Chemical substance evaluation system using multicolor luminescent cells

Abstract

Provided is a method for evaluating the immunotoxicity of a chemical substance to mammals, the method including the following steps. Step 1: A step for bringing the chemical substance into contact with immunotoxicity evaluation mammalian cells produced by introducing a first reporter gene that is under the control of a constitutive expression promoter and a second reporter gene that is under the control of an immunotoxicity evaluation promoter into mammalian cells such that the genes can be transiently or stably expressed. Step 2: A step for evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene and the increase or decrease in expression of the first reporter gene in the presence and absence of the chemical substance.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating immunotoxicity by directly comparing multiple transcriptional activities using cultured mammalian cells into which multiple reporter genes are introduced so that they can be expressed.The present invention also relates to a method for screening chemical substances, particularly drugs, that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity. [Background technology]

[0002] Humanity has discovered or created tens of millions of chemicals to date, and this number is said to be increasing exponentially. Some of these chemicals have harmful effects on the human body, such as allergies, autoimmunity, and immunosuppression, due to their toxic effects on the immune system. Therefore, screening chemicals for their immunotoxic effects is an essential process for minimizing health damage caused by chemical exposure in modern society, where humans live in a chemical-intensive world. Currently, animal testing is the gold standard for immunotoxicity assessment, but the development of in vitro high-throughput and in silico evaluation systems is essential for comprehensively evaluating and managing the tens of thousands of chemicals.

[0003] The present inventors have previously constructed a multi-item immunotoxicity assay (MITA) for chemical substances (Patent Document 1). The MITA includes: 1) 2H4 cells, in which Jurkat cells are transfected with SLG luciferase under the control of the IL-2 promoter, SLO luciferase under the control of the IFN-γ promoter, and SLR luciferase under the control of the G3PDH promoter; 2) THP-G1b cells, in which THP-1 cells are transfected with SLG under the control of the IL-1β promoter and SLR under the control of the G3PDH promoter; and 3) THP-G8 cells, in which THP-1 cells are transfected with SLO under the control of the IL-8 promoter and SLR under the control of the G3PDH promoter. The MITA is a test system capable of high-throughput evaluation of the effects of chemical substances, including drugs, on cytokine production in T cells, macrophages, and dendritic cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2012 / 002507 issue Summary of the Invention [Problem to be solved by the invention]

[0005] Among immunotoxicities, immunosuppression can be caused not only by suppressing cytokine production but also by inhibiting cell proliferation and / or metabolic activity of immunocompetent cells. However, the method of Patent Document 1 was unable to evaluate chemical substances that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity.

[0006] The object of the present invention is to provide a technology for high-throughput evaluation of chemical substances that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity using a cultured cell evaluation system, as an alternative to conventional immunotoxicity evaluation methods using laboratory animals. [Means for solving the problem]

[0007] In order to solve the above-mentioned problems, the present inventors have conducted extensive research and have found that in mammalian cells for immunotoxicity evaluation, in which a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation are introduced into mammalian cells so as to be transiently or stably expressible, the expression level of the second reporter gene relative to the expression level of the first reporter gene, and the expression level of the first reporter gene, can serve as indicators of chemical substances that induce immunosuppression by inhibiting cell proliferation and metabolic activity.

[0008] They also demonstrated that by combining this with an evaluation system that uses the increase or decrease in expression of a second reporter gene as an indicator, chemical substances can be evaluated with greater precision.

[0009] The present invention has been completed based on these findings and includes the following broad aspects.

