WO1989010959A1

WO1989010959A1 - Supertransformants for high expression rates in eukaryotic cells

Info

Publication number: WO1989010959A1
Application number: PCT/US1989/001816
Authority: WO
Inventors: Joel Hedgpeth; Linda Cashion; Wendy Colby; John Morser
Original assignee: Codon
Priority date: 1988-05-06
Filing date: 1989-04-28
Publication date: 1989-11-16

Abstract

This invention relates generally to recombinant DNA techniques and to the expression of mammalian polypeptides in genetically engineered eukaryotic cells. The invention specifically relates to transfection methods that increase levels of expression of a cloned gene product and eukaryotic cell lines derived from these methods. The disclosed invention teaches that multiple transfections with expression vectors each containing different selectable markers and an expression cassette encoding the same structural gene can produce unexpectedly high expression of that structural gene.

Description

SUPERTRANSFORMANTS FOR HIGH EXPRESSION RATES IN EUKARYOTE CELLS

BACKGROUND OF THE INVENTION

Field of the Invention

Background and Prior Art The ability to produce cloned gene products in eukaryotic cell culture is often desirable, particularly when the cloned gene encodes a eukaryotic polypeptide, since prokaryotes lack a number of elements contained in eukaryotic cells. Such elements include the eukaryotic signals for transport, modification and glycosylation. Proper eukaryotic post-translational modification is often necessary for normal function of the final recovered protein. The eukaryotic gene product is most similar to or the same as the natural gene product when the cloned gene is expressed in a eukaryotic cell.

Early transfection experiments of Wigler, M. et al (Cell 11: 223-232 1977) were effective but inefficient. Since Axel first described co-transformation using two plasmids to increase the efficiency of transfecting foreign DNA into host cells (United States Patent No. 4,399,216 and Wigler et al. Cell 16: 777-788 (1979)), it has become common to have a drug resistance selection marker and a non-selectable gene of interest on the same plasmid as separate transcription units, each with their own promoter

(Girli, I., Jouanneau, J. and Yabiv, M. , 1983, Virology 127: 385-396 and Southern, P.J., Berg, P., 1982, J. Mol. Appl. Genet. 1:327-341). Expression levels of the non- selectable gene vary greatly in the individual transfected clones arising from experiment to experiment.

The current methodology usually includes first selecting a population of cells containing the transfected plasmid on the basis of drug resistance. This population is then screened for the introduction of a non-selectable gene, either by assaying for the non-selectable gene product, or for the presence of the genetic material comprising the coding sequence of the non-selectable gene. This method allows for the stable introduction into cultured mammalian cells of any cloned gene, and the systematic isolation of such cells.

A major issue in this area of research is the need to develop systems which can provide high levels of expression of the gene of interest in a reliable and reproducible way. Several methods to improve gene expression have been used successfully.

Gene amplification is proven to be a method frequently used to increase expression levels. Gene amplification is often induced by exposure of sensitive cells to stepwise increases in antifolates, such as methotrexate (MTX), in the growth medium. This amplification can yield cells which are resistant to high levels of MTX and have increased levels of production of other associated genes as well (European Patent Application Nos. 0117059, and 00117060 and

Kaufman et al. (1985) Mol. Cell. Biol. 5: 1750-1759).

However, gene amplification has several drawbacks including the instability of resultant clones, and the need to grow cultures in presence of high level of carcinogens (Kaufman and Sharp (1982) J. Mol. Biol., 159:601-621). In addition, the increased production of foreign proteins in a host through gene amplification techniques has been primarily limited to the use of mutant hosts. This is often undesirable for a number of reasons, most notably, that the necessary mutant for the cell host most suitable for production of a specific foreign protein is typically unavailable. The ability to produce cloned gene products in eukaryotic cell culture is often desirable, particularly when the cloned gene encodes a eukaryotic polypeptide, since prokaryotes lack a number of elements contained in eukaryotic cells. Such elements include the eukaryotic signals for transport, modification and glycosylation. Proper eukaryotic post-translational modification is often necessary for normal function of the final recovered protein. The eukaryotic gene product is most similar to or the same as the natural gene product when the cloned gene is expressed in a eukaryotic cell.

Early transfection experiments of Wigler, M. et al (Cell 11: 223-232, 1977) were effective but inefficient. Since Axel first described co-transformation to increase the efficiency of transfecting foreign DNA. into host cells (United States Patent No. 4,399,216 and Wigler et.al. Cell 16: 777-788, 1979), it has become common to have a drug resistance selection marker and a non-selectable gene of interest on the same plasmid as separate transcription units, each with their own promoter (Girli, I., Jouanneau, J. and Yabiv, M., 1983, Virology 127: 385-396 and Southern, P.J., Berg, P., 1982, J. Mol. Appl. Genet. 1:327-341). Expression levels of the non-selectable gene vary greatly in the individual transfected clones arising from experiment to experiment.

Gene amplification is proven to be a method frequently used to increase expression levels. Gene amplification is often induced by exposure of sensitive cells to stepwise increases in antifolates, such as methotrexate (MTX), in the growth medium. This amplification can yield cells which are resistant to high levels of MTX and have increased levels of production of other associated genes as well (European

Patent Application Nos. 0117059, 00117060 and Kaufman et al. (1985) Mol. Cell. Biol. 5: 1750-1759).

However, gene amplification has several drawbacks including the instability of resultant clones, and the need to grow cultures in presence of high level of carcinogens (Kaufman and Sharp (1982) J. Mol. Biol., 159: 601-621).

