JP5209693B2 - Methods for expressing recombinant proteins in CHO cells - Google Patents

Description

The present invention relates to methods for expressing recombinant product genes in CHO cell lines, as well as recombinant CHO host cells and novel expression vector constructs.
[Background technology]

The Chinese hamster ovary cell (CHO) mammalian expression system is widely used for the production of recombinant proteins. With the exception of lymphoid cell lines, such as hybridoma cell lines, this is one of the few cell types that allows simple and effective high-density suspension batch culture of animal cells. . In addition, they are capable of very high product yields and are relatively resistant to metabolic stress, but lymphocytes are difficult to culture on an industrial scale. In view of the considerable costs incurred, it is most important to maximize the yield of recombinant protein per bioreactor run. Media composition selection and bioreactor design and operation are parameters that affect yield and optimization will be very complex. Furthermore, as expected, an increase in the strength or transcriptional activity of the promoter that controls the expression of the product protein also increases the yield. Increments at the single cell level would translate into a significant improvement in product yield in dense batches or fed-batch cultures, with stationary densities at cell densities in the range of 10 ⁶ to 10 ⁷ cells / ml The gene expression of is shown.

  US Pat. No. 5,866,359 describes a method for enhancing expression from an originally strong hCMV promoter in CHO and NSO cells by co-expressing adenoviral E1A protein from a weak promoter. E1A is a multifunctional transcriptional regulator that may act on cell cycle regulation and has functional domains that independently activate and repress transcription. Fine-tuning E1A expression to an appropriate low level of expression achieves an ideal balance during gene transactivation while co-expression to avoid negative effects on cell cycle progression Critical for the success of the approach. Disadvantages, apart from careful selection of promoters that drive E1A expression, in addition to expressing the recombinant protein of interest, this system blocks some of the protein synthesis capacity of cells expressing E1A. End up.

  WO 95/17516 describes mice for targeting expression vector constructs to highly active loci of B-cell lineage lymphocytes, such as the widely used NSO myeloma cells. Describes the use of the immunoglobulin gamma 2A locus. NSO cells are essentially mouse plasma cells or B cell tumor cell lines. The chromatin that houses the immunoglobulin loci is in its fully active open state in B cells only, and has a higher natural immunoglobulin promoter or recombinant expression construct incorporated into these loci. It can be transcriptional activity.

  Disadvantageously, because of the principle of homologous recombination, the targeting sequence is efficiently only in mouse cell lines that match the sequence of the gamma 2A targeting sequence that accommodates recombinatorial hot spots. For high levels of expression, the gamma 2A locus region must be a transcriptionally active genomic region, limiting its effectiveness for homologous recombination against B cell types The

The object of the present invention is to invent another expression system for CHO protein expression in biotechnology that can enhance expression from standard promoters. Surprisingly, according to the present invention, this object is achieved by providing a gene expression vector for CHO cells with gene targeting sequences originally invented for homologous recombination of mouse B cells. Is done.
[Disclosure of the Invention]

[Best Mode for Carrying Out the Invention]

  According to the present invention, the DNA sequence for expression of the recombinant gene in mammalian cells includes a recombinant product gene and a promoter for expressing the recombinant product gene, preferably a CMV promoter, and expression from the promoter. Further comprising a DNA sequence of a mouse immunoglobulin gamma 2A locus or a fragment or sequence variant thereof that can enhance According to the present invention, such DNA sequences are useful expression vector constructs for the expression of recombinant product genes in CHO cells.

According to the present invention, a method for expressing a recombinant protein comprises:
a mouse IgG2A locus DNA or DNA sequence variant or DNA fragment thereof that is active in CHO cells and is transfected with an expression vector containing a promoter that drives expression of the recombinant product protein and enhances the activity of the promoter; A step of culturing further CHO cells,
b. collecting the product protein;
including.

  A recombinant product gene according to the present invention is a product protein that is required to be expressed or collected in large quantities. This may be a subunit of any protein of interest, for example a therapeutic protein such as an interleukin or enzyme, or a multimeric protein such as an antibody or fragment thereof. The recombinant product gene may include a sequence portion encoding a signal sequence capable of secreting a polypeptide once expressed from the host-producing strain cell. In a further preferred embodiment of the invention, the product protein is a secreted protein. More preferably, the first or product protein is an antibody or engineered antibody, or a fragment thereof, most preferably an immunoglobulin G (IgG) antibody.

  The DNA sequence of the mouse immunoglobulin gamma 2A locus (IgG2A) is originally used as a genomic targeting sequence to generate stable recombinant lymphoid B cell lines that show high expression of the recombinant gene product. Invented in International Publication No. 95/17516. B lymphocytes or plasma cells usually express very high levels of immunoglobulin RNA from the Ig heavy chain locus, presumably due to cell type-specific enhancer / transcription regulator activity and open chromatin structure. The preferred mouse immunoglobulin gamma 2A gene sequence of the present invention is the same as the targeting sequence used in WO 95/17516. This is a 5.1 kb BamHI genomic fragment containing all of the mouse Ig gamma 2A coding region except for the most 5 'part of the CH1 exon (Yamawaki-Kataoka, Y. et al., Proc. Natl. Acad. Sci). USA (1982) 79: 2623-2627; Hall, B. et al., Molecular Immunology (1989) 26: 819-826; Yamawaki-Kataoka, Y. et al., Nucleic Acid Research (1981) 9: 1365-1381). According to the present invention, the promotion of site-specific homologous gene recombination is not a property related to the immunoglobulin gamma 2A gene sequence (IgG2A). Accordingly, it is preferably functional in enhancing expression of the recombinant product gene from a promoter, preferably from the hCMV promoter described below, either under transient or stable expression conditions of CHO cells, or Any variant sequence or sequence fragment or variant sequence fragment of an IgG2A gene sequence that can be enhanced is encompassed by the present invention.

