PROMOTER WITH AN ENRICHED CYTOSINE-GUANINE DINUCLEOTIDE REGION, VECTORS, CELLULAR LINES, METHOD FOR PRODUCING RECOMBINANT PROTEIN

Abstract

The present invention relates to the field of genetic engineering, preferably the expression of recombinant proteins (RP). In particular, the invention relates to a promoter and variants thereof having an equal function and more than 90% sequence identity. The promoter comprises a fragment of 1147 base pairs (bp) of a first promoter, promoter of the -actin gene of the Cricetulus griseus genome, enriched in cytosine-guanine dinucleotides (RegCG). The first promoter can be upstream of a second promoter, cytomegalovirus (CMV) promoter. The invention also relates to vectors, transfected cellular lines and a method for producing RP in mammal cells that have been transfected with vectors containing said promoter or variants thereof.

Claims

1. A promoter for the expression of recombinant proteins (RP), comprising: a first promoter sequence SEQ ID NO: 5 or a variant having the same function and more than 90% sequence identity; a second promoter cytomegalovirus (CMV) having sequence SEQ ID No.: 3 or a variant having more than 90% sequence identity, located downstream of the first promoter; and five (5) tandem glucocorticoid-responsive elements (GRE) having sequence SEQ ID NO.: 8 located between said first and second promoters.

2. The promoter of claim 1, further comprising an additional five (5) tandem glucocorticoid-responsive elements (GRE) located between sequence SEQ ID Nos.: 29 and sequence SEQ ID Nos.: 30.

3.-7. (canceled)

8. A vector comprising a promoter for the expression of recombinant proteins (RP), comprising: a first promoter sequence SEQ ID NO: 5 or a variant having the same function and more than 90% sequence identity; a second promoter cytomegalovirus (CMV) having sequence SEQ ID No.: 3 or a variant having more than 90% sequence identity, located downstream of the first promoter; five (5) tandem glucocorticoid-responsive elements (GRE) having sequence SEQ ID NO.: 8 located between said first and second promoters; and an additional five (5) tandem glucocorticoid-responsive elements (GRE) located between sequence SEQ ID Nos.: 29 and sequence SEQ ID Nos.: 30.

9. The vector of claim 8, wherein the vector encodes a protein such as an antibody and a chain of antibody.

10.-12. (canceled)

13. The vector of claim 8, wherein the vector encodes the antibody heavy chain (Ab2) having sequence SEQ ID No. 22 and the light chart of the antibody (Ab2) having sequence SEQ ID No.: 23.

14. The vector of claim 8, wherein the vector encodes the antibody heavy chain (Ab1) having sequence SEQ ID No. 15 and the light chain of the antibody (Ab1) having sequence SEQ ID No.: 16.

15. The vector of claim 8, further comprising sequence SEQ ID No.: 17.

16. The vector of claim 8, wherein the vector comprises an encoding sequence to enzyme dihydrofolate reductase (DHFR) having sequence SEQ ID No.: 26.

17. The vector of claim 8, wherein the vector responds in a dose-dependent manner when dexamethasone is present.

18.-20. (canceled)

21. A host cell, comprising the vector of claim 8.

22. The host cell of claim 21, wherein the host cell is a mammalian cell.

23. The host cell of claim 22, wherein the host cell is a Chinese hamster ovary cell (CHO).

24.-26. (canceled)

27. A process of producing recombinant proteins (RP), comprising culturing in suspension the host cell of claim 21.

28. The vector of claim 8, wherein the vector encodes a protein such as an antibody or a chain of antibody.

29. The vector of claim 8, wherein the vector encodes the antibody heavy chain (Ab2) having sequence SEQ ID No. 22 or the light chain of the antibody (Ab2) having sequence SEQ ID No.: 23.

30. The vector of claim 8, wherein the vector encodes the antibody heavy chain (Ab1) having sequence SEQ ID No. 15 or the light chain of the antibody (Ab1) having sequence SEQ ID No.: 16.

31. A host cell, comprising the vector of claim 15.

32. A host cell, comprising the vector of claim 16.

33. A process of producing recombinant proteins (RP), comprising culturing in suspension the host cell of claim 31.

34. A process of producing recombinant proteins (RP), comprising culturing in suspension the host cell of claim 32.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1 shows a diagram of promoter variants in reporter vectors, cloned in vector pGL4.17 (SEQ ID No.: 1) encoding for the luciferase protein (Luc2). Seq1-Luc vector (SEQ ID No.: 2) comprising the promoter region CMV (SEQ ID No.: 3), used as a control; the Seq2-Luc vector (SEQ ID No.: 4) comprises RegCG (SEQ ID No.: 5) upstream of the CMV promoter region to form the RegCG-CMV promoter (SEQ ID No.: 6); Seq3a-Luc vector (SEQ ID No.: 7) comprises a tandem of 5 glucocorticoid response elements (GRE) (SEQ ID No.: 8) incorporated in the RegCG-CMV promoter, and CMV between RegCG forming RegCG-GRE-CMV (SEQ ID No.: 9). The vector Seq3b-Luc (SEQ ID No.: 10), contains GRE between enhancer (SEQ ID No.: 29) and the core CMV (SEQ ID No.: 30), forming RegCG-CMV-GRE (SEQ ID No.: 11). Seq3c-Luc (SEQ ID No.: 12) containing a GRE between RegCG and CMV; and other GRE between enhancer and core CMV forming RegCG-GRE-CMV-GRE (SEQ ID No.: 13).

