Method for selection of cell line expressing high levels of recombinant protein using glutamine synthetase split expression vector

Abstract

The present invention relates to a recombinant vector for expression of a target protein or target gene containing a glutamine synthetase split fragment, and a method for selection of a cell line expressing high levels of a recombinant protein using same. According to the present invention, the efficiency of selecting cell lines for biopharmaceutical production dramatically increases,, and it can be used free from stability problems because it is an improvement on the conventionally used selectable marker GS (glutamine synthetase).

Description

METHOD FOR SELECTION OF CELL LINE EXPRESSING HIGH LEVELS OF RECOMBINANT PROTEIN USING GLUTAMINE SYNTHETASE SPLIT EXPRESSION VECTOR

[0001] The present invention relates to a method for selection of a cell line expressing high levels of recombinant protein using a glutamine synthetase split expression vector, and more particularly to a recombinant vector for the expression of a target protein containing a glutamine synthetase split fragment and a method for selection of a cell line expressing high levels of recombinant protein using the same.

[0002]

[0003] The global biopharmaceutical market was valued at approximately KRW 412 trillion in 2020, with an average annual growth rate of 12%. The market size of therapeutic proteins by domestic companies was estimated at 3.3 trillion won in 2020, and the growth rate of production value increased by 54.9% compared to 2019, making it the most active growth sector among pharmaceuticals. As the global market for biopharmaceuticals continues to grow, the number of biopharmaceuticals licensed in each country is also steadily increasing, and domestic biopharmaceutical companies have recently expanded into the contract development manufacturing organization (CDMO) market in which the development is added to the contract manufacturing organization (CMO).

[0004] Recombinant proteins, which form a major part of biopharmaceuticals, can be expressed in many different types of host cells, including prokaryotic and eukaryotic cells. However, glycoproteins, such as antibodies or Fc fusion proteins, in which sugars bind to specific amino acids in the polypeptide to affect efficacy or safety, are often produced in cells that can undergo glycosylation in a post-translational modification of the protein, i.e. animal cells. In particular, in the production of recombinant protein drugs, it is important to produce glycoproteins with the same or similar sugar chains to human glycoproteins because the similarity of the sugar chains to those of human (native) glycoproteins is closely related to the immunogenicity of the protein drug.

[0005] Animal cells that have been used to express and produce recombinant protein drugs include hybridoma, mouse myeloma, and CHO cells. However, although the expression of proteins using these animal cells is excellent in terms of similarity to proteins in the body, they have the disadvantage that the expression amount is significantly lower and it is difficult to cultivate in large quantities. In particular, in the case of therapeutic antibodies, it is not easy to secure a sufficient amount of protein by simply culturing animal cells, as it is usually required to be expressed in kilogram quantities. Therefore, in order to express high levels of a specific gene in these host cells, it is necessary to introduce the gene into the region having a high transcription rate in the animal cell DNA when the DNA to be expressed is randomly introduced into the animal cell genome. The foreign gene is then replicated along with the host cell's genome, but the technology of introducing to a specific region having a good transcription rate (homologous recombination technology) is not yet widely available.

[0006] The currently widely used gene expression system for recombinant proteins is the GS system (U.S. Patent No. 5122464), which was first developed by Celltech, which improved the shortcomings of the gene expression system using DHFR, which was not only time-consuming to obtain a single cell line but also not very productive. The GS system utilizes glutamine synthetase, an enzyme involved in the sole metabolic pathway that utilizes glutamate and ammonia to produce glutamine, and takes advantage of the problem on culturing animal cells in a glutamine-free environment. This GS system requires fewer copies of DNA per cell than conventional DHFR systems, and has the advantage of enabling high expression cell lines to be obtained at an early stage of production cell line screening.

[0007] Therefore, there is an increasing number of companies that are introducing the GS system as an expression system for protein drugs. The cell lines mainly used in the GS system are NS0 cell line and CHO cell line, and among them, NS0 cell line, which is a mouse myeloma cell, does not express enough GS, so it has the advantage of easily selecting only the cells into which the target protein gene is introduced without adding glutamine to the cell culture medium. Unlike the NS0 cell line, CHO cells express enough GS, so they can survive in the absence of glutamine in the culture.

[0008] However, when CHO cells are treated with excessive concentrations of a GS-specific inhibitor such as methionine sulphoximine (MSX), it becomes impossible for the CHO cells to survive on GS produced by the cells themselves, so only cells that have been transfected with a vector containing the GS gene and the target protein gene can survive. This allows the selection of only those cells that have the target protein gene, and furthermore, the production of the target protein. In other words, the more GS-specific inhibitors are applied to the cells, the more the cells will amplify the target gene introduced along with the GS in order to survive, thus increasing the production of the target protein. In the case of NS0 cells, which are resistant and survive at MSX concentrations ranging of 10 to 100 uM, the GS gene and the recombinant expression gene are amplified together. The amplification process results in enormous differences in the protein expression of each clone. Therefore, the process of selecting a high expression cell line is a very tedious and laborious task, requiring selection of hundreds of clones. The most widely used selection method of clones is limiting dilution using multi-well plates, or physically separating clones using glass or metal cloning rings, then enzymatically treating the cells and removing them from the cloning rings using a micropipette. The clones can also be separated by swiping a cotton swab over the clustered cells, but in industrial applications, cell sorting or robotics are now used to select cell lines instead of these traditional methods.

[0009] These methods significantly reduce the labor required to select candidate cell lines. In the case of monoclonal antibodies that require high concentrations, clones with a productivity of 20 pg / cell / day or less are already excluded at the beginning of selection. Highly productive cell lines often require multiple transfections to select, as only a small number of cells can be transfected correctly in a single transfection. It is also difficult to generate 500 to 1,000 stable cell lines on a single transformation plate. When selecting production cell lines, the growth rate of the cells should also be considered. In general, if a cell line is very productive, it will grow slowly, but if it is fast-growing and highly productive, it will be easier to scale up production industrially (Kyu-Heum Na, BIOSAFETY Vol. 8, pp. 56-69, 2007).

[0010] As described earlier, in order to identify cell clones expressing very high levels of the desired foreign gene, it is necessary to examine and test a large number of clones, which is time-consuming, labor-intensive, and costly, and due to the low selection efficiency of glutamine synthetase, which is used as a marker gene, only a small percentage of the selected cells become high expression cell lines. This makes the selection of a small number of high expression cells time-consuming and costly.

[0011] In order to improve the low selection efficiency of glutamine synthetase, which is used as a marker for selecting recombinant proteins, the inventors of the present invention have found that when glutamine synthetase is divided into N-domain and C-domain regions and expressed, and then combined intracellularly, the selection efficiency of cells expressing high levels of recombinant protein is significantly improved; and completed the present invention.

[0012]

[0013] SUMMARY OF INVENTION

[0014] It is an object of the present invention to provide a first vector comprising a nucleic acid encoding an N-domain region of glutamine synthetase as a selectable marker to effectively select cells expressing high levels of a target protein or target gene.

