Novel glutamine synthetase mutants as selection markers

Abstract

The present invention relates to a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q; and The present invention relates to expression vectors, nucleic acids and eukaryotic host cells encoding said modified mammalian glutamine synthetase and its use as a selection marker.The present invention further relates to methods for preparing suitable cell lines or for the production of a protein of interest using said modified mammalian glutamine synthetase for selection.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to modified mammalian glutamine synthetases comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetases, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q, as well as expression vectors, nucleic acids and eukaryotic host cells encoding said modified glutamine synthetases and their use as selection markers.The present invention further relates to methods for producing stable cell lines or for producing a protein of interest using said modified mammalian glutamine synthetases for selection.

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

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

[0004] Glutamine synthetase (EC 6.3.1.2, also known as gamma-glutamyl ammonia ligase) catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine. Glutamine synthetases are classified into three subgroups: GSI, GSII, and GSIII. CHO GS is a class II enzyme, which is the subclass expressed primarily by eukaryotic cells, while bacterial GS proteins are typically members of the GSI class. Although GSI and GSII catalyze the same reaction, they show no or very little sequence similarity, except for residues that form part of the active site, and are quite different overall. For example, bacterial type I GS is a 12-subunit complex (Eisenberg et al. (2000), Biochim. Biophys. Acta 1477, 122-145), and GSII is reported to form stacks of two or three pentameric rings (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228). The overall sequence identity between CHO GS and bacterial GS, e.g., E. coli, is less than 30%.

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

[0006] Also, the transfected glutamine synthetase selection marker has a considerable effect on the selection process and the phenotypic stability as well as productivity of CHO-based cell lines. For example, the use of attenuated mutants of CHO glutamine synthetase has been shown to further improve stability, selection behavior, and productivity (Lin et al. (2019), mAbs, 11:5, 965-976; WO 2018093331, US 20190352631, WO 2017197098). Most of the attenuated mutants described had mutations in conserved substrate binding residues. For example, two attenuated GS mutants containing R324C and R341C mutations were first identified in two unrelated infants with congenital GS deficiency (Lin et al. (2019), mAbs, 11:5, 965-976). Residues reported to be involved in glutamate binding are E134, E136, E196, E203, N248, G249, H253, R299, R319, E338, and R340; residues reported to be involved in ATP binding are W130, A191, G192, P208, N255, S257, R262, R324, and Y336; and residues involved in ammonia binding are D63, S66, D162, and E305 (amino acid numbering refers to human GS) (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228 and WO 2018 / 093331). Respective coordination and structure factor data are available via PDB accession codes 2UU7, 2OJW, and 2QC8.Furthermore, in Chinese Patent No. 114085819A, it was reported that residues within 4.5 Å of MSX (methionine sulfoximine) or ADP / ATP in the predicted complex structure of CHO glutamine synthetase, namely V197, Q201, Q205, A250, G251, H304, T306 and R324, were identified in wild-type glutamine synthetases of human, dog and CHO, among which only the single mutant R324C, especially the triple mutant V197W, A250F and R324K (GSm1), conferred attenuated GS and stable clones.

[0007] Attenuated GS mutants carrying mutations in the active site showed increased selection stringency, but the duration of the selection process and the overall cell growth characteristics may be compromised, negatively impacting the bioprocess performance and making these mutants often unsuitable for therapeutic antibody production. Attenuation of a selection marker is a fine balance between selection stringency, integrated copy number, and cell culture performance (e.g., growth behavior) suitable to support recombinant protein production. Therefore, there is a need to discover novel attenuated GS mutants to improve mammalian expression vectors in cell line development and to generate a set of selection marker mutants with finely balanced selection stringency, especially in the absence of GS inhibitors such as MSX.

[0008] Summary of the Invention The present invention demonstrates that three mutants of mammalian glutamine synthetase with single point mutations generate highly productive and stable host cell pools expressing therapeutic proteins, especially therapeutic antibodies, that show a more stringent selection behavior and beneficial cell culture performance in CHO cells due to their attenuated activity compared to wild-type glutamine synthetase. All three mutants confer increased selection stringency and unexpectedly result in stable host cell pools with higher productivity and / or stability compared to wild-type GS, making these mutants superior for cell line development. The positions of the mutated amino acids have not yet been described and are not directly related to the catalytic binding pocket of the enzyme, but rather appear to affect the stability of the protein by driving structural interactions. These protein mutants are particularly advantageous because they have mutations outside the substrate binding site, e.g. at the protein interface, which is important for the assembly of the multimeric GS protein complex, providing an additional means of modulating attenuation in a more gentle manner.

[0009] More specifically, the present invention relates to a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q. In particular, the mutation does not interfere with binding to a substrate.

[0010] In certain embodiments, a modified mammalian glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q (a) has a decreased enzymatic activity compared to the same mammalian glutamine synthetase not comprising said mutation; and / or (b) mediates increased selection stringency and / or genetic stability upon transfection with a polynucleotide and transgene encoding said modified mammalian glutamine synthetase compared to the same mammalian glutamine synthetase not comprising said mutation.

[0011] In another aspect, the present invention relates to an expression vector comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to the present invention. In a particular embodiment, the expression vector further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably wherein the protein of interest is a therapeutic protein selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

[0012] In yet another aspect, the present invention relates to a nucleic acid sequence comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to the present invention operably linked to a eukaryotic promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0013] In yet another aspect, the present invention relates to a eukaryotic host cell comprising a polynucleotide sequence encoding a modified mammalian glutamine synthetase according to the present invention, an expression vector according to the present invention, or a nucleic acid sequence according to the present invention. The eukaryotic host cell is preferably a mammalian host cell, more preferably a rodent cell, even more preferably a CHO cell. Additionally or alternatively, the eukaryotic host cell is a GS knockout mutant.

[0014] The present invention further relates to a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising the steps of: (a) introducing an expression vector according to the present invention, or a nucleic acid according to the present invention, optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, into a eukaryotic host cell, preferably a mammalian host cell, more preferably a CHO cell; and (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions selecting for the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding the optional protein and / or non-coding RNA of interest is simultaneously integrated into the host cell genome together with the polynucleotide encoding the modified mammalian glutamine synthetase. The method may further comprise the step of isolating a single clone for clonal amplification to prepare a monoclonal cell line.

[0015] In yet another aspect, the present invention provides a method for producing a protein of interest comprising the steps of: (a) introducing an expression vector comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to the present invention, comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q, and further comprising at least one polynucleotide encoding a protein of interest, or a nucleic acid according to the present invention, into a eukaryotic host cell, preferably a mammalian host cell, more preferably a CHO cell; (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions that select for the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome with the polynucleotide encoding the modified mammalian glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the eukaryotic host cell under conditions that produce the protein of interest; and (e) collecting and optionally purifying the protein of interest. The expression vector can be introduced by transfection or transduction.

[0016] In yet another aspect, the present invention provides a method for producing a protein of interest, comprising the steps of: (a) providing a eukaryotic host cell according to the present invention, comprising a polynucleotide encoding a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, the polynucleotide comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetase, the mutation being selected from the group consisting of R298K, N10S, N10T and N10Q, and at least one polynucleotide encoding a protein of interest; (b) culturing the eukaryotic host cell under conditions for producing the protein of interest; and (c) harvesting and optionally purifying the protein of interest. The eukaryotic host cell of the method of the present invention is (a) a mammalian host cell, preferably a rodent cell, more preferably a CHO cell; and / or (b) a GS knockout mutant.

[0017] The present invention also provides a kit comprising an expression vector according to the present invention and a glutamine-free cell culture medium.

[0018] In yet another aspect, the present invention relates to the use of a modified mammalian glutamine synthetase according to the present invention as a selection marker for the generation of monoclonal eukaryotic cell lines, preferably for the generation of mammalian cell lines, more preferably for the generation of rodent cell lines, even more preferably for the generation of CHO cell lines.

[0019] In yet another aspect, the present invention relates to the use of a vector according to the invention for producing a protein of interest in a eukaryotic host cell, preferably a mammalian host cell, more preferably a rodent cell, even more preferably a CHO cell. [Brief description of the drawings]

