MXPA01001238A - Overexpression of desired proteins in eukaryotic cells mediated by cyclin d1 overexpression - Google Patents

Abstract

The invention concerns eukaryotic cells useful in protein expression comprising (a) an inserted nucleic acid encoding a cyclin D gene product and (b) an inserted nucleic acid encoding a protein of interest, wherein the cyclin D gene product and the protein of interest are expressed in the cell. The invention also concerns a process for producinga protein of interest, which comprises (a) inserting into a eukaryotic cell a nucleic acid encoding a cyclin D gene product and a nucleic acid encoding a protein of interest;(b) culturing the cell under conditions permitting the expression of the protein of interest;and (c) isolating the protein of interest. The cells are preferably mammalian, with CHO cells most preferred. The cyclin D gene product is preferably of human origin. Suitable proteins of interest include erythropoietin (EPO), osteoprotegerin (OPG), OPG-Fc, leptin, Fc-leptin, and Novel Erythropoiesis Stimulating Protein (NESP).

Description

OVEREXPRESSION OF DESIRED PROTEINS IN EUCARIÓTIC CELLS MEDIATED BY THE OVEREXPRESSION OF CICLINA GAVE Background of the Invention The progression through the mammalian cell cycle is driven by the ordered activation of cyclin-dependent kinases (CDKs). An active CDK is composed of a catalytic subunit and a regulatory subunit called cyclin. The activity of CDK is regulated by interactions with cyclins and CDK inhibitors (CKIs) and by post-translational modifications (for example, phosphorylation). The transition between cell cycle states is regulated by the control points defined by different cyclin subunits: Gl cyclins for the Gl / S transition, S cyclins for progression through the S phase, and Fungal cyclins or G2 to enter into mitosis. The assignment of a cell to enter the S phase occurs at a restriction point (R) delayed in Gl, after the mitogenic growth factors in the cells are no longer required to complete the division.

REF. DO NOT. 127020 Cyclins D and E are synthesized sequentially during Gl and the speed is limited to enter the S phase, so that they can be seen as Gl cyclins. At least three mammalian genes encode type D cyclins (DI, D2 and D3). Type D cyclins are progressively induced as part of the later response in mitogenic stimulation, and are expressed in a specific model of the cell lineage. The assembly of type D cyclins with CDK4 and CDK6 is regulated post-translationally by mitogens. Once assembled, cyclin D bound to the CDKs must be phosphorylated by a kinase that activates the CDK (CAK) to acquire the catalytic activity. The cyclin D genes are in a different branch of the evolutionary tree of type A-, B-, or E cyclins, and type D cyclins have some properties different from other cyclins. Being short-lived proteins (t? / 2 < 25 min). The withdrawal of the growth factors during Gl prevents the constant accumulation of cyclin D, correlating with the lack of cells deprived of the growth factor to progress beyond the point R. Thus, the expression of cyclin D is regulated by the signals extracellular, unlike the periodic expression of cyclins A, B and E.

Overexpression of human DI and E cyclins in reduced fibroblasts of rodent or human Gl, decreases cell size, and reduces the serum requirement for the transition from Gl to S (Resnitzky et al., MCB 14, 1669-79 (1994) Quelle et al, Genes &Development 7, 1559-71 (1993), Ohtsubo &Roberts, Science 259, 1908-12 (1992)). These results suggest that cyclins D may cancel a physiologically regulated function by cyclin E or vice versa. However, overexpression of cyclin D or E does not lead the cells to the transformation of fibroblasts that remain serum-dependent, inhibited on contact, and unable to form the colonies in the semi-solid medium. Overexpression of cyclin DI has also been found to improve the amplification of the endogenous gene, suggesting that it plays a role in genomic instability during tumor development. Zhou et al., Cancer Res. 56: 36-9 (nineteen ninety six) .

Brief Description of the Invention The present invention involves a eukaryotic cell comprising: (a) a cyclin D gene product, wherein the cyclin D gene product is functionally expressed in the cell at a level greater than any native level of expression; and (b) a protein of interest, wherein the protein of interest is expressed in the cell at a level greater than any native level of expression. The present invention also involves a process for producing a protein of interest comprising: A. creating a eukaryotic cell that expresses 1. a cyclin D gene product at a level greater than any native level of expression and 2. a protein of interest at a level higher than any native level of expression; B. culturing the cell under conditions that allow expression of the protein of interest and functional expression of the cyclin D gene product; and C. isolate the protein of interest. The cells of this invention are preferably mammalian, more preferably CHO cells. The cyclin D gene product is preferably mammalian, more preferably with the gene of human origin. The cyclin D gene or gene encoding the protein of interest (or both) can be comprised in an expression vector or vectors within the cell or can be integrated into the cell gene. Any number of proteins of interest can be used in the present invention. Specifically, EPO, OPG, leptin and NESP, and any derivatives thereof can be employed.