[0027] FIG. 2 shows a diagram of promoter variants into expression vectors, cloned in pcDNA 3.1 vector () (SEQ ID No.: 14) encoding the Ab1 antibody. The Ab1 antibody is encoded by the genes of the heavy (SEQ ID No.: 15) and light (SEQ ID No.: 16) separated by an IRES (SEQ ID No.: 17), forming aCD20-IRES-CH-CL-aCD20 (SEQ ID No.: 18). The vector Seq1-Ab1 (SEQ ID No: 19) comprising the promoter region CMV (SEQ ID No.: 3), used as a control; vector Seq2-Ab1 (SEQ ID No.: 20) comprises RegCG (SEQ ID No.: 5) upstream of the CMV promoter region to form the RegCG-CMV promoter (SEQ ID No.: 6); the vector Seq3a-Ab1 (SEQ ID No.: 21) comprises a tandem of 5 glucocorticoid response elements (GRE) (SEQ ID No.: 8) incorporated in the RegCG-CMV promoter, and CMV between RegCG forming RegCG-GRE-CMV (SEQ ID No.: 9).

[0028] FIG. 3 shows a scheme of a variant RegCG-GRE-CMV promoter (SEQ ID No: 9) expression vectors encoding Ab2 antibody. Ab2 antibody is encoded by the genes of the heavy (SEQ ID No: 22) and light (SEQ ID No: 23) encoded in Seq3a-HAb2 (SEQ ID No: 24) vector and Seq3a-LAb2 (SEQ ID No.: 25), respectively. The Seq3a-Lab2 vector contains downstream of the light chain and separated by a IRES (SEQ ID No: 17) coding sequence for the enzyme dihydrofolate reductase (dhfr) (SEQ ID No: 26).

[0029] FIG. 4 shows the effect of RegCG in combination with GRE on transcriptional activity of the CMV promoter in stable cell lines. The promoter activity was expressed in relative luminescence units (RLU) calculated as the ratio of the luminescence and total protein concentration in cell lysates. The dots indicate the activity of each clone, also showing the average activity for each measurement. The empty circles correspond to clones having higher average clones with Seq1-Luc plus 3 standard deviations activity.

[0030] FIG. 5 shows analysis of induction by dexamethasone of stable clones with promoters having GRE. luciferase activity (RLU) of clones shown with GRE-containing promoters and RegCG by the CMV promoter. Relative Luminescence Units corresponds to the ratio between luciferase activity and protein concentration in the lysate, normalized to value obtained without dexamethasone (* P<0.05, ** P<0.01; *** P<0.001).

[0031] FIG. 6 shows Ab1 antibody production by clones containing the Seq1-Ab1, Seq2-Ab1 and Seq3a-Ab1 CHO-KI vectors in cells. Cells were transfected with Seq3a-Ab1, Seq2-Ab1 and Seq1-Ab1 vectors; and cloned by limit dilution, then sub cultured for 15 days in the presence of the antibiotic G418 selection at 1 mg/mL. A) Distribution of clones based on their decreasing production level Ab1. B) Concentration of Ab1 produced by each clone according to the promoter. Mean values (horizontal bar) were Seq2-Ab1: 25.3 ug/L, Seq3a-Ab1: 24.3 ug/L and Seq1-Ab1: 6.2 ug/L. (*** P<0.001).

[0032] FIG. 7 shows the analysis of induction by dexamethasone of a stable clone with Seq3a-Ab1 promoter (SEQ ID NO: 21) having GRE. Ab1 concentration at different dexamethasone concentrations is shown. (* P<0.05; *** P<0.001).

[0033] FIG. 8 shows the growth curves, production and productivity levels of specific Ab2 producing clones. Seven clones (4, 8, 17, 23, 93, 141 and 156) of CHO-DG44 cells co-transfected with vectors Seq3a-HAb2 (SEQ ID No.: 24) and Seq3a-LAb2 (SEQ ID No.: 25) selected for the production of Ab2 antibody.

