Patent Application
20190309323
The present invention relates to the field of genetic engineering, preferably the expression of recombinant proteins (RP). Particularly, the invention relates to a promoter and variants thereof having same function and having more than 90% sequence identity. The promoter comprises a fragment of 1147 base pairs (bp) of a first promoter, gene β-actin of the genome Cricetulus griseus, enriched in Cytosine-Guanine dinucleotides (RegCG), which may be upstream of a second promoter, cytomegalovirus (CMV). The invention also comprises vectors, transfected cell lines, and method for producing RP, in mammalian cells which have been transfected with vectors containing the aforementioned promoter or variants thereof.
By genetic engineering different RP have been generated with therapeutic application, also known as biopharmaceuticals. Within the RP, therapeutic antibodies (TpAb) have developed, whose most successful applications are in the treatment of cancer and autoimmune diseases.
Although the marketing of TpAb generates high economic activity, with the discovery of new therapeutic targets, it expected an explosion in the generation of new TpAb. To this adds the fact that TpAb should be administered in high doses for long periods, further increasing the demand for them. Although there has been a great development of bioprocesses and bioreactors for RP expression in mammalian cells (CHO-Chinese hamster ovary, NS02 and HEK) in recent years, significantly increasing yields, this has not been enough to meet the growing demand for RP. It is for this reason that many efforts have focused on improving the productive capacity of the cells at different levels. Thus, it is now known that protein expression can be increased, therefore changes at the genetic level of cell lines such increase their viability, gene expression, folding efficiency and secretory capacity, as well as optimizing the conditions of the cell cultures, allowing increased production RP along with increase survival and cell density thereof. Among the documents closest patents can be mentioned CL200400189 (Immuno Japan Inc.) that show an expression vector which allows easily obtain strains of high level of recombinant protein production using a mammalian cells, especially chinese hamster ovary cells as a guest. The vector comprises a promoter with highly inducible expression, a multiple cloning site for insertion of a gene and a polyadenylation signal sequence in that order, from above to below, them further comprising a drug resistant gene.
CL19890089 teaches a process for the preparation of hybrid fusion proteins composed of human interleukin-2 (IL-2) and pseudomonas exotoxin by recombinant DNA technology. Exotoxin is at the carboxyl end and IL-2 at the amino terminus, and comprises transforming a host organism with a recombinant vector. The host thus transformed is cultivated in a suitable medium and the hybrid protein is purified. The hybrid protein (IL2-pe 40) can be used to treat autoimmune diseases and to suppress the immune response in cases of graft rejection and transplants. Production of fusion proteins IL-2 recombinant human, can be substantially enhanced by utilizing a vector construction capable of expressing the hybrid protein at high levels. Moreover, the resulting protein can be purified in high yield using an affinity column with the receptor. In a specific embodiment, the fusion protein of the IL-2 and Pseudomonas exotoxin is expressed at high levels in E. coli. The vector is useful for treating autoimmune diseases such as thyroiditis, type 1 diabetes, rheumatoid arthritis.
US201139339 teaches vectors, cell lines transfected, mammalian host cells (particularly Chinese hamster ovary (CHO) cells) for protein production, where transfection efficiency is based on the use of: a single vector, sequences functional in oriP, codons optimized Epstein-Barr nuclear antigen-1 virus (EBNA1), poly(ethyleneimine) 40 kDa fully deacetylated as a transfection reagent, co-expression of a fibroblast growth factor (FGF) and/or using protein kinase B to enhance and improve heterologous gene expression by valproic acid (VPA).
JP2011019509 (TOYOBO CO LTD) provides an expression vector containing a multi-cloning site optimized to allow cloning of the gene of a target protein with good efficiency, by introducing the expression vector into animal cells and establishment of cell showing a high productivity of the recombinant protein. This expression vector has a multi-cloning site with a restriction enzyme recognition sequence having a low frequency of occurrence in the gene of the target protein, especially an antibody gene, a cassette selection marker gene expression resistance to a mRNA-destabilizing, and at least one gene expression sequence of stabilization.
US20110081708 relates to a method for boosting recombinant gene expression in bacteria, yeast, insect and mammalian cells. This method generally considered most or all of the parameters and factors affecting protein expression including codon usage, use of tRNA, GC content, ribosome binding sequences, promoter, 5′-UTR sequences, ORF and 3′-UTR of genes to increase protein expression of genes in bacteria, yeast, insect and mammalian cells. In particular, the invention relates to a system and method for optimizing the sequence of recombinant protein expression improves with the use of algorithms, and therefore the method can be incorporated into software.
US2011097798 discloses the use of expression vectors of a target cDNA of at least one selective marker gene of a single cassette of mammalian expression is under the control of a promoter compound, said cDNA and at least one marker gene are associated by IRES element. an expression vector comprising also protects; a promoter compound, at least one cloning site the gene target insert, an IRES, a bacterial expression promoter EM7 a selective marker gene, a transcription terminator, a pUC origin of high amplification copy and at least one element a region associated with the nuclear matrix (MAR). Also they mentioned as elements to optimize promoter, the start site of transcription (TSS), the TATA box in TFIID complex element recognition factor II B, TFIIB, among others.
