AU4845297A - Expression system for production of therapeutic proteins - Google Patents

Description

PIO101C 1 Regulation 3-2

AUSTRALIA

Patents A ct 1990

ORIGINAL

COMPLETE

SPECIFICATION

STANDARD

PATENT

I nvention Title: Expression system for production of therapeutic proteins- The following statement is a lull description of this invention, including the best method of performing it known to us: ~caaicau~as~s~-i~ EXPRESSION SYSTEM FOR PRODUCTION OF THERAPEUTIC

PROTEINS

The U.S. Government has a paid-up license in this invention and the right S* 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 produce protein efficiently without toxic effects to the expression system itself and perform the required posttranslational 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 high production 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 posttranslational modification of the desired protein may be more effectively 1A Li accomplished, plasmids encoding the protein of interest are often replicated under control of a replication activator such as the SV40 large T antigen. Although the 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 that fun.tion only for a short time.

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

SUMMARY OF THE INVENTION It is an object of the invention to provide a DNA construct encoding a 1071402-T antigen.

It is another object of the invention to provide a vector for expressing a 107/402-T antigen.

5 It is yet another object of the invention to provide a human cell for use as Sa recipient of an episome encoding a desired protein.

i It is another object of the invention to provide a kit for expressing a desired i protein.

It is still another object of the invention to provide a method of expressing 0 a desired protein.

S'It is even another object of the invention to provide a fusion protein for use in regulating the replication of an episome.

It is another object of the invention to provide a DNA sequence encoding .a fusion protein for regulating the replication of a vector.

It is still another object of the invention to provide a vector for expressing a fusion protein for regulating the replication of an episome.

It is yet another object of the invention to provide a cell comprising a DNA sequence encoding a fusion protein for regulating the replication of an episome.

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 DNA construct encoding a 1071402-T antigen. The construct comprises a DNA sequence encoding an 2 1 inducible transcriptional transactivator, a minimally active promoter, and a DNA sequence encoding a 1071402-T antigen. The inducible transcriptional transactivator activates the minimally active promoter, and the minimally active promoter regulates transcription of the 107/402-T DNA sequence.

Another embodiment of the invention provides a vector for expressing a 107/402-T antigen. The vector comprises a construct encoding a 107/402-T antigen. The construct comprises a DNA sequence encoding an inducible transcriptional transactivator, a minimally active promoter, and a DNA sequence encoding a 107/402-T antigen. The inducible transcriptional transactivator activates the minimally active promoter, and the minimally active promoter regulates transcription of the 107/402-T DNA sequence.

Yet another embodiment of the invention provides a human cell for use as a recipient of an episome comprising a papovavirus origin of replication and a coding sequence for a protein whose expression is desired. The human cell also comprises one or more integrated copies of a DNA sequence encoding a 107/402-T antigen. The 107/402-T antigen drives replication of the episome. Transcription of the DNA sequence encoding the 107/402-T antigen is controlled by one or more integrated copies of a minimally active promoter. The human cell also comprises one or more integrated copies of a DNA sequence encoding an inducible .2Q transcriptional transactivator. The transactivator activates the minimally active promoter.

Another embodiment of the invention provides a kit for expressing a desired protein. The kit comprises a human cell and a episome. The human cell comprises one or more integrated copies of a DNA sequence encoding a 107/402-T antigen. The 107/402-T antigen drives replication of the episome. Transcription of the DNA sequence encoding the 107/402-T antigen is controlled by one or more Sintegrated copies of a minimally active promoter. The human cell also comprises one or more integrated copies of a DNA sequence encoding an inducible transcriptional transactivator. The transactivator activates the minimally active promoter. The episome comprises a papovavirus origin of replication.

Still another embodiment of the invention provides a method of expressing a desired protein. A human cell comprises an episome comprising a papovavirus origin of replication and a coding sequence for a desired protein. The cell also comprises one or more integrated copies of a DNA sequence encoding a 107/402-T antigen. The 107/402-T antigen drives replication of the episome. Transcription of the DNA sequence encoding the 107/402-T antigen is controlled by one or more integrated copies of a minimally active promoter. The cell also comprises one or more integrated copies of a DNA sequence encoding an inducible transcriptional transactivator. The transactivator activates the minimally active promoter. The human cell is cultured under conditions whereby the desired protein is expressed.

Even another embodiment of the invention provides 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 consists of a 107/402-T antigen. The second protein segment consists of a mutant progesterone receptor. The mutant progesterone receptor binds an antiprogestin and does not bind progesterone.

