Factor capable of affecting production of substance using cho cell

Abstract

The present invention provides a mammalian cell capable of producing a target protein, the mammalian cell being characterized in that the expression of a protein associated with endoplasmic reticulum stress and / or a protein associated with transcription is regulated in the cell.

Description

Factors affecting substance production using CHO cells

[0001] The present invention relates to mammalian cells with improved ability to produce a target protein.

[0002] In the field of life science, the technology for producing useful proteins on an industrial scale by mass-cultivating cells capable of producing the desired useful proteins has become extremely important. Examples of useful proteins include physiologically active substances such as insulin and erythropoietin, and protein pharmaceuticals such as antibodies.

[0003] When the above-mentioned physiologically active substances are used as protein drugs, they can be effective even when administered in relatively small doses to patients, but in the case of antibody drugs, single doses of several hundred milligrams are required. Therefore, there remains a high demand for the development of technologies for the efficient production of antibody drugs.

[0004] Various cell lines have been used in the production of antibody pharmaceuticals, but the most frequently used today are CHO (Chinese hamster ovary) cells. CHO cells have also been used for biopharmaceuticals other than antibodies, but are said to be the most compatible production cells for antibody pharmaceuticals in terms of high productivity, quality of glycosylation structure, and safety (Non-Patent Document 1). Several techniques for improving the antibody production ability of CHO cells have been reported (Non-Patent Documents 2 and 3), and many modified strains have been created to date, but there is still a high demand for the creation of more preferable modified strains.

[0005] Kunert R et al., Appl Microbiol Biotechnol. 2016 Apr;100(8):3451-61.Porter AJ et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1446-54.Feary M et al., Biotechnol Prog. 2021 Jul;37(4):e3137.

[0006] Generally, cell lines capable of producing a target protein are constructed using host cells stably transfected with a target gene. However, because the plasmid containing the target gene is randomly inserted into the chromosome of the host cell after transfection, the expression level of the target protein varies greatly among producing clones. Therefore, the process of selecting clones with high expression levels requires a significant amount of time and effort. Various factors are thought to determine the expression level of a target protein in cells, and it is assumed that this is influenced by the efficiency of each process, such as the number of copies of the gene inserted into the chromosome, the transcription process into mRNA, the translation process into polypeptides, protein modification / folding, and even extracellular secretion. However, the characteristics of cells that produce high levels of protein are not yet fully understood. Therefore, the inventors focused on low-producing clones in a cell pool after transfection with a target gene-containing plasmid and compared the results of proteomic analysis of these clones with those of high-producing clones to elucidate the characteristics of cells capable of high protein production at the genetic and cellular levels. As a result, they aimed to develop a method for easily selecting or producing cells capable of efficiently producing a target protein.

[0007] As a result of intensive research into the above-mentioned problems, the present inventors found that the expression levels of specific proteins related to endoplasmic reticulum stress (PERK, Erp44, Txndc12, Pdia5, Pedx4, Edem3, Erlec1, and P3h4) were increased in low-producing clones compared to high-producing clones. They also found that the expression level of a specific transcription-related protein (TAF8) was reduced in low-producing clones. Based on these findings, the present inventors further pursued research and completed the present invention. Specifically, the present invention is as follows:

[0008] [1] A mammalian cell producing a target protein, wherein expression of a protein associated with endoplasmic reticulum stress is regulated in the cell. [2] The cell according to [1], wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. [3] The cell according to [1] or [2], wherein the target protein is an antibody. [4] The cell according to any one of [1] to [3], wherein expression of the protein associated with endoplasmic reticulum stress is inhibited. [5] The cell according to any one of [1] to [4], wherein the protein associated with endoplasmic reticulum stress is an endoplasmic reticulum stress sensor protein. [6] The cell according to [5], wherein the endoplasmic reticulum stress sensor protein is PERK. [7] The cell according to any one of [1] to [4], wherein the protein associated with endoplasmic reticulum stress is a protein that promotes disulfide bond formation in the target protein. [8] The cell according to [7], wherein the protein that promotes disulfide bond formation in the target protein is at least one selected from the group consisting of Erp44, Txndc12, and Pdia5. [9] The cell according to any one of [1] to [4], wherein the protein associated with endoplasmic reticulum stress is a protein having the function of reducing oxidative stress.

[10] The cell according to [9], wherein the protein having the function of reducing oxidative stress is Prdx4.

[11] The cell according to any one of [1] to [4], wherein the protein associated with endoplasmic reticulum stress is a protein having the function of degrading denatured proteins.

[12] The cell according to

[11] , wherein the protein having the function of degrading denatured proteins is at least one selected from the group consisting of Edem3 and Erlec1.

[13] The cell according to any one of [1] to [4], wherein the protein associated with endoplasmic reticulum stress is a protein associated with endoplasmic reticulum phagy.

[14] The cell according to

[13] , wherein the protein associated with endoplasmic reticulum phagy is P3h4.

[15] A mammalian cell producing a target protein, wherein expression of a transcription-related protein is regulated in the cell.

