WO1998059059A1 - Systeme d'expression pour la production de proteines therapeutiques - Google Patents
Systeme d'expression pour la production de proteines therapeutiques Download PDFInfo
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- WO1998059059A1 WO1998059059A1 PCT/US1998/012777 US9812777W WO9859059A1 WO 1998059059 A1 WO1998059059 A1 WO 1998059059A1 US 9812777 W US9812777 W US 9812777W WO 9859059 A1 WO9859059 A1 WO 9859059A1
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- protein
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- antigen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention relates to the area of protein expression. More particularly, the invention relates to human systems for expressing proteins of therapeutic value.
- plasmids encoding the protein of interest are often replicated under control of a replication activator such as the SV40 large T antigen.
- a replication activator such as the SV40 large T antigen.
- the SV40 large T antigen is an efficient replication activator, high levels of extrachromosomal DNA replicating under the control of SV40 large T antigen normally are toxic to host cells (Gerard and Gluzman, 1985). This toxicity results in expression systems which function for only a short time.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/ 402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- Even another embodiment of the invention provides a vector comprising a DNA sequence encoding a fusion protein for use in regulating replication of an episome.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- Still another embodiment of the invention provides a human cell.
- the human cell comprises a DNA sequence encoding a fusion protein for use in regulating replication of an episome.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- kits for expressing a desired protein comprises a human cell comprising a DNA sequence encoding a fusion protein and an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- Yet another embodiment of the invention provides a method of expressing a desired protein.
- a human cell is cultured under conditions whereby the desired protein is expressed.
- the cell comprises a DNA sequence encoding a fusion protein and an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.
- the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond.
- the first protein segment comprises a 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.
- Figure 1 shows point mutations in replication-competent, safety modified SV40 large T antigen mutants. Domains of T antigen that bind to RB, p53, and the SV40 DNA origin are highlighted. The codon 107 mutation substitutes lysine for glutamic acid, and the codon 402 mutation substitutes glutamic acid for aspartic acid.
- Figure 2 demonstrates the presence of point mutations in codons 107 and 402 of replication-competent safety-modified SV40 large T antigen mutants.
- Figure 3 shows co-immunoprecipitation analysis of binding of wild-type and mutant T antigens to human tumor suppressor gene products.
- In vitro translated T antigen (2 x 10 5 dpm) was mixed with CV-1 extracts over producing human RB protein and anti-RB monoclonal antibody G3-245 ( Figure 3 A, lanes 3-6), p53, and anti-p53 monoclonal antibody 1801 ( Figure 3A lanes 7-10), and pi 07 and anti-pl07 monoclonal antibody SD9 ( Figure 3B, lanes 3-6).
- wild-type T antigen is immunoprecipitated with either anti-chromogranin A monoclonal antibody LKH210 (lane 1 of Figure 3 A and Figure 3B) or anti-T antigen monoclonal antibody 416 (lane 2 of Figure 3 A and Figure 3B).
- Figure 4 demonstrates that 107/402-T is replication-competent.
- FIG. 4A HepG2 hepatoma cells were transfected with wild-type and mutant T antigen expression vectors, and total DNA was harvested 2 days post-transfection. DNA samples were sequentially digested with Apal to linearize vector DNA and then with Dpnl to distinguish amplified DNA from the input DNA used to transfect these cells. Since human cells lack adenine methylase activity, newly replicated
- Figure 6 illustrates transgene (alkaline phosphatase) expression mediated by 107/402-T or wild-type T antigen in transiently transfected HepG2 cells.
- Figure 7 depicts the time course of induction of 107/402-T expression vectors in an HT- 1376 tet-off clone by removal of doxycycline.
- Figure 8 shows dependence of 107/402-T expression on doxycycline concentration. Cells were harvested for Western blot analysis of T antigen expression 4 days after exposure to doxycycline.
- Figure 9 shows the half-life of 107/402-T expression after addition of 3 ng/ml of doxycycline.
- Figure 10A shows cyclic production of secreted alkaline phosphatase (SEAP).
- Figure 10B is a Western blot of protein extracts demonstrating 107/402-T antigen expression.