15 Yet another embodiment of the invention provides a DNA sequence S, 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 consists of a 107/402-T antigen. The second protein segment consists of a mutant progesterone receptor. The mutant progesterone receptor binds an antiprogestin Sand does not bind progesterone.

Another embodiment of the invention provides a vector for expressing a fusion protein for use in regulating the replication of an episome. The vector comprises a DNA sequence encoding a fusion 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 consists of a 107/402-T I antigen. The second protein segment consists of a mutant progesterone receptor.

The mutant progesterone receptor binds an antiprogestin and does not bind progesterone.

Still another embodiment of the invention provides a human cell for use as a recipient of an episome comprising a papovavirus origin of replication and a coding sequence for a protein whose expression is desired. The human cell S4 comprises 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 consists of a 107/402-T antigen. The second protein segment consists of a mutant progesterone receptor. The mutant progesterone receptor binds an antiprogestin and does not bind progesterone.

Yet another embodiment of the invention provides a kit for expressing a desired protein. The kit comprises a human cell and an episome. The human cell comprises 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 consists of a 107/402-T antigen. The second protein segment consists of a mutant progesterone receptor. The mutant progesterone receptor binds an antiprogestin and does not bind progesterone. The episome comprises a papovavirus origin of replication.

Even another embodiment of the invention provides method of expressing a desired protein. A human cell comprises 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 .2Q by means of a peptide bond. The first protein segment consists of a 107/402-T antigen. The second protein segment consists of a mutant progesterone receptor.

The mutant progesterone receptor binds an antiprogestin and does not bind progesterone. The human cell is cultured under conditions whereby the desired protein is expressed.

The present invention overcomes the significant limitations of the prior art.

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 have integrated copies of an large T antigen mutant (107/402-T) that retain high levels of replication transactivator activity in dividing human cells. Surprisingly, 107/402-T antigen is an exceptionally efficient replication transactivator in human cells when compared with wild-type T antigen. In the present invention, either the production -s~CICIC or activity of 107/402-T antigen in the human cells is cyclically controlled by the presence of varying concentrations of exogenous agents in the culture medium.

The cyclical control of replication described herein permits amplification of an episome to a level which yields high gene expression without induction of cellular toxicity. The protein is then produced at high levels. Furthermore, because human cells are used in this expression system, post-translational modification of the proteins proceeds normally. Thus, the present invention provides the art with an expression system for therapeutic proteins which is useful to the pharmaceutical and biotechnology industries.

DETAILED 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 for proteins of therapeutic utility. Use of the 1071402-T antigen permits expression which continues for long periods of time and which produces large Squantities of biologically active proteins.

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 et al., 1987; Michalovitz et al., 1987; DeCaprio et al., 1988; Hanahan et al., 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 p107, 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 (see Example 3, infra).

6 ___P____Rl__sq4lsaa~B~DI1I In one embodiment of the present invention, the replication of an episome encoding the protein to be expressed is controlled by regulating the 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 transactivator. 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 transactivator. 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. Several inducible transcriptional transactivators have been constructed, such as the hybrid tetracycline-controlled transcriptional transactivator (Gossen et al., 1992; Gossen et al. 1995), the rapamycin-controlled "gene switch" (Rivera et al., 1996), and the RU486-induced TAXI/UAS "molecular switch" (DeLort and Capecchi, 1996).

SEach transactivator contains a binding site for its inducer and a transcription factor domain. These inducible transcriptional transactivators reversibly bind to specificbinding 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. The transcriptional transactivator may be constructed to be either functional ("inducer-on") or nonfunctional ("inducer-off") in the presence 20 of inducer. An "inducer-on" transcriptional transactivator is not functional in the absence of inducer. In the presence of inducer, the transcription factor domain of the "inducer-on" transcriptional transactivator binds to the specific-binding DNA region and activates the minimally active promoter. An "inducer-off" transcriptional transactivator functions in the absence of inducer. In the presence of inducer, the transcription factor domain of the "inducer-off" transcriptional transactivator does not bind to the specific-binding DNA region and does not activate the minimally active promoter. DNA sequences encoding any of these inducible transcriptional transactivators can be used to practice this invention.

DNA sequences encoding the 107/402-T mutant, the minimally active promoter, and the inducible transcriptional transactivator may be located on the same DNA construct and introduced into the human cell together or the inducible transcriptional transactivator can be located on a separate piece of DNA and 7 -I-YX~5~n~4 e2e 20 a introduced into the cell separately. Expression vectors can be constructed containing one or more copies of the DNA construct or containing elements of the construct separately. Many suitable vectors are available from commercial suppliers, such as Stratagene, GIBCO-BRL, United States Biochemical, and Promega, and from noncommercial sources such as the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852. Suitable vectors may also be constructed in the laboratory using standard recombinant DNA techniques (Sambrook et al., 1989; Glover, 1985; Perbal, 1984). The sequences may be synthesized chemically or 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 transactivator and the 107/402-T antigen into the host cell's DNA may 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 may be integrated.

