EP0517768A1 - Vecteurs d'expressions de cellules de mammiferes - Google Patents

Vecteurs d'expressions de cellules de mammiferes

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Publication number
EP0517768A1
EP0517768A1 EP19910905143 EP91905143A EP0517768A1 EP 0517768 A1 EP0517768 A1 EP 0517768A1 EP 19910905143 EP19910905143 EP 19910905143 EP 91905143 A EP91905143 A EP 91905143A EP 0517768 A1 EP0517768 A1 EP 0517768A1
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Prior art keywords
plasmid
recombinant plasmid
cells
delimited
hindlll
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EP19910905143
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German (de)
English (en)
Inventor
Kevin W. Moore
Paul J. Zavodny
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Merck Sharp and Dohme LLC
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5409—IL-5
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • yeast cloning systems are available, and through the use of such systems the efficient expression in yeast of a human interferon gene has been achieved [Hitzeman et al., Nature (London) 293:717 (1981 )]. It has been found, however, that yeast cells do not correctly transcribe at least one heterologous mammalian gene that contains introns, the rabbit ⁇ -globin gene [Beggs et al., Nature (London) 2 ⁇ S:835 (1980)].
  • heterologous genes have been inserted into mammalian cells by means of direct uptake. This has been accomplished, for example, by calcium phosphate co-precipitation of cloned genes, by which procedure about 1-2% of the cells can generally be induced to take up the DNA. Such a low level of uptake, however, produces only a very low level of expression of the desired gene product.
  • mammalian cells can be found which lack the thymidine kinase gene (tk- cells), better results can be obtained by co- transformation. Tk * cells, which cannot grow in selective HAT
  • hypoxanthine-aminoterin-thymidine can regain this lost enzymatic activity by taking up exogeneous DNA (such as herpes simplex viral DNA) containing the tk gene through calcium phosphate co-precipitation.
  • exogeneous DNA such as herpes simplex viral DNA
  • Other DNA covalently ligated to the tk DNA or merely mixed with it will also be taken up by the cells and will often be co- expressed [see Scangos et al., Gene 14:1 (1981)].
  • a number of investigators have developed expression vectors for use in eukaryotic cells. For example, Okayama et al. [Mol. Cell. Biol.
  • This invention provides recombinant plasmids which can be used to clone heterologous genes in £. c and to clone and express such genes in a variety of mammalian cells.
  • the plasmids are characterized by strategically placed genetic elements and unique restriction sites, the nature of which facilitates the excision and/or substitution of elements and the restoration of the original restriction sites.
  • the recombinant plasmids of the invention comprise DNA sequences which, in the direction of transcription, contain:
  • the polyadenylation signal sequence is a human ⁇ -globin or an SV-40 early or late region polyadenylation signal sequence.
  • DHFR dihydrofolate reductase
  • MTV-LTR mouse mammary tumor virus long terminal repeat
  • splicing signal sequence is a small t antigen intron
  • polyadenylation signal sequence is an SV-40 early region polyadenylation signal sequence.
  • the DHFR transcription unit When present in the plasmids, the DHFR transcription unit is positioned between the DNA sequences containing the ⁇ -lactamase gene and pBR322 origin of replication and the SV-40 or SR ⁇ promoter.
  • the DHFR transcription unit is delimited, in the direction of transcription, by a ⁇ glll and a Hindlll restriction site.
  • the DNA sequence containing the MMTV-LTR promoter within the transcription unit is delimited by two ⁇ gjll restriction sites.
  • the recombinant plasmids comprise DNA sequences containing a ⁇ -galactosidase
  • ⁇ -gal ⁇ -gal
  • CAT chloramphenicol acetyltransferase
  • a splicing signal sequence is present between the DNA sequences containing the SV-40 or SR ⁇ promoter and the polylinker and is delimited, in the direction of transcription, by an Xhol and a E ⁇ tJ restriction site.
  • This splicing signal sequence is preferably a 16S/19S intron.
  • Fig. 1 is a schematic representation of recombinant plasmid pDSVS, showing unique restriction sites and elements.
  • Fig. 2 panels A-H, is a schematic representation showing the construction of plasmid pDSVS from various starting plasmids.
  • Fig. 3 is a schematic representation of plasmid pDSRS. This plasmid is similar to pDSVS except for the substitution of an SR ⁇ promoter for the SV-40 promoter of pDSVS.
  • Fig. 4 is a schematic representation of plasmid pSRS. This plasmid is similar to pDSRS except that the DHFR transcription unit has been deleted in pSRS.
  • Fig. 5, panels A and B, is a schematic representation of the construction of plasmids pDSRG and pSRG.
  • Plasmids are similar to pDSRS and pSRS, respectively, except that plasmids pDSRG and pSRG contain a human ⁇ -globin polyadenylation signal sequence instead of an SV-40 late region polyadenylation signal sequence.
