Novel luciferases with improved properties

Abstract

The present invention relates to a novel luciferase gene that exhibits improved characteristics when expressed in cells as a reporter gene, and to a method of using it to detect and optionally quantify the activity of pharmacologically active molecules in a test sample.The present invention further relates to a method for detecting and optionally quantifying neutralizing antibodies against pharmacologically active molecules present in a test sample, using the reporter cell line of the present invention.The present invention further relates to a method for high throughput screening in drug discovery, using said luciferase gene as a reporter gene, for detecting and optimally monitoring biological processes with optimal sensitivity, signal strength and biological fidelity.

Description

[Technical Field]

[0001] The present invention relates to a luciferase gene derived from the naturally occurring marine copepod Metridia longa luciferase gene, reporter gene cell lines incorporating said luciferase as a reporter gene, and methods of using them to detect and optimally quantitate gene expression in a test sample. The present invention further relates to methods for detecting and optimally quantitating the activity of pharmacologically active molecules present in a test sample, as well as neutralizing antibody responses to said molecules, using the reporter cell lines of the present invention. The present invention further relates to a non-toxic method for high-throughput screening in drug discovery that allows multiplexed sampling to detect and optimally monitor biological processes with optimal sensitivity, signal strength, and biological fidelity. Also disclosed are novel vectors that contain both bacterial and mammalian selectable markers, multiple cloning sites, transposon-specific inverted terminal repeats, efficient minimal promoters, and are ideally suited for the expression of a wide range of naturally occurring or engineered reporter genes, including fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), or enzymes such as luciferases, including but not limited to, firefly luciferase, Renilla luciferase, Gaussia luciferase, or the novel luciferase from Metridia longa disclosed herein. [Background technology]

[0002] Bioluminescent and fluorescent reporter genes are widely used to study gene expression and are widely applied in high-throughput screening (HTS) in drug discovery (Inglese et al., 2007) and quantification of drug activity (Lallemand et al., 2010, 2011, 2017). One of the main advantages of luminescence is that, in contrast to fluorescence, it does not require excitation by an external light source, resulting in a high signal-to-background ratio. Bioluminescence assays use the luciferase enzyme, which catalyzes the oxidation of a luciferin substrate to oxyluciferin, resulting in light emission that can be quantified using a luminometer. Naturally occurring luciferases can be divided into two major groups based on the use of D-luciferin / ATP or coelenterazine as substrates (Thorne et al., 2010). Naturally occurring luciferases can be expressed intracellularly, as in the case of firefly luciferase (FL), or secreted, as in Metridia luciferase (Markova et al., 2004). Luciferases, such as FL or Renilla luciferase, have relatively short half-lives compared to fluorescent proteins such as GFP (Corish and Tyler-Smith 1999) and can be used to quantify dynamic changes in reporter gene transcription levels, allowing for quantification of intracellular gene expression or the activity of various extracellular signals, as shown by the present inventors and others (Lallemand et al. 2008, 2010, 2011, 2017). Luciferases can be combined in dual reporter gene assays, whereby the activity of an extracellular signal, such as after interaction of a drug with a cell surface receptor, can be quantified using FL, for example, under the control of a drug-responsive promoter, and results can be normalized to the expression of a second luciferase, such as Renilla luciferase (RL), under the control of a constitutive promoter, either during transient transfection experiments or after stable transfection of cells with both reporter gene constructs (Lallemand et al., 2010, 2011, 2017).This allows, for example, FL to be used to quantify drug activity, and results can be normalized for effects such as differences in cytotoxicity or cell number after quenching of FL expression and quantification of RL levels in the same well of a microtiter plate using commercially available substrates ( Lallemand et al., 2010 , 2011 , 2017 ).

[0003] Naturally occurring luciferases have found widespread application as reporter genes for studying gene expression and in high-throughput screening in drug discovery; however, improved luciferases are needed that ideally should be very bright so that the product of a single-copy gene is easily detectable, exhibit a large dynamic range, be secreted to give low background levels, allow continuous sampling, use a "glo" substrate that does not require the use of a luminometer with an injector, are small in size, facilitate expression in transfected cells, and are non-toxic to cells when expressed at high levels.

[0004] From the above, it can be seen that there is a need for engineered luciferases that exhibit optimal characteristics for use as reporter genes for quantitating the activity of pharmacologically active molecules and for high-throughput screening in drug discovery.

[0005] There is also a need for universal dual bacterial-mammalian expression with both conventional multiple cloning sites and transposon-specific integration sites, and improved minimal promoters. Summary of the Invention

[0006] The present invention has been made in view of the above-mentioned prior art, and an object of the present invention is to provide a novel luciferase gene that is optimized for use as a reporter gene for quantifying the activity of pharmacologically active molecules and for use in high-throughput screening in drug discovery.

[0007] In particular, it is an object of the present invention to provide optimized luciferase genes that encode luciferase proteins that exhibit superior properties compared to those encoded by the native gene, including, but not limited to, one or more of the following: (i): Increased light output (brightness), (ii): enhanced signal stability and / or signal duration as reflected by high relative light units (RLU); (iii): exhibit a large dynamic range compared to control samples in the absence of pharmacologically active substances; (iv): secreted to give low background levels and allow continuous sampling; (v): have a half-life long enough to obviate the need for a luminometer with an injector; (vi): small molecular size and codon optimization to facilitate expression in heterologous transfected cells; (vii): Exhibits enhanced biocompatibility, including at least one of the following characteristics: improved expression in cells, and reduced toxicity when expressed at high levels. (viii): Allows reconstitution as two subunits, SL1-N-Ter and SL-1-C-Ter, separated by a protease cleavage site, circularized during splicing, and cleaved by proteases entering the cell, leading to reconstitution of SL1 and luminescence, and thus used to detect cell death. (ix) allows for division into two subunits, SL1-N-Ter and SL-1-C-Ter, which can reanneal in the presence of second messengers, such as the calmodulin-dependent serine-threonine phosphatase calcineurin, thus catalyzing Ca 2+ Used to detect changes in flux.

[0008] The elements of the present invention provide one or more of the advantages discussed above.

[0009] In various embodiments, the present invention includes novel luciferases present in solution as soluble active monomers or with other proteins, including luciferases, or fluorescent proteins such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and variants thereof that exhibit different excitation / emission spectra, or as fusion proteins with various linker proteins, or attached to solid surfaces such as particles, assay plates, or tubes.

[0010] In order to solve the problems, in one embodiment of the present invention, there is provided a novel luciferase gene comprising at least one of (i) to (iii): (i) an open reading frame encoding a luciferase protein that has been codon-optimized for expression in said cell;

[0011] In certain embodiments, the open reading frame may comprise or consist of a nucleotide sequence set forth in SEQ ID NO:1 and capable of encoding the amino acid sequence set forth in SEQ ID NO:AA1.

[0012] In a further embodiment, the open reading frame may comprise or consist of a nucleotide sequence set forth in SEQ ID NO:2 and capable of encoding the amino acid sequence set forth in SEQ ID NO:AA2. (ii) a signal peptide to ensure efficient secretion from cells, whether of homologous or heterologous origin;

[0013] In one embodiment, the open reading frame comprises or consists of the sequence shown in SEQ ID NO: 3, which may allow for efficient secretion from the cell. (iii) Novel bacterial expression vectors containing a minimal promoter derived from the 5'UTR of the human interferon beta gene, a codon-optimized signal peptide of the Gaussia luciferase gene, and 5' and 3' transposable repeats recognized by a transposase for insertion of a transgene or reporter gene, which may comprise the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a nourseothricin antibiotic resistance gene, or other antibiotic resistance genes such as ampicillin or kanamycin, for example, under the control of the EM7 minimal promoter or a composite CMV T7 promoter that is functional in both bacterial and mammalian cells, and a codon-optimized hygromycin resistance gene comprising or consisting of the sequence shown in SEQ ID NO: 4, or other mammalian antibiotic resistance genes such as neomycin, puromycin, zeocin or blasticidin.

[0014] In one aspect, the bacterial expression vector may be a plasmid which may comprise or consist entirely of the sequence shown in SEQ ID NO:5.