[0010] [Section 1] 1. A method for assessing the immunotoxicity of a chemical substance to a mammal, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method comprising: [Section 2] Item 2. The method according to Item 1, wherein, in step 2, if the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance, the chemical substance is determined to be highly likely to be a chemical substance having an inhibitory effect on cell proliferation and / or metabolic activity. [Section 3] 3. The method according to Item 1 or 2, wherein, in step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance, it is determined that the chemical substance is unlikely to be a chemical substance having an inhibitory effect on cell proliferation and / or metabolic activity. [Section 4] A method for screening an immunosuppressant, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method comprising: [Section 5] Item 5. The method according to Item 4, wherein, in step 2, if the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance, the chemical substance is determined to be highly likely to be an effective immunosuppressant. [Section 6] Item 6. The method according to Item 4 or 5, wherein, in step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance, it is determined that the chemical substance is unlikely to be an effective immunosuppressant mediated by an inhibitory effect on cell proliferation and / or metabolic activity. [Section 7] A method for screening an anticancer drug, comprising the following steps: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method comprising: [Section 8] Item 8. The method according to Item 7, wherein in step 2, if the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance, the chemical substance is determined to be highly likely to be an effective anticancer agent. [Section 9] Item 9. The method according to Item 7 or 8, wherein, in step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance, it is determined that the chemical substance is unlikely to be an effective anticancer agent mediated by an inhibitory effect on cell proliferation and / or metabolic activity. [Section 10] Step 3: comparing and evaluating the increase or decrease in expression of the second reporter gene in the presence and absence of the chemical substance; Item 10. The method according to any one of Items 1 to 9, further comprising: [Section 11] Item 11. The method according to any one of Items 1 to 10, wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells. [Section 12] 12. The method according to claim 1, wherein the reporter genes are luciferases, and the maximum light emission wavelengths of the reporter genes differ from each other by 20 nm or more. [Section 13] Item 13. The method according to any one of Items 1 to 12, wherein the chemical substance is at least one chemical substance selected from the group consisting of anticancer drugs, drugs that induce immunosuppression by inhibiting cell proliferation or metabolic activity, immunosuppressants, and non-immunosuppressants. [Section 14] Item 8. A kit for evaluating the immunotoxicity of chemical substances in mammals, which is used in the method of Item 1, 4, or 7, and which includes a mammalian cell for immunotoxicity evaluation, into which a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation have been introduced so as to be capable of transient or stable expression. [Effects of the Invention]

[0011] The present invention enables high-throughput evaluation of chemical substances that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity, and also provides a method for screening immunosuppressants and anticancer agents that have cell proliferation and metabolic activity inhibitory effects. DETAILED DESCRIPTION OF THE INVENTION

[0012] According to one aspect of the present invention, there is provided a method for assessing the immunotoxicity of a chemical substance to a mammal, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method is provided, comprising:

[0013] In this specification, mammals include humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, and dogs, with humans being preferred.

[0014] Examples of mammalian cells used in the evaluation method of the embodiment of the present invention include Jurkat cells, U937 cells, and THP-1 cells, with Jurkat cells being preferred.

[0015] The promoter for immunotoxicity evaluation, which is one of the evaluation promoters, may be any gene that is induced in response to a stimulus, such as a cytokine, chemokine, adhesion molecule, costimulatory molecule, or histocompatibility antigen. Examples of cytokines include promoters of genes such as IL-4, IL-2, IL-8, IL-22, IL-12p40, HO-1, IL-1β, and / or IFN-γ, with IL-2 being particularly preferred. Examples of chemokines include promoters of genes such as CXCL1, CXCL2, CXCL3, CXCL10, CCL19, and CCL21. Examples of adhesion molecules include promoters of genes such as CD54 and integrin. Examples of costimulatory molecules include promoters of genes such as CD80, CD86, and PD-L1. Examples of histocompatibility antigens include promoters of genes such as class II MHC. These promoters may be used alone or in combination. A preferred promoter for Jurkat cells is the combination of IL-2 and IFN-γ.

[0016] Other examples of constitutive expression promoters for evaluation include G3PDH, TK, and β-actin, with G3PDH being preferred. While one constitutive expression promoter is typically introduced into a mammalian cell, two or more constitutive expression promoters may also be introduced into the cell. An example of a G3PDH promoter is SEQ ID NO: 1.

[0017] The promoter sequence is exemplified by a sequence 1 kb or more upstream from the vicinity of the transcription start site optimized for immunotoxicity evaluation. More specifically, for example, an IL-2 gene promoter is shown in SEQ ID NO: 2 (putative transcription start site at position 3,003), and an IFN-γ gene promoter is shown in SEQ ID NO: 3 (putative transcription start site at position 4,986). Desirably, the promoter sequence optimized for immunotoxicity evaluation includes a sequence extending from 1 kb upstream from the transcription start site to the transcription start site. The putative transcription start site is defined as the 5'-terminal base of the Reference Sequence of the mRNA of each gene.

