WO2018229767A1 - Procédés pour améliorer les rendements de production de protéines cellulaires - Google Patents

Procédés pour améliorer les rendements de production de protéines cellulaires Download PDF

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WO2018229767A1
WO2018229767A1 PCT/IL2018/050651 IL2018050651W WO2018229767A1 WO 2018229767 A1 WO2018229767 A1 WO 2018229767A1 IL 2018050651 W IL2018050651 W IL 2018050651W WO 2018229767 A1 WO2018229767 A1 WO 2018229767A1
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cell
kit
cells
peptide
onco
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PCT/IL2018/050651
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English (en)
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Boaz Tirosh
Mohamed MAHAMEED
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2018229767A1 publication Critical patent/WO2018229767A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells

Definitions

  • the present invention is in the field of cell biology and biomanufacturing.
  • glycoproteins for therpeutic applications are performed in bioreactors, which can reach a capacity of a few tons.
  • the ability to grow cells in constant conditions of temperature, pH, available nutrients and dissolved oxygen is a daunting task that gets more complicated as the bioreactor size increases. Because fluctuations in growth conditions influence all steps of the biosynthetic pathway, and thus influence the quantity and the quality of the product, it is important to use cells that exhibit stability with respect to their genetic material, growth and metabolic properties.
  • RTK tyrosine kinase receptors
  • the c-KIT RTK plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form.
  • the present invention is directed to cells comprising a mutated KIT tyrosine kinase receptor and compositions comprising same.
  • the invention further provides a method comprising expression of a mutated KIT tyrosine kinase receptor in a cell for improving protein production yields.
  • a cell comprising: (i) a polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to the SEQ ID NO: 1, and (ii) an exogenous polynucleotide encoding a peptide of interest.
  • the polynucleotide sequence encoding SEQ ID NO: 1 is an exogenous polynucleotide.
  • the cell is a eukaryotic cell having increased peptide production efficacy compared to control.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is selected from a Chinese hamster ovary cell (CHO) or a human embryonic kidney cell 293 (HEK293).
  • the peptide is a glycopeptide.
  • the cell comprises a glycopeptide translated from the exogenous polynucleotide.
  • the glycopeptide has human-like glycans.
  • the glycopeptide comprises a polypeptide selected from the group consisting of: an antibody, an immunogen, and a growth factor.
  • the glycopeptide comprises a signal peptide sequence.
  • composition comprising the cell and a carrier.
  • the carrier is a chemically defined animal serum-free medium.
  • a method for improving protein production yields comprising: culturing a cell comprising an exogenous polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to the SEQ ID NO: 1.
  • improving protein production yields is improving global protein production.
  • improving protein production yields is of a protein of interest.
  • the cell has increased peptide production efficacy compared to control.
  • the method further comprises a step of isolating the secreted glycopeptide.
  • the culturing comprises growing of the cell in a medium.
  • the medium is a chemically defined serum-free medium.
  • FIGs. 1A-1C demonstrate the expression of onco-KIT enhances protein translation in a serum- supplemented DMEM medium.
  • A is a graph showing a typical flow cytometry analysis of onco-KIT and hKIT expressing CHO-Kl/GPF-Fc compared to parental untransfected cells. On the right is vertical bar graph depicting a quantification of intracellular GFP-Fc levels indicative for transgene expression. Shown is the average of three independent analyses + SD. GFP-Fc expression was found to be significantly elevated in cells expressing onco-KIT (p ⁇ 0.05).
  • (B) is a vertical bar graph showing qPCR analysis of mRNA levels of GFP-Fc relative to RPLP0 (a housekeeping gene) in CHO-K1 or in cells that express either onco-KIT or hKIT.
  • (C) comprises images of immunoblots of puromycin-labeled proteins and the assessment of KIT and mTOR activity (P-S6, P-4EBP1) in cells that expressed KIT variants.
  • FIGs. 2A-2B demonstrate translation-promoting effects of onco-KIT are maintained in chemically-defined serum-free medium.
  • A comprises a graph showing a typical flow cytometry analysis of intracellular GFP-Fc of the various cell lines following the adaptation to growth in chemically-defined serum-free medium (upper panel). Quantification of fluorescence levels is shown as the average of three independent measurements + SD (lower panel). GFP-Fc expression was significantly elevated in cells expressing onco-KIT (p ⁇ 0.05).
