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  • 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
  • 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
  • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
• • 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
  • C12N15/67—General methods for enhancing the expression
  • C12N15/69—Increasing the copy number of the vector

Definitions

• the invention relates to a selection process for high-producing recombinant cells, a process for the production of heterologous gene products and expression vectors and thus transfected host cells which can be used in these processes.
• Mammalian cells are the preferred host cells for the production of complex biopharmaceutical proteins since the post-translational ones are carried out
• Modifications are human-compatible in both functional and pharmacokinetic terms.
• Commercially relevant cell types are hybridomas,
• Myelomas CHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) -
• Host cells are increasingly cultivated under serum and protein-free production conditions. The reasons for this are the related
• CHO cells Protein as well as the reduction of the potential to introduce pathogens (e.g. prions, viruses).
• pathogens e.g. prions, viruses.
• the heterologous gene is usually introduced into the desired cell line together with a selectable marker gene, such as neomycin phosphotransferase, by transfection.
• a selectable marker gene such as neomycin phosphotransferase
• the heterologous gene and the selectable marker gene can either be expressed together from a single vector or from separate vectors that are co-transfected become.
• the cells are transferred to medium which contains a selection agent, for example G418 using the neomycin phosphotransferase gene, and cultured for a few weeks under these selective conditions.
• the high-growing resistant cells can then be isolated and examined for the expression of the desired gene product.
• a population of cells Due to the arbitrary and undirected integration into the host cell genome, a population of cells is obtained which have completely different expression rates of the heterologous gene. These may also include non-expressing cells in which the selection marker is expressed, but not the gene of interest. To identify cell clones that have a very high expression of the heterologous gene of interest, a large number of clones must therefore be checked and tested, resulting in a high expenditure of time, labor and costs.
• DHFR dihydrofolate reductase
• CHO Chinese Hamster Ovary
• the transfectants are then selected in a medium without glycine, hypoxanthine and thymidine.
• the amplification and thus the establishment of high-producing cell lines is achieved by the increasing addition of methotrexate (MTX), an inhibitor of dihydrofolate reductase (Kaufman et al., 1982; US 4,656,134).
• MTX methotrexate
• the subsequent selection of the highly producing cells obtained is also subject to the random principle and is based on probabilities, which makes this selection step extremely labor-intensive and time-consuming.
• Various methods have been developed for better and faster tracking of gene transformation and expression.
• reporter molecules such as chloramphenicol acetyl transferase, luciferase, ß-galactosidase or fusion proteins which contain the coding regions of ß-galactosidase or luciferase.
• the disadvantage of these corresponding reporter gene assays is that the cells have to be fixed or lysed and incubated with exogenously added substrates and co-factors. Further cultivation of the analyzed cells is therefore excluded.
• a newer method based on the co-expression of the E.
• coli enzyme ß-galactosidase enables a sorting of living cells using a FACS device (Nolan et al., 1988), but here the cell is loaded with the fluorogenic substrate requires hypotonic pretreatment. This activity has to be inhibited before the FACS-based sorting.
• GFP green fluorescent protein
• This method involves the co-transfection of cells by a DNA molecule that contains the coding sequence for the protein of interest and a second DNA molecule that encodes the GFP gene.
• the GFP-expressing cells are then selected.
• Gubin et al. (1997) examined the stability of GFP expression in CHO cells in the absence of selective growth conditions. The cells were transfected with a plasmid that contained both GFP and neomycin phosphotransferase.
• Mosser et al. (1997) used a plasmid that contains a bicistronic expression cassette to identify and select cells that expressed inducible product, coding for GFP and a target gene (or also called gene of interest). The target gene was under the control of an adjustable promoter.
• Coupling of GFP and target gene expression was accomplished using a viral IRES ("Infernal Ribosome Entry Site") element, which expressed a bicistronic mRNA encoding GFP and the protein of interest.
• the plasmid used contained the plasmid used itself no selectable marker gene, which was therefore introduced by a second plasmid in a co-transfection or in a subsequent transfection, whereas Levenson et al.
• Vectors have also been described which contain an IRES element from the Picornavirus family, the IRES element being positioned between the product gene and a selectable marker gene (Pelletier et al., 1988; Jang et al., 1989; Davies et al., 1992).
• GFP has also been successfully merged with resistance marker genes.
• Bennett et al. (1998) a GFP / Zeomycin fusion protein. This bifunctional selection marker was successfully used to identify and select transfected mammalian cells.
• Primig et al. (1998), on the other hand, used a fusion protein made from GFP and neomycin phosphotransferase for their enhancer studies.
• the best clones which were isolated using the combined selection using an amplifiable DHFR selection marker and GFP-based FACS sorting, expressed the protein of interest in a range of at most 3 to 4.5 pg per cell and day.
• the experiments were carried out with adherent cells and in a medium containing serum, i.e. with cells and under conditions that are known to be significantly more robust and characterized by higher basic productivity.
• the object of the present invention was therefore to develop a selection system for recombinant cells with increased productivity, which meets the following requirements:
• Another object of the invention was to provide expression vectors and thus transfected host cells which can be used in this clone selection system are, and a method for producing heterologous gene products using these host cells.
• these objects are achieved with the aid of an expression vector which has a gene which codes for a protein of interest (hereinafter also referred to as "gene of interest”) in functional linkage with a hamster ubiquitin / S27a promoter and a gene encoding a fluorescent protein.
• gene of interest a protein of interest
• the expression vector preferably also contains an amplifiable selection marker gene, e.g. the gene for dihydropholate reductase (DHFR). Furthermore, a preferred expression vector contains further regulatory elements, for example an enhancer in functional connection with the promoter. Furthermore, the expression vector preferably additionally contains an internal ribosome binding site (IRES) which enables the bicistronic expression of the gene which codes for a fluorescent protein and of the gene of interest.
• IRES internal ribosome binding site
• the invention also relates to base vectors which, instead of the gene of interest, have a multiple cloning site for the incorporation of such a gene, i.e. a sequence region with multiple interfaces for restriction endonucleases.
• host cells that have been transfected with one of the expression vectors mentioned.
• These are eukaryotic host cells, preferably mammalian cells, rodent cells such as hamster cells and in particular CHO cells or BHK cells being particularly preferred.
• Another aspect of the present invention is a method for producing a heterologous gene product, in which a host cell transfected with the expression vector according to the invention is cultivated under conditions which allow expression of the gene product and the gene product is isolated from the culture or the culture medium.
• the host cell is transfected, preferably co-transfected, with the expression vector according to the invention and additionally with one or more vectors with genes which code for one or more other proteins of interest.
• the present invention provides a method for producing a heterodimeric protein, in which such a host cell, which has been co-transfected with expression vectors which code for different subunits of the heterodimeric protein, under conditions which allow expression of the enable heterodimeric protein, cultured and isolated the heterodimeric protein from the culture or the culture medium.
• a special application for such a method is the production of antibodies and their subunits.
• Another aspect of the present invention is a method for selecting a host cell that expresses a protein of interest, in which a population of host cells that have been transfected with an expression vector according to the invention is cultivated under conditions that allow expression of the protein of interest and of the fluorescent protein and identifying and / or selecting the cell (s) which show the highest expression levels of fluorescent protein.
• the selection is preferably carried out with the aid of a fluorescence-activated line sorting device (FACS).
• FACS fluorescence-activated line sorting device
• Figure 1 shows a comparison of the achieved expression levels of recombinant cell clones in which the heterologous gene product is expressed either under the control of the CMV promoter or under that of the hamster ubiquitin / S27a promoter. Both promoters are functionally linked to the CMV enhancer and the termination sequence, BGH poly A, is identical in all cases.
• CMV 1 it is a pcDNA3 (Invitrogen, Kaisruhe, DE)
• CMV 2 it is a pBluescript (Stratagene, La Jolla, CA, US) and in CHO it is a pAD-CMV-based expression vector (Werner et al., 1998).
• P / E is a combination of CMV enhancer and hamster ubiquitin / S27a promoter, "P” is only a promoter element and “T” is a termination signal for transcription that is used to polyadenylate the transcribed The position and direction of the transcription initiation within each transcription unit is indicated by an arrow.
• mcs restriction endonucleases
• the amplifiable selection marker dihydrofolate Reductase is abbreviated to "dhfr” and the selection marker neomycin phosphotransferase is abbreviated to "neo”.