  • Fig. 6, panels A-C is a schematic representation of the construction of plasmid pGSRG-hlL5, which is capable of directing the production of recombinant human interleukin-5 in mammalian cells.
  • transcription means the synthesis of messenger RNA (mRNA) from a DNA template.
  • a “promoter” is a DNA sequence that directs the binding of RNA polymerase and thus “promotes” transcription. "In the direction of transcription” means from upstream to downstream (i.e., from 5' to 3'), or in the direction of movement of RNA polymerase as it carries out the transcription of DNA.
  • the SR ⁇ promoter used in this invention has been described by Takebe et al. [Mol. Cell. Biol. 2:466 (1988)].
  • the SV-40 promoter has been described by Okayama et al. [Mol. Cell. Biol. 2: 80 (1983)].
  • an “enhancer” is a DNA sequence that can potentiate the transcription of a gene without regard to the nature of the gene. Although promoters must be positioned upstream of genes that are to be expressed, the positioning of enhancers is not critical. They can function whether upstream or downstream of a gene, and even when they are inverted with respect to the other DNA sequences (i.e., in the 3' to 5' orientation). "Splicing signals” are DNA sequences which can also enhance the transcription of genes encoding some proteins.
  • An “origin of replication” is a point in a plasmid at which replication of the plasmid by a host cell is initiated.
  • a "polyadenylation signal sequence” is a DNA sequence located downstream of the translated regions of a gene at which adenine ribonucleotides are added to form a polyadenylate tail at the 3' end of the mRNA.
  • polylinker is a DNA sequence which contains one or more unique restriction sites. Polylinkers facilitate the insertion of heterologous genes (i.e., genes not normally present in a host cell) that are to be expressed in the plasmids of the invention.
  • heterologous genes i.e., genes not normally present in a host cell
  • heterologous “gene” includes both isolated genes and cDNAs prepared from mRNA.
  • Restriction sites are DNA sequences which define cleavage points for restriction endonucleases. Such sites are "unique" when only one of a given site is present in a plasmid.
  • the plasmids of this invention may contain unique Xbal. ECQRI, Smal. BamHI. Sail. Pstl. Xhol and Hindlll sites.
  • the plasmids of the invention comprise, in the direction of translation:
  • elements (b) - (d) together comprise a heterologous gene transcription unit.
  • the ⁇ -lactamase gene encodes an enzyme which degrades the antibiotic ampiciliin. Because of this activity, the presence of the ⁇ -lactamase gene in a host cell confers on the cell resistance to ampiciliin in the culture medium. As a result, host cells harboring the plasmids of the invention can be selected by growth in such medium, while other cells lacking such resistance will not survive. In the accompanying figures, the presence of the ⁇ -lactamase gene is indicated by "AMP" or "AMP f .
  • a recombinant plasmid containing only the foregoing elements can be used for the transient expression of a heterologous gene inserted into the polylinker. Insertion of a gene into the polylinker is facilitated by the presence in the polylinker of unique restriction sites, including E ⁇ ll, Sail, BamHl. Smal and EcoRI sites. Gene insertion is easily achieved if the termini of the gene following isolation are compatible with the EsJJ and/or Sail, BamHl or EcoRI sites. If they are not, they can readily be modified using standard methods to be compatible. Alternatively, the gene termini can simply be blunt ended by standard methods and inserted into the Smal-cleaved plasmid, because Smal digestion produces blunt-ended cleavage.
  • the polylinker need not contain all of the restriction sites mentioned above; any undesired sites can be deleted by standard methods. Conversely, other sites can be added if need be, as long as they are unique sites so that cleavage with the corresponding restriction endonuclease does not also cut the plasmid elsewhere.
  • a ⁇ -gal or CAT transcription unit is inserted between the DNA sequences containing the ⁇ -lactamase gene and pBR322 origin of replication and the SV-40 or SR ⁇ promoter.
  • Both the ⁇ -gal gene and the CAT gene are well known in the art, the former having been described by Hall et al. [J. Mol. Appl. Gen. 2:101 (1983)], the latter by Gorman et al. [Mol. Cell. Biol. 2:1044 (1982)]. When present in the plasmids, these genes provide useful marker activities for transformant/transfectant screening.
  • the foregoing plasmids are useful for transient expression in mammalian cells, but longer-term expression cannot be achieved with such plasmids. That is because there is no selective pressure to maintain the plasmids in host cells. Over time, they are lost.
  • the plasmids of the invention preferably contain a DHFR transcription unit in place of a ⁇ -gal or CAT transcription unit. The elements of the DHFR transcription unit have been described by Lee et al. [Nature 224:228 (1981 )].