  • B comprises images of immunoblots of puromycin-labeled proteins and the assessment of KIT and mTOR activity (P-S6, P- 4EBP1) in cells that expressed KIT variants.
  • Figs. 3A-3D demonstrate onco-KIT promotes cell proliferation and mitochondrial activity.
  • a and B are graphs showing proliferation analysis for four days in serum- supplemented DMEM medium or in chemically defined serum-free medium, respectively.
  • C and D are vertical bar graphs of MTT analysis (representing cell viability and mitochondrial activity) which was performed at the end of the proliferation experiment for cells growing in serum-supplemented DMEM or chemically-defined serum-free medium, respectively. Shown is the average of three independent measurements + SD. For both conditions, MTT readouts were significantly elevated for cells expressing the onco-KIT (*p ⁇ 0.05).
  • FIGs. 4A-4C demonstrate onco-KIT improves cellular resilience to serum deprivation.
  • A comprises micrographs of cells plated at an equal density and documented for up to four days in serum-deprived DMEM medium (lOOx resolution).
  • B is a vertical bar graph summarizing the percentage of viable cells and
  • C is a vertical bar graph summarizing the viable cells' density (millions/well), both as were recorded on the fourth day. Shown is the average of three independent measurements + SD. Expression of onco-KIT significantly improved both parameters (*p ⁇ 0.05).
  • Figs. 5A-5B demonstrate onco-KIT protects cells from hypoxia stress.
  • A is a vertical bar graph demonstrating percentages of viable cells as measured 6 hours after hypoxia initiation in serum- supplemented DMEM (black) and Fusion medium (gray).
  • B is a vertical bar graph demonstrating the fluorescence intensity of the supernatants of the indicated samples at the end of the hypoxia stress. Shown is the average of three independent measurements + SD. Expression of onco-KIT significantly improved secretion (*p ⁇ 0.05).
  • FIGs. 6A-6C demonstrate onco-KIT enhances unfolded protein response (UPR) activity following ER stress.
  • a and B are expression profiling images comprising western blot analysis of P-IRE1 (top) and RT-PCR of XBP1 splicing (bottom) in CHO- Kl and KIT mutant expressing cell-lines, after thapsigargin or tunicamycin induced ER stress, respectively.
  • C is a vertical bar graph demonstrating quantitative analysis of ERdj4 mRNA levels, as indicative for XBP-ls activity in thapsigargin (TG)-mediated ER stress. Shown are results of a typical experiment out of three.
  • Figs. 7A-7C demonstrate expression of onco-KIT improves protein secretion.
  • A is a vertical bar graph demonstrating fluorescence analysis of cell culture media following cells incubation with cycloheximide for 2 and 4 hours.
  • B is a graph demonstrating cell viability and
  • C is a corresponding graph of the fluorescence intensity of the cell culture media following a seven-day secretion assay in chemically- defined serum-free medium.
  • Figs. 8A-8B are illustrations of the PI3 K/AKT/mTOR pathway.
  • A is an illustration depicting activation in a ligand-dependent fashion in a serum-supplemented medium.
  • B is an illustration of onco-KIT mediated constitutive activation in a ligand- independent manner in serum-free chemically-defined medium. In both scenarios, cells demonstrate proliferation and survival, increase in protein synthesis and stress resilience improvement.
  • the present invention is directed to cells comprising a mutated KIT tyrosine kinase receptor, compositions comprising same and methods for improving protein production yields.
  • the present invention is based, in part, on the finding that cells expressing a mutated KIT provide had significantly elevated levels of protein production yields, both of total protein and/or of a protein of interest which was transected into the cells, such as a glycopeptide.
  • the invention is directed to improving protein production yields, the method comprises culturing a cell comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1.
  • the cell further comprises an exogenous polynucleotide encoding a peptide of interest.
  • the invention is directed to improving protein production yields of a peptide of interest
  • the method comprises culturing a cell comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1, and an exogenous polynucleotide encoding a peptide of interest, thereby improving protein production yields of the peptide of interest.
  • the method of the present invention further improves culture performance of a cell of the invention compared to control.
  • a cell performance comprises global protein synthesis.
  • a cell performance comprises expression of a peptide of interest.
  • a cell performance comprises cell proliferation.
  • a cell performance comprises stress resistance.
  • stress comprises oxygen stress, including but not limited to hypoxia, or ER stress.
