Inducible promoters, vectors and host cells based thereon

Abstract

The present invention relates to the fields of biotechnology and molecular biology, in particular to a universal inducible promoter, a vector and a host cell based thereon, as well as a method for producing said host cell. The proposed invention makes it possible to analyze the activity of a target protein, such as a receptor ligand, for example a cytokine.

Description

[Technical field]

[0001] The present invention relates to the fields of biotechnology and molecular biology, in particular to a universal inducible promoter, a vector and a host cell based thereon, as well as a method for producing said host cell. The proposed invention makes it possible to analyze the activity of a target protein, such as a receptor ligand, for example a cytokine. [Background technology]

[0002] A variety of therapeutically active polypeptides (eg, monoclonal antibodies, fusion proteins, various cytokines, receptor ligands, etc.) have demonstrated their value as effective pharmaceutical products for the treatment of several disorders and diseases.

[0003] To develop new therapeutically active polypeptides, it is necessary to perform functional tests to determine the biological activity of a given therapeutically active polypeptide. Biological activity tests for therapeutically active polypeptides are used to evaluate candidate pharmaceutical products and are a set of techniques based on analyzing the biological activity of molecules in vitro, using both primary cell cultures and specialized cell lines, or in vivo, using experimental animals. Data from functional tests provide information about the activity of candidate therapeutically active polypeptides.

[0004] Functional tests are divided into the following groups: 1) Testing for specific activities (stimulation and inhibition of cell proliferation, cytotoxicity / apoptosis, antiviral activity, differentiation, migration, etc.); 2) Testing for reporter gene expression.

[0005] Proliferation / cytotoxicity modulation tests are the most common tests for most therapeutically active polypeptides. These tests measure the levels and activity of therapeutically active polypeptides by their ability to increase or decrease cell proliferation. The methods are based on tracking the growth or death of cells in response to a test sample (Banks RE. Measurement of cytokines in clinical samples using immunoassays: problems and pitfalls. Crit Rev Clin Lab Sci. 2000 Apr;37(2):131-82. doi: 10.1080 / 10408360091174187. PMID: 10811142).

[0006] Such tests have been applied in antiproliferative activity assays, where, for example, researchers evaluated the ability of IFN-β-1a to reduce the growth of WISH cells (Renato Mastrangeli et al., In vitro biological characterization of IFN-β-1a major glycoforms, Glycobiology, Vol. 25, No. 1, January 2015, pp. 21-29, https: / / doi.org / 10.1093 / glycob / cwu082).

[0007] Another notable example is the lymphocyte proliferation assay, which is widely used to evaluate cellular immunity (Nikbakht, M. et al., Evaluation of a new lymphocyte proliferation assay based on cyclic voltammetry; an alternative method. Sci Rep 9, 4503(2019). https: / / doi.org / 10.1038 / s41598-019-41171-8). To date, cell proliferation assays are commercially available for multiple cytokines: IFN-γ, IL-1α, IL-1β, IL-2, 3, 4, 5, 6, 7, 8, 10, 13, 15, 19, 21, 33.

[0008] Reporter genes are commonly used in cellular assays to follow receptor-mediated changes in expression at the transcriptional and / or translational levels. These optimized genes are expressed under the control of sensitive sequences in the promoter to which transcription factors bind.

[0009] Phyllis A. Rees and R. Joel Lowy reported the use of reporter cell lines to measure type I interferon as an alternative to time-consuming and labor-intensive proliferation assays (Phyllis A. et al., Measuring type I interferon using reporter gene assays based on readily available cell lines, Journal of Immunological Methods, Vol. 461, 2018, pp. 63-72, ISSN 0022-1759, https: / / doi.org / 10.1016 / j.jim.2018.06.007).

[0010] Jacqueline Mock and colleagues developed a reporter line containing a luciferase gene under the control of an NF-kB-dependent promoter. Analogs were generated from traditional tests, such as the CTLL-2 proliferation assay, an ELISA to detect INF-γ-induced IL-12, and measuring TNF cytotoxicity (Mock J. et al., A universal reporter cell line for bioactivity evaluation of engineered cytokine products. Sci Rep. 2020;10(1):3234. Published February 24, 2020. doi:10.1038 / s41598-020-60182-4).

[0011] Zeuner MT et al. reported the use of a reporter cell line containing an NF-kB-sensitive promoter and a luciferase gene to investigate neuroinflammation. This assay allows the evaluation of pro-inflammatory molecules and peptides, as well as the effectiveness of anti-inflammatory drugs, in neuronal cells (Marie-Theres Zeuner et al., Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation, Mediators Inflamm, 2017, doi: 10.1155 / 2017 / 6209865).

[0012] Wang et al. developed an analogue of the proliferation assay based on a cell line expressing luciferase under the control of SIE (serum response element) (Wang L. et al., Development of reporter gene assays to determine the bioactivity of biopharmaceuticals, Biotechnology Advances (2018), https: / / doi.org / 10.1016 / j.biotechadv.2019.107466).

[0013] Advantages of reporter cells compared to traditional specific activity assays include: 1) Easily reproducible results, automated and scalable; 2) specificity for the target pharmaceutical product; 3) high level of sensitivity, low level of background activation; 4) Ease of use, easy detection of the reporter signal.

[0014] A disadvantage of reporter cells compared to traditional specific activity assays is that they involve a lengthy process of developing individual reporter cells for the target pharmaceutical product, a process that can last up to several months.