[16] The cell according to

[15] , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[17] The cell according to

[15] or

[16] , wherein the target protein is an antibody.

[18] The cell according to any of

[15] to

[17] , wherein expression of the transcription-related protein is enhanced.

[19] The cell according to any of

[15] to

[18] , wherein the transcription-related protein is TAF8.

[20] A method for producing a target protein, comprising a step of culturing the cell according to any of [1] to

[19] .

[21] A target protein produced by the cell according to any of [1] to

[19] .

[0009] According to the present invention, cells capable of efficiently producing a target protein can be easily selected or produced.

[0010] FIG. 1 shows proteins whose expression levels differed between high-producing (HP) clones and low-producing (LP) clones. Compared to standard conditions, red indicates upregulation of proteins, and blue indicates downregulation of proteins. The left half of the figure shows high-producing clones (HP), and the right half shows low-producing clones (LP). FIG. 2 shows a vector map of an shRNA expression vector targeting the TAF8 gene, prepared in Example 2. FIG. 3 shows changes in antibody production levels in CHO cells upon knockdown of each target gene (Day 1). FIG. 4 shows changes in antibody production levels in CHO cells upon knockdown of each target gene (Day 2). FIG. 5 shows changes in antibody production levels in CHO cells upon knockdown of each target gene (Day 3). FIG. 6 shows changes in antibody production levels in CHO cells upon knockdown of each target gene (Day 4).

[0011] The present invention will be described in detail below.

[0012] 1. Mammalian cell 1 producing a target protein The present invention provides a mammalian cell producing a target protein, characterized in that expression of a protein associated with endoplasmic reticulum stress is regulated in the cell (hereinafter, sometimes referred to as "mammalian cell 1 of the present invention").

[0013] The mammalian cell 1 of the present invention is characterized in that the expression of a protein associated with endoplasmic reticulum stress is regulated. As used herein, "regulated protein expression" refers to "increased protein expression" or "decreased protein expression." In one aspect, "regulated protein expression" refers to "at least one protein associated with endoplasmic reticulum stress being increased by 1.1-fold or more (e.g., 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 3.0-fold or more) during the production of a protein of interest compared to its expression in the cell before the regulation" or "at least one protein associated with endoplasmic reticulum stress being decreased by 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more) during the production of a protein of interest compared to its expression in the cell before the regulation." Furthermore, "protein expression is regulated" may be considered as "protein activity is regulated."

[0014] In a preferred embodiment of the present invention, the expression of a protein associated with endoplasmic reticulum stress is suppressed in the mammalian cell 1 of the present invention. In this case, the expression level of at least one of the proteins associated with endoplasmic reticulum stress can be reduced by 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more) compared to the expression level of the protein in the cell before suppression.

[0015] Proteins associated with endoplasmic reticulum stress may include, but are not limited to, proteins with the following functions: (1) endoplasmic reticulum stress sensor proteins; (2) proteins with the function of promoting disulfide bond formation in a target protein; (3) proteins with the function of reducing oxidative stress in cells; (4) proteins with the function of degrading denatured proteins; and (5) proteins associated with endoplasmic reticulum phagy.

[0016] (1) A specific example of an endoplasmic reticulum stress sensor protein is protein kinase RNA-like ER kinase (PERK, sometimes referred to as "Eif2ak3"). PERK is a type I membrane protein with kinase activity in its cytoplasmic domain and is activated by autophosphorylation through dimerization. Activated PERK phosphorylates the translation initiation factor subunit eIF2α. It has been reported that phosphorylation of eIF2α results in suppression of cap-dependent translation, resulting in a decrease in the translation rate and a decrease in the synthesis of new proteins inserted into the endoplasmic reticulum (B'chir W et al., Nucleic Acids Res. 2013 Sep;41(16):7683-99.).

[0017] Specific examples of (2) proteins that promote disulfide bond formation in target proteins (i.e., protein disulfide isomerases) include ER protein 44 (Erp44), Thioredoxin domain containing 12 (Txndc12), and Protein disulfide isomerase family A member 5 (Pdia5). Erp44, an endoplasmic reticulum protein, is known to regulate the oligomerization and secretion of various proteins and to be involved in the quality control of secretory proteins (Hisatsune C et al., Molecular cell, 58(6), 1015-1027). It has been reported that Txndc12 expression is increased under ER stress due to unfolded protein response (UPR) (Patricia Inacio et al., EMBO Rep. 2015 Aug; 16(8): 955-964.), and Pdia5 is a protein required for ATF6α response to ER stress (Arisa Higa et al., Mol Cell Biol. 2014 May; 34(10): 1839-1849.).