- 107/402-T antigen is an exceptionally efficient replication transactivator in human cells. This property of 107/402-T antigen can be employed in expression systems to produce proteins of therapeutic utility. Use of the 107/402-T antigen permits expression which continues for long periods of time and which produces large quantities of biologically active proteins.
- human cells are genetically modified to produce very high levels of biologically functional proteins and to continue this production over long periods of time without significant cell toxicity.
- These human cells comprise copies of 107/402-T antigen which retain high levels of replication transactivator activity in dividing human cells.
- the copies of the 107/402-T antigen are integrated.
- 107/402-T antigen is an exceptionally efficient replication transactivator in human cells when compared with wild-type T antigen.
- 107/402-T antigen in human cells can be cyclically controlled by the presence of varying concentrations of exogenous agents in the culture medium.
- the method of cyclically controlling replication described herein permits amplification of an episome to a level which yields high gene expression without induction of cellular toxicity.
- a desired protein can then be produced at high levels.
- post-translational modification of the desired protein(s) proceeds normally.
- the present invention provides the art with an expression system for therapeutic proteins which is useful in the pharmaceutical and biotechnology industries.
- the 107/402-T antigen mutant is described in U. S. Patent No. 5,624,820.
- the mutant protein contains substitutions of amino acid residues 107 (glutamic acid to lysine) and 402 (aspartic acid to glutamic acid). These amino acid substitutions prevent the 107/402-T antigen from binding to the oncogenes p53, RB, and pl07, yet the mutant antigen retains the ability to activate replication of a papovavirus-based episome.
- the 107/402-T antigen binds to the papovavirus origin of replication and activates the replication of adjacent DNA sequences. Under control of the 107/402-T antigen, papovavirus-based episomes replicate to thousands of copies by
- episomal copy number can range from at least 2-, 5-, 10-, 25-, 50-, 100-, 125-, 150-, 200- or 500- fold higher than episomal copy number obtained under control of a wild-type T antigen.
- replication of an episome encoding the protein to be expressed is controlled by regulating transcription of the 107/402-T DNA sequence. Transcription of the DNA sequence is controlled by a minimally active promoter, which can be activated by an inducible transcriptional transregulator. The minimally active promoter prevents large amounts of 107/402-
- T antigen from being transcribed in the absence of an exogenous inducer of the transcriptional transregulator Suitable minimally active promoters are, for example, the minimal CMV promoter (Boshart et al., 1985) and the promoters for TK (Nordeen, 1988), IL-2, and MMTV.
- An inducible transcriptional transregulator can be either a transactivator or a transrepressor.
- Several inducible transcriptional transactivators have been constructed, such as the hybrid tetracycline-controUed transcriptional transactivator (Gossen et al., 1992; Gossen etal.
- rapamycin-controlled "gene switch” (Rivera etal, 1996)
- RU486-induced TAXI/UAS “molecular switch”
- Each transactivator contains a binding site for its inducer and a transcription factor domain.
- These inducible transcriptional transactivators bind reversibly to specific-binding regions of DNA, such as operators, and regulate an adjacent minimal promoter which is functional only when the transcription factor binds to the specific region of DNA.
- Inducible repressor systems have also been developed by substituting the
- KRAB transcriptional repressor domain for the VP16 transactivation domain in hybrid transcription factors (Wang etal. 1997).
- repression of gene transcription is linked to binding of the transcriptional repressor to the target DNA binding consensus sequence, and binding of the transcriptional repressor is controlled by suitable inducer molecules.
- a transcriptional transregulator can be constructed to be either functional ("inducer-on”) or nonfunctional ("inducer-off”) in the presence of inducer.
- An "inducer-on” transcriptional transregulator is not functional in the absence of inducer.
- the transcription factor domain of the "inducer- on” transcriptional transregulator binds to the specific-binding DNA region and activates the minimally active promoter.
- An "inducer-off” transcriptional transregulator functions in the absence of inducer.
- the transcription factor domain of the "inducer-ofT transcriptional transregulator does not bind to the specific-binding DNA region and does not activate the minimally active promoter.