  Such “functional” variants include, for example, base insertions, deletions, or point mutations, and may be performed by methods well known in the art, eg, primer-directed PCR, “error” -Library transfer following PCR-reassembly of overlapping DNA fragments called "error-prone" PCR, "gene-shuffling" or following random mutagenesis of bacterial clones in vitro And functional selection in CHO cells. For example, random mutagenesis can be achieved by alkylation chemistry or UV irradiation as described in Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory 1972). Optionally, a natural displacement-inducing strain of the host bacterium may be used.

  Preferably, such variant sequences or sequence fragments are at least 65%, more preferably 75%, most preferably 90% homologous to the corresponding portion of the native mouse immunoglobulin gamma 2A locus. Is. For example, a SalI restriction site is inserted into the naturally occurring StuI site 39 base pairs upstream of membrane exon 2 (M2) to provide a unique site for linearization within the immunoglobulin gamma 2A sequence Such sequence variants were originally invented for site-specific recombination targeting, but can be used with the present invention as well.

  A “promoter” is defined as a DNA sequence that causes RNA polymerase to bind to DNA and initiate RNA synthesis. According to the present invention, this is a promoter that is active in CHO cells. Such a promoter is preferably a strong promoter. Strong promoters are those that cause mRNA initiation at high frequency in CHO-KI cells, equivalent to or higher than the hCMV core promoter / enhancer fragment (as described in US Pat. No. 5,168,062). Such a promoter may be a cell type dependent strong promoter, as cited in US Pat. No. 5,858,932, or preferably a ubiquitously active strong promoter, more preferably, For example, SV40 virus early and late promoters, human cytomegalovirus (hCMV) or mouse cytomegalovirus (mCMV) immediate early promoter, herpes simplex virus thymidine kinase promoter (TK), or Rous sarcoma virus terminal repeats A constitutively active viral promoter such as a promoter (RS-LTR), more preferably by a 2.1 kb PstI fragment as described in US Pat. No. 5,385,839 and / or European Patent No. -323997-A1 HCMV-MIE promoters defined or functional with these promoter activities A minute. A hCMV promoter construct having the complete first functional intron of the major immediate early (MIE) gene of hCMV, as described in European Patent No. 323997-A1, is particularly preferred according to the present invention. It is an aspect.

  Preferably, the hCMV promoter used in the present invention lacks the “modulator” sequence portion of the upstream / enhancer portion of the promoter. "Modulator" sequences have been found to be deleterious in the range of position -750 to position -1150 with respect to hCMV promoter activity and MIE transcription start site in CHO cells, especially in transient transfection (Meier et al., 1996). , Intervirology 39: 331-342, Regulation of hCMV immediate-early gene expression). Without the modulator sequence, the enhancing effect of the presence of the IgG2A host spot sequence on the (CM-negative or short mod-) hCMV promoter is even more pronounced.

  Transient transfection is characterized by not applying any selection pressure against the selectable marker carried by the vector. A pool or batch of cells resulting from transient transfection is a pool of cells that contain cells that have taken up and expressed foreign DNA and cells that have not taken up. Cells expressing the foreign expression cassette usually do not yet have the DNA transfected into these genomes yet, and transiently transfected cell pools lose the foreign DNA and transfect in the population. There is a tendency to overgrow the affected cells. Thus, expression is strongest in the period immediately after transfection and decreases with time. Preferably, transient transfectants according to the present invention are understood to be cells that are maintained in cell culture in the absence of selective pressure for up to 90 hours after transfection.

  Preferably, the transfected CHO host cell according to the present invention is a stably transfected host cell, especially in combination with the hCMV promoter as described above. Stable transfection means that newly introduced foreign DNA is usually integrated into genomic DNA by a random, non-homologous recombination event; in the case of vector sequences, Transfection may result in the loss of portions of the vector sequence that are not directly involved in the expression of the recombinant product gene, such as bacterial copy number control regions that are redundant due to genomic integration. The transfected host cell has incorporated at least a portion of the expression vector or a different portion within the genome. Similarly, in CHO cells with two or several DNA fragments that at least in vivo yield the essential elements of the expression vector of the invention, i.e. the product gene under the control of a suitable promoter and the functional equivalent of a hot spot IgG2A sequence. Transfection is also included in the definition of such transfected host cells. In vivo construction of functional DNA sequences after transfection of fragmented DNA is described, for example, in WO 99/53046. Such stable integration can also result in double minute chromosomes in CHO cells upon exposure to additional selective pressure for gene amplification. This is included in “stable” in the present sense. Due to the random genomic integration of the expression vector of the invention in CHO, the presence of the targeting sequence enhances the promoter activity for the expression of the recombinant product protein. Such an effect has not been observed and cannot be predicted by homologous gene targeting of mature mouse B cell lines including plasmacytoma / myeloma cell lines; where the IgG2A locus is recombination-inducible The IgG2A targeting sequence only served to increase the frequency of high yielding homologous components, since it was found to be a “recombinatorial” “hot spot”. As stated previously, chromatin in the genomic region of immunoglobulins is an open and highly active state in optimally targeted B cell lines.

