WO1988009809A2

WO1988009809A2 - Cell system for expression of recombinant protein without production of virus

Info

Publication number: WO1988009809A2
Application number: PCT/US1988/001957
Authority: WO
Inventors: John F. Weaver; M. Michele Manos; Yakov Gluzman
Original assignee: Cetus Corporation; Cold Spring Harbor Laboratory
Priority date: 1987-06-12
Filing date: 1988-06-09
Publication date: 1988-12-15
Also published as: WO1988009809A3; IL86699A0

Abstract

The invention pertains to mammalian cells stably transformed with a compatible expression system for expression of a recombinant protein in which a factor required for expression of the recombinant protein is absent from the stably transformed cell, into which mammalian cells is introduced a replication-defective vector which expresses the required factor. The invention also pertains to methods of obtaining expression of recombinant proteins from such cell.

Description

CELL SYSTEM FOR EXPRESSION OF RECOMBINANT PROTEIN WITHOUT PRODUCTION OF VIRUS

The invention relates to the recombinant production of proteins in mammalian cell systems. More particularly, it concerns reproducible, stable cell lines capable of expressing recombinant proteins without producing virus, and methods for the use thereof for the expression of recombinant proteins.

Convenient transient expression systems for mammalian cloning vectors using viral control factors are available. For example, the widely used cDNA library construction technique disclosed by Okayama and Berg (Okayama, H., et al., Mol. Cell Biol. (1983)3:280-289) results in a plasmid containing an insert under the control of the SV40 promoter and the viral replicon. The inserted DNA can be expressed in COS cells (Gluzman, Y., Cell (1981) 23: 175-182) because these cells provide T antigen, a factor necessary for the function of the viral replicon, and the plasmid is thus replicated about a thousand fold in these cells. However, the plasmid is not stably maintained as the cells divide, and the ability of the cells to produce protein is rather quickly lost. To restore this ability requires the transfection of COS cells with additional plasmid.

This system is useful for assay for the presence of a correct cDNA insert, but is clearly in appropriate as a production tool for recombinant proteins. Scale-up requires proportional additional amounts of plasmid DNA, and the transfection must be continually repeated.

Stably transformed mammalian cells containing a compatible expression system for recombinant proteins has been described in published PCT Application W086/04607 published August 14, 1986. In this system, illustrated using an expression system for the colony stimulating factor CSF-1, an Okayama and Berg plasmid constructed as described in Kawasaki et al., Science (1985) 230:291-296 and W086/04607, contained a plasmid encoding CSF-1. The vector, based on CDX-1, is co-transfected into African Green monkey kidney cell line CV-1 (ATCC CCL 70 (Cell Lines and Hybridomas, American Type Culture Collection, Rockville, Maryland 16th ed. 1985). The co-transfecting vector is pRSV-NEO-2 (Gorman, C., et al. Science (1983) 221:551-553) and a transfection frequency of 10^{-5. 12} colonies is obtained as determined by resistance to the antibiotic G418 of these transformants. About 25 to 50% of the transformants are expected to have integrated the DNA of the CSF-1-containing vector.

Since the CV-1 cell line does not contain factors required for the expression of genes under SV40 promoter control, expression in this system requires infection of the transformed CV-1 cell line with SV40 virus to supply the required factor, in this case T- antigen.

One drawback of the system is that in using this SV40 induction system, CSF-1 plasmid replication and expression occur in the presence of a productive SV40 infection. The SV40 virions produced by the induced cells are clearly an undesirable by-product. Furthermore, SV40 viral replication and expression far exceed those of the recombinant plasmid. It would be desirable to achieve expression in an SV40 based inducible system without producing SV40 virus.

Defective virus, containing some elements of the SV40 viral genome are known. Gluzman, et al. "Origin Defective Mutants of SV40", Cold Spring Harbor Symposia on Quantitative Biology (1980) 44:293-300 describe the propogation of SV40 DNA cloned into a plasmid vector in which the SV40 origin of replication has been site specifically mutagenized to yield SV40 mutants that are non-viable in their conventional eukaryotic hosts. The mutants were shown to produce functional T antigen but did not cause cytopathic effects in CV-1 cells. Such cytopathic effects are indicative of a productive SV40 infection.

A recombinant viral genome comprising adenovirus type 5 (Ad5) in which most of the early region 1A nucleotides 455-3330 of the virus Ad5 ΔEl/dl309 is deleted has been described in Van Doren et al., J. Virol. (1984) 50,2:606-614 and is as shown to be effective in transfecting CV-1 host cells to resistance to G418 when a gene encoding aminoglycoside 3'-phosphotransferase II (APHII) is ligated into the Xbal site of adenovirus vector ΔE1/X under the control of the SV40 early promoter. This viral genome is readily integrated in CV-1 cells.