[0018] Examples of reporter genes include luciferase, fluorescent proteins, and colored proteins. Examples of luciferase include luciferases derived from various luminous organisms such as sea fireflies, giant shrimp, luminous insects (fireflies, click beetles, etc.), luminous earthworms, sea pansies, and Aequorea victoria (aequorin).

[0019] Fluorescent proteins include genetically modified fluorescent proteins, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), DsRED, and red fluorescent protein (RFP).

[0020] Colored proteins include phycocyanin and phycoerythrin. Among these, luciferase genes are preferred because it is necessary to distinguish and detect signals from two or more, preferably three or more, reporter genes.

[0021] As used herein, expression of a reporter gene refers to the expression of the reporter gene mRNA or protein.

[0022] The following description will be given taking the luciferase gene as an example of the reporter gene.

[0023] It is important to measure the amounts of luminescence from two or more luciferases and calculate their relative ratios, so two or more, preferably three or more, luciferases must emit light whose emission wavelength is substantially independent of the measurement conditions (e.g., pH). Furthermore, if the luciferases emit light that is independent of the measurement conditions and the ratio of their relative light emission amounts can be measured using a color filter or the like, each emission can be quantified from the transmittance of each emission before and after the filter, so two or more (preferably three or more) luciferases that emit light that can be distinguished from each other are required.

[0024] As used herein, "emission wavelength substantially independent of measurement conditions" means that the maximum emission wavelength varies by 3 nm or less, preferably 2 nm or less, more preferably 1 nm or less, and particularly preferably 0.5 nm or less, even when changes in pH, temperature, concentration, etc. are made. If the amount of change in maximum emission wavelength is within this range, it is preferable because when the expression levels of multiple luciferase substances are separated using a filter or the like and quantified, the ratio of the luciferase substances to each other will hardly change.

[0025] When two or more luciferases emit light using the same luciferin, the condition "the emission wavelength is substantially independent of the measurement conditions" is met. When the luciferins used for two or more luciferases are different, it is desirable that the emission wavelengths of all luciferases be substantially independent of the measurement conditions.

[0026] As used herein, "two or more (preferably three or more) luciferases that emit light that can be distinguished from one another" means that the ratio of the relative amounts of light emitted from each luciferase can be measured using, for example, a filter (such as a color filter, bandpass filter, longpass filter, or shortpass filter). For example, the red and green luciferases derived from railroad worms and the orange and green luciferases derived from Iriomote fireflies can be used to measure the ratio of the relative amounts of light emitted from each luciferase, excluding the green luciferases. To be able to measure the ratio of the relative amounts of light emitted from each luciferase, the maximum emission wavelengths are typically separated by at least 20 nm, preferably at least 30 nm, more preferably at least 40 nm, and particularly preferably at least 50 nm, depending on the filter performance and the peak shape of each emission spectrum. With such a large separation in the maximum emission wavelengths, the light emission amounts of each emission can be simultaneously quantified, for example, by using a filter between the maximum wavelengths and measuring and converting the transmittance of each emission before and after the filter.

[0027] Preferred luciferases for use in the present invention include green-to-red luciferases derived from railroad worms (including mutants thereof, maximum emission wavelength: 535-635 nm, e.g., 540-630 nm), orange-to-green luciferases derived from click beetles (including mutants thereof, maximum emission wavelength: 530-600 nm), and orange-to-green luciferases derived from Iriomote fireflies (including mutants thereof, maximum emission wavelength: 550-590 nm). For example, in the case of railroad worms, luciferases with a maximum red emission wavelength of 622 nm and a maximum green emission wavelength of 545 nm are known (US2002 / 0119542-A1). However, the present inventors have confirmed that in addition to these two species, numerous other luciferases exist that emit light in the 540-635 nm range, and all of these luciferases can be used. For example, the present inventors have confirmed that red luciferase derived from railroad worms, which has a maximum emission wavelength of 622 nm (expressed in insects or E. coli), shifts its maximum emission wavelength to 630 nm when expressed in mammalian cells.

