WO1998059059A1

WO1998059059A1 - Expression system for production of therapeutic proteins

Info

Publication number: WO1998059059A1
Application number: PCT/US1998/012777
Authority: WO
Inventors: Mark J. Cooper; Peter Brunovskis
Original assignee: Case Western Reserve University
Priority date: 1997-06-20
Filing date: 1998-06-19
Publication date: 1998-12-30
Also published as: CA2218852A1; AU4845297A; AU743329B2; AU8074498A; EP0994953A1; JP2002506350A; CA2294119A1

Abstract

A novel method is described for expressing biologically functional therapeutic proteins without host cell toxicity. This method takes advantage of the surprising replication activating ability of the 107/402-T antigen. The invention also provides expression vectors, human cells, and fusion proteins for practicing the method. The invention thus provides the art with an expression system for therapeutic proteins which is useful to the pharmaceutical and biotechnology industries.

Description

EXPRESSION SYSTEM FOR PRODUCTION OF THERAPEUTIC PROTEINS

This application claims the benefit of copending U.S. provisional application Serial No. 60/050,356, filed June 20, 1997, which is incorporated herein by reference.

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of IR55CA/OD66780, CA72737, and IR43CA73376 awarded by the National Institutes of Health.

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of protein expression. More particularly, the invention relates to human systems for expressing proteins of therapeutic value.

BACKGROUND OF THE INVENTION

The production of large quantities of biologically functional therapeutic proteins requires an expression system which can both produce protein efficiently without toxic effects to the expression system itself and perform the required post- translational modifications. One approach to in vitro protein production is to transfect a bacterial or yeast cell with a plasmid encoding the protein of interest and culture the cell under conditions where the plasmid replicates to a high copy number, resulting in the potential for the production of large amounts of the desired protein. Due to differences in the biology of bacterial, yeast, and human cells, however, many non-human expression systems have very low efficiencies of producing functional product when the desired protein requires post-translational modification to be functional (Yarranton, 1990; Geisse et al. 1996). In mammalian cells, where post-translational modification of the desired protein may be accomplished more effectively, plasmids encoding the protein of interest are often replicated under control of a replication activator such as the SV40 large T antigen. Although 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.

Thus there is a need in the art for new systems for producing functional proteins for therapeutic uses.

SUMMARY OF THE INVENTION

It is an object of the invention to provide tools and methods for producing functional proteins for therapeutic uses. These and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention provides 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.

Another embodiment of the invention provides a DNA sequence encoding a fusion protein for use in regulating the 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.

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.

Even another embodiment of the invention provides a kit for expressing a desired protein. The kit 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. Thus, the present invention provides the art with expression vectors, human cells, and fusion proteins for practicing a method of producing therapeutic proteins which is useful to the pharmaceutical and biotechnology industries.

BRTEF DESCRI^PTION OF THE DRAWINGS

Figure 1. 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. 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. 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 10⁵ 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). As controls, 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. Figure 4 demonstrates that 107/402-T is replication-competent.

Figure 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

DNA is resistant to Dpnl digestion. Hence, the presence of unit length, linearized plasmid DNA (as indicated by the arrow) demonstrates newly replicated episome. Hybridization probe: pRC/CMV.107/402-T. Figure 4B. Replication activity of wild-type S V40 large T antigen and SV40 T antigen mutants. Figure 5. Figure 5 shows enhanced replication activity of 107/402-T in HepG2 cells. Figures 5 A and 5B show FACS analysis of propidium iodide-stained HepG2 cells. Figure 5C is a Southern blot which shows episomal copy number of wild-type and 107/402-T expression vectors in transfected HepG2 cells. Figure 5D shows normalized replication activity of 107/402-T and wild-type T antigen expression vectors.

Figure 6. Figure 6 illustrates transgene (alkaline phosphatase) expression mediated by 107/402-T or wild-type T antigen in transiently transfected HepG2 cells.

Figure 7. 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. 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. Figure 9 shows the half-life of 107/402-T expression after addition of 3 ng/ml of doxycycline.

Figure 10. 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.

DETAπ-ED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is a discovery of the present invention that the mutant large T antigen,

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.

The present invention overcomes significant limitations of the prior art. According to the present invention, 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. Preferably, the copies of the 107/402-T antigen are integrated. Surprisingly, 107/402-T antigen is an exceptionally efficient replication transactivator in human cells when compared with wild-type T antigen. Also according to the present invention, either the production or activity of

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. Furthermore, because human cells are used in this expression system, post-translational modification of the desired protein(s) proceeds normally. Thus, 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.