The ease of integration of this vector into the cell is used to transform human cells with a vector similar to the one described in Van Dorin et al., supra. In this case, SV40 early region which encodes T antigen was cloned into Ad5 ΔE1/X. (See Van Doren et al., "Efficient Transformation of Human Fibroblasts by Adenovirus-Simian Virus 40 Recombinants", Mol. and Cell Biol. (1984) 4.8:1653-1656.) The SV40 genome had either a wild-type origin of replication or the deletion of 6 nucleotides at the Bgll site of the SV40 origin of replication as described in Gluzman et al., Cold Spring Harbor Symp., Quant. Biol., supra.

Cells transformed with the chimeric virus having the intact SV40 origin of replication synthesized SV40 DNA but those with the mutant SV40 origin of replication did not produce SV40 DNA. The authors suggest that the origin defective chemeric virus is a convenient system for establishing SV40 transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5. in none of the foregoing references do the authors suggest the use of replication-defective virus as a source of factors required for the expression of recombinant protein in a stably transformed cell.

The invention provides stably transformed mammalian cells containing a compatible expression system for recombinant proteins in which a factor required for expression of the recombinant protein is absent from the stably transformed cell, but is provided by introducing into the mammalian cell a replication-defective vector which expresses the required factor. In one aspect, the invention pertains to the foregoing stably transformed mammalian cell line containing a compatible expression system for recombinant proteins in which a factor required for expression of the recombinant protein is absent from the stably transformed cell, wherein a replication-defective vector that provides the required factor has been introduced into the cell. In another embodiment, the invention pertains to a method of producing a recombinant protein in a mammalian cell which comprises the steps of providing mammalian cells stably transformed with an expression system for said recombinant protein, in which a factor required for expression of the recombinant protein is absent from the stably transformed cell and introducing into said mammalian cell a replication-defective vector which expresses in said cell said factor required for expression of the recombinant protein whereby said recombinant protein is expressed without the production of virus. Figure 1 is a schematic representation of plasmid Ad5

ΔEl/dl309 which is the precursor of plasmid ΔEX/1 the replication- defective adenovirus precursor into which DNA sequences encoding SV40 T antigen are ligated.

Figure 2 is a schematic representation of the viral genome ΔEX/1 into which the early region of SV40 mutant 6-1 has been li gated. Nucleotide numbers at the Xbal linker-junctions are at the left 454 Ad/346 SV40 and the right 2533 SV40/3333 Ad. This virus is designated herein as Ad5.SVR3.

Figure 3A and 3B show the kinetics of CSF-1 expression in stably transformed mammalian cells at different multipliaties of infection (MOI) using SV40 or Ad5.SVR3 as the source of T antigen. In

3A the SV40 MOI is = 0.5, Δ = 3, 0 = 10, = 25, and is mock

infected. In Figure 3B the Ad5.SVR3 MOI is = 2, Δ = 6, 0 = 15,

= 30 and ♤ is mock infected. In the invention, mammalian cells stably transformed with a vector providing an expression system for a desired protein compatible with the cell, but which lacks a factor required for the functioning of the expression system in the mammalian cell are used. By introducing into the cell a replication-defective vector which expresses the required factor expression of the desired recombinant protein is obtained.

In the mammalian cells, according to the invention, it is preferred that the cells are stably transformed with a vector which contains a first origin of replication and control sequences for the expression of the recombinant protein. The first origin of replication is inoperable in the transformed cell because it requires a factor which is not endogenously expressed by the stably transformed cell. By introducing into the cell a vector that is itself replication-defective, but which expresses the factor required for operability of the first origin of replication, expression of the recombinant protein in the stably transformed host is obtained. In general, the control sequences for the expression of the recombinant protein and the control sequences for the expression of the factor required for operability of the first origin of replication, may be any promoter that functions in the particular stably transformed mammalian cell. Mammalian viral promoters such as SV40, RSV, or adenovirus promoters, may be used, for example, as control sequences. Non-viral mammalian promoters that function in the cell may also be used.

In more detail, the first origin of replication is preferably viral origin of replication which requires the presence of a factor that is not endogenously produced by the mammalian cell by endogeneously expressed means that the cell prior to and/or after stable transformation does not express the required factor. Viral origins of replication of this type are exemplified by the SV40 viral replicon in which the SV40 origin of replication requires the expression of SV40 T antigen to function. If the SV40 origin of replication is used, the mammalian host cell will be one which does not express T antigen. For example, CHO cells would be inappropriate when the stably transforming expression system comprises the SV40 origin of replication. In general, any SV40 origin-containing plasmid that can stably transform the host cell may be used so long as T antigen is not expressed by cells transformed with the plasmid.