In general, increased production of foreign proteins in a host through gene amplification techniques has been primarily limited to the use of mutant hosts. This is often undesirable for a number of reasons, most notably, that the mutant cell host most suitable for production of the foreign protein is unavailable. The expression of genes into fully active proteins commonly requires a specific cell type, which may not be available with the necessary mutation. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagrammatic gene map of plasmids pPA003, pPA502, pPA509, pPA510.

Figure 2 illustrates the steps leading to the construction of transfection plasmid pPA003. a. The DNA sequence of the amino terminal region of the t-PA gene extending from intron A through the coding region and 5' - untranslated region to intron A'. The dashed line shows the structure and sequence of the amino terminal region of the t-PA chromosomal gene as it is fused to t-PA cDNA to generate a hybrid genomic DNA-cDNA gene. b. The Nar I fragment of pPA103 extending from intron A' to a Nar I site in t-PA cDNA was inserted into pPA104. This allowed removal of a 3.3 Kb Bel I-Bgl II fragment containing the Bgl II site. This fragment was ligated into pneo5 resulting in pPA003, the genomic hybrid t-PA expression plasmid.

SUMMARY OF THE INVENTION This invention provides for supertransfected eukaryotic cell lines which express a desired gene product, comprising host cells transfected with a first expression vector, and a second expression vector, wherein the first expression vector comprises: a first selectable gene cassette and a gene cassette encoding the desired gene product; wherein the second expression vector comprises: a second selectable gene cassette and a gene cassette encoding said the desired gene product; with the proviso that the first and second selectable genes are different. Preferred selectable gene cassettes are those that contain sequences that encode for a protein selected from the group consisting of dihydrofolate reductase, neomycin phosphotransferase, and hygromycin phosphotransferase. The eukaryotic cell line may express desired gene products that are either non-selectable or selectable and the desired gene products can either be heterologous or endogenous to the host cell. The preferred non-selectable gene encodes tissue plasminogen activator preferably of the human variety. The eukaryotic cell lines are preferably selected from the group consisting of mammalian cells, insect cells or yeast cells. Most preferred are the cells designated CHL-1 (CRL 9446 ) and CHL-2 (CRL 9451). Even more preferred are those cells producing t-PA at a rate of at least about 4 milliunits per cell per day of t-PA. In addition to the above cell lines there is disclosed herein methods for producing protein comprising the step of transfecting of an eukaryotic host cell with: a) a first expression vector comprising: i) a first selectable gene cassette; and ii) a gene cassette encoding the protein and b) a second expression vector comprising: i) a second selectable gene cassette and ii) a gene cassette encoding the protein; and with the proviso that the first and second selectable genes are different and the step of culturing the cells under conditions which permit expression of the protein. The transfecting step can be either a co-transformation or preferably a sequential transformation wherein the cells .are transfected with the first expression vector, cultured and transfected a second time with the second expression vector. The preferred selectable genes are as described above. Where the transfections are sequential it is preferred that the culturing of the cells is under conditions selective for the particular selectable marker contained in the first expression vector. After the second transfection, the cells are recultured preferably under conditions selective for the particular selectable marker contained in the second expression vector. The preferred desired genes are those encoding t-PA, preferably human t-PA. The desired genes encoding t-PA can optionally, but preferably contained introns such as a cDNA/genomic hybrid gene. The preferred first expression vector for t-PA expression is pPA003.

The preferred cell lines for this method are those described above. For production t-PA it is preferred that the cell line contain an endogenous t-PA gene and most preferred as host cells are human melanoma cells.

DETAILED DESCRIPTION Novel methods using recombinant expression vectors are provided for increasing the expression levels of a desired protein in a eukaryotic cell. In accordance with the present invention, eukaryotic cells capable of such are obtained by transformation with at least two vectors each containing at least one selectable gene in addition to a structural gene encoding the desired polypeptide. Such vectors will also include appropriate regulatory sequences for self-replication, and selection in the appropriate host systems. The use of these vectors and cell lines transfected with these vectors represent a significant improvement over prior expression systems. By supertransfecting the cells using expression plasmids having expression cassettes encoding both the desired protein and a selectable marker which is different from the selectable markers previously used, one can achieve levels of expression that exceed the sum of the levels of desire protein produced under single transformations. The described method is operable and useful in a number of host cells which are adapted to tissue culture. Typically, the cells are eukaryotic, preferably human cells, that can grow rapidly in standard media preparations. Substantially any non-microbial cell, whether or not transformed, and which is prevented from growth by a selective agent will find use in the present invention. This invention involves a series of molecular genetic manipulations that can be achieved in a variety of known ways. Prototype vectors and cell lines have been deposited in accordance with the Budapest Treaty. Plasmids pPA003 and pPA509 maintained in an E. coli host and is illustrated in Figure 1. Plasmid pPA003 was transfected into the cell line CHL-1 and a stable transfectant, CHL-2 was isolated. These plasmids and cell lines were deposited with the American Type Culture Collection in Rockville, Maryland U.S.A. and given the accession numbers: pPA003 deposited on January 13, 1987 and assigned ATCC 67293; pPA509 deposited on June 18, 1987 and assigned ATCC 67443; CHL-1 deposited on June 18, 1987 and assigned CRL 9446; and CHL-2 deposited on June 19, 1987 and assigned CRL 945-1. The deposited plasmids and cell lines should not be construed as a limitation of this invention in any manner. Although a convenient starting material, the following will detail various methods available to create equally suitable expression cassettes from alternative starting materials.

A. General Methods

Generally, the definitions of nomenclature and descriptions of general laboratory procedures used in this application can be found in Maniatis, T. et al.,

Molecular Cloning, A Laboratory Manual, Cold Spring

Harbor Laboratory, Cold Spring Harbor, new York, 1982.

The manual is hereinafter referred to as Maniatis. All enzymes were used according to the manufacturer's instructions.