  • a cell of the invention has performance level equal to or greater than the performance level of a control cell. Methods for determining cell performance as defined above are well known to one of ordinary skill in the art. Non-limiting examples include, but are not limited to, qPCR, western blotting, proliferation assays, metabolism assays (such as MTT) and flow cytometry, all as described herein below.
  • a method of the present invention comprises culturing a cell in a medium.
  • a cell is grown in the medium.
  • medium is a cell culture medium suitable for growth and maintenance of a cell having increased peptide production yields.
  • cell culture medium is optimized for cell growth.
  • cell culture medium is optimized for protein synthesis.
  • “cell culture medium” refers to any liquid medium which enables cells proliferation. Growth media are known in the art and can be selected depending of the type of cell to be grown.
  • cell of the invention is cultured under effective conditions, which allow for increased yield of production from the cultured cell.
  • Non-limiting example for increased yield include, but not limited to, increased gene expression, protein production and secretion, molecule biosynthesis, proliferation, stress resistance and others.
  • effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit for increased production yield.
  • an effective medium refers to any medium in which a cell is cultured to produce a peptide of interest of the present invention.
  • a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • growth medium of the present invention is chemically defined so as to not include any animal-derived molecule or compound, such as animal serum.
  • a cell of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates.
  • culturing is carried out at a temperature, pH and oxygen content appropriate for a mammalian cell.
  • culturing conditions are within the expertise of one of ordinary skill in the art.
  • transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide.
  • effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention.
  • a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.
  • medium of the present invention is chemically defined. In some embodiments, a chemically defined medium is devoid of animal products. In some embodiments, a medium devoid of animal products comprises a serum free medium. In some embodiments, a chemically defined serum free medium is optionally supplemented with hormones, including but not limited to insulin.
  • resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium or secreted into a space between two cellular membranes. In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is affected.
  • the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
  • polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety.
  • a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety.
  • a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].
  • polypeptide of the present invention is retrieved in "substantially pure" form that allows for the effective use of the protein in subsequent applications, such as for therapy or diagnosis.
  • substantially pure describes a peptide/polypeptide or other material which has been separated from its native contaminants.
  • a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence.
  • a substantially pure peptide can comprise over about 85 to 90% of a peptide sample, and can be over 95% pure, over 97% pure, or over about 99% pure. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes high resolution may be necessary and HPLC or a similar means for purification can be used. For most purposes, a simple chromatography column or polyacrylamide gel can be used to determine purity.
  • purified does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a peptide is in the "purified” state after purification of the starting material or of the natural material by at least one order of magnitude, 2 or 3, or 4 or 5 orders of magnitude.
  • the polypeptides of the present invention are substantially free of naturally-associated host cell components.
  • the term "substantially free of naturally- associated host cell components" describes a peptide or other material which is separated from the native contaminants which accompany it in its natural host cell state.
  • a peptide which is chemically synthesized or synthesized in a cellular system different from the host cell from which it naturally originates will be free from its naturally-associated host cell components.
  • a cell of the disclosed invention has increased peptide production efficacy compared to control.
  • the term "increased peptide production efficacy" as used herein refers to an endogenous polypeptide or an exogenous polynucleotide encoding a protein of interest.
  • a cell having increased production efficacy of a peptide comprises increased mRNA transcription levels, compared to a control cell.
  • a cell having increased production efficacy of a peptide comprises increased mRNA translation rates, compared to a control cell.
  • a cell having increased production efficacy of a peptide comprises increased translated peptide levels, compared to a control cell.
  • a cell having increased production efficacy of a peptide comprises increased stability of a translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased levels of properly folded translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased level of properly post-translationally modified translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased secretion levels of a translated peptide, compared to a control cell.
  • increasing is by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 60%, by at least 75%, by at least 80%, by at least 90%, by at least 95%, or by at least 100%, compared to control. In some embodiments, increasing is by 1-5%, 3-8%, 7-12%, 10-15%, 13-20%, 18-25%, 22-30%, 26-35%, 33-45%, 40-55%, 50-65%, 60-75%, 70-85%, 80-90%, 90-99%, or 95-100%, compared to control.
  • increasing is by at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to control.
  • Each possibility represents a separate embodiment of the present invention.
  • prokaryotic or eukaryotic cells can be used as host-expression systems to multiply or express the polynucleotide or polypeptide of the present invention.
  • these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.