[0015] Thus, there is a need for reporter cells that can be used to determine the biological activity of a plurality of different therapeutically active polypeptides, rather than just one individual therapeutically active polypeptide. Summary of the Invention [Problem to be solved by the invention]

[0016] The authors of the present invention have developed a universal inducible promoter, containing binding sites for the transcription factors STAT3, STAT5 and AP-1, as well as a minimal promoter, and vectors and host cells based thereon, which make it possible to determine the biological activity of multiple different therapeutically active polypeptides, rather than just one individual therapeutically active polypeptide. [Means for solving the problem]

[0017] In one aspect, the invention relates to an inducible promoter comprising binding sites for the transcription factors STAT3, STAT5 and AP-1, and a minimal promoter. In some embodiments of the invention, the inducible promoter comprises a binding site for the transcription factor STAT3, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0018] In some embodiments of the invention, the inducible promoter comprises a binding site for the transcription factor STAT5, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0019] In some embodiments of the invention, the inducible promoter comprises a binding site for an AP-1 family transcription factor, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0020] In some embodiments of the invention, the inducible promoter contains binding sites for the transcription factors STAT3, STAT5 and AP-1, which are arranged from the 5' to the 3' end in the following order: STAT3-STAT5-AP-1.

[0021] In some embodiments of the invention, the inducible promoter comprises binding sites for the transcription factors STAT3, STAT5 and AP-1, which are arranged from the 5' end to the 3' end in the following order: STAT3-STAT5-AP-1, and comprises a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0022] In some embodiments of the invention, the inducible promoter comprises binding sites for the transcription factors STAT3, STAT5 and AP-1, which comprise the nucleotide sequence of SEQ ID NO:13. In some embodiments of the invention, the inducible promoter comprises a minimal promoter containing a TATA box.

[0023] In some embodiments of the invention, the inducible promoter comprises a minimal promoter comprising the nucleotide sequence of SEQ ID NO:14. In one aspect, the invention relates to an inducible expression vector comprising, from the 5' to 3' end, any of the inducible promoters described above and a reporter gene.

[0024] In some embodiments of the invention, the inducible expression vector comprises a reporter gene that is the gene for firefly luciferase protein. In some embodiments of the present invention, the inducible expression vector comprises the gene for firefly luciferase protein, which comprises the nucleotide sequence of SEQ ID NO:15.

[0025] In some embodiments of the invention, the inducible expression vector comprises the nucleotide sequence of SEQ ID NO:16. In one aspect, the invention relates to a method for generating a host cell for analyzing the activity of a target protein, said method comprising transforming a cell with any of the inducible expression vectors described above.

[0026] In one aspect, the invention relates to a host cell for analyzing the activity of a target protein, said host cell comprising any of the inducible promoters described above and a reporter gene.

[0027] In some embodiments of the invention, the host cell contains a reporter gene that is the gene for firefly luciferase protein. In some embodiments of the invention, the host cell contains a gene for firefly luciferase protein, which comprises the nucleotide sequence of SEQ ID NO:15.

[0028] In some embodiments of the invention, the host cell comprises the nucleotide sequence of SEQ ID NO:16. In some embodiments of the invention, the host cells are used to assay the activity of a target protein, where the target protein is a receptor ligand.

[0029] In some embodiments of the invention, the host cells are used to assay the activity of a target protein, and the target protein is a cytokine. [Brief description of the drawings]