Compared with the wild-type SV40 large T antigen (see Shin et al., 1975; Christian etal, 1987; Michalovitz etα/., 1987; DeCaprio ef α/., 1988; Hanahan et /., 1989; Chen et al., 1990; Chen et al., 1992), 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

2-4 days after transfection in many human cell lines. This replication is greatly enhanced compared with that observed in the presence of wild-type T antigen (Examples 4 and 5). Under control of the 107/402-T antigen, 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. In one embodiment of the present invention, 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. 1995), the rapamycin-controlled "gene switch" (Rivera etal, 1996), and the RU486-induced TAXI/UAS "molecular switch" (DeLort and Capecchi, 1996). 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). In these systems, 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. In the presence 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. In the presence 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. Optionally, the DNA sequences encoding the transcriptional transregulator and the DNA sequence encoding the

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. Alternatively, 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. Many 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 Blvd., 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. Preferably, 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), or HuNSl (myeloma) cells. 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).

In addition to containing the minimally active promoter, the DNA sequences encoding the 107/402-T antigen, and an inducible transcriptional transregulator, 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). According to the invention, 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- lα (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

107/402-T antigen binds. In a preferred embodiment, 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).

Those of skill in the art can select suitable 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.

In one embodiment of the invention, 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.

In a preferred embodiment, 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). For tetracycline or its derivatives, effective concentrations range from at least 1 pg/ml to at least 1 μg/ml. For rapamycin, 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. For example, inducer can be present continuously for 1-3 days or for 1-6 days and then removed entirely, for example by changing the medium.

Alternatively, medium can be changed every 2-3 days and the 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- lα. 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.

In another embodiment of the invention, 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.

In the absence of antiprogestin, the mutant progesterone receptor in the fusion protein interferes with the ability of the 107/402-T antigen to function as a replication transactivator. In the presence of RU486, however, 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. Thus, the fusion protein functions as a protein switch which regulates the replication activating activity of 107/402-T antigen. Within the fusion protein, 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- lα, can be used for this purpose. Alternatively, promoters which are specifically active in tumor ceUs, for example oncofetal promoters such as the α-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. Alternatively, the episome can contain a separate promoter for regulating transcription of the DNA sequence encoding the desired protein. For in vitro protein production, the host cell is grown in an appropriate culture medium. In a preferred embodiment, 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

50,000 to at least 100,000, or at least 100,000 to at least 500,000 or more copies of the plasmid per cell. 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. Optionally, 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. Optionally, 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.

The following are provided for exemplification purposes only and are not intended to limit the scope of the invention that has been described in broad terms above.

EXAMPLE 1

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. In a similar fashion, 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).

EXAMPLE 2

This example demonstrates that 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). To evaluate directly the ability of 107/402-T to bind to wild-type RB, pi 07, and p53, 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 ³⁵S-methionine, using a reticulocyte lysate system as described by the manufacturer (Promega). Labeled T antigen (2 x 10⁵ 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.

Approximately eighteen hours later, cells were harvested using a lysis buffer as described in Cooper et al (1994). Immunoprecipitation analysis was performed using monoclonal antibodies to RB (clones G3-245, Pharmingen), pi 07 (clone SD9, Oncogene Science), and p53 (clone 1801, Oncogene Science), as described in DeCaprio et al, 1988. Band intensities were scanned using a phosphorimager to quantitate binding interactions. The results of these experiments are shown in Figures 3 A and 3B.

As shown in Table I, little or no binding of 107/402-T was detected in these experiments, demonstrating that 107/402-T does not bind significantly to either RB, pl07, or p53.

Table 1. Binding of wild-type and mutant SV40 large T antigens to RB pl07, and p53 tumor suppressor gene products

Observed signal compared to T

Tumor suppressor gene product T,% 107-T,% 402-T,% 107/402-T,%

RB 100 0.03 67 0.07 pl07 100 0 79 0 p53 100 36.2 0 0

EXAMPLE 3

This example demonstrates that 107/402-T is replication-competent and is a more effective replication activator than wild-type large T antigen.

The replication activities of wUd-type and mutant SV40 large T antigens were evaluated in a panel of human cell lines, including HT-1376 (bladder carcinoma), 5637 (bladder carcinoma), MCF-7 (breast carcinoma), SW480 (colon cancer), Hs68 (fibroblast), HepG2 (hepatoma), and 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.