  • microorganisms such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence
  • cells of the invention include mammalian cells to express the polypeptide of the present invention.
  • mammalian cells are derived of human origin.
  • mammalian cells are derived of murine origin.
  • Non- limiting examples for mammalian cells include, but are not limited to 3T3-L1, 4T1, 9L, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, A549, AHL-1, ALC, B 16, B53, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT-20, BxPC3, C2C12, C3H- 10T1/2, C6, Caco-2, Cal-27, Calu-3, CGR8, CHO, CML Tl, CMT12, COR-L23, COR- L23/5010, COR-L23/CPR, COR-L23/R23-, COS-7, COV-434, CT26, D17, DAOY, DH82, DU145, DuCaP, E14Tg2a, EL4, EM-2, EM-3, EMT6/AR10.0, FM3, GL261, H1299, HaCaT,
  • a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed.
  • large quantities of polypeptide are desired.
  • vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired.
  • vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].
  • polynucleotides of the present invention are prepared using PCR techniques as described in Example 1, or any other method or procedure known to one skilled in the art.
  • the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).
  • polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide.
  • the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes.
  • the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes.
  • the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes.
  • cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).
  • yeast expression systems are used.
  • a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447.
  • vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.
  • the expression vector of the present invention may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • recombinant viral vectors which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression of the polynucleotides of the present invention.
  • lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • Various methods can be used to introduce the expression vector of the present invention into cells, in some embodiments, cells introduced with any exogenous polynucleotide, as abovementioned are termed herein "transformed cells” or "recombinant cells".
  • Methods for introducing polynucleotide vectors are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich.
  • nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture.
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
  • the invention is directed to a composition
  • a composition comprising a cell having increased protein production yield and comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1, an exogenous polynucleotide encoding a peptide of interest; and a carrier or diluent.
  • the disclosed invention is directed to a cell comprising: (i) a polynucleotide sequence encoding a mutated c-KIT tyrosine kinase receptor (onco- KIT), and (ii) an exogenous polynucleotide encoding a peptide of interest.
  • Wild type (WT) and mutated (Onco) KIT transcripts and proteins of the invention are specified herein below (Table 1).
  • an onco-KIT of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 1, or an analogue thereof having at least 80% sequence identity thereto.
  • an onco-KIT protein (SEQ ID NO: 1) has a ligand-independent phosphorylation activity as described herein.
  • an onco-KIT protein is a mutant of SEQ ID NO: 9 having ligand- independent KIT protein activity as described herein.
  • an onco- KIT protein (SEQ ID NO: 2) is a fragment of SEQ ID NO: 1 having a ligand-independent phosphorylation activity.
  • an onco-KIT protein is a mutant of SEQ ID NO: 9 or an analogue thereof having at least 80% sequence identity thereto and having an Aspartic acid residue at position 816 substituted by a Valine (D816V).
  • an onco-KIT protein of the invention is encoded by a DNA sequence which comprises or consists the nucleic acid sequence as set forth in SEQ ID Nos.: 3-4 or an analogue thereof having at least 75% sequence identity thereto.
  • an onco-KIT of the invention is a murine onco-KIT which comprises or consists the amino acid sequence as set forth in SEQ ID NO: 5, or an analogue thereof having at least 80% sequence identity thereto.
  • a murine onco-KIT protein (SEQ ID NO: 5) has a ligand-independent phosphorylation activity as described herein.
  • a murine onco-KIT protein is a mutant of SEQ ID NO: 10 having ligand-independent KIT protein activity as described herein.
  • a murine onco-KIT protein (SEQ ID NO: 6) is a fragment of SEQ ID NO: 5 having a ligand-independent phosphorylation activity.
  • a murine onco-KIT protein is a mutant of SEQ ID NO: 10 or an analogue thereof having at least 80% sequence identity thereto and having an Aspartic acid residue at position 818 substituted by a Tyrosine (D818Y).
  • a murine onco-KIT protein of the invention is encoded by a DNA sequence which comprises or consists the nucleic acid sequence as set forth in SEQ ID Nos.: 7-8 or an analogue thereof having at least 75% sequence identity thereto.
  • analogue includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
  • one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine
  • substitution of one basic residue such as lysine, arginine or histidine for another
  • substitution of one acidic residue such as aspartic acid or glutamic acid for another
  • the phrase "conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein.