Nucleotide sizes are given in either kilobases

(kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis or from published DNA sequences. Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage S.L. and Caruthers, M.H. Tetrahedron Letts. 22 (20) :1859-1862 (1981) using an automated synthesizer, as described in Needham-VanDevanter, D.R., et al., Nucleic Acids Res., 12:6159- 6168 (1984). Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J.D. and Regnier, F.E., J. Chrom., 255:137-149 (1983).

The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam, A.M. and Gilbert, W., Grossman, L. and Moldave, D. , eds., Academic Press, New York, Methods in Enzymology, 65:499-560 (1980). Alternatively, the sequence can be confirmed after the assembly of the oligonucleotide fragments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double-stranded templates of Wallace, R.B., et al., Gene, 16:21-26 (1981). This invention relates to cloning and use of expression vectors in eukaryotic cells. Intermediate vectors are cloned for amplification in prokaryotes such as Escherchia, Bacillus and Streptomyces. Most preferred is E. coli because that organism is easy to culture and more fully understood than other species of prokaryotes. The Maniatis manual contains methodology sufficient to conduct all subsequently described clonings in E. coli. Strain MH-1 is preferred unless otherwise stated . All E. coli strains are grown on Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids. Strains with resistance to antibiotics were maintained at the drug concentrations described in Maniatis. Transformations were performed according to the method described by Morrison, D.A. (1977), J. of Bact.,

132:349-351 or by Clark-Curtiss, J.E. and Curtiss, R., 1983, in Methods in Enzymology, 101:347-362, Wu, R., Grossman, L. and Moldave, K., eds., Academic Press, New York. Representative vectors include pBr322 and the pUC series which are available from commercial sources. The host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, electroporation and microinjection of the DNA directly into the cells.

B. Cloning Vectors

Cloning vectors suitable for replication in prokaryotes or eukaryotes and containing transcription terminators useful for regulation of the expression of downstream structural proteins are described herein. The disclosed vectors are comprised of expression cassettes containing at least one independent terminator sequence; sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors; and selection markers for both the prokaryote and eukaryote systems.

In order to select the transformed bacteria, selectable markers must be incorporated into the cloning vectors. These markers permit the selection of bacterial colonies containing the vectors which one desire to replicate. Examples of selectable markers include for E. coli: genes specifying resistance to antibiotics, i.e., ampicillin, tetracycline, chloramphenicol , kanamycin, erythromycin, or genes conferring selectable enzymatic activities such as b- galactosidase. There are numerous other markers both known and unknown which embody the above scientific principles, all of which would be useful as markers to detect those bacteria transformed with the vectors embraced by this invention. C. Selectable Markers for Eukaryotic Cells

In order to select the transfected eukaryotic cells, suitable eukaryote markers must be incorporated into the cloning vectors. These markers may permit selection of transfected cells by virtue of survival in an otherwise lethal environment utilizing the same principles described for the prokaryote markers or the selectable markers may visibly alter the host cells allowing for easy detection. A general overview of this art is found in P.J. Southern P.J. and Berg, P. J. Mol. App. Gen. 1:327-41 (1982).

Examples of selectable markers include the dihydrofolate reductase gene (dhfr), the hygromycin B resistance gene (hmb) , the neomycin phosphotransferase II gene (neo) and the adenosine deaminase gene for the Bowes cells, Chinese hamster cells, C127 murine cells and other higher eukaryotic cells; and the enzyme, b-galactosidase and the nuclear polyhedral virus from Autographa californica for insect cell lines from Spodoptera frugiperda and Bombyx mori; and for yeast, Lue-2, URA-3, Trp-1, and His-3 are known selectable markers (Gene 8:17-24, 1979). There are numerous other markers both known and unknown which embody the above scientific principles, all of which would be useful as markers to detect those eukaryotic cells transfected with the vectors embraced by this invention.

It is also conceivable that the desired structural gene might also operate as a selectable marker and eliminate the need for a separate selectable marker in eukaryotic cell hosts.

For the preferred cell hosts Bowes cells expressing tissue plasminogen, the preferred selectable markers are neomycin phosphotransferase(neo) and hygromycin phosphotransferase (hmb). The neo gene confers resistance to kanomycin in bacteria and to the aminoglycoside antibiotic G418 in eukaryotes (Southern, P.J. et al., (1981) J. Mol. Biol. 150: 1-14). The hmb gene confers resistance to hygromycin B. (Biochlinger and Dinglemann, Mol Cell. Biol 2929-2931, 1984)

In another aspect, the selectable genes may be heterologous or endogenous to the host cell. Examples of heterologous selectable genes would be dhfr in CHO cells, neo in Bowes cells, CHO cells or Vero, or hmb in Bowes, CHO, or Vero cells. The selectable gene may also be an amplifiable gene. Alternatively, amplifiable genes can be used in addition. DHFR is an example of such a selectable/amplifiable gene.

In other instances, the expression vectors may comprise two or more selectable genes. This allows selection of transfectants using first one selection medium, and then a second selection medium. The level of expression of the non-selectable desired gene product increases with each selection procedure.

Whatever selectable gene is chosen, it will be operably linked to one or more regulatory sequences, e.g., the gene will be placed in the expression vector adjacent to a promoter so that expression can occur. Typically, the structural gene will have its own host cell compatible promoter. These promoter/gene cassettes will be adjacent to each other, preferably such that transcription occurs in a divergent manner. The eukaryotic cell lines useful in the practice of the present invention are wild-type or auxotrophic, such choice is governed by the particular selection marker used in the transfection plasmid. For example wild-type cells can be used with a dhfr selection marker although the wild-type cells have an endogenous dhfr gene, but drug resistance selection markers require a host cell which is not resistant to that drug.