[0020] [Figure 1]CHO-K1-GS cell pool expressing monoclonal antibody 1 and wild-type CHO GS as a selection marker. CHO-K1-GS cell pools (n=5) stably transfected with a mammalian expression vector to express monoclonal antibody 1 and CHO wild-type GS as a metabolic selection marker were cultured in medium lacking L-glutamine. The titer [mg / L] of monoclonal antibody 1 was determined on the indicated days after transfection. [Diagram 2] Viability of CHO-K1-GS cells expressing monoclonal antibody 1 and CHO wild-type GS or R298 mutant. CHO-K1-GS cell pools stably transfected with expression cassettes to express monoclonal antibody 1 and CHO wild-type GS (wtGS) or one of six mutants of CHO GS with various mutations at R298 were cultured in medium without L-glutamine. Viability [%] was determined on the indicated days during selection. [Figure 3A] Productivity and average productivity of CHO-K1-GS cell pools expressing monoclonal antibody 1 and CHO wild-type GS or CHO GS R298K mutant. CHO-K1-GS cell pools stably transfected with expression cassettes to express monoclonal antibody 1 and CHO wild-type GS or CHO GS R298K mutant cultured in medium without L-glutamine. The titer [μg / L] of monoclonal antibody 1 was determined on the indicated days during culture. A: Productivity of CHO GS R298K mutant and wild-type CHO GS over the course of 20 days. [Figure 3B] B: Mean productivity of CHO WT GS and CHO GS R298K mutant (**p<0.01). [Figure 4]Viability of CHO-K1-GS cells expressing monoclonal antibody 1 and CHO wild-type GS or CHO GS N10S, N10Q or N10W mutants. CHO-K1-GS cell pools stably transfected with expression cassettes to express monoclonal antibody 1 and CHO wild-type GS or single point mutants CHO GS N10S, N10Q or N10W were cultured in medium without L-glutamine. Viability [%] was determined on the indicated days after transfection. [Diagram 5] Productivity of CHO-K1-GS cells expressing monoclonal antibody 1 and CHO wild type GS compared to N10S, N10Q or N10W CHO GS mutants as selection markers. CHO-K1-GS cell pools stably transfected with expression cassettes to express monoclonal antibody 1 and CHO wild type GS or CHO GS N10S, N10Q or N10W mutants cultured in medium without L-glutamine. Monoclonal antibody 1 titers [mg / L] were determined on the indicated days once cells reached >70% viability. [Figure 6] Viability of CHO-K1-GS cells expressing monoclonal antibody 1 and CHO wild-type GS or additional CHO GS N10 mutants. CHO-K1-GS cell pools stably transfected with expression cassettes to express monoclonal antibody 1 and CHO wild-type GS or single point mutants CHO GS N10S, N10Q, N10W, N10F, N10Y or N10G cultured in medium without L-glutamine. CHO-K1-GS cells carrying N10 mutations were found to be phenotypically unstable, except for cells carrying CHO GS N10S or N10Q. Viability [%] was determined on the indicated days after transfection. [Figure 7]Productivity of CHO-K1-GS cells expressing monoclonal antibody 1 and CHO wild type GS or CHO GS N10S and R298K. GS CHO containing the two mutations N10S and R298K combined was tested in 384 screening steps for the generation of stable pools and for evaluation of productivity at the clonal level. The titer [mg / L] of monoclonal antibody 1 was determined at the end of the screening step. [Figure 8] The sequence of wild-type glutamine synthetase from Cricetulus griseus (SEQ ID NO:1). [Figure 9] The sequence of wild-type glutamine synthetase of Homo sapiens (SEQ ID NO:2). [Figure 10] The sequence of wild-type glutamine synthetase from Mus musculus (SEQ ID NO:3). [Figure 11] The sequence of wild-type glutamine synthetase from the brown rat (Rattus norvegicus) (SEQ ID NO: 4). [Figure 12] Sequence of wild-type glutamine synthetase R298K from Chryseturus griseus (SEQ ID NO:5). [Figure 13] Sequence of wild-type glutamine synthetase N10S from Chryseturus griseus (SEQ ID NO:6). [Figure 14] Sequence of wild-type glutamine synthetase N10T from Chryseturus griseus (SEQ ID NO: 7). [Figure 15] Sequence of wild-type glutamine synthetase N10Q from Chryseturus griseus (SEQ ID NO:8). [Figure 16] Sequences of wild-type glutamine synthetase 298K and N10S from Chryseturus griseus (SEQ ID NO:9). [Figure 17] The sequence of wild-type glutamine synthetase R298K and N10T from Chryseturus griseus (SEQ ID NO: 10). [Figure 18] Sequence of wild-type glutamine synthetase R298K and N10Q from Chryseturus griseus (SEQ ID NO:11).

[0021] Detailed Description The term "comprise" or "comprising" means "including, but not limited to". The term is open-ended and is intended to specify the presence of any stated feature, element, integer, step, or component, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "essentially consisting of". With respect to sequences, the terms "having an amino acid sequence of" and "comprising amino acids of" are used synonymously and include the embodiment "consisting of an amino acid sequence of". Similarly, the term "encoding" or "encoding" is open-ended and allows for the presence or addition of one or more other features, elements, or components. Furthermore, the singular and plural forms are not used restrictively. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

[0022] The term "protein" is used synonymously with "amino acid sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes, such as fusion to other proteins, amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, substitutions of specific amino acid sequences can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with the same properties.

[0023] The term "nucleic acid sequence" is used synonymously with "polynucleotide" and refers to DNA or RNA of any length. In the context of expression vectors, particularly plasmids, and integration into a host cell genome, those skilled in the art will understand it to refer to a DNA sequence or DNA molecule.

[0024] The terms "encoding" and "encoding" broadly refer to any process that uses information within a polymeric macromolecule to direct the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, in some embodiments, the term "encoding" describes a semi-conservative DNA replication process in which one strand of a double-stranded DNA molecule is used as a template to code for a complementary sister strand that is de novo synthesized by a DNA-dependent DNA polymerase. In other embodiments, a DNA molecule may encode an RNA molecule (e.g., by a transcription process using a DNA-dependent RNA polymerase enzyme). An RNA molecule may also encode a polypeptide, as in the process of translation. When used to describe the translation process, the term "encoding" also extends to triple-base codons that code for amino acids. In some embodiments, an RNA molecule may encode a DNA molecule, for example, by a reverse transcription process incorporating an RNA-dependent DNA polymerase. In another embodiment, a DNA molecule may encode a polypeptide, and "encoding" as used in this case is understood to incorporate both the processes of transcription and translation.

[0025] The term "expression" as used herein refers to the transcription and / or translation of a nucleic acid sequence, typically a heterologous nucleic acid sequence, in a host cell. The expression level of a gene product of interest in a host cell can be determined based on either the amount of the corresponding RNA present in the cell or the amount of the polypeptide encoded by the selected sequence. For example, the RNA transcribed from the selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to intracellular RNA, or PCR, such as quantitative PCR. The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by Western blot, by radioimmunoassay, by immunoprecipitation, by assay for biological activity of the protein, by FACS analysis after immunostaining of the protein, or by homogeneous time-resolved fluorescence (HTRF) assay. The expression level of non-coding RNA, such as microRNA, long non-coding RNA, or small hairpin RNA, can be quantified by PCR, such as quantitative PCR.

[0026] As used herein, the term "stable transfection" refers to the transfection of a polynucleotide, which includes the integration of the polynucleotide into the host cell genome, as opposed to a transiently introduced polynucleotide that remains isolated from the genomic DNA of the host cell. Stable integration can be achieved by homologous recombination or other types of recombination. Stable integration can include a step of transiently introducing a heterologous polynucleotide into a host cell. Stable integration of a polynucleotide can also be achieved by transduction using a viral vector, such as a lentivector.

[0027] The term "eukaryotic cell" as used herein refers to a cell having a nucleus within a nuclear envelope, including animal cells, human cells, plant cells, and yeast cells. In the present invention, "eukaryotic cell" particularly encompasses mammalian cells, such as Chinese Hamster Ovary (CHO) cells or HEK293 cell-derived cells, and yeast cells. Mammalian cells as used herein refer to any cell or cell line of mammalian origin, such as human cells or rodent cells. Cells as referred to herein are cells maintained in culture and do not relate to primary cells, but to cell lines or cell line-derived cells, i.e. immortalized cells.

[0028] The term "mutation" as used herein refers to the substitution of one amino acid in a nucleic acid sequence.Those skilled in the art will understand that the mutation at a specific amino acid position, which is referred to by the original amino acid (e.g., R298), allows any amino acid to be substituted other than the original amino acid (R298X).Amino acid substitution can also be defined by one-letter code, e.g., R298K.

[0029] As used herein, the term "about" refers to a 10% variation of the specified numerical value, for example, about 50% has a variation of 45-55%.

[0030] As used herein, the term "stringency of selection" refers to the time period to reach more than 70% viability and doubling time within 48 hours of cell culture after transfection. The longer the period, the more stringent the selection behavior. Typically, attenuated glutamine synthetase exhibits a more stringent selection behavior compared to CHO wild-type glutamine synthetase.

[0031] Modified mammalian glutamine synthetase and its expression by vectors or eukaryotic host cells The present invention demonstrates that mutants of CHO glutamine synthetase, each with one or two single amino acid mutations (groups), can be applied to generate highly productive and stable CHO pools expressing therapeutic antibodies, which show a more stringent selection behavior due to their attenuated activity compared to wild-type glutamine synthetase in CHO. All mutants confer increased selection stringency after transfection, and unexpectedly, stable cell pools with higher productivity and / or stability compared to wild-type GS are obtained, making these mutants better in the cell line development process. In particular, these mutants are advantageous in the absence of glutamine synthetase inhibitors such as MSX.

[0032] The present invention provides a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q, and the mammalian glutamine synthetase preferably has the amino acid sequence of hamster glutamine synthetase (SEQ ID NO:1), human glutamine synthetase (SEQ ID NO:2), mouse glutamine synthetase (SEQ ID NO:3) or rat glutamine synthetase (SEQ ID NO:4), or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3 or 4.

