METHOD FOR THE PRODUCTION OF PROTEINS BY MEANS OF INDUCIBLE EXPRESSION SYSTEMS IN GENETICALLY MODIFIED EUKARYOTIC HOST-CELLS MULTIPLICATED IN-VIVO

Abstract: In the production of proteins and biological interest by means of a stress inducible gene expression unit/eukaryotic host cell system, the transformed cell lines are multiplicated by tumour growing in immunodeficient warm-blooded animals, after which the multiplicated cells are cultured in vitro and subjected to stress, whereby expression occurs in high yield. In vivo multiplication rates of 10<5>-10<6> the innoculated quantity/2 weeks are reported without any loss of the latent inducible expression capacity.

Description: METHOD FOR THE PRODUCTION OF PROTEINS BY MEANS OF INDUCIBLE EXPRESSION SYSTEMS IN GENETICALLY MODIFIED EUKARYOTIC HOST-CELLS MULTIPLICATED IN-VIVO INTRODUCTION The present invention concerns the biochemical production of proteins of interest by the techniques of genetic engineer¬ ing and, more especially, by inducibly expressing genes of interest in appropriate genetically modified eukaryotic host cells. The use of and the advantages of inducively expressing genes under the control, for instance, of heat shock expres¬ sion elements for the production of gene-products oi interest in a variety of cell types, have previously been described (see International Applications PCT/EP86/00451 and EP 84 810 066.5; DKEANO et al. Gene (1987) 49, 1-8). Being applied to a eukaryotic expression • sy * stem, this technique has the intrinsic advantages of having an efficient, general and highly induciblel character. These factors are of considerable interest for the commercial production of pro¬ teins of biological and pharmaceutical interest, particularly where these proteins are complex, modified, unstable or are potentially toxic to the producer cell. However, their econo¬ mical performance will rely to a large degree on the use of host/vector models able Lo ensure a high level of production of fully competent proteins, associated with low cost cell growth and protein purification. For instance, the use of micro organisms as host-cell systems has demonstrated that the ideal quality which should be expected from such a system cannot be found in a single micro organism species, and more appropriate expression systems and vectors are needed. More¬ over, most proteins of therapeutic interest are secreted pro¬ teins, which frequently require post-translational modifica¬ tions for activity and immunolog cal specificity, such modifi¬ cations possibly lacking when translation occurs in bacteria. Hence, the development of genetically engineered eukaryo¬ tic cell lines harbouring recombinant genes constitutes the most promising prospect, despite the relatively primitive state of. eukaryotic cell culture technology as compared to that of bacterial systems. However " , although recent developments in tissue culture, employing chemically defined media, constitute an important breakthrough in the adaptation of tissue culture methods to industrial production, there is presently a need of improved culturing means for multiplication and mass culture of cells transformed with genetically engineered gene expression sys¬ tems. Some of the major problems associated with mass cultures are: - to obtain a large number of cells (especially sterili¬ ty, materials, qualified personnel, space) - to reduce the cost of production, essentially due to manpower but also of culture media containing fetal calf serum (FCS can represent up to 80%.of the 'total production cost). - to purify the expected proteins of industrial interest and particularly in the case of secre able .proteins, to iso¬ late them from the whole suspension media used for cell culture. SUMMARY OF THE INVENTION Thus, in an effort to develop the inducible expression of genes of interest harboured by suitable host cells on an industrial scale, the present inventors have uncovered the method disclosed in annexed claim 1. Briefly stated, appropriate eukaryotic host cell lines capable of generating tumours when innoculated to immunodefi- clent animals are transfected with nucleic acid constructions containing at least one gene of Interest under the control of a stress inducible promoter. With "normal" cells, a transform¬ ing gene, e.g. an oncogene, is also added by co-transfection to impart to the cells unrestricted multiplication capacity. Then the transformed cells which are capable of expressing said gene of interest upon induction are innoculated to im- munodeficient animals whereby they are transiently multipli¬ cated in' the form of tumours developed by the animals. Sur¬ prisingly, and despite the presence in the cells of transcrip¬ tion units not expressed under normal growth conditions, the rates of multiplication were very high (about 10 -10 times the innoculated quantity in 1-2 weeks) without cell degeneracy or loss of the capacity of expressing the gene of interest under stress. This method is also particularly useful for obtaining rapidly and at relatively little expense identifica¬ tion and testing quantities of engineered protein variants of natural proteins. It should be noted at this stage that the multiplication of certain cell lines by tumoral transplantation in warm¬ blooded animals is not novel per-se. For instance, GB-A-. 