[0015] Another object of the present invention is to provide a second vector comprising a nucleic acid encoding a C-domain region of glutamine synthetase as a selectable marker, in order to effectively select cells expressing high levels of a target protein or target gene.

[0016] Another object of the present invention is to provide cells for the production of a target protein or target gene transformed from the first vector and the second vector.

[0017] Another object of the present invention is to provide a method for effectively selecting cells expressing high levels of target protein or target gene.

[0018] Another object of the present invention is to provide a method for producing a target protein by culturing cells for the production of the target protein or target gene.

[0019] To accomplish the above objectives, the present invention provides a first vector comprising as a selectable marker a first marker nucleic acid encoding an N-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID NOs: 1-17; SEQ ID NOs: 35-51; and SEQ ID NOs: 69-85.

[0020] The present invention also provides a second vector comprising as a selectable marker a second marker nucleic acid encoding a C-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID NOs: 18-34; SEQ ID NOs: 52-68; and SEQ ID NOs: 86-102.

[0021] The present invention provides a cell for the production of a target protein or target gene, wherein the first vector and the second vector are introduced, wherein the first vector and / or the second vector further comprises a gene encoding a target protein, wherein the first vector and the second vector are introduced such that when combined with each other, the N-domain region of the first vector and the C-domain region of the second vector result in an intact glutamine synthetase, wherein the first vector and / or the second vector comprises an intein, a leucine zipper or a coiled coil.

[0022] The present invention also provides a method for selection of cells expressing a target protein or target gene, comprising the following steps

[0023] (a) creating a library of cells for the production of the target protein or target gene;

[0024] (b) culturing the library in a medium comprising a glutamine synthetase inhibitor, thereby selecting growing recombinant cells; and

[0025] (C) confirming expression of the target protein or target gene in the selected recombinant cells, and obtaining cells expressing the target protein or target gene.

[0026] The present invention also provides a method of producing a target protein or target gene, comprising the following steps:

[0027] (a) culturing cells for the production of the target protein or target gene, and producing the target protein or target gene; and

[0028] (b) obtaining the generated target protein or target gene.

[0029]

[0030] FIG. 1 illustrates the three-dimensional structure of glutamine synthetase and the three split positions employed in the present invention.

[0031] FIG. 2 shows a recombinant protein expression vector comprising a glutamine synthetase 2 split fragment according to the present invention, wherein GSN represents the N-domain split fragment of glutamine synthetase and GSC represents the C-domain split fragment of glutamine synthetase.

[0032] FIG. 3 shows the results of cell growth upon selection according to the three split positions of glutamine synthetase.

[0033] FIG. 4 shows the results of cell viability upon selection according to the three split positions of glutamine synthetase.

[0034] FIG. 5 shows the results of determining the difference in expression of mCherry when expressed as a recombinant protein using the expression system according to the present invention. It was incubated with different concentrations of methionine sulfoximine (MSX). The efficiency of selecting cell lines expressing the recombinant protein according to glutamine synthetase split positions 1 (NC1), 2 (NC2), and 3 (NC3) relative to the wild type (WT) is shown.

[0035] FIG. 6 shows the results of determining the difference in expression when EGFP is expressed as a recombinant protein using the expression system according to the present invention. The efficiency of selecting cell lines expressing the recombinant protein according to glutamine synthetase split positions 1 (NC1), 2 (NC2), and 3 (NC3) relative to the wild type (WT) is shown.

[0036] FIG. 7 shows the results of identifying differences in expression when Etanercept, an FC fusion protein, is expressed using the expression system according to the present invention.

[0037] FIG. 8 shows the results of selecting cells expressing Etanercept, an FC fusion protein, using the expression system according to the present invention, and identifying high production of Etanercept in a pool of cells, as determined by mean fluorescence intensity (MFI) analysis using fluorescent labeling.

[0038] FIG. 9 shows the results of selecting cells expressing Etanercept, an FC fusion protein, using the expression system according to the present invention to determine the percentage of cell lines expressing high levels of Etanercept in a cell pool.

[0039] FIG. 10 shows the results of determining Etanercept production by batch culture of a pool of cells expressing Etanercept, an FC fusion protein, using cell lines selected by the methods of the present invention.

[0040] FIG. 11 illustrates a recombinant vector for the expression of Rituximab made in accordance with the present invention.

[0041] FIG. 12 shows the results of selecting cells expressing Rituximab, a monoclonal antibody (mAb), with a selecting system according to the present invention, wherein high Rituximab production in a pool of cells was confirmed by a mean fluorescence intensity (MFI) analysis using fluorescent labeling.

[0042] FIG. 13 shows the results of selecting cells expressing a monoclonal antibody (mAb), Rituximab, using a selectable system according to the present invention to determine the proportion of high-producing cell lines in a cell pool.

[0043] FIG. 14 shows the results of determining Etanercept production by batch culture of a pool of cells expressing Rituximab using cell lines selected by the methods of the present invention.

[0044] FIG. 15 shows the structure of a recombinant vector for Rituximab expression with IRES added to the vector, and four vectors were produced by adjusting the positions of the heavy and light chains of Rituximab as follows: (i) first vector: heavy chain:GS-N; (ii) second vector: light chain:GS-C; (iii) first' vector: light chain:GS-N; (iv) second' vector: heavy chain:GS-C.

[0045] FIG. 16 shows the results of selecting cells expressing Rituximab, a monoclonal antibody (mAb), using a selectable system with the addition of IRES, and then confirming by mean fluorescence intensity (MFI) analysis that they exhibit high Rituximab production in a cell pool.

[0046] FIG. 17 shows the results of further utilization of IRES in a Rituximab expression vector produced by the method according to the present invention, as determined by comparing antibody expression in selected cell lines.

[0047] FIG. 18 illustrates a recombinant vector for the expression of Etanercept using gp41-1, SspGyrB or MjaKlbA as an intein.

[0048] FIG. 19 shows the results of selecting cells expressing Etanercept using an expression system using different types of inteins (gp41-1, SspGyrB, or MjaKlbA), and then confirming by MFI (mean fluorescence intensity) analysis that the cells in the pool exhibit high Etanercept production.

[0049] FIG. 20 shows the results of selecting cells expressing Etanercept, an FC fusion protein, using an expression system using different types of inteins (gp41-1, SspGyrB or MjaKlbA) to determine the percentage of cell lines expressing high levels of Etanercept in the cell pool.

[0050] FIG. 21 shows a recombinant vector for Rituximab expression with an additional terminal inverted repeat (TIR) inserted for the transposon system.

[0051] FIG. 22 shows the results of selecting cells expressing Rituximab, a monoclonal antibody (mAb), using a transposon-based selectable system, and then confirming by MFI (mean fluorescence intensity) analysis that the cells in the pool exhibit high Rituximab production.

[0052] FIG. 23 shows the results of selecting cells expressing Rituximab, a monoclonal antibody (mAb), using a transposon-based selectable system to determine the proportion of high expression cell lines in a cell pool.

[0053] FIG. 24 shows the results of cell viability upon selecting according to the 17 split sites of glutamine synthetase.