The host cell may be any human cell 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), or MCF-7 (breast carcinoma) cells. Preferred host cells are those which are particularly 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 and the DNA sequences encoding the 107/402-T antigen, the host cell may contain an episome.

The episome comprises a DNA sequence encoding the desired protein to be expressed, a promoter which is functional in the host cell, and a multiple cloning 8

I

site for insertion of the protein coding sequence. The protein may be, for example, any protein of therapeutic use, including a structural protein, 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 may be one encoded by an endogenous gene in the host cell or may be a protein not normally found in the host cell. The protein may be identical to a naturally occurring protein or may 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 may also be a fusion protein consisting of two or more protein fragments fused together by means of a peptide bond. The fusion protein may include signal peptide sequences to cause secretion of the protein into the culture medium. Such sequences are well known in the art (see, for example, Walter and Blobel, 1982; Caras and Weddell, 1989).

i.1 The promoter may be any promoter which is functional in the selected host cell. Highly active promoters, such as the regulatory region of elongation factorcla (Guo et al., 1996), are preferred. Multiple cloning sites are well known in the art and may be inserted into the episome using standard recombinant

DNA

techniques.

The episome (episome) also comprises a papovavirus origin of replication Sto 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 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 may synthesize an episome in the laboratory using standard recombinant DNA techniques. The episomes may also contain a selectable marker, such as the neomycin phosphotransferase gene or antibiotic resistance genes.

9- i _i In order to express the protein, the cell is cultured in a medium which is suitable to maintain the particular type being used. The inducer of the inducible transcriptional transactivator is then added to the cell. The inducer may be a component of the cell culture medium or may be added separately. In a preferred embodiment, the inducible transcriptional transactivator is the hybrid tetracyclinecontrolled transcriptional transactivator. Tetracycline or a tetracycline 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 the inducer is selected by routine experimentation to result in an episome copy number for the particular cell line which results in maximal expression of the protein without cellular toxicity. Appropriate copy numbers range from 10 to 100, 100 to 1,000, 1,000 to 10,000, 10,000 to 50,000, 50,000 to 100,000, or 100,000 to h 500,000 copies of the plasmid per cell. Plasmid copy number may be measured, 5 for example, by Southern blot (Cooper and Miron, 1993). For tetracycline or its derivatives, effective concentrations range from 100 pg/ml to 1 pig/ml. For rapamycin, suitable concentrations range from 500 pM to 2 nM to 10 nM to 100 SinM. Concentrations of RU486 which may be used effectively range from 1 nM to 100 nM. The inducer concentration may be varied over time to achieve suitable copy numbers per cell. For example, inducer may be present continuously for 1-3 days or for 1-6 days and then removed entirely, for example by changing the medium. Alternatively, medium may be changed every 2-3 days and the concentration of inducer may 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 may be determined by routine experimentation.

The invention also provides a kit for expressing a desired protein. The kit comprises a human cell and an episome. The human cell may be any of the cells described above and contains one or more integrated copies of an inducible transcriptional transactivator, a minimally active promoter, and a DNA sequence encoding the 107/402-T antigen. The plasmid comprises a papovavirus origin of replication, such as the SV40 or BK origins of replication, to which the 1071402-T -1 repliatio~sucasth antigen binds. The plasmid is used as a vector for a coding sequence for the desired protein. The coding sequence for the desired protein may be inserted into the plasmid using standard recombinant DNA techniques. The plasmid also contains an active promoter, for example the regulatory region from elongation S 5 factor-le. A multiple cloning site may be included in the plasmid to permit incorporation of the protein coding sequence.

In another embodiment of the invention, replication of the plasmid 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 cell 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 sequence of the mutant receptor is taught in Vegeto et al. (1992).

The mutant progesterone receptor lacks 54 authentic C-terminal amino acids and includes 12 novel amino acids at the C-terminal (Vegeto et al., 1992). The mutant receptor includes a hormone binding domain that binds only synthetic antiprogestins, such as RU486 (Vegeto et al., 1992). In the absence of the antiprogestin, the mutant progesterone receptor in the fusion protein interferes with Sthe 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 the papovavirus origin can then take place. Thus, the fusion protein functions as a protein switch which regulates the replication activating activity of 107/402-T. Within the fusion protein, the mutant progesterone receptor's hormone binding domain may be located at either the Co terminal or the N-terminal of the 107/402-T antigen, or in the middle of the f 107/402-T antigen molecule.