2,083,826 (HAYASHIBARA) discloses the multiplication of in¬ sulin producing, human oncogenic cells by transplantation into warm-blooded animals such as nude mice or immunosuppressed mammals (rats, hamsters, etc.) and birds. However, the types of cell lines involved in this prior art only include easily preservable stable cell lines such as insuloma, lung tissue carcinoma, lymphoblast or hybridoma ceLls. No corresponding multiplication of cells incorporating cjenes dominated by heat- shock elements has been previously reported, to the knowledge of the present inventors. Furthermore, the rapid in-vivo multiplications of cells of the type involved here, i.e. containing a reco binanl stress-inducible expression system, in very high yield, without the production of undesirable inhibitory products and full preservation of heat-shock in- ducibility, constitutes a very important technical step vis-a¬ vis the previous techniques. In short, the experiments which support the claimed me¬ thod include the following embodiments: Genes coding for products of interest (hormones, enzymes and other proteins of interest for diagnoses and pharmaceuti¬ cal applications) were placed into suitable vectors under the transcriptional and Lranslational control of heat-shock promo¬ ter elements of eukariotic origin (human, Drosophila, etc.). Optionally, other precursor or post-translational sequences and variants thereof were also introduced into the nucleic acids of concern, all this being carried out by usual reco bi- nant DNA techniques. Most of this has been disclosed in a previous application (PCT/EP86/00451) incorporated herein by reference. Then, suitable host cells were transfected with recombi- nant DNA's including a hybrid gene of interest under icontrol of a hsp 70 sequence and an oncogenic transforming gene. When tumoral host-cells were used, no additional oncogene to pro¬ mote multiplication was necessary. Contrastingly, when host cells such as NIH-3T3 were used, co-transfection was brought about using an oncogenic transformant sequence of cellular or viral origin. Then, after further in vitro expansion and testing for expression of the gene under heat shock, the • transformed cell lines were injected into immunodeficient animals, far instance nude mice or immunodepressed rats and Q in the tumors were allowed to grow to about 10 -10 cells. The tumours were removed, minced and the cells were dissociated, for instance trypsinized, and placed into culture media whereby they coild subsequently be stressed. The gene of interest driven by a heat-shock control element, is induced and the cells expressed the protein of Interest. The product was then collected by usual means, for example affinity chro- matαgraphy. Details on the variotis embodiments of this invention are found hereafter and in the sub-claims. These details will be better understood with reference to the annexed figures , a list of which is summarized below. ! BRIEF DESCRIPTION OF THE ANNEXED FIGURES (Figs 1-8 and 11-13 are photographs)- Fig 1 shows a nude mouse carrying a sub-cutaneous (s.c.) tumour produced by cell line (C1.6) injection. Fig 2 shows a Sencar mouse (Swiss-derived) with an intra¬ muscular (i.m.) tumour induced by transplantation of C1.6 tumour cells from nude mice. Fig 3 shows a cyclosporine-immunosuppressed rat with a s.c. tumour induced by transplantation of C1.6 cells from a nude mouse". Fig 4 shows the histological appearance of a s.c. tumour induced by C1.6 cells injection in nude mice. Fig 5 shows a high magni ication of the s.c. tumour shown in fig.4. Note the high incidence of mitotic index. Fig 6 shows a s.c. tumour induced by Cl.6 cells injec¬ tions in nude mice. Note the vascular spaces. Fig 7 shows a pulmonary tumour (fibrosarcoma) induced by intratracheal instillation of HBN2 cells in a nude mouse. Fig 8 shows a high magnification of the intrapulmonary tumour shown in figure 7. Fig 9 represents schematically the construction of plas- mid pl7HBN. Fig 9a represents plasmid pl7hGH dhfr (Dreano • et al. Gene, in press) containing a hsp70-hGH hybrid gene and the mouse dihydrofolate reductase gene under the control of the SV40 early promotor sequence, and terminated by the SV40 terminator sequence. * Fig. 9b represents plasmid pV69 (Meneguzzi et al. 1984, ' EMBO J.j 3_; 365-371) containing the eomycin resistanc^ gene under the control of the herpes simplex thymidine kinase promotor and terminator, and the 5.4kb bovine papilloma virus subgenomic fragment. Fig 9c represents plasmid pl7hGH NEO (PCT/EP86/00451) containing ' the hsp70-hGH hybrid gene and the neomycin resi¬ stance gene under the control of the SV40 early promotor sequence and terminated by the SV40 terminator sequence. Fig 9d represents plasmid phGH BPV which results form the ligation of a Hindlll-BamHI fragment derived from pl7hGH dhfr and a 7.5 Kb fragment resulting from the digestion of pV69 with Hindlll and partial digestion with BamHI. Fig 9e represents plasmid pl7K NEO which results from the ligation of a 1.8 Kb Hindlll-BamHI fragment derived from pV69 (the Hindlll extremity has been filled-in with DNA poly erase I (large fragment); and a 3.4 Kb fragment resulting from the digestion of pl7hGH NEO with PvuII and BamHI. Fig 9f represents plasmid pl7HBN which results from the ligation of a Hindi.TI-Ba HI fragment derived from pl7K NEO, and a 6.8 Kb fragment resulting from digestion of phGH BPV with Hindlll and partial digestion with. BamHI. Fig 10 is a key to the symbols used in Fig 9 to define the various DNA sequences involved. The approximate length (in base-pairs) of such sequences in the plasmids schematized in Fig 9 are given in Table I, below. Fig 11 shows C1.6 tumour cells after dissociation and inoculation in a Petri dish incubated in serum supplemented medium. Fig 12 shows C1.6 tumour cells after dissociation and inoculation in a 1 liter biofermentor with incubation in serum free medium. Fig 13 shows C1.6 tumour cells after dissociation