- DNA sequences encoding either type of inducible transcriptional transregulator can be used to practice this invention.
- DNA sequences encoding the 107/402-T antigen, a minimally active promoter, and an inducible transcriptional transregulator can be located on the same DNA construct or can be encoded by separate DNA constructs.
- a DNA construct can also encode any two of the three elements.
- 107/402-T antigen can be on an episome.
- the episome can comprise a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.
- the papovavirus origin of replication and restriction enzyme site can be on an episome separate from the DNA constructs encoding the 107/402 antigen, the minimally active promoter, and the inducible transcriptional transregulator.
- the episome can also comprise a promoter which regulates transcription of the coding sequence of the desired protein.
- Individual DNA constructs or episomes can be introduced into a cell together or separately, as is desired.
- Expression vectors can be constructed containing one or more copies of a particular DNA construct.
- Suitable vectors are available from commercial suppliers, such as Stratagene, GTBCO-BRL, Amersham, and Promega, as well as from noncommercial sources such as the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209. Suitable vectors may also be constructed in the laboratory using standard recombinant DNA techniques (Sambrook et al., 1989; Glover, 1985; Perbal, 1984). The sequences can be synthesized chemically or can produced by recombinant DNA methods.
- Methods of transfecting DNA into human cells are well known in the art. These methods include, but are not limited to, transferrin-polycation-mediated DNA transfer, transfer with naked or encapsulated nucleic acids, liposome-mediated cell fusion, intracellular uptake of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, and calcium phosphate-mediated transfection. Integration of the DNA sequences encoding the inducible transcription transregulator and the 107/402-T antigen into the host cell's DNA can be facilitated by providing nucleotides at the 3' or 5' ends of these DNA sequences which are homologous to and therefore recombine with the host cell DNA. One or more copies of each DNA sequence or episome can be integrated into the genome of the host cell, as desired.
- the host cell can be any human cell.
- the host cell is capable of dividing and being maintained in vitro, such as HT-1376 (bladder carcinoma), HepG2 (hepatoma), HEK 293 (human embryonic kidney), HT1080 (fibrosarcoma),
- HeLa cervical carcinoma
- Hs68 fibroblasts
- RAJI lymphoma
- SW480 colon cancer
- 5637 bladder carcinoma
- MCF-7 breast carcinoma
- HuNSl myeloma
- Preferred host cells are those which are particularly well-suited for protein secretion, such as myeloma cell lines. Many of these cell lines, together with instructions on how to culture them, are available from the ATCC. Suitable methods for maintaining cell lines in culture are also well known in the art (see Freshney, 1986).
- the host cell can contain an episome.
- the episome comprises a papovavirus origin of replication, a DNA sequence encoding the desired protein to be expressed, a promoter which is functional in the host cell, and a multiple cloning site for insertion of the protein coding sequence, or transgene (see, for example, Walter and Blobel, 1982; Caras and Weddell, 1989).
- transgene expression can be increased at least 2-, 3-, 4-, or 5- fold or more over expression levels achieved using an expression vector encoding wild-type T antigen.
- the protein encoded by the transgene or protein coding sequence can be, for example, any protein of therapeutic utility, including but not limited to a structural protein, an anti-angiogenic or pro-angiogenic factor, a transcription factor, a cytokine, a neuropeptide, a ligand for a cell surface receptor, an enzyme, a growth factor, a receptor for a ligand, an antibody, a hormone, a transport protein, a storage protein, a contractile protein, or a novel engineered protein.
- the protein can be one which is normally encoded by an endogenous gene in the host cell or can be a protein not normally found in the host cell.
- the protein can be identical to a naturally occurring protein or can contain modifications to alter its physicochemical properties, such as stability, activity, affinity for a particular ligand or receptor, antigenicity, therapeutic utility, or ability to be secreted from the host cell.
- the protein can also be a fusion protein comprising two or more protein fragments fused together by means of a peptide bond.
- the fusion protein can include signal peptide sequences to cause secretion of the protein into the culture medium. Such sequences are well known in the art.