• the IRES element derived from the encephalomyocarditis virus serves as an internal ribosome binding site within the bicistronic transcription unit and enables the translation of the subsequent green fluorescent protein "GFP".
• FIG. 3 shows a schematic representation of the eukaryotic expression vectors, each coding for a biopharmaceutical protein and used for the transfection of CHO-DG44 cells.
• P / E is a combination of CMV enhancer and hamster ubiquitin / S27a promoter
• P is only a promoter element
• T is a termination signal for transcription that is required for polyadenylation of the transcribed mRNA
• the position and direction of the transcription initiation within each transcription unit is indicated by an arrow.
• the amplifiable selection marker dihydrofolate reductase is abbreviated to "dhfr” and the selection marker neomycin phosphotransferase to "neo”.
• the IRES element originating from the encephalomyocarditis virus serves as an internal ribosome binding site within the bicistronic transcription unit and enables the translation of the subsequent green fluorescent protein "GFP".
• GFP green fluorescent protein
• "SICAM” codes for the soluble intracellular adhesion molecule (US 5,412,216), whereas "F19HC” and “F19LC” code for the heavy or light chain of the humanized antibody F19 (EP 953 639).
• Figure 4 shows the correlation between sICAM productivity and GFP fluorescence using the cell pool ZB1 as an example. This cell pool was obtained from the transfection with the vector pBIDG-sICAM, in which the therapeutic protein sICAM and GFP are expressed together by a bicistronic transcription unit.
• the pool was subjected to a sequential GFP-based FACS sorting. After each sorting step (Sort), the concentration of the sICAM in the cell culture supernatant of the pool was determined by ELISA and the specific productivity per cell and day (pg / c * d) was calculated. Each data point represents the average of at least three cultivation passages. A total of six sorts were carried out.
• Figure 5 shows the isolation of highly expressive sICAM cells by GFP-based FACS sorting using the example of the cell pool ZB1.
• This cell pool was obtained from the transfection with the vector pBIDG-sICAM, in which the therapeutic protein sICAM and GFP are expressed together by a bicistronic transcription unit.
• the pool was subjected to a sequential GFP-based FACS sorting. After each sort, the concentration of the sICAM in the cell culture supernatant of the pool was determined by ELISA and the specific productivity per cell and day (pg / c * d) was calculated. Each data point represents the average of at least three cultivation passages. A total of six sorts were carried out.
• Figure 6 shows the increases in sICAM productivity achieved by combining a GFP-based selection with an MTX amplification step using the cell pool ZB1 as an example.
• This cell pool which was obtained from the transfection with the vector pBIDG-sICAM, was subjected to a sequential GFP-based FACS sorting.
• DHFR-mediated gene amplification was carried out by adding methotrexate (MTX) to the culture medium (5 nM, 50 nM, 500 nM or 2 // M MTX).
• MTX methotrexate
• the concentration of the sICAM in the cell culture supernatant of the pool was determined by ELISA and the specific productivity per cell and day (pg / c * d) was calculated.
• FIG. 7 shows the viability curve of cell pools after adding different high doses of methotrexate to the culture medium.
• the cell pool ZB1 which was obtained from the transfection with the vector pBIDG-sICAM (Fig. 3), was subjected to a sequential GFP-based FACS sorting. After the fourth sort or sixth sort, a DHFR-mediated gene amplification was carried out by adding methotrexate (MTX) to the culture medium. The cell numbers and the viability were determined during the selection phase by trypan blue staining and monitored over several days of cultivation.
• MTX methotrexate
• Figure 8 shows the correlation between antibody productivity (mAb F19) and GFP fluorescence using the cell pool ZB1 as an example.
• This cell pool was obtained from transfection with the vector combination pBIDG-F19HC and pBIN-F19LC (Fig.3).
• the pool was subjected to a sequential GFP-based FACS sorting. After each sort, the concentration of the antibody F19 in the cell culture supernatant of the pool was determined by ELISA and the specific productivity per cell and day (pg / c * d) was calculated. Each data point represents the average of at least three cultivation passages. A total of six sorts were carried out.
• Figure 9 shows the isolation of highly expressing mAb F19 cell pools by GFP-based selection using FACS using the cell pool ZB1 as an example.
• This cell pool which was obtained from the co-transfection with the vectors pBIDG-F19HC and pBIN-F19LC (FIG. 3), was subjected to a sequential GFP-based FACS sorting.
• the concentration of the antibody F19 in the cell culture supernatant of the pool was determined after each sort by ELISA and the specific productivity per cell and day (pg / c * d) was calculated.
• Each data point represents the average of at least three cultivation passages.
• the expression vector according to the invention contains a gene which codes for a protein of interest (“gene of interest”), in functional association with a hamster ubiquitin / S27a promoter and a gene which codes for a fluorescent protein.
• the expression vector preferably also contains an amplifiable selection marker gene.
• the hamster ubiquitin / S27a promoter is a strong homologous promoter which is described in WO 97/15664.
• a promoter preferably has at least one of the following features: GC-rich sequence region, Sp1 binding site, polypyrimidine element, absence of a TATA box.
• a promoter which has an Sp1 binding site in the absence of a TATA box is particularly preferred.
• such a promoter is preferred which is constitutively activated and is equally active, in particular, under serum-containing, serum-poor and serum-free cell culture conditions.
• it is an inducible promoter, in particular a promoter that is activated by serum withdrawal.
• a particularly advantageous embodiment is a promoter with a nucleotide sequence which is contained in FIG. 5 of WO 97/15664.
• Promoter sequences are particularly preferred in which the sequence from position -161 to - 45 of FIG. 5 is contained.
• the promoters used in the examples of the present patent description each contain a DNA molecule with the sequence from positions 1923 to 2406 of SEQ ID NO: 1 of the enclosed sequence listing. This sequence corresponds to fragment - 372 to + 111 from FIG. 5 of WO 97/15664 and represents the preferred promoter, ie a preferred promoter should encompass this sequence region.
• Another suitable promoter fragment contains the sequence from positions 2134 to 2406 (corresponds to - 161 to + 111 in FIG. 5 of WO) 97/15664).
• a promoter which only contains the sequence from position 2251 to 2406 is no longer functional (corresponds to position - 45 to + 111 in FIG. 5 of WO 97/15664).
• An extension of the promoter sequence in the 5 'direction starting from position 2134 is possible.
• a modified promoter preferably has a transcription activity which corresponds to that of the promoter fragment from positions 1923 to 2406 of the nucleotide sequence given in SEQ ID NO: 1 (- 372 to + 111 from FIG. 5 of WO 97/15664 ) corresponds.
• a modified promoter proves to be suitable for the purposes of the invention if it has a transcription activity which is at least 50%, better at least 80%, even better at least 90%, and even more preferably at least 100% of the activity of the 1923 to 2406 fragment ( - 372 to + 111 fragments) in a comparative reporter gene assay.
• Modified promoters which have a minimal sequence homology to the wild-type sequence SEQ ID NO: 1 of the hamster ubiquitin / S27a promoter of at least 80%, better at least 85%, preferably at least 90%, more preferably at least 95% and particularly preferred are particularly preferred have at least 97% and have a corresponding promoter activity in a comparative reporter gene assay.
• the promoter fragments to be tested are each cloned in front of a promoterless reporter gene which codes, for example, for luciferase, secreted alkaline phosphatase or green fluorescent protein (GFP).
• a promoterless reporter gene which codes, for example, for luciferase, secreted alkaline phosphatase or green fluorescent protein (GFP).
• GFP green fluorescent protein
• These constructs are then introduced into the test cells, for example CHO-DG44, by means of transfection and the induction of reporter gene expression determined by the respective promoter fragment by determining the protein content of the reporter gene.
• a corresponding test can also be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, 1994, updated.
• the promoter sequence of the hamster ubiquitin / S27a promoter as well as the modified promoters, which can also include, for example, the 5'-untranslated region or selected fragments thereof, and the coding region of the ubiquitin / S27a gene or selected fragments thereof, can be from a person skilled in the art with knowledge of the sequence described in WO 97/15664 can be obtained using various standard methods, such as, for example, in Sambrook et al., 1989; Ausubel et al., 1994. Starting from the sequence described in WO 97/15664, for example a suitable section can be selected and an oligonucleotide probe can be chemically synthesized which contains the sequence of this section.
• the ubiquitin / S27a gene or its 5'-untranslated region or other fragments can be cloned, for example by hybridization from a hamster genomic library.