[0015] Thus, in one aspect SEQ ID NO:5 comprises or consists of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4.

[0016] Another aspect of the present invention provides a method for detecting and optionally quantifying the activity of a pharmacologically active molecule in a test sample, the method comprising the steps of: (i) providing a test sample; (ii) contacting the test sample with a cell line according to the present invention, wherein the cell line contains a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a pharmacologically active molecule present in the test sample, operably linked to a downstream promoter sequence, the promoter being operably linked to an open reading frame encoding a first reporter protein comprising one of the sequences set forth in SEQ ID NO: AA1 and SEQ ID NO: AA2. (iii) In a preferred embodiment, to provide for normalization of the assay, the cell line according to the invention further comprises a construct for the constitutive production of a luciferase different from that used in the reporter gene construct responsive to the pharmacologically active molecule. For example, the constitutive production can be the production of a second luciferase, such as Renilla luciferase or firefly luciferase. (iv) determining the activity of the first reporter protein in said cell line using a coelenterazine-based substrate, such as one whose composition is shown in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or any suitable commercially available coelenterazine-based substrate; (v) determining the activity of a second reporter protein in said cell line after measuring the reporter gene luciferase in the same sample using Stop&Glo reagent from the Dual-Glo system (Promega), which efficiently inhibits the activity of a first reporter protein comprising one of the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the absence of coelenterazine. (vi) The activity of a first luciferase normalized to the activity of a second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety. (vii) In a further preferred embodiment, to provide a dual-luciferase assay using different substrates, the cell according to the invention further comprises a double or polycistronic construct operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and a second open reading frame encoding firefly luciferase or any other luciferase that uses luciferin as a substrate, separated by the coding sequence for the self-cleaving 2A peptide F2A set forth in SEQ ID NO: 6. (viii) determining the activity of the first reporter protein in said cell line using a coelenterazine-based substrate, such as one whose composition is shown in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or any suitable commercially available coelenterazine-based substrate; (ix) determining the activity of a second reporter protein in said cell line after reporter gene luciferase has been measured in the same sample using BrightGlo reagent (Promega) or another commercially available luciferin-based substrate; (x) In a further preferred embodiment, to provide a triple luciferase assay using different substrates, the cell of the present invention further comprises three constructs, each operably linked to a different ligand-responsive promoter, that are operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, a second open reading frame encoding firefly luciferase or any other luciferase that uses luciferin as a substrate, and a third open reading frame encoding Renilla luciferase or any other luciferase that uses coelenterazine as a substrate. Alternatively, the Renilla luciferase may be operably linked to a constitutive promoter to enable normalization of the activities of the two ligand-responsive luciferases to the activity of a luciferase under the control of a constitutive promoter as described in U.S. Patent Application Publication No. 2011 / 0189658, the entire contents of which are incorporated herein by reference. (xi) determining the activity of the first reporter protein, the open reading frame of which comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 under the control of a signal peptide ensuring efficient secretion of the first reporter protein into the culture medium or supernatant of the cell line, using a coelenterazine-based substrate, such as one whose composition is set forth in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate; (xii) determining the activity of a second reporter protein requiring a luciferin-based substrate, such as firefly luciferase, in said cell line after cell lysis using DualGlo Reagent (Promega), and then measuring the activity of a third reporter gene, such as Renilla luciferase, requiring a coelenterazine-based substrate, in the same sample using DualGlo Reagent (Promega) or a commercially available two-component substrate, the first component of which is a luciferin-based substrate that allows quantification of firefly luciferase activity, and the second component of which contains a coelenterazine-based substrate that quenches firefly luciferase activity and allows quantification of Renilla luciferase activity. (xiii) In a further preferred embodiment, to provide an improved method for detecting cell death mediated by a protease entering a cell, Svar luciferase is reconstituted as two subunits, SL1-N-Ter and SL-1-C-Ter, separated by a protease cleavage site and comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 7, which encodes SEQ ID NO: AA5, which is circularized during splicing and cleaved by a protease entering the cell, resulting in reconstitution of SL1 and light emission, and therefore quantification of cell death. (xiv) In a further preferred embodiment, activation of intracellular second messengers and the resulting changes in ion flux, such as the calmodulin-dependent serine-threonine phosphatase calcineurin and Ca 2+To provide an improved method for detecting changes in flux, Svar luciferase is reconstituted as two subunits, SL1-N-Ter and SL-1-C-Ter, comprising or consisting of the nucleotide sequence shown in SEQ ID NO:7, which encodes SEQ ID NO:AA:5, which is circularized during splicing and cleaved by a protease entering the cell, resulting in reconstitution of SL1 and light emission, and therefore quantification of cell death. (xv) a protein that can be divided into two subunits, SL1-N-Ter and SL-1-C-Ter, and that comprises or consists of the nucleotide sequence shown in SEQ ID NO:7, which encodes SEQ ID NO:AA:5, and the two subunits can reanneal in the presence of a second messenger, such as the calmodulin-dependent serine-threonine phosphatase calcineurin, thus activating Ca 2+ It can be used to detect changes in flux.

[0017] A further aspect of the present invention relates to a method for high throughput screening in drug discovery, comprising the steps of: (i) providing a test sample consisting of a library of compounds to be screened; (ii) contacting said test sample of (i) with a cell line according to the invention, wherein said cell line contains a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a pharmacologically active molecule present in the test sample, operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein comprising one of the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2. In a preferred embodiment, the cell line according to the invention is seeded into a well plate or assay plate. (iii) Treatment of a cell line according to the invention with an agent used to screen the library. (iv) In a preferred embodiment, to provide for normalization of the assay, the cell line according to the invention further comprises a construct for the constitutive production of a luciferase different from that used in the reporter gene construct. For example, the constitutive production can be the production of a second luciferase, such as Renilla luciferase or firefly luciferase. (v) determining the activity of the first reporter protein in said cell line using a coelenterazine-based substrate, such as one whose composition is shown in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate; (vi) determining the activity of a second reporter protein in said cell line after measuring reporter gene luciferase in the same sample using Bright Glo (Promega), or a commercially available luciferin-based substrate that efficiently inhibits the activity of a first reporter protein comprising one of the sequences shown in SEQ ID NO: AA1 and SEQ ID NO: AA2 in the absence of coelenterazine. (vii) The activity of a first luciferase normalized to the activity of a second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety.

[0018] A further aspect of the present invention is a method for detecting and optionally quantifying neutralizing antibodies to a pharmacologically active molecule present in a test sample, comprising the steps of: (i) providing a test sample containing a pharmacologically active molecule; (ii) providing first and second cell samples, said cell samples comprising a cell line according to the invention; (iii) contacting the first cell sample with a test sample, followed by contacting with a pharmacologically active molecule; (iv) contacting the second cell sample with a pharmacologically active molecule; (iv) determining the activity of the first reporter protein in cells of the first cell sample and determining the activity of the first reporter protein in cells of the second cell sample; (v) providing a ratio between reporter activity in the first and second cell samples, wherein a ratio (first / second) lower than 1 indicates the presence of an antibody to the pharmacologically active molecule in said samples. The present invention relates to a method comprising:

[0019] Consequently, the present invention provides a method for producing a luciferase gene and cell lines transfected with said gene, ECs of cells transfected according to the present invention. 50 The present invention provides for the use thereof in various contexts, which allows for detection of the activity of pharmacologically active molecules with increased sensitivity, such that the activity of a pharmacologically active molecule is at least reduced compared to cells transfected with one or more native genes. The gene may be, for example, a native Metridia longa luciferase gene, but may also include other and / or additional genes. The reduction may be about 2-fold, such as about 3-fold, such as about 4-fold, such as about 5-fold, such as about 6-fold, such as about 7-fold, such as about 8-fold, such as about 9-fold, such as about 10-fold, such as about 50-fold, such as about 100-fold, or such as about 1000-fold. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows a nucleotide sequence alignment of the wild-type Metridia longa luciferase gene and a deletion mutant (nt. 51 to nt. 228) of the 5′ coding region of the gene. [Figure 2] FIG. 1 shows the amino acid sequence of native Metridia longa luciferase, showing the location of the signal peptide and putative catalytic domains 1 and 2. [Figure 3A]Figure 1 shows the response of HEK293 cells after transient transfection of cells with novel Svar vectors encoding the indicated luciferase genes, each under the control of a constitutive CMV promoter, and cotransfection of cells with firefly luciferase under the control of a constitutive thymidine kinase (TK) promoter. Luciferase activity was quantified in cell supernatants after incubating cells at 37°C for 18 hours using a commercially available coelenterazine-based "glo" substrate (QuantiLuc Gold, Invivogen). Cells were then lysed, and firefly luciferase activity was quantified with BrightGlo (Promega). Results are normalized to the expression of firefly luciferase, which does not cross-react with coelenterazine-dependent luciferases. [Figure 3B] Figure 1 shows the response of HEK293 cells after cells were transiently transfected with novel Svar vectors encoding either the wild-type Metridia longa luciferase gene or the Svar luciferase 1 or Svar luciferase 2 genes under the control of a chimeric promoter consisting of an SV40 minimal promoter and a 6-fold tandem repeat of the canonical NFkB recognition sequence, and treated with 100 ng / ml TNFα for 6 hours at 37°C, followed by quantification of the luciferase signal using a commercially available coelenterazine-based "glo" substrate (QuantiLuc Gold, Invivogen). [Figure 4A] Figure 1 shows the coding sequence of the Metridia longa luciferase gene carrying a 177-nucleotide deletion in the 5' translated region of the gene, and a multiple sequence alignment of the coding sequences of four mutant luciferase genes. [Figure 4B]Figure 1 shows a multiple sequence alignment of the coding sequence of the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region of the gene and the coding sequences of four mutant luciferase genes. Nucleotides 1-51 in both the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region and the four mutant luciferase genes represent the nucleotide sequence of the Gaussia luciferase signal peptide. 1. Nucleotide sequence of the coding region of the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region. 2. Nucleotide sequence of the coding region of the SVAR Luc-1 luciferase gene. 3. Nucleotide sequence of the coding region of the SVAR Luc-2 luciferase gene. 4. Nucleotide sequence of the coding region of the SVAR Luc-3 luciferase gene. 5. Nucleotide sequence of the coding region of the SVAR Luc-4 luciferase gene. [Figure 5] Figure 1 shows a multiple sequence alignment of the amino acid sequence of the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region of the gene and the amino acid sequences of four mutant luciferase genes. Amino acids 1-17 in both the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region and the four mutant luciferase proteins represent the amino acid sequence of the Gaussia luciferase signal peptide. 1. Amino acid sequence of the coding region of the Metridia longa luciferase gene with a 177-nucleotide deletion in the 5' translated region. 2. Amino acid sequence of the coding region of the SVAR Luc-1 luciferase gene. 3. Amino acid sequence of the coding region of the SVAR Luc-2 luciferase gene. 4. Amino acid sequence of the coding region of the SVAR Luc-3 luciferase gene. 5. Amino acid sequence of the coding region of the SVAR Luc-4 luciferase gene. [Figure 6A]Figure 1 shows the percent loss of luciferase activity in the supernatant of HEK293 cells after cells were transiently transfected with the indicated luciferase genes, each under the control of a constitutive CMV promoter, and luciferase activity was quantified at increasing times at room temperature using a commercially available coelenterazine-based "glo" substrate (QuantiLuc, Gold Invivogen). [Figure 6B] FIG. 6B shows the relative luciferase activity at 15 minutes for the same samples shown in FIG. 6A. [Figure 7] Figure 1 shows the response of a clonal cell line of HEK293 cells stably transfected with a reporter gene comprising the sequence shown in SEQ ID NO: 1 under the control of a chimeric promoter consisting of an SV40 minimal promoter and a 6-fold tandem repeat of the canonical NFkB recognition sequence, treated with increasing concentrations of TNFα. Panel A. RLU values. Panel B. Fold induction compared to controls not treated with TNFα. [Figure 8] FIG. 1 shows a comparison of the responses of K562 cells stably transfected with either luciferase gene containing the sequence shown in SEQ ID NO: 1 under the control of a chimeric promoter consisting of an SV40 minimal promoter and a 6-fold tandem repeat of the canonical NFkB recognition sequence, and K562 cells stably transfected with a firefly luciferase gene containing an open reading frame encoding a firefly luciferase protein under the control of the same NFkB-responsive promoter, and treatment of the cells with increasing concentrations of TNFα for 6 hours at 37°C. [Figure 9]FIG. 1 shows the response of HEK293 cells after cells were transiently transfected with either a plasmid expressing Svar luciferase 1 under the control of a chimeric promoter consisting of an SV40 minimal promoter and a 6-fold tandem repeat of the canonical NFkB recognition sequence, or pGL4 (Promega), expressing firefly luciferase under the control of the same chimeric promoter, and treated with 200 ng / ml TNFα for 4 hours at 37°C, followed by quantification of Svar luciferase activity using a commercially available coelenterazine-based "glo" substrate (QuantiLuc Plus, Invivogen) or firefly luciferase activity using Bright-Glo (Promega). [Figure 10] The vector contained the EM7 promoter nt. 5072-5137, the codon-optimized hygromycin resistance gene nt. 2532-3557 (Hygro OPTI), the SV40 minimal promoter nt. 2138-2495, and the multiple cloning site nt. 1110-1134 (MCS), and the bacterial replication origin nt. 5954-217 under the control of Svar luciferase-1 (SVL1) nt. 1330-1779, the Gaussia luciferase signal peptide nt. 1276-1329 (GLuc). This figure shows the key features of the new vector (6,325 bp), which contains the codon-optimized coding sequence of the 5'-UTR of the human interferon beta gene (natMx6_OPTI), a minimal promoter from nt. 1141 to 1263, a transposon-specific 5' inverted terminal repeat from nt. 652 to 682, a transposon-specific 3' inverted terminal repeat from nt. 4567 to 4601, and a codon-optimized nourseothricin antibiotic resistance gene from nt. 5138 to 5710 (natMx6_OPTI). Key restriction sites include KpnI, NheI, and XhoI for insertion of a consensus response element, Bcul / SalI for replacement of a mammalian selectable marker, BglII / XbaI for replacement of a reporter gene, and XhoI / BglII for replacement of the minimal promoter sequence. [Figure 11] Table 1 shows the compositions of common coelenterazine luciferase substrates. [Figure 12]Table 2 shows a comparison of the properties of the luciferase encoded by the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and comprising the amino acid sequence encoded by the sequences shown in AA1 and AA2 to the properties of other natural or engineered luciferases reported in the literature. [Figure 13] 1 shows the response of a clonal cell line of HEK293 cells according to the invention transfected with a double or polycistronic construct operably linked to an open reading frame encoding a codon-optimized luciferase reporter protein under the control of a 6-fold tandem repeat of an AP1-responsive promoter, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and a second open reading frame encoding a codon-optimized firefly luciferase separated by the coding sequence for the self-cleaving 2A peptide F2A set forth in SEQ ID NO: 6. Cells were treated with 100 ng / ml PMA for 6 hours at 37° C., after which dual-luciferase activity was quantified. [Figure 14] FIG. 1 shows the N-terminal and C-terminal fragments of circularized Svar luciferase Luc. [Figure 15] FIG. 1 shows functional circularized luciferase Luc-1 when reconstituted.

[0021] Brief description of the nucleotide sequence SEQ ID NO: 1. Codon-optimized SVAR luciferase Luc-1 SEQ ID NO: 2. Codon-optimized SVAR luciferase Luc-2 SEQ ID NO: 3. Gaussia signal peptide, codon optimized and encoding Gaussia signal peptide. SEQ ID NO: 4. Codon-optimized nourseothricin resistance gene SEQ ID NO: 5. Complete nucleotide sequence of the SVAR plasmid SEQ ID NO: 6. Nucleotide sequence of the F2A self-cleaving peptide and the codon-optimized SVAR luciferase Luc-1 upstream of the firefly luciferase gene. Nucleotides 1-54 Gaussia luciferase signal peptide Nucleotides 55-501 Codon-optimized Svar luciferase Luc-1 Nucleotides 517 to 582 encode the F2A self-cleaving peptide Nucleotides 582 to 2235 encode the codon-optimized firefly luciferase SEQ ID NO: 7 encodes the N-terminal and C-terminal fragments of SEQ ID NO: AA5 and thus the circularized luciferase Luc-1.