Brief Description of the Figures Figure 1 shows a western blot of used AM-1 cells transfected with pcDNA3.1 / cyclin DI and treated with a specific antibody of human cyclin DI (see Materials and Methods). This spot demonstrates the expression of human cyclin DI in AM-1 / D cells. Figures 2A, 2B, 2C, and 2D are histograms showing the relative DNA content (x-axis) and the cell number (y-axis) of unsynchronized cells of AM-l / D and AM-1 cell lines stained with iodide of propidium and analyzed by FACScan (Becton Dickinson, see Materials and Methods). These figures show that it speaks significantly more cells in the S phase in the AM-l / D cells. Figure 3 is a graph showing the number of clones expressing OPG-Fc at various levels. AM-1 / D cells were transfected with plasmid pDSRa2 / 0PG-Fc as described in Example 1 below. This graph shows that the highest expression clones were derived from the AM-1 / D cell line. Figure 4 is a graph showing the expression of EPO plotted against the concentration of MTX used for amplification. AM-1 / D cells were transfected with plasmid pDSRa2 / hEPO as described in Example 2 below. Two of the clones show high EPO production with increasing concentrations of MTX, demonstrating the successful amplification of the gene. Figure 5 shows a comparison of OPG (22-194) -cys-Fc expression levels in 46 individual clones derived from AM-1 CHO cells or AM-1 / cyclin D CHO cells. Clones derived from AM-1 / cyclin D CHO cell line show significantly higher expression levels of OPG (22-19) -cys-Fc than "derived" clones of AM-1 CHO cells.

Figure 6 shows a comparison of OPG (22-201) -Fc expression in the 24 highest expression clones as previously determined by western blotting. In each case, the clones derived from the cell line AM-1 / cyclin D CHO show significantly higher expression levels of OPG (22-201) -Fc than the clones derived from CHO (SF) cells. Figure 7 shows the average of NESP expression determined by EIA in the best 14 clones derived from each main CHO cell line. Samples were collected from the conditioned medium of 24-well plates, 2 collections per day. The samples were pre-protected with the western blot. Figure 8 shows the western IEF transfers from the centrifuge and bioreactor collections, which compare the isoform distributions of the secreted NESP protein from the centrifuge and bioreactor cultures. The centrifuge collections are of non-amplified clones. The normal NESP protein, purified from the conditioned medium of the rotating bottle, which shows the desired high molecular weight isoforms. The collections of the conditioned medium contain all the isoforms. The spots show the presence of high isoforms in all the samples.

Detailed Description of the Invention The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific cases. The terms "inserted" and "insert" of nucleic acids means that the nucleic acid is introduced into a cell or organism by external methods (eg, transfection). The inserted nucleic acid may have a sequence foreign to or may already be present in the genome of the cell. In the latter case, the inserted nucleic acid allows for greater expression. or differentially regulated protein encoded by the inserted nucleic acid. The term "EPO related protein" refers to erythropoietin and derivatives and analogs thereof that can be formed by recombinant DNA and other methods. The derivatives include proteins that have terminal truncations, the elimination of the internal amino acids (for example, by restriction and religation of the associated DNA), substitutions of the amino acid and the like. Derivatives also include fusion proteins (e.g., with an Fc region). The exemplary derivatives of erythropoietin are described in International Patent Applications WO 91/05867, 94/09257, 88/03808, and 86/07594, each of which is incorporated by reference herein. The term "leptin-related protein" refers to leptin, preferably human leptin, and derivatives thereof which can be formed by recombinant DNA and other methods. Derivatives include proteins that have terminal truncations, elimination of internal amino acids, amino acid substitutions, and the like. Also included within this definition are fusion proteins comprising leptin, such as a fusion protein comprising leptin and an Fc fragment. Suitable leptin derivatives are described in patent applications WO 96/40912 (filed December 19, 1996), WO 96/05309 (filed on February 22, 1996), WO 97/06816 (filed on October 27, 1996). February 1997), and WO 97/18833 (filed May 29, 1997), which are incorporated by reference herein The term "OPG-related protein" refers to OPG and derivatives thereof that can be formed by recombinant DNA and other methods Derivatives include proteins that have terminal truncations, elimination of internal amino acids (for example, by restriction and re-ligation of the associated DNA), substitutions of the amino acid and the like Derivatives also include fusion proteins ( for example, with an Fc region.) Exemplary OPG derivatives are described in International Patent Application WO 97/23614, which is incorporated herein by reference.