[0018] (3) A specific example of a protein that functions to reduce intracellular oxidative stress is peroxiredoxin-4 (Prdx4). Prdx4 is an antioxidant enzyme belonging to the peroxiredoxin family. It reduces hydrogen peroxide and alkyl hydroperoxides using the reducing power of glutathione and other enzymes. It may be used to remove peroxides that accompany the formation of disulfide bonds during antibody formation. Meanwhile, Prdx4, like PERK, is known to contribute to the rearrangement of disulfide bonds in ATF6α, an endoplasmic reticulum stress sensor, under stress conditions, thereby promoting the export of ATF6α from the ER and activating target genes (Arisa Higa et al., (supra)). Prdx4 has also been reported to be involved in oxidative stress, ER stress, and protein folding (Evan A Elko et al., J Biol Chem. 2021 Jan-Jun; 296: 100665 and Zito E et al., Mol Cell. 2010 Dec 10; 40(5): 787-797.).

[0019] (4) Specific examples of proteins capable of degrading denatured proteins include ER degradation enhancing alpha-mannosidase like protein 3 (Edem3) and ER lectin 1 (Erlec1). Edem3 has alpha 1,2-mannosidase activity and is said to be involved in the trimming of mannose attached to proteins in the endoplasmic reticulum and in the ER-associated degradation of structurally abnormal glycoproteins (Hirao K et al., J. Biol. Chem. 2006 Apr 7;281(14):9650-8). Erlec1 is a lectin that belongs to the endoplasmic reticulum-associated degradation surveillance system and is involved in the degradation of misfolded glycoproteins in the endoplasmic reticulum (Yoshida Y et al., Biochim Biophys Acta. 2010 Feb;1800(2):172-80).

[0020] (5) A specific example of a protein related to endoplasmic reticulum phagy is Prolyl 3-hydroxylase family member 4 (P3h4). P3h4 belongs to the Leprecan (leucine proline-enriched proteoglycans) family localized in the endoplasmic reticulum and is known to form a complex with Prolyl 3-hydroxylase to control the hydroxylation of lysine in collagen (Ochs RL et al., Mol Biol Cell. 1996 Jul;7(7):1015-24.; Gruenwald K et al., J Bone Miner Res. 2014 Mar;29(3):666-75.). It is also known that P3h4 is involved in selective ER-phagy (Ishii S et al., Mol Biol Cell. 2023 Apr 1;34(4):ar29.).

[0021] The means for regulating the expression of a protein associated with endoplasmic reticulum stress in mammalian cells is not particularly limited as long as it can increase or decrease the expression level of the protein to be regulated compared to the expression level of the protein in the mammalian cells before regulation, and any known method can be used. For example, a method for increasing the expression level of a protein associated with endoplasmic reticulum stress in mammalian cells includes, but is not limited to, transfecting an expression vector containing a nucleic acid encoding the protein into mammalian cells and overexpressing the protein in the mammalian cells. Furthermore, a method for decreasing the expression level of a protein associated with endoplasmic reticulum stress in mammalian cells includes, but is not limited to, a method for suppressing gene expression of the protein associated with endoplasmic reticulum stress by gene knockdown. Gene knockdown methods are known per se, and include, but are not limited to, antisense methods and RNAi methods using siRNA, shRNA, microRNA, etc. The knockdown efficiency of the contemplated protein is not particularly limited, and may be, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%. Methods for assessing whether the expression level of a protein has been regulated are also known per se, and include, but are not limited to, Western blotting, ELISA, and the like.

[0022] In a preferred embodiment, the mammalian cell 1 of the present invention is characterized in that the expression level of at least one selected from the group consisting of PERK, Erp44, Txndc12, Pdia5, Prdx4, Edem3, Erlec1, and P3h4 is suppressed. More preferably, the mammalian cell 1 of the present invention is characterized in that the expression level of at least one selected from the group consisting of Erp44, Edem3, Erlec1, and P3h4 is suppressed, and even more preferably, the expression level of at least one selected from the group consisting of Erp44, Edem3, and Erlec1 is suppressed.

[0023] In the mammalian cell 1 of the present invention, the mammalian cell refers to a cell derived from a mammal. Mammalian species from which the mammalian cell of the present invention is derived include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. Preferably, the mammal is a hamster or a human.

[0024] In one embodiment of the mammalian cell 1 of the present invention, the mammalian cell may be, but is not limited to, Chinese hamster ovary (CHO) cells, monkey cells COS-7, human embryonic kidney cells (e.g., HEK293 cells), etc. In a preferred embodiment, the mammalian cell may be a CHO cell.

[0025] In the mammalian cell 1 of the present invention, the target protein may be an endogenous protein or an exogenous protein. In a preferred embodiment, the target protein may be an exogenous protein. The exogenous target protein is not particularly limited, but is preferably a high-value-added protein. Examples of such proteins include antibodies (natural antibodies, antibody fragments, minibodies, chimeric antibodies, humanized antibodies, human antibodies, etc.), physiologically active proteins (granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, interferons, interleukins such as IL-1 and IL-6, t-PA, urokinase, serum albumin, blood coagulation factors, etc.), and cytokines. In a particularly preferred embodiment, the target protein is an exogenous protein and may be an antibody or an antigen-binding fragment thereof.