  • a DHFR transcription unit in the plasmids provides two advantages. Firstly, when introduced into a host cell lacking dihydrofolate reductase (e.g.. into CHO-dhfr cells), the unit provides a means of selection. Cells harboring the plasmids can easily be selected from other cells in medium lacking hypoxanthine and thymidine. Secondly, cells containing high levels of the dhfr cDNA will survive culture in medium containing high levels of methotrexate, an inhibitor of de novo purine synthesis, while cells expressing low levels of the cDNA will not. Such conditions will thus enable selection for cells which have increased or amplified the number of copies of the dhfr gene.
  • heterologous gene that is to be expressed When a heterologous gene that is to be expressed is also present in a plasmid with the dhfr gene, expression of the heterologous gene will be co-amplified as the host cell makes many copies of the dhfr gene.
  • culturing cells harboring a plasmid containing the DHFR transcription unit in the presence of methotrexate caused increased production of both dihydrofolate reductase and interleukin-5 (IL-5).
  • an SV-40 late region [Okayama et al., Mol. Cell. Biol. 2:280 (1983)] or human ⁇ -globin [Lawn et al., Cell 21:647 (1980)] polyadenylation signal sequence was used downstream of the polylinker. These sequences are interchangeable and can also be used in the DHFR transcription unit in place of the SV-40 early region sequence (Lee et al., supra). The SV-40 early region sequence could also be used downstream of the polylinker. The only requirement for such substitutions is that the sequences used in such positions in the plasmids must be terminated by the restriction sites indicated in Fig. 1, if it is desired to later remove the polyadenylation signal sequence. Since the nucleotide sequences of all three are known, they can be chemically synthesized and/or adapted for such use using conventional methods.
  • the plasmids of the invention may optionally contain enhancer regions.
  • Useful enhancers are obtained from animal viruses such as SV-40, polyoma virus, bovine papilloma virus, cytomegalovirus, retroviruses or adenoviruses.
  • the enhancer used should be from a virus for which the host cells are permissive (i.e., from a virus which normally infects cells of the host type).
  • the enhancer regions of a number of viruses are known [see, e.g.. Luciew et al., Cell 22:705 (1983)]. It would be a routine matter to excise these regions based upon published restriction maps for a virus selected and, if necessary, to modify the termini to enable splicing the enhancer into the recombinant plasmids.
  • the enhancers can be synthesized from published sequence data. The only requirement for a selected enhancer is that it preferably not contain a restriction site(s) that would destroy the uniqueness of the above-mentioned sites. One of skill in the art could readily determine this from the published sequence data and the known restriction site sequences.
  • the enhancers from many viruses can be used, the enhancer from SV-40 (Takebe et al., supra) is preferred for use in this invention.
  • the location of the enhancers within the plasmids is not critical, the Hindlll restriction site is a convenient point for insertion.
  • heterologous genes can be inserted into the plasmids, including but not limited to genes encoding blood clotting or fibrinolytic enzymes, regulatory proteins such as the various lymphokines or hormones, growth factors, oncogene products and soluble or cell-surface antigens and receptors.
  • One of the plasmids of the invention has been used to express a gene encoding a soluble form of a gamma interferon receptor.
  • the use of another plasmid to express a gene encoding IL-5 is illustrated.
  • genes will often contain wild-type translated sequences encoding prepro-polypeptides (e.g.. secretory leaders), which may be desirable for the structure, stability and/or secretion of the mature proteins.
  • prepro-polypeptides e.g.. secretory leaders
  • Such polypeptides are removed during post- translational processing by the host cells.
  • prepro- sequences can be deleted by well known methods prior to insertion of the genes into the plasmids.
  • the genes may also contain 5' and/or 3' noncoding sequences.
  • genes or cDNAs to be expressed must contain translation initiation and stop codons, although the SV-40 or SR ⁇ promoter used provide the necessary transcription initiation signals.
  • the preferred order of elements in the plasmids is as described above, any element inserted upstream of the promoter for the heterologous gene, such as the ⁇ -gal or CAT transcription unit or the DHFR transcription unit, can be inserted downstream of the heterologous gene transcription unit instead. This can conveniently be done by the use of the Xbal restriction site.
  • the placement of the genetic elements and unique restriction sites within the plasmids facilitates the substitution of elements or the removal of elements for use in other plasmids or vectors. It also permits easy restoration of the original restriction sites after cleavage has been carried out.
  • the DHFR transcription unit is easily removed. ⁇ gill/Klenow followed by Hindlll/exoVII regenerates the Bglll site. Hindlll/Klenow followed by Bglll/exoVII regenerates the Hindlll site.
  • Xhol/Klenow followed by E ⁇ JJ/exoVII and religation removes the Xhol/Pstl fragment splicing signals and regenerates an Xhol site.
  • the plasmids of the invention can be used in many mammalian host cells, including primary explants from various tissues.