  An “expression vector” is defined herein as the DNA sequence required for transcription and translation of these mRNAs in a suitable mammalian host cell after transfection of the vector. A properly constructed expression vector should usually contain: at least one expressible marker selectable in animal cells, an expression cassette for the recombinant product gene under the control of the upstream promoter region. A limited number of useful restriction sites for insertion. In particular, when used only for transient / episomal expression, this is an Epstein-Barr virus (EBV) or SV40 virus for self-renewal / episomal maintenance in eukaryotic host cells It may further include an origin of replication such as origin, but may lack a selectable marker. Expression vectors can, for example, reciprocate and produce in linear DNA fragments, DNA fragments that contain nuclear targeting sequences or are specifically optimized for interaction with transfection reagents, animal viruses, or bacteria. Suitable plasmids that can be (but are not limited to). Any commonly used selectable marker such as thymidine kinase (tk), dihydrofolate reductase (DHFR), or glutamine synthetase (GS) may be used. In a preferred embodiment, an expressible GS selectable marker is used (Bebbington et al., 1992, High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker, Bio / Technology 10: 169-175 1990, High level expression of tissue inhibitor of metalloproteinases in Chinese Hamster Ovary (CHO) cells using Glutamine synthetase gene amplification, Bio / Technology 8: 662-667). The GS system is one of only two systems that are particularly important for the production of therapeutic proteins. Compared to dihydrofolate reductase (DHFR), the GS system is often able to generate highly productive cell lines from early transfectants and is therefore selected to achieve gene amplification Increased concentrations of agents are present, avoiding the need for multiple selections and providing many times the benefits during development (Brown et al., 1992, Process development for the production of recombinant antibodies using the glutamine synthetase ( GS) system, Cytotechnology 9: 231-236). Similar to the second transcription unit for the expression of the marker gene, the expression unit can be used, for example, by the use of internal ribosome entory sites commonly used in the art. Of course, a monocistronic expression cassette can be used for both. Vice versa, the expression cassette for the product protein comprising the hotspot IgG2A sequence of the invention and the promoter and / or marker cassette need not act cis in a single expression vector; It goes without saying that this could be done successfully with separate co-transfected vectors, or the DNA fragment could then be chromosomally integrated at a single, chained integration site.

  A further object of the present invention is a CHO host cell transfected with the DNA sequence of the present invention. A further object is a method for transfection of such host cells and a method for the expression of recombinant product genes in such host cells. The descriptions and references made in the preferred embodiments of the invention herein relate to all these further objects of the invention as well. It should be noted that a host cell transfected with a DNA sequence or vector of the invention should be construed as a transient or stably transfected cell line. Any transfection technique known to those skilled in the art, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc., is used according to the present invention, if appropriate for a given host cell type. be able to.

A suitable host cell line can be any Chinese hamster ovary (CHO) cell line (Puck et al., 1958, J. Exp. Med. 108: 945-955). The term “host cell” refers to a cell that can proliferate in culture and expresses the desired protein recombinant product protein. Suitable cell lines include, for example, CHOK1 (ATCC CCL-61), CHO pro3-, CHO DG44, CHOP12, or dhfr-CHO cell line DUK-BII (Chassin et al., PNAS 77,1980, 4216-4220), or DUXB11 (Simonsen et al., PNAS 80, 1983, 2495-249). In CHO cells, the immunoglobulin locus is inactive, and thus chromatin is packed densely or closed. Thus, any genetic construct incorporated into an immunoglobulin locus, due to the specific state of chromatin, unless it itself contains flanking locus control regions that facilitate opening of the chromatin on both sides of the expression cassette, Inability to produce high levels of expression of the recombinant protein. Furthermore, the immunoglobulin gene sequences and in particular their intron parts show considerable differences between species, for example between mice and hamsters.
The promoter or enhancer element of the immunoglobulin locus is species and tissue specific and should be active only in B cells. The mouse IgG2A sequences of the present invention also enhance CHO cell gene expression in the absence of any native immunoglobulin promoter that causes full-length transcripts encoding complete IgG heavy chains. Preferably, the IgG2A sequence of the present invention lacks such a promoter. Surprisingly, the mouse IgG2A targeting sequence further improved gene expression in CHO cells by transient transfection of CHO cells with an expression vector according to the present invention (FIG. 1); Transient expression is a further preferred embodiment of the method according to the invention. In transient expression assays that generally occur about 20-50 hours after transfection, the transfected vector is maintained as an episomal element and has not yet been integrated into the genome.