• the reporter gene assay described above the person skilled in the art is able to identify promoter-active fragments without considerable effort and to use them in the sense of the present invention.
• the 5'-untranslated region or special fragments thereof can also easily be obtained by PCR amplification with appropriate primers from genomic DNA or a genomic library. Fragments of the 5 ' untranslated region can also be obtained from larger DNA fragments by limited exonuclease III digestion.
• Such DNA molecules can also be chemically synthesized or generated from chemically synthesized fragments by ligation.
• “Homologous” in the sense of the invention are genes, provided that their nucleotide sequence shows at least 70%, better at least 80%, preferably at least 90%, more preferably at least 95% and particularly preferably at least 97% agreement with the nucleotide sequence of the gene to which it is homologous is.
• the gene of interest contained in the expression vector according to the invention comprises a nucleotide sequence of any length which codes for a product of interest.
• the gene product or "product of interest” is usually a protein, polypeptide, peptide or fragment or derivative thereof. However, it can also be RNA or antisense RNA.
• the gene of interest can be in its entirety, in shortened form, as Fusion gene or labeled gene are present. It can be genomic DNA or preferably cDNA or corresponding fragments or fusions.
• the gene of interest can represent the native gene sequence, be mutated or be modified in some other way. Such modifications include codon optimizations for adaptation a specific host cell and a humanization
• the gene of interest can code, for example, for a secreted, cytoplasmic, nucleus-localized, membrane-bound or cell-surface-bound polypeptide.
• nucleotide sequence or “nucleic acid sequence” denotes an oligonucleotide, nucleotides, polynucleotides and their fragments as well as DNA or RNA of genomic or synthetic origin, which are present as a single or double strand and can represent the coding or the non-coding strand of a gene.
• Standard techniques such as, for example, site-specific mutagenesis or PCR-mediated mutagenesis (for example described in Sambrook et al., 1989 or Ausubel et al., 1994) can be used to modify nucleic acid sequences.
• Coding means the property or capability of a specific sequence of nucleotides in a nucleic acid, for example a gene in a chromosome or an mRNA, as a template for the synthesis of other polymers and macromolecules such as rRNA, tRNA, mRNA, other RNA To serve molecules, cDNA or polypeptides in a biological process. Accordingly, a gene codes for a protein if the desired protein is produced in a cell or another biological system by transcription and subsequent translation of the mRNA.
• Nucleic acid encoding a protein also includes nucleins acids with one, which, because of the degeneracy of the genetic code, another of nucleotide sequence, but result in the same amino acid sequence of the protein. Nucleic acid sequences that code for proteins can also contain introns.
• cDNA denotes deoxyribonucleic acids which are produced by reverse transcription and synthesis of the second strand of DNA from an mRNA or other RNA produced by a gene. If the cDNA is present as a double-stranded DNA molecule, then it contains both a coding as well as a non-coding strand.
• intron denotes non-coding nucleotide sequences of any length. They occur naturally in many eukyaryotic genes and are removed from a previously transcribed mRNA precursor by a process called splicing. For this, an exact cutting out of the intron at 5 ' - and 3 ' end and a correct connection of the resulting mRNA ends is required so that a mature processed mRNA is produced with the correct reading frame for the successful protein synthesis. Many of the splice donor and splice acceptor involved in this splicing process Locations are those that are present at the exon-intron or intron-exon boundaries Sequences have now been characterized. For an overview, see Ohshima et al., 1987.
• Biopharmaceutical proteins / polypeptides include e.g. Antibodies, enzymes, cytokines, lymphokines, adhesion molecules, receptors and their derivatives or fragments are, however, not restricted to these. In general, all polypeptides are important which act as agonists or antagonists and / or can find therapeutic or diagnostic use.
• polypeptides is used for amino acid sequences or proteins and denotes polymers of amino acids of any length. This term also includes proteins which are post-translationally modified by reactions such as, for example, glycosylation, phosphorylation, acetylation or protein processing.
• the structure of the polypeptide can be, for example, by substitutions , Deletions or insertion of amino acids, fusion with other proteins, while maintaining its biological activity.
• therapeutic proteins are insulin, insulin-like growth factor, human growth hormone (hGH) and other growth factors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines, for example interleukins (IL) such as IL-1, IL-2, IL -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 , IL-16, IL-17, IL-18, interferon (IFN) -alpha, beta, gamma, omega or tau, tumor necrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF, GM- CSF, M-CSF, MCP-1 and VEGF.
• IL interleukins
• IFN interferon
• TNF tumor necrosis factor
• TNF tumor necrosis factor
• the antibodies can be of human or non-human origin. Humanized and chimeric antibodies are also suitable.
• Fab fragments (fragment antigen-binding Fab) consist of the variable regions of both chains, which are held together by the adjacent constant regions. They can, for example, be generated from conventional antibodies by treatment with a protease, such as papain, or else by DNA cloning.
• Other antibody fragments are F (ab ') 2 fragments, which can be produced by proteolytic digestion with pepsin.
• variable region of the heavy and light chain are frequently linked to one another by means of a short peptide fragment of approx. 10-30 amino acids, particularly preferably 15 amino acids. This creates a single polypeptide chain in which VH and VL are linked by a peptide linker.
• scFv single-chain Fv fragment
• multimeric scFv derivatives have been developed in recent years to produce multimeric scFv derivatives.
• the intention is to generate recombinant antibodies with improved pharmacokinetic properties and increased binding avidity.
• these are produced as fusion proteins with multimerization domains.
• multimerization domains e.g. the CH3 region of an IgG or coiled coil structure act like the leucine zipper domains.
• the interaction between the VH and VL regions of the scFv fragment is used for multimerization (eg slide , Tri- and pentabodies).
• a person skilled in the art refers to a bivalent homodimeric scFv derivative as “diabody”.
• the shortening of the peptide linker in the scFv molecule to 5-10 amino acids results in the formation of homodimers through the superposition of VH / VL chains.
• the diabodies can also be stabilized by the introduction of disulfide bridges. Examples of diabodies can be found in the literature, for example in Perisic et al. (1994).
• minibody as a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, particularly preferably IgG1, as a dimerization region. This connects the scFv fragments via a hinge. Region, also from IgG, and a linker region, examples of such minibodies are described in Hu et al. (1996).
• trimers the person skilled in the art describes a trivalent homotrimeric scFv derivative (Kortt et al., 1997). The direct fusion of VH-VL without using a linker sequence leads to the formation of trimers.
• fragments which the person skilled in the art calls mini-antibodies and which have a bi-, tri- or tetravalent structure are also derivatives of scFv fragments.
• the multimerization is achieved via di-, tri- or tetrameric “coiled coil” structures (Pack et al., 1993 and 1995; Lovejoy et al., 1993).
• the expression vector according to the invention contains a gene coding for a fluorescent protein in functional association with the gene of interest and under the control of the hamster ubiquitin / S27a promoter, a modified hamster ubiquitin / S27a promoter or a homologue thereof.
• the fluorescent protein can be, for example, a green, blue-green, blue, yellow or other-colored fluorescent protein.
• GFP green fluorescent protein
• a special example is the green fluorescent protein (GFP) from Aequorea victoria or Renilla reniformis and mutants developed from it; see, for example, Bennet et al. (1998); Chalfie et al. (1994); WO 01/04306 and the literature cited there. Further fluorescent proteins and genes coding therefor are described in WO 00/34318, WO 00/34326, WO 00/34526 and WO 01/27150, which are incorporated herein by reference.
• fluorescent proteins are fluorophores from non-bioluminescent organisms of the species Anthozoa, for example from Anemonia majano, Clavularia sp., Zoanthus sp. I, Zoanthus sp. II, Discosoma striata, Discosoma sp. "Red”, Discosoma sp. "Green”, Discosoma sp. “Magenta”, Anemonia sulcata.
• the fluorescence proteins used according to the invention also include natural or genetically engineered mutants and variants, their fragments, derivatives or e.g. Variants fused with other proteins or peptides.
• the mutations introduced can change, for example, the excitation or emission spectrum, the formation of chromophores, the extinction coefficient or the stability of the protein. Codon optimization can also improve expression in mammalian cells or other species.
• the fluorescent protein can also be used in fusion with a selection marker, preferably with an amplifiable selection marker such as, for example, dihydrofolate reductase (DHFR).