- the promoter can be any promoter which is functional in the selected host cell. Highly active promoters, such as the regulatory region of elongation factor- l ⁇ (Guo et al, 1996), are preferred. Multiple cloning sites are well known in the art and can be inserted into the episome using standard recombinant DNA techniques.
- the episome also comprises a papovavirus origin of replication to which the
- the origin of replication is an SV40 or a BK origin of replication.
- the sequence of the SV40 origin of replication is taught in Subramanian et al, 1977; Reddy et al 1978; Fiers et al, 1978; and Van Heuverswyn et al, 1978.
- the sequence of the BK origin of replication is disclosed in Yang et al. (1979) and Deyerle et al (1989).
- episomes for use in this protein expression system from those available commercially or noncommercially, such as from the ATCC. Alternatively, one can synthesize an episome in the laboratory using standard recombinant DNA techniques. Episomes can also contain a selectable marker, such as the neomycin phosphotransferase gene or antibiotic resistance genes.
- the host cell is cultured in a medium which is suitable to maintain the particular cell type being used.
- the cell is contacted with an inducer of the inducible transcriptional transregulator.
- the inducer can be a component of the cell culture medium or can be added separately.
- the inducible transcriptional transregulator is a hybrid tetracycline-controUed transcriptional transactivator.
- TetracycUne or a tetracycUne derivative such as oxytetracycline, chlortetracycline, anhydrotetracycline, or doxycycline, is added to the culture medium to cause the transactivator to regulate transcription of the DNA sequence encoding the 107/402-T antigen.
- the concentration of inducer is selected by routine experimentation to result in an episome copy number for the particular ceU Une which results in maximal expression of the protein without cellular toxicity.
- Appropriate copy numbers range from at least 10 to at least 100, at least 100 to at least 1,000, at least 1,000 to at least 10,000, at least 10,000 to at least 50,000, at least 50,000 to at least 100,000, or at least 100,000 to at least 500,000 copies or more of the plasmid per cell.
- Plasmid copy number can be measured, for example, by Southern blot (Cooper and Miron, 1993).
- effective concentrations range from at least 1 pg/ml to at least 1 ⁇ g/ml.
- suitable concentrations range from at least 500 pM to at least 2 nM to at least 10 nM to at least 100 nM.
- the half-maximal concentration for inhibition using doxycycline for example is approximately 0.01 ng/ml ( Figure 8).
- Concentrations of RU486 which can be used effectively range from at least 1 nM to at least 100 nM.
- Inducer concentration can be varied over time to achieve suitable copy numbers per cell.
- inducer can be present continuously for 1-3 days or for 1-6 days and then removed entirely, for example by changing the medium.
- medium can be changed every 2-3 days and the concentration of inducer can be varied, for example, by one-half or one-tenth.
- concentration of inducer can be varied, for example, by one-half or one-tenth.
- the precise variation regimen will depend on the cell being used and the stability of the inducer under particular culture conditions. These parameters can be determined by routine experimentation. Thus, one skilled in the art can empirically vary the inducer regimen to maximize the output of transgene expression for any given construct of interest.
- the optimal regimen will be based, in part, on potential toxicities of the desired protein to the producer cell line, the extent to which transcription factors are in limited concentration as they bind to amplified promoter regions in episomes encoding the desired protein, and other factors which may limit the inherent production capabilities of the producer cell line.
- the invention also provides a kit for expressing a desired protein by regulating transcription of the 107/402-T antigen.
- the kit comprises a human cell and a first episome.
- the human cell can be any of the cells described above.
- the first episome comprises a papovavirus origin of replication, such as the SV40 or BK origins of replication, to which the 107/402-T antigen binds.
- the first episome is used as a vector for a coding sequence for the desired protein.
- the coding sequence for the desired protein can be inserted into the first episome using standard recombinant DNA techniques.
- the first episome can also contain an active promoter, for example the regulatory region from elongation factor- l ⁇ .
- a restriction enzyme site or multiple cloning site can be included in the first episome to permit incorporation of the protein coding sequence, or the first episome can be provided with a coding sequence for a desired protein already inserted.
- the human cell also contains one or more copies of a first DNA sequence encoding an inducible transcriptional transregulator, a minimally active promoter, and a second DNA sequence encoding the 107/402-T antigen.