For example, as described in US Pat. No. 5,563,162, suitable media and culture methods for mammalian cell lines are well known in the art. Examples of standard cell culture media for laboratory flasks or low density cell cultures and suitable for specific cell type requirements are: Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, lp. 173 ff, 1963), Dulbecco Eagle modified medium (DMEM) ), Eagle's minimum essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p288 ff. 1965), or modification of Iscoves lacking albumin, transferrin, and lecithin DMEM (Iscoves et al., J. Exp. Med. 1, p. 923 ff., 1978). For example, Ham's F10 or F12 media was specifically designed for CHO cell culture. Other media specifically adapted for CHO cell culture are described in EP-481791. Such media can be supplemented with fetal calf serum (FBS, also called fetal calf serum FCS), which provides a natural source of a plethora of hormones and growth factors. It is known that it can be done. Mammalian cell culture is today a routine procedure well documented in scientific textbooks and manuals, such as R. Ian Fresney, Culture of Animal cells, a manual, 4 ^th edition, Wiley- Covered in detail in Liss / NY, 2000.

  Preferably, the cell culture medium according to the present invention lacks fetal calf serum (FCS or FBS) and is called “serum free”. Cells in serum-free medium generally require insulin and transferrin in serum-free medium for optimal growth. Transferrin may be at least partially substituted with a non-peptide chelating agent such as tropolone or siderophore or combined with an antioxidant such as vitamin C, as described in WO 94/02592 Advantageously, the level of inorganic iron source may be increased. Most cell lines are composed of one or more synthetic cells including, for example, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factors I and II (IGFI, IGFII), etc. Growth factors (including recombinant polypeptides) are required. Other classes of factors that may be required include: prostaglandins, transport and binding proteins (eg, ceruloplasmin, high and low density lipoproteins, bovine serum albumin (BSA)), hormones including steroid hormones, and fatty acids . Testing of polypeptide factors is best done by testing new polypeptide factors in a step-wise fashion in the presence of what has been found to be growth stimulating. These growth factors are synthetic or recombinant. There are several methodological approaches that are well known in animal cell culture and typical examples are described below. The first step is to transfer the cells from serum-supplemented medium to obtain conditions that survive and / or grow slowly after 3-6 days. For most cell types, this is due at least in part to the effect of seeding density. Once optimal hormone / growth factor / polypeptide supplementation is found, the inoculation density for survival will decrease. In a more preferred embodiment, the cell culture medium is protein-free without both fetal serum and other proteins such as individual protein growth factor supplementation or recombinant transferrin.

A possible embodiment of one method of the invention, ie the expression and recovery of the recombinant product protein is a high density culture of animal host cells, for example in a fed-batch bioreactor. Conventional downstream processing may then be applied. As a result, a high density culture medium must be used. Such high density culture media are usually defined as all amino acids, energy sources such as glucose in the range given above, inorganic salts, vitamins, trace elements (inorganic compounds normally present at final concentrations in the micromolar range). Buffer, 4 nucleosides or their equivalent nucleotides, antioxidants such as glutathione (reduced), vitamin C, and critical membrane lipids such as cholesterol or phosphatidylcholine, or lipid precursors such as choline or inositol Can supplement the nutritional properties of other ingredients. A high density medium is thought to be enriched in most or all of these compounds and is GB2251249 compared to RPMI1640, except for inorganic salts where the osmolarity of the isotonic medium is regulated. These contain higher amounts (enhanced) than the standard medium from previous surgery that can be received from. Preferably, the high density medium according to the present invention has an enhanced balance in that all amino acids except tryptophan are above 75 mg / l medium. Preferably, in conjunction with general amino acid requirements, glutamine and / or asparagine is greater than 1 g / l, more preferably greater than 2 g / l in high density media. In the context of the present invention, high density cell culture is defined as a population of animal cells having a temporary density of live cells of at least 10 ⁵ cells / ml or more, or preferably at least 10 ⁶ cells / ml or more, This population was grown continuously from single cells or lower viable cell density in cell culture medium at constant or increased culture volume.

In a further preferred embodiment, the fed-batch method controls glucose concentration with a separate feed, as described in GB2251249, with at least glutamine, optionally with one or several other amino acids, preferably glycine. Separately, it is a culture system that supplies cell cultures in a medium to maintain these concentrations. Preferably, the feed of more glutamine and optionally one or several other amino acids is one or more energies such as glucose to the cell culture as described in EP-229,809-A. Combined with feeding sources. Feeding is usually initiated 25-60 hours after the start of culture; for example, it is useful to begin feeding when cells reach a density of about 10 ⁶ cells / ml. In cultured animal cells, "glutaminolysis" (McKeehan et al., 1984, Glutaminolysis in animal cells, in: Carbohydrate Metabolism in Cultured Cells, ed. MJ Morgan, Plenum Press, New York, pp. 11-150) Is well known in the art to be an important source of energy during the growth phase. Total glutamine and / or asparagine feed (see Kurano, N. et al., 1990, J. Biotechnology 15,113-128 for replacement of glutamine with asparagine) is usually 0.5-10 g per culture volume, preferably Range from 1-2 g per culture volume; other amino acids that can be present in the feed are 10-300 mg total feed per liter of culture, especially glycine, lysine, arginine, valine, isoleucine, and leucine Is usually higher than at least 150-200 mg of other amino acids. The feed can be added as a shot-addition or continuously as a pump feed, preferably the feed is pumped almost continuously into the bioreactor. It will be appreciated that the pH is carefully controlled during the fed-batch culture in the bioreactor by adding a base or buffer to an approximately physiological pH that is optimal for a given cell line. When glucose is used as an energy source, the total glucose feed is usually 1-10, preferably 3-6 grams per liter of culture. Apart from the amino acid content, the feed preferably contains a low amount of choline in the range of 5-20 mg per liter of culture. More preferably, such a choline feed is combined with ethanolamine supplementation, particularly in combination with glutamine feeding, essentially as described in US Pat. No. 6,048,728. The use of the GS-marker system requires a small amount of glutamine compared to non-GS expression systems because the accumulation of excess glutamine in addition to that produced endogenously results in ammonia production and associated toxicity. Needless to say. For GS, glutamine in the medium or feed is largely replaced by its equivalents and / or precursors (ie, asparagine and / or glutamic acid).