In the preferred embodiment examples are given using either a monocistronic or discistronic expression arrangement. The essential difference is whether one promoter is operably linked to both the selectable gene and the non-selectable gene or whether the genes are transcribed from separate promoters. In the case of a monocistronic arrangement the expression vectors are constructed as follows: promoter - non-selectable gene - selectable gene - polyadenylation sequence. In the case of a distronic arrangement the expression vectors are constructed as follows : promoter- non-selectable or selectable gene- polyadenylation sequence.

D. Replication Origins

The cloning vectors must contain an origin of replication suitable for directing replication in prokaryotes. For maintenance in eukaryotic hosts the vectors must either contain an origin of replication usually of viral origin or have the capacity to integrate into the host genome. There are numerous examples of origin of replications for prokaryotes. E. coli replicons, which are the most closely studied, have origins of replication which are temperature dependent, high copy mutations or those which constitutively sustain plasmid copies at only lower moderate levels. Examples of E. coli origins of replication are Co1E1 ori, R1 ori R, or pSC101 ori. After transfection into eukaryotic cells,the plasmids will either integrate into the host's genome or remain extrachromosomally replicating. In the embodiment exemplified, the plasmids described integrate into the host cell. Examples of non-integrating vectors are those derivatives from the Epstein Bar virus and bovine papilloma virus. Yates et al., Nature, 313: 812-814 (1985). There are numerous origins of replication both known and unknown which embody the above scientific principles, all of which would be useful to maintain the plasmids within the prokaryotic and eukaryotic hosts transformed, transfected or infected with the vectors embraced by this invention. E. Eukaryote Host Cells

Of the higher eukaryotic cell systems useful for the expression of desired proteins, there are numerous cell systems to select from. Illustrative examples of mammalian cell lines include Bowes, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines. Cells suitable for use in this invention are commercially available from the American Type Culture Collection. Illustrative insect cell lines include Spodoptera frugiperda (fall Armyworm) and Bombyx mori (silkworm).

The disclosed example makes use of CHL-1 cells. These are derived from RPMI 7932 (Bowes melanoma cells), a readily available human cell line. The CHL-1 line is improved over the parental RPMI 7932 cell line. The CHL-1 cells, unlike the parental cells, have been cured of mycoplasma. contamination, which is an opportunistic organism that frequently contaminates cells in long term culture and interferes with large scale, i.e. commercial, use in a production scheme.

Further, the CHL-1 derivative can grow at high cell density than the parental cell line. CHL-1 cells are capable of growth to a density of 5 X 10⁷ cells/ml; whereas the parental line will grow to only 5 X 10⁶ cells/ml under the same conditions.

F. Eukaryotic Expression Cassettes

The eukaryotic expression cassettes are sequences of DNA which are functionally capable of directing the expression of proteins in a eukaryotic host. The cassettes are generically comprised of a functional promoter that permits the initiation of transcription, an operator, a ribosomal initiation site, at least one structural gene and an appropriate 3' portion encoding the polyadenylation signal sequences necessary to terminate the transcription process. An example of a terminator sequence is the polyadenylation sequence from SV40. The cassettes may optionally contain a signal sequence in the 5' region of the structural gene that will direct post-translational processing of the structural gene, i. Structural Genes The structural gene may be essentially any DNA sequence (e.g., cDNA or genomic clone with introns) that upon expression codes for a desired polypeptide, including heterologous proteins or endogenous proteins or proteins naturally produced by the host cell. Typically, it will be a eukaryotic structural gene, the protein product of which has a commercial utility, particularly in medical applications. Thus, the polypeptides (typically from about 5,000 to 300,000 MW) can be any variety of proteins, such as enzymes, immunoglobulins, hormones, vaccines, receptors and the like. Examples of proteins falling within the above categories include tissue plasminogen activator, Factor VIII:C, interferons, insulin, growth hormone, growth factors, erythropietin, interleukins 1, 2, and 3, and others, as new genes are cloned and expressed. Importantly, the entire structural gene for the naturally occurring protein need not be expressed, as fragments or subunits may be produced as desired. If necessary, the proteins will contain leader sequences, transmembrance sequences or the like, depending upon the particular ultimate utility.

The precise structural gene incorporated into the expression vector is not crucial to the practice of the present invention. In the preferred embodiment the gene for tissue plasminogen activator (t-PA) has been used to describe the current invention. The structural gene encode tissue plasminogen activator is not selectable by growth in any defined medium. T-PA production must be determined by assay of surviving transfeetants. Other suitable non-selectable genes include are human growth hormone, bovine growth hormone, erythropoetin, IgE, calf chymosin, glycosylation inhibiting factor (GIF), and urokinase. ii. Promoters

There are an unlimited number of promoters available for expression in eukaryotes and the following is not meant to be a limitation of this invention nor a list of preferred promoters. The following promoters are simply some of the more well studied promoters available for expression in various hosts and alternative promoters would work equally well in this invention. Promoters useful for regulating the expression of heterologous proteins in mammalian or insect systems include but are not limited to the following: the retroviral long terminal repeat promoters (Nature, 297:479-483, 1982), SV-40 promoter (Science, 222:524-527, 1983), thymidine kinase promoter (Banerji, J., Rosconi,S., Schaffner, W., 1982 Cell

27:299 - 308 ) , or the beta-globin promoter (Luciw,P.A., Bishop,J.M., Varmus, H.E., and Capecchi, M.R., 1983 Cel 33: 705 -716). For yeast promoters, the following are useful for expression: GAL1,10 (Mol. and Cell. Biol., 4:1440-48, 1984); ADH2 (J. Biol. Chem. 258 : -2674-2682, 1983); PHO5 (EMBOJ. 6:675-680, 1982); and MFα1 (Herskowitz, I. & Oshima, Y. (1982).