[0026] Methods for expressing exogenous proteins in mammalian cells are well known to those skilled in the art. For example, an expression vector containing a nucleic acid encoding the protein of interest can be transfected into mammalian cells, and the resulting mammalian cells can be cultured to produce the protein of interest.

[0027] The culture conditions for producing a target protein in mammalian cells vary depending on the type of cells used, so suitable conditions can be determined as appropriate. For example, in the case of CHO cells, gas-phase CO ２ The culture can be carried out in an atmosphere with a concentration of 0% to 40%, preferably 2% to 10%, at a pH of 6.5 to 7.2, preferably 6.7 to 7.0, more preferably 6.8 to 6.9, at 30°C to 39°C, preferably about 37°C, for 1 to 50 days, more preferably 1 to 14 days.

[0028] The medium for culturing mammalian cells is not particularly limited as long as it allows the mammalian cells to survive and produce the target protein. Examples include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagle's Minimum Essential Medium (αMEM)), and MEM medium (Minimum Essential Medium (MEM)). Medium), RPMI 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, E-RDF medium, UltraCHO® medium, EX-CELL® 302 medium, EX-CELL® 325-PF medium, EX-CELL® ACF CHO medium, EX-CELL® CHO DHFR medium, EX-CELL® CD CHO medium, EX-CELL® CD CHO-2 medium, EX-CELL® CD CHO-3 medium, CD OptiCHO® medium, CD Examples of the medium include, but are not limited to, CHO medium, CD CHO AGT (registered trademark) medium, CD FortiCHO (registered trademark) medium, CHO-S-SFMII medium, CD DG44 medium, HyClone CDM4CHO medium, HyClone SFM4CHO medium, CD-ACP-CHO, HyClone SFM4CHO-Utility medium, KBM270 medium, KBM240 medium, KBM230 medium, KBM306 medium, BD Select CHO medium, CHOMACS CD medium, ESF SFM medium, Daigo T medium, IS CHO-V medium (registered trademark), IS CHO-V-GS medium (registered trademark), and Opti-Pro.

[0029] Those skilled in the art can add to the above-mentioned medium one or more combinations of various amino acids, various vitamins, energy sources, osmotic pressure regulators, iron sources, pH buffers, antibiotics, etc. Furthermore, those skilled in the art can add one or more combinations of other chemical components or biological components depending on the purpose.

[0030] Various amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Various vitamins include biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and the like. Examples of suitable additives include sodium, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, ascorbic acid, etc.; energy sources include glucose, galactose, mannose, fructose, etc.; osmotic pressure adjusters include sodium chloride, potassium chloride, potassium nitrate, etc.; iron sources include iron EDTA, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, etc.; pH buffers include sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc. The concentration of additives may be adjusted appropriately depending on the type of cells to be cultured, etc.

[0031] Examples of antibiotics added to the medium include sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexlocillin, mecillinam, andinocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, off Examples of suitable amines include, but are not limited to, loxacin, enoxacin, pipemidic acid, sulbactam, clavulic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, adinocillin, tazobactam, aztreonam, sulfazetine, isosulfazetine, norcadicine, m-carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline. The concentration of the amines to be added may be adjusted appropriately depending on the type of cells to be cultured, etc.

[0032] Furthermore, various culture apparatuses for mammalian cell culture include, but are not limited to, a fermenter-type tank culture apparatus, an airlift-type culture apparatus, a culture flask-type culture apparatus, a spinner flask-type culture apparatus, a microcarrier-type culture apparatus, a fluidized bed-type culture apparatus, a hollow fiber-type culture apparatus, a roller bottle-type culture apparatus, a packed tank-type culture apparatus, and a bioreactor-type culture apparatus.

[0033] 2. Mammalian Cell 2 Producing a Target Protein The present invention provides a mammalian cell producing a target protein, characterized in that expression of a transcription-related protein is regulated in the cell (hereinafter, sometimes referred to as "Mammalian Cell 2 of the Present Invention").

[0034] The mammalian cell 1 of the present invention described above is characterized by regulated expression of a protein associated with endoplasmic reticulum stress, while the mammalian cell 2 of the present invention is characterized by regulated expression of a protein associated with transcription. In other words, the only difference between the mammalian cell 1 of the present invention and the mammalian cell 2 of the present invention is the protein whose expression is regulated. Therefore, the type of mammalian cell, the type of target protein, the method for regulating protein expression, the culture conditions of the mammalian cell, and the like for the mammalian cell 2 of the present invention can be the same as those described for the mammalian cell 1 of the present invention, and in terms of definitions, "ER stress" can be replaced with "transcription."

[0035] In the mammalian cell 2 of the present invention, the expression of transcription-related proteins is regulated. The transcription-related proteins are not particularly limited as long as they are proteins that can affect the amount of gene transcription. Examples of such proteins include proteins that constitute general transcription factors such as TFIID. TFIID is composed of a TATA-binding protein (TBP) and several subunits called TBP-associated factors (TAFs).