[0054] FIG. 25 shows the results of selecting cells expressing Etanercept, an FC fusion protein, using an expression system based on the 17 split sites of glutamine synthetase, and determining the percentage of cell lines expressing high levels of Etanercept in the cell pool.

[0055]

[0056] DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EXAMPLES

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. In general, the nomenclature used herein is well known and in common use in the art.

[0058] Current methods of biopharmaceutical production cell line development are characterized by inefficient selectable systems that result in a very small number of cells among the total cell pool (single digits in approximately 10,000) being selected as cell lines expressing high levels of the target protein, and significant time and cost are required to obtain these few high expression cell lines.

[0059] In the present invention, a method of dramatically increasing the efficiency of selecting cell lines that highly express target proteins by splitting glutamin synthetase (GS), an existing selectable marker for biopharmaceutical production cell line selecting, into two fragments and using them as selectable markers was developed.

[0060] The production of medical proteins or other recombinant proteins using recombinant protein-producing cell lines selected using the method of the present invention was found to exhibit up to 27-fold recombinant protein productivity compared to cells selected using conventional methods using wild-type glutamine synthetase as a selectable marker (see FIGs. 5-7).

[0061] Thus, in one aspect, the present invention relates to a first vector comprising as a selectable marker a first marker nucleic acid encoding an N-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID NOs: 1-17; SEQ ID NOs: 35-51; and SEQ ID NOs: 69-85;

[0062] In the present invention, the N-domain region of the glutamine synthetase gene may be represented by any one of SEQ ID NOs: 1 to 17.

[0063] Examples of N-domain region sequences of glutamine synthetase genes that can be used in the present invention are shown in Table 1.

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] In the present invention, "glutamine synthetase (EC 6.3.1.2)" refers to an enzyme that synthesizes glutamine from glutamte and ammonia (NH3), wherein the glutamine synthetase may be any enzyme having the above activity, regardless of any source thereof, e.g., human, mouse, Chinese hamster, microorganism, bacteria, filamentous fungi (e.g., Thalaromyces).

[0084] In one example, the N-terminal domain of the glutamine synthetase may consist of an amino acid sequence of any one of SEQ ID Nos: 1-17; SEQ ID Nos: 35-51; and SEQ ID Nos: 69-85, respectively, and the C-terminal domain of the glutamine synthetase may consist of an amino acid sequence of any one of SEQ ID Nos: 18-34; SEQ ID Nos: 52-68; and SEQ ID Nos: 86-102, respectively, and if the N-terminal domain and C-terminal domain have the same or corresponding conversion activity as the glutamine synthetase when spliced or combined, the present invention may include a polypeptide having an amino acid sequence of SEQ ID Nos: 1 to 102 in which some sequences are deleted, modified, substituted or added.

[0085] The present invention also may include within the scope thereof a polypeptide having the same or corresponding conversion activity as the glutamine synthetase, as a polypeptide having at least 90%, 92%, 94%, 96%, 98%, or 99%, 99.5%, or 99.8% homology or identity to the amino acid sequence of SEQ ID NOs: 1-102, regardless of the source of the enzyme.

[0086] In this present invention, although it is stated that a "polypeptide (or protein) comprising an amino acid sequence of a particular sequence number" or "a polypeptide (or protein) consisting of an amino acid sequence of a particular sequence number", a polypeptide (or protein) having an amino acid sequence in which some sequences are deleted, modified, substituted or added may also be used as a protein or polypeptide to be modified in the present application, provided that it has the same or corresponding activity as the polypeptide (or protein) having the amino acid sequence of the corresponding sequence number. For example, a polypeptide (or protein) in which some amino acid sequences of any one of amino acid sequences of SEQ ID Nos: 1-102 are deleted, modified or substituted, or some sequences are added to any one of amino acid sequences of SEQ ID Nos: 1-102 can fall under a polypeptide consisting of any one of amino acid sequences of SEQ ID Nos: 1-102 if it has the same or corresponding activity as the polypeptide consisting of any one of amino acid sequences of SEQ ID Nos: 1-102.

[0087] The amino acid sequence of the glutamine synthetase and the sequence of the gene encoding the glutamine synthetase can be readily obtained from databases known in the art, such as the National Center for Biotechnology Information (NCBI) and the DNA Data Bank of Japan (DDBJ), for example, GenBank Accession No. NP_001028216.1.

[0088] In the present invention, a polynucleotide or polypeptide "comprising a particular nucleic acid sequence (sequence) or amino acid sequence" may mean that the polynucleotide or polypeptide consists of or essentially comprises the particular nucleic acid sequence (sequence) or amino acid sequence, and may be construed to include a sequence in which variations (deletions, substitutions, modifications, and / or additions) have been made to the specific nucleic acid sequence (sequence) or amino acid sequence to the extent that the polynucleotide or polypeptide retains its original function and / or intended function (or not exclude the variations). In one example, a polynucleotide or polypeptide "comprising a particular nucleic acid sequence (sequence) or amino acid sequence" means that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleic acid sequence (sequence) or amino acid sequence, or (ii) consists of or essentially comprises a nucleic acid or an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99.5% or more, or 99.9% or more homology to the particular nucleic acid sequence (sequence) or an amino acid sequence, retains its original function and / or intended function.

[0089] As used herein, "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or sequences, which may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0090] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may be used in conjunction with default gap penalties established by the program used. In practical terms, homologous or identical sequences can generally be hybridized, in whole or in part, under moderate or high stringent conditions. It would be obvious that hybridization also includes hybridization with polynucleotides containing normal codons or codon-retractable codons.

[0091] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity may be determined by a known computer algorithm, for example, the "FASTA" program, using default parameters, such as, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al.: 276-277) (version 5.0.0 or later), can be used and determined (comprising the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073)). For example, homology, similarity, or identity can be determined using BLAST from the National Center for Biotechnology Information Database, or ClustalW.

[0092] The homology, similarity, or identity of a polynucleotide or polypeptide may be determined by comparing sequence information using a GAP computer program, such as, Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv.Appl. Math (1981) 2:482. In summary, a GAP program can be defined as the number of similarly arranged symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. The default parameters for the GAP program may include (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14: 6745 as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0093] It is also obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also encompassed by the glutamine synthetase used in the present invention, provided that the amino acid sequence has such homology or identity and exhibits corresponding efficacy to the glutamine synthetase used in the present invention. For example, this case includes having sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant of the present invention at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0094] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

[0095] As used herein, a "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified so that the amino acid sequence of the variant is different from that before modification but the functions or properties thereof retain. Such variants are typically identified by modifying one or more amino acids among the amino acid sequences of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased relative to the polypeptide before modification. In addition, some variants may include variants with one or more parts removed, such as an N-terminal leader sequence or transmembrane domain. Other variants may comprise variants in which portions of the N- and / or C-terminus of the mature protein have been removed. The term "variant" may be used interchangeably with the terms modification, modified polypeptide, modified protein, mutant, mutein, divergent, and the like, and may include, but is not limited to, the terms used in a modified sense.