The promoters of preferred for Bowes cells which are the preferred cell type for expressing tissue plasminogen activator will typically be any of the well known viral promoters derived from Retroviruses, Adenovirus or Simian Virus 40 (SV40). (See, generally, Winnacker, E.-L., Introduction to Gene Technology, VCH Publishers, N.Y. (1987), which is incorporated herein by reference.)

G. Regulation of Expression

This invention relates to the expression of desired proteins in eukaryotic host cells. It is expected that those of skill in the art are knowledgeable in the expression systems chosen for ultimate expression of any chosen protein and no attempt to describe in detail the various methods known for the expression of proteins in eukaryotes will be made. However, several general references are available which describe in detail the processes for expression of proteins in eukaryotic cell systems. These references cite additional references which give even greater detail. For example, the expression of proteins in yeast is generally described in Methods in yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory (1982). Methodology for protein expression in higher eukaryotes is generally described in

Eukaryotic Viral Vectors, Ed. Yakov Gluzman, Cold Spring Harbor Laboratory, 1982; and in B. Howard and M. McCormick, Vector Mediated Gene Transfer (pp. 211-233); I. Abraham, DNA mediated gene transfer (pp. 181-210) in Molecular Cell Genetics, Ed. Michael M. Gottesmann, John Wiley & Sons (1985) and J.H. Miller and M.P. Calos, Gene Transfer Vectors for Mammalian Cells, Cold Spring Harbor Lab. NY (1987).

H. Culturing of Cells It is preferred that the host cell is capable of rapid growth in cell culture and able to glycosylate expressed gene products to ensure that the protein is produced in high quantity and resembles the naturally occurring material. Cells known to be suitable for dense growth in tissue culture are particularly desirable and in the art a variety of invertebrate or vertebrate cells have been employed whether normal or transformed.

The transfected cells are cultured by means well known in the art. For examples, see: Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc. (1977). The expression products are harvested from the cell medium in those systems where the protein is excreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or enzymatic means, which are well known in the art. I. Supertransfection

The disclosed supertransfeetion methods provide enhanced production of a structural gene product in a eukaryotic host cell. According to the method, a eukaryotic host cell is transfected with at least two vectors each containing at least one selectable gene and at least one structural gene of interest. The preferred embodiment describes a first expression vector comprising at least one structural gene of interest and at least one selectable gene. Transfectants are selected by growth under suitable selective conditions for identifying transfected clones. Such a transfection is referred to herein as a primary transfection. A transfected cell line is isolated and characterized. It is then used as a recipient host in a second transfection with a second expression vector comprising at least one structural gene of interest and at least one selectable gene. Such a transfection is called a supertransfeetion. Supertransfectants are selected by growth under suitable selective conditions according to the selectable gene present on the second expression vector. A transfected cell is then isolated and characterized as a supertransfeeted cell, giving rise to a supertransfected cell line. Each of the structural genes of interest and the selectable genes are operably linked to one or more regulatory DNA sequences. The supertransfected cell line is then grown under appropriate conditions to allow for expression of the structural gene of interest, and subsequent recovery of that structural gene product.

In the disclosed examples, the first and second expression vectors carry the same structural gene of interest encoding a desired polypeptide product. The plasmid encoded structural gene of the examples is endogenous to the host cell. It is also possible to practice the current invention to express proteins that are heterologous, that is, proteins whose DNA sequence is not normally present in the host cell.

The general method used to isolate clones has been to introduce purified plasmid DNA into cells that had been plated 24 hours previously by the calcium phosphate precipitation technique (Wigler PNAS 76: 1376-1376 1979).

It will be recognized by those of skill that the transformation with additional plasmids having the desired gene and a third and perhaps fourth selectable markers might yield even higher yields of desired proteins. However, the absolute levels of production for any one protein is limited by the detrimental effect that such expression will have on a cell. There needs to be a balance for each cell between the numbers of times the cell is supertransfected and its ability to withstand the directing of its protein making machinery to high levels of the desired protein. The optimum number of times a cell can be supertransfected will be dependent upon the host cell chosen and the desired protein being expressed. This is an emperical determination within the skill of the ordinary artisan when following the invention described herein.

In the disclosed example when plasmids containing only one selection marker were used, clones were selected with the appropriate selective agent and then assayed for expression of t-PA. When plasmids containing two selection markers (hmb and dhfr) were used, a population of clones was isolated first using resistance to hygromycin B and then, subsequently, clones were identified by their ability to grow in MTX. To compare the expression levels of transfectants generated with the plasmids, disclosed herein, a population of clones selected from a single transfection experiment was analyzed to determine t-PA expression as a mass culture value. Individual clones were then isolated from the population to determine the distribution of expression levels of clones within the population.

Definitions The phrase "non-selectable gene" refers to a structural gene encoding a gene product for which no selective growth medium is known which would allow the experimenter to distinguish between clones which express the gene and clones which do not.

The phrase "operably linked" refers to sequences of DNA wherein one domain is capable of effecting, as a promoter, the transcription of a second domain which encodes a structural gene.

The phrase "expression vector" includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operably linked to other sequences capable of effecting their expression. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeable. The invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

The term "host cells" refers to cells which can be grown in culture and are capable of being transformed or transfecting using plasmids and vectors as herein described. The term "recombinant host cells" refers to cells which have been transformed with vectors constructed using recombinant DNA techniques.

"CHL-2" refers to a stable eukaryotic cell line derived from CHL-1 by transfection with a plasmid encoding t-PA, pPA003. This is a plasmid designation. "P" written before a set of letters and indicates that what is being referred to is a plasmid; e.g., pPA525. t-PA (mu/c/d). Refers to a standardized amount of t-PA produced by a defined number of cells.