SA vector for expressing the fusion protein can be constructed using recombinant DNA techniquws available in the art. The vector preferably comprises an active promoter, for expressing large quantities of the fusion protein. In a preferred embodiment, a promoter such as the CMV immediate early promoterenhancer, or a highly active human promoter such as the regulatory region from %1 Selongation factor-la, is used. The vector may 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 may I contain or later be a recipient of an episome containing a papovavirus origin of replication and a DNA sequence encoding the protein of interest, as described supra.

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, and ZK112993, may also be used. The antiprogestin may be a component of the culture medium or may be added separately. The concentration of antiprogestin is selected by routine experimentation to result in an episome copy number for the particular cell line which results in maximal expression of the protein without cellular toxicity. Appropriate copy numbers, as measured, for example, by Southern blot (Cooper and Miron, 1993), range from 5 10 to 100, 100 to 1,000, 1,000 to 10,000, 10,000 to 50,000, 50,000 to 100,000, or 100,000 to 500,000 copies of the plasmid per cell. The concentration of antiprogestin which results in appropriate plasmid copy numbers for a particular Si" cell type ranges from 1 nM to 100 nM. The concentration of antiprogestin may be varied over time to achieve suitable copy numbers per cell.

O. 0 The invention also provides a kit for expressing a desired protein. The kit comprises a human cell and an episome. The human cell may be any of the cells described above and contains a vector for expressing the 107/402-T-mutant i" -progesterone receptor fusion protein. 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 as for insertion of a coding sequence for the desired protein. The coding sequence for the desired protein may be inserted into the plasmid using standard recombinant DNA techniques. One or more restriction enzyme sites may be included in the plasmid to permit incorporation of the protein coding sequence.

12 The following are provided for exemplification purposes only and are not iniended 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 as a 2.1 kb BamHI fragment. After XbaI 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. pRCICMV contains an S:i" DNA origin; pRCCMV-T therefore contains a complete SV40 replicon.

'In a similar fashion, pRC/CMV.107-T was constructed from pSG5-K1, 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 i ~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).

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.

EXAMPLE 2 This example demonstrates that 107/402-T antigen does not bind to wild- Stype RB, p10 7 and p53 proteins.

The biochemical correlate of SV40 large T antigen-mediated induction of S tumorigenicity is complex formation with p53, RB, and possibly RB-related proteins such as pl0 7 (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 13

L

bind to wild-type RB, p107, and p53, in vitro translated wild-type and mutant T antigens were added to extracts from CV-1 cells in which human RB, p10 7 or p5 3 were transiently expressed at high levels.

Wild-type and mutant T antigens were translated in vitro in the presence of "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, p10 7 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 pTM1 plasmid (Moss et al., 1990) containing a T7 polymerase site immediately upstream of either human RB, p107, 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), p107 (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.

Little or no binding of 107/402-T was detected in these experiments, S" demonstrating that 107/402-T does not bind significantly to either RB, p10 7 or p 53 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 wild-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 i 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 toxicities. The day after gene transfer, cell 14 t 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 DpnI digestion, as described in Cooper and Miron (1993).

Significant replication activity was observed in the human cell lines. In Hep G2 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. Furthermore, in the Hep G2 cell line the replication activating ability of 107/402-T was increased over that of wild-type SV40 large T antigen by a factor of one hundred.

REFERENCES

Boshart et al. (1985) Cell 41: 521-30 Caras and Weddell (1989) Science 243: 1196-98 Chen et al. (1990) J. Virol. 64, 3350-57 Chen et al. (1992) Oncogene 7 1167-75 Christian et al. (1987) Cancer Res. 47, 6066-73 Claudio et al. (1994) Cancer Res. 54: 5556-60 Cooper et al. (1994) Oncol. Res. 6: 569-79 Cooper and Miron (1993) Human Gene Ther. 4: 557-66 DeLort and Capecchi (1996) Human Gene Therapy 7, 809-20 DeCaprio et al. (1988) Cell 54, 275-83 Deyerle et al. (1989) J. Virol. 63: 356-65 Ewen et al. (1991) Cell 66, 1155-64 Fiers et al. (1978) Nature 273, 113-20 C _Freshney, ed. (1986) ANIMAL CELL CULTURE Gerard and Gluzman (1985) Mol. Cell. Biol. 5: 3231-40 I, Glover, ed. (1985) DNA CLONING: A PRACTICAL APPROACH, vols. 1 and 2 Gossen and Bujard (1992) PNAS 89: 5547-51 Gossen (1995) Science 268: 1766-69 Guo et al. (1996) Gene Ther. 3: 802-10 Hanahan et al. (1989) Science 246: 1265-75 Kalderon and Smith (1984) Virol. 139: 109-37 Lin and Simmons (1991) J. Virol. 65: 2066-72 Linzer and Levin (1979) Cell 17: 43-52 Michalovitz et al. (1987) J. Virol. 61: 2648-54 Moss et al. (1990) nATURE 348: 91-92 Nordeen, S.K. (1988) BioTechniques 6: 454-48 Perbal (1984) A PRACTICAL GUIDE TO MOLECULAR CLONING Reddy et al. (1978) Science 200, 494-502 Rivera et al. (1996) Nature Med. 2: 1028-32 Roberts et al. (1986) Cell 46, 741-52 Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL Shin et al. (1975) PNAS 72: 4435-39 Subramanian et al. (1977) J. Biol. Chem. 252: 355-67 Van Heuverswyn et al. (1978) Eur. J. Biochem. 100: 51-60 Vegeto et al. (1992) Cell 69: 703-13 Yang et al. (1979) Science 206: 456-61 Walter and Blobel (1982) Nature 299: 691-98 ee It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