[0034] FIG. 9 shows luciferase activity expressed as relative luminescence units of different promoters in expression vectors Seq4-Luc (SEQ ID No.: 35), Seq5-Luc (SEQ ID No.: 36) and Seq1-Luc, transiently transfected in CHO K1 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention relates to a promoter and variants thereof having the same function and more than 90% sequence identity. Preferably, such variants have greater than 95% sequence identity. Even more preferably, such variants have more than 98% sequence identity. Even more preferably, such variants have greater than 99% sequence identity. The promoter comprises a promoter which is a fragment of 1147 bp of promoter sequence of -actin of the genome Cricetulus griseus, enriched in citosine-guanine dinucleotides (RegCG) sequence SEQ ID No.: 5, which may be upstream of the CMV promoter. The invention also comprises vectors, transfected cell lines, methods for producing such vectors and cell lines, and method for producing PR, in mammalian cells which have been transfected with vectors containing the aforementioned promoter or variants. The present invention optionally proposes incorporating RegCG (SEQ ID NO: 5), upstream of the CMV promoter expression vectors, to prevent silencing of the latter, thus improving the PR expression levels in mammalian cells, CHO preferably cells. Thus, an expression system more efficient (4.5 times), with respect to commercial viral vectors, the production yield of Recombinant Antibodies (ACR) developed.

[0036] Thus, the present invention incorporates a 1147 bp fragment of the promoter sequence of -actin of the genome Cricetulus griseus, enriched in cotisine-guanine dinucleotides (RegCG) sequence SEQ ID No. 5 and Ab2 in CHO cells transfected with the vector generated in the present invention, where Ab1, corresponds to an anti-CD20 antibody whose human light chain sequence (SEQ ID No: 31) and heavy chain sequence (SEQ ID No.: 32) are identical to those of the commercial antibody Rituximab, and where Ab2 corresponds to a human anti-TNF antibody whose light chain sequence (SEQ ID NO: 33) and heavy chain sequence (SEQ ID No.: 34) correspond to the sequences of an antibody registered in Chile patent 50500.

[0037] For incorporation of the expression vector to a mammalian cell, you can use any technique of inserting DNA known in the art, such as transfection, viral transduction among others, including those widely established techniques, such as electroporation, coprecipitation phosphate calcium, lipofection, and other. In addition, transfection can be performed in transiently, or stably, transfecting defined as one in which a selective pressure on the cells is performed, maintaining the vector episomally therein.

[0038] On the other hand, stable transfection involves the integration of vector to genomic DNA of the cells, either by non-homologous recombination, or by site-directed additions. Cells performing this addition can be selected using a marker in the vector, which can also allow the amplification of the copy number of the vector in the genome of the host cell.

[0039] For the generation of this promoter, was used as the basis of early cytomegalovirus promoter sequence (CMV) (SEQ ID No.: 3) because it has high transcriptional activity and is widely used for expression in cells PR animal. However, this promoter silencing frequently suffer in production lines, resulting in low productivity clones of PR. Therefore, in order to avoid such silencing, river was incorporated upstream of the promoter region of 1147 bp (SEQ ID No.: 5) derived from a CpG island, which was designated RegCG. RegCG can be immediately upstream of the promoter or alternatively separated from this by a segment, such as segment 187 bp (SEQ ID No.: 8). In the latter case, the spacer segment contains glucocorticoid recognition elements (GREs) in order to generate an adjustable mechanism protein production by the addition of a glucocortidoide such as dexamethasone in the culture medium; whose presence does not affect the decrease of promoter silencing, maintaining its basal activity in the absence of the inducer.

[0040] In order to find sequences capable of maintaining the transcriptional activity of the CMV promoter sequence, genomic DNA sequences (gDNA) of promoter regions of genes housekeeping analyzed identifying a region with 67.7% content CG corresponding to the promoter region and part of the first exon of beta actin gene (ACTB) of the Cricetulus griseus of (RegCG) (SEQ ID No.: 5). Briefly, the promoter sequence from gDNA of cells Chinese hamster ovary (CHO) was amplified by PCR using the F1 (SEQ ID No: 27) and R1 primers (SEQ ID No.: 28) containing sites Cloning restriction for. Then this amplicon was purified and cloned into pGEM-T Easy (Promega). Positive clones which were sequenced were selected, checking the construction designed. This fragment was subcloned into the expression vector Seq1-Luc (SEQ ID No.: 2), to generate Seq2-Luc (SEQ ID No.: 4). Stable cell lines by transfection of CHO-K1 (ATCC CCL-61) cells with reporters vectors Seq1-Luc, Seq2-Luc, Seq3a-Luc, Seq3b-Luc and Seq3c-Luc were generated and clones were isolated in the presence of the antibiotic G418 by limit dilution. Then, 16 clones were sub cultured and luciferase activity was measured on day 41 after the removal of antibiotic selection. Luciferase activity Seq2-Luc, Seq3a-Luc, Seq3b-Luc and Seq3c-Luc, is higher promoter activity Seq1-Luc culture day 41 (FIG. 4).