[0036] In a preferred embodiment of the mammalian cell 2 of the present invention, the transcription-related protein may be a TAF. Examples of TAFs include, but are not limited to, TAF1 (TAFII250), TAF2 (CIF150), TAF3 (TAFII140), TAF4 (TAFII130 / 135), TAF4B (TAFII105), TAF5 (TAFII100), TAF6 (TAFII70 / 80), TAF7 (TAFII55), TAF8 (TAFII43), TAF9 (TAFII31 / 32), TAF9B (TAFII31L), TAF10 (TAFII30), TAF11 (TAFII28), TAF12 (TAFII20 / 15), TAF13 (TAFII18), and TAF15 (TAFII68). In a preferred embodiment, the transcription-related protein may be TAF8.

[0037] In a preferred embodiment, the mammalian cell 2 of the present invention has an enhanced expression level of a transcription-associated protein. In this case, the expression level of the transcription-associated protein can be increased by 1.1-fold or more (e.g., 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, or 3.0-fold or more) compared to the expression level in the cell before enhancement.

[0038] In a more preferred embodiment, the mammalian cell 2 of the present invention is characterized in that the expression level of TAF8 is enhanced.

[0039] Although the mammalian cells 1 and 2 of the present invention have been described as separate entities based on the function of the protein whose expression level is intended to be regulated (i.e., a protein associated with endoplasmic reticulum stress or a protein associated with transcription), in one aspect of the present invention, an embodiment in which the expression levels of both a protein associated with endoplasmic reticulum stress and a protein associated with transcription in a mammalian cell are simultaneously regulated can also be included in the present invention, as long as the desired effect of the present invention can be obtained. In a preferred embodiment, the mammalian cells of the present invention are characterized in that the expression level of at least one selected from the group consisting of PERK, Erp44, Txndc12, Pdia5, Prdx4, Edem3, Erlec1, and P3h4 is suppressed, and / or the expression level of TAF8 is enhanced.

[0040] Furthermore, by utilizing the above-described characteristics of the mammalian cells of the present invention, mammalian cells that highly produce a protein of interest can be easily selected. That is, the present invention also provides a method for selecting mammalian cells that highly produce a protein of interest (hereinafter, sometimes referred to as the "method of the present invention").

[0041] The method of the present invention comprises quantifying the expression level of at least one selected from the group consisting of PERK, Erp44, Txndc12, Pdia5, Prdx4, Edem3, Erlec1, P3h4, and TAF8 in mammalian cells. The protein quantification method may be a method known per se.

[0042] The lower the expression level of at least one selected from the group consisting of PERK, Erp44, Txndc12, Pdia5, Prdx4, Edem3, Erlec1, and P3h4, the higher the mammalian cell's production of the target protein.Furthermore, the higher the expression level of TAF8, the higher the mammalian cell's production of the target protein.

[0043] Furthermore, by utilizing the mammalian cells of the present invention, it is possible to efficiently produce a target protein. In this respect, the present invention also provides a method for producing a target protein (hereinafter, sometimes referred to as the "production method of the present invention").

[0044] The production method of the present invention is characterized by culturing mammalian cell 1 of the present invention and / or mammalian cell 2 of the present invention under conditions under which these cells produce a target protein. Suitable culture conditions for culturing mammalian cell 1 of the present invention or mammalian cell 2 of the present invention to produce a target protein may be the conditions described for mammalian cell 1 of the present invention, but are not limited to these as long as the target protein is produced.

[0045] In another aspect, the present invention provides a target protein produced by mammalian cell 1 of the present invention or mammalian cell 2 of the present invention (hereinafter, sometimes referred to as "target protein of the present invention"). Without wishing to be bound by theory, mammalian cell 1 of the present invention and mammalian cell 2 of the present invention are thought to have an intracellular environment that is favorable for the production of a target protein. Therefore, the target protein produced in this favorable intracellular environment can be of high quality.