  • the chimeras and/or peptides of the invention encompass variant thereof.
  • variant refers to a polypeptide or nucleotide sequence which comprises a modification of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively.
  • the modifications are substitution, deletion, and/or insertion of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively.
  • the changes may be of minor nature, such as conservative amino acid substitutions or for nucleotide sequence resulting in conservative amino acid substitutions that do not significantly affect the activity of the polypeptide.
  • the changes may be substitution of an amino acid molecule, resulting in an addition of a glycosylation site, thereby increasing glycosylation of the polypeptide.
  • the invention further encompasses a polynucleotide sequence comprising a nucleic acid encoding a peptide of interest.
  • a peptide of interest in an exogenous peptide.
  • polynucleotide encoding a peptide of interest is transformed into a cell of the invention.
  • Non-limiting examples for methods of transforming exogenous polynucleotide molecule into a cell of the disclosed invention include, but not limited to: membrane permeabilization, electroporation, viral transformation, transfection, among others, all of which are known to a person of ordinary skill in the art.
  • the peptide of interest comprises a signal peptide sequence for secretion.
  • signal peptide sequence refers to an approximately 16-40 amino acid stretch present on the amino -terminus of a protein which directs the nascent protein to the periplasm (prokaryotic cells) or permits the secretion of the protein (eukaryotic cells).
  • the signal peptide can be cleaved from the protein once the protein has been directed to its desired location (i.e., periplasm, secretory granule, etc.).
  • signal peptide signal peptide sequence
  • leader sequence peptide are used interchangeably in the art.
  • a control cell is any cell having a native KIT activity.
  • a native KIT activity comprises ligand dependent phosphorylation activity.
  • a control cell comprises SEQ ID Nos.: 9 or 10.
  • endogenous is used to refer to a polypeptide that is naturally expressed or produced by a cell, a tissue or an organism.
  • exogenous is used to refer to any polynucleotide or polypeptide that originate outside of the organism of concern or study and is transformed into the cell.
  • chimeric is used to refer to a polypeptide formed by the joining of two or more peptides through a peptide bond formed between the amino terminus of one peptide and the carboxyl terminus of another peptide.
  • the chimeric polypeptide may be expressed as a single polypeptide fusion protein from a nucleic acid sequence encoding the single contiguous conjugate.
  • the polypeptide of interest of the disclosed invention is a glycosylated polypeptide.
  • glycosylated polypeptide and “glycopeptide” are interchangeable.
  • a glycopeptide of the present invention requires glycosylation for rendering activity.
  • glycosylation refers to the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to the polypeptide.
  • the oligosaccharide side chains are linked to the backbone of the polypeptide through either N- or O-linkages.
  • N-linked glycosylation refers to the attachment of the carbohydrate moiety to an asparagine (i.e., N) residue in a glycoprotein chain.
  • a glycopeptide of the invention is selected from the group consisting of: antibodies, growth factors, immunogens, enzymes, coagulation or anticoagulation proteins.
  • the polynucleotide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis -regulatory sequence (e.g., promoter sequence).
  • a cis -regulatory sequence e.g., promoter sequence
  • the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention.
  • the cis-regulatory sequence is suitable for directing tissue- specific expression of the polypeptide of the present invention.
  • the cis- regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.
  • polynucleotide refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide.
  • a polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase.
  • the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.
  • composite polynucleotide sequence refers to a sequence, which is at least partially complementary and at least partially genomic.
  • a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences.
  • intronic sequences include cis acting expression regulatory elements.
  • SEQ ID NO: 1 comprises or consists of the acid sequence as set forth:
  • SEQ ID NO: 2 comprises or consists of the acid sequence as set forth:
  • SEQ ID NO: 9 comprises or consists of the acid sequence as set forth:
  • a ALYKNLLHS KES S C S DSTNE YMDMKPG VS YV VPTKADKRRS VR IGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAAR NILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFES D VWS YGIFLWELFS LGS S PYPGMP VDS KFYKMIKEGFRMLS PEH AP AEM YDIM KTC WD ADPLKRPTFKQIVQLIEKQIS ES TNHI YS NLANCS PNRQKP V VDHS VRI NSVGSTASSS QPLLVHDD V .