[0041] Furthermore, the effect of addition of the promoter on luciferase activity in transient transfections was determined. CHO-K1 cells were grown to reach 90% confluence were co-transfected using Lipofectamine CD (Invitrogen. EEU U), with the normalization vector pGL4.73 (Promega. USA) expressing Renilla luciferase reniformis (Ren), under the control of SV40 promoter, and with experimental vectors Seq1-Luc (SEQ ID No.: 2), Seq4-Luc (SEQ ID No.: 35) and Seq5-Luc (SEQ ID No.: 36) expressing Photinus pyralis luciferase (Luc), under the control of the promoters studied and pGL4.17 (base vector without promoter). Cotransfected cells were cultured for 18 hours at 37 C. and 5% CO.sub.2, and then the product luminicencia reactions Luc and Ren independently analyzed using the Dual-Glo Luciferase Assay System (Promega system. USA) in 96-well white. luminicencia relative units for the reason Luc/Ren was calculated. it is noted that RegCG has transcriptional activity (FIG. 9).

[0042] Similarly, a DNA sequence (SEQ ID No.: 8) was synthesized containing a tandem of 5 repeats of the consensus sequence elements glucocorticoid response (GRE), separated by 25 nucleotides of irrelevant sequence, i.e., which may be any sequence. Subsequently, GRE was incorporated into the vector carrying the CMV promoter with RegCG, between both elements, and Seq3a-Luc vector (SEQ ID No.: 7) was generated. The luciferase activity of clones transfected CHO-K1 cells with Seq2-Luc vectors and selected with G418 antibiotic selection was then analyzed. It was compared with the activity of clones of cells transfected with the vector Seq1-Luc Control (SEQ ID No.: 2), generated in the same way. To perform the analysis, clones were grown to a level of confluence of 50 to 80%, induced with dexamethasone at concentrations of 0; 0.1; 1 and 10 M, respectively, and luciferase activity measured after 48 hours of treatment. Regarding the promoter activity he was found at day 41, that Seq2-Luc and Seq3a-Luc vectors presented Seq3a-13 and 7.5 times, respectively, the promoter activity control (FIG. 4). Furthermore, Seq3a-Luc clones showed that there is a significant increase in luminescence of 30 to 40% compared to stimulation with dexamethasone (FIG. 5), where a Seq3c-Luc clone, whose promoter contains two tandem GRE, achieved an increase 70%.

[0043] Tests were performed to measure the expression of recombinant antibodies in stable transfections, for that several vectors were constructed and light (SEQ ID No.: 16) anti-CD20 (Ab1) of an antibody: first bicistronic expression system where the luciferase gene was replaced by genes of the heavy chain (SEQ ID No. 15) was generated separated by a IRES (Internal Ribosomal Entry Site) (SEQ ID NO: 17) downstream of Seq1, Seq2 and Seq3a promoters. Stable expression clones were obtained by G418 selection, and limit dilution, randomly selected 39 clones of each construct. The concentration supernatants were obtained corresponding passage to day 35 after removal selection antibiotic and antibody production was measured by ELISA, it reached values higher than 300 g/L in 13 thereof being 4.1 times average Seq2-Ab1 respect to Seq1-Ab1 (FIG. 6).

[0044] Finally, clones generated using the construction Seg3a-Ab1 (SEQ ID No.: 21) having glucocorticoid response elements are able to respond to dexamethasone. Cells were grown to confluency and treated with dexamethasone at different concentrations (0, 0.8, 3.2 and 12.8 NM) and Abl concentration was measured after 48 hours by capture ELISA (FIG. 7).

[0045] The above results were developed in CHO-K1 cells, easy to clone grown in adhesion. The next step was to analyze the promoter activity in conditions of industrial application mainly suspension growth, unlike the CHO-K1 cell line. The cell line CHO-DG44 (A1097101 Gibco by Life Technologies) is adapted to grow in suspension and has mutated enzyme gene dihydrofolate reductase (DHFR), so the expression vector of the recombinant protein contains the dhfr gene (SEQ ID No.: 26) which allows the growth of these cells when grown in the absence of hypoxanthine and thymidine (HT). This deficiency is used to induce gene amplification of which are co-expressed in the expression vector together with the dhfr gene when grown in the presence of methotrexate, an inhibitor of DHFR at concentrations toxic sub recombinant genes. Under these conditions the cells respond by generating the dhfr gene amplifications, increasing the production of this enzyme and increased proliferation dependent on the number of copies generated. Along with it not only the dhfr gene is amplified, but also its surrounding regions. In this regard, the vector Seq3a-LAb2, a vector with the Seq3a promoter using IRES system to express the light chain gene of the Ab2 AcR downstream dhfr (FIG. 2) was constructed. For expression of heavy chain of the RAb Ab3 under the Seq3a promoter control, the Seq3a-Hab2 vector, subject to selection with G418 antibiotic, was generated. CHODG-44 suspension cells transfected with both vectors and then cells stably transformed with both vectors were selected on medium lacking HT and G418. Thus, during the process of gene amplification, dhfr co-amplified the recombinant gene of the heavy chain (SEQ ID No.: 24) and light chain (SEQ ID No.: 25) of the Ab2 antibody. Cultures were grown in suspension generated in 6-well plates with 3 mL of CDOptiCHO medium with 2 mM glutamine and 0.1% Pluronic at 140 rpm for antibody production.