[0096] In addition, variants can include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of a variant may be conjugated with a signal (or leader) sequence that is involved in the translocation of the protein, either co-translationally or post-translationally.

[0097] The variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.

[0098] In the present application, "-th in the amino acid sequence of SEQ ID NO: -" may be used interchangeably with "-th at the N-terminus of a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: -".

[0099] As used herein, "corresponding to" refers to an amino acid residue at a position in a polypeptide, or an amino acid residue that is similar to, identical to, or homologous to a residue in a polypeptide. Identifying an amino acid at a corresponding position may be to determine a specific amino acid in a sequence referenced to a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0100] In one aspect of the present invention, a glutamic acid synthase consisting of 377 amino acids is split into an N-domain region and a C-domain region for expression, wherein the split site is (1) S73:N74, a split site at the 73rd amino acid from the N-terminus, (2) R106:R107, a split site at the 106th amino acid from the N-terminus, or (3) D143:G144, a split site at the 143rd amino acid from the N-terminus; however, when using a glutamic acid synthase variant in which the amino acids corresponding to the above split sites are conservatively substituted, the same sequence from the N-terminus may be selected as the split site.

[0101] The vectors of the present invention may comprise an intein sequence, or a leucine zipper or coiled coil sequence, for joining or splicing an N-domain fragment of a glutamine synthetase gene and a C-domain fragment of a glutamine synthetase gene expressed in splice, wherein the intein, leucine zipper or coil is selected from the group consisting of gp41-1, SspGyrB, Mja-KlbA, Cth-Ter, NpuDnaE, NrdA-2, SspDnaX, gp41-8, NrdJ-1, IMPDH-1, M86, coiled coil, leucine zipper, SH3, and PRM.

[0102] Intein sequences that can be used in the present invention are shown in Table 2.

[0103]

[0104]

[0105]

[0106] The vectors of the present invention further comprises an internal ribosomal entry site (IRES) between the gene encoding the target protein and a split fragment of the glutamine synthetase gene as a selectable marker.

[0107] In the present invention, the target protein may be a protein selected from the group consisting of Fc-fusion proteins, antibodies, and bispecific antibodies, and the vector of the present invention may comprise a gene encoding the target protein.

[0108] Examples of backbone vectors that can be used for expression vectors of the present invention include, but are not limited to, expression vectors such as pcDNA™, pTargeT™, and UCOE; viral vectors such as lentiviral (LV), pAAV, pRetro, and others; and transposon vectors such as piggyBac (PB), sleeping beauty (SB), and Leap-In™.

[0109] In another aspect, the present invention also relates to a second vector comprising as a selectable marker a second marker nucleic acid encoding a C-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID Nos: 18-34; SEQ ID Nos: 52-68; and SEQ ID Nos: 86-102.

[0110] In the present invention, the C-domain region of the glutamine synthetase gene may be represented by any one of SEQ ID Nos: 18 to 34.

[0111] Examples of C-domain region sequences of glutamine synthetase genes that can be used in the present invention are shown in Table 1.

[0112] The vectors of the present invention may comprise an intein sequence, a leucine zipper or coiled-coil sequence for joining or splicing an N-domain fragment of a glutamine synthetase gene and a C-domain fragment of a glutamine synthetase gene expressed in splice, wherein the intein or zipper may be selected from the group consisting of gp41-1, SspGyrB, Mja-KlbA, Cth-Ter, NpuDnaE, NrdA-2, SspDnaX, gp41-8, NrdJ-1, IMPDH-1, M86, coiled coil, leucine zipper, SH3, and PRM.

[0113] Intein sequences that can be used in the present invention are shown in Table 2.

[0114] The vectors of the present invention can further comprises an internal ribosomal entry site (IRES) between the gene encoding the target protein and a split fragment of the glutamine synthetase gene as a selectable marker.

[0115] In the present invention, a target gene may be, but is not limited to, a gene encoding a first or second monomer of a protein selected from the group consisting of proteins, Fc-fusion proteins, antibodies, and bispecific antibodies, a transgene, a gene encoding mRNA and RNAi.

[0116] The vector of the present invention may comprise the target gene.

[0117] Examples of backbone vectors that can be used for expression vectors of the present invention include, but are not limited to, expression vectors such as pcDNA™, pTargeT™, and UCOE; viral vectors such as lentiviral (LV), pAAV, pRetro, and others; and transposon vectors such as piggyBac (PB), sleeping beauty (SB), and Leap-In™.

[0118] In the present invention, a "vector" may comprise a DNA construct comprising a sequence of polynucleotides encoding a target polypeptide operably linked to an expression control region (or expression control sequence) suitable for expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating termination of transcription and detoxification. After transformation into a suitable host cell, the vector may replicate or function regardless of the host genome, or may be integrated into the genome itself.

[0119] The vectors used in the present invention are not particularly limited and any vector known in the art can be utilized. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state.

[0120] As used herein, the term "transformation" means introducing a vector comprising a polynucleotide encoding a target polypeptide into a host cell such that the polypeptide encoded by the polynucleotide is expressed in the host cell. The transformed polynucleotide may be inserted and located within a chromosome of the host cell, or may be located outside the chromosome, or both, as long as it can be expressed in the host cell. Further, the polynucleotide may comprise DNA and / or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form, provided that it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements necessary to express itself. The expression cassette may typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Alternatively, the polynucleotide may be, but not limited to, one introduced into a host cell in its native form and operably linked to a sequence required for expression in the host cell.

[0121] As used herein, the term "transformation" means introducing a vector comprising a polynucleotide encoding a target polypeptide into a host cell or microorganism such that the polypeptide encoded by the polynucleotide is expressed in the host cell. The transformed polynucleotide may be inserted and located within a chromosome of the host cell, or may be located outside the chromosome, or both, as long as it can be expressed in the host cell. Further, the polynucleotide may comprise DNA and / or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form, provided that it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements necessary to express itself. The expression cassette may typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Alternatively, the polynucleotide may be, but not limited to, one introduced into a host cell in its native form and operably linked to a sequence required for expression in the host cell.

[0122] Further, as used herein, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present invention.

[0123] In another aspect, the present invention relates to a cell for the production of a target protein or target gene, wherein the first vector and the second vector are introduced, wherein the first vector and / or the second vector further comprises a gene encoding a target protein, wherein the first vector and the second vector are introduced such that when combined with each other, the N-domain region of the first vector and the C-domain region of the second vector result in an intact glutamine synthetase, wherein the first vector and / or the second vector comprises an intein, a leucine zipper or a coiled coil.