[0028] In particular, when using luciferases derived from railroad worms, Iriomote fireflies, and other insects that have multiple luciferases with wavelength maxima that are somewhat different from each other, a single luminescent substrate (e.g., firefly luciferin can be used for luciferases from railroad worms, Iriomote fireflies, and click beetles) can be used to simultaneously quantify the luminescence from multiple co-expressed luciferases, allowing accurate measurement of the expression ratio of each promoter. Furthermore, as luciferases whose emission wavelengths are independent of measurement conditions (e.g., pH), such as blue-emitting Renilla luciferase, various dinoflagellate luciferases (full sequence or including luminescent domains such as domain 1, domain 2, and domain 3; JP 2002-335961; Li L., Hong R., Hasting JW., Proc. Natl. Acad. Sci. USA (1997) 94, 8954), Renilla luciferase, and Gaussia luciferase can also be used in combination. Using luciferases derived from railroad worms, Iriomote fireflies, and click beetles, as well as firefly luciferin, can reduce background noise. Combinations of dinoflagellate luciferase and luciferin also reduce background noise. Furthermore, combinations of Cypridina luciferase and Gaussia luciferase, which secrete luciferase extracellularly, are also preferred because they do not affect background noise, as cell activity is measured in a portion of the culture medium.

[0029] In one preferred embodiment of the present invention, the use of luciferases from railroad worms and Iriomote fireflies allows for quantification of the expression levels of at least three promoters using a single luciferin (e.g., the red luciferase from railroad worms and the orange and green luciferases from Iriomote fireflies) (V.R. Viviani, A. Uchida, N. Suenaga, M. Ryufuku & Y. Ohmiya: Thr-226 is a key residue for bioluminescence spectra determination in beetle luciferases (2001) Biochem. Biophys. Res. Communi. 280, 1286-1291). Furthermore, the use of blue luciferases (renilla, dinoflagellate, or sea firefly luciferases) allows for quantification of four or more promoters. By using appropriate filter settings, multiple expression analyses are possible within the 540-635 nm (green to red) wavelength range, preferably 540-630 nm. Additionally, one type of blue luciferase with a different substrate can be added. Therefore, simultaneous luciferase assays are possible, with three or more luciferases using the same luciferin, or four or more luciferases using different luciferins.

[0030] A preferred embodiment of the present invention involves, for example, increasing the copy number of reporter gene mRNA. For example, changing the cDNA sequence from insect codon usage (biased codon usage) to that of mammals, altering the cDNA sequence to prevent binding of extra transcription factors, and altering the cDNA to address the limitations of its applications due to the large number of restriction enzyme sites. These techniques have also been effective in expressing railroad worm luciferase and Iriomote firefly luciferase in mammalian cells. Changing the codon usage (biased codon usage) to that of mammals and altering the cDNA sequence to prevent binding of extra transcription factors are particularly effective.

[0031] The cDNA sequence can be modified by considering the following points in the order 1) to 4): 1) The amino acid sequence of luciferase should be changed as little as possible (preferably not at all); 2) Next, the cDNA sequence is altered so that extra transcription factors do not bind; 3) Furthermore, in the cDNA sequence, the insect codon usage is changed to that of mammals; 4) Furthermore, the cDNA sequence is modified to eliminate restriction enzyme sites.

[0032] The above describes the expression of luciferase derived from railroad worms and Iriomote firefly luciferase, but the same applies to luciferases derived from other organisms such as click beetles.

[0033] As used herein, the term "luciferase" encompasses a group of luminescent enzymes, such as luciferase, that catalyze the photochemical reaction of luciferin, including aequorin. Furthermore, proteins with weak catalytic activity (the ability to oxidize luciferin and convert it into a luminescent substance), which emit light by changing the structure of luciferin, are also included in the luciferases of the present invention, as long as the emission wavelength is not substantially dependent on the measurement conditions (e.g., pH).