[0030] [Figure 1]Figure 1 is a diagram of the gene reporter construct. The construct contains the nucleotide sequence of a reporter firefly luciferase protein gene under the control of a STAT3, STAT5, and AP-1 dependent promoter. The non-coding region contains a minimal promoter consisting of a TATA box, at the 5' end of which are located cis-regulatory sequences consisting of alternating binding sites for the transcription factors STAT3, STAT5, and AP-1 as three repeated sequences as shown in the diagram. The flanking sequences are complementary to the region surrounding the expression cassette in the targeting vector and contain recognition sites for the restriction endonucleases Acc65I and BglII. [Diagram 2] Figure 2 is a map of the reporter plasmid vector E6.90_STAT3_STAT5_AP-1_FLuc, which encodes the firefly luciferase gene under the control of STAT3, STAT5, and AP-1 dependent promoters. AmpR is a beta-lactamase gene that confers resistance to ampicillin, allowing selection in E. coli cells, polyA is a polyadenylation signal, transcription terminator, STAT3-STAT5-AP-1 is a regulatory sequence that controls FLuc gene expression, MinP is a minimal promoter containing a TATA box, FLuc is the Photinus pyralis (firefly) luciferase gene, HygR is a gene that confers resistance to hygromycin, allowing selection in eukaryotic cell cultures, and SV40 promoter / enhancer is the eukaryotic promoter / enhancer of the SV40 virus. [Diagram 3]Figure 3 is a map of the expression plasmid vector E4.244_IL10RA_PR, encoding the human IL10RA gene. HS4 is the HS4 insulator, pCMVe / EF1alpha is the constitutive hybrid promoter of CMV / EF1alpha, Kozak is the Kozak sequence, IL10RA is the human IL10RA gene, polyA is the polyadenylation signal, transcription terminator, SV40 enhancer is the SV40 viral enhancer, b-globin MAR is the MAR sequence (M5) of the human b-globin gene, Rep origin 1 is the replication origin site, AmpR is the beta-lactamase gene that confers resistance to ampicillin, which allows selection in E. coli cell culture, F1 origin is the bacteriophage f1 replication origin, and SV40 promoter is the eukaryotic promoter of the SV40 virus. PuroR is eukaryotic resistance to puromycin. This gene confers resistance to puromycin, which allows for selection in eukaryotic cell culture. [Figure 4] Figure 4 is a map of the expression plasmid vector E4.245_IL10RB_BR, encoding the human IL10RB gene. HS4 is the HS4 insulator, pCMVe / EF1alpha is the constitutive hybrid promoter of CMV / EF1alpha, Kozak is the Kozak sequence, IL10RA is the human IL10RA gene, polyA is the polyadenylation signal, transcription terminator, SV40 enhancer is the SV40 viral enhancer, b-globin MAR is the MAR sequence (M5) of the human b-globin gene, Rep origin 1 is the replication origin site, AmpR is the beta-lactamase gene that confers resistance to ampicillin, which allows selection in E. coli cell culture, F1 origin is the bacteriophage f1 replication origin, SV40 promoter is the eukaryotic promoter of the SV40 virus, and BlastiR is eukaryotic resistance to blasticidin. This gene confers resistance to blasticidin, which allows for selection in eukaryotic cell culture. [Diagram 5] FIG. 5 is a histogram showing the degree of activation multiplicity of HEK293 Nf-kB reporter cells in response to various doses of PMA. [Figure 6] Figure 6 shows histograms of the fold increase in activation of reporter clone line HEK293_STAT3-STAT5-AP1_FLuc in response to PMA at a concentration of 33 nM. A2, A4, A5, B1, B3, B5, B6, C4, C6, D1, D4, C4#2, B0>, B1>, B2>, B3> are the names of the various clone reporter lines, initial - untransfected parental cells, pool - cell population after transfection. Pool and untransfected cells were used as controls. [Figure 7] 7 is a histogram showing the degree of activation fold increase of reporter clone line HEK293_STAT3-STAT5-AP1_FLuc expressing IL10R in response to 100 ng / ml IL10. A1.2, A1.4, A1.5, A1.7, C1.1, C1.2, C1.6, C1.15, C1.11, C1.14, C1.16, A4_1, A4_3, A4_9, A4_10, C4_3, C4_12, C4_13, A5, A8, A9, A12, A15, A23, A31, C44, C4.4, C12, C16, C20, C27, C28, C29, C31, C33, C40, and C41 are the names of various clone lines of IL10 reporter. [Figure 8] FIG. 8 is a graph showing the dose-dependent response of HEK293-STAT3-STAT5-AP1_FLuc_IL10R cells to IL10 activation. [Figure 9]Figure 9 is a graph of GAS6-dependent activation of the HEK293_STAT3-STAT5-AP1_FLuc_AXL pool generated based on three different clones (Cl.D1, cl.B3, cl.C4#2) of the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc. GAS6 is an activator of AXL-dependent cell signaling. Cl.D1, cl.B3, cl.C4#2) are clones of the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc. [Figure 10] Figure 10 is a graph showing the activation of the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc_AXL in response to the concentration of various GAS6 products. GAS6 products #1, #2 and #3 are three different Gas6 products. [Figure 11] FIG. 11 is a graph showing activation of the reporter cell line HEK293_AXL_STAT3-STAT5-AP1 as a function of cell incubation time in the presence of GAS6. [Figure 12] FIG. 12 is a graph showing DU145 cell proliferation as a function of GAS6 concentration. [Figure 13] Figure 13 is a graph showing the GAS6-dependent activation of various clones of DU145_STAT3-STAT5-AP1_FLuc (clone C2 in Figure 13, clone H4 in Figure 14, clone E8 in Figure 15). Clones C2, H4 and E8 are clones of the DU145_STAT3-STAT5-AP1 cell line, which were generated based on the DU145 cell line, a natural AXL expresser, using a construct encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter. These clones showed the highest response levels to GAS6 processing. [Figure 14]Figure 14 is a graph showing the GAS6-dependent activation of various clones of DU145_STAT3-STAT5-AP1_FLuc (clone C2 in Figure 13, clone H4 in Figure 14, clone E8 in Figure 15). Clones C2, H4 and E8 are clones of the DU145_STAT3-STAT5-AP1 cell line, which were generated based on the DU145 cell line, a natural AXL expresser, using a construct encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter. These clones showed the highest response levels to GAS6 processing. [Figure 15] Figure 15 is a graph showing the GAS6-dependent activation of various clones of DU145_STAT3-STAT5-AP1_FLuc (clone C2 in Figure 13, clone H4 in Figure 14, clone E8 in Figure 15). Clones C2, H4 and E8 are clones of the DU145_STAT3-STAT5-AP1 cell line, which were generated based on the DU145 cell line, a natural AXL expresser, using a construct encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter. These clones showed the highest response levels to GAS6 processing. [Figure 16] Figure 16 is a graph of GAS6-dependent activation of pools and individual clones of HEK293_AXL_STAT3-STAT5-AP1_FLuc. Cl.1, cl.8, cl.10, cl.14 are clones of the HEK293_AXL_STAT3-STAT5-AP1_FLuc cell line generated from the HEK293_AXL_STAT3-STAT5-AP1_FLuc pool. The pool is the cell line HEK293_AXL_STAT3-STAT5-AP1_FLuc generated from the AXL overexpresser cell line HEK293_AXL, in which the gene encoding firefly luciferase was introduced under the control of a STAT3-STAT5-AP1-dependent promoter. [Figure 17] FIG. 17 is a graph showing the activation of the reporter cell line HEK293_AXL_STAT3-STAT5-AP1 as a function of cell number. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Definitions and general methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0032] Furthermore, singular terms shall include plural terms and plural terms shall include the singular term, unless otherwise required by context. In general, the current classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal chemistry and pharmaceutical chemistry, as well as protein and nucleic acid hybridization and chemical reactions described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are performed according to manufacturer's guidelines, as is common in the art, or as described herein.