  • Cell lines that can be used include but are not limited to the African green monkey kidney (COS), Chinese hamster ovary (CHO), NS-1, SP2/0, NIH 3T3, NIH 3T6, C127, CV-1 , HeLa, mouse L and Bowes cell lines.
  • COS African green monkey kidney
  • CHO Chinese hamster ovary
  • NS-1 African green monkey kidney
  • SP2/0 Chinese hamster ovary
  • NS-1 SP2/0
  • NIH 3T3T6 NIH 3T3T6, C127, CV-1
  • HeLa HeLa
  • mouse L mouse L
  • Bowes cell lines where co-amplification using the DHFR transcription unit is desired, dihydrofolate reductase-deficient cells such as CHO-dhfr cells [Urlaub e
  • Plasmid DNA can be introduced into mammalian cells in a number of ways. Transient expression studies in mammalian cells such as COS cells can be carried out using the DEAE-dextran method with chloroquine boost [Yokota et al.,Proc. Natl. Acad. Sci. USA 22:68 (1985)]. Transfection of suspension cells (e.g.. NS-1) can be accomplished using the method of electroporation [Potter et al., Proc. Natl. Acad. Sci. USA 21:7161 (1984)]. Fibroblasts such as CHO cells can be transfected using the calcium phosphate precipitation method [Graham et al., Virology 52:456 (1973)]. All three of these methods are described below in detail.
  • Identification of cells harboring the plasmids can be made using standard methods. For example, host cell genetic material can be probed by Southern blot analysis. Expression of the desired heterologous gene can be detected by standard immunochemical or enzymatic analysis, or by bioassay.
  • the DNA was then concentrated using a Elutip-D column (Schleicher and Schuell Inc., Keene, NH) following the manufacturer's instructions and precipitated at -20°C with ethanol in the presence of 10 ⁇ g of yeast tRNA carrier (Bethesda Research Laboratories, Bethesda, MD).
  • T4 DNA ligation was carried out for 16 hours at 4°C in a buffer containing 50 mM Tris- HCl, pH 7.8, 10 mM MgCI 2 , 20 mM dithiothreitol, 1 mM ATP and 50 mg/ml bovine serum albumin. Klenow blunt-ending of single-stranded DNA ends was carried out in restriction enzyme buffer which had been adjusted to contain 1 mM dGTP, dATP, dCTP and TTP.
  • Oligonucleotides used as linkers were either purchased from New England Biolabs, Beverly, MA, or prepared by standard methods using a Model 380A DNA synthesizer (Applied BioSystems, Foster City, CA). Transformation and Transfection
  • the plasmid DNAs were introducted by DEAE-dextran treatment with a chloroquine boost [Yokota et al., Proc. Natl. Acad. Sci. USA 22:68 (1985)]. Briefly, 10 ml of a suspension of COS cells were plated onto 100 mm culture dishes at approximately 10 5 cells/ml in DME medium, penicillin/streptomycin (pen/strep) and 2 mM glutamine (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). After the cells reached approximately 50% confluence, the medium was removed and the cells were washed with 10 ml of serum-free DME.
  • a plasmid DNA-DME-DEAE-dextran mixture was prepared as follows: for each plate, DME medium (4.2 ml) supplemented with 50 mM Tris-HCI, pH 7.4, glutamine and antibiotics as described above was mixed with 80 ⁇ l of DEAE-dextran (20 mg/ml in sterile, distilled water; Pharmacia, Piscataway, NJ). Plasmid DNA (10 ⁇ g/dish) was added to 4.2 ml of the DME-DEAE-dextran mixture, and the mixture was added to the washed cells. Each 100 mm culture dish was incubated for 4 hours, the DNA solution was aspirated from the plate, and the plate was washed with 5 ml of serum-free DME.
  • the cells were incubated for 3 hours. After this incubation, the chloroquine was removed by aspiration and the plate washed with 5 ml of DME and aspirated. Each plate then received 10 ml of DME (10% fetal bovine serum, antibiotics and glutamine as above) and was incubated for approximately 72 hours. The cells were collected or the conditioned medium was harvested for bioassay. For stable transfection of myeloma cells, the plasmid DNAs were introducted by electroporation [Potter et al., Proc. Natl. Acad. Sci.
  • the cells were held at room temperature for 10 minutes and then the four aliquots were combined and placed in a plastic T75 tissue culture flask in a total volume of 10 ml of complete DME. After a 3-day incubation period, the cells were diluted with selection medium to a final concentration of 0.5 ⁇ g/ml mycophenolic acid (BRL Life Technologies, Gaithersburg, MD), 100 ⁇ g/ml Xanthine (Sigma, St. Louis, MO), 15 ⁇ g/ml Hypoxanthine (Sigma), 10% fetal bovine serum and 880 ⁇ g/ml glutamine. The cells were diluted to give approximately 1 ,000 cells/0.2 ml/well of a 96-well tissue culture plate. Colonies of transfected cells could usually be seen forming in 7-14 days. Cells were collected or conditioned medium was harvested for assay as described above.