[0033] More specifically, in a first aspect, the present invention relates to a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q. The mutation in the modified mammalian glutamine synthetase according to the present invention does not interfere with substrate binding. In particular, the mutation is not present in a residue that binds to a substrate. This means that the residue has not been reported (or has not yet been reported) to be involved in glutamate, ATP or ammonia binding. In a particular embodiment, the mutation is R298K and / or N10S or N10T or N10Q. Thus, the mutation is R298K, or the mutation is N10X, where X is S, T or Q (R298K or N10S or N10T or N10Q), or the mutation is R298K and N10X, where X is S, T or Q (R298K and N10S or N10T or N10Q). Preferably, the mutation N10X is N10S or N10Q. In a particular embodiment, the mutation is R298K, or N10S, or N10Q, or R298K and N10S, or R298K and N10Q.

[0034] The mammalian glutamine synthetase according to the invention comprises a mutation in a mammalian glutamine synthetase having at least 95% amino acid sequence identity to SEQ ID NO: 1, 2, 3 or 4. Preferably, the glutamine synthetase has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3 or 4. In a particular embodiment, the glutamine synthetase is a Chryseturus griseus glutamine synthetase, preferably having the amino acid sequence of SEQ ID NO: 1, a human glutamine synthetase, preferably having the amino acid sequence of SEQ ID NO: 2, a mouse glutamine synthetase, preferably having the amino acid sequence of SEQ ID NO: 3, or a rat glutamine synthetase, having the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the mammalian glutamine synthetase is a Chryseturus griseus glutamine synthetase having at least 95% sequence identity to SEQ ID NO:1, or at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1.

[0035] In the context of the present invention, the term "having at least 95% sequence identity to" refers to a protein comprising a sequence that shares at least 95% of amino acid residues with the sequence of a reference sequence. Sequence identity can be readily determined by sequence alignment. The sequence can be a naturally occurring sequence, such as a sequence from a different species than the reference sequence, or an allelic variant of the reference sequence, or an engineered sequence that contains one or more modifications throughout the reference sequence.

[0036] The modified mammalian glutamine synthetase provided herein, comprising a mutation at amino acid position 10 and / or 298 of a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q, may further comprise one or more additional mutations that attenuate glutamine synthetase activity or enhance catalytic activity for glutamine synthetase. The additional mutation may be a substitution with another amino acid, as described by Lin et al. (2019) (mAbs, 11:5, 965-976) or WO2018093331, US20190352631, or WO2017197098.

[0037] In certain embodiments, the modified glutamine synthetase of the invention (wherein the modified glutamine synthetase comprises a mutation at amino acid position 10 and / or 298 of a mammalian glutamine synthetase) has the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10 or 11.

[0038] In certain embodiments, the modified glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q has a decreased enzymatic activity compared to the same mammalian glutamine synthetase not comprising said mutation. In alternative or additional embodiments, the modified mammalian glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q mediates increased selection stringency and / or genetic stability upon transfection with a polynucleotide and transgene encoding the modified mammalian glutamine synthetase compared to the same mammalian glutamine synthetase not comprising said mutation. In yet another embodiment, the modified mammalian glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q has a reduced enzymatic activity compared to a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO: 1 and / or mediates increased selection stringency and / or genetic stability upon transfection with a polypeptide encoding the modified mammalian glutamine synthetase and a transgene compared to a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO: 1. The increased selection stringency may be determined by the time to reach a survival rate of more than 70%. The transgene may, for example, encode a protein of interest and / or a non-coding RNA. The term reduced enzyme activity refers to reduced mammalian glutamine synthetase activity compared to the same mammalian glutamine synthetase not containing the mutations of the modified mammalian glutamine synthetase of the present invention or compared to a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO: 1, preferably reduced here means reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. This can be determined, for example, by the duration of the selection procedure as indirect output information.

[0039] In a related aspect, the present invention relates to the use of a modified mammalian glutamine synthetase according to the present invention (first aspect) as a selection marker for the generation of monoclonal eukaryotic cell lines, preferably for the generation of mammalian cell lines, more preferably for the generation of rodent cell lines, even more preferably for the generation of CHO cell lines.

[0040] In a second aspect, the present invention relates to an expression vector comprising a polynucleotide encoding a modified mammalian glutamine synthetase of the present invention. The embodiments and examples specified for the first aspect apply analogously to the second aspect. The expression vector according to the present invention is for the expression of a heterologous sequence in a eukaryotic host cell, preferably a mammalian host cell, i.e. it is adapted for expression in a eukaryotic or mammalian cell, respectively. Thus, the expression vector according to the present invention is characterized in that it comprises a polynucleotide encoding a modified mammalian glutamine synthetase, preferably wherein the polynucleotide encoding the modified mammalian glutamine synthetase is operably linked to a promoter. Typically, the expression vector comprises an expression cassette comprising a polynucleotide encoding a modified mammalian glutamine synthetase operably linked to a promoter. Preferably, the expression vector is a eukaryotic expression vector and the promoter is a eukaryotic promoter, more preferably the expression vector is a mammalian expression vector and the promoter is a mammalian promoter. The mammalian promoter controls transcription in mammalian cells. Exemplary mammalian promoters include, but are not limited to, simian virus 40 early promoter (SV40), cytomegalovirus immediate early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-actin promoter linked to the cytomegalovirus early enhancer (CAGG). The expression vector may further include bacterial sequences, such as an origin of replication and resistance genes, for amplification of the vector in bacterial cells.

[0041] Said expression vector typically further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest. The polynucleotide encoding the protein and / or the non-coding RNA of interest is operably linked to a promoter. The protein and / or the non-coding RNA of interest and the modified glutamine synthetase of the present invention may be encoded by the same expression cassette (polycistronic) or by separate expression cassettes. In certain embodiments, the non-coding RNA is an RNA that mediates RNA interference (RNAi), such as a microRNA, a small interfering RNA, a long non-coding RNA, or a small hairpin RNA.

[0042] The term "expression cassette" as used herein refers to a separate piece of DNA, particularly vector DNA, consisting of one or more coding polynucleotide sequences and regulatory sequences that control their expression in transfected or transduced cells. An expression cassette contains at least three components: a promoter sequence, an open reading frame, and a transcription termination sequence. In mammalian expression vectors containing a polynucleotide encoding a protein of interest, the transcription termination sequence is called the 3' untranslated region and usually contains a polyadenylation site. An expression cassette directs the cell's machinery to make RNA, and therefore may also be referred to as a transcription cassette. The RNA may be a coding RNA that is further processed into an mRNA that encodes a protein sequence, such as glutamine synthetase or a protein of interest, or it may be a non-coding RNA, such as an RNA that mediates RNA interference (RNAi), such as a microRNA, a small interfering RNA, a long non-coding RNA, or a small hairpin RNA.

[0043] The protein of interest can be any protein, but is typically a therapeutic protein. These include, but are not limited to, cytokines, growth factors, hormones, blood clotting factors, vaccines, interferons, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics. In certain embodiments, the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics.

[0044] In a particular embodiment, the protein of interest is an antibody. When the protein of interest is an antibody, the eukaryotic expression vector (particularly a mammalian expression vector) comprises a polynucleotide comprising the coding sequence of the variable region of the heavy chain and / or the coding sequence of the variable region of the light chain of the antibody. In a particular embodiment, the eukaryotic expression vector comprises a polynucleotide comprising the coding sequence of the heavy chain and / or the coding sequence of the light chain of the antibody. Thus, the polynucleotide comprising the coding sequence of the variable region of the heavy chain and the polynucleotide comprising the coding sequence of the variable region of the light chain may be expressed on the same eukaryotic expression vector or on separate eukaryotic expression vectors. The expression vector may comprise a polycistronic expression cassette, e.g. a bicistronic expression cassette, and / or a plurality of expression cassettes. A polycistronic expression cassette comprises more than one open reading frame separated by a sequence encoding an RNA sequence allowing initiation of translation, such as an internal ribosome entry site (IRES). In a polycistronic expression cassette, two or more open reading frames are under the control of the same promoter. The polynucleotide encoding at least one variable region of the heavy chain and the polynucleotide encoding at least one variable region of the light chain may therefore be expressed in the same expression cassette (e.g. separated by an IRES sequence) or by two separate expression cassettes. Furthermore, the bacterial glutamine synthetase and the protein of interest and / or the non-coding RNA may be expressed by the same or different expression cassette(s). If the protein of interest is an antibody, the bacterial glutamine synthetase and the polynucleotide encoding at least one variable region of the heavy chain and / or the polynucleotide encoding at least one variable region of the light chain may be expressed by the same or different expression cassettes or a mixture thereof.

[0045] In a preferred embodiment, the eukaryotic expression vector is for stable integration into the host cell genome (e.g. for stable transfection), and the integration part of the vector comprises a polynucleotide encoding bacterial glutamine synthetase and at least one polynucleotide encoding a protein and / or non-coding RNA of interest. The eukaryotic expression vector according to the present invention is a plasmid, a bacterial artificial chromosome (BAC) or a viral vector. The plasmid, bacterial artificial chromosome or viral vector (e.g. lentiviral vector) can be introduced into a eukaryotic host cell (e.g. a mammalian host cell) via transfection or transduction, respectively, and is preferably stably integrated into the host cell genome. The skilled artisan knows suitable plasmids, BAC or viral vectors, and knows that the plasmid may further comprise transposon recognition sequences upstream and downstream of the polynucleotide encoding bacterial synthetase as a selection marker, and optionally at least one polynucleotide encoding a protein and / or non-coding RNA of interest.