[0033] The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence," as used interchangeably herein, refer to an exact sequence of nucleotides, modified or not, defining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0034] As used herein, polynucleotides include, by way of non-limiting example, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR and the like, and synthetic means.

[0035] It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The sequences of the present invention are isolated and / or purified, i.e. they have been sampled directly or indirectly, for example by copying an at least partially modified environment. Thus, isolated nucleic acids, obtained for example by recombinant genetics using a host cell or obtained by chemical synthesis, should also be mentioned here.

[0036] Unless otherwise indicated, the term nucleotide sequence encompasses its complement. Thus, a nucleic acid having a particular sequence should be understood as a nucleic acid that encompasses its complementary strand, with its complementary sequence.

[0037] The term "vector," as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0038] Detailed Description of the Invention Inducible promoters In one aspect, the invention relates to an inducible promoter comprising binding sites for the transcription factors STAT3, STAT5 and AP-1, and a minimal promoter.

[0039] The inducible promoters according to the present invention are versatile for use in reporter cell systems to determine the activity of receptor ligands, such as cytokines. An inducible promoter refers to a promoter that effects gene expression in response to a particular signal.

[0040] The authors of the present invention have surprisingly found that the presence of binding sites for the transcription factors STAT3, STAT5 and AP-1 in an inducible promoter makes it possible to determine the level of expression of a reporter gene in response to a specific signal generated from the interaction between a ligand and its receptor, the receptor ligands being IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, GM-CSF, IL-3, IL-5, IL-6, IL-11, IL-12, IL-27, LIF, OSM, CNTF, CT-1, CLC, NP, leptin, NNT-1 / BSF-3, IFN-α, IFN-β, IFN-γ, IL-23, IL-10, IL-20, IL-22, IL-28, IL-29, IL-1 The inducible promoter may be selected from a number of cytokines and other receptor ligands including, for example, IL-1, IL-24, IL-26, IL37, IL18, M-CSF, CSF1, G-CSF, CSF3, IL-31, IL-35, growth hormone, prolactin, THPO, MGDF, EPO, TSLP, TNFSF18, IL-1, IL-17, TNF, TNF-α, RANKL (OPGL / TRANCE), TRAIL, VEGI, CD153 (CD30L), CD154 (CD40L), CD70 (CD27L), TNF-C, CD70, 4-1BB ligand or GAS6, thus making the inducible promoter versatile for use in reporter cell lines to determine the activity of a ligand, e.g., a cytokine, for a receptor.

[0041] The binding site for the transcription factor STAT3 contains a core region that is TTCnnnGAA. In some embodiments of the invention, the inducible promoter comprises a binding site for the transcription factor STAT3, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0042] The binding site for the transcription factor STAT5 contains a core region that is TTCnnnGAA. In some embodiments of the invention, the inducible promoter comprises a binding site for the transcription factor STAT5, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0043] The binding site for the transcription factor AP-1 contains a core region that is T[G / T]A[C / G]TCA. In some embodiments of the invention, the inducible promoter comprises a binding site for an AP-1 family transcription factor, which is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0044] In some embodiments of the invention, the inducible promoter comprises binding sites for the transcription factors STAT3, STAT5 and AP-1, which are arranged from the 5' to the 3' end in the following order: STAT3-STAT5-AP-1.

[0045] In some embodiments of the present invention, the inducible promoter comprises the binding sites of the transcription factors STAT3, STAT5 and AP-1, which are arranged in the following order from 5' to 3' end: STAT3-STAT5-AP-1, and STAT3-STAT5-AP-1 is represented as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more repeat sequences. The maximum amount of STAT3-STAT5-AP-1 repeat sequence is limited by the capacity of the expression vector. The amount of STAT3-STAT5-AP-1 repeat sequence does not affect the performance of the inducible promoter according to the present invention.

[0046] In some embodiments of the invention, the inducible promoter comprises binding sites for the transcription factors STAT3, STAT5 and AP-1, which are arranged from the 5' end to the 3' end in the following order: STAT3-STAT5-AP-1, and comprises a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0047] In some embodiments of the invention, the inducible promoter comprises binding sites for the transcription factors STAT3, STAT5 and AP-1, which comprise the nucleotide sequence of SEQ ID NO:13. In some embodiments of the invention, the inducible promoter comprises a minimal promoter containing a TATA box.

[0048] A minimal promoter refers to a promoter that has a minimal content of regulatory sequences, including a TATA box (Hogness box) and a transcription start site, and the promoter will not function unless special regulatory sequences are added in front of it.

[0049] In some embodiments of the invention, the inducible promoter comprises a minimal promoter comprising the nucleotide sequence of SEQ ID NO:14. Inducible expression vectors In one aspect, the invention relates to an inducible expression vector comprising, from the 5' to 3' end, any of the inducible promoters described above and a reporter gene.

[0050] The inducible expression vectors according to the invention are versatile for generating reporter cell lines for determining the activity of a ligand, such as a cytokine, for its receptor. The inducible expression vector is a recombinant vector.

[0051] In some embodiments of the present invention, the inducible expression vector comprises a reporter gene that is the gene for luciferase protein. In some embodiments of the invention, the inducible expression vector comprises a reporter gene that is the gene for firefly luciferase protein.