  • SEQ ID NO: 5 comprises or consists of the acid sequence as set forth:
  • SEQ ID NO: 6 comprises or consists of the acid sequence as set forth:
  • SEQ ID NO: 10 comprises or consists of the acid sequence as set forth:
  • each of the verbs, "comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • CHO-K1 (ATCC) cells were cultured in Dulbecco's Modified Eagle medium (Biological Industries, Israel): Nutrient Mixture F-12 (F-12 DMEM, Sigma- Aldrich) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine (Biological Industries, Israel), 1% penicillin- streptomycin solution (Biological Industries, Israel), and 1 mM sodium pyruvate (Biological Industries, Israel) at 37 °C under 5% C0 2 .
  • Chemically-induced ER stress was performed by treating cells with either thapsigargin or tunicamycin (purchased from Fermentek, Israel), at a concentration of 2.5 ⁇ g/mL for 8 hours. Compounds were dissolved in DMSO to 2.5 mg/mL stock solution.
  • GFP-Fc was cloned into pcDNA3.1(+) between EcoRI and EcoRV restriction sites.
  • CHO-K1 cells were transfected using TransIT ® 2020 (3 ⁇ ⁇ of transfection reagent ⁇ g of DNA). The transfected cells were recovered for 24 hours followed by FACS sorting of GFP positive cells. Following one week of culturing, sorting was preformed, and the process was then repeated three more times until a stable pool of GFP-positive cells was obtained. From this bulk, single-cell cloning was performed by limiting dilution. The expanded clones were validated for GFP-Fc expression by flow cytometry and immunoblotting, and the same clone was used for further engineering (termed 'CHO-Kl/GFP-Fc').
  • Onco-KIT and hKIT were cloned into pcDNA 3.
  • KIT variants were separately transfected into the same clone of the CHO-Kl/GFP-Fc cells following two weeks of selection with Hygromycin B (A.G. Scientific, Inc.) at a concentration of 500 ⁇ g/mL. KIT expression was verified by immunoblotting .
  • Blots were blocked in 10% skim milk in TBST buffer for 1 hour at room temperature.
  • Primary antibodies were rabbit anti-phospho S6 Ribosomal Protein (Ser240/244) (cell signaling #2215), c-Kit (D13A2) (cell signaling #3074), Phospho-c-Kit (Tyr719) (cell signaling #3391), Phospho-4E-BPl (Thr37/46) (236B4) (cell signaling #2855), Anti-IREl (phospho S724) Rabbit [EPR5253] (abeam ab 124945), mouse anti-puromycin Antibody, clone 12D10 (Millipore #MABE343). For GFP and p97 polyclonal antibodies were used.
  • GFP-Fc forward- 5'- TGAAGTTC ATCTGC ACC ACCG-3 ' (SEQ ID NO: 11), reverse - 5'- AGTCGTGCTGCTTC ATGTGGT-3 ' (SEQ ID NO: 12); for RPLP0 forward: 5'- CCAACTACTTCCTTAAGATCATCCAACT-'3 (SEQ ID NO: 13), reverse: 5'- ACATGCGGATCTGCTGCA-'3 (SEQ ID NO: 14).
  • RT-PCR was used for the detection of XBP1 mRNA splicing using 5x Red Load Taq (LAROVA) with CHO-XBP1 splicing primers: forward- 5 '-CCTTGT AATTGAGAACC AGG-3 ' (SEQ ID NO: 15), reverse - 5'-CCAAAAGGATATCAGACTCGG-3' (SEQ ID NO: 16).
  • ERdj4 forward-5- GGTGTGCCAAAATCGGCATC-3' (SEQ ID NO: 17), reverse: 5'- GCACTGTGTCCAAGTGTATCA-3' (SEQ ID NO: 18).
  • MTT assay was performed according to the supplier's instructions (Vybrant® MTT Cell Proliferation Assay Kit, ThermoFisher).
  • GFP-Fc a CHO-K1 single-cell clone that stably expresses an Ig fusion composed of a GFP domain, preceded by a signal peptide, fused to the constant region of a human IgGl, termed GFP-Fc.
  • WT wild type
  • SEQ ID NO: 2 also known in the art to comprise a D816V substitution
  • Flow cytometry analysis of the intracellular levels of GFP-Fc indicated a significantly higher expression in CHO-Kl cells that co-expressed onco-KIT compared to parental cells or hKIT (Fig. 1A).
  • levels of the GFP-Fc encoding mPvNA were of similar values (Fig. IB).
  • the discrepancy between the mRNA levels and the measured fluorescence indicated a regulation at the post-transcription level.