The t-PA is measured by a fibrin plate assay. The assay is described in detailed disclosure of this application. These units are defined by World Health Organization as international units. The specific activity of the purified protein is 630,000 units/mg.

The following examples are offered by way of illustration and not by way of limitation.

Examples A primary object of the present invention is to provide conditions and plasmids that lead to high levels of production of a predetermined polypeptide. In the following examples, four parameters have been described: the selectable gene marker, the selection conditions, amplification conditions and host cells. The end result from the following experimental work is a production system that will economically produce the desired protein on a large scale. It will be readily apparent to those skilled in the art that a wide variety of genes of interest may be substituted in place of t-PA.

A. Design of the Expression Plasmids

The t-PA expression plasmids were constructed with dhfr, neo or hmb genes as selection markers. Promoters used on the described plasmids include LTR, SVE, and TK. These plasmids were used to determine if the level of t-PA expression could be increased by repeatedly transfecting a cell line. B. Plasmid Construction i. PPA003

All base numbering for nucleotide positions of the t-PA encoding sequences correspond to the sequence numbering published by Pennica et al., "Cloning and Expression of Human Tissue-Type Plasminogen Activator cDNA in E. coli," Nature 301:214-221 (1983).

Plasmid PA003 (Figure 1) contains the t-PA gene and the neo gene both operably linked to the LTR promoter. This plasmid is constructed so that the LTR promoter controls the dicistronic expression of t-PA. A description of the steps used to assemble this plasmid is provided below and is depicted in Fig. 2A and B. pPA003 has been deposited with the ATCC and has accession number 67293.

PNeo-5 (Lusky and Botchan, Cell 36:391-401, 1984) was used as the starting material for expression vector construction for t-PA. PNeo-5 contains the triple LTR promoter the neo gene and a SV-40 late polyadenylation sequence. The DNA sequences coding for t-PA were assembled from two sources: the 5'-end of the gene was from a human DNA gene library and the 3'-end was selected from a cDNA library made from CHL-1 mRNA. The isolation and assembly of the DNA sequence encoding the t-PA gene is depicted in Fig. 2 and described below.

Initially, a cDNA library was constructed mRNA isolated from to CHL-1 mRNA. The CHL-1 cDNA was digested with Bgl II and xho II and inserted in Charon 4A, a lambda phage cloning vector (Maniatis, et al, Molecular Cloning, Cold Spring Harbor, 1982). A clone containing the Bgl II/xho II fragment which extends from bp 187 to bp 2161 of the coding sequence of cDNA t-PA was identified by hybridization to oligonucleotide probes.

The Bgl II/xho II fragment corresponding to bp 187 to 2161 disclosed in the published t-PA cDNA sequence was removed from the Charon A4 clone and subcloned into pUC19 to yield pPA104. The subcloned Bgl II/xho II cDNA fragment from pPA104, contains sequences which code for the mature form of the t-PA protein but do not contain sequences for the pre- and pro- peptides of t-PA, that are required for secretion of the active protein. In order to express and secrete the mature and active form of t-PA in transfected cells, it was necessary to provide sequences coding for the secretory process.

The sequences that encode the pre- and pro- peptides were obtained by isolating a genomic fragment from a human genomic lambda library (Figure 2a). This library was screened using the 414 bp Pst I fragment from the t-PA cDNA as the target sequence on πVX (Maniatis et al). A 4kb Bgl II genomic fragment was identified that contained the 5' end of the t-PA structural gene including a portion of the 5' untranslated sequences, and 105 nucleotides encoding the pre- and pro- peptides, separated by a large intron (Figure 2b). This 4kb Bgl II genomic fragment was subcloned from the lambda phage identified into plasmid pPA104, which already contained the cDNA Bgl II/xho II fragment. The new intermediate was called pPA103. Plasmid pPA103 contains a genomic/cDNA junction which is redundant in that subclone. This junction was removed by digesting pPA103 with Nar I since both the cDNA and the genomic 5' region contain a Nar I site. This Nar I fragment was then moved into the cDNA subclone, pPA104 at its equivalent Nar I site ( Figure 3 ) . This new intermediate, pPA115, then contained genomic sequences fused to the cDNA coding region of t-PA.

The genomic/cDNA t-PA gene was removed by digesting pPA115 with Bel I and partial digestion with Bgl II. The Bel I - Bgl II cassette was then inserted into the unique Bgl II site of pneo5 thus generating pPA003. Because the t-PA coding region contains both genomic (including introns and exons) and cDNA sequences, this gene construct is referred to as a hybrid t-PA gene.

This hybrid t-PA gene in pPA003 contains the 5' end of the gene including the 5' untranslated sequences of t-PA, the pre- and pro- coding regions, the intron between these regions, and the coding region for mature t-PA. ii. Intermediate Plasmid PPA502

Plasmid pPA502 (Figure 1) contains a genomic- cDNA hybrid gene coding for t-PA under control of the triple LTR promoter and the gene for hygromycin B resistance under control of the TK promoter. Plasmid pPA102 was used as the source of the LTR-t-PA sequences and was constructed as follows: pPA102 was made by cutting pPA003 at the Bel I and Xho I sites which flank the neomycin resistance gene. The ends of the resulting DNA fragment were treated with the Klenow fragment of DNA polymerase. The blunt ended DNA fragment was religated, thus deleting the neo gene. The source of the TK-hygromycin sequences was pHMR272 (BioChlinger and Dinglemann, Mol Cell. Biol 2929-2931, 1984) which contains: a bacterial PI promoter followed by the TK promoter, the gene for hygromycin resistance, and finally the polyadenylation region of the TK gene. HMR272 was digested at its unique Hind III site located at the 3' end of the TK terminator. The DNA was treated with Klenow fragment and BamH I linkers were ligated to the linearized DNA. Upon digestion with BamH I, a DNA fragment 2.7kb in length containing the promoter and hygromycin gene was generated. pPA102 was partially digested with BamH I.The BamH I fragment containing TK-hmb was then cloned into the BamH I site of pPA102 that follows the t-PA gene and the SV40 polyadenylation region. iii. Intermediate Plasmid pSC662

