C for at least 24 h.
  • cell culture medium supernatant was analyzed following centrifugation for antibody titers using a ForteBio Octet ⁇ device with protein A Biosensors. Dilution of samples and standard curve were processed in the same production medium.
  • Example 1 Antibody production in CHO cells using CHO wildtype glutamine synthetase as metabolic selection marker
  • CHO-K1-GS cells once transfected with a vector carrying a glutamine synthetase from Cricetulus griseus (CHO wildtype glutamine synthetase (GS), SEQ ID NO: 2) selection marker can survive selection under cultivation in medium without supplementation of L-glutamine, when the transgene vector is stably integrated into the genome of the cell.
  • CHO-K1-GS cells were transfected with a vector carrying the expression cassettes of a monoclonal antibody 1 (mAb1) and a CHO wildtype GS. Following selection, stable CHO pools were passaged as described above and samples for titer measurements of mAb1 were taken regularly. Under the applied experimental conditions, the stable CHO pools (transfected with wildtype CHO GS) remained stable and productive for ⁇ 20 days post transfection with decreasing levels of productivity (titer) thereafter ( Figure 1).
  • mAb1 monoclonal antibody 1
  • Example 2 Antibody production in CHO cells using Providencia vermicola GS and other bacterial GS as selection marker
  • CHO-K1-GS cells were transfected with a vector carrying the expression cassette of mAb1 and the glutamine synthetase from Cricetulus griseus (SEQ ID NO: 2) or Providencia vermicola (SEQ ID NO: 1) and viability, viable cell count (VCD) and productivity were measured over time during the selection phase.
  • SEQ ID NO: 2 the glutamine synthetase from Cricetulus griseus
  • SEQ ID NO: 1 the glutamine synthetase from Cricetulus griseus
  • VCD viable cell count
  • productivity viable cell count
  • CHO cells transfected with the bacterial Class-I GS from Providencia vermicola survived the selection process, suggesting that this prokaryotic metabolic selection marker is functional in CHO cells.
  • Cells transfected with the Providencia GS took longer to fully recover from selection ( Figure 2A) and cell growth was slower ( Figure 2B) in comparison to the CHO GS, suggesting that the selection stringency is increased.
  • CHO cells transfected with the Providencia vermicola glutamine synthetase showed an increased selection stringency and significantly higher productivity. Moreover, cell pools show an increased phenotypic stability and remain productive for at least 40 days, whereas cells transfected with CHO GS completely lost their productivity after ⁇ 20 days post transfection.
  • Table B Selection stringency is analysed by measuring recovery of viability of > 70% and genetic stability is demonstrated by stable productivity over a certain period of time, such as more than 20 days post-
  • Stable pools transfected with GS from Photorhabdus luminescens recovered 16 to 17 days post-transfection (reaching a viability of > 70 %), compared to 13 days post-transfection for GS from Providencia vermicola.
  • Productivity was comparable for GS from Photorhabdus luminescens and Providencia vermicola, although slightly delayed for GS from Photorhabdus luminescens, and strongly reduced for GS from Budvicia aquatica ( Figure 4B).
  • bacterial GS such as from Providencia vermicola
  • SEQ ID NO: 1 amino acid sequence of GS from Providencia vermicola
  • Example 3 Antibody production in CHO cells using Providencia vermicola wildtype GS and E130F or E130G mutants as selection marker
  • CHO-K1-GS cells were transfected with a vector carrying expression cassettes for mAb1 and wildtype glutamine synthetase from Providencia vermicola (SEQ ID NO: 1) or glutamine synthetase from Providencia vermicola comprising mutation E130F (SEQ ID NO: 4) or E130G (SEQ ID NO: 5).
  • Example 4 Antibody production and VCD in CHO cells using Providencia vermicola wildtype GS and F226Y mutant as selection marker.
  • CHO-K1-GS cells were transfected with a vector carrying the expression cassettes for mAb1 and CHO wildtype GS (CHO WT GS), Providencia wildtype GS (SEQ ID NO: 1) or Providencia GS F226Y mutant (SEQ ID NO: 6). After stable pool generation and single cell deposition, top clones were subjected to shaking flask experiment head-to-head with CHO WT GS. Both Providencia wildtype GS and Providencia GS F226 outperformed CHO wildtype GS, showing about 50% higher titers (mg/L) compared to CHO wildtype GS clones ( Figure 6A). Compared Providencia wildtype GS, cell clones with Providencia GS F226Y showed further increased growth behaviorwith higher viable cell densities ( Figure 6B). This may provide an advantage for biopharmaceutical production and particularly for combinations with E130 mutations.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un vecteur d'expression de mammifère comprenant un polynucléotide codant pour une glutamine synthétase bactérienne en tant que marqueur de sélection et un acide nucléique et une cellule hôte de mammifère codant pour ladite glutamine synthétase bactérienne. Plus particulièrement, la glutamine synthétase bactérienne est issue de bactéries de l'ordre des Enterobacterales et de la famille des Morganellaceae, de préférence la glutamine synthétase bactérienne est une glutamine synthétase de l'espèce Providencia, et comprend éventuellement une mutation en position E130 et/ou F226 et/ou R345A. L'invention concerne également des procédés de préparation d'une cellule de mammifère exprimant de manière stable une protéine d'intérêt et/ou un ARN non codant, ainsi qu'un procédé de production d'une protéine dans une cellule de mammifère à l'aide de cette glutamine synthétase bactérienne.
PCT/EP2023/057369 2022-03-23 2023-03-22 Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère WO2023180398A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22163849 2022-03-23
EP22163849.7 2022-03-23