  • the calcium phosphate precipitation method was used for the stable transfection of fibroblasts with plasmid DNAs.
  • the protocol included in the Mammalian Transfection Kit (Stratagene, LaJolla, CA) was followed. Briefly, fibroblasts were plated into a 100 mm tissue culture dish at 5 x 10 5 cells dish in complete DME medium prepared as described above plus 1X nonessential amino acids (100X stock from Gibco), and 4 ⁇ g/ml thymidine and 15 ⁇ g/ml hypoxanthine (both contained in 100X stock from Gibco). After a 24 hour incubation, 10 ⁇ g of plasmid DNA (as a calcium phosphate precipitate) were added per plate.
  • the cells were incubated for 24 hours, and the plates were washed with serum-free DME medium and aspirated. The cells were incubated for 24 hours, split 1 :10 and allowed to grow for 24 hours before the addition of selection medium.
  • the selection medium contained DME, glutamine and nonessential amino acids as described above, plus 5% dialyzed fetal bovine serum (Gibco) without antibiotics. After colonies began to form, medium containing methotrexate was added stepwise until cells were obtained which could grow at a 1 ⁇ M level of methotrexate. This produced amplification of the integrated locus [Kaufman et al., J. Mol. Biol. 152:601 (1982)]. Cells were harvested or conditioned medium was collected for assay as described above.
  • £. coli strain C600 (ATCC 23724) was used for plasmid constructions.
  • the cells were grown in 20:10:5 TYE medium (20 g tryptone, 10 g yeast extract, 5 g NaCI per liter).
  • COS cells (ATCC CRL 1650) were cultured in DME medium (Gibco, Grand Island, NY) plus antibiotics, 2 mM glutamine (Gibco) and 10% fetal bovine serum (Hyclone, Logan, UT) as described above.
  • NS-1 cells (ATCC TIB 18) were cultured in DME medium like the COS cells.
  • CHO dhfr cells (clone DXB-11 ) were obtained from Dr. L Chasin, Columbia University, NY. These cells were routinely carried in complete DME medium with added 1 X nonessential amino acids, thymidine and hypoxanthine (from 100X stock, Gibco).
  • the fragment containing the pcD-derived SV-40 splicing signal sequence and murine gamma interferon cDNA was removed from plasmid pMgammal 8 by Xhol digestion, and the plasmid backbone was religated to form plasmid pL27B.
  • the DNA fragment containing the SV-40 early promoter and SV-40 late region polyadenylation signal of plasmid pl_27B was removed by Accl restriction and Klenow polymerase fill-in of the 5' overhangs, ligation of P ⁇ t linkers (GCTGCAGC; No. 1024, New England Biolabs), and Pstl/Hindlll simultaneous digestion.
  • Hindlll/PstI fragment containing the SV-40 DNAs described above was ligated to Hindlll/Pstl-di ⁇ ested pL14R to generate plasmid pL15R-B.
  • the BamHl restriction site immediately adjacent to the unique Hindlll site of pL15R-B was removed by BamHl partial digestion to generate linearized DNA. This was followed by Klenow polymerase fill- in of the 5' overhangs and religation of the plasmid to generate plasmid pL602.
  • the small EcoRI/Pstl fragment of pL602 was removed by EcoRI/Pstl digestion and replaced with a synthetic DNA containing compatible EcoRI/Pstl overhangs but which resulted in the loss of both sites, due to lack of reconstitution of the complete recognition sequence.
  • the synthetic DNA contained the recognition sequence for Xbal.
  • the nucleotide sequences of the synthetic DNAs, AB176 and AB177, are shown in Fig. 2C.
  • the SV-40 late region polyadenylation signal was removed from plasmid pCDV-1 by Accl/Xhol digestion, Klenow polymerase fill-in of the 5' overhangs, ligation to EcoRI linkers
  • the DNA fragment containing the SV-40 early promoter and intron from plasmid pL1 was removed by £sll digestion, S1 Nuclease digestion of the 3' overhang, Klenow polymerase fill-in (of any 5' overhangs produced by S1 digestion), ligation of Xbal linkers (CTCTAGAG; No. 1032, New England Biolabs), and simultaneous restriction with Xbal and Hindlll.
  • the resulting Hindlll-Xbal-linkered fragment was inserted into Hindlll/Xbal-restricted plasmid pMT11 S to form plasmid pKML-1.
  • the DNA fragment containing the SV-40 early promoter and intron was removed from plasmid pKML-1 by Hindlll/Bglll digestion and inserted into Hindlll/BamHI-di ⁇ ested pUC13 to create plasmid pUCL1-E.