[0022] A brief description of the amino acid sequence SEQ ID NO: AA1; SVAR luciferase Luc-1 encoded by SEQ ID NO: 1. SEQ ID NO: AA2; SVAR luciferase Luc-2 encoded by SEQ ID NO: 2. SEQ ID NO: AA3: Amino acid sequence of Gaussia signal peptide Sequence number AA4: Amino acid sequences of Gaussia signal peptide (amino acids 1-18), SVAR luciferase Luc-1 (amino acids 19-167), F2A self-cleaving peptide (amino acids 168-194), and firefly luciferase (amino acids 195-745). SEQ ID NO: AA5; the amino acid sequence corresponds to the N- and C-terminal fragments of the circularized luciferase Luc-1 that is functional when reconstituted, as shown in FIG. 14 DETAILED DESCRIPTION OF THE INVENTION

[0023] In describing embodiments of the present invention, specific terminology is used for the sake of clarity, however, the present invention is not intended to be limited to the specific terminology so selected, and it is understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish the same or equivalent purpose.

[0024] The inventors provide, inter alia, an open reading frame derived from the luciferase gene of Metridia longa that encodes a luciferase protein that has improved properties compared to the native protein, is codon-optimized for expression in mammalian cells, and exhibits optimal characteristics for use as a reporter gene for quantification of the activity of pharmacologically active molecules, as well as methods of using it to detect / quantitate antibodies to pharmacologically active molecules and for high-throughput screening in drug discovery.

[0025] In certain embodiments, the present invention relates to an open reading frame which may comprise or consist of an amino acid sequence set forth in SEQ ID NO: AA1, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: AA1, such as at least 80% sequence identity to the sequence set forth in SEQ ID NO: AA1, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity. In one aspect, the nucleotide sequence is capable of encoding an amino acid sequence identical to the sequence set forth in SEQ ID NO: AA1.

[0026] In one embodiment, SEQ ID NO: AA1 MGVKVLFALICIAVAEAKRGKSPGKKLPLAVIMEIEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMEPMEQFIAQVDRCASCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQKEVHNIKGMAGDR may be.

[0027] In one aspect, the present invention relates to an open reading frame which may comprise or consist of a nucleotide sequence as set forth in SEQ ID NO: 1 encoding the amino acid sequence as set forth in SEQ ID NO: AA1, or a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 1, such as at least 80% sequence identity to the sequence as set forth in SEQ ID NO: 1, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0028] In certain embodiments, the present invention relates to an open reading frame comprising or consisting of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: AA2, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: AA2, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity, to the sequence set forth in SEQ ID NO: AA2. In one aspect, the nucleotide sequence is capable of encoding an amino acid sequence identical to the sequence set forth in SEQ ID NO: AA2.

[0029] In one embodiment, SEQ ID NO: AA2 is MGVKVLFALICIAVAEAKRGKSPGKKLPLAVIMEIEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMEPMEQFVAQVDRCASCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQKEVHNIKGMAGDR may be.

[0030] In one aspect, the invention relates to a nucleotide sequence as set forth in SEQ ID NO: 2 encoding the amino acid sequence as set forth in SEQ ID NO: AA2, or a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 2, such as at least 80% sequence identity to the sequence as set forth in SEQ ID NO: 2, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0031] In another aspect, the present invention relates to an open reading frame which may comprise or consist of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: AA1, and which may further comprise or consist of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: AA2.

[0032] In a particular embodiment, the present invention relates to an open reading frame which may comprise or consist of a nucleotide sequence as set forth in SEQ ID NO: 1 and which may encode the amino acid sequence as set forth in SEQ ID NO: AA1, and which may further comprise or consist of a nucleotide sequence as set forth in SEQ ID NO: 2 and which may encode the amino acid sequence as set forth in SEQ ID NO: AA2.

[0033] In a further aspect of the invention, SEQ ID NO: AA3 is MGVKVLFALICIAVAEAK, or a sequence having at least 75% sequence identity to SEQ ID NO: AA3, such as at least 80% sequence identity to the sequence shown in SEQ ID NO: AA3, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0034] In a still further aspect of the invention, SEQ ID NO: AA4 is * or a sequence having at least 75% sequence identity to SEQ ID NO: AA4, such as at least 80% sequence identity to the sequence shown in SEQ ID NO: AA4, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0035] In one aspect, the present invention provides a method for producing a medicament comprising the steps of: MEAEAERGKSPGKKLPLAVIMEIEANAFKAGCTRGCLICLSKIKCTAKMKV YIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMEPMEQFIAQVDR CASCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQKEVHNIKGMAGDR or which may comprise or consist of a sequence having at least 75% sequence identity to SEQ ID NO: AA5, such as at least 80% sequence identity to the sequence shown in SEQ ID NO: AA5, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0036] In another aspect, the present invention relates to a cell or cell line according to the present invention, wherein said cell may comprise a polynucleotide comprising a promoter operably linked to a downstream open reading frame encoding at least one amino acid according to the present invention.

[0037] The cell or cell line may comprise an open reading frame which may comprise or consist of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: AA1, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: AA1, such as at least 80% sequence identity to the sequence set forth in SEQ ID NO: AA1, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity. In one embodiment, the nucleotide sequence is capable of encoding an amino acid sequence identical to the sequence set forth in SEQ ID NO: AA1.

[0038] In another embodiment, the cell or cell line according to the invention may comprise an open reading frame comprising or consisting of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: AA2, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO: AA2, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity, to the sequence set forth in SEQ ID NO: AA2. In one embodiment, the nucleotide sequence is capable of encoding an amino acid sequence identical to the sequence set forth in SEQ ID NO: AA2.

[0039] In another embodiment, the cell or cell line may comprise an open reading frame that may comprise or consist of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:AA1, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO:AA1, and an open reading frame that comprises or consists of a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:AA2, or an amino acid sequence having at least 75% sequence identity to SEQ ID NO:AA2. In one non-limiting example, the cell or cell line may comprise SEQ ID NO:1, which encodes SEQ ID NO:AA1, and SEQ ID NO:2, which encodes SEQ ID NO:AA2.

[0040] The present invention also relates to one or more of SEQ ID NOs: 3 to 7, or any such sequences having at least 75% sequence identity to SEQ ID NOs: 3 to 7, such as at least 80% sequence identity to the sequences shown in SEQ ID NOs: 3 to 7, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity.

[0041] In another aspect, the present invention relates to a cell or cell line which may further comprise or consist of an open reading frame which may comprise or consist of the nucleotide sequence set forth in SEQ ID NO: 6, or any such sequence having at least 75% sequence identity with SEQ ID NO: 6, such as at least 80% sequence identity with the sequence set forth in SEQ ID NO: 6, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, or such as 100% sequence identity. Thus, a cell or cell line according to the invention may comprise one of SEQ ID NO: 1 or 2, or may comprise both SEQ ID NO: 1 or 2, and may further comprise SEQ ID NO: 6.

[0042] In a further aspect, the present invention relates to a cell or cell line which may comprise or consist of an open reading frame which may comprise or consist of at least one or more of the nucleotide sequences set out in SEQ ID NOs: 1 to 4.

[0043] In one aspect, the invention relates to a cell or cell line that may comprise or consist of the nucleotide sequence set forth in SEQ ID NO: 1 and may comprise or consist of an open reading frame that may further comprise one or more of SEQ ID NOs: 3-4, and the entire collected sequence may be set forth in SEQ ID NO: 5.

[0044] In another aspect, the present invention relates to a cell or cell line which may comprise or consist of the nucleotide sequence set forth in SEQ ID NO: 2 and may comprise or consist of an open reading frame which may further comprise one or more of SEQ ID NOs: 3-4.

