FR2657880A1 - Production of recombinant proteins from human lymphoblastoid cells and heteromyelomas - Google Patents

Abstract

The presents invention relates to a lymphoblastoid cell line producing a determined protein, characterised in that it is transformed by a vector for expressing the said protein containing: - a promoter, efficient in human cells of haematopoietic origin, - a leader sequence ligated in 5' of the sequence encoding the said protein, - a polyadenylation signal in 3' of the coding sequence. The invention also relates to a process for producing proteins by culturing a cell line according to the invention in an appropriate culture medium.

Description

The invention relates to the expression of protein products using recombinant DNA techniques, and more particularly to the expression of biologically active human derythropoietin from human lymphocytes stably transfected.

The production of proteins with high added value by culturing eukaryotic cells is currently undergoing significant development. The interest of eukaryotic cells in the production of recombinant proteins lies essentially in the capacity of these cells to carry out a maturation of proteins allowing both the obtaining of biologically active molecules as well as their compatibility with a recipient organism. This is the case for example for proteins such as factor VIII, factor IX or Epo.

On an industrial scale, the improvement of mass culture processes is mainly based on technological innovation in the field of bioreactors (Mizrahi, Biotechnology 4, 123, 1986;
Arathoon and Birch, Science 232, 1390, 1986). However, the cultured cells and the secreted proteins have very diverse characteristics and a systematic optimization of the culture parameters must be undertaken for each line in order to achieve the desired productivity (Martin et al,
Biotechnology, 5, 838, 1987). It is clear that considerable efforts must be made to develop systems with high expression potential and to define the methods which allow the early choice of efficient clones.

Most of the expression systems currently used have been developed using eukaryotic cells of animal origin (CHO, BHK, Vero cells, etc.) and relatively few relate to human cells.

The use of human lymphoblastoid lines as host cells is an advantageous method given the high protein synthesis capacity of this cell type. On the other hand, lymphoblastoid cells are widely used for the production of human monoclonal antibodies and their culture technique has been the subject of major research efforts at INTS.

In the INTS laboratories, a defined culture medium has been developed (Drouet et al, Biosciences 5, 75, 1986; patent 8608494) and its use extended to continuous culture.

The present invention therefore proposes to use human lymphoblastoid lines immortalized by the Epstein virus
Barr or heteromyelomas expressing or not the EBNA1 nuclear antigen of EBV for the expression of cloned genes by developing both expression vectors with autonomous replication in these cells and stable integration systems in DNA cellular. The proposed episomal expression system comprises two elements of the genome of the Epstein-Barr virus, the OriP region which plays the role of origin of replication by cis action on sequences carrying this element and the gene coding for the EBNA1 antigen. which promotes and maintains autonomous replication by acting in trans on OriP.

Epo is a 36 kDa glycoprotein comprising 40 to 50% carbohydrates, which regulates the production of erythrocytes by stimulating the proliferation, differentiation and maturation of erythroid progenitors (Goldwasser et al, Blood Cells, 10, 147, 1984; Browne et al, Cold Spring Harbor Symposia on Quantitative Biology, L 1 693, 1986; Zanjani et al, Transfusion, 29, 46, 1989).

Human Epo is produced in the kidney of healthy adult humans and is present in very low concentration plasma (10 to 20 mU / ml). The mature protein consists of a single polypeptide chain of 166 amino acids but the urinary Epo (and the recombinant) only comprises 165 amino acids by elimination of the C-terminal arginine at position 166 (Recni et al, J. Biol. Chem.

262, 17156, 1987).

The stimulating effect of Epo on the formation of erythrocytes makes this factor a major therapeutic agent in the treatment of anemia in renal failure, in hemodialysis patients as well as for the postoperative recovery of patients.

Although there are known methods of producing human PO, for example a method of extracting urine from aplastic patients (Mikake et al, J, Biol. Chem. 252, 5558, 1977), and a method by tissue culture of human renal tumor cells (Sherwood et al, Endocrinology 99, 504, 1976), these methods do not allow the mass production of highly purified Epo
The recombinant DNA technology associated with the use of animal cells as host cells today makes it possible to solve this problem of erythropoietin production.

The cloning of erythropoietin cDNA from a human fetal liver cDNA library, as well as the cloning of the Epo gene from a genomic library have been described by Jacobs et al (Nature 3 13, 806, 1985 ) then by Lin et al (Proc. Natl. Acad. Sci.

USA 82, 7580, 1985) and Powel et al (Proc. Natl. Acad. Sci., USA 83, 6465, 1986).

The expression of recombinant human Epo (Epo HR) could be obtained using a replicative vector in COS cells (monkey kidney) (Kirwin-Amgen patent, Inc nO 0148605 A2, Patent
Genetics Institute, Inc no. WO 86/03520). In this system the expression of Epo is transient. Stable expression of Epo has been described in CHO dhfr (Chinese hamster ovary) cells using an integrative vector containing the DHFR gene (Patent
Kirin-Amgen, Inc nO 0148605 A2, Brevet Genetics Institute, Inc n "
WO 86/03520, University of Washington patent no. 0255231A1).

Epo has also been produced in BHK (hamster kidney) cells (University of Washington patent n00255231A1).

In both cases, stable lines, selected for their ability to resist methotrexate (MTX) and express a high level of Epo, were obtained. The authors also show that the transfected cell lines express and secrete biologically active Epo in vitro and in vivo.

Epo HR has also been produced in hamster kidney cells (BHK) and in murine fibroblasts NIH / 3T3 (Patent
University of Washington, no 0225 231 Al) and Goto et al,
Biotechnology 6 67 1988 (Snow Brand Milk Products Co patent
Ltd No. 0236059 A2), as well as in mouse epithelial cells (C127) using a viral replicon (BPV) (Integrated Patent
Genetics, Inc no 0267678 A1). Epo HR has been produced in insect cells infected with a recombinant baculovirus (Wojchowski et al, Biochim. Biophys. Acta 910, 224, 1987) and in yeasts (Elliot et al, Gene 79, 167, 1989).