and inoculation in a 2 liter biofermentor with incubation in serum-free medium in the presence of 3g/l of cytodex 1 micro- carriers. TABLE 1 Identification of the sequences in Figs 9a-9f and restriction sites . Plasmid Sequences :kb p!7hGH dhfr D: 0.6; B: <0. ; 1:0.7; C: 0.7(termina or ) :0.4(promotor ' , K:0.7; A: 2.3 pV69 L: 0.5(promotor) : 0 :3( erminato ) : 1.0; M:5.4:A:2.2 pl7hGH NEO D: 0.6; B: <0.1 ; 1:0.7; C: 0.4 (promoto ) : 0.7 ( terminator) ; J : 1.5; A: 2.8 phGH BPV C:0.7 ( terminator) ; 1:0.7; M:5.4; A:2.2 pl7K NEO D: 0.6; B: <0.1; : 0. ( promotor) : 0.3 ( terminator) ; J:1.0; A:2.8 pl7HBN D:0.6; B:<0.1; 1:0.7 C : .7( terminator ) ; : 0.5(promotor ) : 0.3 ( terminator) ; :1.0; M-.5.4; A: 2.8 BI : RamHI BII : Bgl II HIII : Hind III PII : PvuII DETAILED DESCRIPTION OF THE INVENTION For harbouring the gene recombinant DNA constructions, suitable -tell lines had to be selected. Cell lines (NIH3T3) producing high-levels of human growth hormone (hGH) under heat shock control, have been described by Dreano et al., (Gene, (1987) 49_, 1-8). In brief, one tij-ansformed 3T3 mouse cell line clone 6 (Cl. 6) can secrete 2 toj5 μg hGH per 10 cells over a 20 hour period following a 2 hours heat shock at 42°C. This concentration is 1200 time the hGH concentration measured in the medium of non heat-treated C1.6 cells. In addition, these cells can be utilized for repeated induction cycles and they were therefore highly suitable for use in the present inven¬ tion. Wc report below the results of transplantation procedures of three genetically engineered mouse cells lines: clone 6 (C1.6), HBN2 and Clone 18 (C1.18). These three cell lines expressed the human hsp70-hGH hybrid gene, and contain the following additional genes; in C1.6, the human Harvey c-ras oncogene (Tabiή et al. 1982, Nature, 300; 143-149); in HBN2 the bovine papilloma virus (BPV) subgenomlc fragment, and a neomycin resistance gene (used for G418 resistance selection), and in C1.18 the neomycin resistance gene (C1.18 Is used as a control). Obviously, other genes of interest can also be placed under the control of the hsp70 promotor sequences in similar DNA constructions and expressed similarly. Such genes of interest include for instance viral proteins, hormones, enzymes, blood proteins and others. Regarding heat-shock control sequences, it is obvious that other heat-shock control elements can be used in place of the sequences mentioned in the specific experiments illustrat¬ ing the present invention. Genetically engineered variants obtained by nucleotide deletion, mutation and insertion, as disclosed in our application PCT/EP86/00451 , are also suit¬ able. Transplantation of the two first cell lines into nude mice, induced tumour in injected animals. After prαteolytic dissociation, or treatment with collagen the tumour cells were able to produce, after heat induction, the same quantity of hGH as the. parental cells. Using this methodology we were able to obtain approxima ive!y 10 tumour cells per tumour. Final¬ ly we showed that these cells retrieved from tumours in ani¬ mals, can be easily cultivated on cytodex microcarriers in 1 liter or 2 liter bioreactors and, in addition, in chemically defined media serum free medium (SFM), or serum and protein free medium (SPFM) (see CHESSEBEUF and PADIEU. , PCT/84/901363.6; FR-Λ-8 /04843) . Repeated heat regulated pro¬ duction of hGH was once again obtained. 1. Clone 6 The BALB NIH-3T3 cells (obt ined from the ATCC organiza¬ tion) were c'o-transfectt.d with plasmid pl7hGHdhfr, see fig 9a, carrying the human growth ho_mone (hGH) driven by the human heat shock protein 70 (hsp70) promotor (plasmid p!7hGH dhfr is disclosed in PCT/EP86/004 ) , and by a plasmid carry¬ ing the human c-ras oneogene. Two weeks later, foci were isolated, cultured and analyzed for hGH production. Clone 6 was found to secrete 3 μg of hGH per 10 cells'in a 15 hour period following a single heat treatment of 2 hours at 42°C. 1.1 Transplanta ion into nude mice Spontaneously transformed 3TJ cells injected into nude mice are able to induce tumour formation (Rubin and Arnstein, 1982, Cancer Res., 300; 143-149, Rubin 1984, J. Natl. Cancer Inst. , 2 . 375-381) . 1.1.1 Subcutaneous ansplanta ion 10 b cells in 0.1 ml of culture medium without serum were injected subcutaneously in the dorsal area of nude mice. The first tumour was clearly visible 3 weeks after inoculation (fig. 1) when the tumour reached a diameter of 20 mm (weighing about 3g) , it was dissected. A part of this 1st generation tumour (designated Gl/n) was minced in culture medium and one portion was resuspended to inoculate a monolayer culture for subsequent, heat shock, and the other portion was reinjected subcutaneously into other nude mice (to produce G2/n) , finally the remaining part of the tumour was fixed with formaldehyde for histological examination. These tumour cells had a great capacity to anchor on plastic dishes. Indeed, 30 min. after seeding In complete- medium most of the cells were attached, and presented a fibrobLastic aspect. After one night, cells covered the dish and only few dead cells were observed (fig. 11) . After heat treatment of tumour cells (from Gl/n) in cell culture, they were shown to secrete 2,5 to 5 μg of hGH per 10 cells; this is within the same range of production as that of the original C1.6 cells. In addition, tumour slices (0.5 to Ig) were found, after heat treatment, to secrete 20 to 100 μg of hGH in suspension medium. Identical experiments were per¬ formed in cells from tumours passaged up to 8 times in nude mice (G8/n) . Table II (see Experimental part) shows that, with C1.6 the heat