• DHFR dihydrofolate reductase
• the fluorescence emitted by the fluorescent proteins enables the detection of the proteins e.g. by flow cytometry with a fluorescence activated cell sorter (FACS) or by fluorescence microscopy.
• FACS fluorescence activated cell sorter
• the hamster ubiquitin / S27a promoter can be brought into functional relationship with other regulatory sequences in order to increase / regulate the transcription activity in an expression cassette.
• the promoter can be functionally linked to enhancer sequences in order to increase the transcription activity. This can be done by one or more Enhancers and / or multiple copies of an enhancer sequence can be used, for example a CMV or SV40 enhancer.
• enhancer denotes a polynucleotide sequence which acts in cis localization on the activity of a promoter and thus stimulates the transcription of a gene functionally linked to this promoter.
• the action of the enhancers is position and orientation independent and can thus be positioned in front of or behind a transcription unit, within an intron or even within the coding region.
• the enhancer can be located both in the immediate vicinity of the transcription unit and at a considerable distance from the promoter. A physical and functional overlap with the promoter is also possible.
• enhancers are known to the person skilled in the art from various sources (and are stored in databases such as GenBank, for example SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer) and are available as independent elements or elements cloned within polynucleotide sequences (e.g. deposited at ATCC or from commercial and individual sources).
• a variety of promoter sequences also include enhancer sequences, e.g. the commonly used CMV promoter.
• the human CMV enhancer is one of the strongest enhancers identified to date.
• An example of an inducible enhancer is the metallothionein enhancer, which can be stimulated by glucocorticoids or heavy metals.
• the promoter sequences can also be combined with regulatory sequences which allow control / regulation of the transcription activity. In this way, the promoter can be made repressible / inducible. This can be done, for example, by linking to sequences that represent binding sites for positive or negative regulating transcription factors.
• the above-mentioned transcription factor SP-1 for example, has a positive influence on the transcription activity.
• Another example is the binding site for the activator protein AP-1, which can have a positive as well as a negative effect on the transcription.
• the activity of the AP-1 can be determined by various factors, such as growth factors, cytokines and serum, can be controlled (Faisst et al., 1992, and references therein).
• the transcription efficiency can also be increased by changing the promoter sequence by mutation (substitution, insertion or deletion) of one, two, three or more bases and then determining in a reporter gene assay whether this increases the promoter activity.
• the additional regulatory elements include promoters other than the hamster ubiquitin / S27a promoter, enhancers, termination and polyadenylation signals and other expression control elements. Both inducible and constitutive regulatory sequences are known for the different cell types. "Transcription regulatory elements" usually include a promoter upstream of the gene sequence to be expressed, transcription initiation and termination sites, and a polyadenylation signal.
• a “promoter” is a polynucleotide sequence that enables and controls the transcription of the genes or sequences functionally linked to it.
• a promoter contains recognition sequences for the binding of the RNA polymerase and the initiation site of the transcription (transcription initiation site).
• transcription initiation site For the expression of a desired sequence in A suitable, functional promoter must be selected in each case for a specific cell type or a host cell.
• the person skilled in the art knows a large number of promoters from various sources, including constitutive, inducible and repressible promoters. They are stored in databases, for example GenBank, and can be used as stand-alone or within elements cloned from polynucleotide sequences can be obtained from commercial or individual sources.
• inducible promoters the activity of the promoter can be reduced or enhanced in response to a signal.
• An example of an inducible pro motor is the tetracycline (tet) promoter.
• tet tetracycline
• tetO tetracycline operator sequences
• tTA tetracycline-regulated transactivator protein
• jun, fos, metallothionine and heat shock promoters see also Sambrook et al., 1989; Gossen et al., 1994).
• the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionin-I promoter, the long terminal repeat region of the Rous Sarcoma virus and the early promoter of the human cytomegalovirus are particularly well suited for high expression in eukaryotes.
• heterologous mammalian promoters are the actin, immunoglobulin, or heat shock promoter (s).
• transcription initiation site refers to a nucleic acid in the construct that corresponds to the first nucleic acid that is incorporated into the primary transcript, i.e., the mRNA precursor.
• the transcription initiation site may overlap with the promoter sequences.
• transcription termination site refers to a nucleotide sequence that is normally present at the 3 'end of the gene of interest or of the gene segment to be transcribed and which causes the transcription to be terminated by RNA polymerase.
• polyadenylation signal is a signal sequence which causes the cleavage at a specific site at the 3 'end of the eukaryotic mRNA and the post-transcriptional incorporation of a sequence of about 100-200 adenine nucleotides (polyA tail) at the cleaved 3' end.
• the polyadenylation signal comprises the sequence AATAAA about 10-30 nucleotides upstream of the cleavage site and a downstream sequence.
• Various polyadenylation elements are known, for example tk polyA, SV40 late and early polyA or BGH polyA (described for example in US Pat. No. 5,122,458).
• each transcription unit has a promoter or a promoter / enhancer element, a gene of interest and / or a marker gene, and also a transcription termination element.
• the transcription unit contains further translation regulatory units.
• “Translation regulatory elements” include a translation initiation site (AUG), a stop codon, and a polyA signal for each polypeptide to be expressed.
• the gene of interest usually contains a signal sequence encoding a signal precursor peptide that transports the synthesized polypeptide to and through the ER membrane, often, but not always, on the amine oterminus of the secreted protein and is cleaved by signal peptidases after the protein has been passed through the ER membrane.
• the gene sequence will usually, but not necessarily, contain its own signal sequence. If the native signal sequence is not present, a heterologous signal sequence can be introduced in a known manner. Numerous such signal sequences are known to the person skilled in the art and are stored in sequence databases such as GenBank and EMBL.
• IRES element comprises a sequence which functions the translation initiation independently of a 5'-terminal methylguanosinium cap (CAP structure) and the upstream gene and in an animal cell the translation of two cistrons (open reading frame) from a single transcript
• the IRES element provides an independent ribosome binding site for the translation of the open reading frame immediately downstream, in contrast to bacterial mRNA, which can be multicistronic, ie can code for several different polypeptides or products that are successively translated by the mRNA , most animal cell mRNAs are monocistronic and encode only a single protein or product, and a multicistronic transcript in a eukaryotic cell would translate from upstream of the closest translation initiation site and terminated by the first stop codon, whereupon the transcript would be released from the ribosome.
• a multicistronic transcript with an IRES element that is functionally linked to the second or further open reading frames in the transcript enables the subsequent translation of the downstream open reading frame so that in the eukaryotic cell two or more polypeptides encoded by the same transcript or products are produced.
• the IRES element can be of different lengths and of different origins and e.g. from the encephalomyocarditis virus (EMCV) or other picornaviruses.
• EMCV encephalomyocarditis virus
• Various IRES sequences and their use in the construction of vectors have been described in the literature; see e.g. Pelletier et al., 1988; Jang et al., 1989; Davies et al., 1992; Adam et al., 1991; Morgan et al., 1992; Sugimoto et al., 1994; Ramesh et al., 1996, Mosser et al., 1997.
• the downstream gene sequence is functionally linked to the 3 'end of the IRES element, i.e. the distance is chosen so that the expression of the gene is not influenced or is influenced only marginally or has an expression which is sufficient for the purpose.
• the optimal and permissible distance between the IRES element and the start codon of the downstream gene for sufficient expression can be determined in simple experiments by varying the distance and determining the expression rate as a function of the distance with the aid of reporter gene assays.
• a preferred vector according to the invention additionally contains an amplifiable selection marker gene which enables an amplification of the amplifiable marker gene and preferably the co-amplification of a transcription unit consisting of the hamster ubiquitin / S27a gene, the gene of interest and the gene for the fluorescent protein.
• the host cells transfected with a corresponding expression vector are cultivated in the presence of a suitable selection agent, so that only those host cells can be reproduced which have several gene copies of at least the amplifiable selection marker gene. This is preferably achieved by gradually culturing the cells in the presence of increasing amounts of selection agent.
• the amplifiable selection marker gene usually encodes an enzyme necessary for the growth of eukaryotic cells under certain cultivation conditions.
• the amplifiable selection marker gene can code for dihydrofolate reductase (DHFR).
• DHFR dihydrofolate reductase
• the gene is amplified if a host cell transfected with it is cultivated in the presence of the selection agent methotrexate (MTX).
• MTX methotrexate
• Table 1 below gives examples of further amplifiable selection marker genes which can be used according to the invention and the associated selection agents, which are described in an overview by Kaufman, Methods in Enzymology, 185: 537-566 (1990).