  An expression vector comprising at least a (first) transcription unit for a product gene that yields a product protein upon expression in a host cell, the transcription unit being under the control of a mouse cytomegalovirus promoter (mCMV promoter) It is a further independent object of the present invention to invent an expression vector that further comprises a second transcription unit comprising a glutamine synthetase (GS) marker gene. Such a product gene or gene of interest (GOI), which may be named, can be, for example, an immunoglobulin coding sequence. A glutamine synthetase marker gene is any enzymatically active GS coding sequence, which is a natural gene sequence or a variant thereof. The above definition of “functional variant” as described above applies here as well and includes the preferred range of sequence homology. Preferably, the GS marker gene is a mammalian GS marker gene or a derivative thereof. Surprisingly, such an expression vector is much higher for transfection in CHO cells than, for example, an expression vector in which the first transcription unit containing the gene of interest is under the control of the hCMV promoter Transfection rate can be achieved. In CHO cells, despite the fact that the transcriptional activity of the mCMV promoter is much higher than that of the hCMV promoter; usually, transfection reduces the survival of clones by transfection at higher metabolic loads and is less It is believed to produce a number of transfectants. Thus, there is no possibility that this effect correlates in an obvious manner with the amount of product protein expressed or unexpected toxicity, the latter probably adversely affecting the growth of transfectants. Indeed, this finding is the exact opposite of any prediction of those skilled in the art.

  A further object according to the invention is such an animal host cell, in particular a CHO cell, which has been transfected with such an expression vector, which can be maintained episomally or can be stably integrated into the genome. , And transfection methods. Similarly, transfection of animal cells, particularly CHO cells, in vivo with two or more gene fragments that yields the functional equivalent of the transcription unit of interest of the present invention in vivo is possible for such transfected host cells. Within the definition. Preferably, the host cell is a stably transfected cell meaning that the first and second transcription units are chromosomally integrated.

  A further object is preferably a first transcription unit for at least the product gene, wherein the first unit yields a product protein by expression in a host cell, and the first transcription unit comprises: When using an expression vector further comprising a first transcription unit under the control of the mouse cytomegalovirus promoter (mCMV promoter), and further comprising a second transcription unit comprising a glutamine synthetase (GS) marker gene, Use of the mCMV promoter to increase the transfection rate of CHO cells. It could also be transfected as first and second expression in different vectors or as isolated gene fragments containing individual expression units. Furthermore, CHO cells that have been pre-recombined and expressed with a first transcription unit containing mCMV could be transfected. According to the present invention, “increasing the transfection rate” is defined as the transfection rate in the presence of the mCMV promoter and expression vector according to the present invention, wherein the expression vector mCMV promoter is defined in US Pat. No. 5,658,759. And, for example, the same transfection rate under the same transfection and cell culture conditions except that it has been replaced with the hCMV-first intron enhancer / promoter construct as described in SEQ ID NO: 3 of the present invention Defined by comparison with the expression vector and host cell. For a given identical product gene, this hCMV-intron MIE-promoter construct serves as a standard for determining the required effect of enhanced transfection rates. Preferably, use of the mCMV promoter results in at least a 10-fold increase in transfection rate.

  Furthermore, all relevant definitions above apply equally to the independent object of the invention. It should be emphasized that the object of the present invention does not require the presence of the murine IgG2A targeting sequence as a prerequisite.

Murine cytomegalovirus (mCMV) is a member of a very diverse group of herpesviridae. There may be wide variation even between cytomegaloviruses of different host species. For example, mCMV differs significantly from human cytomegalovirus (hCMV) with respect to biological properties, immediate early (IE) gene organization, and overall nucleotide sequence. In addition, the 235 kbp genome of mCMV lacks most internal and terminal repeat features of hCMV. Thus, the isomeric form of the MCMV genome does not exist (Ebeling, A. et al. (1983), J. Virol. 47, 421-433; Mercer, JA et al. (1983), Virology 129, 94-106). In accordance with the present invention, it is possible to use a promoter region that is essentially identical to the large approximately 2.1 kb PstI fragment described in US Pat. No. 4,686,615 or any functional fragment thereof. In a more preferred embodiment, the mCMV promoter fragment used includes a transcription start site (+0) and extends from the upstream region to about position −500 . Surprisingly, such a fragment was found to promote expression more potently than a promoter cassette extending 800 base pairs further upstream from position -500 . In a most preferred embodiment, the core promoter region extends from the upstream region of the transcription start site, but extends from the natural transcription start site to the XhoI restriction site at about position -150, or is located at position -100 from the natural transcription start site The one that can be extended to the upstream region is used. It goes without saying that the transcription initiation site may be designed to include appropriate restriction sites for insertion of the recombinant product gene.