EP2316955 relates to a system and method for the production of proteins comprising the use of animals or plants which modify protein folding or processing capacity, and involves the co-expression of a polypeptide significantly increases the yield of cells the protein of interest in cells. Such polypeptide coexpressed may be a component or modulator XBP1 or ATF6.
WO2010092335 relates to a modified recombinant host cell to increase expression levels of Ero1 and XBP1 relative to expression levels in XBP1 and Ero1 in an unmodified cell. It also relates to a method for producing a recombinant protein of interest, wherein the yield of the protein of interest is increased, increasing the ability of the cell to perform post-translational modifications. The method may be initiated or positively regulated through UPR. Also it mentions that the modified cell increases expression of Ero1 and XBP1. The method used to produce recombinant antibodies such as proteins or fragments thereof.
US20100120089 describes an inducible expression cassette comprising a high promoter gene dihydrofolate reductase (DHFR) with GC-rich repeat sequences, which are partially or completely removed, to enhance the effectiveness of a gene amplification system. An expression vector comprising such a cassette and optionally a gene encoding a recombinant protein of interest, a line of animals transformed with the vector cells, and a method of mass production and purification of a recombinant protein by also describes culturing the transformant. The invention enables reduction of time needed to establish a cell line that produces high levels, a recombinant protein of interest with a low concentration of DHFR inhibitor, which allows more efficient production of the recombinant protein.
KR20080016871 relates to a mammalian host cell with a high expression level of a recombinant antibody containing a double gene vector sequences optimized nucleotide encoding the light chain and the antibody heavy chain, and a process for improving the production of recombinant antibodies in a mammalian host cell. The vector comprises at least a first transcription unit containing a first sequence of synthetic nucleotides encoding a first polypeptide chain and a second transcription unit containing a second synthetic nucleotide sequence encoding a second polypeptide chain where both synthetic sequences They are based on the nucleotide sequences of natural origin, wherein the first and second nucleotide sequence is adapted to genes of CHO cells, wherein the expression vector mammals, the two synthetic nucleotide sequences have a GC content and/or distribution GC; which is different from the sequences corresponding natural nucleotides.
US20070141557 relates to a method for optimizing expression of a nucleotide sequence based on the amino acid sequence of a protein, and device for carrying out said method. An important in the invention variable, GC content (guanine and cytosine bases), DNA motifs that are repeated or the reverse complementary sequence repeats. WO0069881 refers to a binding protein to an Estrogen Response Element call (ERE-BP), and isolated polynucleotides encoding said protein. Vectors, host cells, recombinant methods for producing the protein, transgenic animals and antibody also provided. The invention relates to therapeutic methods and treatment of related disorders mentioned protein. It also provides an isolated nucleic acid encoding said protein binding to a response element mammalian estrogen (ERE-BPs), and fragments thereof.
U.S. Pat. No. 8,298,816B2 provides a hybrid promoter comprising a combination of a CMV enhancer promoter and a beta-actin or mammalian transcriptional regulatory region of the genomic sequence post Woodchuck Hepatitis Virus (WPRE) and a beta-actin promoter mammalian it is stronger than existing promoters. In addition, the activities of beta-actin promoters could be to coexpress enhancers Ras oncogene product, which is a transactivator.
US20130174284A1 relates to a recombinant vector and a transgenic mouse expressing human ferritin in tissue nonspecifically, and more particularly, to a vector prepared for operably linking a gene of human ferritin to an enhancer early cytomegalovirus (CMV) and beta-actin promoter. Furthermore, the invention relates to a method for preparing a transgenic mouse and a method for monitoring cell or tissue therapy using the transgenic mouse.
US20130273634A1 discloses a method for mass production of human coagulation factor VII. The method includes a) providing an expression vector which carries i) a promoter dihydrofolate reductase vacuum one or more sequences CCGCC repeated a GC rich region of the same gene and a dihydrofolate reductase (DHFR) operably linked to the and ii) a cytomegalovirus (CMV) and factor VII gene of this human coagulation; b) obtaining a transformed host cell line containing the expression vector; c) culturing the transfected host cell in the presence of an inhibitor of dihydrofolate reductase to select cells expressing the factor VII human coagulation with high efficiency; d) adding sodium butyrate to the selected host cells.
US20130324593A1 relates to a hybrid promoter that all or part of a CMV enhancer, all or part of a promoter, beta-actin, all or part of a CMV promoter, and all or part of a beta-actin intron are operatively connected to each other, a recombinant vector comprising the same, a transformant, a pharmaceutical composition comprising the recombinant vector or the transformant, and a method for preparing a target protein using the recombinant vector or the transformant. The hybrid promoter of the invention is capable of inducing high expression of a target protein in a eukaryotic cell. Then, the hybrid promoter of the invention can be effectively used for the development of an antibody or the production of a DNA vaccine.