[0052] In some embodiments of the present invention, the inducible expression vector comprises the gene for firefly luciferase protein, which comprises the nucleotide sequence of SEQ ID NO:15. In some embodiments of the invention, the inducible expression vector comprises the nucleotide sequence of SEQ ID NO:16.

[0053] In some embodiments of the invention, the inducible expression vector comprises a reporter gene, which is a gene encoding green fluorescent protein (GFP), or the red fluorescent protein gene (DsRed), or the LacZ gene encoding the enzyme beta-galactosidase, or any other gene encoding a fluorescent or luminescent protein that can be used as a reporter gene.

[0054] In some embodiments of the present invention, the inducible expression vector comprises a heterologous nucleic acid sequence that comprises a regulatory sequence that effects expression of a gene for a reporter polypeptide. "Regulatory sequences that effect expression of products encoded by heterologous nucleic acid sequences in target cells" as used herein means polynucleotide sequences necessary to affect expression and processing of the coding sequence into which it is cloned. Expression control sequences include appropriate transcription initiation, termination, inducible promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and sequences that improve protein secretion, if necessary. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences, and in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "regulatory sequences" encompasses at least all components whose presence is important for expression and processing.

[0055] The term "enhancers" or "enhancer" as used herein can refer to a DNA sequence located adjacent to a DNA sequence that encodes a recombinant product. Enhancer sequences are generally located 5' from a promoter sequence, or can be located downstream or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into a recombinant product or products). Thus, enhancer sequences can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence that encodes a recombinant product. Enhancer sequences can increase the amount of recombinant product expressed from a DNA sequence beyond the expression level associated with a single promoter sequence. Multiple enhancer sequences are readily available to one of skill in the art.

[0056] In some embodiments of the invention, the universal inducible expression vector is an inducible expression vector capable of expressing any of the following: IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, GM-CSF, IL-3, IL-5, IL-6, IL-11, IL-12, IL-27, LIF, OSM, CNTF, CT-1, CLC, NP, leptin, NNT-1 / BSF-3, IFN-α, IFN-β, IFN-γ, IL-23, IL-10, IL-20, IL-22, IL-28, IL-29, IL-19, IL-24, IL-26, IL37, IL18, M-CSF, CSF1, GSK1, GSK2, GSK3, GSK4, GSK5, GSK6, GSK7, GSK8, GSK9, GSK10, GSK11, GSK12, GSK13, GSK14, GSK15, GSK16, GSK17, GSK18, GSK19, GSK20, GSK21, GSK30, GSK40, GSK5, GSK60, GSK70, GSK8, GSK9, GSK15, GSK16, GSK17, GSK18, GSK19, GSK19, GSK20 ...19, GSK20, GSK19, - may be used to generate host cells to be used as universal reporter cell lines to analyze the activity of several different receptor ligands, such as CSF, CSF3, IL-31, IL-35, growth hormone, prolactin, THPO, MGDF, EPO, TSLP, TNFSF18, IL-1, IL-17, TNF, TNF-α, RANKL (OPGL / TRANCE), TRAIL, VEGI, CD153 (CD30L), CD154 (CD40L), CD70 (CD27L), TNF-C, CD70, 4-1BB ligand or GAS6.

[0057] Host cells and methods for producing same In one aspect, the invention relates to a method for generating a host cell for analyzing the activity of a target protein, said method comprising transforming a cell with any of the inducible expression vectors described above.

[0058] In one aspect, the invention relates to a host cell for analyzing the activity of a target protein, said host cell comprising any of the inducible promoters described above and a reporter gene.

[0059] Due to the above-mentioned characteristics of the inducible promoter according to the invention, the host cell according to the invention can be used to express, for example, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, GM-CSF, IL-3, IL-5, IL-6, IL-11, IL-12, IL-27, LIF, OSM, CNTF, CT-1, CLC, NP, leptin, NNT-1 / BSF-3, IFN-α, IFN-β, IFN-γ, IL-23, IL-10, IL-20, IL-22, IL-28, IL-29, IL-19, IL-24, IL-26 ...8, IL-29, IL-28, IL-29, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-49, IL-50, IL-51, IL-52, IL-53, The antibodies may be used to analyze the activity of several different receptor ligands, including L37, IL18, M-CSF, CSF1, G-CSF, CSF3, IL-31, IL-35, growth hormone, prolactin, THPO, MGDF, EPO, TSLP, TNFSF18, IL-1, IL-17, TNF, TNF-α, RANKL (OPGL / TRANCE), TRAIL, VEGI, CD153 (CD30L), CD154 (CD40L), CD70 (CD27L), TNF-C, CD70, 4-1BB ligand, or GAS6.

[0060] The host cell according to the invention is a recombinant host cell. In some embodiments of the invention, the host cell comprises a reporter gene which is a gene for a luciferase protein.

[0061] In some embodiments of the invention, the host cell contains a reporter gene that is the gene for firefly luciferase protein. In some embodiments of the invention, the host cell contains a gene for firefly luciferase protein, which comprises the nucleotide sequence of SEQ ID NO:15.

[0062] In some embodiments of the invention, the host cell comprises the nucleotide sequence of SEQ ID NO:16. In some embodiments of the invention, the host cell comprises a reporter gene, which is a gene encoding green fluorescent protein (GFP), or the red fluorescent protein gene (DsRed), or the LacZ gene encoding the enzyme beta-galactosidase, or any other gene encoding a fluorescent or luminescent protein that can be used as a reporter gene.