- the DNA sequences encoding the inducer transcriptional transregulator and the 107/402-T antigen can be integrated into the genome of the cells or can be on the first episome or a second episome.
- replication of the episome encoding the protein to be expressed is controlled by regulating the activity of the 107/402-T antigen, by means of a "protein switch.”
- This regulation is accomplished by providing the ceU with a fusion protein comprising two protein segments fused together by means of a peptide bond.
- the first protein segment comprises the 107/402-T antigen.
- the second protein segment comprises a mutant progesterone receptor.
- the mutant receptor includes a hormone binding domain that binds only synthetic antiprogestins, such as RU486.
- Other segments of the human progesterone receptor have comparable properties (DeLort and Capecchi, 1996).
- Mutant progesterone receptors include progesterone receptors which comprise amino acids not normally present in a progesterone receptor, truncated progesterones, and the like.
- One sequence of a mutant receptor is taught in Vegeto et al. (1992). This particular mutant progesterone receptor lacks 54 authentic C- terminal amino acids and includes 12 novel amino acids at the C-terminal.
- the mutant progesterone receptor in the fusion protein interferes with the ability of the 107/402-T antigen to function as a replication transactivator.
- the conformation of the mutant progesterone receptor changes and 107/402-T antigen becomes functional. Replication of an episome which contains a papovavirus origin of repUcation can then take place.
- the fusion protein functions as a protein switch which regulates the replication activating activity of 107/402-T antigen.
- the hormone binding domain of mutant progesterone receptor can be located at either the C-terminal or the N-terminal of the 107/402-T antigen, or in the middle of the 107/402-T antigen molecule.
- a vector for expressing the fusion protein can be constructed using recombinant DNA techniques available in the art.
- the vector preferably comprises an active promoter for expressing large quantities of the fusion protein.
- a promoter such as the CMV immediate early promoter-enhancer, or a highly active human promoter such as the regulatory region from elongation factor- l ⁇ , can be used for this purpose.
- promoters which are specifically active in tumor ceUs for example oncofetal promoters such as the ⁇ -fetoprotein promoter (Huber et al, 1991) or CEA promoter (Osaki et al, 1994), can be used to regulate expression of the fusion protein.
- the vector can be introduced into a human cell and stably integrated into the host DNA using the methods described above.
- Suitable host cells for use in this embodiment are those described above.
- the host cell can contain or can later be a recipient of an episome containing a papovavirus origin of replication and a DNA sequence encoding a desired protein, as described above.
- the promoter which regulates transcription of the DNA sequence encoding the fusion protein can also regulate transcription of the DNA sequence encoding the desired protein, for example, by including between the two coding sequences an internal ribosome entry site, as is known in the art.
- the episome can contain a separate promoter for regulating transcription of the DNA sequence encoding the desired protein.
- the host cell is grown in an appropriate culture medium.
- RU486 is added to the cell.
- Other antiprogestins such as Onapristone, Org31710, or ZK112993, can also be used.
- the antiprogestin can be a component of the culture medium or can be added separately.
- the concentration of antiprogestin is selected by routine experimentation to result in an episome copy number for the particular cell Une which results in maximal expression of the protein without ceUular toxicity.
- Appropriate copy numbers as measured, for example, by Southern blot (Cooper and Miron, 1993), range from at least 10 to at least 100, at least 100 to at least 1,000, at least 1,000 to at least 10,000, at least 10,000 to at least 50,000, at least
- the concentration of antiprogestin which results in appropriate plasmid copy numbers for a particular cell type ranges from at least 1 nM to at least 10, 25, 50, 75, or 100 nM.
- the concentration of antiprogestin can be varied over time to achieve suitable copy numbers per cell.
- the invention also provides a kit for expressing a desired protein by regulating activity of the 107/402-T antigen.
- the kit comprises a human cell and an episome.
- the human cell can be any of the cells described above and contains a one or more copies of a DNA sequence encoding a 107/402-T-mutant progesterone receptor fusion protein.
- the DNA sequence encoding the fusion protein can be integrated into the cells' s genome. Expression of the fusion protein is controlled by an active promoter, as described above.