Claims (26)

1. A DNA construct encoding a 107/402-T antigen, comprising: a first DNA sequence encoding an inducible transcriptional -i transactivator; a minimally active promoter; and a second DNA sequence encoding a 107/402-T antigen, wherein the inducible transcriptional transactivator activates the minimally active promoter and the minimally active promoter regulates transcription of the second DNA sequence.

2. The DNA construct of claim 1 wherein the inducible transcriptional 1 transactivator is induced by a compound selected from the group consisting of: tetracycline, doxycycline, rapamycin, and RU486.

3. A vector for expressing a 107/402-T antigen, comprising one or more copies of the DNA construct of claim 1.

4. A human cell for use as a recipient of an episome encoding a desired protein, comprising: one or more integrated copies of a DNA sequence encoding a 107/402-T antigen; one or more integrated copies of a minimally active promoter wherein the promoter controls the transcription of the DNA sequence encoding the 107/402-T antigen; and one or more integrated copies of a DNA sequence encoding an inducible transcriptional transactivator, wherein the transactivator activates the minimally active promoter.

The human cell of claim 4 wherein the inducible transcriptional transactivator is induced by a compound selected from the group consisting of: tetracycline, doxycycline, rapamycin, and RU486.

6. The human cell of claim 4 further comprising an episome comprising papovavirus origin of replication and a coding sequence for a protein whose expression is desired.

7. A kit for expressing a desired protein, comprising: the human cell of claim 4; and an episome comprising a papovavirus origin of replication for insertion of a coding sequence of a desired protein.

8. The kit of claim 7 wherein the episome further comprises a promoter.

9. A method of expressing a desired protein, comprising the step of culturing the human cell of claim 6 under conditions whereby the desired protein is expressed.

The method of claim 9 wherein the desired protein is secreted.

11. The method of claim 9 further comprising the step of contacting the the cell with an inducer of the transcriptional transactivator.

12. The method of claim 9 further comprising the step of varying the .:concentration of an inducer of the transcriptional transactivator over time. :i

13. 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 consists of a 107/402-T antigen and wherein the second protein segment consists of a mutant progesterone receptor which binds antiprogestin and does not bind to progesterone.

14. The fusion protein of claim 13 wherein the first protein segment is N-terminal to the second protein segment.

15. The fusion protein of claim 13 wherein the first protein segment is C-terminal to the second protein segment.

16. A DNA sequence encoding the fusion protein of claim 13.

17. A vector comprising the DNA sequence of claim 16.

18. A human cell comprising the DNA sequence of claim 16.

19. The human cell of claim 18 further comprising an episome comprising a papovavirus origin of replication and a second DNA sequence encoding a desired protein.

The human cell of claim 19 wherein the plasmid further comprises a promoter.

21. A kit for expressing a desired protein, comprising: the human cell of claim 19; and an episome comprising a papovavirus origin of replication. 18

22. The kit of claim 21 wherein the episome further comprises a promoter.

23. A method of expressing a desired protein, comprising the step of culturing the human cell of claim 19 under conditions whereby the desired protein is expressed.

24. The method of claim 23 wherein the desired protein is secreted.

The method of claim 23 further comprising the step of adding to the cell an antiprogestin.

26. The method of claim 23 further comprising the step of varying the concentration of the antiprogestin over time. *e CASE WESITEN RESERVE UNIVERSITY by Freehills Patent Attorneys Registered Patent Attorneys for the Applicant 17 Decenber 1997 *19 919