[0063] In some embodiments of the invention, the host cells are used to assay the activity of a target protein, where the target protein is a receptor ligand. In some embodiments of the invention, the host cells are used to assay the activity of a target protein, and the target protein is a cytokine.

[0064] In some embodiments of the invention, the host cell is a eukaryotic cell. In some embodiments of the invention, the host cell is a mammalian cell. Mammalian cell lines used as hosts for transformation are well known in the art and include several immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and several other cell lines. The cell line is selected by determining which cell line has high expression levels and provides the required characteristics of the protein produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. Plant host cells include, for example, tobacco, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include species of the genera Escherichia and Streptomyces. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0065] The above host cells of the present invention do not refer to host cells generated using human embryos. The above host cells of the present invention do not refer to host cells produced by modifying the genetic integrity of human germline cells. EXAMPLES

[0066] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any manner.

[0067] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications may be made therein without departing from the spirit or scope of the accompanying embodiments.

[0068] Materials and General Methods Recombinant DNA techniques DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to manufacturer's protocols.

[0069] Gene synthesis The desired gene segments were prepared from oligonucleotides made by chemical synthesis. Gene segments of 300-1400 bp in length flanked by single restriction sites were constructed by annealing and ligation of oligonucleotides containing PCR amplification, followed by cloning via the restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0070] DNA sequencing The DNA sequence was determined by Sanger sequencing. DNA sequence analysis and sequence data management Unipro's UGENE suite version 1.29 and SnapGene Viewer were used for sequence generation, mapping, analysis, annotation and illustration.

[0071] Expression vector The vector containing the reporter gene contained the reporter construct: a gene conferring resistance to the selective antibiotic (ampicillin) in E. coli, in addition to an origin of replication that results in replication of the plasmid in E. coli, as described in the application.

[0072] The genetic constructs of the vectors described below were constructed by PCR and / or gene synthesis and construction using known recombinant methods and techniques, for example by joining the corresponding nucleic acid segments using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger quantities of plasmids were prepared by plasmid preparation from transformed E. coli cultures.

[0073] Example 1. Generation of a reporter plasmid vector encoding the firefly luciferase gene under the control of a STAT3-STAT5-AP-1 sensitive promoter. To generate a universal reporter construct, we chose the transcription factors STAT3, STAT5, and AP-1, which mediate the activity of many different signaling molecules.

[0074] We created a regulatory sequence that contains a minimal promoter consisting of a TATA box and three repeats of the transcription factors STAT3, STAT5, and AP-1 (doi:10.1128 / JVI.01713-10; doi:10.1074 / jbc.M001748200) at the 5' end of the promoter (Figure 1). This combination of transcription factor binding sites allows us to generate inducible promoters with broad applicability.

[0075] To construct the array, we synthesized six 60 bp long partially complementary oligonucleotides with 20 bp overlap. The assembly was performed by synthesis from the oligonucleotides, resulting in a 249 bp long fragment flanked by regions complementary to the plasmid vector. The resulting sequence was cloned into the vector E6.90 by ligation-independent cloning (Figure 2). The cloned nucleic acid sequences were verified by DNA sequencing.

[0076] Example 2. Generation of plasmid vectors encoding IL-10 receptor subunits to create stable expressing cell lines. To evaluate the reporter lines in specific cellular assays, we expressed the IL-10 receptor on the surface of the reporter cells.

[0077] To generate the native sequences of the human IL-10Ra gene (https: / / www.uniprot.org / uniprot / Q13651) and the human IL-10Rb gene (https: / / www.uniprot.org / uniprot / Q08334), we isolated total RNA from peripheral blood mononuclear cells of donors and synthesized cDNA on its basis. The resulting cDNA library was used as a template for PCR amplification of the target sequences flanked by restriction sites. The target genes in expression vectors were cloned by the restriction-ligation method (Figure 3, Figure 4). The cloned nucleic acid sequences were verified by DNA sequencing.

[0078] Example 3. Activation of HEK293 Nf-kB. We screened the clonal reporter lines using the PMA activator, which directly affects protein kinase C and thus triggers a signaling cascade that activates reporter gene expression (https: / / doi.org / 10.1007 / s11373-005-1210-5;doi:10.1093 / carcin / bgm261). To determine the best dose of PMA, we performed experiments involving the activation of the HEK293 Nf-kB reporter line. Cells were cultured at 3*10 per well. 5Cells were seeded in white 96-well culture plates for adherent cultures and PMA activator was added to the wells at the concentrations indicated in the graph, up to 150 μl. All solutions were prepared in DMEM growth medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 10 μg / ml gentamicin. Plates were incubated for 24 hours at 37° C. in a humid atmosphere in the presence of 5% CO2. After incubation, wells were drained and 100 μl of luciferin substrate with soluble components was added, plates were left in the dark for 10 minutes, after which luminescence levels were detected using a plate reader using the SparkControl V2.1 software package (Tecan, Austria).

[0079] Thus, the best concentration of PMA activator was selected as 33 nM (Figure 5). Example 4. Generation of stable reporter cell lines. Important endpoints for the reporter cell line are the degree of fold activation in response to the test molecule and the level of background activation.