- the episome comprises a papovavirus origin of replication to which the 107/402-T antigen binds, such as an SV40 or BK origin of replication.
- the episome is used for insertion of a coding sequence for the desired protein and can also be integrated into the genome of the cell if desired.
- the coding sequence for the desired protein can be inserted into the episome using standard recombinant DNA techniques.
- One or more restriction enzyme sites or a multiple cloning site can be included in the episome to permit incorporation of the protein coding sequence.
- the human cell of the kit can comprise the episome. Transcription of the coding sequence of the desired protein can be regulated by the promoter which regulates expression of the fusion protein or by a separate promoter, as described above.
- This example demonstrates the construction of the 107/402-T antigen mutant.
- Wild-type SV40 large T antigen cDNA was isolated from plasmid pSG5-T as a 2.1 kb BamHI fragment. After Xbal linker addition, T antigen cDNA was ligated in the unique Xbal site of pRC/CMV (Invitrogen) to form pRC/CMV-T. In this vector, T antigen cDNA is transcriptionally controlled by the cytomegalovirus (CMV) immediate-early promoter. pRC/CMV contains an S V40 DNA origin; pRC/CMV-T therefore contains a complete SV40 replicon.
- CMV cytomegalovirus
- pRC/CMV.107-T was constructed from pSG5-Kl, which encodes a mutant T antigen substituting lysine for glutamic acid at codon 107 (Kalderon and Smith, 1984).
- pRC/CMV.402-T and pRC/CMV.107/402-T were constructed by substituting a 1067 base pair Hpal C-terminal fragment of T antigen from pRC/CMV-T and pRC/CMV.107-T, respectively, with the corresponding T antigen fragment from a mutant SV40 virus clone that encodes a point mutation which substitutes glutamic acid for aspartic acid at codon 402 (clone 402DE) (Lin and Simmons, 1991).
- These point mutations are shown schematically in Figure 1.
- DNA sequence analysis confirmed in-frame ligation of the Hpal fragment, and also verified presence or absence of point mutations in codons 107 and 402 for each plasmid construct ( Figure 2).
- 107/402-T antigen does not bind to wild- type RB, pi 07, and p53 proteins.
- the biochemical correlate of S V40 large T antigen-mediated induction of tumorigenicity is complex formation with p53, RB, and possibly RB-related proteins such as pl07 (Linzer and Levine, 1979; DeCaprio et al, 1988; Ewen et al, 1991; Claudio et al, 1994).
- pl07 Linzer and Levine, 1979; DeCaprio et al, 1988; Ewen et al, 1991; Claudio et al, 1994.
- in vitro translated wild-type and mutant T antigens were added to extracts from CV-1 cells in which human RB, pi 07, or p53 were transiently expressed at high levels.
- Wild-type and mutant T antigens were translated in vitro in the presence of 35 S-methionine, using a reticulocyte lysate system as described by the manufacturer (Promega). Labeled T antigen (2 x 10 5 dpm) was added to extracts from CV-1 cells transiently expressing human RB, pi 07, or p53 at high levels. CV-1 cells were infected with a vaccinia virus vector encoding T7 RNA polymerase. One hour later cells were transfected with derivatives of the pTMl plasmid (Moss et al, 1990) containing a T7 polymerase site immediately upstream of either human RB, pi 07, or p53 cDNA.
- This example demonstrates that 107/402-T is replication-competent and is a more effective replication activator than wild-type large T antigen.
- HT-1376 blade carcinoma
- 5637 bladedder carcinoma
- MCF-7 breast carcinoma
- SW480 colon cancer
- Hs68 fibroblast
- HepG2 Hepatoma
- RAJI lymphoma
- Cells were transfected using either lipofectin (GIBCO) (Cooper and Miron, 1993), calcium phosphate DNA precipitation (Graham and Van der Eb, 1973), or electroporation. Specific transfection conditions were optimized to achieve a transfection efficiency of at least 1% while minimizing cell toxicity. The day after gene transfer, cell cultures were split to maintain log phase growth for the duration of the experiment.