  • the inventors pulsed the cells for a short time with puromycin and performed immunoblotting with an anti-puromycin antibody that resolves only the newly synthesized proteins. Expression of onco-KIT, but not hKIT, elevated the global protein translation (Fig. 1C, top panel).
  • KIT When expressed in cells, KIT yields two major polypeptides that are distinct in their glycan types and are readily separated on SDS-PAGE. The heavier one, which contains complex N-linked carbohydrate modifications and resides at the cell surface, and a lighter one that express the high mannose N-linked glycans, which is found in the ER.
  • the WT hKIT allele When expressed in CHO cells, the WT hKIT allele was mostly expressed in the heavier form, while the onco-KIT was mostly expressed in the lighter form, corresponding to its typical intracellular localization. Analysis of phosphorylated KIT status indicated that both KIT variants, when cultured in the presence of serum, were active (Fig. 1C).
  • KIT activates the PI3K pathway, which subsequently activates the mammalian target of rapamycin (mTOR) pathway.
  • mTOR when activated, increases protein translation by multiple pathways.
  • the inventors measured the levels of phosphorylated 4-EBP1 and ribosomal S6 proteins by immunoblotting. As both of these targets are phosphorylated in an mTOR-dependent fashion, an elevation in the activity of mTOR in the onco-KIT expressing cells was indicated (Fig. 1C).
  • the inventors adapted the cells for growth in the chemically-defined animal-free medium, EX-CELL® CD CHO Fusion.
  • Examination of the intracellular GFP-Fc levels by flow cytometry displayed a higher expression for cells expressing the onco-KIT than those expressing hKIT and the CHO- Kl controls (Fig. 2A).
  • Translation analysis indicated that the onco-KIT expressing cells increased the levels of protein synthesis using the puromycin-pulse method (Fig. 2B, upper panel). This was consistent with the analysis of phospho KIT levels, which was detected only for the onco-KIT expressing cells and not for the hKIT expressing cells.
  • Onco-KIT promotes cell proliferation and mitochondrial activity
  • Onco-KIT triggered a strong RTK signal in the absence of the ligand.
  • the inventors followed cell number and viability under serum deprivation conditions. While the parental CHO-Kl and the hKIT expressing CHO-Kl cells showed clear morphological signs of apoptosis after 4 days without the serum, the onco-KIT expressing cells continued to proliferate and reached 100% confluence (Fig. 4A). Total cell numbers were three times more than the controls and the onco-KIT cells maintained higher viability (Fig. 4B and 4C). The continuing proliferation of onco-KIT transduced CHO-Kl cells, even in the absence of serum, indicated that the ligand-independent signal emitted by onco-KIT can replace the growth supporting elements that are provided by the serum.
  • onco-KIT is mainly found in the ER and its trafficking to the cell surface can be expedited by manipulating its phosphorylation status.
  • the difference in the intracellular localization urged the inventors to investigate whether onco-KIT has any effect on ER function in stressful conditions.
  • the stable expression of onco-KIT or hKIT did not result in a strong unfolded protein response (UPR) as assessed by the expression of P-IRE1 and the splicing of XBP1, a hallmark of the UPR.
  • UPR unfolded protein response
  • onco-KIT may have improved the ability to sustain UPR signaling under ER stress conditions.