[0124] In one example, the cells for producing the target protein may be transfected with a first vector comprising a GS N-domain region of SEQ ID NO: 1 and a second vector comprising a GS C-domain region of SEQ ID NO: 18,

[0125] a first vector comprising a GS N-domain region of SEQ ID NO: 2 can be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 19,

[0126] a first vector comprising a GS N-domain region of SEQ ID NO: 3 may be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 20,

[0127] a first vector comprising a GS N-domain region of SEQ ID NO: 4 can be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 21,

[0128] a first vector comprising a GS N-domain region of SEQ ID NO: 5 may be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 22,

[0129] a first vector comprising a GS N-domain region of SEQ ID NO: 6 can be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 23,

[0130] a first vector comprising a GS N-domain region of SEQ ID NO: 7 may be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 24,

[0131] a first vector comprising a GS N-domain region of SEQ ID NO: 8 can be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 25, and

[0132] a first vector comprising a GS N-domain region of SEQ ID NO: 9 can be transfected with a second vector comprising a GS C-domain region of SEQ ID NO: 26.

[0133] Any type of cell can be used for the production of a target protein or target gene in the present invention, for example, prokaryotic, insect, yeast, or animal cells, preferably myeloma cell lines, CHO, HEK293, NS0, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38 cells, human-derived primary cells, immune cells, stem cells, peripheral blood mononuclear cell-derived T-cells, B-cells, NK-cells, macrophages, and the like, and more preferably CHO, HEK293.

[0134] In another aspect, the present invention relates to a method for selection of cells expressing a target protein, comprising the following steps

[0135] (a) creating a library of cells for producing the target protein or target gene;

[0136] (b) culturing the library in a medium comprising a glutamine synthetase inhibitor, thereby selecting the growing recombinant cells; and

[0137] (C) confirming expression of the target protein in the selected recombinant cells, and obtaining cells expressing the target protein.

[0138] In the present invention, the glutamine synthetase inhibitor may be, but is not limited to, methionine sulphoximine (MSX).

[0139] In the present invention, the target protein may be a protein, an Fc-fusion protein, an antibody, or a bispecific antibody, but is not limited thereto.

[0140] In another aspect, the present invention is directed to a method of producing a target protein, comprising the following steps:

[0141] (a) culturing cells for producing the target protein or target gene, and producing the target protein; and

[0142] (b) obtaining the generated target protein.

[0143] In the present invention, the target protein may be a protein, an Fc-fusion protein, an antibody, or a bispecific antibody, but is not limited thereto.

[0144] As used herein, the term "glutamine synthetase (GS)" refers to an enzyme present in mammalian organs or microorganisms that catalyzes the synthesis that glutamic acid and ammonia becomes glutamine in the presence of ATP. Divalent metal ions are required to activate the enzyme, and its activity is inhibited by glycine, alanine, tryptophan, histidine, glucosamine hexaphosphate, cytidine triphosphate, and the like. For the purposes of the present invention, the glutamine synthetase may mean a protein used for the purpose of selecting cells into which a vector comprising a gene encoding a target protein has been introduced in a target protein expression system, or used for the purpose of increasing the expression of a gene encoding a target protein in a polycistronic relationship with a gene encoding a glutamine synthetase, by inhibition against a glutamine synthetase inhibitor. Preferably, it may be, but not limited to, a protein that can be inhibited by a glutamine synthetase inhibitor to increase the expression of a target protein.

[0145] As used in the present invention, the term "glutamine synthetase inhibitor" refers to an external factor capable of inhibiting the enzymatic activity of the glutamine synthetase. Examples of such glutamine synthetase inhibitors include, but are not limited to, glycine, alanine, tryptophan, histidine, glucosamine hexaphosphate, cytidine triphosphate, or methionine sulphoximine (MSX).

[0146] As used in the present invention, the term "susceptibility" generally refers to the property of being receptive to external stimuli, and in terms of enzyme, to the property of having enzyme activity increased or decreased by external factors that regulate the activity of the enzyme. For the purposes of the present invention, susceptibility is used to mean, but is not limited to, the property that the activity of a GS is inhibited by a GS inhibitor that inhibits the activity of the GS.

[0147] As used herein, the term "target protein" refers to a protein to be produced by a host cell, and for purposes of the present invention, includes, but is not limited to, a protein whose expression is amplified by a modified glutamine synthetase. The target protein is not particularly limited to any protein that can be expressed using the vectors of the present invention.

[0148] As used herein, the term "target gene" refers to a gene, transgene, mRNA encoding the target protein, or RNA for RNA interference (RNAi, shRNA, siRNA, etc.).

[0149] As used in the present invention, the term "expression vector" refers to a genetic construct comprising essential regulatory elements operably linked to an insert such that the insert is expressed when present in the cells of an organism. The expression vectors may be prepared and purified using standard recombinant DNA techniques. The type of expression vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic or eukaryotic cells, but it is preferably a vector having a strongly active promoter, while being capable of producing a large amount of the foreign protein in a form similar to its natural state while retaining strong expression. Preferably, the expression vector comprises at least a promoter, an initiation codon, a gene encoding the desired protein, and a termination codon terminator. Other suitable components may include DNA encoding a signal peptide, enhancer sequences, untranslated regions on the 5' and 3' sides of the desired gene, selectable marker regions, or replicable units. Furthermore, the expression vector may be in the form of a mono-cistronic vector comprising a polynucleotide encoding a single protein, a poly-cistronic vector comprising a polynucleotide encoding two or more recombinant proteins, and in particular a bicistronic vector, wherein the target protein, a modified glutamine synthetase, is expressed by a single promoter. However, for the purposes of the present invention, the expression vector may use, but not limited to, preferably, a monocistronic vector comprising an SV40 promoter, or a policistronic vector comprising an IRES sequence, more preferably, an expression cassette in which the gene encoding the desired protein, the IRES and the gene encoding the modified glutamine synthetase are operably linked in sequence; or an expression vector comprising an expression cassette in which the gene encoding a modified glutamine synthetase, an IRES and a gene encoding the desired protein are operably linked sequentially.

[0150] As used in the present invention, "IRES" refers to an internal ribosomal entry site that facilitates the initiation of translation of an mRNA from an internal site (i.e., a site other than the 5' end of the mRNA). An example of a suitable IRES is the IRES of encephalomyocarditis virus (ECMV), as described in the document [Jang and Wimmer Genes &Development 4 1560 (1990) and Jang, Davies, Kaufman and Wimmer J. Vir. 63 1651 (1989)]. Residues 335-848 of EMCV form a suitable IRES; other variants or portions of the ECMV IRES are known in the art and would be suitable for use in the present invention. Suitable portions or variants of the IRES will confer sufficient translation of the second open reading frame (ORF).

[0151] Expression of the target protein in the expression vector of the present invention can be linked to the GS fragment via an IRES, and when linked to an IRES, the target protein must be expressed in order for the GS fragment to be expressed.

[0152] Similar to the present invention, Amgen has developed and patented (KR102439719B1) a technology for the preparation of heterocomplex proteins using a vector containing GS fragments, and in the case of the above patent, the improvement on the selection of cell lines expressing high levels of target gene was about 2 times higher compared to using wild-type GS, while the present invention was found to have an improvement of about 20 times (FIGs. 5-7).