regulated production is maintained at a constant rate during 24 generations in nude mice, and the 8 generations in sencar mice; with HBN2, another clone to be described below, a similar result was also observed with cells from the first 2 generations in nude mice. After successive subcuta¬ neous passages into nude mice, the time to tumour onset was reduced to only 2 weeks for the production of a 30 mm tumour, this when the tumour cells came from another nude mouse, compared to the 4 weeks needed to obtain tumours derived • from cell cultures. The histological appearance (fig. 4 to 6) of the subcutaneous tumours remained the same (a fibrosarcoma with a high mitotic index) during passage in nude mice. In the tumours up to a diameter of 30 mm produced in nude mice with C1.6, no major tumour necrosis was observed, Moreover, the periphery of the tumour was well irrigated by blood capil- laires (fig. 6) . 1.1.2 Intraperitoneal injection C1.6 cells, derived from tissue culture (10 cells), were also injected intraperitoneally into a nude mouse. Three weeks after injection, when a swelling of the abdomen was observed, the mouse .was sacrificed. At. necrosy, tumours with a marked ascite were noted, and tumour metastases were found in the peritoneal lymph nodes and around the kidneys, as well as in the liver. Microscopically this tumour was similar to the tumours obtained by subcutaneous injection of C1.6 cells. 1.2 Transplantation into sencar mice The * Sencar strain of mice (Swiss derived strain, as ex¬ plained in the experimental section), which is not genetically immunodeficient, was used as a host for genetically engineered transformed cells, >-„s a test of whether these cells can grow in normal mice, after previous passage in nude mice. An at¬ tempt to produce tumours in Sencar mice directly from cell cultures was unsuccessful. However, Cl.b cells obtained after a first generation Lumpur in nude mice. (Gl/n), injected subcutaneously (s.c.) or ' intraperitoneally (i.p.) into Sencar mice, produced respectively s.c. and i.p. tumours in Sencar mice (denominated Gl/s/sc and Gl/.s/ip fig. 2). This experiment was repeated with a 2nd generation tumour in nude mice (G2/n) which, injected s.c. into a Sencar mouse, also produced an s.c. tumour in Sencar mice. A direct passage of C1.6 tumours from Sencar to Sencar was also successful since an i.p. tumour in Sencar mouse (Gl/s/ip) was transplanted into several other Sencar mice by s.c, i.p. and intramuscular (i.m.) routes, to produce the G2/s tumours. I.m. and s.c. tumours have been observed and Sencar to Sencar transplantations can be continu¬ ed. The histological structure of the tumours in Sencar mice is similar to that of those induced in the nude mice. 1.3 Transplantation into rats In order to verify if interspecies xenografts of geneti¬ cally engineered, transformed cells could be produced, tumours grown in nude mice (G4/n) were subcutaneously transplanted (pieces of about 75 mm ) into three albino rats. One rat received cyclosporine for immunosuppression (see Experimental part) , the two other rats did not received the drug. Two weeks after transplantation, the tumour had regressed in the two normal rats, ' while a large tumour (12 cm diameter after 3 weeks) was developed In the cyclosporine-immunosuppressed rat (fig. 3). ■ At necropsy this tumour was found to consist of a large necrotic area surrounded by a layer of about 2 cm of living tujmour cells (totalling about 40 g) . Its histological structure ; was once again that of a fibrosarcoma, similar to the tumours induced in mice previously. The heat-induced pro¬ duction of hGH from the cell of this tumour was similar to that from nude mice tumors. Immunosuppressed animals are inte¬ resting hosts for growing genetically engineered cells and, in addition to rat, other warm-blooded animals are also usable. 2 Clone HBN 2 HBN2 is also a clone derived from the NIH-3T3 cell line, with incorporation of a plasmid pl7HBN, whose construction is described in fig. 9a-9f, containing 3 transcription units: (1) the hGH gene driven by the human hsp70 promotor, (2) the neomycin resistance gene under control of the herpes simplex virus thymidine kinase promotor (used for cell selection via G418 resistance) and (3) a subgenomic fragment of bovine papiloma virus carryin sequences responsible for the mainte¬ nance of multicopy plasmids in a stable form in mouse fibro- blasts (Meneguzzi et al . 1984, EMBO J. , 3, 365-371). 2.1 Transplantat on into nude mice Two types of transplantation of HBN2 cells from culture (using 10 cells in 0.1 ml of culture medium without serum) were performed. In one case, the cells were injected subcuta¬ neously in the footpad of nude mice. One month later, a growth was noted, this first generation tumour (HBN2/Gl/ri) was taken, minced with scissors, and tumour cells were injected into the back of nude mice to produce the second ' generation tumours (HBN2/G2/n) . In the second case, 10 HBN2 cells in 0.5 ml of medium without serum were introduced intra-tracheally into the lungs of a nude, mouse. When the animal was necrυpsied two months later, a large pulmonary tumour (10 mm diameter) invading a pulmonary lobe was found. The tumour (HBN2/Gl/s/i ) was asses¬ sed microscopically to be a sarcoma (fig. 7 and 8). Again, cells isolated from tumour derived from clone HBN2 were stable and provided the desired protein in high • yield under heat induction in culture medium, (see Table III) 3 Clone 18 (control) NIH-3T3 cells were co-transfected with plasmid p!7hGH dhfr together with a plasmid carrying the neomycin resistance gene under control of the early SV40 promotor (Southern and Berg 1982, J. Mol. Appl . Gen . I ; 327-341). Two weeks later G418-resistant (200 μg per ml of suspension medium) clones were isolated and expanded. Clone 18 was found to secrete approximately 1,5 μg of hGH per 10 cells after heat treat¬ ment. Two nude .mice were injected s.c in the foot paw as disclosed above. Three months later no tumour had grown at the injection site which indicated that the transplantation was unsuccessful. 