- This example demonstrates that 107/402-T has enhanced replication activity compared to wild-type T antigen during S-phase of the cell cycle.
- HepG2 hepatoma cells were co-transfected with pCMVSEAP (CMV immediately-early promoter transcribing secreted alkaline phosphatase) and either pRSVwt-T, pRSV.107/402-T, or pRSV (no insert).
- pCMVSEAP CMV immediately-early promoter transcribing secreted alkaline phosphatase
- pRSVwt-T pRSV.107/402-T
- pRSV no insert
- RS V expression vectors lack an SV40 DNA origin and hence will not replicate in transiently transfected cells; the duration of T antigen expression will therefore be limited.
- pCMVSEAP contains an SV40 DNA origin and will repUcate extrachromosomally in cells co-expressing T antigen.
- HepG2 cells in 100 mm dishes were cotransfected with 10 ng of pCMVSEAP and 14 ⁇ g of the RSV-based
- HT-1376 human bladder carcinoma cells were sequentially transfected with three plasmid constructs: (a) pTET-OFF, which encodes the tetracycline-controUed transcriptional transactivator (tTA) under control of the CMV immediate-early promoter and the neomycin resistance gene under control of the SV40 early promoter, (b) pTRE.107/402-T, which encodes 107/402-T under control of the CMV minimal promoter and contains the tetracycline operon (binding site of tTA just upstream of the CMV minimal promoter), and (c) pCMVhygro, which encodes the hygromycin resistance gene under control of the CMV promoter.
- pTET-OFF which encodes the tetracycline-controUed transcriptional transactivator (tTA) under control of the CMV immediate-early promoter and the neomycin resistance gene under control of the SV40 early promoter
- HT-1376 cells were first transfected with pTET-OFF, and neomycin resistant clones of stable transfectants were characterized by transiently transfecting clones with pTRE.luciferase in the presence or absence of doxycycline. Clones which yielded significant luciferase activity only in the absence of doxycycline (but no detectable luciferase activity in the presence of doxycycline) were then co-transfected with pTRE.107/402-T and pCMVhygro. Again, single cell clones of stable transfectants were screened for high basal levels of 107/402-T and complete turn-off of 107/402-T expression in the presence of doxycycline.
- Figure 7 shows the time course of induction of 107/402-T expression upon washout of saturating amounts of doxycycline (3 ng/ml). Steady-state levels of 107/402-T are achieved by 3 days.
- the doxycycline concentration-dependence of 107/402-T expression is presented in Figure 8.
- the half-maximal inhibitory concentration of doxycycline is approximately 0.01 ng/ml.
- the half-life of 107/402-T expression after addition of 3 ng/ml doxycycline is presented in Figure 9.
- the observed decrease of 107/402-T expression yields a half-life of 22.7 hours in this cell line.
- pCMVSEAP an expression plasmid in which the CMV immediate-early promoter regulates transcription of a secreted alkaline phosphatase reporter gene.
- pCMVSEAP contains the S V40 DNA origin and hence will replicate extrachromosomally in the presence of 107/402-T antigen.
- ceUs were incubated without doxycycline for 4 days to produce maximal levels of 107/402-T antigen expression.
- Duplicate dishes of cells were then transfected with pCMVSEAP (day 0) and replated in a series of 60 mm weUs for analysis of alkaline phosphatase expression at a series of time points.
- Doxycycline 50 ng/ml was added back to the cells between days 2-5 to block production of 107/402-T antigen. Media was changed every 24 hours to determine daily alkaline phosphatase activity.
- levels of transgene expression can be optimized for a given appUcation by simply altering the regimen of doxycycline exposure to yield appropriate levels of episomal amplification.
- This modular and flexible system permits optimization of expression for a given transgene based on potential toxicities of the transgene to the host production cell as weU as the inherent synthetic capabilities of the producer cell.