In order to induce expresson of the desired recombinant protein by the stably transformed host cell comprising the first viral origin of replication, a replication-defective vector which expresses the factor required by the first viral origin of replication is introduced into the stably transformed host cell. It is preferred hat the replication-defective vector is one which easily transforms the host cell, and in general, viral vectors that are highly infectious for the host cell are preferred. The mechanism of the replication defect of the replication-defective vector may vary and any replication-defective vector that easily transforms or transfects the host cell may be used. However, it is required that the defect that makes the vector replication-defective, is not complemented by the host cell, the first viral origin of replication or the factor required for expression of the first viral origin of replication carried by the replication-defective vector.

In one embodiment of the invention, the first viral origin of replication is the SV40 viral origin of replication. T antigen, required for proper function of the SV40 viral origin of replication, is encoded by a DNA sequence ligated with control sequences for the expression thereof into a replication-defective adenovirus vector. As mentioned above, it is necessary that the stably transformed host cell does not produce the factor required by the first viral origin of replication. Thus, when the first viral origin of replication (ori) is an SV40 ori, the cell lines into which it is stably transformed will be chosen from those which does not endogenously express T antigen. In general, SV40-permissive cell lines which have not been modified to provide T antigen expression, are appropriate hosts. Parti culary preferred are primate cell lines that have not been so modified. One such primate cell line is the CV-1 cell line, ATCC #CCL 70.

Adenovirus vectors are particularly useful in infecting primate cell lines including those of both human beings and non-human primates. The ability of adenovirus vectors to infect human and non- human primate cells makes adenovirus vectors particularly useful as vectors to provide the expression of the factor required for the function of the first viral origin of replication. Since it is required that the cells of the invention do not produce virus, replication-defective adenovirus vectors are preferred. Such replication-defective adenovirus vectors are known. One is described in Van Doren et al., Molecular and Cellular Biology, surpa, and designated therein as ori-ΔEl/X. Ori-ΔEl/X is a chimeric virus containing the SV40 early region Hpall-BamHI fragment which encodes T antigen and a defective SV40 origin of replication, cloned into the helper independent adenovirus vector ΔEI/X. ΔE1/X is an adenovirus type in which most of the early region 1A, i.e., nucleotides 455-3330, of Ad5 ΔEl/dl309 is deleted. Early region 1 encodes the functions responsible for adenovirus-induced transformation and these functions are required for adenovirus replication. The early region SV40 fragment cloned into ΔEl/X is origin defective (ori-mutant 6-1, having 6 nucleotides deleted from the Bgll site).

The invention will be better understood in connection with the following exmaples which are intended by the inventor to be exemplary and not limiting. The method of the invention is illustrated below using an expression system for colony stimulating factor. The expression vector is constructed as described by Kawasaki et al., Science 230:291-296, and in published PCT Application No. WO/04607.

Example I

Producti on of Stabl e Cel l Li ne Encodi ng Recombi nant CSF-1 Pl asmid DNA.

The human CSF-1 gene was cloned using the Okyama and Berg cloning and expression vector CDX-1, as described in Kawasaki et al., (1985) Science 230:296. This CSF-1 cDNA containing plasmid pCSF-17 was used for co-transfection into CV-1 cells along with the dominant selectable genetic marker for cell transformation, aminoglycoside phosphotransferase (neo) contained in plasmid pFC62. Plasmid pFC62 was derived from plasmid RSVneo 5.74 kb whic is described in Gorman et al., Science 221:551-553 as follows (1980). The 320 bp of Tn5 DNA between the unique Hindlll and Bglll sites (containing the aphB promoter and several ATG's, Tn5 coordinates 1195-1515, was deleted following digestion, repair and religation (pFC55). The 880 bp between the unique Smal and Hpal sites (containing extraneous Tn5 sequences, coordinates 2519-2685, the SV40 T splice signals, SV40 coordinates 4710-4100, and the "late" SV40 polyA signal, SV40 coordinates 2770-2669) was similarly deleted (pFC61). Finally, the 180 bp between the Ndel site (in pBR322) and the Nrul site (in the 5'- end of the RSV LTR) was deleted following digestion with Ndel and Nrul, repair, and religation in the presence of an Xhol linker. Plasmid pFC62 (4.3 kb) contains a Xhol site at the junction of the pBR322 sequence and the RSV LTR sequence. Plasmid DNA's were prepared for transfection by established methods.