Although generally effective in quantitative terms, the expression systems used all have various drawbacks. For example, the Epo HR produced in insect cells is not correctly glycosylated (absence of sialic acids) and the hormone produced is biologically inactive in vivo (Quelle et al, Blood 74, 652, 1989).

This observation agrees well with the role of carbohydrates, and more particularly of sialic acid, on the biological activity of the hormone which has been known for a long time (Lowg et al, Nature 185, 102, 1960; Goldwasser and Kung , J. Biol. Chem. 247, 5159, 1972).

Although the Epo HR produced by CHO cells is biologically active and used in human therapy (Eschbach et aI, N.

Engl. J. Med. 316, 73, 1987), these cells as well as the BHK cells generate complications of exploitation on an industrial scale because of their mode of culture (attached cells). Furthermore, glycosylation heterogeneity is observed, in particular the sialylation rate which is crucial for biological activity in vivo (Sasaki et al, Biochemistry 27, 8618, 1988; Takeuchi et al, J. Biol.

Chem. 263, 3657, 1988; Tsuda et al, Biochemistry 27, 5646, 1988
Fukuda et al, Blood, 84, 1989).

To avoid the difficulties linked to the culture mode dependent on the support of the abovementioned cells, the Sumitomo Chemical group
Compagny Ltd (patent no. 0232034) uses human cells, in particular Namalwa cells, which have a high cell multiplication rate and whose culture method is suspension. The Epo HR produced by these cells has characteristics close to those of the urinary Epo and is biologically active in vitro and in vivo (Yanagi et al, DNA, 8,419, 1989; Gene 76, 19, 1989). One drawback of this system is the tumorigenicity of these cells, derived from Burkitt's lymphoma.

This is why the present invention provides a line of lymphoblastoid cells producing a specific protein, characterized in that it has been transformed by an expression vector for said protein comprising - an effective promoter in human cells of hematopoietic origin, - a leader sequence ligated in 5 ′ of the sequence coding for said protein, - a polyadenylation signal in 3 ′ of the coding sequence.

Indeed, considering that the host cells most suitable for the production of human erythropoietin remain human cells, the use, for production purposes, of human lymphoblastoid cells which are either established from leukocytes of peripheral blood of a healthy individual, either obtained by in vitro transformation using the Epstein-Barr virus (EBV), seems the most satisfactory. Such cells proliferate indefinitely in culture but they are not malignant and in particular they are not clonogenic in agar or tumorigenic in nude mice (Tursz, MédecinelSciences, 6 (suppl.), 42, 1989). In both cases, these cells synthesize the EBNA-1 nuclear antigen of EBV. A third type of line can also be used, for example a heteromyeloma obtained by fusion of a line of human B lymphocytes transformed in vitro. by EBV and mouse myeloma. As a result of the loss of certain human chromosomes, this cell is
EBNA-.

INTS has acquired excellent experience in the development and production of human monoclonal antibodies produced by lymphoid cells immortalized in vitro by EBV. The production is facilitated by the fact that the cells grow in suspension and the culture is carried out according to the batch mode or in continuous culture. In addition, a culture medium poor in proteins has been established in order to facilitate the purification of the immunoglobulins secreted by these cells (INTS patent n
F2600076).

On the other hand, the capacity for synthesis and high secretion of immunoglobulins retained after immortalization of these cells accounts for their secretory potentialities for the production of a foreign protein. Such a system can therefore be advantageously used for the production of recombinant proteins.

Since human-mouse heteromyeloma lines have proven to be of great interest in stabilizing B lymphoid cells producing human monoclonal antibodies (Teng et al, PNAS 80, 7308, 1983), we will use such cells both as fusion partner of the B lymphoid lines producing recombinant proteins and as host cell for the stable production of the recombinant proteins after transfection with the expression vectors.

The invention therefore relates to human lymphoblastoid cell lines and heteromyelomas capable of producing a large number of proteins, in particular human Epo, the mode of culture of these cells and the purification of a
Epo active from culture supernatants.

The plasmids specifically described in this invention are vectors for the expression of the Epo gene but can also be used as vectors for expression of the Epo cDNA whose isolation is described in this invention, same as for the expression of other proteins.

To prepare an Epo-producing cell line, an expression vector must first be constructed. This expression vector must contain a functional transcription unit in the chosen host cell. The promoter of a virus such as Adenovirus 5 (Ad5) capable of replicating in human cells of hematopoietic origin (Iymphoids and myeloids) is suitable. One can also provide a leader sequence, for example, the late major promoter (MLP) of Ad2, coupled to a cDNA copy of the tripartite leader sequence of Ad2 (which allows an increase in the translation efficiency of 1 ' Messenger RNA) is ligated at the 5 ′ end of the DNA coding for Epo and a polyadenylation signal, in particular the signal derived from the SV40 virus, is ligated at the 3 ′ end.

An efficient transcription activator sequence in human lymphocytes is preferably added upstream of the MLP.

The activator sequence of the Ad5 E1A gene is used.

The vector will also include a marker gene, for example a dominant selection gene of bacterial origin; it is the gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) which is present on the vectors specifically described to allow selection of the transformed cells (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 98, 2072, 1981 ).

For an optimal translation of the messenger RNA, in particular of the Epo, the region of Ad5 coding for the RNA VAI and VAII is added to the expression vector. The structure of this basic plasmid pTS17 is shown in Figure 1.

The invention also proposes two methods which make it possible to obtain the establishment of stable lines producing Epo.