  According to the present invention, the first transcription unit under the control of the mCMV promoter can also accommodate at least one intron sequence. Such treatment is well known in the art to stabilize RNA transcripts and to promote efficient protein synthesis from the corresponding mRNA. However, it is not desirable to include the first natural intron of mCMV in the mCMV promoter construct in order to effectively synthesize proteins without considering the required effect on transfection rates. In contrast to the situation in the hCMV promoter (see US Pat. No. 5,916,639), such a natural first intron of mCMV was found to reduce the expression of the recombinant gene from the mCMV promoter. Are therefore excluded in a further preferred embodiment.

Examples of preferred possible embodiments of the GS marker gene cassette are listed in the sequence listing. SEQ ID NO: 1 (pEE15.1hCMV / GFP + hotspot) + 2 (pEE14.4hCMV / GFP) is an example of a suitable GS gene cassette expressed from the SV40 (early and late) promoter, a weak to moderate promoter, respectively And further includes an expression cassette for GFP (green fluorescent protein) under the control of the hCMV promoter. SEQ ID NO: 1 describes the GS cDNA sequence described in more detail in FIG. 3 under the control of the SV40 early promoter. SEQ ID NO: 2 identifies an artificial GS-minigene cassette containing an intron under the control of the late SV40 promoter. CHO cells are not naturally glutamine auxotrophs, and thus, for example, Cockett et al., 1990, High level expression of tissue inhibitor of metalloproteinases in Chinese Hamster Ovary (CHO) cells using Glutamine synthetase gene amplification, Bio / Technology 8: 662 The selection scheme described in -667 can be applied. Examples of suitable transfection methods for CHO cells are listed therein as well; for example, classical calcium phosphate precipitation or more modern lipofection techniques can be used. Transfection rates are usually expressed as the number of positively transfected cells (transient transfections) or clones (stable transfections after the selection period), obtained from a pool of cells subjected to transfection. Defined. The effect meant for the purposes of the present invention is for example SEQ ID NO: 3 (pEE12.4 hCMV-GFP + SV40 early promoter / GS cDNA) or SEQ ID NO: 4 (pEE12.4 mCMV-GFP + SV40 early promoter / GS cDNA) Either plasmid can be seen by transfecting CHO-K1 cells by lipofection (any commercial reagent and manufacturer protocol). Transfected cells may be cultured in any conventional medium. The medium may be supplemented with fetal serum as defined above or a serum-free medium. Preferably, the cell culture medium is a serum-supplemented medium, more preferably at least 1% (v / v) fetal serum, such as fetal bovine serum or fetal calf serum, most preferably at least 5% (v / v ) Cell culture medium supplemented with fetal serum. As another preferred example, the transfection method is performed by electroporation.

Experiment 1
Transient and stable expression of GFP vectors containing hot spot sequences in CHO-K1 cells
CHO-K1 cells (ATCCCCL-61) were adapted and cultured in normal cell culture medium GMEM-S (Gibco, UK) supplemented with 10% FCS. For GS selection, the medium must be completely glutamine free as described in Table 1 below; this requires the use of dialyzed FCS. All cultures were performed in rotating shake flasks at 125 rpm at 36.5 ° C. Lipofectin (Superfectin ™, Gibco, UK) was used for transfection and the green fluorescence of the transfectant pool was measured by FACS with 488 nm excitation. For all GS / GFP vector constructs, 5 transfections were performed independently and all data were averages from 5 independently analyzed pools. Starting with transient transfectants 48 hours after transfection, select 10% of the top-scoring of highly expressing cells in the live cell pool in the number of cells against the fluorescence diagram and average Fluorescence was determined (Figure 1). Viable cell populations were preselected by gating on the forward diagram gates for lateral scatter.

  To generate stable transfectants, GS markers are regularly supplemented with glutamine-free medium with 25 μM MSX (methionine sulphoximine, Crockett et al., Ibd.) For 26 days. Selection was made 24 hours after transfection by continuing cell culture while dividing the culture. Note the effect of medium levels of other amino acids on the ability of MSX for selection. See Bebbington et al., US Pat. No. 5,827,739. Then, fluorescence analysis was performed again as outlined above (FIG. 2).