Preparation Process Gene Structures The nucleic acids used in the present invention can be prepared by recombinant nucleic acid methods. See, for example, the recombinant DNA methods of Nelles et al., J. Biol. Chem., 262, 10855 (1987). The nucleic acids can be derived from a variety of sources, including genomic DNA, subgenomic DNA, cDNA, synthetic DNA, and combinations thereof. Genomic and cDNA can be obtained in several ways. The encoded cells can be isolated by the desired sequence, fragmented genomic DNA (for example, by treatment with one or more restriction endonucleases), and the resulting fragments cloned, identified with a probe complementary to the desired sequence, and protected by the presence of a sequence encoded for the desired activity. For the cDNA, the cDNA can be cloned and the resulting clone is protected with a probe by the cDNA which is encoded for the desired region. In isolating the desired clone, the cDNA can be manipulated in substantially the same manner as genomic DNA. In addition to the cadmified sequence of the protein of interest, the gene structure must contain several regulatory regions. For expression, transcriptional and translational signals recognized by an appropriate host are necessary. The encoded sequence must also be linked to a promoter compatible with the host or host cell. The promoter can be inducible, allowing also the control of expression, or constitutive. Several of the appropriate promoters are known in the art. Alternatively, the promoter region of the genomic D can be obtained in association with the coding sequence. For extension the host cells recognizing the regulatory transcript and the translation initiation signals associated with the coding region, the 5 'region adjacent to the coding sequence can be retained and used for transcriptional and translational regulation. This region will normally include 'the sequences involved with the initiation of transcription and translation, such as the TATA box, the coronation sequence, CAAT sequence, and the like. Typically, this region will have at least about 150 long base pairs, more usually at about 200 bp, and rarely exceeding about 1 to 2 kb. The 3 'non-coding region can also be retained, especially especially for its transcriptional termination regulatory sequences, such as the stop signal and the polyadenylated region. In addition, the 3 'non-coding region may also contain an enhancer. Where the transcriptional termination signals are not satisfactorily functional in the host cell, then a functional 3 'region of a different gene can be substituted. The alternative of the substituted 3 'region will depend on the cellular system chosen for the expression. A wide variety of transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. The transcriptional and translational regulatory sequences can be derived from viral sources (eg, adenovirus, bovine papilloma virus, Simian virus, and the like) where the regulatory signals are derived from a gene that has a high level of expression in the host. Alternatively, promoters of mammalian expression products (eg, actin, collagen, myosin and the like) can be employed. Transcriptional initiation regulatory signals allowing repression or activation can be selected, so that the expression of the genes can be modulated. A controllable modulation technique is the use of regulatory signals that are sensitive to temperature, so that the expression can be repressed or initiated by changing the temperature. Another controllable modulation technique is the use of regulatory signals that are sensitive to certain chemicals. The structures may comprise an endogenous nucleic acid sequence for the host cell together with the promoter, the increaser, and other regulatory regions that may or may not be endogenous to the cell. The structures allow the expression of the endogenous sequence to be increased (for example, through operable coupling to a constitutive promoter) or controlled (for example, through operable coupling to regulatory signals). To form the structures of the gene, the DNA fragments can be ligated according to conventional techniques known in the art. Techniques include the use of restriction enzymes to convert fragments with sticky terminals to blunt ends (or vice versa), polymerases and nucleotides to fill sticky ends to form blunt ends, alkaline phosphatase to avoid unwanted ligatures, and links to join the fragments. The structures can be introduced into a cell for transformation along with a gene that allows selection where the structure will be integrated into the host genome (eg, homologous recombination). Normally, the structure will be part of a vector that has a repeating system recognized by the host cell.