In a first method, the vector containing the Epo gene is integrated into the chromosomes of the host cell. In the second method, the vector containing the Epo gene replicates autonomously in the cell nucleus. The advantage of this second system is to avoid placing the Epo gene under the influence of cis sequences, as can happen after chromosomal integration.

In the first case, a murine DNA fragment of mitochondrial origin is added to the vector to give plasmids such as the plasmid pTS39 (Figure 2). This fragment has been described as allowing tandem multimerization of the vector during integration into the genome of mammalian cells (Lutfalla et al. Somatic Cell. And Mol. Genetics 11, 223, 1985) and therefore should allow higher expression of Epo.

In the second case, the oriP region of the EB virus or else the region
OriP and the EBNA-1 EBV gene are added to the vector to give plasmids such as plasmids pTS53 and pTS59 respectively (Figure 3). The EBNA-1 protein acts in trans on the oriP region. It promotes and maintains the autonomous replication of the DNA sequence that carries oriP.

This autonomous replication mechanism has been partially elucidated by Sugden and collaborators (Yates et al, Proc. NatI. Acad. Sci. USA 8 1 3806, 1984 (Sugden) et al, Mol. Cell. Biol. 5, 410, 1985;
Reissman et al, Mol. Cell. Biol. 5, 1822, 1985, Yates et al, Nature 313, 812, 1985). The effectiveness of such a system in directing the synthesis of recombinant proteins has recently been demonstrated, for example in the case of TNF (Kioussis et al, EMBO J. 6, 355, 1987), INF-t and EGF-R (Young et al. Gene 62 171, 1988) and hemagglutinin of the influenza virus (Jalanko et al, Gene 7 287, 1989). This system can also be used to build libraries of CDNA and direct cloning of genes by expression techniques (Margolskee et al, Mol. Cell. Biol. 8, 2837, 1988) In general, the number of copies of the vector is from 20 to 200 per cell depending on the promoters and activator sequences used (Jalanko et al, Biochem. Biophys. Acta 949, 206, 1988; Hauer et al,
Nucleic Acids Res. 1 7, 1989, 1989). A variant of the episomal system consists in using a cell expressing the protein
EBNA-1 constitutively and expression vectors containing only the oriP sequence. Such a cell can be constructed by integration of the EBNA-1 gene into a HepG2 line (Luftalla et al, Gene ff6, 27, 1989) or else , as in this invention, by immortalization of human B lymphoid cells by the Epstein-Barr virus (Thoda et al, Cancer Res. 38, 3560, 1978; Anderson and Gusetta, In vitro 20, 856, 1984).

The method used to introduce DNA into lymphoblastoid cells is electroporation (Potter et al, Proc. Nat.

Acad. Sci. USA; 81, 7161, 1984). Human lymphoblastoid cells used as host cells come from three separate sources - from ATCC under the reference CCL156 RPMI 1788 for cells established from leukocytes in the peripheral blood of a healthy individual (Huang and Moore, J. Natl. Cancer Inst. 34, 1119, 1969) - from INTS with regard to B lymphocytes taken from a healthy donor and immortalized in vitro by EBV according to a protocol described at INTS for the production of human monoclonal antibodies (Goossens et al, J. Immunol. Methods, 101, 193, 1987).

- INTS with regard to heteromyeloma 1C5E2 obtained by fusion of a line of human B lymphocytes transformed in vitro by EBV (H2D5D2B6) and a mouse myeloma (B6Ag) As a result of the loss of certain human chromosomes, this cell is EBNA-.

- ATCC under the reference CRL 1668 as regards the heteromyeloma SHMD33 (EBNA-) obtained by fusion of a human myeloma cell (FU-266 cloned EI) with the mouse myeloma P3X63Ag 8,653 (Teng et al , PNAS 80, 7308, 1983).

- INTS with regard to heteromyeloma obtained by fusion of the clone producing Epo 156 0.5 F8 with heteromyeloma
SHMD33.

The transformed cells producing Epo HR are propagated in flasks or culture dishes in Iscove medium, 10% SVF.

The Epo HR secreted into the culture medium is purified by methods which derive from those described, for the purification of Epo obtained from natural sources (Miyake et al, J. Biol. Chem. 252, 5558, 1977; Krystal et al, Blood 67, 71, 1986).

The human Epo gene used in the invention was isolated from a genomic DNA library using a synthetic oligonucleotide deduced from the gene sequence. Epo cDNA was isolated from total RNA from a human kidney tumor producing Epo by the chain polymerization method (PCR), using two synthetic oligonucleotides framing the RNA messenger from Epo.

The following paragraphs give a detailed description of the invention. It is understood that this invention is not restricted to the production of recombinant Epo by human cells but also extends to the production of any recombinant protein using the expression systems described in invention.

The present invention also relates to a process for producing proteins, characterized in that a cell line as described above is cultivated in an appropriate culture medium and in that the desired protein is recovered.

This process is more particularly useful in the preparation of Epo and its analogs, i.e. active fragments and variants of Epo.

The examples below will better illustrate other characteristics and advantages of the present invention.

In the attached figures,
Figure 1: Basic plasmid (pTS17) used for the construction of integrative (pTS39) and replicative (pTS53 and pTS59) vectors.

E: sequence activating the Ad5 ElA gene; MLP: late major promoter of Ad2; L: cDNA copy of the tripartite leader sequence of Ad2; Epo coding region of the Epo gene; pA: SV40 virus polyadenylation signal; XGPRT: bacterial gene coding for xanthine guanine phosphorybosyl transferase; VA: AdS genes encoding VA RNAs.

Figure 2: pTS39 integrative type expression vector.