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Abstract
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98929103A EP0994953A1 (fr) | 1997-06-20 | 1998-06-19 | Systeme d'expression pour la production de proteines therapeutiques |
AU80744/98A AU743329B2 (en) | 1997-06-20 | 1998-06-19 | Expression system for production of therapeutic proteins |
JP50484699A JP2002506350A (ja) | 1997-06-20 | 1998-06-19 | 治療用蛋白質の製造のための発現系 |
CA002294119A CA2294119A1 (fr) | 1997-06-20 | 1998-06-19 | Systeme d'expression pour la production de proteines therapeutiques |
US09/935,368 US20020031803A1 (en) | 1997-06-20 | 2001-08-24 | Expression system for production of therapeutic proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5035697P | 1997-06-20 | 1997-06-20 | |
US60/050,356 | 1997-06-20 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09473646 A-371-Of-International | 1999-12-28 | ||
US09/935,368 Continuation-In-Part US20020031803A1 (en) | 1993-11-12 | 2001-08-24 | Expression system for production of therapeutic proteins |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998059059A1 true WO1998059059A1 (fr) | 1998-12-30 |
Family
ID=21964794
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/012777 WO1998059059A1 (fr) | 1997-06-20 | 1998-06-19 | Systeme d'expression pour la production de proteines therapeutiques |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0994953A1 (fr) |
JP (1) | JP2002506350A (fr) |
AU (2) | AU4845297A (fr) |
CA (2) | CA2218852A1 (fr) |
WO (1) | WO1998059059A1 (fr) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993023431A1 (fr) * | 1992-05-14 | 1993-11-25 | Baylor College Of Medicine | Recepteurs d'hormones steroides mutees, procede d'utilisation et commutateur moleculaire pour therapie genique |
WO1995013377A1 (fr) * | 1993-11-12 | 1995-05-18 | Case Western Reserve University | Vecteur d'expression episomique destine a la therapie genique humaine |
WO1996040911A1 (fr) * | 1995-06-07 | 1996-12-19 | Baylor College Of Medicine | Hormones steroides modifiees pour therapie genique et leurs methodes d'utilisation |
-
1997
- 1997-12-17 AU AU48452/97A patent/AU4845297A/en not_active Abandoned
- 1997-12-18 CA CA 2218852 patent/CA2218852A1/fr not_active Abandoned
-
1998
- 1998-06-19 AU AU80744/98A patent/AU743329B2/en not_active Ceased
- 1998-06-19 WO PCT/US1998/012777 patent/WO1998059059A1/fr not_active Application Discontinuation
- 1998-06-19 CA CA002294119A patent/CA2294119A1/fr not_active Abandoned
- 1998-06-19 JP JP50484699A patent/JP2002506350A/ja active Pending
- 1998-06-19 EP EP98929103A patent/EP0994953A1/fr not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993023431A1 (fr) * | 1992-05-14 | 1993-11-25 | Baylor College Of Medicine | Recepteurs d'hormones steroides mutees, procede d'utilisation et commutateur moleculaire pour therapie genique |
WO1995013377A1 (fr) * | 1993-11-12 | 1995-05-18 | Case Western Reserve University | Vecteur d'expression episomique destine a la therapie genique humaine |
US5624820A (en) * | 1993-11-12 | 1997-04-29 | Case Western Reserve University | Episomal expression vector for human gene therapy |
WO1996040911A1 (fr) * | 1995-06-07 | 1996-12-19 | Baylor College Of Medicine | Hormones steroides modifiees pour therapie genique et leurs methodes d'utilisation |
Non-Patent Citations (2)
Title |
---|
E. VEGETO ET AL.,: "The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor", CELL, vol. 69, 1992, Cambridge, MA, US, pages 703 - 713, XP002073903 * |
M.J. COOPER ET AL.,: "Safety-modified episomal vectors for human gene therapy", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 94, no. 12, 10 June 1997 (1997-06-10), Washington, DC, US, pages 6450 - 6455, XP002073902 * |
Also Published As
Publication number | Publication date |
---|---|
EP0994953A1 (fr) | 2000-04-26 |
CA2218852A1 (fr) | 1998-12-20 |
AU4845297A (en) | 1998-12-24 |
CA2294119A1 (fr) | 1998-12-30 |
AU743329B2 (en) | 2002-01-24 |
AU8074498A (en) | 1999-01-04 |
JP2002506350A (ja) | 2002-02-26 |
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