[0046] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0047] [Example 1] Experimental Materials and Methods [Creation and Culture of Cell Clones] CHO-DG44 cells (Urlaub G, et al. Cell. 1983 Jun;33(2):405-12.) adapted to serum-free suspension culture were transfected with vectors (Shibafuji Y, et al. Biotechnol J. 2023 Jul;18(7):e2200638.) containing genes for two types of human monoclonal antibodies (designated IgG1A and IgG1B), and cloning was performed. From the resulting clones, a total of 12 clones were selected for testing, including three highly productive clones and three less productive clones expressing IgG1A and IgG1B, respectively. For cell passage, basal medium preheated to 37°C in a water bath was added to a 125 mL Elman-Meyer flask, and the medium was conditioned in a shaker incubator. The cryopreservation vials containing the cells were thawed in a hot water bath, placed in a sterile centrifuge tube containing basal medium, and centrifuged (220 g x 5 min). The supernatant was discarded, and the cell pellet was suspended in a flask containing basal medium to initiate cell culture. The shaker incubator conditions were 37.0°C, 125 rpm (25 mm revolution shake diameter), and 5% CO. ２ Three days after the start of culture, the viable cell count (VCD) and viability were measured using a Vi-cell BLU (Beckman Coulter). Fed-batch culture of CHO cells: Culture conditions were 37.0°C, 125 rpm (25 mm revolution shake diameter), 5% CO ２ Each clone (N=3) was cultured in a starting volume of 30 mL using a chemically defined basal medium. The initial cell density at the start of the fed-batch culture was 0.5 x 10 ６The concentration was measured as cells / mL. A chemically defined feed medium was added from day 3 of culture, and a fixed amount of fed-batch medium was added every three days (Days 3, 6, 9, and 12). Harvesting was performed on day 14 of culture. Sampling was performed on Days 3, 6, 9, 12, and 14, and culture trend data, titer data, metabolite data, pH, and osmolality were measured. VCD and viability were measured using a Vi-cell BLU (Beckman Coulter). For titer analysis and metabolite measurement, culture samples were centrifuged (9390 g x 1 min) to remove cells, and analysis was performed using a Cedex Bio HT Analyzer (Roche). The pH and osmotic pressure were measured using a pH meter S2K333 (Toyorika) and an automatic osmotic pressure meter OM-6060 (Arkray), respectively.

[0048] [Sample preparation for proteomics analysis] Sampling was performed on Day 6 fed-batch cultured CHO cells. After centrifugation (14,000 g × 30 sec), the supernatant was removed. The cell pellet was washed with 2 mL of ice-cold phosphate-buffered saline (PBS) and centrifuged again (14,000 g × 30 sec) to remove the PBS. The obtained cell pellet was immediately stored at -80°C.

[0049] Protein extraction and digestion was performed with modifications to a previously published method (Kawashima Y, et al. J Proteome Res. 2022 Jun 3;21(6):1418-1427). The cell pellet was added to 2% SDS-100 mM Tris-HCl (pH 8.5) and sonicated using a Bioruptor (CosmoBio). Protein concentration was measured by BCA assay. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to 20 μg of protein at a final concentration of 23.8 mM and incubated at 80°C for 10 minutes. Subsequently, iodoacetamide (IAA) was added to a final concentration of 31.3 mM and alkylated by incubation at room temperature for 30 minutes in the dark. To this reaction solution, 20 μL of 13.3 μg-solid / μL Sera-mag SpeedBead Carboxylate-Modified Magnetic Particles (a 1:1 mixture of hydrophilic and hydrophobic particles, Cytiva) washed with water was added and mixed. Next, 99.5% ethanol was added to a final concentration of 71% and mixed for 10 minutes. The beads were separated on a magnetic stand, the supernatant discarded, and the beads were washed with 80% ethanol and then with acetonitrile. The air-dried beads were suspended in 100 μL of 50 mM Tris-HCl (pH 8.0) and sonicated for 5 minutes. 2 μg of a trypsin / LysC mixture (Promega) was added, and the mixture was incubated at 37°C for 20 hours. The mixture was quenched by adding 20 μL of 5% TFA and mixing, and the beads were separated using a magnetic stand. The supernatant was collected, desalted using a GL-Tip SDB (GL Sciences), concentrated using a SpeedVac (Thermo Fisher Scientific), and resuspended in 2% acetonitrile-0.1% TFA. The peptide concentration was measured by absorbance.

[0050] [LC-MS Measurement for Proteomics Analysis] Measurements were performed using a Thermo Scientific EASY-nLC1200 (Thermo Fisher Scientific) and an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). 0.1% formic acid was used as mobile phase A, and 0.1% formic acid-80% acetonitrile was used as mobile phase B. An Acclaim PepMap 100 C18 (75 μm × 12 cm, 3 μm, Thermo Fisher Scientific) was used as the trap column. The sample load was 500 ng, and separation was performed using a NANO HPLC capillary column (75 μm x 12 cm, Nikkyo Technos) at a column temperature of 35°C and a flow rate of 200 nL / min. The gradient conditions were as follows: 6% (0-1 min) - 47% (91 min) - 75% (96 min) - 90% (100-115 min). The ion source spray was set to positive mode and the voltage was 2.0 kV. Mass spectra were acquired in overlapping DIA mode. The MS1 scan range was m / z 495-745, the resolution was 15,000, and the target was 4 x 10 ５ The MS2 spectrum had a precursor range of m / z 500-740 or 502-738, a scan range of m / z 200-1800, a resolution of 60,000, and a target of 1.6 × 10 ６ The isolation window width was set to 4 m / z.

[0051] [Proteomic Data Analysis] Data acquired by LC-MS was analyzed using DIA-NN version 1.8.1 (Demichev V et al., Nat Methods. 2020 Jan;17(1):41-44.). First, the measurement result file (raw file) was converted to an mzML file using MSconvert version 3.0.20293 (Kessner D et al., Bioinformatics. 2008 Nov 1;24(21):2534-6.), and then converted to a dia file using DIA-NN. A spectral library was created from the amino acid sequence obtained from Ensembl (Cricetulus griseus CHOK1GS, Ensembl release 104). The analysis parameters of DIA-NN were set as follows: charge range 2-4, peptide length range 7-45, precursor ion m / z range 495-745, fragment ion m / z range 200-1800, and MS1 and MS2 precision 10 ppm.