Mito DNA: murine DNA fragment of mitochondrial origin. A second selection gene (DHFR) is placed in a bicistronic position downstream of the coding region of the Epo gene. It is not used as a selection agent in the invention. However, such a vector has the advantage of being also usable in dhfr- or even dhfr + cells, in order to allow gene amplification in the presence of MTX.

Figure 3: pTS53 and pTS59 replicative type expression vectors.

 SV: enhancer and early promoter of the SV40 virus; oriP: origin of replication of the EBV virus; EBNA: EBNA1 gene of the EBV virus.

Figure 4: Epo gene cloning protocol.

Figure 5: Epo cDNA cloning protocol.

Figure 6: Construction of the vectors pTS39, pTS53 and pTS59.

E; activator sequence of the Ad5 E1A gene; MLP: late major promoter of Ad2; L123: tripartite leader sequence of Ad2
CAT. bacterial gene encoding chloramphenicol acetyl transferase; pA: SV40 virus polyadenylation signal; The polylinker of pUC18; L2: synthetic polylinker; VA: AdS VA genes encoding VA RNAs; XGPRT: bacterial gene coding for xanthine-guanine-phosphoribosyl transferase; Epo: coding region of the Epo gene; mito: murine DNA fragment of mitochondrial origin; DHFR: murine cDNA coding for dihydrofolate reductase; oriP: origin of replication of the EBV virus; EBNA: gene
EBNA1 of the EBV virus.

ISOLATION OF THE HUMAN GENE OF ERYTHROPOIETIN
We have chosen to screen a genomic DNA library with a 30-mer oligonucleotide: 5'-CGTGATATTCTCGGCCICCTTGGCCTCCAA- 3 'deduced from the gene sequence (Lin et al, Proc. Natl. Acad. Sci.

USA X, 7580, 1985). This probe is complementary inverted to the messenger RNA and it recognizes the sequence of the Epo at position 129-159 (the base nO 1 representing the initiation of translation).

Before using this probe to screen a library, the hybridization conditions were tested in Southern. These conditions being controlled, we screened a genomic DNA library from Clontech (ref ML 10006). The cloning vector is EMBL 3, it contains inserted fragments of human DNA of 12 to 20 kb. We displayed 107 clones distributed over 10 boxes 15 cm in diameter. Three sets of filters, replicas of these dishes, were washed and hybridized with the oligonucleotide probes labeled with T4 polynucleotide kinase. The specific activity of this probe was 109 cpm / g.

The hybridization conditions were as follows -Pre-hybridization: 6 SSC, 2 X Denhardt's, 0.005% Tetra-sodium disphosphate (Nappi), 100 llg / ml salmon sperm, 520C for 4 h.

-Hybridization: 6 SSC, 2 X Denhardt's, 0.05% Nappi, 20 Rg / ml tRNA 520C overnight with the Epo probe (106 cpm / ml per filter).

-Washing: 2XSSC laboratory temperature, 2 X SSC 0.005% Napi 3 x 20 min. at 520C.

Under these conventional conditions approximately 1/20 to 1/30 of the clones per filter are revealed, all in triplicate. Statistically, depending on the representativeness of the bank, we should have a maximum of 3 clones per filter which hybridize specifically with this probe. In order to obtain this result, different hybridization and washing conditions were carried out on 9 clones taken at the end of the primary screening - pre-hybridization and hybridization in 3xSSC instead of 6 X SSC, - hybridization temperature and of washing at 550C and 570C - additional washing in 1 X SSC, 0.05% Nappi at 520C for 20 min and / or 0.1 SSC, 0.05% Nappi at 520C for 20 min.

Tertiary then quaternary screening was then carried out to lead to the characterization of 3 pure clones hybridizing very strongly with the probe.

The DNA of these 3 phages was amplified, then analyzed. In order to control the presence of the Epo gene in its entirety, we constructed the restriction map of these 3 clones, looked for the presence of the 3 'and 5' ends of the Epo gene using two oligonucleotide probes recognizing the terminals of this gene. This work allowed us to show that two of the three clones contained the entire Epo gene.

Restriction fragments containing the entire Epo gene were subcloned into an amplification vector of the pUC type. Thus the two restriction fragments B amHI-HindIII of 5.4 kb and BglII-Bg; III of 4 kb were subcloned according to the procedure summarized in Figure 4.

ISOLATION OF EPO HUMAN cDNA
We have isolated the total RNA from a human renal tumor constitutively expressing Epo. From these total RNAs, the poly A + RNA fraction was isolated by passage through an oligo-dT column. We then controlled by the Northern technique that this population of poly (A +) RNA expressed the messenger coding for Epo. To clone the Epo cDNA, two techniques can be used: either the construction of a cDNA library and screening with a specific Epo probe, or the direct cloning of the Epo cDNA by the PCR method. We chose the second method. For this, two probes framing the Epo cDNA were chosen.
INTS 10: 5'-CTGGAGTGTCCATGGGACAG-3 '
This oligonucleotide (20-mer) is in position 694 with respect to ATG adenosine 1 (initiation of messenger translation
Epo). This oligonucleotide recognizes the reverse complementary sequence of the Epo mRNA and bounds the 3 'part of the cDNA.

.INTS 9: 5'-AGGCGGGAGATGGGGTGC-3 '
This oligonucleotide (20-mer) is in the same direction as the mRNA and bounds the 5 ′ part of the Epo cDNA (position-10 relative to adenosine 1 of the initiation codon). The INTS 10 probe allows us to copy the Epo mRNA matrix using reverse transcriptase. The mRNA is then degraded with sodium hydroxide for 1 hour at 650C. This single-stranded cDNA can then be amplified by the PCR technique using the two probes limiting the cDNA: INTS 10 and INTS 9. We thus amplified a DNA fragment of 723 bp which was, after electrophoresis, cloned into an amplification vector of the pUC type then sequenced. The protocol for cloning Epo cDNA is summarized in Figure 5.