[0034] A combination of two or more luciferases that emit light with the same luminescent substrate is desirable. Preferred luciferases whose emission wavelength does not substantially change depending on the measurement conditions and that emit light with the same luminescent substrate include red luciferase derived from railroad worms and green luciferase derived from railroad worms, or other luciferases derived from railroad worms that have an emission wavelength in the range of about 540 to 635 nm, preferably about 540 to 630 nm, as well as green luciferase derived from Iriomote fireflies and orange luciferase derived from Iriomote fireflies. Other examples include luciferase derived from click beetles (about 530 to 600 nm). Since the luminescence intensities of the red / green luciferase derived from railroad worms and the orange / green luciferase derived from Iriomote fireflies are comparable when the amount of luciferase is the same, these luciferases are particularly suitable for quantifying the transcriptional activity of promoters in multiple ways.

[0035] It is preferable that the at least two luciferase genes emit light of different colors using the same luminescent substrate and have similar intracellular life spans.In this respect, red / green luciferase derived from railroad worms and orange / green luciferase derived from Iriomote fireflies are preferred, and red luciferase derived from railroad worms and orange / green luciferase derived from Iriomote fireflies are particularly preferred.

[0036] Furthermore, it is preferable that the emission colors of at least one, preferably at least two luciferases used in the present invention whose emission wavelengths do not change depending on measurement conditions (e.g., pH) and other luciferases for this standardization can be separated using filters, allowing for simple quantification of each emission color using a device.

[0037] A preferred combination of promoter and luciferase of the present invention is to monitor the constant expression promoter using railroad worm red luciferase and the immunotoxicity evaluation promoter using Iriomote firefly orange or green luciferase. The red, orange, and green luciferases can be interchanged.

[0038] As the luciferase gene of the present invention, a wild-type or mutant luciferase gene can be used as is, or DNA that can hybridize with the luciferase gene under stringent conditions, or DNA in which one or more amino acids of the luciferase have been substituted, added, deleted, or inserted and that encodes a polypeptide having luciferase activity, can be used as the luciferase gene.

[0039] Through investigation of various expression systems, the present inventors have found that for stable expression of luciferase in mammalian cells, it is important to introduce elements that increase translation efficiency and / or mRNA stabilizing elements into a gene construct. Accordingly, in one preferred embodiment, an example of an element that increases translation efficiency is the Kozak sequence (Ko), and an example of an mRNA stabilizing element is the β-globin intron II. Furthermore, the inventors have confirmed that changing the cDNA sequence from insect codon usage (biased codon usage) to that for mammals, and further changing the cDNA sequence to prevent the binding of extra transcription factors, are also preferable for stable expression of luciferase in mammalian cells.

[0040] In one preferred embodiment, the gene construct of the present invention comprises a luciferase gene, a promoter upstream of the gene, an element that improves translation efficiency, and / or an mRNA stabilizing element, and further comprises an enhancer, an IRES, SV40pA, a drug resistance gene (Neo r etc.).

[0041] Examples of preferred genetic constructs of the present invention are shown below. (1) (Promoter for immunotoxicity evaluation)-(Kozak sequence)-(Red, Green, or Orange luciferase)-(SV40 poly(A) sequence) (2) (Promoter for immunotoxicity evaluation)-(Kozak sequence)-(Red, Green, or Orange luciferase)-(IRES)-(Neo r Drug resistance genes (e.g., SV40 polyA sequence)

[0042] The gene construct of the present invention may be introduced directly into mammalian cells, but is preferably incorporated into a vector (including, for example, a plasmid or viral vector) and then introduced into mammalian cells. When multiple luciferases are incorporated into a gene construct so that they can be expressed, it is sufficient to introduce one gene construct or expression vector into mammalian cells, but when one luciferase is incorporated into one gene construct, it is sufficient to introduce multiple gene constructs or expression vectors into mammalian cells simultaneously or sequentially according to standard methods.