4. Tumour cells grown on petri dishes in serum free media Tumour cells (C1.6) were dissu iat.ed by successive tryp- sin treatments; one portion from each trypsination step was inoculated on Petri dishes in three different types of media: - serum supplemented medium (SSM) constituted of Williams 'E solution plus 5% of fetal calf serum and 5% new born calf serum (see fig.11) - serum free medium (SFM) constituted of the Williams 'E medium plus 4 g/1 of bovine serum albumin, and 7.6 μmole /l of a mixture of 6 free fatty acids (FFA) . These six FFA were found indispensable to insure the Krebs cycle functions providing the necessary acetyl-CoA needed in the abscence of serum lipids, -serum and protein free medium (SPFM) constituted of the Williams'E medium and containing 50 mg/1 of dextran and 7.6 μmole/1 of 6 FFA. After cultivation, the cells were subjected to stress as usual. Results are summarized in Table II (see Experimental part). The advantages associated with the use of SFM and SPFM are : (1) Easy selection of a basal synthetic medium for optimal growth or cell function (2) Reduction of the cost by elemin ing the use of fetal calf serum (3) Use of a defined culture medium which eliminate the many known and unknown components of serum (4) Supplementation with known amounts of effectors and inhi¬ bitors (5) Simplification of the extraction process especially for secreted proteins such as hGH (6) Elimination of the extrinsic effects of the serum (7) Easy formulation a d use of a selective media (8) Elimination of many contaminating substances bound to albumin (9) Continuation of fundamental research on phenotypic expres¬ sion of diploid cell ine (10) Innovation in the applied research of new synthetic basal medium which meets the WHO and FDA requirements when normal or reco binant eukaryotic cells are cultured for the production of therapeutic or biologically active molecules for human administration. These advantages are taught by CHESEBEUF and PADIEU W084/03710). After reinoculation on Petri dishes, C1.6 had exactly the same behaviour in SFM and in SPFM as in SSM, i.e. very rapid anchorage, recovering a fibroblastic pattern and complete colonization of the surface of the dish. Tumour cells were similar to the parental C1.6 cells, without any visible contaminating cells. Therefore using SFM and SPFM culture media for cultivating the cells expanded according to the present method were technically and economically advantageous . 5 - C1 . 6 Cel l s in a b iof ermentor 5 . 1 Bi of ermen tator wi th in i crocar ri ers q After each trypsinization step portion of about 3.10 cells were introduced into 3 biofermentators containing re¬ spectively 250ml of one of each of the 3 different media, together with 3 to 5 g/1 of cytodex inicrocarriers. 12 hours after inoculation, the inicrocarriers were fully covered with tumour cells. Trypan blue coloration indicated the presence of less than 3% of dead cells in any medium (fig 13) . Cells can be maintained in biofermentors for more than 23 days and are subjected, during this period, to several 4 hrs treatment at 42°C. The production of hGH was noted using an R1A (Radioim- mune assay) procedure. Table TIT (see the experimental part) shows that cells secreted hGH after each heat treatments; the production in SSM was more important i han in SFM (see fig 12) and even more so than in PFM. It is interesting to note that in the SSM biofermentor, about 3 mg of hGH wus secreted into the medium. It should be noted that these cells were derived from less than 1 complete tumour, and that a heat treatment cuuld be applied daily. 5.2 Biofermentator without inicrocarriers Dissociated turnout cells can also be maintained alive in biofermentator without mi r_ocarriers although the conditions there are less favorable. For instance, it was observed that the cells were still alive 5 days after innoculation. EXPERIMENTAL DETAILS Method to construct PI 7HBN (fig 9) Plasmid pBPV hGH : pi 7hGH dhfr (Dreano et al. , ibid) was digested with Hindlll , and then partially with Bglll. A 1.4 kb fragment containing the human hsp70 promotor, the hGH gene and the SV40 termination signal was extracted from low melting agarose (Sigma, type VII). Plasmid V69 (Meneguzzi et al., ibid) was digested wi h Hindlll and partially with BamHI, a 7.6 kb fragment containing plasmid vector sequences and the 5.4kb bovine papllloma v rus (BPV) subgeno ic fragment was isolated and ligated with the above fragment. Plasmid pl7K NEO: pV69 was digested with Hindlll, treated with the DNA polymerase large fragment and then digested with' BamHI. A 1.8 kb resulting fragment carrying the neomycin resistance gene controlled by the herpes simplex' thymidine kinase transcription signals (promotor and terminator), was purified, and ligated to a PvuII BamHI purified fragment from pl7hGH NEO containing the human hsp70 promotor and pBR322 derived sequences . pl7HBN : pl7K NEO was linearised by digestion with Hindlll and BamHI. This fragment including the hsp70 promotor and the neomycin resistance transcription unit, was ligated with, a 6.8 kb fragment from pBPV hGH (obtained by total diges¬ tion with Hindlll and partial digestion with BamHI) containing the hGH gene, SV40 termination signals, and the BPV subgeono- rrtic fragment. The resulting plasmid p!7HBN contains three complete "transcription units" : - the human hsp70 -hGH hybrid gene with SV40 terminator - the neomycin resistance gene with TK transcription signals - "the BPV subgenomic fragment" 2. Procedure used to obtain cell lines 2.1 Selection via focus formation Cells were co-transfected with pl7hGH dhfr (Dreano et al. 1986) and pEJ (Tabin et al . 