[0080] A stable reporter cell line was generated using a HEK293 cell line (ATCC CRL-1573, USA) transfected with a plasmid vector containing the reporter construct of Example 1. The resulting stable pool was cloned by limiting dilution cloning. To screen the clonal lines HEK293_STAT3-STAT5-AP1_FLuc, we performed a luciferase expression activation test. Cells were plated at 1*10 per well in a 96-well plate. 5Cells were seeded and 33 nM PMA activator was added to the wells in a maximum final volume of 150 μl. All solutions were prepared in DMEM growth medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 10 μg / ml gentamicin. Plates were incubated for 24 h at 37° C. in a humid atmosphere in the presence of 5% CO2. After incubation, wells were drained and 100 μl of luciferin substrate with soluble components was added and plates were left in the dark for 10 min, after which luminescence levels were detected using a plate reader using the SparkControl V2.1 software package (Tecan, Austria).

[0081] A reporter cell line was thus generated that exhibits a high degree of fold activation and a low level of background activation (Figure 6). Example 5. Generation of an IL10-specific reporter line.

[0082] To evaluate the reporter lines in a specific cell test, we expressed the IL-10 receptor on the surface of the reporter cells. For this purpose, the HEK293_STAT3-STAT5-AP1_FLuc reporter cell line generated in Example 4 was transfected with a plasmid vector encoding the IL-10 receptor subunit generated in Example 2. To screen the clonal reporter lines expressing the IL10 receptor, we performed the luciferase expression activation test described in Example 3. Recombinant human IL10 at a concentration of 100 ng / ml was used as an activator.

[0083] Screening and selection of clones based on the degree of doubling of activation led to an IL10-specific reporter line based on the universal line (Figure 7). Example 6. IL10 activity determination test Reporter strains are used to evaluate the activity of candidate pharmaceutical products. We carried out luciferase expression activation assays in response to various concentrations of IL10 activators, as shown in the graph. The assay design is described in Example 3.

[0084] We finally observed a dose-dependent response of the reporter cell line to specific activators (Figure 8). Example 7. Creation of the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc_AXL to assess AXL-mediated activation of intracellular signaling by the Gas6 STAT3-STAT5-AP1 product, based on the universal reporter cell line HEK293_STAT3-STAT5-AP1_FLuc.

[0085] To generate the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc_AXL, we used three clones of the universal reporter cell line HEK293_STAT3-STAT5-AP1_FLuc: cl.D1, cl.B3 and cl.C4#2, on the basis of which we generated three pools of cells, each with an embedded and constitutively expressed AXL receptor gene. The AXL-mediated activation of these pools was tested. The tests were carried out in 96-well culture plates designed for luminescence assays. The suspension contained 30,000 HEK293_STAT3-STAT5-AP1_FLuc_AXL cells in each well and the AXL ligand GAS6 at the concentrations shown in FIG. 9. The final volume of the cell suspension in the well was 100 μl, and all the components of the suspension were prepared in DMEM medium without fetal bovine serum. After adding all the components, the plate was incubated at 37°C, 5% CO2 for 16 hours, and then using a luminescence assay kit, we measured the luminescence intensity in the wells. Measurements were performed using a plate reader. The activation level was calculated as the ratio of the luminescence level for a given pool in the presence of GAS6 to the luminescence level in the absence of GAS6.

[0086] Example 8. Testing of different GAS6 products for AXL-mediated activation of STAT3-STAT5-AP1 in the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc_AXL.

[0087] The test was carried out similarly to the procedure described in Example 7 (in the presence of GAS6 at a concentration of 2 μg / ml) and for activation we used HEK293_STAT3-STAT5-AP1_FLuc_AXL (pool generated based on the cell line HEK293_STAT3-STAT5-AP1_FLuc:cl.D1, see Example 7) and various GAS6 preparations at the concentrations indicated in the graph (Figure 10).

[0088] Example 9. Analysis of AXL-mediated activation of HEK293_STAT3-STAT5-AP1_FLuc_AXL clones. From the pools generated on the basis of the cell lines HEK293_STAT3-STAT5-AP1_FLuc:cl.D1, cl.B3 and cl.C4#2 (see Example 7), we selected individual clones that showed GAS6-dependent activation in the test (performed as in Example 7, in the presence of GAS6 at a concentration of 2 μg / ml). The clone that showed the highest level of activation was selected from the cell pool generated on the basis of cl.D1. The activation levels in one of the 96-well plates containing the various clones of a given pool and the initial pool itself are shown in Table 1, which shows the activation relative to non-activated cells, the H11 wells corresponding to the pool. Grey indicates the wells corresponding to the clone HEK293_STAT3-STAT5-AP1_FLuc_AXL (cl.13) that was used for further studies.

[0089] [Table 1]

[0090] Example 10. Study of the activation of the reporter cell line HEK293_STAT3-STAT5-AP1_FLuc_AXL as a function of incubation time in the presence of GAS6. The test was carried out similarly to the procedure described in Example 7, we used cells of the reporter strain HEK293_STAT3-STAT5-AP1_FLuc_AXL (cl.13) and GAS6 at a concentration of 2 μg / ml for activation (Figure 11), after adding all the components, the plate was incubated for the time indicated in the graph, and then the luminescence intensity in the wells was determined. The level of activation was calculated as the ratio of the luminescence value in the presence of GAS6 at said concentration to the luminescence value in the absence of GAS6 variant at the same incubation time. The maximum level of activation was observed during 24 hours of incubation.

[0091] Example 11. Development of a reporter cell line based on the naturally occurring Du145 cell line to assess AXL-mediated activation of STAT3-STAT5-AP1 intracellular signaling.