Cells that were not transfected served as negative controls. A hotspot vector (pEE15.1 “hCMV + hotspot”) that drives GFP expression under the control of the hCMV promoter containing the first complete intron of CMV is given in SEQ ID NO: 1, The pEE15.1 vector is shown in FIG. 3, in which the GFP sequence is inserted into the EcoRI restriction site of the polylinker. PEE12.4 “hCMV” corresponding to SEQ ID NO: 3 is the same as “pEE15.1hCMV + hotspot” except that it does not contain a 5.1 kb BamH1 fragment containing the IgG2A sequence. pEE12.4 served as a vector control. An additional vector control, pEE12.4 “hCMV (Kozak-)”, contains a Kozak sequence at the cloning site that contains the translation initiation site (GCCGCCACCATGG), a primer-directed mutation (Sambrook et al., Molecular cloning, Cold Spring Harbor 1983). ) To weaken the original Kozak sequence and translation initiation site (ACCATGGTCCATGG) by mutating to a frame-shifted functional Kozak sequence. The vector of SEQ ID NO: 1 further deletes the 400 base pair modulator region of the hCMV enhancer portion, deletes the enhancer element ˜750 upstream from the transcription start site, and pEE15.1 “hCMV (mod −) / GS cDNA Was designed to produce. By exchanging the GS minigene of pEE14.4 “hCMV (mod-)” / GFP and the GS cDNA cassette of pEE15.1 (s. FIG. 3) corresponding to SEQ ID NO: 2, the vector pEE15.1 “hCMV (mod-) ) “/ GS minigene”. Thus, all transfected cells contained a plasmid vector containing the GFP coding sequence. The GS minigene contains a single, first intron of the GS gene and approximately 1 kb of 3 ′ flanking DNA under the control of the late SV40 promoter; the 3 ′ portion of the genomic GS DNA is transfected into cells Are believed to produce high copy number vector DNA, and thus GS (see US Pat. No. 4,770,359, Bebbington et al.). All hCMV vectors used in this study express the GS marker gene from its cDNA sequence, but the use of the GS minigene is included as an additional control to eliminate the strong effects of GS copy number and expression level. It is done.

  Transfection was performed with the linearized hotspot vector pEE15.1 “hCMV + hotspot” vector for generation and expression analysis of stably transfected CHO cells. A plasmid linearized with SalI is cleaved with IgG2A, which contains a free DNA-terminal sequence that potentially stimulates recombination with a genomic region that shares a certain degree of homology with the flanking DNA portion, and then hamster CHO cells The potential targeting effect of mouse IgG2A was tested. PvuI can be cleaved with a bacterial lactamase marker gene and thus promote heterogeneous random recombination. In fact, the mean fluorescence was high in transfectants linearized with PvuI, so that vector linearization has some influence and targeting to the immunoglobulin locus of CHO cells is not responsible for the effects of the present invention. Show both things. In addition, the effect of enhanced promoter activity was observed consistently in the transiently transfected population of cells and correlated well with the relative strength of the individual vector constructs. Clearly, genomic integration is not involved in this early stage of transfection.

  FIG. 3 shows the pEE15.1 vector of about 12830 BP. Detailed descriptions of GS markers and hCMV-p / intron expression cassettes can be found in US Pat. No. 5,827,739 and US Pat. No. 5,916,39. pEE15.1 is a possible embodiment of an expression vector according to the present invention, except that the DNA sequence encoding the recombinant product protein has not yet been inserted into the polylinker site. The complete 13535 base pair sequence of the pEE15.1 construct containing GFP is given in SEQ ID NO: 1 where the GFP coding sequence is in frame to the EcoRI restriction site centered at base position 12814. The introduction of a unique restriction site that accommodates the ATG start codon and optimizes the Kozak sequence environment of the start codon is detailed in US Pat. No. 5,916,393. Thus, expression of the GFP protein is under the control of the hCMV-major immediate early gene promoter (hCMV-MIE or hCMV for short), followed immediately by the first intron of the hCMV-MIE gene, followed by the NeoI site (US Pat. No. 5,916,939). issue). Polyadenylation is ensured by the SV40 polyA site further downstream of the polylinker insertion site. pEE15.1 further contains a cDNA sequence that is under the control of the SV40 early promoter and encodes a hamster-derived glutamine synthetase (GS) following the SV40 intron + polyA. IgG2A locus or “hot spot” sequence (shaded box representing heavy chain constant region, hinge, membrane anchor CH1, Hi, CH2, CH3, M1, M2) has already been published in WO 9517516 and Is a 5.1 kb BamHI fragment of the mouse IgG2A locus described in the literature cited in. Unique restriction sites PvuI and SalI are indicated.

Experiment 2
Electroporation of CHO cells with mCMVp12.4-GFP construct (SEQ ID NO: 4) Adherent CHO-K1 cells (ATCCCCL-61) contain 2 mM glutamine and are essentially EP supplemented with 10% FCS. Cultured in Isakov DMEM medium described in -481791. Optionally, prior to the GS marker selection described in Experiment 1, the G-MEM medium described in Table 1 and further containing 2 mM glutamine can be used. Cells were detached and pelleted and finally resuspended twice in serum-free medium at a density of 5.3 × 10 ⁶ cells / ml. A total of 4 × 10 ⁶ cells were electroporated per 750 μl electroporation batch. Electroporation was performed as described in Methods in Molecular Biology, ed. JA Nickoloffed, Humana Press 1995, Vol. 48 / Chap. 8: Animal cell electroporation and electrofusion protocols. p12.4 mCMV-GFP vector DNA (SEQ ID NO: 4) was linearized. 50 μl (20 μg) DNA was added to 750 μL cells in an electroporation cuvette and electroporated with -300 Volts / 750 μFd—expected electroporation time of approximately 12-14 milliseconds. After electroporation, in a T75 flask (containing 10% fetal serum but no glutamine, see Table 1 for details), select a GS with an 800 μl volume of 25 ml modified Glasgow- Transferred to MEM (GMEM, Gibco) medium. Divide into 2 × T75 flasks by transferring 12.9 ml to the second flask and incubate at 37 ° C. in 10% CO ₂ overnight.