  • the overhangs created by BamHl and Bgill are identical, so the DNA fragments annealed. Both sites, however, were destroyed because the entire recognition sequences for both enzymes were lost.
  • the BamHl site of pUCL1-E was destroyed by BamHl digestion, Klenow polymerase fill-in of the 5' overhangs and religation to create pUCL2-A.
  • the SV-40-containing Hindlll/Xbal fragment of pUCL2-A was inserted into Hindlll/Xbal-cut plasmid pKMP-2 to generate plasmid pcDL-4.
  • the J2gill and Xbal sites of plasmid pcDL-4 were destroyed by Bglll/Xbal restriction followed by exonuclease VII digestion of the protruding 5' overhangs and religation to generate plasmid pL64G.
  • the Xhol/BamHI fragment of plasmid pL64G containing the SV-40 intron was inserted into Xhol/BamHl-digested pL603 to generate plasmid pT443-2.
  • the unique Ndel site in plasmid pcDV-1 was converted to an Xbal site by Ndel digestion, Klenow polymerase fill-in of the 5' overhangs, ligation to Xbal linkers (CTCTAGAG; No. 1032, New England Biolabs), Xbal digestion and religation to form plasmid pT512- 2.
  • CTCTAGAG ligation to Xbal linkers
  • Xbal digestion ligation to Xbal linkers
  • a synthetic DNA fragment containing restriction sites for BamHl, Smal and Ej&RI (the original BamHl site of pT514-2 was lost on cloning) was inserted between the BamHl and Bail sites of plasmid pT514-2 to create plasmid pT519-1.
  • the nucleotide sequences of the DNAs used, AB199 and AB200, are shown in Fig. 2G.
  • the two Hindlll sites of plasmid pl_23 were converted to Bfllll sites by Hindlll digestion, Klenow fill-in of the 5' overhangs on both DNA fragments, ligation of fJflill linkers (CAGATCTG; No.
  • the SR ⁇ promoter was excised from plasmid pcDL-SR ⁇ 296 by digestion with Hindlll and and P_sl I.
  • the DNA fragment containing the SR ⁇ promoter was electroeluted from an agarose gel and ligated to Hindlll/PstI - digested pDSVS to generate pDSRS. This construction is shown schematically in Fig. 3.
  • Synthetic oligonucleotides AB361 and AB362 (Fig. 4) were synthesized as described above. When annealed, these oligonucleotides produced a double-stranded DNA with a Bglll overhang at one end and a Hindlll overhang at the other. The synthetic DNAs were annealed by heating to 95°C in a volume of 20 microliters and then allowed to reach room temperature.
  • the general expression plasmid pDSVS was digested with Bfllll and Hindlll and electroeluted from an agarose gel. The annealed oligonucleotides were ligated to Bglll/Hindlll-di ⁇ ested pDSVS to produce the plasmid pSVS. The result was the removal of the dhfr transcription unit, with the regeneration of both restriction sites. The SR ⁇ promoter was then substituted for the SV40 promoter of pSVS. The SR ⁇ promoter was isolated from plasmid pcDL-SR ⁇ 296 by digestion with Hindlll and Xhol. The promoter element was ligated to Hindlll/Xhol-di ⁇ ested pSVS to generate plasmid pSRS. Construction of Plasmids pDSRG and pSRG
  • Synthetic oligonucleotides AB523 and AB524 (Fig. 5) were synthesized as described above. When annealed, these olibonucleotides introduced restriction sites for Xhol. BamHl. Bglll. and Sail between the existing EcoRI and Hindlll sites of pUC19. The AB523 and AB524 synthetic oligonucleotides were annealed as above and ligated to EcoRI/Hindlll-digested pUC19 to produce plasmid p679-2.
  • the human beta globin 3' untranslated region was obtained by BamHl digestion of plasmid pBut-7 and ligated to Bglll- digested p679, to produce plasmid p658J.
  • This plasmid was used at the source for the human beta globin polyadenylation signal in the SR ⁇ promoter containing expression plasmids.
  • Plasmid p658J was digested with Hindlll. end-filled with DNA Klenow fragment, the Klenow was inactivated, and the plasmid was digested with EcoRI. This fragment was ligated to Xbal-digested. Klenow-treated and EcoRI-digested pDSRS and pSRS, to produce pDSRG and pSRG, respectively.
  • Human IL-5 cDNA was removed from plasmid pcD-SR ⁇ -hlL5 by the polymerase chain reaction (PCR) [Saiki et al., Science 239:487 (1988)].
  • the forward primer, AB790 (Fig. 6), was designed to introduce Ba l restriction site upstream of a modified initiation region representing an optimum Kozak configuration [Kozak, J. Cell. Biol. 108:229 (1989)].