1982, Nature 30_0_, 143-149) using CaCl- procedure (Graham and van der Eb, 1973, Virol. J> , 456- 467) . After two weeks, clones that were capable of focus formation were isolated, and expanded. 10° cells of each clone were seeded into 20 cm i dishes, and after 24 hrs in culture were heat treated for 2 hours at 42°C, and subsequently in¬ cubated at 37° C for 15 hours. Clone 6 produced 3 μg of hGH 6 per 10 cells under these conditions of heat shock. 2.2 G418 Selection Cells were co-transfected with pl7hGH dhfr and pSV2 NEO (Southern and Berg. , 1982 J. Mol . Appl.Genet. 1, 327-341), or were transfected with ρl.7HBN using the CaCl„ procedure. After two weeks of culture in complete suspension medium, containing 200 mg of G418 (GIBCO), visible resultant clones were observ¬ ed after 2 weeks; they were isolated and analyzed further as above. C1.18 and HBN2 were found to inducibly secrete respec¬ tively 1.4 and 0.7 μg of hGH per 10 6 cells. 3 Experiments " using mice and rats 3.1 Genetir l 1 y engineered el 1 transplantations into nude mice Nude (nu/nu) mice aged 4 to 6 weeks, from Iffa-Credo (L'Arbresle, France) were housed in Macrolon cages with a cover filter and placed in an air-filtered cabinet. They received an autoclaved diet and normal tap water ad lib. Portions of 10 genetically modified cells were trypsi- nated, rinced twice in Dulbecco's modified Eagle's medium without serum and resuspended in about 0.1 ml of the same medium for intramuscular (i.m. ), intra peritoneal (i.p.) and subcutaneous (s.c) injections, and in 0.5 ml for intratracheal instillation. mice Clone 6 cells, s.c. transplanted into nude mice", deve¬ loped s.c tumours. When a tumour reached a size of 20 mm in diameter (about 4 weeks after inoculation) , the mouse was sacrificed and the tumour (about 3g) was dissected, minced in culture medium without serum, and a part of the minced tumour (1/10) was rein ected into nude or sencar mice. Battelle-Geneva breeds the Senca-έ- strain of mice, and albino mouse derived from the Swiss mouse by selection for its sensitivity to skin carcinogens. (Sencar results from "Sensi¬ tive to Carcinogens"). Contrary to the nude mice, this strain of mouse is not genetically immunodeprived. 3.3 Transplantation into rats 3 Pieces of about 75mm of a Cl.b tumour, which was deve¬ loped in nude mice (4th generation), were .transplanted subcutaneously into Sprague Dawley albino rats (IFFA-CREDO, France). The transplantation was performed in 3 rats; 2 normal rats, and one rat chemically immunosuppressed, by ten daily s.c injection of 60 mg/kg of cyclosporine (Sandoz, Bennet et al. , % Cancer Res. , 4_5, 4963-4969). 3.4 Histology When a tumour was taken for retransplantation, a portion was placed into a fixative solution (10% buffered formalin) then processed a d sections were stained by haemalin-phloxin- saffron for 6 microscopic observations as described in M. GABE (1968), Techniques Histologiques Ed. Masson et Cie Paris. 4 Detailed method to transfer tumour cells from mice to biofermentors 4.1 Description of the three culture medium The composition of the three culture media used in this section are : - serum supplemented medium (SSM) constituted of Williams E medium (Gibco) plus 5% of fetal calf serum (FCS) and 5% new born calf serum (both from Boehringer). - serum free medium (SFM) consisting of the same medium, with the addition of 4 g/1 of bovine serum albumin (BSA) fraction V (Sigma) (equivalent to 10% FCS) and 7.6 μ mole/1 of a mixture of 6 long chain free fatty acids (FFA) in molar proportions close to those of rat plasma (except increased cis-linolenic acid) i.e. palmitic acid 31.0%, ci s-palmitoleic acid 2.8%, stearic acid 11.6%, cis-oleic acid 13.4%, cis-linoleic acid 35.6%, cis-linolenic acid 5.6% (Sigma). - serum and protein free medium (SPFM) constituted of the same medium containing 50 mg/1 of 2.10 b dalton dextran (Pharmacia) which was as efficient as 4 g/1 BSA to solubilize 7.6 μmole/1 of 6 FFA. Each medium contains 50μg/ml of gentamicin (gental- line, Unilabo) as antibiotic 4.2 Transfer of t mour cells from mj_ce to biofermentors The mouse was sac_ i iced, the skin was cut and the tumour easily extracted. After finely mincing with scissors, tumour fragment were transferred to a trypsinization bottle of 50 ml, provided with a magnetic stirrer. To this was added an equal volume of a solution diluted 3 times by trypsin rinsing medium 1:250 (equivalent to trypsin which hydrolyses 250 times its weight of casein) obtained from the Microbiological Associates Company, Bethesda, MD, USA. It was stirred for 10 minutes at 100 _pm, at 37°C, then the suspension of cells was decanted into "Ham F10 medium" cooled on ice (2 volumes of medium per volume of cells) and was cenLrifuged t 30 g. In parallel, the trypsinization procedure was renewed in the bottle. The se¬ quential digestion with the trypsin was repeated until the dissociation was complete, in general 10 to 13 times. After each centrifugation step, cells were suspended in about 4.3 ml 7 of the culture medium. 