The cassette that contains the SV40 promoter and dhfr gene was prepared by removing the dhfr gene from pSV2-dhfr (Subramani, Mulligan and Berg, (1982) Op. Cit.) by digestion with Hind III and BamH I. This dhfr gene was ligated into the Hind III and BamH I sites of pBR328 (BRL, Bethesda, Md). SV40 DNA (BRL Bethesda, Maryland) was cut at the Hpa II and Hind III sites flanking the SV40 early promoter. This fragment was ligated to the Klenow treated Cla I site and the Hind III site of pBR328.

Excision of the SV40 promoter and dhfr gene cassette was accomplished by digestion with Pvu II and BamH I and subcloned into pBR327 which had been cut with EcoRv and BamHI. This intermediate, pSC661, was then cut with EcoRI and BamH I in order to subclone the SV40- dhfr cassette into a pUC18 derivative, pSC652, which had also been digested with EcoRI and BamH I. Plasmid pSC652 was made from pUC18 by converting the BamH I site into a Cla I site using a BamH I-Cla I linker. This final subclone was called pSC662 and has Cla I sites flanking the entire SV40 promoter and dhfr cassette, iv. Plasmids PPA509 and PPA510 Plasmids pPA509 and 510 (Figure 1) contain; the dhfr gene under the control of a truncated SV40 early promoter starting at the SV40 Pvu II site; the t-PA genomic-cDNA hybrid gene operably linked to the LTR promoter; and the gene for hygromycin selection operably linked to the TK promoter. Using pSC662 digested with Cla I, the SV40-dhfr genecassette was cloned into the Cla I site of pPA502 (as described above) directly 5' to the triple LTR promoter and t-PA gene. In pPA509 the dhfr gene is transcribed in the divergent direction from the t-PA gene, while in pPA510 transcription is in the same direction. The plasmid pPA509 has been deposited in the ATCC to be maintained for a period of thirty years and is designated as ATCC 67443.

C. Host Cells Two different cell lines are utilized. CHL-1

(A.T.C.C. Accession No. CRL 9446) is the cell line used for most of these experiments. CHL-1 is a derivative of RPMI 7932 (Bowes) cells described in the detailed description. A second cell line, CHL-2 (A.T.C.C. Accession No. CRL 9451) is derived from CHL-1 by a previous transfection with pPA003. This stably transfected cell line produces t-PA at a rate of 0.20-0.35 mU/cell/day; about 2-3 times more than the parent CHL-1.

D. Transfection and Selection i. Primary Transfection with PPA003 Plasmid pPA003 was transfected into CHL-1 cells according to Wigler et al. Five micrograms of plasmid DNA was precipitated with CaCl₂ and added to a monolayer of 5 X 10⁵ CHL1 cells in 6 well plates. Forty eight hours after transfection, a known number of cells were plated onto selective media containing 1.0 mg/ml G418. The cells were incubated in the selective media until colonies appeared; generally 3 - 4 weeks. The pPA003 transfected clones were then transferred to 24 well microtiter plates and incubated until confluent. Cells were trypsinized and counted, and 1 ml was seeded at 10e5 cells/ml in a 24 well plates. Fresh medium was placed in the wells for a given period of time (usually 72 hours) before the number of cells was estimated and the medium was assayed for t-PA activity. Parental CHL- 1 cells were included as a control. Production of t-PA could then be calculated in micro units per cell per day (mU/c/d). Individual colonies were then chosen and grown under standard conditions in culture flasks so that an accurate estimate of t-PA production could be obtained. From this experiment a clone producing approximately 0.3 mU/c/d. This clone was called CHL-2 and was used in subsequent transfection experiments. The transfection or selection frequency is determined as the number of colonies arising after selection, divided by the total number of cells plated. ii. Supertransfection Using PPA509 and PPA510 Plasmids pPA509 and pPA510 were used in separate experiments for primary transfection into CHL-1 or supertransfection in CHL-2. The transfections were carried out according to the methods described above, however, the selection media contained 0.3 mg/ml hygromycin B. Transfectants were isolated as described above. T-PA production was determined.

E. Amplification of the t-PA Gene Clones arising from selection in hygromycin were subjected to a second round of selective pressure. 500nM of methotrexate was added to the growth medium to select for transfectants that expressed the dhfr gene. Clones which had amplified the dhfr gene would be able to grow in this high level of MTX. In the process of gene amplification, other plasmid sequences will be co-amplified with the dhfr gene and thus lead to increased gene expression of the non-selectable gene as well. Individual resistant clones appeared after 4 - 6 weeks. These were isolated and assayed for t-PA production. Additionally, the level of t-PA production was determined as a mass culture.