• a gene which codes for a polypeptide with the function of DHFR is preferred as the amplifiable selection marker gene Fusion protein from the fluorescent protein and DHFR.
• DHFR is required for the biosynthesis of purines. Cells that lack the DHFR gene cannot grow in purine-deficient media. The DHFR gene is therefore a useful selection marker for the selection and amplification of genes in cells which are cultivated in a purine-free medium.
• the selection agent used in conjunction with the DHFR gene is methotrexate (MTX).
• the present invention therefore includes a method of producing high-production recombinant host cells, comprising the following steps: (i) transfection of the host cells with genes encoding at least one protein of interest, a fluorescent protein and DHFR, (ii) culturing the Cells under conditions that allow expression of the various genes, and (iii) amplification of these co-integrated genes by culturing the cells in the presence of a selection agent that allows the amplification of at least the amplifiable selectable marker gene, such as methotrexate.
• the transfected cells are preferably cultivated in hypoxanthine / thymidine-free medium in the absence of serum and with the addition of increasing concentrations of MTX.
• the concentration of MTX in the first amplification step is preferably at least 200 nM, in an even more preferred embodiment at least 500 nM and can be increased in steps up to 1 / M. In individual cases, concentrations of over 1 ⁇ M can also be used.
• Mammalian cells preferably mouse myeloma and hamster cells, are preferred host cells for the use of DHFR as an amplifiable selection marker.
• the cell lines CHO-DUKX (ATCC CRL-9096) and CHO-DG44 (Urlaub et al., 1983) are particularly preferred since, due to the mutation, they do not have their own DHFR activity.
• a mutated DHFR gene can be used in the transfection, which codes for a protein with a reduced sensitivity to methotrexate (Simonson et al., 1983; Wigler et al., 1980; Haber et al., 1982).
• the expression vector according to the invention can in principle be prepared by conventional methods known to the person skilled in the art, such as, for example, in Sambrock et al. (1989). There is also a description of the functional components of a vector, eg suitable promoters (in addition to the hamster ubiquitin / S27a promoter), enhancers, termination and polyadenylation signals, antibiotic resistance genes, selection markers, replication starting points and splice signals.
• cloning vectors can be used for the production, for example plasmids, bacteriophages, phagemids, cosmids or viral vectors such as baculovirus, retroviruses, adenoviruses, adeno-associated viruses and herpes simplex virus, but also artificial chromosomes / mini-chromosomes.
• the eukaryotic expression vectors typically also contain prokaryotic sequences such as, for example, the origin of replication and antibiotic resistance genes, which enable the multiplication and selection of the vector in bacteria.
• a variety of eukaryotic expression vectors containing multiple cloning sites for the introduction of a polynucleotide sequence are known and some are commercially available from various companies such as Stratagene, La Jolla, CA, USA; Invitrogen, Carlsbad, CA, USA; Promega, Madison, Wl, USA or BD Biosciences Clontech, Palo Alto, CA, USA available.
• the hamster ubiquitin / S27a promoter, the gene of interest, the gene coding for a fluorescent protein preferably also the amplifiable selection marker gene, for example dihydrofolate reductase, and optionally additional regulatory elements such as the internal ribosome binding site ( IRES), enhancer or a polyadenylation signal introduced into the expression vector.
• An expression vector according to the invention minimally contains an ubiquitin / S27a promoter, the gene of interest, and the gene coding for a fluorescent protein.
• the expression vector preferably also contains an amplifiable selection marker gene.
• modified ubiquitin / S27a promoters for example like the modified ubiquitin / S27a promoters described in the present application, is also in accordance with the invention.
• a promoter / enhancer is functionally linked to a coding gene sequence if it can control or modulate the transcription of the linked gene sequence in the cis position.
• functionally linked DNA sequences are in close proximity and, if two coding gene sequences are linked or in the case of a secretion signal sequence, in the same reading frame.
• a functionally linked promoter is generally upstream of the coding gene sequence, it does not necessarily have to be closely adjacent.
• Enhancers also do not have to be in close proximity, as long as they favor transcription of the gene sequence. For this purpose, they can be both upstream and downstream of the gene sequence, possibly at some distance.
• a polyadenylation site is functionally linked to a gene sequence if it is positioned at the 3 'end of the gene sequence in such a way that the transcription proceeds via the coding sequence up to the polyadenylation signal.
• the linkage can be carried out using conventional recombinant methods, e.g. by means of the PCR technique, by ligation at suitable restriction sites or by splicing. If no suitable restriction sites are available, synthetic oligonucleotide linkers or adapters can be used in a manner known per se. According to the invention, the functional linkage preferably does not take place via intron sequences.
• the ubiquitin / S27a promoter or a modified form thereof, the gene of interest and the gene coding for a fluorescent protein are functionally linked to one another.
• the functional linkage takes place via an IRES element, so that a bicistronic mRNA is synthesized by both genes.
• the expression vector according to the invention can additionally contain enhancer elements which act functionally on one or more promoters.
• An expression vector in which the ubiquitin / S27a promoter or a modified form thereof is linked to an enhancer element for example an SV40 enhancer or a CMV enhancer element, is particularly preferred.
• the genes can be expressed within an expression vector from one or more transcription units.
• a region that contains one or more genes to be transcribed is defined as a “transcription unit”.
• the genes within a transcription unit are functionally linked to one another such that all genes within such a unit are under the transcriptional control of the same promoter or promoter / enhancer.
• IRES elements or introns can be used for the functional linking of the genes within a transcription unit.
• the expression vector can contain a single transcription unit for expressing the gene of interest, the gene for the fluorescent protein and the amplifiable selection marker. Alternatively, these genes can also be arranged in two or more transcription units. Different combinations of the genes are possible within a transcription unit. In a further embodiment of the present invention, more than one expression vector, consisting of one, two or more transcription units, can be introduced into a host cell by co-transfection or in successive transfections in any order. Any combination of regulatory elements and genes on each vector can be chosen as long as sufficient expression of the transcription units is ensured. If necessary, further regulatory elements and genes, such as additional genes of interest or selection markers, can be positioned on the expression vectors.
• the expression vector according to the invention can contain the gene which codes for a fluorescent protein and the amplifiable selection marker gene in one or in two separate transcription units.
• Each transcription unit can transcribe and express one or more gene products. If both genes are contained in a transcription unit, they are under the control of the same promoter or promoter / enhancer, preferably using an IRES element to ensure the functional linkage of all components.
• the gene encoding a fluorescent protein and the amplifiable selection marker gene are contained in two separate transcription units, they can be under the control of the same or different promoters / enhancers.
• the one (bicistronic) transcription unit contains the gene of interest and the gene coding for a fluorescent protein, while the other transcription unit contains the amplifiable selection marker gene.
• Each transcription unit is preferably delimited at the 3 'end by a sequence which codes for a polyA signal, preferably tk polyA, BGH polyA or SV40 polyA.
• nucleotide sequences for a wide variety of restriction endonucleases and the associated restriction endonucleases are known in the prior art. Sequences are preferably used which consist of at least 6 nucleotides exist as a recognition sequence. A list of suitable recognition sequences can be found, for example, in Sambrook et al. (1989).
• Eukaryotic host cells are used for transfection with the expression vector according to the invention, preferably mammalian cells and in particular rodent cells such as e.g. Mouse, rat and hamster cell lines.
• rodent cells such as e.g. Mouse, rat and hamster cell lines.
• the successful transfection of the corresponding cells with an expression vector according to the invention results in transformed, genetically modified, recombinant or transgenic cells, which are also the subject of the present invention.
• Host cells preferred in the context of the invention are hamster cells such as, for. BHK21, BHK TK “ , CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-DG44 cells or derivatives / descendants of these cell lines.
• CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cells are particularly preferred, in particular CHO-DG44 and CHO-DUKX cells, mouse myeloma cells, preferably NS0 and Sp2 / 0 cells and derivatives / descendants of these cell lines are also suitable.
• hamster and mouse cells examples include human, mouse, rat, monkey, rodent, or eukaryotic cells, including but not limited to yeast, insect, and plant cells, can also be used as host cells Production of biopharmaceutical proteins can be used.