CONSTRUCTION OF EXPRESSION VEGIBURS - INTEGRATIVE VECTORS
The expression vector of integrative type pTS39 represented on the
Figure 2 was constructed as follows
The starting plasmid is pMLP10 (Ballay et al, Hepadna
Viruses, 481, 1987, Alan R. Liss, Inc.) shown schematically in Figure 6.

The plasmid pMLPCAT is derived from pMLP 10 by insertion of the HindIII-BamHI fragment of pHp34-CAT containing the CAT gene and the SV40 processing signals (P. Gilardi et al, Nucleic Acid
Research 20, 7877-7887, 1984) between the HindIII and BamHI sites of pMLP10. The plasmid pTS1 was then obtained by insertion of the EcoRI-HindIII polylinker of pUC18 at the NruI site of pMLPCAT. The plasmid pTS2 is derived from pTS1 by insertion of the following synthetic oligonucleotide -AGCTTGCGGCCGCGGTTACCAGATCTACGCGTGTT-3 '
-ACGCCGGCGCCAATGGTCTAGATGCGCACAA-5 'containing the rare sites NotI, SacII, BstEII, BglII, MluI between the HindIII-HDaI sites of pTS1. pTS5 is then obtained by inserting the HindIII fragment of pAG60VA (containing the VAI and VAII genes of AdS) at the XbaI site of pTS2. The plasmid pTS17 is derived from pTS5 by insertion of the PvuII fragment of pAGT40 (containing the XGPRT gene under the control of the promoter and of the maturation signals of the Tk gene of the HSV1 virus) at the SmaI site of pTS5. The plasmid pTS29 was then obtained by inserting the BstEII-BglII genomic fragment (containing the Epo gene) between the HindIII and BglIII sites of pTS17. pTS37 is derived from pTS29 by insertion of the EcoRI-PvuI fragment from p81 (containing a mitochondrial DNA sequence) at the SacI site of pTS29.

Finally, pTS39 was obtained by inserting the BamHI-BglII fragment from pTG1086 (containing the coding region of the murine DHFR cDNA) at the BglII site of pTS37 (FIG. 6).

- REPLICATIVE TYPE VECTORS
Two replicative type vectors were constructed -pTS53, in which the OriP sequence necessary for obtaining a replicon was isolated from the plasmid p220.2 (Sugden et al.

Mol. Cell. Biol. 5, 410, 1985) using the enzyme AccI and inserted into the SacI site of the vector pTS29 (Figures 3 and 6).

- pTS59 in which the OriP and EBNA-1 sequences of EBV were isolated from the plasmid pTS48 (variant of p220.2 containing the EBNA-1 gene under the control of the enhancer and of the early promoter of the SV40 virus) using the enzymes Eco RI and BamHI then inserted into the SacI site of the vector pTS29 (Figures 3 and 6). This last vector was constructed in order to observe the effect of an overexpression of EBNA-1 on the number of replicons copies in an EBNA + cell, and also to obtain a replicon in an EBNA cell.
CELL TRANSFECTION AND SELECTION OF
TRANSFORMING
The transfection is carried out with a 68701 "Bioblock" electropulser.

Two aqueous solutions are prepared beforehand
The fusion medium: Inositol 250 mM, KH2PO4 1 mM, Mg Acetate 0.5 mM, Ca Acetate 0.1 mM; pH 7.4.

The post-fusion medium: NaCl 132 mM, KCI 8 mM, KH2PO4 1 mM,
Mg 0.1 mM acetate, Ca 0.1 mM acetate; pH 7.4.

After 2 days of culture, the cells are washed and adjusted to 4.107 / ml in the fusion medium. The suspension is incubated for 10 min at 0 ° C. in the presence of the DNA to be transfected (5 μg / 106 cells).

A sample of the suspension is placed between the electrodes of the device and the electroporation is carried out under the following conditions: 4 rectangular pulses at 1 second intervals of duration 100 uS, with an electric field of 1,500 V / cm2.

Immediately after the electric shock, the suspension is diluted 1/20 and then incubated for 20 min at 370C. The cell viability is between 20 and 60%. The cells are then cultured at a concentration of 0.5106 cells / ml.

48 hours after transfection an aliquot of the supernatant is removed for the determination of Epo. The cells are then cultured in a selective medium (Iscove, 10% SVF dialysed, mycophenolic acid 1 ug / ml, xanthine 250 ug / ml).

After 14 to 21 days of selection from the pool, cloning by limiting dilution is carried out in 96-well microplates in the presence of feeder cells (total human lymphocytes, irradiated at 4000 rds, from healthy donors).

PRODUCTION OF EPO HR BY TRANSFORMED CELLS * CCL156 cells - with the expression vector pTS39 (integrative system)
About 6.106 CCL156 cells are transfected by electroporation with 30 μg of plasmid DNA, then subjected 48 h after transfection, in the selective medium. The resistant population is cloned by limiting dilution in the presence of feeder cells (irradiated human lymphocytes). Among the different clones obtained, clone 156 0.5 AF8 secretes approximately 100 units of Epo per ml of culture medium / 72 h and was followed long term in the presence and in the absence of selection pressure. The results presented in Table 1 show that this clone is stable over 48 passages, ie approximately 7 months of culture in the absence of selection pressure.