[0046] A preferred protein of interest is an antibody, including fragments and derivatives thereof. Typically, antibodies are monospecific, although antibodies may be multispecific. Thus, the present invention may be used to generate monospecific antibodies, multispecific antibodies, or fragments thereof, preferably antibodies (monospecific), bispecific antibodies, trispecific antibodies, or fragments thereof, preferably antigen-binding fragments thereof. Exemplary antibodies within the scope of the present invention include anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD44v6 antibodies, anti-CD49d antibodies, anti-CD52 antibodies, anti-EGFR (epidermal growth factor receptor) 1 (HER1) antibodies, anti-EGFR2 (HER2) antibodies, anti-GD (ganglioside) 3 antibodies, anti-IGF (insulin growth factor) antibodies, anti-VEGF (endovascular Examples of the antibody include, but are not limited to, anti-CD20 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD40 antibody, anti-CD44 antibody, anti-CD52 antibody, anti-HER2 / neu (erbB2) antibody, anti-EGFR antibody, anti-IGF antibody, anti-VEGF antibody, anti-TNFα antibody, anti-IL-2 antibody, and anti-IgE antibody.

[0047] The terms "antibody", "antibodies" or "immunoglobulin(s)" are used herein in the broadest sense to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g. bispecific antibodies) and antibody fragments, so long as they exhibit the desired antigen-binding activity. There are various immunoglobulin classes: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, the antibody is an IgG antibody, more preferably an IgG1 antibody or an IgG4 antibody.

[0048] The antibody may be of any species, including chimeric and humanized antibodies. A "chimeric" antibody is a molecule in which the domains or regions of the antibody are derived from different species. For example, the variable regions of the heavy and light chains may be derived from a rat or mouse antibody, and the constant region may be derived from a human antibody. In a "humanized" antibody, only minimal sequences are derived from a species other than human. Often, only amino acid residues of the CDRs of a human antibody are replaced with amino acid residues of the CDRs of a species other than human, such as mouse, rat, rabbit, or llama. Sometimes, some important framework amino acid residues that affect the binding specificity and affinity to the antigen are also replaced by non-human amino acid residues.

[0049] Typically, antibodies are tetrameric polypeptides composed of two heterodimeric pairs, each formed by a heavy chain and a light chain. Stabilization of both the heterodimeric as well as the tetrameric polypeptide structures occurs via disulfide bridges between the chains. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", the terms "domain" and "region" being used synonymously herein. Each domain contains approximately 70-110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain at their N-terminus "variable domains" or "variable regions" with less conserved sequences that are involved in antigen recognition and binding. The variable region of the light chain is also referred to as "VL" and the variable region of the heavy chain is also referred to as "VH".

[0050] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Fab fragments consist of the variable regions of both chains, which are held together by adjacent constant regions. They can be formed from conventional antibodies by digestion with proteases, such as with papain, but Fab fragments can also be generated by genetic engineering as well. Still other antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0051] Using genetic engineering techniques, it is possible to generate shortened antibody fragments consisting only of the variable regions of the heavy (VH) and light (VL) chains. These are called Fv fragments (Fragment variable = fragment of the variable part). These Fv fragments are often stabilized, since they lack the covalent bond between the two chains by the cysteines of the constant chains. Advantageously, the variable region of the heavy chain and the variable region of the light chain are linked by a short peptide fragment, for example of 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This type of antibody protein is known as single chain Fv (scFv). Examples of scFv antibody proteins are known to those skilled in the art. Thus, antibody fragments and antigen-binding fragments further include Fv fragments and especially scFv.

[0052] In recent years, various strategies have been developed to prepare scFv as multimeric derivatives. This is aimed in particular at resulting in recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as increased avidity. To achieve multimerization of scFv, scFv have been prepared as fusion proteins with multimerization domains. The multimerization domain can be, for example, the CH3 region of IgG or coiled-coil structures (helical structures), such as leucine-zipper domains. However, there are also strategies in which interactions between the VH / VL regions of scFv are used for multimerization (e.g. diabodies, tribodies and pentabodies). By diabody, the skilled person means a bivalent homodimeric scFv derivative. Homodimers are formed by shortening the linker in the scFv molecule to 5-10 amino acids, leading to the superposition of the interchain VH / VL. Diabodies can be further stabilized by the incorporation of disulfide bridges. Examples of diabody-antibody proteins are known from the prior art.

[0053] By minibody the skilled person means a bivalent homodimeric scFv derivative, which consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably an IgG, most preferably an IgG1, as a dimerization region connected to the scFv via a hinge region (for example also derived from IgG1) and a linker region. Examples of minibody-antibody proteins are known from the prior art.

[0054] By triabody the skilled person means a trivalent homotrimeric scFv derivative in which the VH-VL are directly fused without a linker sequence to form a trimer.

[0055] The skilled person will also be familiar with the so-called mini-antibodies, which have a bivalent, trivalent or tetravalent structure and are derived from scFv. The multimerization is achieved by dimeric, trimeric or tetrameric coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest encodes any of these above desired polypeptides, preferably a monoclonal antibody, derivative or fragment thereof.

[0056] Additionally, single domain antibodies (sdAbs), also called nanobodies, which are antibody fragments of a single monomeric antibody variable domain, are encompassed. Single domain antibodies are typically derived from camelids (V H H fragment) or cartilaginous fish (V NAR The heavy chains are engineered from antibodies found in the human IgG1 heavy chains (HbA1, HbA2, HbA3, HbA4, HbA5, HbA6, HbA7, HbA8, HbA9, HbA10, HbA11, HbA12, HbA13, HbA14, HbA15, HbA16, HbA17, HbA18, HbA19, Hb

[0057] Immunoglobulin fragments consisting of the CH2 and CH3 domains of the antibody heavy chain are called "Fc fragments", "Fc regions" or "Fc" because of their crystallization properties (Fc = fragment crystallizable). They can be formed from conventional antibodies by digestion with proteases such as papain or pepsin, but can also be produced by genetic engineering. The N-terminal part of the Fc fragment can vary depending on how many amino acids of the hinge region are still present.

[0058] An antibody comprising an antigen-binding fragment and an Fc region may also be referred to as a full-length antibody. Full-length antibodies may be monospecific and multispecific antibodies. Multispecific antibodies are antibodies that have at least two different antigen-binding sites, each of which binds to a different epitope. Multispecific antibodies include bispecific and trispecific antibodies. Bispecific antibodies have two different binding sites. Multispecific antibodies also include antibody formats other than full-length antibodies, such as antibody-derived molecules.

[0059] Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, natural killer cells, or macrophages) in one molecule. Exemplary bispecific antibodies are, but are not limited to, diabodies, BiTE (bispecific T cell targeting) formats, and DART (dual affinity retargeting reagent) formats. The diabody format separates the cognate variable domains of heavy and light chains with two antigen-binding specificities on two separate polypeptide chains, which are non-covalently linked. The DART format is based on the diabody format, but it provides additional stabilization through a C-terminal disulfide bridge. Trispecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. They can be constructed with bispecific antibody technology, which reconstitutes the antigen recognition domains of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been generated that target CD38 on cancer cells and CD3 and CD28 on T cells. Multispecific antibodies are particularly difficult to produce with high production quality.

[0060] As used herein, the term "antibody-derived molecule" refers to any molecule that contains at least one antigen-binding portion that is structurally related to an antibody, including modified full-length monospecific or bispecific antibodies that have been further modified with additional antigen-binding portions, or smaller antibody formats, including those described herein.

[0061] As used herein, the term "antibody mimic" refers to a protein that binds to a specific antigen in a manner similar to an antibody, but is not structurally related to an antibody. Antibody mimics include, but are not limited to, anticalins, affibodies, adnectins, monobodies, DARPins (designed ankyrin repeat proteins), affimers, and affitins.

[0062] Single domain antibodies (sdAbs) may also be referred to as nanobodies. The skilled artisan will understand that the protein may comprise more than one antigen-binding domain and therefore may be multivalent, preferably bivalent (e.g. a bivalent sdAb or a bivalent anticalin or any other bivalent antibody mimetic).

[0063] Another preferred therapeutic protein is a fusion protein, such as an Fc-fusion protein. Thus, the present invention may advantageously be used for the creation of fusion proteins, such as Fc-fusion proteins. The effector portion of the fusion protein may be the complete sequence or any part of the sequence of a native or modified heterologous protein. The immunoglobulin constant domain sequences may be obtained from any immunoglobulin subtype, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtype or class, such as IgA, IgE, IgD or IgM. Preferentially, they are derived from human immunoglobulins, more preferably from human IgG, even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc-fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc and scFv fragments linked to the CH2 domain of the constant region of an immunoglobulin heavy chain, containing an N-linked glycosylation site. Fc-fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of the constant region of an immunoglobulin heavy chain, which contains an N-linked glycosylation site, into another expression construct, which contains, for example, another immunoglobulin domain, an enzymatically active protein part, or an effector domain. Thus, Fc-fusion proteins according to the invention also include single chain Fv fragments linked to the CH2 domain of the constant region of an immunoglobulin heavy chain, which contains, for example, an N-linked glycosylation site.