Cell lines. CV-1 African Green monkey kidney cells, ATCC CCL 70 and 293 cells (adenovirus type 5-transformed human embryonic kidney cells) which provide adenovirus early region 1 gene function, ATCC CRL 1573, were obtained from the American Type Culture Collection.

Cultures were grown in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum, 10 μg/ml pennicillin, and 10 μg/ml streptomycin.

Transfection and isolation of stable transformants.

CV-1 cells were co-transfected with mixtures of pCSF17 DNA and pFC62 using the calcium phosphate co-precipitation method including glycerol shock as described in Frost et al., Virology (1978)91: 39-50. CV-1 cells were seeded in T-25 flasks at 1 x 10⁴ cells/cm² the day before transfection. A total of 10 micrograms of precipitated DNA was added to each culture to be transfected at a target (cPCSF-17) to marker (pFC62) DNA ratio of 10:1 or 20:1. Three days after transfection mock treated and transfected cultures were subcultured into growth medium containing 400 μg/ml G418 (Gibco, Grand Island, NY). Colonies of G418-resistant cells were observed within 10 days. Individual colonies were picked with a pasteur pipette and transferred to 24-well plates. Clones were expanded and tested for CSF-1 expression following infection with SV40. Induction of CSF-1 expression.

Cultures were grown to confluence (approximately 6 x 10⁴ cells/cm²) in 60 ran plates in growth medium consisting of Dulbeccos minimal essential medium (DME) with 10% fetal bovine serum (FBS). Immediately prior to induction, a control culture was used to determine cell number. SV40 or Ad5.SVR3 dilutions were calculated based upon cell number and were prepared in growth medium containing 5% FBS for SV40 or no serum for Ad5.SVR3. Growth medium was removed from the monolayer, replaced with 1 ml of virus inoculum and virus allowed to adsorb at 37°C for approximately one hour with occasional shaking to redistrubute virus. The viral inoculum was calculated to yield multiplicities of infection of 0.5, 3, 10 or 25 with SV40, and 2, 6, 15 and 30 using Ad5.SVR3. Mock infected controls were run for both inducing viruses. After adsorption, the inoculum was removed and the cells were washed one time with serum-free DME. Growth medium (6 ml DME, 5% FBS) was replaced and the cultures were further incubated until harvest. Growth medium supernatants were harvested daily for 7 days for analysis and were replaced by an equivalent amount of fresh growth medium. Samples were analyzed for CSF-1 activity by RIA and or in a bone marrow proliferation assay. The results are shown in Figure 3.

CSF-1 Assays.

Expression of CSF-1 was measured either in a radioimmunoassay specific for human CSF-1 or in a murine bone marrow colony formation assay, both as described in Das, S. K. , et al., Blood (1981) 58: 630-641). The RIA measures the ability of a CSF-1 sample to complete with highly purified ¹²⁵I labeled CSF-1 from MIA PaCa-2 cells for binding to a rabbit polyclonal antiserum raised .against partially purified human urinary CSF-1. CSF-1 titers from the RIA expressed in U/ml were calculated from a MIA PaCa-2 derived CSF-1 standard calibrated in the murine bone marrow colony assay. This bone marrow assay measures colony formation (>50 cells) of mononuclear bone marrow cells from Balb/c mice in a seven day assay in MEM-alpha medium containing 10% FBS and 0.3 agar. Cells were prepared and diluted to 1 x 10⁵ in 900 μl, mixed with 100 μl of diluted sample and placed in six well tissue culture plates. A unit of activity corresponds to one colony. Colony morphology was determined by removing individual colonies from the agar, cytocentrifuging the cells and staining.

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 4 : (11) International Publication Number: WO 88/ 09 C12N 5/00, 15/00, C12P 21/02 A3 (43) International Publication Date

15 December 1988 (15.12

(21) International Application Number: PCT/US88/01957 (74) Agent: HALLUIN, Albert, P. ; Cetus Corporation, 1 Fifty-Third Street, Emeryville, CA 94608 (US).

(22) International Filing Date: 9 June 1988 (09.06.88)

(81) Designated States: AT (European patent), BE (E

(31) Priority Application Number: 061,684 pean patent), CH (European patent), DE (Europ patent), DK, FI, FR (European patent), GB (E

(32) Priority Date: 12 June 1987 (12.06.87) pean patent), IT (European patent), JP, LU (E pean patent), NL (European patent), NO, SE (E

(33) Priority Country: US pean patent).