[0045] In one aspect, the invention relates to the use of the amino acid sequences, or polynucleotides capable of encoding any one of the amino acid sequences of the invention, or cells or cell lines in any diagnostic method.

[0046] In one aspect, the invention relates to the use of an amino acid sequence, or a polynucleotide capable of encoding any one of the amino acid sequences of the invention, or a cell or cell line, for determining the presence of neutralizing antibodies in a biological sample.

[0047] In another aspect, the invention relates to the use of the amino acid sequences, or polynucleotides capable of encoding any one of the amino acid sequences of the invention, or cells or cell lines in drug discovery, such as in screening drugs or drug candidates. In certain aspects, the screening may involve high-throughput screening.

[0048] Thus, the present invention also relates to a method for detecting and optionally quantifying the activity of a pharmacologically active molecule in a test sample, which in one aspect may comprise the steps of: (i) providing a test sample; (ii) contacting said test sample with a cell line according to the invention, wherein said cell line contains a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a pharmacologically active molecule present in the test sample, operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame comprising one of the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2 encoding a first reporter protein; (iii) optionally providing normalization of the assay using a cell line according to the invention further comprising a construct for the constitutive production of a luciferase different from that used in the reporter gene construct responsive to the pharmacologically active molecule in (ii). In one aspect, the constitutive production may be of a second luciferase different from the reporter protein in (ii), which in principle may be any suitable reporter protein, such as Renilla luciferase or firefly luciferase. (iv) determining the activity of the first reporter protein (in (ii)) in said cell line using a coelenterazine-based substrate such as one whose composition is shown in Table 1, or a commercially available substrate, e.g., Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate. (v) determining the activity of the second reporter protein (in (iii)) in said cell line after reporter gene luciferase has been measured in the same sample using a detection system comprising the Stop&Glo reagent from the Dual-Glo system (Promega) or a second component of a dual luciferase buffer containing a coelenterazine-based substrate that quenches firefly luciferase activity and allows subsequent quantification of Renilla luciferase activity, and which, in the absence of coelenterazine, efficiently inhibits the activity of the first reporter protein encoded by one of the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. (vi) The activity of a first luciferase normalized to the activity of a second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated herein by reference in its entirety.

[0049] In a further preferred embodiment, to provide a dual luciferase assay using different substrates, the cells according to the present invention may further comprise a double or polycistronic construct operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and a second open reading frame encoding firefly luciferase or any other luciferase that uses luciferin as a substrate, separated by the coding sequence for the self-cleaving 2A peptide F2A set forth in SEQ ID NO: 6.

[0050] The method according to the invention may also alternatively comprise the following steps: (vii) determining the activity of the first reporter protein in said cell line using a coelenterazine-based substrate such as that whose composition is shown in Table 1, or a commercially available substrate such as Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate. (viii) Determining the activity of a second reporter protein in said cell line after measuring the reporter gene luciferase in the same sample using BrightGlo reagent (Promega) or another commercially available luciferin-based substrate.

[0051] In a further preferred embodiment, to provide a triple luciferase assay using different substrates, the cells of the present invention further comprise three constructs, each operably linked to a different ligand-responsive promoter, that are operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, a second open reading frame encoding firefly luciferase or any other luciferase that uses luciferin as a substrate, and a third open reading frame encoding Renilla luciferase or any other luciferase that uses coelenterazine as a substrate. Alternatively, the Renilla luciferase may be operably linked to a constitutive promoter, allowing the activity of the two ligand-responsive luciferases to be normalized to the activity of a luciferase under the control of a constitutive promoter, as described in U.S. Patent Application Publication No. 2011 / 0189658, the entire contents of which are incorporated herein by reference.

[0052] The method according to the invention may alternatively comprise the following steps: (ix) determining the activity of the first reporter protein, wherein the open reading frame comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 under the control of a signal peptide ensuring efficient secretion of the first reporter protein into the culture medium or supernatant of the cell line, using a coelenterazine-based substrate, such as one whose composition is shown in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate. (x) determining the activity of a second reporter protein requiring a luciferin-based substrate, such as firefly luciferase, in said cell line after cell lysis using DualGlo Reagent (Promega), a commercially available two-component substrate whose first component is a luciferin-based substrate that allows quantification of firefly luciferase activity and whose second component contains a coelenterazine-based substrate that quenches firefly luciferase activity and allows quantification of Renilla luciferase activity; and then measuring the activity of a third reporter gene, such as Renilla luciferase, that requires a coelenterazine-based substrate, in the same sample using DualGlo Reagent (Promega) or another commercially available equivalent.

[0053] As mentioned above, the present invention also relates to a method for high throughput screening in drug discovery, comprising the steps of: (i) providing a test sample consisting of a library of compounds to be screened; (ii) contacting said test sample with a cell line according to the invention, wherein said cell line contains a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a pharmacologically active molecule present in the test sample, operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein comprising one of the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. In one aspect, the cell line according to the invention may be seeded into a 96, 384 or 1536 well assay plate. (iii) Treatment of a cell line according to the invention with an agent or drug candidate used to screen the library. (iv) Optionally, to provide normalization of the assay, the cell line according to the invention further comprises a construct for the constitutive production of a luciferase different from that used in the reporter gene construct and therefore different from the reporter protein encoded by SEQ ID NO: 1 or SEQ ID NO: 2. For example, the constitutive production can be the production of a second luciferase, such as Renilla luciferase or firefly luciferase. (v) determining the activity of the first reporter protein in the cell line using a coelenterazine-based substrate, such as one whose composition is shown in Table 1, or a commercially available substrate, such as Quanti-Luc Gold (InvivoGen), or another commercially available coelenterazine-based substrate. (vi) determining the activity of a second reporter protein in said cell line after measuring reporter gene luciferase in the same sample using Bright Glo (Promega), or a commercially available luciferin-based substrate that efficiently inhibits the activity of a first reporter protein comprising one of the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the absence of coelenterazine. (vii) Determining the activity of the first luciferase normalized to the activity of the second luciferase is described in U.S. Patent Application Publication No. 2011 / 0189658, which is incorporated by reference in its entirety.

[0054] A further aspect of the present invention relates to a method for detecting and optionally quantifying neutralizing antibodies to a pharmacologically active molecule present in a test sample.

[0055] The method according to the present invention may comprise the following steps: (i) providing a test sample containing a pharmacologically active molecule; (ii) providing first and second cell samples, said cell samples comprising a cell line according to the invention; (iii) contacting the first cell sample in (ii) with the test sample (in (i)) and subsequently with a pharmacologically active molecule; (iv) contacting the second cell sample with a pharmacologically active molecule; (iv) determining the activity of the first reporter protein in cells of the first cell sample and determining the activity of the first reporter protein in cells of the second cell sample; (v) providing a ratio between reporter activity in the first and second cell samples, wherein a ratio (1 / 2) lower than 1 indicates the presence of an antibody to the pharmacologically active molecule in said samples.

[0056] definition vector The term "vector" or "vector construct" refers to a DNA molecule used as a vehicle for transferring recombinant genetic material to a host cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. The vector itself is generally a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector transferring genetic information to a host is typically to isolate, propagate, or express the insert in target cells. Vectors called expression vectors (expression constructs) are specifically adapted for expression of heterologous sequences in target cells and generally contain a promoter sequence that drives expression of the heterologous sequence. The choice of vector used in embodiments of the present invention depends on the particular use of the vector encoding a polypeptide or polynucleotide.

[0057] operably linked The term "operably linked" refers to the connection of elements that are part of a functional unit, such as a gene or open reading frame. Thus, by operably linking a promoter to a nucleic acid sequence (open reading frame, ORF) that encodes a polypeptide, the two elements become part of a functional unit, i.e., a gene. Linking an expression control sequence (promoter) to a nucleic acid sequence enables transcription of the nucleic acid sequence as directed by the promoter. By operably linking two heterologous nucleic acid sequences that encode a polypeptide, the sequences become part of a functional unit, i.e., an open reading frame that encodes a fusion protein containing the amino acid sequences encoded by the heterologous nucleic acid sequences. By operably linking two amino acid sequences, the sequences become part of the same functional unit, i.e., a polypeptide. By operably linking two heterologous amino acid sequences, a hybrid (fusion) polypeptide is generated.