[0153] Recombinant vectors comprising a split GS as a selectable marker according to the present invention can improve the selection efficiency of cell lines containing a recombinant vector, and such improved selectable markers can be used as selectable markers for vectors comprising, for example, secreted protein expression genes, endogenous genes, transgenes, RNAs, and the like. Suitable examples include selecting vectors containing Fc-fusion proteins, antibodies, biosimilars, bispecific antibodies, cytokines, growth factors, reporter genes, T-cell receptors, chimeric antigen receptors, messenger RNA (mRNA), RNA interference, and the like.

[0154]

[0155] [Examples]

[0156] The present invention is described in more detail with reference to the following examples. These examples are intended solely to illustrate the present invention, and it would be apparent to one of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

[0157]

[0158] Example 1: Selecting a split site for a split expression system of glutamine synthetase

[0159] To select a split site for split expression of glutamine synthetase, the root mean square fluctuation, tertiary structural position of the split site, and evolutionary conservation of the amino acid sequences were analyzed.

[0160] For structural analysis, following three programs were used:

[0161] 1) RMSF calculation (CABS-flex),

[0162] 2) Determination of tertiary structural position (pymol, https: / pymol.org / 2 / ),

[0163] 3) Evolutionary conservation (Evolutionary Trace,)

[0164] As a result, the following three sites were selected as split sites in glutamine synthetase, which consists of 373 amino acids:

[0165] (1) NC1 (N73 / C74): S73:N74

[0166] (2) NC2 (n106 / c107): R106:R107

[0167] (3) NC3 (n143 / c144): D143:G144

[0168] The three selected split sites on the three-dimensional structure of glutamine synthetase are shown in FIG. 1.

[0169]

[0170] Example 2: Preparation of a vector for expressing a recombinant protein comprising a split fragment of glutamine synthetase

[0171] The vector for expressing a recombinant protein was produced by removing the existing selectable markers from the pcDNA vector (pcDNA™3.1 / Hygro(+), ThermoFisher scientific) by polymerase chain reaction and KLD reaction, and introducing the IRES and glutamine synthetase selectable markers using gibson assembly. Then, the target gene to be expressed between the CMV promoter and IRES was introduced using gibson assembly to create an expression vector.

[0172] A recombinant protein expression vector comprising a glutamine synthetase 2 split fragment produced by the method described above is shown in FIG. 2, wherein GSN represents the N-domain split fragment of glutamine synthetase and GSC represents the C-domain split fragment of glutamine synthetase.

[0173]

[0174] Example 3: Determination of cell growth and cell viability by 3 different split positions of GS

[0175] The cell line used in this example is a GS knockout cell line, wherein the GS gene was knocked out in CHO-K1 (ATCC CCL-61) (Noh, S.M., Shin, S. & Lee, G.M., Sci Rep 8:5361, 2018).

[0176] Cell lines were produced by transfecting either vector 1 or vector 2 for expression of the target protein according to the three GS split sites constructed in Example 2, or transfecting vector 1 and vector 2, and each was cultured in glutamine-deficient medium, and cell growth and cell viability were determined.

[0177] As shown in FIG. 3, in cell lines transfected with either the first vector containing the N-domain split fragment of glutamine synthetase or GSC, or the second vector containing the C-domain split fragment of glutamine synthetase, cells did not grow, and only cells transfected with both the first and second vectors grew normally.

[0178] Similarly, as shown in FIG. 4, only cells transfected with both the first vector containing the N-domain split fragment of glutamine synthetase or GSC, and the second vector containing the C-domain split fragment of glutamine synthetase, exhibited normal viability.

[0179]

[0180] Example 4: Determination of a recombinant protein expression in selected cells

[0181] The recombinant protein expression vector was prepared as in Example 2 using various types of target proteins, and the amount of recombinant protein expression in the cell line into which the recombinant protein expression vector was introduced was checked.

[0182]

[0183] (1) mCherry protein expression

[0184] The mCherry protein encoding gene (SEQ ID NO: 125) was introduced using gibson assembly at the GOI position, as shown in FIG. 2. The vector was introduced into the CHO-K1 GS knockout cell line using the transfection reagent. SFM4Transfx-293 liquid medium (hyclone) and Opti-MEM (ThermoFisher scientific) medium were used for transfection.

[0185] Cell lines transfected with these vectors were floating cultured in PowerCHO™ 2 Serum-free Medium (Lonza) for 2 days at 37°C, 110 rpm, and then cultured and selected in glutamine-free medium for selection. Once sorted, the cells were measured for fluorescent protein expression using a flow cytometer.

[0186] The cells were incubated with different concentrations (0 μM, 25 μM, 50 μM, and 100 μM) of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase.

[0187] As a result, NC1, NC2, and NC3 cells exhibited higher mCherry expression than cells selected using wild-type glutamine synthetase as a selectable marker, as shown in FIG. 5.

[0188]

[0189] (2) EGFP protein expression

[0190] The EGFP protein encoding gene (SEQ ID NO: 126) was introduced using gibson assembly at the GOI position, as shown in FIG. 2. The vector was introduced into the CHO-K1 GS knockout cell line using a transfection reagent. The cell lines transfected with these vectors were floating cultured in PowerCHO™ 2 Serum-free Medium (Lonza) for 2 days at 37°C, 110 rpm, and then cultured and selected in glutamine-free medium for selection. Once sorted, the cells were measured for fluorescent protein expression using a flow cytometer.

[0191] The cells were incubated with different concentrations (0 μM, and 25 μM) of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase.

[0192] As a result, NC1, NC2, and NC3 cells exhibited higher EGFP expression than cells selected using wild-type glutamine synthetase as a selectable marker, as shown in FIG. 6.

[0193]

[0194] (3) Expression of FC fusion protein, Etanercept

[0195] The Etanercept protein encoding gene (SEQ ID NO: 127) was introduced at the GOI position, as shown in FIG. 7. The vector was introduced into the CHO-K1 GS knockout cell line using a transfection reagent. The cell lines transfected with these vectors were floating cultured in PowerCHO™ 2 Serum-free Medium (Lonza) medium at 37°C, 110 rpm for 2 days and then cultured and selected in glutamine-free medium. Once sorted, the cells were measured for protein expression in a flow cytometer using fluorescently conjugated antibodies. The amount of Etanercept produced during the culture period was also quantified using an enzyme-linked immunosorbent assay (ELISA).

[0196] The cells were incubated with different concentrations (0 μM, and 25 μM) of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase.

[0197] As a result, NC1, NC2, and NC3 cells exhibited higher etanercept expression than cells selected using wild-type glutamine synthetase as a selectable marker, as shown in FIG. 7.

[0198] Furthermore, after selecting the cells expressing the FC fusion protein, Etanercept, the percentage of cell lines expressing high levels of Etanercept in the cell pool was determined, and it was found that the cell lines in the system using the split GS of the present invention exhibited higher Etanercept production compared to the conventional method (WT), as shown in FIG. 8.

[0199] As shown in FIG. 9, the proportion of high expression cell lines was increased in the system using the split GS of the present invention compared to the conventional method (WT).

[0200] In addition, the production of etanercept was determined over time in batch cultures for selected cell lines.