Publications (1)

Publication Number Publication Date
WO2023180398A1 true WO2023180398A1 (fr) 2023-09-28

Family

ID=80930164

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/057369 WO2023180398A1 (fr) 2022-03-23 2023-03-22 Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère

Country Status (1)

Country Link
WO (1) WO2023180398A1 (fr)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0240792A1 (fr) 1986-03-18 1987-10-14 The General Hospital Corporation Expression de glutamine-synthétase naturelle et mutante dans des hôtes étrangers
WO2004003175A2 (fr) 2002-07-01 2004-01-08 Arkion Life Sciences Llc Procede et materiels servant a la production de glucosamine et de n-acetylglucosamine
WO2006000045A1 (fr) 2004-06-25 2006-01-05 Proteome Systems Intellectual Property Pty Ltd Nouvelles techniques de diagnostic et de traitement d'une infection par m. tuberculosis et reactifs a cet effet
CN1884501A (zh) 2006-06-29 2006-12-27 清华大学 谷氨酰胺合成酶及其专用表达工程菌与应用
CN102625930A (zh) 2009-08-11 2012-08-01 万佳雷射有限公司 电容式触摸面板
WO2017197098A1 (fr) 2016-05-11 2017-11-16 Amgen Inc. Sélection directe de cellules exprimant des teneurs élevées en protéines hétéromères à l'aide de vecteurs de complémentation intragénique de glutamine synthétase
WO2018093331A1 (fr) 2016-11-16 2018-05-24 Agency For Science, Technology And Research Glutamine synthétase atténuée en tant que marqueur de sélection
WO2018144807A2 (fr) 2017-02-02 2018-08-09 Massachusetts Institute Of Technology Levure modifiée en tant que procédé de bioremédiation
CN112625930A (zh) * 2020-11-09 2021-04-09 华南农业大学 一种酵母重组菌及其在减少蛋鸡盲肠氨气产生中的应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0240792A1 (fr) 1986-03-18 1987-10-14 The General Hospital Corporation Expression de glutamine-synthétase naturelle et mutante dans des hôtes étrangers
WO2004003175A2 (fr) 2002-07-01 2004-01-08 Arkion Life Sciences Llc Procede et materiels servant a la production de glucosamine et de n-acetylglucosamine
WO2006000045A1 (fr) 2004-06-25 2006-01-05 Proteome Systems Intellectual Property Pty Ltd Nouvelles techniques de diagnostic et de traitement d'une infection par m. tuberculosis et reactifs a cet effet
CN1884501A (zh) 2006-06-29 2006-12-27 清华大学 谷氨酰胺合成酶及其专用表达工程菌与应用
CN102625930A (zh) 2009-08-11 2012-08-01 万佳雷射有限公司 电容式触摸面板
WO2017197098A1 (fr) 2016-05-11 2017-11-16 Amgen Inc. Sélection directe de cellules exprimant des teneurs élevées en protéines hétéromères à l'aide de vecteurs de complémentation intragénique de glutamine synthétase
WO2018093331A1 (fr) 2016-11-16 2018-05-24 Agency For Science, Technology And Research Glutamine synthétase atténuée en tant que marqueur de sélection
US20190352631A1 (en) 2016-11-16 2019-11-21 Agency For Science Technology And Research Attenuated Glutamine Synthetase as a Selection Marker
WO2018144807A2 (fr) 2017-02-02 2018-08-09 Massachusetts Institute Of Technology Levure modifiée en tant que procédé de bioremédiation
CN112625930A (zh) * 2020-11-09 2021-04-09 华南农业大学 一种酵母重组菌及其在减少蛋鸡盲肠氨气产生中的应用