[0046] As seen in FIG. 8, the Sec3a-Hab2 and Sec3a-Lab2 vector containing RegCG are capable of producing the antibody in a form of suspension culture, and it was possible to isolate a large number of clones with this feature.

Example 1: Preparation of Expression Vectors of an Anti-Human TNF Under Control of the Promoter RegCG/GRE/CMV (SEQ ID No.:9)

[0047] The anti-human TNF (or antibody Ab2) antibody is produced by expression of the genes encoding for the light (Lab2) and heavy (Hab2) chains of Ab2, expressed in independent vectors, both under the control of RegCG/GRE/CMV promoter. The Seq3a-HAb2 vector expresses and contains a selection system based on expression of neomycin resistance gene under the control of the SV40 promoter. The Seq3a-Lab2 vector expresses Lab2 and contains a selection system based on expression of the enzyme dihydrofolate reductase (DHFR), expressed in biscistronics form with Lab2. The RegCG/GRE/CMV promoter contained in both vectors was obtained from Seq3a_Luc vector (FIG. 1c).

[0048] For vector construction Seq3a-HAb2 (FIG. 3b), the gene was replaced Luc2 from Seq3a-Luc vector for gene Hab2. To do this, first from the vector Seq3a-Luc, the fragment containing the Luc2 gene and poly A between restriction sites HindIII and BamHI, was removed by digestion with the corresponding restriction enzymes. Between both sites was inserted sequentially a DNA fragment synthesized by hybridizing oligonucleotides complementary sequence IntF (SEQ ID No.: 37) and IntR (SEQ ID No.: 38) containing the restriction sites MluI, NdeI and EcoRI, leaving cohesive ends for restriction sites HindIII and BamHI, then the site between EcoRI and BamHI was inserted a DNA fragment synthesized by PCR with primers PoA-Fw (SEQ ID No.: 39) and PoA-Rv (SEQ ID No: 40) from the vector Seq3a-Luc, which contains the poly a sequence, wherein the generated amplicon contains at its ends sites EcoRI and BamHI, generating Seq3a-IC vector (SEQ ID No: 41), which has a region downstream of RegCG policlonal/GRE/CMV, which can be inserted gene promoter different recombinant proteins. The fragment containing the gene was obtained HAb2 by PCR amplification with oligonucleotides HTNFHind-Fv (SEQ ID No.: 42) and RitHMlu-Rv (SEQ ID No.: 43), from the vector pHQG9 (SEQ ID No.: 44), where the amplicon leaves its ends cleavage sites for the restriction enzymes HindIII and MluI. The amplicon was cut with the enzymes HindIII and MluI (Fermentas) and then inserted into the Seq3a-IC vector (SEQ ID No.: 41) between the same sites by ligation with T4 ligase enzyme (NEB).

[0049] For vector construction Seq3a-Lab2 (FIG. 3b), first, the CMV promoter in the vector was replaced pKQG9 (SEQ ID No.: 45) by RegCG/GRE/CMV promoter between the restriction sites SalI and HindIII. Then, the DNA fragment containing the RegCG/GRE/CMV promoter and the gene Hab2 between sites SalI and BamHI was cut. In parallel, the pOptiVEC-TOPO vector (Invitrogen) was cut by restriction sites between SalI and BamHI, and the DNA fragment containing the CMV promoter, located before the IRES sequence was removed. Subsequently, the fragment containing the RegCG/GRE/CMV promoter and the gene Lab2, between sites SalI and BamHI pKQG9 vector (SEQ ID No.: 45), was introduced, resulting in the bicistronic system, expressing Lab2 and DHFR on a single transcript where Lab2 genes and DHFR are separated by an IRES sequence.