[0201] For batch culture, 125 mL ERL flasks were incubated at 37°C and 110 rpm in a floating incubator. During the incubation period, 1 mL aliquots were taken at the measurement time and the supernatant was obtained by centrifugation. The quantification of antibodies produced was determined by enzyme-linked immunosorbent assay (ELISA) of the supernatant.

[0202] As a result, it was found that the cell lines using NC2 and NC3 produced large amounts of etanercept (ETN), as shown in FIG. 10.

[0203]

[0204] (4) Monoclonal antibody (mAb) Rituximab expression

[0205] The vector for expressing Rituximab used a vector prepared by the method of Noh et al. (Noh, S.M., Shin, S. & Lee, G.M., Sci Rep 8:5361, 2018) as a backbone vector.

[0206] The Rituximab protein encoding gene (SEQ ID NO: 128 (heavy chain sequence) and SEQ ID NO: 129 (light chain sequence)) was introduced at the GOI position, as shown in FIG. 13. The vector was introduced into the CHO-K1 GS knockout cell line using a transfection reagent. The cell lines transfected with these vectors were floating cultured at 37°C, 110 rpm for 2 days and then cultured and selected in glutamine-free medium. Once sorted, the cells were measured for protein expression in a flow cytometer using fluorescently conjugated antibodies.

[0207] The first and second vectors for Rituximab expression are shown in FIG. 11.

[0208] FIG. 11 illustrates a recombinant vector for the expression of Rituximab made in accordance with the present invention.

[0209] After selecting the cells expressing Rituximab, the Rituximab production of the cell lines expressing Rituximab in the cell pool was determined, and it was found that the cell lines in the system using the split GS of the present invention exhibited higher Rituximab production compared to the conventional method (WT), as shown in FIG. 12.

[0210] Furthermore, after selecting cells expressing the monoclonal antibody (mAb) Rituximab, the proportion of cell lines expressing high levels of Rituximab in the cell pool was determined, and it was found that the proportion of high expression cell line NC3 was increased in the system using the split GS of the present invention compared to the conventional method (WT), as shown in FIG. 13.

[0211] In addition, Rituximab production was determined over time in batch cultures for selected cell lines.

[0212] For batch culture, 125 mL ERL flasks were incubated at 37°C and 110 rpm in a floating incubator. During the incubation period, 1 mL aliquots were taken at the measurement time and the supernatant was obtained by centrifugation. The quantification of antibodies produced was determined by enzyme-linked immunosorbent assay (ELISA) of the supernatant.

[0213] As a result, it was confirmed that the cell lines using NC3 produced large amounts of Rituximab, as shown in FIG. 14.

[0214] In addition, an IRES (internal ribosomal entry site) was added to the recombinant vector for Rituximab expression to increase the proportion of cells expressing the target protein. In the conventional method of using wild-type GS as a selectable marker, it is difficult to regulate expression using IRES because the selectable marker is a single GS, but the present invention uses a fragment of GS, so the heavy chain and light chain of the antibody can be introduced into the first vector and the second vector, respectively, and the IRES can be added. The gene encoding the target protein and the GS fragment are linked through IRES, and when linked by IRES, the target protein must be expressed in order for the GS fragment to be expressed. Therefore, the use of IRES can increase the selection rate of expression cells.

[0215] FIG. 15 shows the structure of a recombinant vector for Rituximab expression with IRES added thereto. Four vectors were prepared by adjusting the positions of the heavy and light chains of Rituximab as shown below.

[0216] (i) First vector : heavy chain:GS-N

[0217] (ii) Second Vector : light Chain:GS-C

[0218] (iii) First' vector : light chain:GS-N

[0219] (iv) Second' vector : heavy chain:GS-C

[0220] The cell lines transfected with the above vectors were cultured, and selected, and the cells expressing Rituximab, a monoclonal antibody (mAb), were selected, and the high Rituximab production in the cell pool was confirmed by MFI (mean fluorescence intensity) analysis using fluorescent labeling, the results of which are shown in FIG. 16, confirming the high Rituximab production in the cell lines selected by the method of the present invention. Furthermore, the percentage of cells expressing Rituximab in the cell pool was determined, and the results are shown in FIG. 17.

[0221]

[0222] Example 5: Method for selection using a vector using SspGyrB and MjaKlbA intein

[0223] In addition to the gp41-1 intein used in the vector constructed in Example 2, to determine whether the split-expressed glutamine synthetase would function properly using other intein, a recombinant vector for Etanercept expression was constructed using the SspGyrB intein (SEQ ID Nos: 105 and 106) and the MjaKlbA intein (SEQ ID Nos: 107 and 108) (FIG. 18).

[0224] Cell lines transformed with the above recombinant vector were selected in the same manner as in Example 4(3), and cells expressing Etanercept were selected through an expression system using several types of inteins (gp41-1, SspGyrB, or MjaKlbA), and the production of Etanercept in the cell pool was confirmed by mean fluorescence intensity (MFI) using fluorescent labeling. As a result, as shown in FIG. 19, the production of Etanercept was confirmed in all three inteins, and the production was higher when MjaKlbA was used.

[0225] Also, as a result of confirming the proportion of cell lines expressing high levels of Etanercept in the selected cell pool, it was found that the proportion of high expression cell lines was high for all three inteins.

[0226]

[0227] Example 6: Cell line selection by chromosomal insertion using transposons

[0228] The efficiency of the selectable system of the present invention was confirmed when the gene encoding the recombinant protein was inserted into the chromosome of a cell line.

[0229] The PiggyBac transposon system (NovoPro Bioscience) was used for chromosomal insertion of the recombinant vector.

[0230] In the Rituximab recombinant vector disclosed in Example 4(4) and FIG. 11, a recombinant vector for Rituximab expression was constructed by introducing additional terminal inverted repeats (TIRs) at both ends of the existing Rituximab expression vector using the gibson assembly method (FIG. 21).

[0231] In addition, together with the recombinant vector for expression, the vector of FIG. 2 was used as a backbone vector, thereby co-transfecting a PiggyBac transposase vector in which the portion from GOI (gene of interest) to int (the beginning of polyA) was removed and a transposase gene was introduced.

[0232] After selecting the cells expressing Rituximab in the same way as in Example 4(4), the amount of Rituximab production of the cell lines expressing Rituximab in the cell pool was checked, and it was found that the cell lines in the system using the split GS of the present invention exhibited higher Rituximab production compared to the conventional method (WT), even when transposons were used, as shown in FIG. 22.

[0233] Furthermore, after selecting cells expressing the monoclonal antibody (mAb) Rituximab, the proportion of cell lines expressing high levels of Rituximab in the cell pool was determined, and as shown in FIG. 23, it was found that the proportion of high expression cell lines was increased in the system using the split GS of the present invention compared to the conventional method (WT), even when transposons were used.