US20140017726A1 relates to a nucleic acid molecule comprising a promoter functional herpesvirus, functional enhancer of herpesvirus, and one or more inner elements of the CpG island of phosphoribosyltransferase gene and/or a functional variant thereof. A method of producing the desired polypeptide using the nucleic acid molecule, a vector and a host cell containing the nucleic acid molecule are also disclosed.
KR20120059222A discloses a fusion promoter that is operably linked partially or completely a CMV promoter, and partially or completely to a beta-actin, intron maintaining high expression by a target gene. The fusion protein is operably linked, partially or completely, to a promoter or a beta actin intron. The CMV promoter contains a DNA fragment of specific sequence. The beta-actin intron containing said DNA fragment specific sequence. recombinant vector contains a fusion promoter containing an ampicillin resistant, chloramphenicol antibiotic kamicina or the acetyl transferase gene. Pharmaceutical composition containing the vector or recombinant host cell transformed with the recombinant vector.
KR20050018720 teaches an oligonucleotide to activate cytomegalovirus (CMV) and method to activate it. The invention solves problems such as difficulty in vivo CMV promoter and CMV inactivation by interferon activation when the CMV promoter is applied to gene therapy.
“High-level protein expression in CHO transient transfection scalable”, Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen J F, Journal: Biotech Bioeng. 2009 Jun. 15: 103 (3); 542-51 reveals of Chinese hamster ovary (CHO) cells have been widely used as the platform production of therapeutic proteins, including monoclonal antibodies in the pharmaceutical industry. For early development, it is advantageous to rapidly produce large amounts of protein in the same cell line. Therefore, it is highly desirable, the development of a platform transiently transfected CHO cells with a high level of protein expression. The publication develops such a platform. Polyethyleneimine (PEI) was used as transfection reagent. It was found that DMSO and lithium acetate (LiAc) levels improve transient expression of transfection significantly in CHO cells. With an optimized process transient transfection, the monoclonal antibody (Mab) was expressed in CHO cells at a high level, with an average of 80 mg/L.
“Endogenous estrogen receptor is transcriptionally active in primary beta cells from ovarian estrogen receptor knockout mice”, S O Mueller, Katzenellenbogen J A, Korach K S., Steroids. 2004 September; 69 (10): 681-6 teaches that an estrogen receptor (ER) alpha is an inducible transcription factor by the hormone plays a key physiological role. Interestingly, a clear and indisputable physiological function of the recently described ERP remains difficult, with the exception of the ovary, where it has been a cooperative role of ERa and ER shown. Investigates, endogenous ERs which act as inducible transcription factors. Also studied selective agonist potency ER indenestrol R-A, the ERa agonist and antagonist pure ERP R, R-diethyl tetrahidrocriseno ERa agonist and pure propylpyrazole triol.
The present invention relates to the field of genetic engineering and protein expression, preferably recombinant proteins (RP). Particularly, the 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 β-actin genome Cricetulus griseus, rich in cytosine-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 RP, 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 RP expression levels in mammalian cells, preferably CHO cells. Thus, an expression system more efficient (4.5 times), with respect to commercial viral vectors, the production yield of Recombinant Antibodies (RAb) developed.
In addition, this system optimized expression may allow the development of new biopharmaceuticals and/or biogenerics.
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 β-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.
Thus, the present invention incorporates a 1147 bp fragment of the promoter sequence of β-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.
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.
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.
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.
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 (
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% CO2, 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 (
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 μM, 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 (
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 (
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 (
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 (
As seen in
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 (
For vector construction Seq3a-HAb2 (
For vector construction Seq3a-Lab2 (
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 3×105 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% CO2. 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% CO2, 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% CO2; for scaling.
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 μg of vector DNA is added, previously linearized by cutting with the restriction enzyme PvuI and purified with 20 μl of Lipofectamine 2000 (Invitrogen) in 500 μL OptiMEM medium (Invitrogen). Cells with this mixture for 4 hours at 37° C. with 5% CO2 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 μl 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 (
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 CO2 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.
In
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 μl of Lipofectamine 2000 (Invitrogen) in added 500 μl OptiMEM medium (Invitrogen). Cells with this mixture for 4 hours at 37° C. with 5% CO2 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 μl 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.
In
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.
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 CO2 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.
In
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% CO2. 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 3×105 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=((N−No)×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.
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.
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% CO2, 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 μl CD Lifectamine 2000 (Invitrogen) in 100 μl OptiMEM medium (Invitrogen) was added, and incubating for 4 hours at 37° C. and 5% CO2. Then the transfection mixture was removed and fed with 500 μl of DMEM-F12 medium, supplemented with 10% FBS and incubated for 48 hours at 37° C. and 5% CO2. 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).
In
| Number | Date | Country | Kind |
|---|---|---|---|
| 1661-2016 | Jun 2016 | CL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CL2017/050024 | 6/9/2017 | WO | 00 |