Example 2: Host Cell Preparation for Expression of the Antibody Ab2

[0050] The cells used correspond to the CHO cell line DG-44 (Gibco A1097101 by Life Technologies). To obtain clones derived from CHO DG44, they were co-transfected cells with Seq3a-LAb2 and Seq3a-HAb2 vectors by lipotransfection. Briefly, cells are routinely cultured in suspension and agitation CD DG44 medium (Gibco), specific for CHO DG44 cells. Being seeded at a cell density of 310.sup.5 cells/mL in 30 mL of culture, supplemented with 2 mM glutamine and 0.1% Pluronic (Sigma Aldrich) and cultured statically at 37 C. and 8% CO.sub.2. Then the reagent for transfection was added Lipofectamine 2000 (ThermoFisher Scientific) diluted in Opti-MEM (Gibco) and subsequently diluted to transfect DNA into this mixture. The DNA was previously linearized by digestion with PvuI restriction enzyme, which cuts at a restriction site element ampicillin resistance. The DNA/lipofectamine complex was added to the cells and 4 hours later the medium was changed by CD OptiCHO (ThermoFisher Scientific) with G418 800 g/mL, supplemented with 2 mM glutamine and Pluronic 0.1% (Sigma Aldrich), and culturing was continued at 37 C. and 5% CO.sub.2, with stirring at 120 rpm. The culture medium was changed every three days until viability reached 90% or more. Then the cells were cultured in the same culture medium but with the addition of methotrexate (ThermoFisher Scientific) at 50 nM with medium change every three days until viability reached 90% or more. This process was repeated for methotrexate concentrations of 100 nM, 250 nM and 500 nM. This latter population clones were obtained by single cell suspension in semisolid medium ClonaCell (StemCell Technologies). The appearance of clones was confirmed two weeks after planting and the clones obtained were transferred to culture plates 96 dishes in medium CD OptiCHO with G418 supplemented with 2 mM glutamine and Pluronic 0.1% (Sigma Aldrich), 37 C. and 5% CO.sub.2; for scaling.

Example 3: Effect of RegCG in Combination with GRE on the Transcriptional Activity of the CMV Promoter in Stable Cell Lines

[0051] Stable cell lines were generated by transfecting CHO-K1 (ATCC CCL-61) cells with vectors reporters Seq1-Luc (SEQ ID NO: 2), Seq2-Luc (SEQ ID No.: 4), Seq3a-Luc (SEQ ID No.: 7), Seq3b-Luc (SEQ ID NO: 10), and Seq3c-Luc (SEQ ID NO: 12), for that the cells were cultured in 6-well plates using DMEM-F12 medium supplemented with 10% FCS to reach 90% confluence. Cells were washed with phosphate buffered saline (PBS) and a mixture containing 1 g of vector DNA is added, previously linearized by cutting with the restriction enzyme PvuI and purified with 20 l of Lipofectamine 2000 (Invitrogen) in 500 L OptiMEM medium (Invitrogen). Cells with this mixture for 4 hours at 37 C. with 5% CO.sub.2 was incubated, after the which the medium was removed and further incubated with 2 mL of medium DMEM-F12 supplemented with 10% FBS in the presence antibiotic selection G418 (Gibco) to 500 g/mL once controls without transfection vector began to die, between 10 to 15 days, clones were isolated by limit dilution. For this purpose the cells were released with trypsin and resuspended in DMEM-F12 supplemented with FBS and G418 800 g/mL, to which the cells were seeded in culture plates 96 wells with 200 l cells at dilutions such to remaining on average 0.5; 1 and 10 cells per well plates. Cell growth was obtained in less than one third of the wells seeded at 0.5 cells per well, indicating that the crops are clonal. 16 random clones were then selected for vector, which sub cultured in the absence of antibiotic selection to promote gene silencing passages made every 3 to 4 days. Finally the luciferase activity was measured on day 41 after the removal of antibiotic selection. For this, the Bright-Glo Luciferase Assay System (Promega) was used following the supplier's instructions, for which the culture cells of each clone were lysed, after which luciferase activity was measured by measuring luminescence generated by the addition of substrate of this enzyme and quantified in a model luminometer Luminoskan Ascent (Thermo Electronic Corporation). In parallel, from each lysate total concentration by the Bradford technique proteins, the reaction was quantified by the mediation of absorbance at 405 nm was quantitated. This absorbance was used to normalize the luciferase activity by reason enters luminescence (Luc) and the absorbance at 405 nm, (Luc/Abs), expressed as relative luminescence units. The data of the relative luminescence units were plotted on a graph of points in the statistical program GraphPad Prism (FIG. 4), in which the average values are indicated by horizontal bar. Seq4-Luc vector whose promoter is RegCG, has 3 times higher average activity than CMV, indicating that RegCG has greater resistance to CMV clones silencing already stabilized. Luciferase activity Seq2-Luc, Seq3a-Luc, Seq3b-Luc, Seq3c-Luc vectors, whose promoters combine RegCG and CMV, are those that have the highest activities, where the highest average activity was obtained with Seq2-Luc vector containing the RegCG/CMV promoter, this being 13 times the average activity Seq1-Luc vector under the control of CMV. Indicating there is a potentiating effect of promoter activity by combining RegCG and CMV, regardless of the presence of GRE.