[0064] The term "cytokine" refers to small proteins released by cells that act as intercellular mediators, e.g., affect the behavior of cells surrounding the secreting cell. Cytokines can be secreted by immune cells or other cells, e.g., T cells, B cells, natural killer cells, and macrophages. Cytokines can be involved in intercellular signaling events, e.g., autocrine signaling, paracrine signaling, and endocrine signaling. They can mediate a range of biological processes, including, but not limited to, immunity, inflammation, and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

[0065] As used herein, "growth factor" refers to a protein or polypeptide capable of stimulating cell growth. They include, but are not limited to, insulin, epidermal growth factor (EGF), ephrin (Eph), erythropoietin, glial cell stimulating factor (GSF); colony stimulating factors (CSF) (including macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF)); stem cell growth factor (SCGF) (also called Steel factor); stromal cell-derived factor (SDF), effective fragments thereof, and combinations thereof; and vascular endothelial growth factor (VEGF). Other growth factors may include hepatocyte growth factor (HGF), angiopoietin-1, angiopoietin-2, b-FGF (basic fibroblast growth factor), and FLT-3 ligand, and effective fragments thereof.

[0066] In a third aspect, the present invention relates to a kit comprising an expression vector according to the invention and optionally a glutamine-free cell culture medium.

[0067] In a fourth aspect, the present invention further relates to the use of the expression vector of the present invention (second aspect) for the expression of a protein of interest and / or a non-coding RNA in a eukaryotic host cell, in particular a mammalian host cell, more preferably a rodent cell, such as a CHO cell. The modified mammalian glutamine synthetase encoded by the expression vector serves as a selection marker in the host cell. In a particular embodiment, the use of the expression vector of the present invention is for the production of a protein of interest in a eukaryotic host cell, in particular a mammalian, such as a rodent cell, such as a CHO cell. The embodiments and examples specified for the second and first aspects apply analogously to this aspect.

[0068] In a fifth aspect, the present invention relates to a nucleic acid sequence comprising a polynucleotide encoding a modified mammalian glutamine synthetase of the present invention operably linked to a promoter, preferably a eukaryotic promoter (e.g. a mammalian promoter), optionally further comprising at least one polypeptide and / or non-coding RNA encoding a protein of interest. In particular, the encoded modified mammalian glutamine synthetase is the modified mammalian glutamine synthetase of the first aspect. The nucleic acid sequence may be part of an expression vector of the second aspect. Thus, the embodiments and examples specified for the first and second aspects apply to this aspect as well. In particular, in certain embodiments, the nucleic acid comprises a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3 or 4, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q, preferably the mutation is R298K and / or N10S / Q.

[0069] In a sixth aspect, the present invention relates to a eukaryotic host cell comprising a polynucleotide encoding a modified mammalian glutamine synthetase of the present invention (first aspect), an expression vector of the present invention (second aspect), or a nucleic acid sequence of the present invention (fifth aspect). In a particular embodiment, the eukaryotic host cell comprises a polynucleotide encoding a modified mammalian glutamine synthetase simultaneously integrated into the host cell genome with at least one polynucleotide encoding a protein and / or a non-coding RNA of interest. The eukaryotic host cell can be any host cell, provided that the host cell is an immortalized cell and not a primary cell. Preferably, the eukaryotic host cell is a mammalian host cell or a yeast host cell, more preferably a mammalian host cell. In a particular embodiment, the mammalian host cell is a mouse, human, or rodent cell, more preferably a rodent cell, even more preferably a CHO cell. Furthermore, the eukaryotic host cell is preferably a GS gene knockout cell (GS knockout mutant) host cell, such as a mammalian GS gene knockout cell. The term "GS gene knockout cells" as used herein refers to cells in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in disruption of the enzymatic function of GS. Such cells may be referred to as GS- / - or GS- / + cells, depending on whether both alleles or only one are deleted or disrupted. The GS gene introduced by exogenous glutamine supplementation or an expression vector is essential for the cell survival of the GS gene knockout cells. In a preferred embodiment, the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS- / -) cell. In a particular embodiment, the eukaryotic or mammalian host cell is a monoclonal cell line generated by a process of single cell cloning and clonal amplification.

[0070] Methods for preparing cell lines or for producing proteins In a seventh aspect, the present invention relates to a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising the steps of: (a) introducing into a eukaryotic host cell, preferably a CHO cell, an expression vector according to the present invention (second aspect) or a nucleic acid of the present invention (fifth aspect), comprising a modified mammalian glutamine synthetase of the present invention (first aspect), optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest; and (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions selecting for the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding the optional protein and / or non-coding RNA of interest is co-integrated into the host cell genome together with the polynucleotide encoding the modified mammalian glutamine synthetase. Optionally, the method may further comprise culturing the resulting pool of cells stably expressing the protein and / or non-coding RNA of interest selected in step (b) and / or isolating or culturing the pool of cells stably expressing the protein and / or non-coding RNA of interest. Cells stably expressing the protein and / or non-coding RNA of interest means that the polynucleotide encoding the protein and / or non-coding RNA of interest is stably integrated into the genome of the host cell and the protein and / or non-coding RNA of interest is stably expressed, i.e. expressed for an extended period of time, e.g. for at least 40 days, preferably for several months or years.

[0071] Said expression vector or nucleic acid can be a plasmid, a bacterial artificial chromosome (BAC) or a viral vector. Thus, the expression vector or nucleic acid of the present invention can be introduced by transfection or transduction, respectively. Preferably, said expression vector or nucleic acid is a plasmid. More preferably, said expression vector or nucleic acid is introduced by stable transfection. Methods for transducing or transfecting expression vectors or nucleic acids into eukaryotic cells are well known in the art and include chemical means, such as calcium phosphate precipitation and lipofection, and physical means, such as electroporation. The polynucleotide encoding the modified mammalian glutamine synthetase and the polynucleotide encoding the protein and / or non-coding RNA of interest are operably linked to a eukaryotic promoter and are therefore adapted for expression in eukaryotic host cells.

[0072] The method of the present invention may further comprise a step (c) of isolating a single clone for clonal propagation to prepare a monoclonal cell line. Those skilled in the art will appreciate that transfection or transduction often requires a large number of cells, resulting in a heterogeneous pool of recombinant cells, e.g., with a diverse population of integration sites. For the generation of a clonal cell line, the cell pool is diluted or selected for the isolation of single cells (monoclonality), and each single clone is subjected to clonal amplification to prepare a monoclonal cell line. The term "cell line" as used herein refers to a cell population derived from a single cell clone and can be propagated for an infinite amount of time. It is therefore also referred to as a monoclonal cell line. Thus, the cell line is genetically stable, and therefore the characteristics of the cell line should not change over time. In particular, phenotypic characteristics such as production levels (titer) and growth rate and density (viable cell density and maximum viable cell density), viability, and genetic integrity as measured via copy number and DNA fingerprinting assays should be maintained when cultured under comparable conditions.

[0073] The cell pool or monoclonal cell line prepared according to the method of the present invention can further be used to stably produce a protein of interest or to stably produce non-coding RNA, such as RNA that mediates RNA interference, such as microRNA, small interfering RNA, long non-coding RNA, or small hairpin RNA. RNA interference can be used to silence genes and therefore to create cell pools or monoclonal cell lines with beneficial properties, such as for the production of proteins. For example, the difficulty of removing host cell proteins (HCPs) can be silenced in the cell pool or monoclonal cell line, or enzymes such as fucosyltransferases can be silenced to modify the glycosylation profile of the cell pool or monoclonal cell line. Furthermore, CHO cells, which are commonly used for large-scale industrial production, are often engineered to improve their characteristics in the production process or to facilitate the selection of recombinant cells. Such engineering includes, but is not limited to, improved resistance to apoptosis, reduced autophagy, increased cell proliferation, altered expression of cell cycle regulatory proteins, chaperone engineering, unfolded protein response (UPR) engineering, secretory pathway engineering, and metabolic engineering. Such engineering may potentially be achieved using RNA interference in eukaryotic host cells generated by the methods of the invention.

[0074] In a related eighth aspect, the invention relates to a method for producing a protein of interest comprising the steps of: (a) introducing into a eukaryotic host cell an expression vector (second aspect) comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to the invention, the polynucleotide comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q, and further comprising at least one polynucleotide encoding a protein of interest; (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions for selection of the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome together with the polynucleotide encoding the modified mammalian glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the mammalian host cell under conditions for production of the protein of interest; and (e) collecting and optionally purifying the protein of interest. The expression vector or nucleic acid of the invention may be introduced by transfection or transduction. Preferably, the expression vector or nucleic acid is introduced by stable transfection.

[0075] Those skilled in the art will understand that for the expression vector, typically only a portion of the vector is integrated into the genome of the host cell. Thus, the integrated portion of the vector comprises a polynucleotide encoding a modified mammalian glutamine synthetase and at least one polynucleotide encoding a protein and / or non-coding RNA of interest. Furthermore, the integrated portion of the expression vector or the integrated nucleic acid sequence of the present invention may be further amplified, for example by increasing the concentration of a glutamine synthetase inhibitor, such as methionine sulfoximine (MSX). Amplification is optional, and may result in higher productivity due to a higher copy number of polynucleotides encoding the protein and / or non-coding RNA of interest, since they become co-amplified together with the modified mammalian glutamine synthetase. The method according to the present invention may include the generation of cell pools or monoclonal cell lines. Furthermore, the eukaryotic host cell generated according to the method of the present invention or the eukaryotic host cell described in the present invention may be further used to produce a protein and / or non-coding RNA of interest or in a method for producing a protein of interest.