(71) Applicants: CETUS CORPORATION [US/US]; 1400 Published

Fifty-Third Street, Emeryville, CA 94608 (US). COLD With international search report SPRING HARBOR LABORATORY [US/US]; P.O. Before the expiration of the time limit for amending Box 100, Cold Spring Harbor, NY 11724 (US). claims and to be republished in the event of the receip amendments.

(72) Inventors: WEAVER, John, F. ; 3385 St. Mary's Road,

Lafayette, CA 94549 (US). MANOS, M., Michele ; (88) Date of publication of the international search report: 288 Whitmore #402, Oakland, CA 9461 1 (US). 12 January 1989 (12.01. GLUZMAN, Yakov ; 20 Downey Drive, Huntington, NY 11743 (US).

(54) Title: CELL SYSTEM FOR EXPRESSION OF RECOMBINANT PROTEIN WITHOUT PRODUCTION OF RUS

Replicon : Adeno* (human cells)

Size : 32 kb

Selectable phenoiype for animal cells: transformed foci Cloning sites : Xb"!

(57) Abstract

The invention pertains to mammalian cells stably transformed with a compatible expression system for expressi of a recombinant protein in which a factor required for expression of the recombinant protein is absent from the stab transformed cell, into which mammalian cells is introduced a replication-defective vector which expresses the required f tor. The invention also pertains to methods of obtaining expression of recombinant proteins from such cell.

FOR THE PURPOSES OFMFORMAπON ONLY

Codes used to identify States parly to the PCT on the front pages of p amphlets publishing international applications under the PCT.

AT Austria FR France ML Mali AU Australia GA Gabon MR Mauritania

BB Barbados GB United Kingdom MW Malawi

BE Belgium HU Hungary NL Netherlands

BG Bulgaria rr Italy NO Norway

BJ Benin JP Japan RO Romania

BR Brazil KP Democratic People's Republic SD Sudaa

CF Central African Republic ofKorea SE Sweden

CG Congo KR Republic ofKorea SN Senegal

CH Switzerland LI Liechtenstein SU Soviet Union

CM Cameroon LK Sri Lanka TD Chad

DE Germany, Federal Republic of LU Luxembourg TG Togo

DK Denmark MC Monaco US United States of America π Finland MG Madagascar

Claims

WHAT IS CLAIMED IS:

1. A stably transformed mammalian cell comprising a compatible expression system for recombinant proteins in which a factor required for expression of the recombinant protein is absent from the stably transformed cell, and introducing into said cell a replication-defective vector that expresses said required factor.

2. The stably transformed mammalian cell of claim 1 wherein said compatible expression system comprises a first origin of replication and control sequences for the expression of the recombinant protein, said first origin of replication requiring a factor for its operability not endogeneously expressed by the mammalian cell, and wherein said replication-defective vector introduced into said cell, expresses in said cell, said factor required operability of said first origin of replication.

3. The cell of claim 2 wherein said first origin of replication is a viral origin of replication.

4. The cell of claim 3 wherein said first viral origin of replication is an SV40 viral origin of replication and said factor is SV40 T antigen.

5. The cell of claim 2 wherein said replication-defective vector is an adenovirus vector.

6. The cell of claim 5 wherein said first origin of replication is an SV40 origin of replication and said factor is SV40 T antigen.

7. The cell of claim 6 wherein the adenovirus early genes Ela and Elb are replaced by the SV40 early region from SV40 origin- defective mutant 6-1.

8. The cel l of cl aim 1 wherei n said recombi nant protein is

CSF-1.

9. The cell of claim 2 wherein said recombinant protein is

CSF-1.

10. The cell of claim 6 wherein said recombinant protein is

CSF-1.

11. A method for expressing a recombinant protein in a mammalian cell comprising providing a stably transformed mammalian cell comprising a compatible expression system for recombinant proteins in which a factor required for expression of the recombinant protein is absent from the stably transformed cell and introducing into said cell a replication-defective virus that expresses said required factor.

12. A method for expressing a recombinant protein in a mammalian cell comprising providing a mammalian cell stably transformed with an expression system for said recombinant protein, said expression system comprising a first origin of replication requiring a factor for operability not endogeneously expressed in the mammalian cell and introducing into said mammalian cell a replication- defective vector which expresses in said cell said factor required for operability of said first origin of replicati on.

13. The method of claim 12 wherein said first origin of replication is an SV40 origin of replication and said factor is SV40 t and said replication-defective vector is an adenovirus vector.

14. The method of claim 12 wherein said recombinant protein is CSF-1.