1/10 was transferred onto a 20c Petri dish (Falcon) , and 9/10 into a 1 liter biofermentor (Techne, Cambridge, U.K. ). After some trypsination steps trypsin so¬ lution was no longer diluted. In another procedure, comparable dissociation was obtained using a collagenase solution (Boeh¬ ringer, 0.55g/1) . When the dissociation was complete, suspension medium (SSM, SFP or SPFM, qsp 250ml) cytodex inicrocarriers (Pharmacia France, F78340, 3 to 5 g/1) were added. Media are treated with CO2 by bubbling a 5% C0 9 /air mixLure for about 10 min. Finally, cells are incubated at 37°C overnight. Media are changed and biofermentors are placed in a 42°C water bath for 3 or 4 hours followed by a 20 hour period of incubation at 37°C. Media are removed, and replaced with fresh medium before heat treatment or not as described in Table II. Table II : quantification of hGH produced by C1.6 cells after passage in nude or sencar mice. Tumours were retrieved from mice, minced finely with scissors, and treated with 2 trypsin. Dissociated cells were seeded in 20cm. Petri dish (Falcon) with 10 cells in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The next day cells were -heat treated for 1 to 2 hours at 42°C, or not treated (controls) . Af er 20 hours at 37°C, hGH secreted in sample medium was quantified by means of a hGH RIA kit from Cambridge Inc. r Mass. U.S.A. The number in the cell identifi¬ cation code indicate the tumor generation order (see section 1.1.1). Mice Cells Heat shock control (hGH μg/20 ml/10 D cells Nude Cl-6 2 to 5 0.01 Gl/n 2 to 5 G2/n L.3 • . ^ G7/n 6 0 .03 G8/n 2.5 0, .03 ' G9/n 2.2 0, .03 Gll/n 1.4 0. .02 G13/n 0.7 0, .01 G14/n 1.0 0. .01 G16/n 1.7 0 G18/n 1.9 0. .04 G20/n 2.9 0 G22/n 1.2 0. .0002 G24/n 0.7 0. .0005 Sencar G2/s 1.1 0. ,01 G2/s 10 0. 08 G5/s > \ .3 0. 04 G7/≤ 4.5 0. 01 G8/s 7.8 0. 02 Table III : hGH secretion in transformed NIH 3T3 cells maintained in a biof rmentor. Each biofermentor was seeded Q with 2.2 10 tumour cells in a volume of 250 ml of each of the three culture media described befote containing 3 to 5 g/1 of cytodex microcarriers. Cells were heat treated, some days after inoculation (see first columm in Table III), for 3 to 4 hours at 42°C and kept at 37°C 20 hours afterwards and, final¬ ly, put in fresh media. The CO-, concentration in the medium was maintained by bubbling a 5% C0„ in air mixture into the medium. Twelve hours after inoculation, the microcarriers were found to be coveted with tumour cells. A trypan blue stain indicated the presence of less than 3% of dead cells in any of the media utilized. In experiment 2, each biofermentor was seeded with 50.10 tumour cells in a volume of 100 ml of the media shown below containing 2 g/1 of citodex microcarriers. The cells were heat treated during-tø h at 43°C, kept at 37°C for 24 h more, and finally put into fresh media. Suspension media were tested for hGH presence by RIA (provided by Institut Pasteur Production), or ElA. The cells were maintained in Williams' E medium (experi¬ ment 1) or .in William's E/HAM F-10 (50:50, v/v) (experiment 2) supplemented with: - SSM; 5% of fetal calf serum and 5% of new born calf serum, - SFM; 4 g/1 of bovine serum albumin and 7.6 μmole/1 of six FFA (free fatty acids) - SPFM; 50 mg/1 of dextran and 7.6 μmole/1- of six FFA. EXPERIMENT 1 Days of HS c >SM • ' • SFM SPϊ ? M after - .' inoculation mg/1 μg/10 6 c mg/1 μg/10 b c mg/1 μg/10 6 c 1 0.35 0.04 0.44 0.05 0.70 0.08 2 0.35 0.04 0.26 • 0.0 0.26 0.03 3 l ' .l . 0.1 0.53 0.06 0.53 0.06 7 2.3 0.26 1.9 0.22 0.79 0.09 9 2.9 0.33 1.7 0.19 0.18 0.02 11 2.1 0.24 1.9 0.22 0.79 0.09 13 4.2 0.48 0.35 0.04 — — EXPERIMENT 2 1 0.25 0.5 0.35 0.7 0.25 0.5 5 2.95 5.9 θ ' .45 0.9 0.30 0.6 7 0.40 0.8 0.6 1.2 2.25 4.5 12 0.25 0.5 1.2 2.4 3.65 7.3 5. In another series of expe iments, a DNA fragment coding for the hepatitis B virus surface antigen (HBsAG) was placed under the control of the human 70 kb heat shock protein promotor. The resulting plasmid construct (pl7MSJneo carrying a neomycin resistance selection gene) was used in trasfection experiments to establish a stable amnion cell line of human origin (Wish), expressing the HBsAg gene in a heat-regulated fashion. Post-translational modifications, such as assembly, glycosylation, secretion and production of both major and middle S proteins appeared to function normally. In addition, the production of HBsAg under various protocols of heat in¬ duction was found to be possible. After inoculation into nude mice, development of tumours was observed at the site of injection. Tumour cells, dispersed by means of collagenase or trypsin treatment from excised tumours and subsequently seeded into Petri dishes id secrete the same quantities of HBsAg after heat induction as cells of the original cell line. Results on the production of the anl igen by the tumoral cell's are given in Table IV below. Table IV: HBsAg secreted by WB4 cells before and after passage as tumours into nude mice. Parental cells (around 10 ) were injected into the back area of nude mice; three weeks later the mice developed tumours. The mice were sacrificed, the tumours were finely minced with scissor and tumour frag¬ ments were either injected into nude mice to produce the next generation of tumours, or sequentially treated with trypsin to dissociate tumour cell which were seeded in Petri dishes. Tumour cells at a density of 10 cells per 25cm dish were heat treated (HS) u_ not (Control) for 2 h at 43°C and post- incubated overnight at 37°C. HBsAg was measured using an ELISA kit obtained from Abbot. Generation WB4 ng HBSsAg/10 6 cells Control. HS parent ;al cells 0 80 Gl 0 100 G2 nd nd G3 0 50 G4 0 38 Complete details on the construction of plasmid pl7MS neo are published in M. DREANO et al.. Virus ' Research (1987) ,8_, 43-59.