[0058] Minimal constitutive promoter The promoter directing the expression of the reporter gene is typically constitutively active in the mammalian host cells used to establish the reporter cell line of the present invention, and does not respond to treatment of the cells with pharmacologically active molecules alone. Useful constitutively active promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter, SV40 promoter, UBC promoter, PGK promoter, human β-actin (hACTB), human elongation factor-1α (hEF-1α), thymidine kinase (TK) promoter, and the cytomegalovirus early enhancer / chicken β-actin (CAG) promoter.

[0059] When describing embodiments of the present invention, not all possible combinations and permutations of embodiments are explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention contemplates all possible combinations and permutations of the described embodiments.

[0060] As used herein, the terms "comprising," "comprise," and "comprises" are intended in all instances to be optionally interchangeable with the terms "consisting of," "consist of," and "consist of," respectively. The present invention will be more fully understood by reference to the detailed description of the invention. However, this should not be construed as limiting the scope of the invention. All literature citations are incorporated herein by reference in their entirety.

[0061] The term "SEQ ID NO: X" is intended to mean the DNA or polynucleotide SEQ ID NO: X. According to the present invention, the definition may refer, for example, to any one of SEQ ID NOs: 1 to 7.

[0062] The term "SEQ ID NO: AAX" is intended to mean the peptide or amino acid sequence SEQ ID NO: X. According to the present invention, the definition may refer, for example, to any one of SEQ ID NOs: AA1-5.

[0063] example The invention will now be further illustrated by the following non-limiting examples.

[0064] To develop luciferase proteins with optimal characteristics for use as reporter genes to quantify the activity of pharmacologically active molecules and for high-throughput screening in drug discovery, it is desirable to make the gene as small as possible to facilitate expression in mammalian cells, especially when the luciferase is expressed as a fusion protein with other proteins or molecules. A series of deletion mutants of the 5' coding region of the native Metridia longa luciferase gene were designed in silico, synthesized in vitro, and tested in transient transfection experiments. The nucleotide sequence of the native Metridia longa luciferase gene is shown in Figure 1, and the amino acid sequence is shown in Figure 2. A deletion mutant containing amino acid 1 of the initiation codon (AUG) through amino acid 76 in the 5' translated region of the native Metridia longa luciferase gene (Figure 1) was found to exhibit activity comparable to that of the native protein, while reducing the size of the luciferase protein by 76 amino acids, corresponding to a reduction in molecular weight of 6.5 kDa, considering that the molecular weight of the SVAR Luc-1 protein is 17.3 kDa compared to the molecular weight of the native Metridia longa protein, 23.8 kDa (Figure 3). To increase the efficiency of secretion, thus reducing background levels of bioluminescence and enabling continuous sampling, we replaced the signal peptide of the luciferase gene from the calanoid copepod Gaussia, which has been shown to be the most efficient signal peptide described to date (Stern, B. et al., 2007), with the native signal peptide of the Metridia longa luciferase gene (Figure 1), encoding 17 amino acids of the signal peptide (Figure 2).To increase secretion efficiency and reduce background levels in cells transfected with a reporter gene construct in response to treatment of cells with pharmacologically active molecules, and thus increase the signal compared to control cells not treated with pharmacologically active molecules, the native signal peptide of the Metridia longa luciferase gene was deleted and replaced with a codon-optimized signal peptide from the gene encoding Gaussia luciferase.

[0065] We used oligonucleotides containing single or multiple mutations to introduce a series of random mutations into the coding sequence of the native Metridia longa luciferase gene (Figure 4). The oligonucleotides were synthesized in vitro and tested in transient transfection experiments in the human HEK293 cell line (ATCC catalog CRL-1573), originally derived from human embryonic kidney cells grown in tissue culture. The specific mutations introduced into the coding sequence of the native Metridia longa luciferase gene were found, individually and together, to encode luciferase proteins superior to those encoded by the native gene in at least one of the following characteristics: enhanced luminescence, including increased light output (brightness), enhanced signal stability and / or signal duration, as reflected by higher relative light units (RLU), and increased dynamic range compared to control samples in the absence of pharmacologically active substances. As shown in Figure 5, in the first putative catalytic domain (Markova, S.V. et al., 2004), it was found that mutating the methionine-encoding nucleotides at positions 66 and 105 to serine (S) and isoleucine (I), respectively, stabilized luminescence, allowing the use of the "glo" substrate and eliminating the need for a luminometer with an injector.

[0066] Mutation of alanine (A) to glutamic acid (E) at position 100 in the second putative catalytic domain of the amino acid sequence encoded by the native Metridia longa luciferase gene (Markova, S.V. et al., 2004) was found to encode luciferase proteins (SVAR Luc-1 with sequence number AA1 and SVAR Luc-2 with sequence number AA2) that exhibited increased luminescence, including increased light output (brightness), increased signal stability and / or signal duration as reflected by higher relative light units (RLU) (Figure 5), and a higher dynamic range compared to control samples in the absence of pharmacologically active substances than those exhibited by the native Metridia longa luciferase gene (Figure 3B).

[0067] It was found that mutating the isoleucine (I) at position 106 in the amino acid sequence to a valine (V) in the first putative catalytic domain resulted in a luciferase protein, SVAR Luc-2 (nucleotide SEQ ID NO:2 and amino acid sequence SEQ ID NO:AA2), that exhibited improved activity compared to native Metridia longa luciferase or luciferase encoded by a Metridia longa luciferase gene having a 177-nucleotide deletion in the 5' translated region, and comparable or slightly reduced activity compared to SVAR Luc-1 luciferase (nucleotide SEQ ID NO:1 and amino acid sequence SEQ ID NO:AA1).

[0068] Mutations from nt 423 to nt 435 in the coding sequence of the native Metridia longa luciferase gene, encoding the amino acids alanine (A) and serine (S) at positions 141 and 142, to the amino acids tyrosine (T) and glycine (G), respectively, were found to encode a luciferase protein (SVAR Luc-3) that exhibited reduced luminescence, including decreased light output (brightness), reduced signal stability and / or signal duration reflected by lower relative light units (RLU) than those exhibited by the native gene, and a lower dynamic range compared to control samples in the absence of pharmacologically active substances than those exhibited by the native Metridia longa luciferase genes, Svar luciferase 1 or Svar luciferase 2 (Figure 3). Similarly, mutations at nt 432 and nt 433, which encode an alanine (A) at position 144 and a methionine at position 145, respectively, in the amino acid sequence encoded by the native Metridia longa luciferase gene, were found to encode a luciferase protein (SVAR Luc-4) that exhibited reduced luminescence, including reduced light output (brightness), reduced signal stability, and / or signal duration, as reflected by lower relative light units (RLU) than those exhibited by the native Metridia longa luciferase genes, Svar Luc-1 or Svar Luc-2 (Figure 3B). Tested in transient transfection experiments in human HEK293 cells at 37°C for 18 hours, followed by quantification of luciferase activity using the commercially available coelenterazine-based "glo" substrate QuantiLuc Gold (Invivogen), both Svar luciferase 1 and Svar luciferase 2 under the control of the strong constitutive CMV promoter exhibited increased signal stability and / or signal duration over that exhibited by wild-type Metridia longa luciferase (Figure 6).Both Svar luciferase 1 and Svar luciferase 2 also exhibited increased light output (brightness), reflected by higher relative light units (RLU) than that exhibited by the native Metridia longa gene, or a Metridia longa gene lacking amino acids 1–76 of the 5′ translated region of the gene, or Svar luciferase 3, or Renilla luciferase, Nano luciferase, or Gaussia luciferase (Figure 3A). Transient transfection experiments in HEK293 cells transfected with the wild-type Metridia longa luciferase gene, Svar luciferase 1, or Svar luciferase 2, each under the control of a chimeric promoter consisting of an SV40 minimal promoter and a six-fold tandem repeat of the canonical NFkB recognition sequence, and treated with or without 100 ng / ml TNFα for 6 h at 37°C showed that both Svar luciferase genes exhibited enhanced luciferase activity compared to the wild-type gene, as reflected by higher relative light units (RLU) using the commercially available coelenterazine-based "glo" substrate QuantiLuc Plus (Invivogen) (Figure 3B).

