WO1988009809A2 - Systeme cellulaire pour l'expression d'une proteine recombinante sans production de virus - Google Patents

Systeme cellulaire pour l'expression d'une proteine recombinante sans production de virus Download PDF

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Publication number
WO1988009809A2
WO1988009809A2 PCT/US1988/001957 US8801957W WO8809809A2 WO 1988009809 A2 WO1988009809 A2 WO 1988009809A2 US 8801957 W US8801957 W US 8801957W WO 8809809 A2 WO8809809 A2 WO 8809809A2
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replication
cell
origin
factor
expression
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PCT/US1988/001957
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English (en)
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WO1988009809A3 (fr
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John F. Weaver
M. Michele Manos
Yakov Gluzman
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Cetus Corporation
Cold Spring Harbor Laboratory
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Publication of WO1988009809A2 publication Critical patent/WO1988009809A2/fr
Publication of WO1988009809A3 publication Critical patent/WO1988009809A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]

Definitions

  • 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.
  • the widely used cDNA library construction technique disclosed by Okayama and Berg 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ El/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 ⁇ E1/X under the control of the SV40 early promoter.
  • AHII aminoglycoside 3'-phosphotransferase II
  • 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.
  • 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.
  • the invention in another embodiment, 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
  • ⁇ El/dl309 which is the precursor of plasmid ⁇ EX/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 ⁇ EX/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.
  • MOI multipliaties of infection
  • 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.
  • a replication-defective vector which expresses the required factor expression of the desired recombinant protein is obtained.
  • 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.
  • 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.
  • 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.
  • 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.
  • a replication-defective vector which expresses the factor required by the first viral origin of replication is introduced into the stably transformed host cell.
  • 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.
  • 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.
  • the stably transformed host cell does not produce the factor required by the first viral origin of replication.
  • the first viral origin of replication ori
  • the cell lines into which it is stably transformed will be chosen from those which does not endogenously express T antigen.
  • 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- ⁇ El/X.
  • Ori- ⁇ El/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 ⁇ EI/X.
  • ⁇ E1/X is an adenovirus type in which most of the early region 1A, i.e., nucleotides 455-3330, of Ad5 ⁇ El/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 ⁇ El/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.
  • 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).
  • Plasmid pFC62 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.
  • 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 ⁇ g/ml pennicillin, and 10 ⁇ g/ml streptomycin.
  • 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 10 4 cells/cm 2 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 ⁇ g/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 10 4 cells/cm 2 ) 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.
  • DME Dulbeccos minimal essential medium
  • FBS fetal bovine serum
  • 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 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 125 I 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 10 5 in 900 ⁇ l, mixed with 100 ⁇ l 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.
  • 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.

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Abstract

L'invention concerne des cellules de mammifères qui sont transformées de manière stable avec un système d'expression compatible pour exprimer une protéine recombinante dans laquelle un facteur nécessaire à l'expression de la protéine recombinante est absent de la cellule transformée de manière stable, cellules mammifères dans lesquelles est introduit un vecteur défectueux au niveau de la réplication et qui exprime le facteur requis. L'invention concerne également des procédés d'obtention de l'expression de protéines recombinantes à partir d'une telle cellule.
PCT/US1988/001957 1987-06-12 1988-06-09 Systeme cellulaire pour l'expression d'une proteine recombinante sans production de virus WO1988009809A2 (fr)

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US6168487A 1987-06-12 1987-06-12
US061,684 1987-06-12

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WO1988009809A3 WO1988009809A3 (fr) 1989-01-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843742A (en) * 1994-12-16 1998-12-01 Avigen Incorporated Adeno-associated derived vector systems for gene delivery and integration into target cells
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Bio/Technology, volume 6, no. 3, March 1988, (New York, US), J.F. Weaver et al.: "Production of recombinant human CSF-1 in an inducible mammalian expression system", pages 287-290 *
Molecular and Cellular Biology, volume 4, no. 8, August 1984, American Society for Microbiology, K. Van Doren et al.: "Efficient transformation of human fibroblast by adeno-virus-Simian Virus 40 recombinants", pages 1653-1656 *
Proceedings of the National Academy of Sciences of the USA, volume 82, no. 11, June 1985, (Washington, D.C., US), M. Yamada et al.: "Overproduction of the protein product of a non-selected foreign gene carried by an adenovirus vector", pages 3567-3571 *
Science, volume 230, 18 October 1985, E.S. Kawasaki et al.: "Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1)", pages 291-296 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843742A (en) * 1994-12-16 1998-12-01 Avigen Incorporated Adeno-associated derived vector systems for gene delivery and integration into target cells
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII

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IL86699A0 (en) 1988-11-30
WO1988009809A3 (fr) 1989-01-12

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