[0076] Thus, in a further ninth aspect, the present invention relates to a method for producing a protein of interest comprising the steps of: (a) providing a eukaryotic host cell of the invention (sixth aspect) comprising a polynucleotide encoding a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetase, the polynucleotide having the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, wherein said mutation is selected from the group consisting of R298K, N10S, N10T and N10Q, and at least one polynucleotide encoding a protein of interest; (b) culturing the eukaryotic host cell under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest.

[0077] In a preferred embodiment of the method of the present invention, the eukaryotic host cell is a GS gene knockout cell. Those skilled in the art will understand that this refers to the endogenous GS gene, whereas the modified mammalian GS gene is present in the host cell after transfection or transduction of the expression vector or nucleic acid sequence of the present invention. The eukaryotic host cell (transfected or transduced with the expression vector or nucleic acid sequence of the present invention) is cultured in a medium that does not contain any glutamine under conditions that select for the modified mammalian glutamine synthetase of step (b). This may further include the addition of the GS inhibitor methionine sulfoximine (MSX).

[0078] In a particular embodiment of the method of the present invention, the cells (cell pools) or monoclonal cell lines are produced with increased selection stringency and / or have increased genetic stability and / or have higher productivity compared to cells or monoclonal cell lines produced with the same mammalian glutamine synthetase that does not contain at least one mutation and / or compared to cells or monoclonal cell lines produced with a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO: 1. Genetic stability can be quantified by measuring the copy number of the integrated transgene. Genetic stability is evaluated by measuring the copy number over a longer culture time. Furthermore, genome rearrangements are monitored, for example, via Southern blot analysis. The increased selection stringency can be determined by the time to reach a survival rate of more than 70%.

[0079] The eukaryotic host cell can be any host cell, provided that the host cell is an immortalized cell and not a primary cell. The methods described herein are in vitro methods, and the eukaryotic host cell described in the present invention is for in vitro use in cell culture. The eukaryotic host cell includes in particular yeast cells and mammalian cells, and is preferably a mammalian cell. The yeast cell can be, but is not limited to, Saccharomyces cerevisiae, Pichia pastoris, Klyveromyces lactis, or Klyveromyces marxianus. The term "mammalian cell" as used herein refers to a mammalian cell line suitable for the production of a product of interest, such as a heterologous protein and / or a non-coding RNA of interest, and may also be referred to as a "host cell" or a "mammalian host cell". The mammalian cell is preferably a transformed and / or immortalized cell line. They are adapted to continuous passage in cell culture, preferably serum-free cell culture and / or preferably suspension culture, and do not contain primary non-transformed cells or cells that are part of an organ structure.

[0080] Preferably, the mammalian host cell is a mouse, human or rodent cell, more preferably a rodent cell, even more preferably a CHO cell. Preferred mammalian cells for heterologous protein production are mouse, rodent or human cells. Preferred examples of mammalian cells or mammalian cell lines are CHO cells (e.g. DG44 and K1), NS0 cells, HEK293 cells (e.g. HEK293 cells or HEK293T cells) and BHK21 cells. Preferably, the mammalian cells or mammalian cell lines are adapted to grow in suspension. In a preferred embodiment, the mammalian cells or mammalian cell lines are CHO cells. In a particular embodiment, the mammalian cells are HEK293 cells or CHO cells or HEK293 cells or CHO cell derived cells, preferably the mammalian cells are CHO cells or CHO derived cells.

[0081] Suitable rodent cells may be, for example, hamster cells, in particular BHK21 cells, BHK TK cells, CHO cells, CHO-K1 cells, CHO-DXB11 cells (also called CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or derivatives / progenies of any of such cell lines. Particularly preferred are CHO cells, such as CHO-DG44 cells, CHO-K1 cells, and BHK21 cells, and even more preferred are CHO-DG44 cells and CHO-K1 cells. Most preferred are CHO-DG44 cells. Glutamine synthetase (GS)-deficient derivatives of mammalian cells, in particular CHO-DG44 cells and CHO-K1 cells, are also included. In one embodiment of the invention, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO DXB11 cell, a CHO-S cell, a CHO GS-deficient cell, or a derivative thereof. A suitable human cell is a HEK293 cell or a HEK293T cell. The host cell may be a mouse cell, such as a mouse myeloma cell, for example a NS0 cell and a Sp2 / 0 cell, or a derivative / descendant of any of such cell lines.

[0082] Moreover, the eukaryotic host cell is preferably a GS gene knockout cell (GS knockout mutant) host cell. As used herein, the term "GS gene knockout cell" refers to a cell in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in disruption of the enzymatic function of GS. Such cells may be referred to as GS- / - or GS- / + cells, depending on whether both alleles or only one are deleted or disrupted. Exogenous glutamine supplementation or the GS gene introduced by the expression vector is essential for cell survival of the GS gene knockout cell. In a preferred embodiment, the mammalian host cell is a CHO-K1 cell, more preferably a CHO-K1-GS (GS- / -) cell.

[0083] Preferably, CHO cells are metabolically engineered, for example by knockout of endogenous glutamine synthetase (GS), to allow for an efficient cell line development process, to facilitate selection with methionine sulfoximine (MSX).

[0084] Non-limiting examples of mammalian cells that may be used in the context of the present invention are also summarized in Table A. However, derivatives / progeny of such cells, other mammalian cells, including but not limited to human, mouse, rat, monkey and rodent cell lines, may also be used in the present invention, particularly for the production of biopharmaceutical proteins.

[0085] [Table 1]

[0086] It is most preferred when the cells are established, adapted and completely cultured under serum-free conditions and, optionally, in a medium completely free of any proteins / peptides of animal origin. Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any of the media may be supplemented with a wide variety of compounds as necessary, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium phosphate, magnesium phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose, or other equivalent energy sources, antibiotics, and trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. For the growth and selection of genetically modified cells expressing the selection gene, an appropriate selection agent is added to the culture medium.

[0087] The protein of interest encoded by a mammalian expression vector or produced by the method of the invention is preferably produced in CHO cells in cell culture. After expression, the recombinant protein is harvested and further purified. The antibody may be recovered from the culture medium as a secreted protein in harvested cell culture fluid (HCCF) or from a cell lysate (i.e., a liquid containing the contents of cells lysed by any means, including but not limited to enzymatic, chemical, osmotic, mechanical, and / or physical disruption of the cell membrane and optionally the cell wall) and purified using the techniques described herein. According to the invention, the method comprises the step of providing a harvested cell culture fluid comprising the protein of interest, e.g., an antibody, as a starting material, wherein the HCCF is derived from a CHO cell culture. Preferably, the protein of interest, e.g., an antibody, is recovered from the harvested cell culture fluid after cell separation, e.g., by filtration and / or centrifugation. Thus, in certain embodiments, harvesting comprises centrifugation and / or filtration to produce a harvested cell culture fluid.

[0088] The modified mammalian glutamine synthetase of the first aspect, the expression vector of the second aspect, the nucleic acid of the fifth aspect, and the eukaryotic host cell of the sixth aspect may be used in methods of the invention, and therefore the embodiments and examples specified with respect to these aspects apply equally to the method-related aspects.

[0089] In view of the above, it will be appreciated that the present invention also encompasses the following:

[0090] Item 1 provides a modified mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3 or 4, comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetase, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q.

[0091] Item 2 specifies the modified mammalian glutamine synthetase of item 2, wherein the mutation does not interfere with binding to a substrate.

[0092] Item 3 specifies the modified mammalian glutamine synthetase of items 1 or 2, wherein the mutation is R298K and / or N10S, or N10T or N10Q.

[0093] Item 4 specifies the modified mammalian glutamine synthetase of any one of items 1 to 3, having the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10 or 11.

[0094] Item 5 describes the modified mammalian glutamine synthetase of any one of items 1 to 4, wherein the modified mammalian glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q (a) has a decreased enzymatic activity compared to the same mammalian glutamine synthetase not comprising the mutation; and / or (b) mediates increased selection stringency and / or genetic stability upon transfection with a polynucleotide encoding the modified mammalian glutamine synthetase and a transgene compared to the same mammalian glutamine synthetase not comprising the mutation.

[0095] Item 6 describes the modified mammalian glutamine synthetase of any one of items 1 to 4, wherein the modified mammalian glutamine synthetase comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q (a) has a decreased enzymatic activity compared to a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO:1; and / or (b) mediates increased selection stringency and / or genetic stability upon transfection with a polynucleotide encoding the modified mammalian glutamine synthetase and a transgene compared to a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO:1.

[0096] Item 7 provides an expression vector comprising a polynucleotide encoding the modified mammalian glutamine synthetase of any one of items 1 to 6.

[0097] Item 8 specifies the expression vector of item 7, wherein the expression vector comprises an expression cassette comprising a polynucleotide encoding a modified mammalian glutamine synthetase, preferably wherein the polynucleotide encoding the modified mammalian glutamine synthetase is operably linked to a promoter.

[0098] Item 9 specifies the expression vector according to item 7 or 8, wherein the expression vector further comprises at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably an expression cassette comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0099] Item 10 specifies the expression vector according to item 9, wherein the protein of interest is a therapeutic protein, preferably selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

[0100] Item 11 specifies the expression vector according to item 10, wherein the protein of interest is an antibody, and preferably wherein the expression vector comprises a polynucleotide comprising a coding sequence for a variable region of an antibody heavy chain and / or a coding sequence for a variable region of an antibody light chain.