DNA harvested from transient transfectants was evaluated for the presence of extrachromosomal plasmid replication by resistance to Dpnl digestion, as described in Cooper and Miron (1993). As shown in Figure 4 A, significant replication activity was observed in human cells. In HepG2 cells, for example, a copy number of approximately 25,000 per cell was noted by two days post-gene transfer, and copy numbers ranging from 80 to 100,000 were observed in other human cell types (Cooper et al. 1997). Furthermore, in the HepG2 cell Une the replication activating abUity of 107/402-T was increased over that of wild-type SV40 large T antigen by a factor of one hundred (Figures 4 A and 4B). EXAMPLE 4

This example demonstrates that 107/402-T has enhanced replication activity compared to wild-type T antigen during S-phase of the cell cycle.

As described in Example 3, the copy number of pRC/CMV.107/402-T in HepG2 human hepatoma ceUs was 100-fold higher than pRC/CMV.T at 2 days post gene transfer. To further investigate the mechanism underlying this difference in episomal copy number, the cell cycle dependence of repUcation activity was evaluated. HepG2 cells were transfected with pRC/CMV.107/402-T or pRC/CMV.T. Twenty-four hours later, cells in early Gl of the ceU cycle were isolated by centrifugal elutriation. The initial population of Gl -enriched cells (time

0) and cells 6, 12, 18, 24, and 30 hours after replating were assayed for cell cycle analysis (FACS analysis of propidium iodide-stained cells, Figures 5 A and 5B) and episomal copy number (Southern blot analysis, Figure 5C). Log-phase growth conditions were maintained during replating. The band intensities in Figure 5C were normalized for transient transfection efficiency by FACS analysis of T antigen expression. The normalized replication activity is presented in Figure 5D.

The cell cycle analysis demonstrated that the peak time period for traversal of S phase was 12 - 18 hours post-replating. By 30 hours post-replating, cells transfected with pRC/CMV.107/402-T had a 3.4-fold increase in copy number compared to pRC/CMV.T. The increase in replication activity appeared to be largely restricted to S-phase of the cell cycle and accounts for the enhanced replication activity of 107/402-T in comparison to wild-type T antigen.

EXAMPLE 5

This example demonstrates that 107/402-T significantly enhances gene expression compared to wild-type T antigen.

To evaluate replication reporter transgene expression mediated by 107/402- T or wild-type T antigen, 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). These 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. In contrast, 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 μg of the RSV-based vectors.

On day one, samples of medium from the cells were saved and the ceUs were trypsinized and replated. At each 24 hour interval, media was harvested and cell extracts were prepared to calculate the total amount of protein per well. Alkaline phosphatase activity was measured in media using a commercial chemiluminescent assay (Tropix).

Data are presented in Figure 6 as relative light units per μg of protein per 24 hours. A two- to five-fold improvement in alkaline phosphatase activity was observed in the 107/402-T co-transfectants compared to the wild-type T antigen co-transfectants. The level of alkaline phosphatase activity in the 107/402-T co-transfectants was greater than an order of magnitude higher than the pRSV co- transfectants (non-replicating standard expression vector control), emphasizing the importance of this replicating expression system for producing high levels of recombinant protein.

EXAMPLE 6

This example demonstrates that gene-modified cells can be prepared to express 107/402-T under transcriptional control of the tetracycline-controUed gene switch.

To prepare a human cell line in which expression of 107/402-T would be under control of doxycycline, 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. 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.

An example of an HT- 1376 tet-off clone that demonstrates precise control of 107/402 -T expression is shown in Figure 7. 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.

The data in Figures 7-9 permit design of a cyclic regimen of doxycyline that wiU fluctuate levels of 107/402 -T expression about a predetermined level of 107/402-T expression. Such a cyclic profile of 107/402-T expression will, in turn, generate sustained and elevated levels of transgene expression derived from a replicating reporter plasmid encoding the S V40 DNA origin.

EXAMPLE 7

This example demonstrates that cyclic regimens of doxycycUne can be used to control transgene expression in the gene-modified cells of Example 6.

The HT-1376 clone described in Example 6 (clone 4A6/E3) was transfected with 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. In this experiment, ceUs were incubated without doxycycline for 4 days to produce maximal levels of 107/402-T antigen expression. Duplicate dishes of cells (A, B) 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.

To measure secreted alkaline phosphatase activity, 25 μl of media were assayed using a chemiluminescent assay as described by the manufacturer (Tropix, Inc.). The light units per well were then calculated, and cells were harvested for protein determination. Activity is expressed as relative light units of alkaline phosphatase activity per μg of protein per 24 hours.

As shown in Figure 10 A, alkaline phosphatase activity peaks on day 3 and then declines. In Figure 10B, the protein extracts were evaluated for 107/402-T expression by Western blot analysis. The same blot was reprobed for β-actin to ensure equal loading of extracts per well. Normalized levels of 107/402-T antigen expression are plotted in Figure 10A and demonstrate that levels of 107/402-T antigen can be cycled by appropriate exposure of the cells to doxycycline.