[0092] To test the activation of Du145 cell proliferation by the AXL ligand GAS6 (Figure 12), we used the Du145 cell line, a natural AXL expressor. The assay was performed in 96-well culture plates for adherent cultures. The suspension contained 5,000 Du145 cells in each well and the AXL ligand GAS6 at the concentrations shown in the graph. The final volume of the cell suspension in the well was 100 μl, and all the components of the suspension were prepared in DMEM medium containing 0.3% fetal bovine serum. After adding all the components, the plate was incubated at 37°C and 5% CO2 for 72 hours, then 10 ml of Alamar Blue reagent was added to each well, then the tablet was shaken on an orbital shaker and incubated at 37°C and 5% CO2 for 6 hours. Fluorescence was measured using a plate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0093] We generated stable clones of Du145 cells containing the STAT3-STAT5-AP1_FLuc gene encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter to generate a reporter cell line sensitive to GAS6-dependent activation of AXL signaling based on the naturally AXL-expressing cell line.

[0094] Clones that showed activation in the test were selected (Figures 13-15). The test was carried out in a 96-well ELISA plate. The day before the experiment, the AXL ligand GAS6 was immobilized on plastic: we prepared a GAS6 solution at a concentration of 5 μg / ml in DPBS and added 100 μL to each plate well. The plate was incubated for 16 hours at 37 °C with 5% CO2. Then we added a suspension containing 50,000 Du145_STAT3-STAT5-AP1_FLuc cells. The final volume of the cell suspension was 100 μL per well. After adding the cell suspension, the plate was incubated for 16 hours at 37 °C with 5% CO2, then using a luminescence assay kit we measured the luminescence intensity in the wells. The measurement was carried out using a plate reader.

[0095] Example 12. Creation of a reporter cell line HEK293_AXL_STAT3-STAT5-AP1_FLuc based on the AXL-overexpressing cell line HEK-293-AXL to assess AXL-mediated activation of STAT3-STAT5-AP1 intracellular signaling.

[0096] To create the reporter cell line HEK293_AXL_STAT3-STAT5-AP1_FLuc, we used the cell line HEK293_AXL, an AXL overexpressor, based on a pool of cells that we generated that contained a gene encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter. We then selected individual clones from this pool that showed activation in the test (Figure 16). The test was carried out at a concentration of GAS6 2 μg / ml, similar to the procedure described in Example 7. The level of activation was calculated as the ratio of the luminescence value for a given clone in the presence of GAS6 to the luminescence value in the absence of GAS6.

[0097] Example 13. Study of the activation of the reporter cell line HEK293_AXL_STAT3-STAT5-AP1 as a function of cell number. Tests were performed similarly to the procedure described in Example 7, with GAS6 at a concentration of 2 μg / ml, and cells of the reporter strain HEK293_AXL_STAT3-STAT5-AP1 were used for activation at the number of cells per well of a 96-well plate as shown in the graph ( FIG. 17 ). The maximum level of activation was observed when the wells contained 25,000-50,000 cells of the reporter strain.

Claims

1. An inducible promoter containing binding sites for the transcription factors STAT3, STAT5 and AP-1, and a minimal promoter.

2. 2. The inducible promoter of claim 1, wherein the binding site for the transcription factor STAT3 is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

3. 2. The inducible promoter of claim 1, wherein the binding site for the transcription factor STAT5 is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

4. 2. The inducible promoter of claim 1, wherein the binding site for an AP-1 family transcription factor is a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

5. 2. The inducible promoter of claim 1, wherein the binding sites for the transcription factors STAT3, STAT5 and AP-1 are arranged in the following order from the 5' end to the 3' end: STAT3-STAT5-AP-1.

6. 6. The inducible promoter of claim 5, wherein the binding sites for the transcription factors STAT3, STAT5, and AP-1 are arranged in the following order from the 5' end to the 3' end: STAT3-STAT5-AP-1, and comprise a nucleotide sequence selected from the group of nucleotide sequences comprising SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

7. 2. The inducible promoter of claim 1, wherein the binding sites for the transcription factors STAT3, STAT5 and AP-1 comprise the nucleotide sequence of SEQ ID NO:

13.

8. The inducible promoter of claim 1 , wherein the minimal promoter comprises a TATA box.

9. The inducible promoter of claim 1 , wherein the minimal promoter comprises the nucleotide sequence of SEQ ID NO:

14.

10. An inducible expression vector comprising, in the 5'- to 3'-direction, the inducible promoter of any one of claims 1 to 9 and a reporter gene.

11. The inducible expression vector of claim 10, wherein the reporter gene is a gene encoding a firefly luciferase protein.

12. 12. The inducible expression vector of claim 11, wherein the gene for the firefly luciferase protein comprises the nucleotide sequence of SEQ ID NO:

15.

13. 11. The inducible expression vector of claim 10, comprising the nucleotide sequence of SEQ ID NO:

16.

14. 11. A method for generating a host cell for analyzing the activity of a target protein, the method comprising transforming the cell with the inducible expression vector of claim 10.

15. A host cell comprising an inducible promoter described in any one of claims 1 to 9, and a reporter gene.

16. 16. The host cell of claim 15, wherein the reporter gene is a gene for firefly luciferase protein.

17. 17. The host cell of claim 16, wherein the gene for the firefly luciferase protein comprises the nucleotide sequence of SEQ ID NO:

15.

18. 16. The host cell of claim 15, comprising the nucleotide sequence of SEQ ID NO:

16.

19. The host cell of claim 15 , wherein the target protein is a receptor ligand.

20. The host cell of claim 19, wherein the receptor ligand is a cytokine.

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