• the eukaryotic host cells are transfected with a polynucleotide or one of the expression vectors according to the invention by customary methods (Sambrook et al., 1989; Ausubel et al., 1994). Suitable transfection methods are e.g. liposome-mediated transfection, calcium phosphate coprecipitation, electroporation, polycations (e.g. DEAE-dextran) -mediated transfection, protoplast fusion, microinjection and viral infections. According to the invention, a stable transfection is preferably carried out, the constructs either being integrated into the genome of the host cell or an artificial chromosome / minichromosome or are contained in a stable manner episomally in the host cell.
• each sequence or gene that is introduced into a host cell is referred to as a “heterologous sequence” or “heterologous gene” with respect to the host cell. Even if the sequence or gene to be introduced is identical to an endogenous sequence or an endogenous gene of the host cell.
• a hamster actin gene that is introduced into a hamster host cell is by definition a heterologous gene.
• heterodimeric proteins such as monoclonal antibodies (mAbs)
• mAbs monoclonal antibodies
• the transfection of suitable host cells can in principle be carried out in two different ways.
• Such mAbs are made up of several subunits, the heavy and light chains. Genes coding for these subunits can be found in independent or in multicistronic transcription units a single plasmid, which is then used to transfect the host cell. This is to ensure the stoichiometric representation of the genes after integration into the genome of the host cell.
• independent transcription units it must be ensured that the mRNAs that code for the different proteins have the same stability, transcription and translation efficiency.
• the expression of the genes within a multicistronic transcription unit is carried out by a single promoter and only one transcript is produced.
• the use of IRES elements enables a quite efficient internal translation initiation of the genes in the second and subsequent cistrons. Nevertheless, the expression rates for these cistrons are lower than that of the first cistron, whose translation initiation via a so-called “cap” -dependent pre-initiation complex is much more efficient than the IRES-dependent translation initiation.
• additional intercistronic elements are introduced which, in cooperation with the IRES elements, ensure uniform expression rates (WO 94/05785).
• Another possibility preferred according to the invention for the simultaneous production of several heterologous proteins is co-transfection, in which the genes are integrated separately into different expression vectors.
• This has the advantage that certain ratios of the genes and gene products to one another can be set, as a result of which differences in the mRNA stability and in the transcription and translation efficiency can be compensated for.
• the expression vectors are more stable and easier to handle both in cloning and in transfection.
• the host cells are therefore additionally transfected, preferably co-transfected, with one or more vectors with genes which code for one or more other proteins of interest.
• the other vector (s) used for the co-transfection code for example, for the other protein (s) of interest under the control of the same Promoter / enhancer combination and for at least one further selection marker, for example neomycin phosphotransferase.
• the host cells are preferably established, adapted and cultivated under serum-free conditions, if appropriate in media which are free from animal proteins / peptides.
• media which are free from animal proteins / peptides.
• Examples of commercially available media are Ham 's F12 (Sigma, Deisenhofen, DE), RPMI-1640 (Sigma), Dulbecco 's Modified Eagle 's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), Iscove ' s Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Ca., USA), CHO-S-SFMII (Invitrogen), serum-free CHO medium (Sigma) and protein-free CHO medium (Sigma).
• Each of these media can optionally be supplemented with various compounds, e.g. hormones and / or other growth factors (e.g. insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g. sodium chloride, calcium, magnesium, phosphate), buffers (e.g. HEPES) , Nucleosides (eg adenosine, thymidine), glutamine, glucose or other equivalent nutrients, antibiotics and / or trace elements.
• growth factors e.g. insulin, transferrin, epidermal growth factor, insulin-like growth factor
• salts e.g. sodium chloride, calcium, magnesium, phosphate
• buffers e.g. HEPES
• Nucleosides eg adenosine, thymidine
• glutamine glucose or other equivalent nutrients
• antibiotics and / or trace elements e.g., antibiotics and / or trace elements.
• serum-free media are preferred according to the invention, media which have been
• a “selection agent” is a substance that affects the growth or survival of host cells with a deficiency for the respective selection marker gene.
• the antibiotic Geneticin G4108 is used as a medium additive to select for the presence of an expressed antibiotic resistance gene such as the neomycin phosphotransferase
• the selection agent can also be a substance which triggers an amplification of the selection marker gene if the gene used is an amplifiable selection marker (see Table 1), for example methotrexate is a selection agent which is suitable for the amplification of the DHFR gene Examples of other selection agents which trigger amplification are listed in Table 1.
• a “selection marker gene” is a gene which enables the specific selection of cells which receive this gene by adding an appropriate selection agent to the culture medium.
• an antibiotic resistance gene can be used as a positive selection marker. Only cells that contain this Genes that have been transformed can grow and therefore be selected in the presence of the corresponding antibiotic. Non-transformed cells, on the other hand, cannot grow or survive under these selection conditions.
• the selection markers used in this invention include genetically engineered mutants and variants, fragments, functional equivalents, derivatives, homologues and fusions with other proteins or peptides, as long as the selection marker maintains its selective properties.
• Such derivatives have considerable homology in the amino acid sequence in the regions or domains to which the selective property is ascribed.
• selection marker genes including bifunctional (positive / negative) markers, have been described in the literature (see, for example, WO 92/08796 and WO 94/28143).
• selection markers commonly used in eukaryotic cells include the genes for aminoglycoside phosphotransferase (APH), hygromycin phosphotransferase (HYG), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase, asparagine synthetase and genes that confer resistance to neomycin (G418), puromycin, histidinol D, bleomycin, phleomycin and zeocin.
• APH aminoglycoside phosphotransferase
• HAG hygromycin phosphotransferase
• DHFR dihydrofolate reductase
• TK thymidine kinase
• G418 puromycin,
• FACS fluorescence-activated cell sorting
• Bacterial ß- Galactosidase, cell surface markers or fluorescent proteins e.g. green fluorescent protein (GFP) and its variants of Aequorea victoria and Renilla reniformis or other species, red fluorescent protein and proteins fluorescent in other colors and their variants of non-bioluminescent organisms such as Discosoma sp., Anemonia sp., Clavularia sp., Zoanthus sp.
• GFP green fluorescent protein
• red fluorescent protein and proteins fluorescent in other colors and their variants of non-bioluminescent organisms such as Discosoma sp., Anemonia sp., Clavularia sp., Zoanthus sp.
• the use of the DHFR gene is preferred for the selection of genetically modified (recombinant) host cells as amplifiable selection marker gene.
• DHFR-negative basic cells such as CHO-DG44 or CHO-DUKX
• this marker is particularly suitable for selection and subsequent amplification, since these cells do not express endogenous DHFR and therefore do not grow in a purine-free medium. Therefore, the DHFR gene can be used as the dominant selection marker and the transformed cells are selected in hypoxanthine / thymidine-free medium.
• Methotrexate (MTX) is used to achieve DHFR-mediated gene amplification. The growth properties are significantly influenced by the addition of MTX.
• recombinant host cells can be enriched by the clone selection system according to the invention, which show a considerably more robust behavior towards high MTX concentrations (see Fig. 7).
• host cells that were identified and sorted out using a fluorescence-activated line sorter (FACS) could be cultivated and amplified in the presence of 500 nM, preferably in the presence of 1 // M MTX, which led to a significant increase in productivity.
• FACS fluorescence-activated line sorter
• a method for the selection of highly producing host cells is considered to be particularly according to the invention, in which host cells which are transfected with an expression vector according to the invention and at least express the gene of interest, the fluorescent protein and a DHFR gene, are sorted out using FACS sorting, and at least be subjected to a gene amplification step in the presence of at least 500 nM, preferably 1 ⁇ M MTX.
• “Fermentation robustness” means growth properties of the cells such as, for example, adherence to certain growth rates, robustness towards “upscaling” (larger dimensions of the bioreactors) and reaching high cell numbers and vitalities in the stock keeping, in order to do justice to the industrial passenger rates during “upscaling”.
• expression refers to the transcription and / or translation of a heterologous gene sequence in a host cell.
• the expression rate can be determined generally, either on the basis of the amount of the corresponding mRNA that is present in the host cell or on the basis of the one produced Amount of
• a gene product encoded by the gene of interest The amount of through
• Transcription of a selected nucleotide sequence generated mRNA can be determined, for example, by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization of cellular RNA or by PCR methods
• Nucleotide sequence can also be encoded by various methods, such as by ELISA, Western blot, radioimmunoassay, immunoprecipitation, detection of the biological activity of the protein or by immunostaining
• Protein with subsequent FACS analysis can be determined (Sambrook et al., 1989;
• High expression level (rate) means the persistent and sufficiently high expression or synthesis of a heterologous sequence introduced into a host cell, for example a gene coding for a therapeutic protein, designated.