[0069] HEK293 cells were transfected with the Svar luciferase Luc-1 gene under the control of a chimeric promoter consisting of an SV40 minimal promoter and a six-fold tandem repeat of the canonical NFkB recognition sequence, and stable clones were isolated and characterized. One such clonal cell line, treated with or without increasing amounts of TNFα for 6 hours at 37°C, elicited higher levels of both relative light units (RLU) and fold induction compared with untreated control samples using the commercially available coelenterazine-based "glo" substrate QuantiLuc Gold (Invivogen) (Figure 7).

[0070] K562 cells (ATCC, catalog no. CCL-243), originally derived from chronic myeloid leukemia, were transfected with either the Svar luciferase Luc-1 gene or the firefly luciferase gene under the control of a chimeric promoter consisting of an SV40 minimal promoter and a six-fold tandem repeat of the canonical NFkB recognition sequence, respectively. Stable clones were isolated and characterized. Clonal cell lines were treated with increasing concentrations of TNFα for 6 hours at 37°C (Figure 8). Cells stably transfected with the Svar Luc-1 gene exhibited significantly higher relative light unit (RLU) values ​​than cells stably transfected with the firefly luciferase gene under the control of the same NFkB-responsive promoter after quantification of luciferase activity using the commercially available coelenterazine-based "glo" substrate QuantiLuc Gold (Invivogen) (Figure 8).

[0071] SVAR Luc-1 luciferase exhibits several improved properties compared to other natural or engineered luciferases reported in the literature, including small size, brightness, and the absence of toxicity to transfected mammalian cells (Table 2).

[0072] To develop a plasmid that exhibits optimal characteristics for use as a vector for expression of SvarLuc-1 and SvarLuc-2 luciferase as reporter genes, for quantitation of the activity of pharmacologically active molecules, and for high-throughput screening in drug discovery, including, but not limited to, the nucleotide sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, the plasmid should be able to easily replicate extrachromosomally in laboratory strains of Escherichia coli (E. coli), contain the necessary DNA sequences required for this purpose, and be relatively small in size, on the order of 3,000-6,000 base pairs, to reduce the probability of interference with host factors and increase yield in bacteria. Therefore, a plasmid (pSVAR001) was designed in silico and synthesized in vitro containing the following: a bacterial replication origin (nt. 5954-217), a minimal promoter (nt. 1141-1263) from the 5'UTR of the human interferon beta gene, a signal peptide (SEQ ID NO: 3) from a codon-optimized Gaussia luciferase gene (nt. 1276-1329), and a coding sequence of a reporter gene (nt. 13) including, but not limited to, the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. The plasmid contains a transposon-specific 5' (nt. 652-682) and 3' (nt. 4567-4601) inverted terminal repeats, a recognition sequence for transposase integration of the transgene, a nourseothricin antibiotic resistance gene (nt. 5138-5710) under the control of the EM7 promoter (nt. 5072-5137), and a codon-optimized hygromycin resistance gene (nt. 2532-3557) under the control of an SV40 minimal promoter (nt. 2138-2495) comprising or consisting of the sequence shown in SEQ ID NO: 4. The plasmid further contains several important restriction sites: KpnI, NheI, and Xhol for insertion of a consensus response element, BcuI-SalI for replacement of a mammalian selectable marker, BglI-Xbal for replacement of a reporter gene, including, but not limited to, the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and Xhol-BglII for replacement of the minimal promoter sequence.To test the performance of the pSVAR001 plasmid, a six-fold tandem repeat of the canonical NFkB recognition sequence was inserted into the Kpn-XhoI restriction site, and the resulting plasmid was tested in transient transfection experiments in human HEK293 cells treated or not with 200 ng / ml TNFα for 4 h at 37°C, after which SVAR1 luciferase activity was quantified using a commercially available coelenterazine-based substrate (QuantiLuc Gold, Invivogen). Comparison of the performance of the pSVAR001 plasmid, which contains a 6-fold tandem repeat of the canonical NFkB recognition sequence, with that of the pGL4 plasmid (Promega), which contains the SVAR Luc-1 luciferase gene inserted into the BgIII-Xbal restriction sites under the control of a 6-fold tandem repeat of the canonical NFkB recognition sequence inserted into the Kpn-XhoI restriction sites, showed that the maximum RLU levels obtained with the pSVAR001 plasmid were similar to those obtained with the pGL4 plasmid containing the SVAR Luc-1 luciferase gene under the control of a 6-fold tandem repeat of the canonical NFkB recognition sequence, but the fold induction was significantly greater with the pSVAR001 plasmid (Figure 9).

[0073] References Inglese, J., et al., Nat. Chem. Biol. 3:466-479, 2007 Thorne, N., et al., Chem. Biol. 17:646-657, 2010 Markova, SV., et al., J. Biol. Chem. 279:3212-3217, 2004 Stern, B., et al. Trends Cell Mol. Biol. 2:1-17, 2007 Corish, P., and Tyler-Smith, C., Protein Eng., 12:1035-1040, 1999 Lallemand et al. J. Interferon & Cytokine Res. 28:393-404, 2008.` Lallemand et al. J.Immunol. Methods. 356: 18-28, 2010. Lallemand et al. J.Immunol. Methods. 373: 229-239, 2011 Lallemand et al. J.Immunol. Res. 390: 1-19, 2017

[0074] In certain embodiments, the present invention also relates to the following: 1. (i) A first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a luciferase reporter protein, said open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. (ii) In a preferred embodiment, to provide assay normalization, the cell line according to the invention further comprises a construct for the constitutive production of a luciferase different from that used in the reporter gene construct responsive to the pharmacologically active molecule. For example, the constitutive production can be the production of a second luciferase, such as Renilla luciferase or firefly luciferase, under the control of a constitutive promoter. (xi) In a further preferred embodiment, to provide a triple luciferase assay using different substrates, the cell according to the invention further comprises a dual construct, each operably linked to a different ligand-responsive promoter, wherein the open reading frame encoding a luciferase reporter protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and a second open reading frame encoding firefly luciferase, or any other luciferase that uses luciferin as a substrate, operably linked to a third open reading frame encoding Renilla luciferase, or any other luciferase that uses coelenterazine as a substrate, under the control of a third ligand-responsive promoter, or under the control of a constitutive promoter that allows the activity of the two ligand-responsive luciferases to be normalized to the activity of a luciferase under the control of a constitutive promoter as described in U.S. Patent Application Publication No. 2011 / 0189658. (iii) determining the activity of a first reporter protein comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the open reading frame is under the control of a signal peptide ensuring efficient secretion of the first reporter protein in the culture medium or supernatant of the cell line, using a coelenterazine-based substrate, such as one whose composition is set forth in Table 1, or a commercially available substrate such as Quanti-Luc Gold (InvivoGen). (iv) determining the activity of a second reporter protein requiring a luciferin-based substrate, such as firefly luciferase, in said cell line after cell lysis using Dual-Glo substrate (Promega); measuring the activity of a third reporter gene, such as Renilla luciferase, requiring a celeranthrize-based substrate, in the same sample after cell lysis using DualGlo reagent (Promega), which efficiently inhibits the activity of the second reporter protein, and then quantifying the activity of the third reporter gene. Metazoan cells containing

[0075] 2. A mammalian cell according to any preceding item, wherein the open reading frame comprises or consists of the sequence set out in SEQ ID NO:1 or SEQ ID NO:2, or a sequence having at least 75% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 79% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 85% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 90% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 95% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 97% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 98% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, such as 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2.10. A mammalian cell according to any preceding claim, wherein the open reading frame comprises or consists of the sequence set out in SEQ ID NO:1.