[0101] Item 12 specifies the expression vector according to any one of items 9 to 11, wherein the expression vector comprises a polycistronic expression cassette and / or a plurality of expression cassettes, preferably wherein the expression vector comprises a plurality of expression cassettes.

[0102] Item 13 specifies the expression vector of any one of items 7 to 12, wherein the expression vector is for stable transfection, and the integrating portion of the vector comprises a polynucleotide encoding a modified mammalian glutamine synthetase and at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0103] Item 14 provides a nucleic acid sequence comprising a polynucleotide encoding the modified mammalian glutamine synthetase of any one of items 1 to 6 operably linked to a mammalian promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

[0104] Item 15 provides a eukaryotic host cell comprising a polynucleotide sequence encoding the modified mammalian glutamine synthetase of any one of Items 1 to 6, an expression vector of any one of Items 7 to 13, or a nucleic acid sequence of Item 14.

[0105] Item 16 specifies the eukaryotic host cell of item 15, wherein the eukaryotic host cell is (a) a mammalian cell, preferably a rodent cell, more preferably a CHO cell, and / or (b) a GS gene knockout cell.

[0106] Item 17 provides a method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising: (a) introducing the expression vector according to any one of items 7 to 13, or the nucleic acid of item 14, optionally further comprising at least one polynucleotide encoding the protein and / or non-coding RNA of interest, into a eukaryotic host cell, preferably a mammalian host cell, more preferably a CHO cell; and (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions selecting for the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding the optional protein and / or non-coding RNA of interest is co-integrated into the host cell genome together with the polynucleotide encoding the modified mammalian glutamine synthetase.

[0107] Item 18 specifies the method of item 17, further comprising the step of (d) isolating a single clone for clonal expansion to prepare a monoclonal cell line.

[0108] Item 19 provides a method for producing a protein of interest, comprising the steps of: (a) introducing an expression vector comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to any one of items 7 to 13, the polynucleotide comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q, and further comprising at least one polynucleotide encoding a protein of interest, or the nucleic acid of item 14, into a eukaryotic host cell, preferably a mammalian host cell, more preferably a CHO cell; (b) culturing the eukaryotic host cell in a medium free of glutamine under conditions that select for the modified mammalian glutamine synthetase, wherein the at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome with the polynucleotide encoding the modified mammalian glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the eukaryotic host cell under conditions that produce the protein of interest; and (e) collecting and optionally purifying the protein of interest.

[0109] Item 20 specifies the method of any one of items 17 to 19, wherein the expression vector is introduced by transfection or transduction.

[0110] Item 21 specifies the method of item 20, wherein the expression vector is introduced by stable transfection.

[0111] Item 22 provides a method for producing a protein of interest, comprising: (a) providing a eukaryotic host cell of item 15 or 16, comprising a polynucleotide encoding a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3 or 4, the polynucleotide comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetase, the mutation being selected from the group consisting of R298K, N10S, N10T and N10Q, and at least one polynucleotide encoding a protein of interest; (b) culturing the eukaryotic host cell under conditions for producing the protein of interest; and (c) collecting and optionally purifying the protein of interest.

[0112] Item 23 specifies the method of any one of items 17 to 22, wherein (a) the eukaryotic host cell is a GS gene knockout cell (GS- / - or GS- / +), and / or (b) the step of culturing the eukaryotic host cell in a medium completely devoid of glutamine under conditions selecting for the modified mammalian glutamine synthetase of step (b) comprises the addition of the GS inhibitor methionine sulfoximine (MSX).

[0113] Item 24 specifies the method of any one of items 17 to 23, wherein the eukaryotic host cell is a mammalian cell, preferably a rodent cell, more preferably a CHO cell.

[0114] Item 25 specifies the method of any one of items 17 to 24, wherein the cell or monoclonal cell line is produced with increased selection stringency and / or has increased genetic stability and / or has higher productivity compared to a cell or monoclonal cell line produced with the same mammalian glutamine synthetase not comprising at least one mutation and / or compared to a cell or monoclonal cell line produced with a mammalian glutamine synthetase having the amino acid sequence of SEQ ID NO:1.

[0115] Item 26 provides a kit comprising the expression vector of any one of Items 7 to 13 and a cell culture medium not containing glutamine.

[0116] Item 27 provides the use of a modified mammalian glutamine synthetase according to items 1 to 6 as a selection marker for the generation of a monoclonal eukaryotic cell line, preferably for the generation of a mammalian cell line, more preferably for the generation of a rodent cell line, even more preferably for the generation of a CHO cell line.

[0117] Item 28 provides the use of the vector according to any one of items 7 to 13 for producing a protein of interest in a eukaryotic host cell, preferably a mammalian cell, more preferably a rodent cell, even more preferably a CHO cell.

[0118] Working Example Linearization of plasmids for transfection The plasmids used for stable transfection contain a cytomegalovirus-driven antibody transcription cassette, an ampicillin transgene, and a glutamine synthetase transgene as a metabolic selection marker. Plasmids were linearized for transfection using PvuI (cuts once in the ampicillin transgene). Restriction enzyme digestion of 20 μg of plasmid DNA was performed with PvuI (NEB) for 3 hours at 37° C. according to the manufacturer's protocol. The linearized plasmid DNA was purified (Qiagen Plasmid Maxi Kit) according to the manufacturer's protocol. Final DNA concentration was determined via a Nanodrop spectrophotometer.

[0119] Host cell culture Host cells of the CHO-K1-GS knockout cell line (also called CHO-K1-GS KO or simply CHO-K1-GS), which has a genomic knockout of the endogenous glutamine synthetase gene, were cultured in host cell medium supplemented with L-glutamine. The host cell culture was at 3 × 10 cells per mL. 5A seeding density of 1000 cells was initiated. Growth conditions were set at 36.5°C and 5% carbon dioxide in a shaking incubator at 120 rpm in shake flasks. Cell density and viability determinations were performed with a Sedex HiRes© cell counter.

[0120] Transfection of linearized plasmids into CHO cells One day before transfection, host cells were cultured at 0.8 × 10 6 Cells were seeded at a cell density of 10 cells / mL. On the day of transfection, cell density and viability were determined and the amount of cells required for transfection was centrifuged at 750 x G for 7 min. The supernatant was discarded. 10 μg of linearized plasmid DNA per transfection was transfected using the Neon© transfection system (Invitrogen) and electroporation. An electroporation cuvette was filled with 3 mL of electroporation buffer E2 and 89 μl of buffer R was added to the cells (5 x 10 6 The cells were resuspended and mixed with 10 μg of linearized plasmid. Transfection was performed using 1500 volts, 10 ms, and 2 pulses. The transfected cells were transferred to 5 ml of pre-warmed host cell medium in a T25 ml flask and incubated at 8% carbon dioxide and 37° C. for at least 24 hours.

[0121] Selection of stable CHO cell pools 24 hours after transfection, cells were transferred to selection medium (medium without L-glutamine). 10 mL of selection medium was pre-warmed for every pool in a T75 flask. Two stable pools were cultured for each transfection. Cells were centrifuged (750×G for 7 min), resuspended in 20 mL selection medium, and incubated at 37° C. with 8% carbon dioxide. Cells were monitored by microscope during selection. Additional selection medium (5 mL) was added after 7 days.

[0122] Stable pool passaging and production process The selection phase was deemed successful once the cells reached at least 70% viability and a doubling time of 48 h or less. After selection, cells were cultured at 3 x 10 per mL in a total volume of 30 mL of medium (containing no L-glutamine) in 125 mL shake flasks. 5 Starting with 1 cell, cells were passaged every 2 to 3 days at 36.5° C. and 5% carbon dioxide with shaking at 120 rpm. Samples for titer determination were taken periodically. If the antibody titer was stable for at least 14 days, indicating phenotypic stability, the production run was started for at least 7 days.

[0123] For the production run, cells were cultured at 7 x 10 per mL in shake flasks. 5 At a density of 1000 viable cells, the cells were seeded in a total volume of 30 mL of basal medium without glutamine and the cells were cultivated at 34.5° C. with 5% carbon dioxide and shaking at 120 rpm. During a production run of at least 7 days, cells were counted, samples were withdrawn once a day for pH, glucose and titer measurements, and pH and glucose feeds were adjusted if necessary. Starting from day 2, a daily feed (glutamine water-in-oil) was supplemented. Parameters such as pH were determined using a RAPIDlab348Ex© and glucose was measured via an EKF diagnostics instrument. On day 7, cell culture medium supernatants were analyzed for antibody titers after centrifugation using a ForteBio Octet© instrument with a Protein A biosensor. Sample dilutions and standard curves were processed during the same production run.

[0124] Example 1: Production of antibodies in CHO cells using CHO wild-type GS as a metabolic selection marker CHO-K1-GS cells, once transfected with a vector carrying the glutamine synthetase derived from Chryseturus griseus (CHO wild-type glutamine synthetase (GS), SEQ ID NO:2) selectable marker, can survive selection under culture in medium not supplemented with L-glutamine if the transgene vector is stably integrated into the genome of the cells.