Example 4: Analysis of Dexamethasone Induction of Stable Clones with Promoters Having GRE (SEQ ID Nos.: 9, 11 and 13)

[0052] Clones increased production were selected from transfections with promoters containing GRE to test whether they are able to respond to dexamethasone increased reporter activity. To do this, the selected clones in 24 well plates in DMEM-F12 medium, supplemented with 10% FBS, seeding 500,000 cells/mL at 37 C. and CO.sub.2 and 5% were cultured. The next days were stimulated by addition of dexamethasone (SIGMA) at different concentrations and cultivation was continued for 48 hours. The luciferase activity was then determined by Luc/Abs relationship, similarly to that performed in Example 3.

[0053] In FIG. 5, the result of two clones Seq3a-Luc and two Seq3c-Luc, which showed a significant increase in average luminance between 20% and 40%, compared to stimulation with dexamethasone is between 0.1 clones and 10 M.

Example 5: Production of Antibody Ab1 by Clones Containing the Seq1-Ab1 (SEQ ID No: 19) Vector Seq2-Ab1 (SEQ ID No: 20) and Seq3a-Ab1 (SEQ ID No: 21) in Cells CHO-K1

[0054] Stable cell lines were generated by transfecting CHO-K1 (ATCC CCL-61) cells with reporters vectors Seq1-Ab1, Seq2-Ab1 and Seq3a-Ab1, for this purpose the cells were plated in 6-well plates in DMEM-F12 medium, supplemented with 10% FCS to reach 90% confluence. Cells were washed with phosphate buffered saline (PBS) and a mixture containing 1 ug of DNA vector, previously linearized by cutting with the restriction enzyme PvuI and purified along with 100 l of Lipofectamine 2000 (Invitrogen) in added 500 l OptiMEM medium (Invitrogen). Cells with this mixture for 4 hours at 37 C. with 5% CO.sub.2 was incubated, after the which the medium was removed and further incubated with 2 mL of DMEM-F12 medium, supplemented with 10% FBS in the presence antibiotic selection G418 (Gibco) to 800 g/mL. Once controls without transfection vector began to die, between 10 to 15 days, clones were isolated by limit dilution. For this purpose the cells were released with trypsin and resuspended in DMEM-F12 medium, supplemented with FBS and G418 800 g/mL, to which the cells were seeded in culture plates 96 wells with 200 l cells at dilutions such so that they are on average 0.5; 1 and 10 cells per well plates. Cell growth was obtained in less than one third of the wells seeded at 0.5 cells per well, indicating that the crops are clonal. Then for each vector clones, 30 clones were randomly selected, which sub cultured in the absence of antibiotic selection to promote gene silencing passages made every 3 to 4 days. Finally the production of secreted antibodies to the supernatant on day 39 was measured after elimination of antibiotic selection. For this purpose, an ELISA capture was used, in which ELISA plates were sensitized with an anti-human IgG Kappa polyclonal antibody (Dako), then dilutions of culture supernatant was added to capture the antibody Ab1. After anti human IgG HRP conjugated antibody (IgG-HRP, eBioscience), which was revealed with the reagent TMB (Thermo Fisher Scientific) was added. Antibody concentration by measuring absorbance at 405 nm and calibration was determined versus absorbance of wells with commercial anti-CD20 (Rituximab) antibody instead of supernatant.

[0055] In FIG. 6 it is shown in the upper panel the Seq1-Ab1, Seq2-Ab1 and Seq3a-Ab1 clones sorted from highest to lowest antibody production. In the bottom panel the same clones are shown in a dot plot, in which the mean values for each group of clones are indicated by a horizontal bar.

[0056] The average concentrations achieved with Seq2-Ab1 and Seq3a-Ab1 clones, whose promoters combine RegCG and CMV, are 4.5 times the average vector activity Seq1-Ab1, under the control of CMV. Indicating that there is a potentiating effect of promoter activity by combining RegCG and CMV, regardless of the presence of GRE. Supporting that shown in Example 4 that has greater resistance RegCG silencing CMV clones already stabilized.

Example 6: Analysis of Induction by Dexamethasone of a Stable Clone with Seq3a-Ab1 Promoter (SEQ ID No.: 21) Having GRE

[0057] High production clone from transfections with Seq3a-Ab1 vector whose promoter contains GRE, to analyze whether it is able to respond to dexamethasone increasing production of antibody was selected. For this, cultured in a 24-well plate in DMEM-F12 medium, supplemented with 10% FBS, seeding 500,000 cells/mL at 37 C. and CO.sub.2 to 5%. Two days after the cells reached confluency, were stimulated by addition of dexamethasone (SIGMA) at different concentrations and cultivation she was continued for 48 hours. The concentration of antibody secreted into the supernatant was then determined using an ELISA capture with anti-human IgG Kappa polyclonal antibody (Dako) and revealed with anti IgG (eBioscience) antibody, similarly to that conducted in Example 5.