Development of culture in large volumes is in progress: on roller, the clone 0.5 A F8 secret 80 to 100 U Epo / ml over three days of culture in medium poor in proteins CK4 or CK4N (basic medium: mixture Iscove-Ham F12 (1: 1); CK4: supplements: human albumin 50 mg / l, human transferrin (saturated at 30% by addition of FeC13) 1 mg / l, bovine insulin 3 mg / l, linoleic acid 1 mg / 1, putrescine 10 1M, ethanolamine 20 I1M; CK4N, idem plus ribonucleotides: Adenosine 100 mg / l, Cytidine 100 mg / l, guanosine 100 mg / l, Uridine 100 mg / l). This secretion rate can be renewed 3 times if the medium is changed every 3 days.

An amplification test with methotrexate (MTX) of the DHFR gene was carried out on this clone maintained without a selective medium. For doses of MTX less than or equal to 20 nM, the wild cell resists as much as the clone. On the other hand, in the presence of 50 nM MTX no clone of wild cell resists while 24 clones of 0.5 AF8 are obtained. A preliminary assay performed by a test
ELISA indicates that 10 clones secrete between 500 and 1000 U
Epo / ml. A secretion test carried out on the 4th pass on the clones cultured in the presence of 50 nM MTX makes it possible to select a clone, 0.5 AF8E2, which secretes 260 U Epo / ml, ie 446 U Epo / 106 cells.

- with the expression vector pTS53 (replicative system)
Autonomous replication of the vector is ensured by the presence of the oriP region of EBV on the plasmid and that of the protein.
EBNA-I synthesized by the cell.

Five clones were obtained according to the technology described above. One of them, clone C2, was followed on 22 passages, ie 3 months in the presence of selective and secret medium 80 to 100 units of Epo per ml of culture / 72 h (Table 2). In the absence of a selective medium (17th passage), the secretion in 72 h is only 18 U Epo / 10 6 cells whereas in selective medium it is 66 U Epo / 106 cells.

- with the vector pTS59 (replicative system)
The autonomous replication of the vector is ensured by the presence of the OriP region on the vector and by the overexpression of the protein.
EBNA-1 by the cell.

Monitoring the expression of Epo HR over 21 days after transfection, in the presence of selective medium, shows that the cells transfected with pTS59 secrete from 0.5 to 29 U Epo / ml / 106 cells from D 2 to D 21 , while the secretion of cells transfected with pTS53 under the same conditions, varies from 0.16 to 3 U of Epo / ml / 10 6 cells. This result suggests that the vector pTS59 which has the EBNA-1 gene would replicate more efficiently than pTS53 which contains only the oriP region. However, cloning of cells transfected with pTS59 proves difficult; currently only two clones have been obtained and they express only a small amount of Epo HR. A first Southern blot analysis of the Hirt extract (low molecular weight DNA) on the clone C2 indicates that the vector pTS53 replicates at the rate of 2 to 3 copies per cell.

* Cells immortalized in vitro by EBV (INTS LE-6B4)
LE6B4 cells are transfected with the 3 vectors pTS39, pTS53 and pTS59. The Epo assays carried out at different times after transfection are positive: in particular for pTS59, 29 U Epo / ml / 106 cells are detected after 21 days of selection.

* Heteromyelomas (INTS 1C5E2)
1C5E2 cells were transfected in a preliminary experiment by the liposome technique (Felgner et al, Proc.

Natl. Acad. Sci., 84, 7413, 1987).

Epo assays performed 48 hr after transfection indicate no detectable activity. However, after 7 days in a selective medium, the supernatants exhibit a low activity detected in
ELISA. Cloning by limiting dilution has been carried out; an initial assay made it possible to detect two positive clones in the RIA test: the clone îC5pTS59-2 (5 mU / ml) and the clone lC5pTS59-B4 (20 mU / ml). It is therefore possible to express Epo HR to heteromyelomas.

As a result, an EBNA- heteromyeloma, the SHMD33 line (ATCC CRL 1668) was transfected with the integrative vector pTS39 and the replicative vector pTS59 and the stable clones are being selected. This heteromyeloma will also be used as a fusion partner with the clone 0.5 AF8 (vector pTS39 + host CCL156) in order to select an EBNA- clone highly producing Epo HR.

DETECTION OF BIOLOGICAL ACTIVITY IN VITRO AND
IMMUNOLOGICAL OF EPO HR
Culture of fetal liver erythroblasts
Epo activity is measured in vitro according to the incorporation of 59Fe by mouse fetal liver erythroblasts (taken on the 13th day of gestation) cultured in 26 hours according to a method derived from that described by Stephenson et al , Endocrinology 88, 1519, 1971.

The embryos are taken sterile from Balb / c mice (Charles River France). In certain cases, splenic erythroblasts are obtained after induction of intense anemia in mice after injection of phenylhydrazine according to the method of Krystal (Exp. Hematol. 11, 649, 1983).

The cells are mechanically dissociated and suspended in RPMI 1640 medium (Tambourin et al, Biomedicine 19, 112, 1983; Goldwasser et al, Endocrinology 97, 315, 1975) at a concentration of 1.6 x 106 cells / ml containing 7% of fetal calf serum, 85 µM albumin and 0.4 µM human transferrin.

Depending on the case, a serum-free medium is used in which purified or synthetic natural phospholipids (soy, ovolecithin) (32 x 10-3 M) and cholesterol (1.6 x 10-3 M) are dissolved in chloroform, secondarily evaporated under current nitrogen. The lipids in albumin solution at 1 mg / ml in RPMI are dispersed by ultrasound in melting ice (Boffa et al,
Exp. Hematol. 10, 675, 1982).

The cell suspension is distributed in volume of 200 μl in the round bottom wells of the "Nunclon" culture plates. After an incubation of 21 hours at 370C in the presence of 0.7% CO 2, the 5 9 Fe-transferrin is incorporated into the wells, the plates are shaken and put back into the incubator for a prolonged incubation of 5 hours.

Each sample is analyzed at 3 or 4 protein concentrations.

The activity of each dose results from a quadruple determination.