[0125] CHO-K1-GS cells were transfected with a vector carrying an expression cassette for monoclonal antibody 1 (mAb1) and CHO wild-type glutamine synthetase. After selection, stable CHO pools were passaged as described above and samples were taken periodically for measurement of the titer of monoclonal antibody 1. Under the applied experimental conditions, stable CHO pools remained stable and productive for approximately 20 days after transfection, after which the level of productivity (titer) decreased (Figure 1).

[0126] Example 2: Production of antibodies in CHO cells using CHO GS with a mutation at position R298 as a selection marker To functionally test the novel CHO-based glutamine synthetase mutants, the stringency of selection (time to reach >70% viability after transfection), the number of productive passages, and the specific productivity were measured over time. For CHO pools that remained productive after extended passages, a 7-day production run in shake flasks was subsequently performed in a controlled process setting to further evaluate the cell culture process parameters.

[0127] CHO-K1-GS cells were transfected with vectors carrying expression cassettes for monoclonal antibody 1 and CHO GS with various mutations at amino acid position 298 (R298 in SEQ ID NO:1). Viable cell density (VCD), viability and productivity were measured over time during the selection period. From all point mutations tested, only CHO GS R298K survived the selection period, suggesting that R298 is a conserved amino acid residue. Cells transfected with the CHO GS R298K mutation took longer to fully recover from selection compared to CHO wild-type GS (wtGS) (Figure 2) and cell growth was slower, suggesting increased stringency of selection.

[0128] After the transfected cells had fully recovered from selection and reached a doubling time of at least 70% and at least 48 hours, the cells were transferred to shake flasks for passage and samples were taken periodically for titration of monoclonal antibody 1. CHO GS R298K further conferred significantly higher productivity, with cell pools remaining more stable over the course of 20 days compared to wild-type CHO GS (CHO WT GS) (Figure 3A), and the average productivity of CHO GS R298K was increased approximately 4-fold compared to CHO wild-type GS (Figure 3B).

[0129] Overall, CHO cells transfected with the CHO GS mutant R298K showed increased selection stringency and significantly higher productivity. Furthermore, cell pools showed increased phenotypic stability and remained productive for at least 45 days (data not shown), whereas cells transfected with CHO WT GS completely lost their productivity about 20 days after transfection.

[0130] Example 3: Production of antibodies in CHO cells using CHO GS with a mutation at position N10 as a selection marker CHO-K1-GS cells were transfected with vectors carrying expression cassettes for monoclonal antibody 1 and glutamine synthetase from Chryseturus griseus carrying various mutations at amino acid position N10 (SEQ ID NO:1). Viable cell density (VCD), viability and productivity were measured over time during the selection period. Cells transfected with CHO GS carrying N10S, N10Q and N10W mutations took longer to fully recover from selection (Figure 4) and cell growth was slower compared to CHO WT GS, suggesting increased stringency of selection.

[0131] All mutants tested (N10S, N10Q and N10W) initially showed similar levels of productivity compared to wild-type CHO GS (CHO WT GS). Surprisingly, CHO GS N10S and N10Q showed a significant increase in phenotypic stability compared to CHO WT GS and N10W, both of which showed a decrease in productivity after 4 days under selection (Figure 5). N10S and N10Q remained stable during further production steps in shake flasks (data not shown).

[0132] Furthermore, CHO GS N10F, N10Y and N10G were tested together with CHO N10Q, N10S and N10W in addition to wild type CHO GS. All mutants were functional and generated stable pools, although the stringency of selection was varied (growth period after transfection was strongly variable) (Figure 6). However, with the exception of CHO GS N10S and CHO GS N10Q, none of the mutants tested showed phenotypic stability (data not shown). However, due to the structural similarity of the amino acids serine and tyrosine, CHO GS N10T, which was not tested, is expected to show a phenotype similar to CHO GS N10S.

[0133] In conclusion, CHO cells transfected with the CHO GS mutants N10S and N10Q showed increased selection stringency and significantly longer phenotypic stability compared to the wild-type selection marker.

[0134] Example 4: Production of antibodies in CHO cells combining N10S and R298K mutations and using CHO GS as a selection marker Examples 2 and 3 demonstrated that specific mutations of R298 and N10 conferred increased selection stringency and significantly longer phenotypic stability compared to the wild-type GS selection marker. Furthermore, CHO GS combining N10 and R298 mutations were tested. Specifically, mutations N10S and R298K were combined and tested at the 384 screening stage at the clone level for the generation of stable pools and evaluation of productivity.

[0135] CHO-K1-GS cells were transfected with a vector carrying an expression cassette for monoclonal antibody 1 and glutamine synthetase from Chryseturus griseus carrying the N10S and R298K mutations. The double mutant was found to generate stable cell pools with increased productivity (Figure 7). Notably, genomic stability was clearly superior compared to CHO WT (data not shown).

[0136] [Table 2]

Claims

1. A modified mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3 or 4, comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q.

2. 3. The modified mammalian glutamine synthetase of claim 2, wherein the mutation does not interfere with binding to a substrate.

3. 3. The modified mammalian glutamine synthetase of claim 1 or 2, comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q, (a) having a decreased enzymatic activity compared to the same mammalian glutamine synthetase not comprising said mutation; and / or (b) mediating increased selection stringency and / or genetic stability upon transfection with a polynucleotide and transgene encoding said modified mammalian glutamine synthetase, compared to the same mammalian glutamine synthetase not comprising said mutation.

4. An expression vector comprising a polynucleotide encoding the modified mammalian glutamine synthetase of any one of claims 1 to 3.

5. The expression vector of claim 4, further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, preferably wherein the protein of interest is a therapeutic protein selected from the group consisting of a cytokine, a hormone, a fusion protein, an antibody, an antibody-derived molecule, and an antibody mimetic.

6. 10. A nucleic acid sequence comprising a polynucleotide encoding the modified mammalian glutamine synthetase of any one of claims 1 to 3 operably linked to a eukaryotic promoter, and optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest.

7. A eukaryotic host cell comprising a polynucleotide sequence encoding a modified mammalian glutamine synthetase of any one of claims 1 to 3, an expression vector of claim 4 or 5, or a nucleic acid sequence of claim 6.

8. (a) introducing an expression vector according to claim 4 or 5, or a nucleic acid according to claim 6, optionally further comprising at least one polynucleotide encoding a protein and / or a non-coding RNA of interest, into a eukaryotic host cell, preferably a mammalian cell, more preferably a Chinese Hamster Ovary (CHO) cell; and (b) culturing a eukaryotic host cell in a medium that is completely devoid of glutamine under conditions that select for the modified mammalian glutamine synthetase, wherein at least one polynucleotide encoding an optional protein of interest and / or a non-coding RNA is co-integrated into the host cell genome with a polynucleotide encoding the modified mammalian glutamine synthetase; 1. A method for preparing a cell stably expressing a protein and / or a non-coding RNA of interest, comprising:

9. (a) introducing an expression vector comprising a polynucleotide encoding a modified mammalian glutamine synthetase according to claim 4 or 5, comprising a mutation selected from the group consisting of R298K, N10S, N10T and N10Q, and further comprising at least one polynucleotide encoding a protein of interest, or a nucleic acid according to claim 6, into a eukaryotic host cell, preferably a mammalian host cell, more preferably a CHO cell; (b) culturing a eukaryotic host cell in a medium free of glutamine under conditions that select for the modified mammalian glutamine synthetase, wherein at least one polynucleotide encoding a protein of interest is co-integrated into the host cell genome with a polynucleotide encoding the modified mammalian glutamine synthetase; (c) optionally isolating a single clone for clonal amplification to prepare a monoclonal cell line; (d) culturing the eukaryotic host cells under conditions to produce the protein of interest; and (e) harvesting and optionally purifying the protein of interest. A method for producing a protein of interest comprising:

10. 10. The method of claim 8 or 9, wherein the expression vector is introduced by transfection or transduction.

11. (a) providing a eukaryotic host cell of claim 7, comprising a polynucleotide encoding a mammalian glutamine synthetase having an amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3 or 4, the polynucleotide comprising a mutation at amino acid position 10 and / or 298 in the mammalian glutamine synthetase, the mutation being selected from the group consisting of R298K, N10S, N10T and N10Q, and at least one polynucleotide encoding a protein of interest; (b) culturing the eukaryotic host cell under conditions to produce the protein of interest; and (c) harvesting and optionally purifying the protein of interest A method for producing a protein of interest comprising:

12. 12. The eukaryotic host cell of claim 7 or the method of any of claims 8 to 11, wherein the eukaryotic host cell is: (a) a mammalian host cell, preferably a rodent cell, more preferably a CHO cell; and / or (b) a GS knockout mutant.

13. A kit comprising an expression vector according to claim 4 or 5 and a cell culture medium that does not contain glutamine.

14. Use of a modified mammalian glutamine synthetase according to claims 1 to 3 as a selection marker for the generation of monoclonal eukaryotic cell lines, preferably for the generation of mammalian cell lines, more preferably for the generation of rodent cell lines, even more preferably for the generation of CHO cell lines.

15. 6. Use of a vector according to any one of claims 4 or 5 for producing a protein of interest in a eukaryotic host cell, preferably a mammalian host cell, more preferably a rodent cell, even more preferably a CHO cell.

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