[0052] [Experimental Results] Factors involved in antibody productivity were comprehensively analyzed by comparing high- and low-producing clones using proteomics analysis. Differences in expression levels were detected between high- and low-producing clones for 287 protein factors. These were categorized by intracellular biological process, resulting in 83 processes. Furthermore, attention was focused on 10 intracellular processes that were statistically likely. Categories related to cell proliferation, glycoprotein metabolism, and protein folding were found.

[0053] Proteins whose expression levels change significantly between low-producing and high-producing clones are shown in Table 1-1 (measured values), Table 1-2 (scores obtained by standardizing the measured values ​​for creating heat maps), and Figure 1 (heat map).

[0054]

[0055]

[0056] As shown in Table 1-1, Table 1-2 and FIG. 1, the expression levels of the following proteins (1) to (8) were increased in the low-producing clones compared to the high-producing clones. (1) Prdx4 (Peroxiredoxin-4) (2) Txndc12 (thioredoxin domain containing 12) (3) Pdia5 (protein disulfide isomerase family A member 5) (4) P3h4 (prolyl 3-hydroxylase family member 4) (5) Erp44 (ER protein 44) (6) Edem3 (ER degradation enhancing alpha-mannosidase like protein 3) (7) Erlec1 (ER lectin 1) (8) PERK (PKR-like ER kinase, also known as "Eif2ak3") Furthermore, the expression levels of the following proteins (9) were lower in the low-producing clones compared to the high-producing clones: (9) TAF8 (TATA-box binding protein associated factor 8)

[0057] (1) to (8) are all proteins related to endoplasmic reticulum stress, and (9) is a protein related to transcription.

[0058] Although not wishing to be bound by theory, the increased expression of proteins related to ER stress in the low-producing clones is thought to be at least partly due to the increased expression of PERK, one of the sensor proteins important in the ER stress response. Activation of PERK appears to increase the expression of ER stress-related proteins downstream of the PERK signaling cascade, such as Prdx4, Erp44, Txndc12, Pdia5, P3h4, Edem3, and Erlec1.

[0059] [Example 2] Experimental Materials and Methods [Preparation of shRNA-Mediated Gene Knockdown Pool] Of the target genes selected by proteomics analysis, eight genes (TAF8, Pdia5, P3h4, Edem3, Prdx4, Txndc12, Erp44, and Erlec1), excluding PERK, were subjected to shRNA-mediated gene knockdown. An shRNA expression vector targeting a sequence not present in the genomic sequence of CHO cells was used as a control. These shRNA expression vectors were commissioned to VectorBuilder Inc. (Chicago, IL, USA). An overview of each of the prepared expression vectors is shown in Tables 2 and 3 below. As an example, a vector map of an shRNA expression vector targeting the TAF8 gene is shown in Figure 2. The nucleic acid sequences of the shRNA expression vector targeting the TAF8 gene and the shRNA expression vector targeting a gene other than TAF8 are identical except for the nucleic acid sequence of the shRNA. Detailed information on each shRNA expression vector can be obtained from vectorbuilder.com using the vector IDs shown in Tables 2 and 3. The day before transfection, the IgG1A and IgG1B expression clones constructed above were transfected into 100 cells with a VCD of 0.5 x 10. ６ After 1 day of culture, the cells were subcultured in a chemically defined basal medium at 10 x 10 cells / mL. ６ The culture medium was separated into 100 μL of cells and centrifuged at 220 × g for 5 minutes. The supernatant was discarded and the cell pellet was collected. D-PBS was added to wash the cells, and the mixture was centrifuged again at 220 × g for 5 minutes, the supernatant was discarded, and the cell pellet was collected. The cell pellet was resuspended in 100 μL of Neon Transfection Buffer R (Thermofisher Scientific), mixed with 10 μg of shRNA expression vector, and transfection by electroporation was performed using a Neon Transfection System (Thermofisher Scientific) to prepare knockdown pools of each gene.

[0060]

[0061]

[0062] [Batch culture of knockdown CHO cell pool] The culture conditions were 37.0°C, 125 rpm (orbital shake diameter 25 mm), 5% CO ２ After electroporation, each pool was cultured in a starting volume of 10 mL using chemically defined basal medium. The day of transfection was designated Day 0, and samples were taken daily until Day 4 to measure VCD, viability, and titer data. VCD and viability were measured using a Vi-cell BLU (Beckman Coulter). Titer analysis was performed using an Octet (SARTORIUS).