The rate of incorporation of 59 fie into the heme extracted with methyl ethyl ketone or into the cells is measured as a function of the logarithm of the dose of the sample and the results are expressed in milliunits / ml or per mg of protein.

The standard is recombinant human Epo grading 100,000
U / mg. Its specific activity was defined according to human Epo samples from the second Reference preparation
International (Annable et al, Bull. Wld. Hlth. Org. 47, 99, 1972).

Culture of erythroid progenitors from bone marrow or peripheral blood.

The effects of Epo HR on the maturation of BFUe and CFUe were determined in culture of erythroid cell precursors on methylcellulose in IMDM serum-free medium (Cormier et al, Cell Differentiation, 17 261, 1985. Epo HR, like human urinary Epo, determines the differentiation and maturation of BFUe from mouse bone marrow or human peripheral blood. The dose-effect of Epo HR on the development of CFUe is compared with that given by 1 'Natural epo.

Detection by radioimmunological tests
The radioimmunoassay of Epo HR was carried out in cell supernatants according to two methods
By precipitation with double antibodies in the soluble phase:
A monospecific human urinary anti-Epo rabbit immune serum (Terry Fox Lab, Vancouver) is incubated with an Epo H standard or a sample to be assayed for 16 h at 40C. After this time, 2000 cpm 1251-Epo HR (Amersham) are added and the preparations are incubated for 2 h at 40C. The dilution of the immune serum chosen is that which gives a fixed radioactivity of 20 to 30%. The final dilution of the immune serum is between 10-4 and 10-5. After an incubation of 2 h at 200C with goat anti-rabbit Fc immunoglobulins, the radioactivity of the pellet is measured.

By precipitation by double antibody in solid phase on
Tachisorb:
The reagents are distributed in a single step on Dynatech 96-well microtiter plates in a final volume of 220 μl.

After an incubation of 2 hours with horizontal rotary shaking at room temperature, the harvest is carried out on a glass fiber filter on a Skatron device.

The distribution successively concerns the Epo of the range or of the sample in a volume of 100 μl, the immune anti-Epo serum 10 μl, 125 I-Epo HR 10 μl and the Tachisorb R 100 μl. After transfer of the glass fiber samples to the polystyrene tube, the radioactivity is measured.

The measurement range of the method is between 1 and 100 mU.

The supernatants of the transformed cells have activities of up to 800 IUml while the cell supernatants with antisense vector are devoid of activity. There is always an excellent correlation between in vitro biological assays and radioimmunoassay.

DETECTION AND MEASUREMENT OF IN VIVO BIOLOGICAL ACTIVITY
EPO HR
The in vivo activity of the Epo produced in the culture supernatants of the transformed lymphoblastoid cells is tested after injection into mice made polyglobular by transfusion in order to inhibit the synthesis of endogenous Epo (Jacobson. Et al Proc. Soc.

Exp. Biol. Med, 94, 243, 1957). The results indicate that the Epo produced by lymphoblastoid cells has a powerful biological activity comparable to that of Epo HR produced by CHO cells (Kirin-Amgen patent nO 0148605 A2).

The results reflect a close correlation with in vitro biological assays according to the second international reference preparation of human urinary Epo.

PURIFICATION AND ISOLATION OF EPO HR 1st example
Cell culture supernatant in a protein-poor environment (CK4).

The culture medium is concentrated by ultrafiltration on a PM10 Amicon membrane or by tangential filtration on Minitan equipped with a PLGC membrane followed by washing in glucose solution 12.5 mM, galactose 12.5 mM, PEG 6000 0.1 mg / ml Beta-mercaptoethanol 10-4 M, up to a resistivity of the filtrate> 5000 Ohms.

The lyophilized concentrate is chromatographed on Ultrogel AcA44 in 10 mM Ca acetate buffer, 100 mM NaCl, 5.6 10-4 M phenol, 12.5 mM glucose, 12.5 mM galactose, PEG 6000 0.1 mg / ml pH 6.8.

The active fractions are concentrated by ultrafiltration and
Freeze-dried. The product is then treated by HPLC on a column
TSK 545 DEAE in 0.048 mM acetate buffer, 3 mM CaC12, 12.5 mM glucose, 12.5 mM galactose, PEG-6000 0.1 mg / ml, 10-4 M Betamercaptoethanol then elution by a discontinuous molarity gradient of NaCl. The activity yield is close to 75-%.

After dialysis and concentration, the product is chromatographed on
CM affigel blue in 10 mM phosphate buffer, 150 mM NaCl pH 7.2. The Epo is eluted with a molarity of 1.15 M NaCl. The specific activity of Epo HR is greater than 100,000 U / mg.

2nd example
The concentrate of culture media obtained on ultrafiltration membranes is treated on DEAE Sephacel at 40C in 48 mM acetate buffer, 3 mM CaCl2, 5.4 10-4 M phenol, pH 4.5. The selected activity is eluted in 0.6 M NaCl.

After concentration and dialysis, the eluate is subjected to filtration on Ultrogel AcA44, then the active fraction detected by RIA is chromatographed by HPLC-DEAE in the presence of reducing agents and stabilizing hexoses. The Epo HR eluted in pH 5.5 buffer, with low NaCl molarity, is concentrated on an Amicon PM10 membrane and then loaded onto a column of WGA Sepharose 6MB in PBS buffer at 40C. Its elution is obtained by N-acetylglucosamine 0.5 M.

In gel electrophoresis in polyacrylamide gradient from 4 to 30% and SDS, a component of 34 KDa is observed in the preparations resulting from WGA sepharose. This component is identified in immunoblotting by anti-Epo polyclonal immunoglobulins
HR and anti Epo HR monoclonal antibodies. A minority component migrating over a 42 kDa area is observed in certain preparations. This component is not recognized in immunoblots by anti-Epo HR polyclonal immune sera. This result is confirmed by radioimmunological detection and by the analysis in culture of mouse fetal erythroblasts of mouse eluates of preparative electrophoresis in SDS polyacrylamide gel.