Expression Vectors Expression vehicles for the production of the molecules of the invention include plasmids or other vectors. In general, vectors contain control sequences that allow expression in several cell types. Appropriate expression vectors containing the desired coding and control sequences can be constructed using recombinant DNA techniques known in the art, several of which are described in Sambrook et al., Molecular Cloning: - A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). An expression vector as contemplated by the present invention is at least capable of directing the replication and expression of the coding of the nucleic acid encoding the protein of interest or the cyclin D gene product. A class of vectors utilizes the elements of DNA that autonomously provide the extrachromosomal replication plasmids derived from the viruses of animals (for example, bovine papillomavirus, polyomavirus, adenovirus, or SV40). A second class of vectors remains on the integration of the desired gene sequences into the host cell chromosome. Expression vectors useful in the present invention typically contain an origin of replication, a 5 'promoter located at (i.e., upstream of) the DNA sequence to be expressed, and a transcription termination sequence. Suitable origins of replication, for example, include the ColEl origins of replication, pSClOl, M13, SV40 and EBV. Suitable termination sequences include, for example, bovine growth hormone, - SV40, lacZ and the polyhedral polyadenylation signals AcMNPV. Suitable promoters include, for example, the cytomegalovirus promoter, the lacZ promoter, the gal 10 promoter and the AcMNPV polyhedral promoter. The promoter sequence may also be inducible, to allow modulation of expression (eg, by the presence or absence of nutrients or other inducers in the growth medium). An example is the lac operon obtained from the bacteriophage lambda plac5, which can be induced by IPTG. Expression vectors may also include other regulatory sequences for optimal expression of the desired product. The sequences include the stability leader sequences, which provide the stability of the expression product; secretory conductive sequences, which maintain the secretion of the expression product; increasers, which increases the regulation of the expression of the DNA sequence; and the sequences that recognize the restriction enzyme, which provide the sites to be divided by the restriction endonucleases. All materials are known in the art and are commercially available. See, for example, Okayama, Mol. Cell Biol., 3, 280 (1983). An appropriate expression vector may also include the labeled sequences, which allow phenotypic selection of the transformed host cells. A label can provide prototrophy in an auxotrophic host, biocidal resistance (e.g., antibiotic resistance) and the like. The selectable marker gene can either be directly linked to the coding sequences to be expressed, or introduced into the same cell by co-transfection. Examples of selectable markers include genes for neomycin resistance, ampicillin, hygromycin resistance and the like. The characteristics of the current expression vectors used must be compatible with the host cells to be used. For a mammalian host, for example, the expression vector may contain promoters isolated from the genome of mammalian cells, (e.g., the mouse metallothionin promoter), or viruses that grow in these cells (e.g., the 7.5 promoter). K of vaccinia virus). Suitable commercially available expression vectors where the encoded sequences of the present invention can be inserted include the mammalian expression vectors pADNc I or pADNc I / Neo (which is preferred), the baculovirus expression vector pBlueBac, the expression vector prokaryotic pADNc 'II and yeast expression vector pYes2, all of which can be obtained from Invitrogen Corp., San Diego, California.

Host Cells The present invention additionally involves host cells that contain the expression vectors comprising the DNA sequences for the protein of interest and the cyclin D gene product. The appropriate host cells are eukaryotic cells; for example, Spodoptera frugiperda insect cells, COS 7 cells, human fibroblasts, and Saccharomyces cerevisiae cells. Mammalian cells that may be useful as hosts include cells of fibroblast origin (eg, VERO or CHO-Kl) or of lymphoid origin (eg, SP2 / 0-AG14, or P3x63Sg8) or derivative thereof. The preferred mammalian host cells are CHO cells. Immortalized cells, such as myeloma or lymphoma cells, are also appropriate host cells. These cells can grow in an appropriate nutrient medium in culture flasks or can be injected into a synergistic host (e.g., mouse or rat) or an immunodeficient host or host site (e.g., nude mouse or hamster pouch). In particular, the cells can be introduced into the abdominal cavity for the production of ascites fluid and collected from the chimeric molecule. Alternatively, cells can be injected subcutaneously and antibodies collected from host blood. The cells can be used in the same way as the hybridoma cells. See Diamond et al., N. Eng. J. Med., 304, 1344 (1981); Monoclonal Antibodies: Hybridomas-A New Dimension in Bioiogic Analysis (Kennatt, et al., Eds.) Plenum (1980). Expression vectors can be introduced into the host cells by various methods known in the art. For example, transfection of host cells with the expression vectors can be carried out by the calcium phosphate precipitation method. However, other methods for introducing expression vectors into host cells (eg, electroporation, liposomal fusion, nuclear injection, and viral or phage infection) can also be employed. Host cells that contain an expression vector can be identified by one or more of the following six general methods: (a) DNA-DNA hybridization; (b) presence or absence of the functions of the marker gene; (c) evaluation of the level of transcription as a measure for the production of mRNA transcripts that encode the gene structures in the host cell; (d) immunologically detecting the gene product; (e) enzyme assay; and (f) PCR. These methods are well known in the art; see, for example, the North American Patent. No. 5,744,314 which is incorporated by reference herein. The expression vectors and DNA molecules of the present invention can also be sequenced. The various sequencing methods are known in the art. See, for example, the dideoxy chain termination method described in Sanger et al., Proc. Nati Acad. Sci. USA 74, 5463-7 (1977), and the Maxam-Gilbert method described in Proc. Nati Acad. Sci USA 74, 560-4 (1977). Once an expression vector has been introduced into an appropriate host cell, the host cell can be cultured under conditions that allow expression of larger amounts of the protein of interest. The protein of interest can be isolated and purified according to conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, and the like. The preferred method is affinity chromatography.

It must, of course, be understood that not all expression vectors and DNA regulatory sequences will work equally well to express the nucleic acids of the present invention. None of all the host cells will work equally well with the same expression system. However, one skilled in the art can make a choice between expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope and spirit of the present invention. invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Further descriptions of the specific embodiments of the present invention are detailed below. These embodiments are exemplary and serve to illustrate the extensive applicability of the present invention. Unless otherwise indicated in the specification, these embodiments comprise preferred elements of the invention.