[0234]

[0235] Example 7: Confirmation of viability of cells transformed with a vector containing a fragment of the altered split site of glutamine synthetase

[0236] 17 split sites were selected by adding 14 split sites around the selected 3 split sites other than the 3 glutamine synthetase split sites selected in Example 1, and a first vector comprising a nucleic acid fragment encoding an N domain protein of SEQ ID Nos: 1 to 17 and a second vector comprising a nucleic acid fragment encoding a C domain protein of SEQ ID Nos: 18 to 34 were constructed in the same manner as in Example 2.

[0237]

[0238] The cell line used in this example is a GS knockout cell line, which is CHO-K1 (ATCC CCL-61) cells with the GS gene knocked out.

[0239] In the same manner as Example 2, cell lines transfected with either vector 1 or vector 2 for expression of the target protein depending on the 17 GS split sites constructed, or transfected with vector 1 and vector 2 were produced, and cultured in glutamine-deficient medium, respectively, and cell growth and cell viability were determined.

[0240] Cell viability was measured on day 11, and as shown in FIG. 24, GS KO host (Negative control) cells did not survive, while all cells transfected with the vector containing the 17 split sites survived.

[0241]

[0242] Example 8: Determination of a recombinant protein expression in cells transfected with a vector containing a fragment of the altered split site of glutamine synthetase

[0243] The Etanercept protein encoding gene was introduced into the 17 selected first and second vectors in Example 7.

[0244] The Etanercept protein encoding gene (SEQ ID NO: 127) was introduced at the GOI position, as shown in FIG. 7. The vector was introduced into the CHO-K1 GS knockout cell line using a transfection reagent. The cell lines transfected with these vectors were floating cultured in PowerCHO™ 2 Serum-free Medium (Lonza) medium at 37°C, 110 rpm for 2 days and then cultured and selected in glutamine-free medium. Once sorted, the cells were measured for protein expression in a flow cytometer using fluorescently conjugated antibodies. The amount of Etanercept produced during the culture period was also quantified using an enzyme-linked immunosorbent assay (ELISA).

[0245] The cells were incubated with different concentrations (0 μM, and 25 μM) of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase.

[0246] Cells were analyzed by flow cytometry to determine the percentage of Etanercept-expressing cells. Since GS KO hosts did not survive in the selection, cells cultured in medium with glutamine were used as a negative control to distinguish Etanercept-expressing cells in flow cytometry.

[0247] As a result, as shown in FIG. 25, the percentage of cell lines expressing high levels of Etanercept was increased in the system using 17 split GS compared to the control.

[0248]

[0249] According to the present invention, the efficiency of selecting cell lines for biopharmaceutical production can be dramatically improved, and since it is an improvement of the existing selectable marker GS (glutamine synthetase), it is free from stability issues.

[0250]

[0251] While the foregoing has described in detail certain aspects of the present invention, it would be apparent to one of ordinary skill in the art that these specific descriptions are merely preferred examples and are not intended to limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0252]

[0253] Attached in an electonic file.

Claims

1.A first vector comprising, as a selectable marker, a first marker nucleic acid encoding an N-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID NOs: 1-17; SEQ ID NOs: 35-51; and SEQ ID NOs: 69-85.2.The first vector for producing a recombinant protein according to claim 1, wherein the N-domain region of the glutamine synthetase gene is represented by any one of SEQ ID Nos: 1-17.3.The first vector according to claim 1, further comprising an intein, a leucine zipper, or a coiled coil.4.The first vector according to claim 3, wherein the intein, leucine zipper, or coiled coil is selected from the group consisting of gp41-1, SspGyrB, Mja-KlbA, Cth-Ter, NpuDnaE, NrdA-2, SspDnaX, gp41-8, NrdJ-1, IMPDH-1, M86, coiled coil, leucine zipper, SH3 and PRM.5.The first vector according to claim 1, further comprising an internal ribosomal entry site (IRES).6.The first vector according to claim 1, comprising a target gene selected from the group consisting of a gene, transgene, mRNA encoding a first monomer of a protein selected from the group consisting of a protein, an Fc-fusion protein, an antibody, and a bivalent antibody, and RNAi thereof.7.A second vector comprising, as a selectable marker, a second marker nucleic acid encoding a C-domain region of a glutamine synthetase gene having an amino acid sequence represented by any one of SEQ ID NOs: 18-34; SEQ ID NOs: 52-68; and SEQ ID NOs: 86-102.8.The second vector according to claim 7, wherein the C-domain region of the glutamine synthetase gene is represented by any one of SEQ ID Nos: 18-34.9.The second vector according to claim 7, further comprising an intein, a leucine zipper, or a coiled coil.10.The second vector according to claim 9, wherein the intein, leucine zipper, or coiled coil is selected from the group consisting of gp41-1, SspGyrB, Mja-KlbA, Cth-Ter, NpuDnaE, NrdA-2, SspDnaX, gp41-8, NrdJ-1, IMPDH-1, M86, coiled coil, leucine zipper, SH3 and PRM.11.The second vector according to claim 7, further comprising an internal ribosomal entry site (IRES).12.The second vector according to claim 7, comprising a target gene selected from the group consisting of a gene, transgene, mRNA encoding a second monomer of a protein selected from the group consisting of a protein, an Fc-fusion protein, an antibody, and a bivalent antibody, and RNAi thereof.13.A cell for producing a target protein or target gene, wherein the first vector according to claim 1 and the second vector according to claim 7 are introduced, wherein the first vector and / or second vector further comprises a gene encoding a target protein,wherein the first vector and the second vector are introduced such that when combined with each other, the N-domain site of the first vector and the C-domain site of the second vector result in an intact glutamine synthetase, wherein the first vector and / or the second vector comprises an intein, a leucine zipper or a coiled coil.14.The cell for producing a target protein or target gene according to claim 13, wherein the cell is a prokaryotic, insect cell, yeast, or animal cell.15.The cell for producing a target protein or target gene according to claim 13, wherein the cell is selected from the group consisting of a myeloma cell line, CHO, NS0, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38 cells.16.A method for selection of cells expressing a target protein or target gene, comprising the following steps;(a) creating a library of cells for producing the target protein or target gene according to claim 13;(b) culturing the library in a medium comprising a glutamine synthetase inhibitor, and selecting the growing recombinant cells; and(C) confirming expression of the target protein or target gene in the selected recombinant cells, and obtaining cells expressing the target protein or target gene.17.The method according to claim 16, wherein the glutamine synthetase inhibitor is methionine sulphoximine (MSX).18.The method according to claim 16, wherein the target gene is selected from the group consisting of a gene, transgene, mRNA encoding a second monomer of a protein selected from the group consisting of a protein, a Fc-fusion protein, an antibody, and a bispecific antibody, and RNAi thereof.19.A method of producing a target protein or target gene, comprising the following steps:(A) culturing cells for producing the target protein according to claim 13, and producing the target protein or target gene; and(b) obtaining the generated target protein or target gene.20.The method according to claim 19, wherein the target gene is selected from the group consisting of a gene, transgene, mRNA encoding a second monomer of a protein selected from the group consisting of a protein, a Fc-fusion protein, an antibody, and a bispecific antibody, and RNAi thereof.

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