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Abstract

La présente invention concerne une cellule comprenant : une séquence polynucléotidique codant pour un onco-KIT, ou un analogue de celle-ci ayant au moins 80% d'homologie avec l'onco-KIT, et un polynucléotide exogène codant pour un peptide d'intérêt. L'invention concerne en outre une méthode d'amélioration des rendements de production de protéines.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5464764A (en) 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5932447A (en) 1994-05-17 1999-08-03 Bristol-Myers Squibb Company Cloning and expression of a gene encoding bryodin 1 from Bryonia dioica

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5464764A (en) 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5487992A (en) 1989-08-22 1996-01-30 University Of Utah Research Foundation Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5932447A (en) 1994-05-17 1999-08-03 Bristol-Myers Squibb Company Cloning and expression of a gene encoding bryodin 1 from Bryonia dioica

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Basic and Clinical Immunology", 1994, APPLETON & LANGE
"Cell Biology: A Laboratory Handbook", vol. I-III, 1994
"Culture of Animal Cells - A Manual of Basic Technique", 1994, WILEY-LISS
"Current Protocols in Immunology", vol. I-III, 1994
"Current Protocols in Molecular Biology", 1992, JOHN WILEY & SONS
"Current Protocols in Molecular Biology", vol. I-III, 1994
"Genome Analysis: A Laboratory Manual Series", vol. 1-4, 1998, COLD SPRING HARBOR LABORATORY PRESS
"Strategies for Protein Purification and Characterization - A Laboratory Course Manual", 1996, CSHL PRESS
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, JOHN WILEY AND SONS
BOOTH ET AL., IMMUNOL. LETT., vol. 19, 1988, pages 65 - 70
CHANG ET AL.: "Somatic Gene Therapy", 1995, CRC PRESS
FISCHER SIMON ET AL: "The art of CHO cell engineering: A comprehensive retrospect and future perspectives", BIOTECHNOLOGY ADVANCES, ELSEVIER PUBLISHING, BARKING, GB, vol. 33, no. 8, 31 October 2015 (2015-10-31), pages 1878 - 1896, XP029328726, ISSN: 0734-9750, DOI: 10.1016/J.BIOTECHADV.2015.10.015 *
GARDELLA ET AL., J. BIOL. CHEM., vol. 265, 1990, pages 15854 - 15859
GILBOA, BIOTECHNIQUES, vol. 4, no. 6, 1986, pages 504 - 512
H. BOUGHERARA ET AL: "The Aberrant Localization of Oncogenic Kit Tyrosine Kinase Receptor Mutants Is Reversed on Specific Inhibitory Treatment", MOLECULAR CANCER RESEARCH, vol. 7, no. 9, 1 September 2009 (2009-09-01), pages 1525 - 1533, XP055142588, ISSN: 1541-7786, DOI: 10.1158/1541-7786.MCR-09-0138 *
JEE YON KIM ET AL: "CHO cells in biotechnology for production of recombinant proteins: current state and further potential", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 93, no. 3, 9 December 2011 (2011-12-09), pages 917 - 930, XP035006081, ISSN: 1432-0614, DOI: 10.1007/S00253-011-3758-5 *
MAHAMEED MOHAMED ET AL: "Engineering CHO Cells With an Oncogenic KIT Improves Cells Growth, Resilience to Stress, and Productivity", BIOTECHNOLOGY AND BIOENGINEERING, vol. 114, no. 11, November 2017 (2017-11-01), pages 2560 - 2570, XP002785537, ISSN: 0006-3592 *
MOHAN CHAYA ET AL: "Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells.", BIOTECHNOLOGY JOURNAL, vol. 3, no. 5, May 2008 (2008-05-01), pages 624 - 630, XP002785535, ISSN: 1860-7314, DOI: 10.1002/biot.200700249 *
NINGFEI AN ET AL: "Pim1 kinase regulates c-Kit gene translation", EXPERIMENTAL HEMATOLOGY & ONCOLOGY, BIOMED CENTRAL LTD, LONDON, UK, vol. 5, no. 1, 30 December 2016 (2016-12-30), pages 1 - 8, XP021241079, DOI: 10.1186/S40164-016-0060-3 *
PERBAL: "A Practical Guide to Molecular Cloning", 1988, JOHN WILEY & SONS
SAMBROOK ET AL.: "Molecular Cloning: A laboratory Manual", 1989
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRINGS HARBOR LABORATORY
STUDIER ET AL., METHODS IN ENZYMOL., vol. 185, 1990, pages 60 - 89
VECTORS: "A Survey of Molecular Cloning Vectors and Their Uses", 1988, BUTTERWORTHS
VEGA ET AL.: "Gene Targeting", 1995, CRC PRESS
WATSON ET AL.: "Recombinant DNA", SCIENTIFIC AMERICAN BOOKS
YOSHINORI KATAKURA ET AL: "Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes", CYTOTECHNOLOGY, KLUWER ACADEMIC PUBLISHERS, DO, vol. 31, no. 1-2, 1 September 1999 (1999-09-01), pages 103 - 109, XP019236654, ISSN: 1573-0778, DOI: 10.1023/A:1008048928053 *
ZUSTIAK MATTHEW P ET AL: "Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL.", BIOTECHNOLOGY JOURNAL SEP 2014, vol. 9, no. 9, September 2014 (2014-09-01), pages 1164 - 1174, XP002785536, ISSN: 1860-7314 *

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