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "A0A356D784 Glutamine synthetase", UNIPROT DATABASE, 19 January 2022 (2022-01-19), XP055958135, Retrieved from the Internet <URL:https://rest.uniprot.org/uniprotkb/A0A356D784.txt> [retrieved on 20220906] *
ANONYMOUS: "Escherichia coli glutamate--ammonia ligase - EFC4589804", EMBL DATABASE, 8 May 2019 (2019-05-08), XP055958128, Retrieved from the Internet <URL:https://www.ebi.ac.uk/ena/browser/api/embl/EFC4589804.1?lineLimit=1000> [retrieved on 20220906] *
ANONYMOUS: "UNIPROT:A0A0A0CL50 Glutamine synthetase from Photorhabdus luminescens", 7 January 2015 (2015-01-07), XP093040003, Retrieved from the Internet <URL:http://ibis.internal.epo.org/exam/dbfetch.jsp?id=UNIPROT:A0A0A0CL50> [retrieved on 20230418] *
EISENBERG ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1477, 2000, pages 122 - 145
KRAJEWSKI ET AL., J. MOL. BIOL., vol. 375, 2008, pages 217 - 228
LIN ET AL., MABS, vol. 11, no. 5, 2019, pages 965 - 976
PAO-CHUN LIN ET AL: "Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics", MABS, vol. 11, no. 5, 4 July 2019 (2019-07-04), US, pages 965 - 976, XP055686551, ISSN: 1942-0862, DOI: 10.1080/19420862.2019.1612690 *
ZUO ET AL., SCIENTIFIC REPORTS, vol. 8, no. 1, 2018, pages 1 - 8
ZUO WU ET AL: "Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis", vol. 8, no. 1, 23 October 2018 (2018-10-23), XP055958405, Retrieved from the Internet <URL:http://www.nature.com/articles/s41598-018-34022-5> DOI: 10.1038/s41598-018-34022-5 *

Similar Documents

Publication Publication Date Title
TWI605123B (zh) Ｃｈｏ表現系統
JP5701061B2 (ja) 哺乳類発現ベクター
JP2010536396A (ja) タンパク質力価を増加させる方法
US20220064690A1 (en) CELL ENGINEERING USING RNAs
EP2938728B1 (fr) Introns artificiels
WO2012081628A1 (fr) Procédé de production d&#39;une protéine
US9243053B2 (en) Heterologous intron within an immunoglobulin domain
TW201009075A (en) Improved production host cell lines
EP2339015B1 (fr) Procédés permettant de modifier les taux de production de protéines
US9340592B2 (en) CHO/CERT cell lines
JP2022177131A (ja) 真核細胞においてポリペプチドを発現させるための発現構築物及び方法
CN107109435B (zh) 用于细胞转染和蛋白生产的新型选择性标记物
AU2012252305A1 (en) Enhancement of protein production yield mediated by a fast shuttling cdc42 gtpase
US20140248665A1 (en) Novel intron sequences
EP2938726B1 (fr) Intron hétérologue dans un peptide signal
WO2023180398A1 (fr) Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère
WO2023180374A1 (fr) Nouveaux variants de glutamine synthétase en tant que marqueur de sélection
WO2024068995A1 (fr) Nouveau système de transposase
JP2009050279A (ja) 新規なネオマイシンホスホトランスフェラーゼ遺伝子及び高生産組換え細胞の選抜方法
Class et al. Patent application title: CELL ENGINEERING USING RNAs Inventors: Lore Florin (Danbury, CT, US) Hitto Kaufman (Ulm, DE) Angelika Hausser (Stuttgart, DE) Monilola Olayioye (Ulm, DE) Michaela Strotbek (Asperg, DE)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23713110

Country of ref document: EP

Kind code of ref document: A1