• Enhanced or high expression or a high expression level (rate) or high productivity is present if a cell according to the invention is cultivated according to a method according to the invention described here and if this cell is at least more than approximately 5 pg of the desired gene product produced per day (5 pg / day / cell).
• An increased or high expression or a high expression level (rate) or high productivity is also present if the cell of the invention produces at least more than about 10 pg of the desired gene product per day (10 pg / day / cell).
• Enhanced or high expression or a high level (s) of expression or high productivity is particularly present when the cell according to the invention produces at least more than about 15 pg of the desired gene product per day (15 pg / day /Cell).
• Enhanced or high expression or a high level (s) of expression or high productivity is present in particular if the cell according to the invention produces at least more than about 20 pg of the desired gene product per day (20 pg / day /Cell).
• a particularly increased or high expression or a particularly high expression level (rate) or a particularly high productivity is present when the cell according to the invention produces at least more than about 30 pg of the desired gene product per day (30 pg / day / cell).
• a high or increased expression, a high productivity or a (e) high expression level (rate) in the sense of the present invention can be achieved in different ways.
• a gene for an amplifiable selection marker By co-expressing the gene of interest with a gene for an amplifiable selection marker, cells can be selected and identified that express the heterologous gene to a high degree.
• the amplifiable selection marker enables not only the selection of stably transfected host cells but also the gene amplification of the heterologous gene of interest.
• the integration of the additional copies of the nucleic acids can take place in the genome of the host cells, in additional artificial / mini-chromosomes or in episomally localized polynucleotides.
• This procedure can be combined with a FACS-assisted selection of recombinant host cells which contain, for example, one (or more) fluorescent protein (s) (eg GFP) or a cell surface marker as a further selection marker.
• fluorescent protein eg GFP
• Other methods of achieving increased expression, and a combination of different methods is also possible, are based, for example, on the use of (artificial) transcription factors, treatment of the cells with natural or synthetic agents for upregulating endogenous or heterologous gene expression, improvement of the stability (half-life) of the mRNA or protein, improvement of mRNA Translation initiation, increasing the gene dose by using episomal plasmids (based on the use of viral sequences as the origin of replication, for example SV40, polyoma, adenovirus, EBV or BPV), using amplification-promoting sequences (Hemann et al., 1994) or on DNA Concatener-based in vitro amplification systems (Monaco et al., 1996).
• a coupled transcription of the gene of interest and the gene which codes for the fluorescent protein Both the protein of interest and the fluorescent protein are expressed from the resulting bicistronic mRNA. Due to this coupling of the expression of the protein of interest and the fluorescent protein, it is easily possible according to the invention to select and isolate high-producing recombinant host cells via the expressed fluorescent protein, e.g. by sorting with the aid of a fluorescence-activated line sorting device (FACS).
• FACS fluorescence-activated line sorting device
• the selection of recombinant host cells that show a high vitality and an increased expression rate of the desired gene product is a multi-stage process.
• the host cells transfected with the expression vector according to the invention or optionally a further vector, for example co-transfected, are examined at least for the expression of the gene coupled to the gene of interest which codes for a fluorescent protein in order to identify and select the cell / cell population, that show the highest expression levels of fluorescent protein.
• Preferably, only the cells that belong to the 10% cells with the highest expression rate of fluorescent protein are sorted out and further cultivated. In practice, this means that the brightest 10% of the fluorescent cells are sorted out and further cultivated.
• the brightest 5%, preferably the brightest 3%, or even the brightest 1% of the fluorescent cells of a cell mixture can be sorted out and multiplied.
• only the brightest 0.5% or the brightest 0.1% of the fluorescent cells are sorted out and multiplied.
• the cells previously transformed with the expression vector according to the invention are cultivated in a selection medium which optionally also contains a selection agent specific for the amplifiable selection marker. Gradually increased concentrations of selection agent can be used to exert a gradually increased selection pressure.
• the selection step can be carried out on cell pools or with pre-sorted cell pools / cell clones.
• One or more, preferably two or more and in particular three or more sorting steps can be carried out, with the cells between the individual sorting steps over a certain period of time, e.g. about two weeks at pools, cultivated and propagated.
• the host cells can be subjected to one or more gene amplification steps in order to increase the number of copies of at least the gene of interest and the amplifiable selectable marker gene.
• Methods for stepwise gene amplification using methotrexate are described by way of example in US Pat. No. 5,179,017. According to the high productivity that can be achieved is not tied to an increased number of gene copies. Rather, it is an expression of the increased stability and fermentation robustness of the high-performance clones. It is therefore possible to reduce the number of gene amplification steps required and e.g. perform only one gene amplification.
• a method for the selection of cells comprises the following steps: i) transformation of suitable host cells at least with one of the vectors according to the invention, the DNA of the expression vectors preferably being stably incorporated into the host cell genome or into artificial chromosomes / minichromosomes; ii) the transformed cells are cultured under conditions that allow expression of the gene of interest and the fluorescent protein; iii) the cells are cultivated in the presence of at least one selection agent, so that only those cells are grown which can grow in the presence of said selection agent; iv) sorting out cells from a cell mixture which show the highest expression rate of fluorescent protein, the cells being detected and sorted with the aid of a fluorescence-activated line sorting device (FACS); v) the cultivation of the sorted cells with the highest expression rates for the fluorescent protein.
• FACS fluorescence-activated line sorting device
• steps ii) - v) can be repeated one or more times with the cells obtained after step v).
• the transformed cells can optionally also be subjected to one or more gene amplification tests in which they are cultivated in the presence of a selection agent which leads to an amplification of the amplifiable selectable marker gene. This step can be carried out both with cells that have not yet been sorted and with cells that have already been pre-sorted once or several times.
• the selected high-producing cells are preferably grown in a serum-free culture medium and preferably in suspension culture under conditions which allow expression of the gene of interest.
• the protein of interest is preferably obtained as a secreted gene product from the cell culture medium. If the protein is expressed without a secretion signal, the gene product can also be isolated from cell lysates. In order to obtain a pure, homogeneous product which is essentially free of other recombinant proteins and host cell proteins, usual cleaning steps are carried out. To do this, cells and cell debris are often first removed from the culture medium or lysate.
• the desired gene product can then be freed from contaminating soluble proteins, polypeptides and nucleic acids, for example by fractionation on immunoaffinity and ion exchange columns, ethanol precipitation, reverse phase HPLC or chromatography on Sephadex, silica or cation exchange resins such as DEAE.
• Methods used to Purification of a heterologous protein expressed by recombinant host cells are known to the person skilled in the art and are described in the literature, for example by Harris et al. (1995) and Scopes (1988).
• alkaline phosphatase bp base pair
• FACS fluorescence-activated cell sorter
• FAP fibroblast activated protein
• GFP green fluorescent protein
• HRPO horseradish peroxidase
• IRES infernal ribosomal entry site kb: Kilobase mAb: monoclonal antibody
• PCR polymerase chain reaction sICAM: soluble intracellular adhesion molecule
• the cells CHO-DG44 / DHFR - / - (Urlaub et al., 1983) were permanently suspended cells in serum-free and hypoxanthine and thymidine supplemented CHO-S-SFMII medium (Invitrogen GmbH, Düsseldorf, DE) in cell culture bottles at 37 ° C cultivated in a humid atmosphere and 5% C0 2 .
• the cell counts as well as the viability were checked with a CASY1 Cell Counter (sharpness System, DE) or by trypan blue staining and the cells are then sown in a concentration of 1 - 3 x10 5 / mL and passaged every 2 - 3 days.
• Lipofectamine Plus reagent (Invitrogen GmbH) was used to transfect CHO-DG44.
• a total of 1 ⁇ g plasmid DNA, 4 ⁇ L lipofectamine and 6 ⁇ L plus reagent according to the manufacturer's instructions were mixed and mixed in a volume of 200 ⁇ L with 6 ⁇ 10 5 exponentially growing CHO-DG44 cells in 0.8 mL HT- supplemented CHO-S-SFMII medium. After three hours of incubation at 37 ° C in a cell incubator, 2 mL of HT-supplemented CHO-S-SFMII medium was added.