Depending on the case, an additional purification step is carried out either on CM Affigel blue, or on HA-Ultrogel. Or in HPLC filtration in 0.1% SDS medium. The apparent molecular weight of Epo HR in SDS polyacrylamide gel is 34 kDa.

The specific activity obtained is greater than 120,000 U / mg in culture of erythroblasts and in RIA with yields of between 20 and 30%.

Table 1 Production of Epo HR by CCL 156 lymphoid cells (clone 156.05AF8) transformed with the integrative vector pTS39.

Selective environment Number Secretion Index
from de d'Epo
passages Multiplication U / ml / 72 h
Yes 22 3.7 38
30 nd 38
38 2 40
50 4.6 160
52 3.8 107
No 13 3.1 42
21 nd 43
29 1.8 52
41 5.2 146
43 6.0 87
48 9.8 86
Table 2: Production of Epo HR by CCL 156 lymphoid cells (clone C2) transformed with replicative vector pTS53 (with selection pressure).

Number of Epo Secretion Index of passages Multiplication U / ml / 72h 5 3.5 70 8 5 90 12 4 115 22 3 99

Claims (28)

 1) Lymphoblastoid cell line producing a specific protein, characterized in that it has been transformed by an expression vector for said protein comprising  - an effective promoter in human cells of hematopoietic origin,  a leader sequence ligated 5 ′ to the sequence coding for said protein,  - a polyadenylation signal 3 'to the coding sequence.  2) Line according to claim 1, characterized in that the promoter is a promoter originating from a virus capable of replicating in human cells of hematopoietic origin.  3) Line according to one of claims 1 and 2, characterized in that an activating transcription sequence effective in human lymphocytes is added upstream of the promoter.  4) Line according to one of claims 1 to 3, characterized in that the promoter, the activator sequence and the leader sequence come from Adenovirus.  5) Line according to one of claims 1 to 4, characterized in that it is a vector comprising, in addition, a marker gene.  6) Line according to one of claims 1 to 5, characterized in that the marker gene is a dominant selection gene of bacterial origin.  7) Line according to one of claims 1 to 6, characterized in that the vector is self-replicating and has an origin of effective replication in lymphoblastoid cells.  8) Line according to claim 7, characterized in that the origin of replication comes from the Epstein-Barr virus: oriP.  9) Line according to claim 8, characterized in that the line also expresses the protein EBNA-1 which acts in trans on oriP.  10) Line according to claim 9, characterized in that the gene coding for EBNA-1 is carried by the expression vector.  11) Line according to one of claims 1 to 6, characterized in that the vector is integrative and does not include an origin of replication, but a murine DNA fragment of mitochondrial origin.  12) Line according to claim 11, characterized in that the integrative vector comprises the murine cDNA coding for dihydrofolate reductase (DHFR), placed in a bicistronic position downstream of the coding sequence of the protein.  13) Line according to one of claims 1 to 12, characterized in that the vector comprises the following sequence  - E - MLP - L123 - P - pA - E being the activating sequence of the transcription of a Adenovirus, - MLP is the promoter of an Adenovirus, - L is the leading sequence of the same Adenovirus, - P is the sequence coding for the protein, - pA is a polyadenylation signal  14) Line according to claim 13, characterized in that: - E is the activating sequence of the ElA gene of Adenovirus 5, - MLP is the major late promoter of Adenovirus 2, - L123 is the tripartite sequence of Adenovirus 2, - pA is the polyadenylation signal derived from the SV40 virus.  15) Line according to one of claims 1 to 14, characterized in that P is the sequence coding for erythropoietin or a protein having the activity of erythropoietin,  16) Line according to one of claims 1 to 15, characterized in that the selection gene is the gene coding for xanthineguanine phosphoribosyl -transferase (XGPRT).  17) Line according to one of claims 1 to 16, characterized in that the vector also comprises the region of AdS coding for the RNA VAI and VAII.  18) Line according to one of claims 1 to 17, characterized in that the lymphoblastoid cell line which is transformed by the expression vector is a line established from leukocytes of the peripheral blood of a healthy individual.  19) Line according to one of claims 1 to 18, characterized in that it is filed with the ATCC under the reference CCL156 RPMI 1788.  20) Line according to one of claims 1 to 7, characterized in that it is a cell line immortalized in vitro by EBV from B lymphocytes from a healthy donor.  21) Line according to one of claims 1 to 17 and 20, characterized in that it comes from the INTS under the reference LE6B4.  22) Line according to one of claims 1 to 17, characterized in that the human B lymphoid line transformed in vitro by EBV (H2D5D2B6) is fused with a mouse myeloma (B6Ag) and that it has lost the gene EBNA1.  23) Line according to one of claims 1 to 17 and 22, characterized in that it comes from the INTS under the reference 1C5E2.  24) Line according to one of claims 1 to 17, characterized in that it is a line of heteromyeloma SHMD33 (EBNA-) obtained by fusion of a line of myeloma with a mouse myeloma (P3X63Ag8, 653 )  25) Line according to one of claims 1 to 17 and 24 characterized in that it is filed with the ATCC under the reference CRL 1668.  26) Line according to one of claims 1 to 23, characterized in that the sequence coding for the Epo used is obtained from a human genomic library.  27) Process for the production of proteins, characterized in that a cell line according to one of claims 1 to 24 is cultivated in an appropriate culture medium and in that the desired protein is recovered.  28) Method according to claim 25, characterized in that the erythropoietin is prepared.