F. t-PA Assay

Cells tha£ had taken up plasmid DNA during transfection and were able to grow in the appropriate selection medium formed clones on 100 mm petri dishes. These clones were either individually isolated or pooled together as a population in order to assay for t-PA expression. The assay for t-PA production was carried out in medium containing 0.1% fetal bovine serum and 10 KlU/ml aprotinin. T-PA production in 24 hours was determined for a known number of cells. Aliquots of cell culture supernatant after a 24 hour production period were saved and the number of cells in the culture was counted. For the t-PA analysis, 5 ul samples of culture supernatant were placed in a circular well excised from an agarose matrix containing sheep fibrinogen, human thrombin and human plasminogen. A clearing around the circle of culture supernatant was interpreted as conversion of plasminogen to plasmin which had digested the fibrin, all initiated by the presence of t-PA. (Suck, D., Kabash, W. , and Mannherz, H.G. PNAS 78, 4324323 (1981).) The size Of the clearing, thus, correlates with the amount of t-PA in the sample. The values given in the tables are obtained by dividing the t-PA units in the supernatant by the number of cells in the culture.

G. T-PA Production in Primary Transfected Cells and Supertransfected Cells The results of the t-PA assays on mass cultures are given in Table 1.

The baseline for t-PA expression in CHL-1 is 0.1 mu/c/d and 0.3- mu/c/d in CHL-2. In CHL-1 transfected with pPA509 the t-PA expression level is 0.3 mu/c/d. The effect of supertransfection would then be expected to result in a level of t-PA expression at '0.6 mu/c/d in CHL-2 cells transfected with pPA509. In fact the actual effect of supertransfection on t-PA expression is clearly much more than additive; 1.4 mu/c/d.

Using hygromycin B selection CHL-1 and CHL-2 cells transfected with pPA510 similar results are apparent. The mass culture t-PA levels in this supertransfection were 3 - 4 fold higher than expected. When methotrexate was included in the growth medium the results of supertransfection were even more dramatic. Comparing primary transfections of CHL-1 as mass culture levels of t-PA expression with supertransfections of CHL-2 using either pPA509 or pPA510 shows the clear and unexpectedly high level of amplified t-PA expression in the supertransfectants. If the level of expression of each consecutive transfection was additive, the expected result for CHL-2 transfections would be 2.4 mu/c/d for pPA509 and 1.5mu/c/d for pPA510. In marked contrast, the actual results for CHL-2 transfections with these plasmids was 7 fold greater than expected; 15.7 for pPA509 and 10.8 for pPA510.

The results of t-PA assays on individual clones are given in Table 2.

T-PA levels for individual clones resulting from these transfections follow the same pattern as the mass culture assays. When hygromycin B was used as the selective agent, t-PA levels in clones arising from transfections with either pPA509 or pPA510 reached higher levels when CHL-2 was the host cell line. The most startling difference in performance of individual clones was achieved when methotrexate was added to the growth medium. Plasmids pPA509 and pPA510 behaved similarly. However, in the primary transfections using CHL-1 cells the maximum t-PA expression of individual clones was in the range of 2.0

- 3,9 mu/c/d. In the supertransfections using CHL-2 the expression of t-PA by individual clones ranged as high as 14.0 - 15.0 mu/c/d. This result was approximately 6

- 7 times higher than expected.

Claims

WHAT IS CLAIMED IS:

1. A supertransfected eukaryotic cell line which expresses a desired gene product, comprising host cells transfected with a first expression vector, and a second expression vector, wherein the first expression vector comprises: a first selectable gene cassette and a gene cassette encoding the desired gene product; wherein the second expression vector comprises: a second selectable gene cassette and a gene cassette encoding said the desired gene product; and wherein said first and second selectable genes are different.

2. The eukaryotic cell line of claim 1 wherein the first and second selectable gene cassettes encode a protein selected from the group consisting of dihydrofolate reductase, neomycin phosphotransferase, and hygromycin phosphotransferase.

3. The eukaryotic cell line of claim 1 wherein the desired gene product is non-selectable.

4. The eukaryotic cell line of claim 3 wherein the non-selectable gene is endogenous to the host cells.

5. The eukaryotic cell line of claim 3 wherein the non-selectable gene encodes tissue plasminogen activator.

6. The eukaryotic cell line of claim 1 wherein the cells are selected from the group consisting of mammalian cells, insect cells or yeast cells.

7. The eukaryotic cell line of claim 1 wherein the host cell is CHL-1 (CRL 9446 ) or CHL-2 (CRL 9451).

8. A eukaryotic cell line of claim 1 wherein said cell line is capable of producing at least 4 milliunits per cell per day of t-PA.

9. A method of producing protein comprising the step of transfecting an eukaryotic host cell with: a) a first expression vector comprising: i) a first selectable gene cassette; and ii) a gene cassette encoding the protein and b) a second expression vector comprising: i) a second selectable gene cassette and ii) a gene cassette encoding the protein; and wherein said first and second selectable genes are different and the step of culturing the cells under conditions which permit expression of the protein.

10. A method according to claim 9 wherein said first and second selectable genes are selected from the group of genes encoding dihydrofolate reductase, neomycin phosphotransferase, and hygromycin phosphotransferase;

11. The method of claim 9 wherein the eukaryotic host cell is transfected with the first expression vector followed by a second transfection with the second expression vector.

12. The method of claim 11 wherein the first transfection step is followed by culturing of the transformants under conditions selective for the expression vector inserted into the cell.

13. The method of claim 12 wherein the second tranfection step is followed by culturing of the transformants under conditions selective for the second expression vector inserted into the cell.

14. The method of claim 13 wherein the protein is t-PA.

15. The method of claim 14 wherein the t-PA encoding gene cassette contains introns.

16. The method of claim 15 wherein the t-PA is encoded by a cDNA/genomic hybrid gene.

17. The method of claim 16 where the first expression vector is pPA003.

18. The method of claim 9 wherein the eukaryote host cells are selected from the group consisting of mammalian cells, insect cells or yeast cells.

19. The method of claim 14 wherein the host cell contains an endogenous t-PA gene.

20. The method of claim 19 where in host cells are human melanoma cells.