These data demonstrate the ability to cycle levels of transgene expression using the method of the invention. Furthermore, 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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Claims

1. A fusion protein for use in regulating replication of an episome, comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402- T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.

2. The fusion protein of claim 1 wherein the first protein segment is N- terminal to the second protein segment.

3. The fusion protein of claim 1 wherein the first protein segment is C- terminal to the second protein segment.

4. A DNA sequence encoding a fusion protein for use in regulating the replication of an episome, comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.

5. A vector comprising a DNA sequence encoding a fusion protein for use in regulating replication of an episome, wherein the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.

6. The vector of claim 5 further comprising a first promoter, wherein the first promoter regulates transcription of the DNA sequence encoding the fusion protein.

7. The vector of claim 5 wherein the vector further comprises an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.

8. The vector of claim 7 wherein the first promoter regulates transcription of the coding sequence of the desired protein.

9. The vector of claim 7 wherein the episome further comprises a second promoter, wherein the second promoter regulates transcription of the coding sequence of the desired protein.

10. A vector comprising a DNA sequence comprising (a) a coding sequence for a fusion protein for use in regulating replication of an episome, wherein the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone; (b) a first promoter, wherein the first promoter regulates transcription of the DNA sequence encoding the fusion protein; and (c) an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein, wherein the first promoter regulates transcription of the coding sequence of the desired protein.

11. A vector comprising a DNA sequence comprising (a) a coding sequence for a fusion protein for use in regulating replication of an episome, wherein the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone; (b) a first promoter, wherein the first promoter regulates transcription of the DNA sequence encoding the fusion protein; and (c) an episome comprising a papovavirus origin of replication, a restriction enzyme site for insertion of a coding sequence of a desired protein, and a second promoter, wherein the second promoter regulates transcription of the coding sequence of the desired protein.

12. A human cell comprising a DNA sequence encoding a fusion protein for use in regulating replication of an episome, wherein the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.

13. The human cell of claim 12 wherein the DNA sequence further comprises a first promoter, wherein the first promoter regulates transcription of the DNA sequence encoding the fusion protein.

14. The human cell of claim 12 wherein the DNA sequence is integrated into the genome of the human cell.

15. The human cell of claim 12 further comprising an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.

16. The human cell of claim 15 wherein the episome is integrated into the genome of the human cell.

17. The human cell of claim 15 wherein the first promoter regulates transcription of the coding sequence of the desired protein.

18. The human cell of claim 15 wherein the episome further comprises a second promoter, wherein the second promoter regulates transcription of the coding sequence of the desired protein.

19. A kit for expressing a desired protein, comprising: a human cell comprising a DNA sequence encoding a fusion protein, wherein the fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone; and an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.

20. The kit of claim 19 wherein the DNA sequence is integrated into the genome of the human cell.

21. The kit of claim 19 wherein the episome is integrated into the genome of the human ceU.

22. The kit of claim 19 wherein the DNA sequence further comprises a first promoter, wherein the first promoter regulates transcription of the DNA sequence encoding the fusion protein.

23. The kit of claim 22 wherein the first promoter regulates transcription of the coding sequence of the desired protein.

24. The kit of claim 19 wherein the episome further comprises a second promoter, wherein the second promoter regulates transcription of the coding sequence of the desired protein.

25. A method of expressing a desired protein, comprising the step of: culturing a human cell under conditions whereby the desired protein is expressed, wherein the cell comprises (a) a DNA sequence encoding a fusion protein comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises a 107/402-T antigen and wherein the second protein segment comprises a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone; and (b) an episome comprising a papovavirus origin of replication and a restriction enzyme site for insertion of a coding sequence of a desired protein.

26. The method of claim 25 wherein the DNA sequence is integrated into the genome of the human cell.

27. The method of claim 25 wherein the episome is integrated into the genome of the human cell.

28. The method of claim 25 wherein the DNA sequence comprises a first promoter, wherein the first promoter regulates transcription of the DNA sequence.

29. The method of claim 28 wherein the first promoter regulates transcription of the coding sequence of the desired protein.

30. The method of claim 25 wherein the episome further comprises a second promoter, wherein the second promoter regulates transcription of the coding sequence of the desired protein.

31. The method of claim 25 wherein the desired protein is secreted.

32. The method of claim 25 further comprising the step of adding to the ceU an antiprogestin.

33. The method of claim 32 further comprising the step of varying the concentration of the antiprogestin over time.