Materials & Methods Plasmids The Human-Cyclin DI gene (access Genbank # M64349) was cloned into pSDNA 3.1 (Invitrogen) and constitutively expressed under the cytomegalovirus promoter / enhancer (CMV) to generate cDNA 3.1 / cyclin Di. The vector also carries the neomycin resistance gene for the selection of stable mammalian cell lines in the presence of G418.

Cell culture CHOd (SF) cells grew in 100 mm plates in the complete medium. { DMEM (Gibco / BRL), 5% of Fetal Bovine Serum (JRH Biosciences), 1% non-essential amino acids (Gibco / BRL), 1% hypoxanthine / thymidine (HT) (Gibco / BRL), and 1% glutamine / penicillin / streptomycin (Gibco / BRL)} . The cells were trypsinized, counted, and cultured in 60 mm plates (Falcon), at cell Ixl0e6 / plate. The AM-1 CHOd cells grew in the centrifuge culture in suspension in the Amgen VM-soybean medium. Cells were counted, centrifuged, resuspended in complete medium, and cultured in 60 mm plates at lxlOed cell / plate. The cells were incubated overnight. Three hours before transfection, the medium in the cells was replaced with 4 ml of fresh medium. Transfection of Ciclina DI. This transfection using the ADNpc3.1 neo® vector (Invitrogen) contains the insertion of cyclin DI cDNA. For each plate of 60 mm of cells, 5 ug of ADNpc3.1 / cyclin DI (2 mg / ml) and 5 μg of mouse genomic DNA carrier (Clontech) (100 μg / ml) were added to 172.5 μl of sterile distilled water. Twenty-five microliters of 2.5 M CaCl2 were added to the DNA solution, giving the final volume of 250 μl. As a control of the vector, 5 μg of the pneumatic DNA vector (1126 mg / ml) was added along with 5 μg of the DNA carrier, to 170.5 μl of sterile water, followed by 25 μl of 2.5 M CaCl; . As a simulated control, 25 μl of 2.5 M CaCl was added to 225 μl of water. The DNA solutions were added dropwise to an equal volume (250 μl) of 2X HBS (280 mM NaCl, 10 mM KCl, 1.5 mm NaHP042 H20, 0.2 mM dextrose, 50 mM HEPES, pH 7.05 ) through which the air was constantly bubbled. The DNA / HBS solutions were incubated at room temperature for 30 minutes. The medium was removed from the CHO cell plates and the DNA / HBS solutions (500 μl total volume per plate) were added dropwise. A total of 3 plates were treated with ADNpc3.1 / cyclin DI, and one plate for each vector and each simulated control for each cell line. The cells were incubated at room temperature - for 30 minutes, after 5 ml of the complete medium was added to each plate. The cells were then incubated overnight at 37 ° C. The next day, the medium was replaced with fresh medium. CHO (SF) cells reached confluence 48 hours after transfection. The cells were trypsinized and electroplated in the complete medium in a ratio of 1x60 mm per plate to 8x100 mm per plate. The next day, the medium was replaced with complete medium containing 1 mg / ml of Geneticin (G418) (Gibco / BRL). AM-1 CHO cells reached confluency 72 hours after transfection, and were similarly reelectroplated. The medium in the cells was replaced with the fresh complete medium + G418 twice a week. Ten days after the initial addition of the G418 selective medium, colonies were isolated from the cyclin DI CHO (SF) and AM-1 CHO transfected plates using glass cloning (War) cylinders. The cells were trypsinized and recultivated in 24-well plates. The frequency of trapsfection of CHO (SF) cells was much higher (>100 colonies / plate) than that of AM-1 CHO cells (20-30 colonies / plate). A total of 72 colonies were isolated from the CHO (SF) / cyclin DI plates, and 68 colonies from the AM-1 CHO / cyclin DI plates. No colony was present in any group of simulated control plates containing the selective medium of G418. After the colonies were collected, the remaining cells from each group of plates were trypsinized and combined into 100 mm plate culture wells (3 wells for CHO (SF) / cyclin DI, and 2 wells for AM-1 CHO / cyclin DI). The transfected cells grew to confluence and then re-electroplated in 6 well plates, 2 wells per clone / well. To reach confluence, one cell well for each clone / well was lysed with 250 ml at 1% Triton lysis buffer £ 1% Triton XlOO, 1 mM NaV03, 50 mM Tris / HCl pH 8, 100 mM NaCl, protease inhibitors). The used cells were centrifuged at 14 K for 15 minutes. The supernatants were collected and stored at -20 ° C. The cells used were analyzed by a western blot for the expression of cyclin DI. 25 μl of each sample plus the control samples of the CHO (SF) and AM-1 CHO lines were mixed with 5 μl of the 5X PAGE gel sample buffer, boiled for 3 minutes, and charged to 12% gel Novex Tris-glycine. The gels were electrotreated in 0.2 μ nitrocellulose filters (Schleicher and Schuell). The filters were probed with the anticycline DI (Calbiochem) Ab-3 monoclonal antibody and developed using the ECL reagents from Amersham. The results indicated vary in degrees of overexpression of cyclin DI in the culture wells and in most of the clones. The two culture wells of AM-1 CHO / cyclin DI and the two highest expressions of the CHO (SF) / cyclin DI wells were combined in the "main wells" for each cell line to be used in the subsequent transfections . Main well cultures were tested to determine whether they have retained the negative DHFR phenotype by growing the cells in the hypoxanthine / thymidine-free selective medium (DMEM, 5% dialyzed fetal bovine serum (Hyclone), non-essential amino acids , glutamine / penicillin / streptomycin). No cell growth was detected in this medium.