  The next day, 37.5 ml of GS-selected GMEM was supplemented with 10% FBS + 33.3 μM MSX (methionine sulfoximine). Therefore, MSX was finally ˜25 μM. After an additional 26 days of incubation, transfectants were counted by the number of colonies per flask. By microscopic visual inspection with a standard inverted microscope for visual inspection of the culture flask, positive colonies become bright and bright green and can be easily counted.

  The mCMV construct of SEQ ID NO: 4 produced 20 times more lesions than cells transfected in parallel with the hCMV construct of SEQ ID NO: 3. The vector construct differed only in the CMV promoter element driving GFP expression, and the remaining vector portion of the vector was the same (GS-marker; including the cDNA GS-marker cassette of p12.4). When cells were diluted into 96-well plates immediately after transfection, more colonies were generated from mCMV transfected cells (> 400 colonies) than from hCMV transfected cells (approximately 45 colonies).

Table 1: Media for GS selection
A. Preservation solution
1. Autoclaved 400ml aliquot of double-distilled water
2. 10 x Glasgow MEM (GMEM) without Glutamine (GIBCO: 042-2541 in UK), stored at 4 ° C.
3. 7.5% sodium bicarbonate (GIBCO: 043-05080 in UK; 670-5080 in US). Store at 4 ° C.
4. 100 × Non-essential amino acids (NEAA) (GIBCO: 043-01140 in UK; 320-1140 in US). Store at 4 ° C.
5. 100 x glutamic acid + asparagine (G + A): Add 600 mg glutamic acid and 600 mg asparagine (Sigma). Make in 100 ml distilled water and sterilize by passing through a 2 μm sterile filter (Nalgene). Store at 4 ° C.
6. 1OOmM sodium pyruvate (GIBCO: 043-01360 in UK; 320-1360 in US)
7. 50 x nucleosides:
35mg adenosine
35mg guanosine
35mg cytidine
35mg uridine
12mg thymidine
(Each from Sigma). Make up to 100 ml with water, filter sterilize and store in aliquots of 10 ml at -20 ° C.
8. Dialyzed FCS (GIBCO: 014-06300). Heat inactivated at 56 ° C for 30 minutes and stored at -20 ° C. When using GS selection, it is important to use dialyzed FCS.
9. Penicillin-streptomycin at 5000 units / ml (P / S: GIBCO: 043-05070 in UK; 600-5070 in US).
10. Prepare 100 mm L.MSX (Sigma): 18 mg / ml PBS solution. Filter sterilized and stored at -20 ℃.
B. Medium preparation
To make GMEM-S Medium 1, add the following in the order given using aseptic technique.
1. 400ml water
2.10 × GMEM 50ml
3. Sodium bicarbonate 18.1ml
4. NEAA 5ml
5. G + A 5ml
6. Sodium pyruvate 5ml
7. Nucleoside 10ml
8. Dialyzed FCS 50ml
9. Penicillin-streptomycin 5ml
GMEM-S contains nonessential amino acids, alanine, aspartic acid, glycine, proline, and serine (100 μM), glutamic acid and asparagine (500 μM), and adenosine, guanosine, cytidine, and uridine (30 μM) and thymidine (10 μM).

  A possible embodiment of the invention is illustrated in the figure. What is shown is as follows.

Relative expression level of green fluorescent protein (GFP) from hCMV promoter in the presence of hCMV promoter and IgG2A hotspot sequence in transient transfection of CHO-K1 cells. Relative expression level of GFP from hCMV promoter in the presence of hCMV promoter and IgG2A hotspot sequence in stably transfected CHO-K1 cells. Plasmid map of hCMV-MIE expression vector with IgG2A targeting sequence.

Claims (7)

And immediate early promoter of the murine cytomegalovirus (mCMV), at least, mouse site and intron sequences not the first intron of the native cytomegalovirus, IgG gamma 2A locus of the mouse is a portion to further enhance the activity of the promoter CHO cells an expression vector transfected containing a 5.1 kb BamHl genomic fragment of DNA. The CHO cell according to claim 1, wherein the vector further comprises a transcription unit encoding a selectable marker . The CHO cell according to claim 1, wherein the vector further comprises a transcription unit encoding a glutamine synthetase (GS) marker . The CHO cell according to any one of claims 1 to 3, wherein the CHO cell is stably transfected. A method for expressing a recombinant protein comprising:
a. culturing CHO cells transfected with an expression vector containing a 5.1 kb BamHl genomic fragment of mouse IgG gamma 2A locus DNA that contains a mouse cytomegalovirus immediate early promoter and further enhances the activity of the promoter ; ,
b. collecting the product protein;
Including methods. 6. A method according to claim 5 , characterized in that the 5.1 kb BamHl genomic fragment of the mouse IgG gamma 2A locus DNA lacks the native immunoglobulin promoter. 6. The method of claim 5, wherein the mouse cytomegalovirus immediate early promoter or functional portion thereof having promoter activity comprises a natural transcription start site (+0) and extends upstream to position −500. A method characterized by that .

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