[0058] In FIG. 7, the result output, showing a significant increase in production Ab1 compared to stimulation with dexamethasone at 3.2 and 12.8 M is increasing by 48% at the highest dose used respect to control without dexamethasone.

Example 7: Growth and Production Levels of Specific Productivity Ab2 Producing Clones

[0059] Clone prepared according to Example 2, derived from CHO DG44 (A1097101 Gibco by Life Technologies) cells were routinely maintained in culture medium CD OptiCHO (ThermoFisher Scientific) supplemented with 2 mM glutamine, Pluronic 0.1% (Sigma Aldrich) G418 and 50 g/mL, at 37 C. and 5% CO.sub.2. As a result of Cloning, initial few cells, so that the clones were initially grown in 96-well plates, to then be scaled as needed, to 24 well plates and eventually to 6-well plates are obtained. After reaching the growth seed in 6-well plates, cells were cultured with shaking at 120 rpm. For the production and growth curves, the clones were seeded in Erlenmeyer flasks with filter ventilation, polycarbonate (Corning) at 310.sup.5 cells/mL in 30 mL of culture medium. every day an aliquot was removed culture for cell count and obtain supernatant for determination of antibody concentration. Cell viability was determined by counting the stained cells with trypan blue microscope. The antibody concentration in supernatant was determined by capture ELISA. This assay involves the capture antibody in an ELISA plate previously sensitized with an antiKappa polyclonal antibody (Dako), followed by addition of an antibody directed against the immunoglobulin heavy chain conjugated to peroxidase. Detection was performed using 3,3,5,5-Tetramethylbenzidine (TMB, ThermoFisher Scientific) and the absorbance at 450 nm was measured. Specific productivity was calculated by the following formula:

qmAb=mmAb/A; A=((NNo)t/ln(N/No)

where mmAb represents the total mass of antibody in the supernatant in picograms; and N and No amount of viable cells in t2 and t1, respectively times.

[0060] Clones were selected sequentially by levels of antibody production from cultures in 96-well plates. Clones with highest values were then cultured in 6-well plates, where the top 7 clones were selected, which finally grown in flasks containing 30 mL of medium and stirring characterizing their parameters; variation of cell density over time, and net production of Ab2 specific productivity. FIG. 8 shows the analysis of selected clones 7, which shows that the high yield and specific productivities were achieved by clones 23 and 156 is shown.

Example 8: Luciferase Activity of Different Promoters in Expression Vectors Seq4-Luc (SEQ ID No: 35), Seq5-Luc (SEQ ID No: 36) and Seq1-Luc, Transiently Transfected CHO K1 in Cells

[0061] CHO-K1 cells were cultured in 24-well plates in DMEM-F12 medium (Gibco) supplemented with fetal bovine serum (FBS) (HyClone) 10%, at 37 C. and 5% CO.sub.2, inoculating 300,000 cells/mL for 24 hours, or up to between 80 to 90% confluency. Then were cotransfected cells independently with 500 ng of reporter vectors Seq4-Luc (SEQ ID No: 35), Seq5-Luc (SEQ ID No: 36), Seq1-Luc and empty vector pGL4.17 (Promega) together with 500 ng of vector normalizer pGL4.73 (Promega). To do this, for each transfection the culture medium was removed and a mixture containing the reporter vector and the normalizer 20 l CD Lifectamine 2000 (Invitrogen) in 100 l OptiMEM medium (Invitrogen) was added, and incubating for 4 hours at 37 C. and 5% CO.sub.2. Then the transfection mixture was removed and fed with 500 l of DMEM-F12 medium, supplemented with 10% FBS and incubated for 48 hours at 37 C. and 5% CO.sub.2. To analyze the activity of the reporter gene transfections, the Dual-Glo Luciferase Assay System (Promega. USA) (Promega) was used following the manufacturer's instruction, where from cell lysate its measurable sequentially luciferase activity firefly expressed as reporters vectors and activity of renilla luciferase expressed by the normalizer vector. Both activities were mediated by quantifying luminiscence read on a Lumininometer, model Luminoskan ASECNT (Thermo Electronic Corporation). The final measure was obtained from the ratio between the luminances of the firefly (LUC) and renilla (Ren), (Lun/Ren).

[0062] In FIG. 9, it was observed that the higher activity was obtained with the vector Seq2-Luc, under the control of CMV, and the lowest activity was obtained with Seq5-Luc vector, under the control of the minimal portion of CMV or Core CMV. The Seq4-Luc vector, whose promoter is RegCG, is intermediate between the activity Seq2-Luc and Seq5-Luc, indicating that the RegCG promoter of the Seq4-Luc vector is functional, reaching 24% of CMV activity.