TRANSFECTION OF EPQ The main well cultures of CHO (SF) / cyclin DI and AM-1 CHO / cyclin DI were maintained in the complete CHOd medium supplemented with 1 mg / ml of G418. 60 mm to 8xl0e5 cells / plate and incubated all the noch, e The cells were transfected using the above protocol with DNA labeled EP0 / pSW19 (a proximal derivative of pDSR 2) /, DNA control vector of pDSRa2, and a DNA carrier of herring sperm (Gibco / BRL) A final concentration of 5 μg / plate of pSWI9 / EP0 DNA was used.Twenty-four hours after transfection, the cells were given to the fresh medium.Seventy-two hours after transfection, the cells were trypsinized and reelectroplated in the selective medium + 1 mg / ml G418 in a ratio of 1: 8, 1:10, and 1:20 (60 mm plate to 100 mm plates). The cells were replaced twice a week with the fresh medium containing G418. After re-electroplating, 48 colonies were isolated from the transfected CHO (SF) / cyclin Dl / EPO plates. The remaining cells were trypsinized and combined in two culture wells. Fifteen days after re-electroplating, 48 colonies were isolated from the transfected plates of AM-1 CHO / cyclin Dl / EPO, and the remaining cells were combined in two wells. EPO expression was analyzed by western blotting in the serum-free conditioned medium that is harvested from the cultures of 24 confluent wells of isolated clones or cultures in 100 mm plates of the wells. The gels were carried out under reducing conditions, and the spots were probed with the 2D8 anti-EPO monoclonal antibody.

OPG TRANSFECTIONS Transfections of the DNA structure of pSWI9 / OPG in the main wells of CHOd- (SF) / cyclin DI and AM-1 CHOdVcycline DI, and the main line of CHOd ~ (SF) were carried out the same way as EPO transfections. Two separate transfections of OPG-Fc (22-201) were carried out in the three host cell lines. A total of 124 CHO (SF), 110 CHO (SF) / cicD, and 147 colonies of AM-1 CHO / cicD were harvested from OPG-Fc transfections (22-201). The expression of OPG was analyzed in the resulting clones and the wells by means of the western blot using the anti-huIgG-Fc-HRP antibody (Pierce), or a rabbit purified affinity polyclonal anti-huOPG.

Propidium iodide staining for DNA The cells were plated in 100 mm dishes and allowed to grow at approximately 50% confluence. The cells must be in the registration phase for the assay. The cells were harvested by trypsinization and pipetted into a centrifuge tube containing an equal volume of DMEM / FBS. An aliquot of cell suspension was counted with a hemocytometer. The cell density should be approximately 10 ^ -107 cells / 5 ml.

The cells were pelleted by centrifugation at 10000g and washed twice with ice cold PBS. The cells must be in a suspension of a single cell. 0.5 ml of PBS was added and the cell suspension was vortexed. The cells were then fixed by adding 2 ml of 70% ethanol dropwise down the side of the tube while mixing the cell suspension gently. The minimum fixing time is 30 minutes. (At this point the cell suspension can be stored for approximately 2 weeks at 4 ° C before staining and analysis). The cells were centrifuged to remove the supernatant of ethanol, washed once with PBS and allowed to rehydrate for 5 minutes at 4 ° C. The cells were centrifuged, the PBS was removed, and the pellets were resuspended in 0.5 ml of propidium iodide solution (Molecular Probing) (50 μg / ml prepared in PB?). 10 μl of DNAse-free RNase (10 mg / ml) (Boehringer Mannheim) was added and the cell suspension was incubated at 37 ° C for 20 minutes. The samples were diluted with 1 ml of PBS and analyzed by FACS for 1 hour.