• DHFR-based selection of stably transfected CHO-DG44 the cells were transferred 2 days after transfection in CHO-S-SFMII medium without addition of hypoxanthine and thymidine, the medium being changed every 3 to 4 days.
• the medium was also G418 (Invitrogen) in a concentration of 400 ⁇ g / mL added.
• DHFR-based gene amplification of the integrated heterologous genes was achieved by adding the selection agent MTX (Sigma, Deisenhofen, DE) in a concentration of 5 - 2000 nM to the HT-free CHO-S-SFMII medium.
• MTX Sigma, Deisenhofen, DE
• Expression Vectors For expression analysis, eukaryotic expression vectors based on the pAD-CMV vector (Werner et al., 1998) and the constitutive expression of a heterologous gene via the combination of CMV enhancer / hamster ubiquitin / S27a promoter (WO 97/15664) were used. convey. While the base vector pBID contains the DHFR minigen, which serves as an amplifiable selection marker (see, for example, EP 0 393 438), the DHFR minigen in the vector pBIN has been replaced by a neomycin resistance gene (Fig. 2).
• the selection marker neomycin phosphotransferase including SV40 early promoter and TK polyadenylation signal, from the commercial plasmid pBK-CMV (Stratagene, La Jolla, CA, USA) isolated as a 1640 bp Bsu36l fragment. After a filling reaction of the fragment ends by Klenow DNA polymerase, the fragment was ligated with the 3750 bp Bsu36l / Stul fragment of the vector pBID, which was also treated with Klenow DNA polymerase. In the bicistronic base vector pBIDG (Fig.
• the IRES-GFP gene region was isolated from the vector plRES2-EGFP (Clontech, Palo Alto, CA, USA) and brought under the control of the CMV enhancer / promoter in the vector pBID, that the multiple cloning site between promoter region and IRES element was preserved.
• the procedure was as follows. In a PCR mutagenesis, the plasmid plRES2-EGFP being used as a template, the HindIII-AAGCTT interface within the IRES sequence was converted into the sequence sequence ATGCTT by the use of mutagenic primers and thus removed.
• an Xbal interface was introduced by means of a primer with complementarity to the 5 ' end of the IRES sequence and a Spel interface with complementarity to the 3 ' end of the GFP sequence.
• the resulting PCR fragment which included the complete IRES and GFP sequence, was digested with Xbal and Spei and cloned into the singular Xbal site at the 3 ' end of the multiple cloning site of the vector pBID.
• the human sICAM gene was isolated as a HindIII / SalI fragment from pAD-sICAM (Werner et al., 1998) and cloned into the corresponding interfaces of the vector pBIDG, resulting in the vector pBIDG-sICAM (Fig. 3).
• the heavy chain was isolated as a 1.5 kb Nael / Hindlll fragment from the plasmid pG1D105F19HC (NAGENESEQ: AAZ32786) and in the vector digested with EcoRI (filled in with Klenow DNA polymerase) and Hindlll pBIDG cloned, resulting in the vector pBIDG-F19HC (Fig.3).
• the flow cytometric analyzes and sorting were carried out with a Coulter Epics Altra device.
• the FACS is equipped with a helium-argon laser with an excitation wavelength of 488 nm.
• the fluorescence intensity is recorded at a wavelength that is adequate for the fluorescence protein and processed using the connected software Coulter Expo32.
• the sorting was carried out at a rate of 8000 - 10000 events / second.
• the suspended cells were centrifuged off (5 min at 180 ⁇ g) and adjusted to a cell concentration of 1-1.5 ⁇ 10 7 / mL in HBSS.
• the cells were then sorted according to their fluorescent protein signal.
• the cells were taken up in tubes with the culture medium provided, centrifuged and, depending on the number of cells sorted out, sown into corresponding culture vessels.
• sICAM titers in supernatants from stably transfected CHO-DG44 cells were quantified by ELISA according to standard protocols (Ausubel et al., 1994, updated), using two sICAM-specific mAbs developed in-house (for example in US Pat. No. 5,284,931 and US 5,475,091). One of the two antibodies is an HRPO-conjugated antibody. Purified sICAM protein was used as the standard.
• the F19 mAb in the supernatants of stably transfected CHO-DG44 cells was quantified using ELISA according to standard protocols (Ausubel et al., 1994, updated), using a goat anti-human IgG Fc fragment (Dianova, Hamburg, DE) and on the other hand an AP-conjugated goat anti human kappa light chain antibody (Sigma) was used. Purified F19 antibodies served as standard. Productivities (pg / cell / day) were calculated using the formula pg / ((Ct-Co) t / In (Ct-Co)), where Co and Ct indicate the number of cells when sowing or harvesting and t the cultivation time.
• Example 1 Comparison of CMV and hamster ubiquitin / S27a promoter activity
• CHO-DG44 cells were transfected with various recombinant vectors.
• the heterologous gene product on the one hand a lysosomal enzyme and on the other hand an IgG1 antibody, was expressed either under the control of the CMV promoter or under that of the hamster ubiquitin / S27a promoter. Both promoters were functionally linked to the CMV enhancer.
• the BGH polyA was used as the termination signal for the heterologous gene.
• the expression vectors which contained the CMV promoter were based either on a modified pcDNA3 ("CMV 1 ", Invitrogen) or pBluescript vector ("CMV 2 ", Stratagene) and additionally coded for the amplifiable selection marker dihydrofolate reductase.
• the expression vector with the hamster promoter is based on the pAD-CVM vector (Werner et al., 1998).
• a co-transfection with a second vector was carried out, which contained a neomycin resistance gene as a selection marker.
• the CMV enhancer can also be replaced by the SV40 enhancer.
• cell clones in HT-free medium were selected and isolated after the transfection.
• Cell clones with the highest productivity with regard to the recombinant protein were subjected to a step-by-step DHFR-based gene amplification by gradually increasing the methotrexate concentration from 5 nM to 50 nM, 500 nM to 2 ⁇ M, in each case combined with a dilution cloning. About 20 to 30 clones with the highest productivity were selected at each amplification stage.
• the hamster promoter proved to be more powerful. Both in the expression of the lysosomal enzyme and in the expression of the antibody, productivities or titers were achieved which were 2 to 5 times higher than in the cells in which the heterologous gene was expressed under the control of the CMV promoter.
• the relative titers and relative are exemplary specific productivities of the best cell clones at the respective amplification level, the expression based on the CMV promoter being set as 1 for the respective heterologous gene (CMV 1 for the lysosomal enzyme, CMV 2 for the antibody).
• Example 2 Isolation of highly expressive sICAM cells by GFP-based FACS sorting
• sICAM intercellular adhesion molecule
• the therapeutic protein sICAM and GFP were expressed together by a bicistronic and the DHFR by a separate transcription unit. Two to three weeks after the first selection in HT-free CHO-S-SFMII medium, the 5% of the cells with the highest GFP fluorescence were sorted out. After approximately two weeks of cultivation, the 5% cells with the highest GFP fluorescence were again isolated. In total, this sequential sorting was carried out six times. A good correlation between sICAM productivity and GFP fluorescence was shown (Fig. 4). The FACS-based selection alone, without any MTX amplification step, quickly isolated cell pools with high specific productivities of up to 16 pg / cell / day (Fig. 5).
• productivity could even be increased to over 30 pg / cell / day (Fig. 6).
• Example 3 Isolation of cells with high expression of the mAb F19 by GFP-based FACS sorting
• CHO-DG44 cells were transfected with the plasmid combination pBIDG-F19HC and pBIN-F19LC (Fig.3).
• the expressed humanized antibody F19 is directed against the surface molecule FAP, which is synthesized by reactive stroma fibroblasts (see also patent EP 0 953 639).
• the two protein chains of the antibody are each expressed by their own vector, which additionally codes for a DHFR or neomycin phosphotransferase selection marker in a separate transcription unit.
• the vector pBIDG-F19HC also contains a further selection marker, the GFP.
• the co-transfection of CHO-DG44 with the vectors pBIDG-F19HC / pBIN-F19LC enabled cells with a high expression of the antibody F19 in a short time are isolated that the cells with a high GFP content were selected by sequential FACS sorting.
• FACS sorting After a first two to three week selection of the transfected cell pools in HT-free CHO-S-SFMII medium with the addition of 400 ⁇ g / mL G418 sorted out the 5% of the cells with the highest GFP fluorescence by FACS. This sorting was carried out up to six times in total, with a cultivation period of approximately 2 weeks between each sorting.

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