Claims: CLAIMS 1. A method for the biochemical production of proteins of interest with an inducible recombinant gene expression unit/host cell system, this method comprising the following steps : (1) Chosing a suitable cell line of eukaryotic origin, (2) Genetically modifying these cells by transforming with recombinant DNA containing the following elements : (a) a gene coding for a protein of interest, this gene being under the control of a stress inducible transcription promotor of eukaryotic origin, (b) an oncogene or an oncogenic transforming gene of cellular or viral origin, (3) Inoculating the transformed ce ls into selected laborato¬ ry warm-blooded animals capable of growing tumors in response to inoculation; (4) Allowing the tumors to grow in the animal until tumors of 10 cells or more are obtained, (5) Removing said tumors and effecting dissociation thereof, introducing the dissociated cells into in-vitro culture medium and subjecting them to stress to induce expression of the gene of interest; (6) Isolating the expressed protein. 2. The method of claim 1, wherein the selected cell lines (1) are tumor cells, whereby element (b) is omitted. 3. The method of claim 1, wherein the transcription promotor (b) is a human 70 Kd heat-shock (hsp70) promoter fragment. 4. The method of claim 3, wherein the oncogene or tran¬ sforming gene is selected from (i) the human c-ras oncogene and (ii) a subgenomic fragment of bovine papilloma virus. 5. The method of claim 3, wherein the gene of interest (a) is a human growth hormone (hGH) gene or a hepatitis B virus surface antigen (HBsAG). 6. "The method of claim 3, wherein said recombinant DNA (2) is selected from (i) plasmid pl7hGH dhfr carrying the hGH gene driven by the hsp70 promotor; (ii) plasmid pl7HBN car- rying the hGH gene under control of the hsp70 promotor, the neomycin resistance gene driven by the herpes simplex virus thymidine kinase promotor and the t'ransformant fragment of bovine papilloma virus (clone HBN2). 7. The method of claim 3, wherein said transformation in step (2) is effected by co-trans ection with plasmid pl7hGH dhfr and plasmid pEJ, the latter carrying the human c-ras oncogene (clone 6). 8. The method of Claim 2, wherein said transformation in step (2) is effected by co-transfection with plasmid pl7hGH dhfr and plasmid pSV2NE0, a plasmid carrying the neomycin resistance gene under control of early SV40 promotor. .9. The . method of claim 1, wherein the selected cell lines, is NIH-3T3. 10. The method of claim 3 wherein the selected animals include nude mice and chemically immunosuppressed animals. 11. The method of claim 3 including after step (4) the further intermediate step of : • (4b) removing the tumor and inπoculating a portion there¬ of Into immune animals such as SENCAR mice. 12. The method of claim 3, i n which step (5) involves cultivation in culture media selected from (i) serum supple¬ mented medium (SSM), (ii) serum free medium (SFM) supplemented with fatty -acids and (iii) serum and protein free medium supplemented with fatty acids and dextran. (SPFM) 13. The method of claim 12, including operating in a fermentator in the presence of homogeneously distributed mi¬ crocarriers (CYTODEX) or hollow fiber culture system. 14. The method of claim 1.3, wherein isolation of the protein of interest is effected by techniques selected from affinity chromatograph , dialysis, ion exchange, and gel chro¬ matography. 15. The method of claim 3, wherein said recombinant DNA (2) Is a plasmid pl7MS Neo carrying the HBsAg gene driven by the hsp70 promotor. 16. The method of claim 3, wherein the host cell lines are selected from NIH-3T3, and WISH ( TCC-CCL25) .

Inventors: BROMLEY PETER, DREANO MICHEL, FISCHBACH MICHEL, FOUILLET XAVIER, PADIEU PRUDENT, VOELLMY RICHARD, BROMLEY, PETER, DREANO, MICHEL, FISCHBACH, MICHEL, FOUILLET, XAVIER, PADIEU, PRUDENT, VOELLMY, RICHARD
Applicants: BATTELLE MEMORIAL INSTITUTE
Application year: 1987
Main classification: C12N15
IPC codes: A01K67, C07K14, C12N5, C12N15, C12P21, C12R1