Gene Amplification The amplification of the gene in the cells was carried out by the cautious addition of the step-wise step of methotrexate (MTX) to the growth medium. The cells were subcultured in a ratio of 1:10 in 100 mm plates in three low concentrations of methotrexate (1 nM, 2.5 nM, and 5 nM). The growth of the cells was monitored and the plate that reached the confluence was first used for the next amplification cycle. If the cells grew equally well at more than one methotrexate concentration, the highest concentration was used for the next cycle. A collection of 48-hour serum-free DMEM (4 ml / plate) was taken for the analysis of the level of protein expression by means of the western blot or EIA. The cells were allowed to recover in the complete medium with methotrexate for 24 hours and then subcultured at a ratio of 1:10 at 3 higher concentrations of methotrexate. The concentration of MTX was increased in increments of 10 nM to 100 nM. In general, if the level of expression of the protein has not reached a peak per 100 nM, amplification is continued in increments of 100 nM until a maximum expression level is reached. The cells were maintained in the presence of MTX once the optimal concentration was determined.

Generation of the AM-l / D cell line The AM-l / D cell line was derived from the CHOd (-) cell line described in U.S. Patent No. 4,703,008, issued October 27, 1987 and Urlaub and collaborators (1980), Proc. Nati Acad. Sci. 77: 4461, the lines are incorporated herein by reference. The cell line of CHOd (-) was adapted passing in the VM-SOJA medium with the decreased levels of and finally in total absence of the fetal bovine serum. The serum-free cell line resulting from AM-1 still retains the dhfr (-) phenotype. The AM-1 cell line was designed for the over-expression of human cyclin DI. The pCp3.1 / cyclin DI was transfected into the AM-1 cell line by the standard procedure of calcium phosphate precipitation for transfection. In the selection with G418, the colonies were trypsinized and combined in two wells. Expression of the human cyclin DI protein was demonstrated by Western blots (Figure 1) of the Used cells prepared from the wells using a cyclin-specific human DI antibody (anti-cyclin DI Ab-3, Calbiochem). Several random colonies collected with cloning rings were picked and the Used cells were also prepared and analyzed. As expected, the expression of human cyclin DI was variable among the clones. Compared with the clones, the two wells exhibited high global levels of human cyclin ID protein (Figure 1). The two wells were combined in a main well culture (AM-l / D), which was the main cell line used in the subsequent experiments.

Functionality of the human Cyclin DI expressed in the cell line AM-l / D We determined that the human cyclin DI was functionally expressed by its alteration of the cell cycle dynamics; for example, see Figure 2 and later. The unsynchronized cells of AM-1 / D and AM-1 cell lines were stained with propidium iodide and analyzed by FACScan (Becton Dickinso?). Figures 2A-2D show a relative DNA content (x-axis) and cell number (y-axis). Hμbo significantly more cells in the S phase for the AM-l / D - cells (40.08%, 40.14%) compared to the AM-1 cells (33.73%, 32.25%). This demonstrated that the overexpression of human cyclin DI in the AM-l / D, a CHO cell line, was functional to significantly alter the dynamics of the cell cycle. Example 1: Plasmid pDSRa2 / 0PG-Fc was constructed to express a fusion protein of OPG (GenBank access # U94332) -Fc human with expression vector of pDSRa2. Using the normal calcium phosphate precipitation, the linearized DNA of pDSRa2 / OPG-Fc was transfected in parallel in the AM-1 / D cells and in the original unmodified AM-1 cells. 44 colonies of each transfection were harvested randomly after the cells were placed on the selective medium lacking HT supplements for two weeks. The colonies were transferred individually into 24-well plates and grown to confluence. After 48 hours, the serum-free conditioned media was collected and analyzed by EIA using the antiserum specific for human OPG. Figure 3 shows the number of clones expressing OPG-Fc at various levels of these two cell lines. It is clear that there are significantly more clones of AM-l / D cell lines expressing higher levels of the recombinant protein, and that all of the higher expression clones expressing more than 20 mg / ml of OPG-Fc were derived from the cell line of AM-l / D.

Example 2: The pDSRa2 / hEpo plasmid was constructed to express the human Epo gene and transfected into MA-l / D cells following the calcium phosphate method described above. Forty-eight colonies were collected at random for further analysis. Four of the high expression clones were identified by the EIA analysis of the conditioned medium and subjected to the amplification of methotrexate starting at 1 nM. Figure 4 shows the expression of EPO during the amplification process. Two of the clones, clone 2 of AM-l / D and clone 33 of AM-l / D showed high Epo production with increasing concentrations of MTX, demonstrating successful gene amplification. These clones were tested frozen and thawed, and the extensive steps without loss of Epo expression, demonstrating the stability of Epo gene expression in these clones.