  • the reverse primer, AB791 (Fig. 6), was designed to introduce an EcoRI restriction site downstream of the termination codon for human IL-5. The directions of the primers are shown by the arrows in Fig. 6. After 30 cycles of PCR (95°C for 2 min., 40°C for 2 min.
  • the amplified DNA fragment was digested with SaH/Hindlll and ligated to Sall/Hindlll-di ⁇ ested pSRS to produce the expression plasmid pSRS-hlL5.
  • the expression cassette for human IL-5 containing the SR ⁇ promoter, splice sites, and hlL-5 cDNA up to the EcoRI site was removed from plasmid pSRS-hlL5 by Hindlll/EcoRI digestion and ligated to Hindlll/EcoRI-dioested pDSRG and pSRG, to produce pDSRG-hlL5 and pSRG-hlL5, respectively.
  • ⁇ ]i xanthine-guanine phosphoribosyltransferase (gpt) was then introduced into the hlL-5 expression plasmid pSRG-hlL5.
  • the region containing the £. ⁇ ⁇ . gpt transcription unit, a portion of the rearranged immunoglobulin in the heavy chain variable region (Vh1 ), amplicillin marker and £. ££ii plasmid origin of replication was removed from plasmid pD8Ch2UK by digestion with Xhol and Bail, end-fill with Klenow polymerase as above, inactivation of the Klenow enzyme and digestion with Xbal.
  • the region from Xbal to Bail containing the elements described above is identical to the Xbal to Bail region in the plasmid pSVD ⁇ tbeta [Schnee et al., Proc. Natl. Acad. Sci. USA 24:6904 (1987)].
  • This Xbal/Sall fragment was inserted into ad Ill-digested, Klenow treated and Xbal-dioested pSRG-hlL5 to produce pGSRG-hlL5.
  • This human IL-5 expression plasmid will express human IL-5 using the SR ⁇ promoter, SV40 splice signals and human beta globin 3' untranslated region.
  • TF-1 The erythroleukemic human cell line TF-1 was obtained from Dr. T. Kitamura (Tokyo University), although the murine BCLi cell line (ATCC TIB 197) described by Dutton et al. [J. Immunol. 122:2451 (1984)] could have been used instead.
  • TF-1 cells were routinely cultured in RPM1 1640 medium (Gibco) containing 10% fetal bovine serum, 1 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF), and pen/strep.
  • RPM1 1640 medium Gibco
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the cells were washed twice in phospate buffered saline (PBS, Gibco) and aliquoted into 96-well plates in RPMI 1640 at 4 x 10 4 cells/well. An equal volume containing test sample in RPM1 1640 and 10% fetal bovine serum was added to the cells. The cells were incubated for 48 hours at 37°C, 10 ⁇ g of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) dye dissolved in PBS were added to each well, and the cells were incubated for 6 hours.
  • PBS phospate buffered saline
  • MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • a 1 :1 dilution in each well was made using acid- isopropanol (0.1 M HCI:95% isopropanol), and the contents of the wells were thoroughly mixed.
  • the colorimetric assay of Mosmann [J. Immunol. Methods 65:55 (1983)] was carried out using a V-max spectrophotometer (Molecular Devices, Palo Alto, CA) set at 570 nm. Blank absorbance at 630 nm was subtracted from the measurements at 570 nm.
  • Activity (expressed in units/ml) was defined as the inverse of the dilution of the sample required to give half-maximal proliferative induction of TF-1 cells when cultured for 48 hours.
  • Human IL-5 expression plasmids described above were transfected into three different cell lines and the conditioned media from the cultures were assayed for human IL-5 biological activity, using the TF-1 cell bioassay.
  • Conditioned medium from COS cells was prepared as described above.
  • Recombinant CHO cells, either unamplified or selected to grow in 1 ⁇ M methotrexate, were seeded into 5 ml of DME medium (plus 10% fetal bovine serum, 1X nonessential amino acids and 880 ⁇ g/ml glutamine without antibiotics) a 5 x 10 5 cells/ml and cultured for 72 hours.
  • Cell culture conditioned medium was harvested and passed through a 0.22 ⁇ Nalgene low protein binding syringe filter in preparation for bioassay.
  • Recombinant myeloma cells (NS-1 , ATCC TIB 18) were seeded into 5 ml of HB101 medium (Hana Biological, Alameda, CA) supplemented with 800 ⁇ g/ml glutamine at 5 x 10 5 cells ml and cultured for 72 hours as described above for CHO cells. After incubation, the conditioned medium was harvested for bioassay, with the results shown in Table I.
  • the data of Table I show that human IL-5 was efficiently produced in all three cell types.
  • the IL-5 biological activity in the conditioned media ranged from 2,000 units/ml in COS cells (transient expression) to 20,000 units/ml in amplified CHO cells. Production in the CHO cells increased more than ten fold upon selection with methotrexate.