[0076] 3. The cell of any of the preceding items, wherein the cell is selected from the group consisting of HEK293 including all variants, HEK293T, K562, U937, Jurkat, Molt-4, HeLa cells, HT1080 cells, ARPE-19, ARPE-19 / HPV-16, insect cells such as Sf9 cells, avian cells such as DT-40, MSB1 or LMH, mouse cells such as L929 cells or LS variants, Chinese Hamster Ovary (CHO) cells such as CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV cells.

[0077] 4. The cell line of any of the preceding items, wherein the cell line is HEK293.

[0078] 5. A method for detecting and optionally quantifying the activity of a pharmacologically active molecule in a test sample, comprising: (i) providing a test sample; (ii) contacting the test sample with the cell line of any one of the preceding items; (iii) determining the activity of a first reporter protein in said cell line. A method comprising:

[0079] 6. The cell line expresses a second reporter protein and the method comprises: (iv) determining the activity of a first reporter protein in said cell line; (v) providing a ratio between the activity of the first reporter protein and the activity of the second reporter protein. Item 10. The method of any of items 1 to 9, further comprising:

[0080] 7. A method for high throughput screening in drug discovery comprising: (i) providing a test sample consisting of a library of compounds to be screened; (ii) contacting said test sample with a cell line according to the invention, said cell line containing a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence responsive to treatment of the cell line with a pharmacologically active molecule present in the test sample, operably linked to a downstream promoter sequence, said promoter being operably linked to an open reading frame encoding a first reporter protein comprising one of the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2. In a preferred embodiment, the cell lines according to the present invention are seeded into 384 or 1536 well assay plates.

[0081] 8. A plasmid exhibiting optimal characteristics for use as a vector for the expression of reporter genes to quantify the activity of pharmacologically active molecules.

[0082] 9. A plasmid exhibiting optimal characteristics for use as a vector for the expression of a reporter gene, said reporter gene comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0083] 10. A plasmid exhibiting optimal characteristics for use as a vector for the expression of reporter genes in mammalian cells, which contains a codon-optimized hygromycin resistance gene comprising or consisting of the nucleotide sequence set forth in SEQ ID NO:4.

[0084] 11. A plasmid exhibiting optimal characteristics for use as a vector for the expression of reporter genes in mammalian cells, comprising or consisting of the nucleotide sequence shown in SEQ ID NO:5. [Sequence List Free Text]

[0085] Sequence Listing 1 <223> Artificial sequences Sequence Listing 1 <223> SEQ ID NO: 1 Sequence Listing 2 <223> Artificial sequences Sequence Listing 2 <223> SEQ ID NO: 2 Sequence Listing 3 <223> Artificial sequences Sequence Listing 3 <223> SEQ ID NO: 3 Sequence Listing 4 <223> Artificial sequences Sequence Listing 4 <223> SEQ ID NO:4 Sequence Listing 5 <223> Artificial sequences Sequence Listing 5 <223> SEQ ID NO:5 Sequence Listing 6 <223> Artificial sequences Sequence Listing 6 <223> SEQ ID NO:6 Sequence Listing 7 <223> Artificial sequences Sequence Listing 7 <223> Sequence number AA1 Sequence Listing 8 <223> Artificial sequences Sequence Listing 8 <223> Sequence number AA2 Sequence Listing 9 <223> Artificial sequences Sequence Listing 9 <223> Sequence number AA3 Sequence Listing 10 <223> Artificial sequences Sequence Listing 10 <223> Sequence number AA4 Sequence Listing 11 <223> artificial Sequence Listing 11 <223> SEQ ID NO:7 Sequence Listing 12 <223> artificial Sequence Listing 12 <223> Sequence number AA5 Sequence Listing 14 <223> artificial Sequence Listing 15 <223> artificial Sequence Listing 16 <223> artificial Sequence Listing 17 <223> artificial Sequence Listing 18 <223> artificial Sequence Listing 19 <223> artificial Sequence Listing 20 <223> artificial Sequence Listing 21 <223> artificial Sequence Listing 22 <223> artificial Sequence Listing 23 <223> artificial Sequence Listing 24 <223> artificial

Claims

1. One or more nucleotides encoding one or more of the peptide sequences shown in SEQ ID NO:7 and / or SEQ ID NO:

8.

2. 2. The one or more nucleotides according to claim 1, wherein the nucleotide sequence is one or more of the sequences set forth in SEQ ID NO:1 and / or SEQ ID NO:

2.

3. A vector comprising one or more of the sequences shown in SEQ ID NO:1 and / or SEQ ID NO:

2.

4. The vector of claim 3, comprising the sequence shown in SEQ ID NO:

6.

5. 4. The vector of claim 3, comprising an open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and a second open reading frame encoding firefly luciferase or any other luciferase that uses luciferin as a substrate, separated by the coding sequence for the self-cleaving 2A peptide F2A set forth in SEQ ID NO:

6.

6. A cell or cell line comprising the vector according to any one of claims 3 to 5.

7. 7. The cell or cell line of claim 6, wherein the cell or cell line comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, the promoter being operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and further comprising a construct for constitutive production of a second reporter protein different from SEQ ID NO: 7 and SEQ ID NO:

8.

8. The cell or cell line of any one of claims 6 to 7, wherein the vector further comprises SEQ ID NO:3 and SEQ ID NO:

4.

9. 9. The cell or cell line of claim 6, wherein the cell or cell line comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, the promoter operably linked to an open reading frame encoding a luciferase reporter protein, the open reading frame comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, a further construct comprising a heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, the promoter operably linked to an open reading frame encoding a luciferase reporter protein that is a firefly luciferase that requires a luciferin-based substrate, and a further construct comprising a third heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, the promoter operably linked to an open reading frame encoding a luciferase reporter protein that is a Renilla luciferase that requires a coelenterazine-based substrate different from SEQ ID NO: 7 and SEQ ID NO:

8.

10. 10. The cell or cell line according to any one of claims 6 to 9, which is a cell or cell line selected from the group comprising insect cells which are HEK293, HEK293T, K562, U937, Jurkat, Molt-4, HeLa cells, HT1080 cells, ARPE-19, ARPE-19 / HPV-16, Sf9 cells, all variants thereof; avian cells selected from DT-40, MSB1 or LMH; murine cells selected from L929 cells or LS variants; and Chinese hamster ovary (CHO) cells selected from CHO-K1, CHO-DXB11, CHO-DG44, CHOK1SV cells.

11. A kit comprising a cell or cell line according to any one of claims 6 to 10 or a nucleotide according to any one of claims 1 to 2 for use in a diagnostic or drug screening method.

12. 12. The kit of claim 11, wherein the diagnostic application comprises a method for detecting or quantifying the activity of a pharmacologically active molecule in a test sample, and wherein the drug screening method is a high-throughput screening method.

13. 1. A method for detecting or quantifying the activity of a pharmacologically active molecule in a test sample, comprising: i) providing a test sample; ii) contacting the test sample with a cell line according to any one of claims 7 to 10, wherein the cell line comprises a first heterologous polynucleotide comprising a heterologous cis-acting regulatory sequence operably linked to a downstream promoter sequence, the heterologous cis-acting regulatory sequence being responsive to treatment of the cell line with the pharmacologically active molecule present in the test sample; the promoter is operably linked to an open reading frame; the open reading frame encodes a first reporter protein comprising one of the sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8; wherein the open reading frame comprises a further construct for the constitutive production of a second reporter protein different from SEQ ID NO:7 and SEQ ID NO:8; iii) determining the activity of said first reporter protein as set forth in SEQ ID NO: 7 or SEQ ID NO: 8 using a coelenterazine-based substrate; iv) determining the activity of said second reporter protein in said cell line after measuring the fluorescence from said reporter protein of SEQ ID NO: 7 or SEQ ID NO: 8 in the same sample by using a commercially available luciferin-based substrate that efficiently inhibits the activity of said first reporter protein encoded by the sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 in the absence of coelenterazine; v) normalizing the activity of the first reporter protein to the activity of the second reporter protein. A method comprising:

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