Expression of OPG in cells of AM-1 / cyclin D CHO Expression of two OPG structures was compared in AM-1 / cyclin D CHO cells and other CHO cell lines. The OPG (22-201) -Fc was transfected into AM-1 / cyclin D CHO cells and the main CHO cell line that has been previously adapted to growth in serum-free medium [CHO (SF)]. The OPG (22-194) »cys-Fc was transfected into the cells of AM-1 cyclin D CHO and its main AM-1 CHO cell line. The transfections were carried out using the standard calcium phosphate method and the selection of DHFR. The transfected colonies were isolated using the glass cloning cylinders. The individual colonies grew for the confluence in plates of 2 wells. The serum free conditioned medium collections were analyzed for OPG secreted by western blot using a polyclonal antiserum against OPG. The conditioned medium samples from the clones showing the highest expression levels by western blotting were further analyzed by ELISA. OPG (22-194S) -cys-Fc expression levels were compared in 46 individual clones derived from AM-1 CHO cells or AM-1 / cyclin D CHO cells (Figure 5). OPG (22-201) -Fc expression was also compared in the 24 highest expression clones as previously determined by western blotting (Figure 6). In each case, clones derived from the AM-1 / cyclin D CHO cell line showed significantly higher levels of expression than clones derived from CHO (SF) or AM-1 CHO cells.

Expression of NESP in CHO cells Adapted to serum-free medium NESP DNA inserted into the expression vector of pDSR * 2 was transfected by the standard calcium phosphate method into three different CHOd-adapted serum-free cell lines: CHOd "(SF), AM-1, and AM-1 / cyclin D. Transfection, selection, and isolation of clones were carried out in the medium containing serum in the adherent culture.The clones expressing high levels of NESP were Initially identified by the western blot using a monoclonal antibody to EPO, the quantification of the expression was elaborated by EIA.The highest expression clones of each cell line were tested for growth in the suspension culture in the serum-free medium. The clones also underwent DNA amplification by growing at gradually increasing concentrations of methotrexate in the medium containing serum in the adherent culture. P during the amplification process was monitored by western blotting and EIA analysis. At various stages during the amplification process, the clones were analyzed for growth and expression in the serum free suspension culture. The clones derived from the AM-1 / cyclin D CHO cell line had a higher overall level of NESP expression than those derived from the other two main CHO cell lines (Figure 7). The clones were also analyzed by isoelectric focusing to determine the isoform distribution. No significant difference was observed in the isoform distributions in the different clones or in a control cell line (61L) derived from the main CHO cell line (Figure 8). The abbreviations used throughout this specification are defined as follows: CHO Chinese hamster ovary DHFR EBV Epstein-Barr virus EIA Immunoassay of enzyme EPO erythropoietin HBS saline buffered with HEPES EF MTX methotrexate NESP new protein that stimulates erythropoiesis OPG osteoprotegerin It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, property is claimed as contained in the following:

Claims (20)

1. A eukaryotic cell characterized in that it comprises a) a cyclin D gene product, wherein the cyclin D gene product is functionally expressed in the cell at a level greater than any native level of expression; and b) a protein of interest, wherein the protein of interest is expressed in the cell at a level greater than any native level of expression.

2. The cell according to claim 1, characterized in that the cell is a mammalian cell.

3. The cell according to claim 1, characterized in that the cell is a CHO cell.

4. The cell according to claim 3, characterized in that the cell is adapted to the serum free medium.

5. The cell according to claim 1, characterized in that the cell is an AM-1 cell.

6. The cell according to claim 1, characterized in that the cyclin D gene product comprises a mammalian cyclin DI.

7. The cell according to claim 1, characterized in that the cyclin D gene product comprises a human cyclin D.

8. The cell according to claim 1, characterized in that the cyclin D gene product comprises a human cyclin DI.

9. The cell according to claim 1, characterized in that the protein of interest is an EPO-related protein, an OPG-related protein, or a leptin-related protein.

10. The cell according to claim 1, characterized in that the protein of interest is EPO, NESP, OPG, OPG-Fc, leptin or Fc-leptin.

11. A process for producing a protein of interest characterized in that it comprises: A. Creates a eukaryotic cell that expresses 1. a cyclin D gene product at a level greater than any native level of expression and 2. a protein of interest at a higher level than any native level of expression; B. culturing the cell under conditions that allow expression of the protein of interest and functional expression of the cyclin D gene product; and C. isolate the protein of interest.

12. The process according to claim 11, characterized in that the cell is a mammalian cell.

13. The process according to claim 11, characterized in that the cell is a CHO cell.

14. The process according to claim 13, characterized in that the cell is adapted to the serum-free medium.

15. The process according to claim 11, characterized in that the cell is an AM-1 cell.

16. The process according to claim 11, characterized in that the cyclin D gene product comprises a mammalian cyclin DI.

17. The process according to claim 11, characterized in that the cyclin D gene product comprises a human cyclin D.

18. The process according to claim 11, characterized in that the cyclin D gene product comprises a human cyclin DI.

19. The process according to claim 18, characterized in that the protein of interest is an EPO-related protein, a protein related to OPG, or a leptin-related protein.

20. The process according to claim 19, characterized in that the protein of interest is EPO, NESP, OPG, OPG-Fc, leptin or Fc-leptin.