  • the unamplified production of IL-5 in the NS-1 cells in view of the variability of the bioassay used, was roughly comparable to the amplified production in the CHO cells.
  • Plasmids pDSVS, pDSRS, pDSRG, pSRS and pSRG were deposited February 21 , 1990 with the American Type Culture Collection, Rockville, MD, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Pu ⁇ oses of Patent Procedures. They have been assigned Accession Nos. ATCC 68231 , 68232, 68233, 68234 and 68235, respectively.

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Abstract

L'invention se rapporte à de nouveaux plasmides recombinants qui peuvent être utlisés pour effectuer le clonage de gènes hétérologues dans des cellules d'E. coli et pour effectuer le clonage et l'expression de tels gènes dans des cellules de mammifères. Ces plasmides contiennent un certain nombre de sites de restriction uniques qui peuvent être utilisés pour faciliter l'élimination ou le remplacement d'éléments génétiques et l'introduction de gènes hétérologues dans les plasmides. La nature et la position de ces sites uniques sont telles que la régénération des sites après l'opération de clivage par endonucléase peut facilement être réalisée avec un minimum de manipulations. Des cellules hôtes contenant ces plasmides recombinants sont également décrites dans la présente invention.
EP19910905143 1990-02-28 1991-02-26 Vecteurs d'expressions de cellules de mammiferes Withdrawn EP0517768A1 (fr)

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WO1992008792A2 (fr) * 1990-11-16 1992-05-29 Schering Corporation PROCEDE D'AUGMENTATION ET D'ACTIVATION DES MONOCYTES ET DES NEUTROPHILES ET D'INDUCTION DE LA MATURATION DE CELLULES MYELOïDES A L'AIDES DE L'INTERLEUKINE-5
EP0732404A4 (fr) * 1993-12-03 1997-07-30 Asahi Chemical Ind Nouveau vecteur de detection d'expression
US5741657A (en) 1995-03-20 1998-04-21 The Regents Of The University Of California Fluorogenic substrates for β-lactamase and methods of use
DE69637484T2 (de) * 1996-08-19 2010-06-24 Morphosys Ip Gmbh Vektoren/DNA-Sequenzen aus humanen kombinatorischen Antikörper-Bibliotheken
US7829084B2 (en) 2001-01-17 2010-11-09 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
CN101684158A (zh) 2001-01-17 2010-03-31 特鲁比昂药品公司 结合域-免疫球蛋白融合蛋白
JPWO2004070030A1 (ja) * 2003-02-03 2006-05-25 株式会社イムノ・ジャパン 動物細胞用高発現ベクター
ATE514783T1 (de) 2003-11-12 2011-07-15 Schering Corp Plasmidsystem zur expression mehrerer gene
PE20050928A1 (es) 2003-11-21 2005-11-08 Schering Corp Combinaciones terapeuticas de anticuerpo anti-igfr1
BRPI0617057A2 (pt) 2005-08-30 2011-07-12 Univ Miami anticorpo isolado, toxina especìfica para receptor de fator de necrose tumoral 25 (tnfr25), método para ativar o receptor de fator de necrose tumoral 25 (tnfr25), método para inibir a sinalização do receptor de fator de necrose tumoral 25 (tnfr25) numa célula, vacina antitumoral, método para imunizar um paciente contra tumor, método para tratar cáncer num paciente, método para tratar e/ou prevenir inflamação intestinal, composição terapêutica para a facilitação de um transplante de órgão, método para transplantar um tecido de um doador para um hospedeiro, método para inibir a expressão clonal de uma população de células t cd8 cognatas, método para tratar e/ou prevenir inflamação pulmonar, antagonista de tnfr25 isolado, composição e vetor de expressão
WO2009006479A2 (fr) 2007-07-02 2009-01-08 Etubics Corporation Procédés et compositions de production d'un vecteur adénoviral destiné a être utilisé dans des vaccinations multiples
CA2741373A1 (fr) 2008-10-30 2010-06-03 Peixuan Guo Biocapteur a connecteur de proteine motrice d'encapsulation d'adn viral a integration membranaire pour sequencage d'adn et autres utilisations
US8137933B2 (en) 2008-11-12 2012-03-20 Schering Corporation Mammalian expression vector pUHAB
DK2462165T3 (en) 2009-08-03 2016-08-29 Univ Miami Method for in vivo proliferation of regulatory T cells
US9605276B2 (en) 2012-08-24 2017-03-28 Etubics Corporation Replication defective adenovirus vector in vaccination
CA2897826C (fr) 2013-01-09 2022-09-27 Taylor H. Schreiber Compositions et procedes pour la regulation de lymphocytes t regulateurs a l'aide d'une proteine d'une fusion tl1a-ig

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