[0063] [Analysis of mRNA quantity] For the cultured cells subjected to batch culture described above, mRNA was extracted using an RNeasy Mini Kit (QIAGEN) according to the manufacturer's protocol. A portion of the resulting RNA solution was taken and the concentration was measured by absorbance using a NanoDrop (Thermofisher Scientific). Furthermore, the degree of RNA degradation was measured using a TapeStation 4200 (Agilent). RNA concentrations were equalized between samples, and cDNA was synthesized using the iScript AdV cDNA Kit for RT-qPCR (Bio-Rad) according to the manufacturer's protocol. Real-time PCR was performed using the prepared cDNA as a template and THUNDERBIRD SYBR qPCR Mix (TOYOBO). Real-time PCR was performed using a CFX96Touch real-time PCR analysis system (Bio-Rad). The amount of Chinese hamster cyclophilin (Cyc) mRNA was measured for each sample, and the measurement results for TAF8, Pdia5, P3h4, Edem3, Prdx4, Txndc12, Erp44, and Erlec1 were normalized.

[0064] [Experimental Results] Knockdown pools were prepared for the target genes selected by proteomics analysis and subjected to culture evaluation. The Pdia5, P3h4, Edem3, Prdx4, Txndc12, Erp44, and Erlec1 knockdown pools showed increased antibody production compared to the control. Furthermore, the TAF8 knockdown pool showed decreased antibody production compared to the control (Figures 3, 4, 5, and 6). Table 4 shows the ratio of the mRNA levels of the corresponding factors in each knockdown pool, relative to the mRNA level of the corresponding factor in the control. As shown in Table 4, cells transfected with shRNA vectors against TAF8, Pdia5, P3h4, Edem3, Prdx4, Txndc12, Erp44, and Erlec1 showed suppression of the expression levels of the corresponding factors compared to the control. These results indicate that suppressing gene expression of Pdia5, P3h4, Edem3, Prdx4, Txndc12, Erp44, and Erlec1 improves antibody production. Furthermore, suppressing gene expression of TAF8 reduced antibody production, suggesting the possibility that enhancing gene expression may improve antibody production.

[0065]

[0066] The results of this example are believed to clearly demonstrate the characteristics of cell lines with low target protein production. The results of this example demonstrate that cells with high target protein production can be easily and accurately selected by using the expression levels of endoplasmic reticulum stress-related proteins and transcription-related proteins as indicators. Furthermore, by artificially reducing the expression level of the above-mentioned endoplasmic reticulum stress-related proteins or artificially increasing the expression level of transcription-related proteins, mammalian cells with high target protein production can be easily produced.

[0067] According to the present invention, cells that highly produce a target protein can be easily selected or produced, and therefore the present invention is extremely useful in the fields of medicine, pharmacology, protein science, and the like.

[0068] This application is based on patent application No. 2023-211379 filed in Japan (filing date: December 14, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A mammalian cell producing a target protein, characterized in that expression of a protein associated with endoplasmic reticulum stress is regulated in said cell.

2. The cell of claim 1, wherein said mammalian cell is a Chinese Hamster Ovary (CHO) cell.

3. The cell according to claim 1 or 2, wherein the target protein is an antibody.

4. The cell according to claim 1 or 2, characterized in that the expression of a protein associated with the endoplasmic reticulum stress is suppressed.

5. The cell according to claim 1 or 2, wherein the protein associated with endoplasmic reticulum stress is an endoplasmic reticulum stress sensor protein.

6. The cell of claim 5, wherein the endoplasmic reticulum stress sensor protein is PERK.

7. The cell according to claim 1 or 2, wherein the protein associated with endoplasmic reticulum stress is a protein having the function of promoting disulfide bond formation in a target protein.

8. The cell according to claim 7, wherein the protein having the function of promoting disulfide bond formation of the target protein is at least one selected from the group consisting of Erp44, Txndc12 and Pdia5.

9. The cell according to claim 1 or 2, wherein the protein associated with endoplasmic reticulum stress is a protein having the function of reducing oxidative stress.

10. The cell according to claim 9, wherein the protein having the function of reducing oxidative stress is Prdx4.

11. The cell according to claim 1 or 2, wherein the protein associated with endoplasmic reticulum stress is a protein having the function of degrading denatured proteins.

12. The cell according to claim 11, wherein the protein having the function of degrading the denatured protein is at least one selected from the group consisting of Edem3 and Erlec1.

13. The cell according to claim 1 or 2, wherein the protein associated with endoplasmic reticulum stress is a protein associated with endoplasmic reticulum phagy.

14. The cell of claim 13, wherein the protein associated with endoplasmic reticulum phagy is P3h4.

15. A mammalian cell producing a target protein, characterized in that expression of a protein associated with transcription is regulated in said cell.

16. The cell of claim 15, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

17. The cell according to claim 15 or 16, wherein the target protein is an antibody.

18. A cell according to claim 15 or 16, characterized in that expression of a protein associated with said transcription is enhanced.

19. The cell according to claim 15 or 16, wherein the transcription-related protein is TAF8.

20. A method for producing a target protein, comprising the step of culturing the cell according to claim 1 or 15.

21. A target protein produced by the cell of claim 1 or 15.