[0043] In one embodiment, the immunotoxicity of a chemical substance can be evaluated by assessing whether the chemical substance inhibits cell proliferation or metabolic activity, or both, in mammalian cells for immunotoxicity evaluation that have been contacted with the chemical substance. The evaluation is based on whether the following two items are reduced when the mammalian cells for immunotoxicity evaluation are contacted with the chemical substance compared to when the cells are not contacted with the chemical substance (hereinafter, these may be referred to as IL-2 Luc LTT): (A) Ratio of mRNA or protein expression level of the second reporter gene under the control of a promoter for immunotoxicity evaluation to that of the first reporter gene under the control of a constant expression promoter. (B) Expression level of mRNA or protein of the first reporter gene under the control of a constitutive promoter If (A) of the two items above increases and (B) decreases, this indicates that the chemical substance has cell proliferation inhibition and / or metabolic activity inhibition. Therefore, it can be determined that the chemical substance is highly likely to be immunotoxic. Furthermore, if (A) of the two items above remains constant or decreases and / or (B) remains constant or increases, it is determined that the chemical substance does not have cell proliferation inhibition and / or metabolic activity inhibition. Therefore, it can be determined that the chemical substance is unlikely to be immunotoxic due to cell proliferation inhibition and / or metabolic activity inhibition.

[0044] The chemical substance to be evaluated by the evaluation method of the embodiment of the present invention is not particularly limited, but examples thereof include at least one chemical substance selected from the group consisting of anticancer drugs, drugs that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity, immunosuppressants, and non-immunosuppressants.

[0045] Examples of anticancer drugs include bleomycin, etoposide, 5-fluorouracil, taxol, etc. Examples of drugs that induce immunosuppression by inhibiting cell proliferation and / or metabolic activity include azathioprine, methotrexate, mizoribine, rapamycin, etc. Examples of immunosuppressants include dexamethasone, cyclosporine, minocycline, chloroquine, etc. Examples of non-immunosuppressants include acetaminophen, indomethacin, warfarin, pravastatin, etc.

[0046] In a preferred embodiment, the method for evaluating chemical substances that inhibit cell proliferation and / or metabolic activity is carried out using Jurkat cells in a long-term stable culture, in which a gene containing a multicolor luciferase is introduced downstream of the gene promoter described below. Specifically, Toyobo's red luciferase vector (pSLR), orange luciferase vector (pSLO), and green luciferase vector (pSLG) can be preferably used. For Jurkat cells, a stable cell line, Jurkat2H4, can be produced by using a group of plasmids in which the G3PDH gene promoter is inserted into pSLR, the IL-2 gene promoter is inserted into pSLG, and the IFN-γ gene promoter is inserted into pSLO. The promoter sequences for the IL-2 gene and the IFN-γ gene include 1 kb, preferably 3 kb or more, upstream of the transcription start site. More specifically, for the IL-2 gene promoter, the sequence 3 kb upstream is set forth in SEQ ID NO: 2, and the transcription start site is predicted to be around position 3,003; therefore, 1 kb upstream refers to the sequence from around position 2,003 downstream, and 3 kb upstream refers to the sequence from around position 3 downstream. On the other hand, for the IFN-γ gene promoter, the sequence 5 kb upstream is set forth in SEQ ID NO: 3, and the transcription start site is predicted to be around position 4,986; therefore, 1 kb upstream refers to the sequence from around position 3,986 downstream, and 3 kb upstream refers to the sequence from around position 1,986 downstream.

[0047] In a preferred embodiment, evaluation of chemical substances involves treating or contacting mammalian cells for immunotoxicity evaluation with a chemical substance at a concentration ranging from 0.1 ng / mL to 2 mg / mL for 18 to 30 hours, stimulating them with PMA (phorbol 12-myristate 13-acetate) and ionomycin, culturing them for 5 to 10 hours, quantifying the luciferase activities of three colors: orange luciferase (SLO), green luciferase (ISLR), and orange luciferase (SLG), and comparing the results with those of cells not contacted with the chemical substance in terms of (A) and (B). More preferably, evaluation is based on the following indicators. In (A-1) and (B-1), "nIL2LA" refers to the value obtained by normalizing SLG luciferase activity (IL2LA) with SLR luciferase activity (GAPLA). (A-1) % suppression = {(nIL2LA in chemical-untreated 2H4 cells) - (nIL2LA in chemical-treated 2H4 cells)} × 100 / (nIL2LA in chemical-untreated 2H4 cells) (B-1) Inh-GAPLA = (GAPLA in chemical-treated 2H4 cells) / (GAPLA in chemical-untreated 2H4 cells) Here, if the minimum values ​​of (A-1) and (B-1) are both below the reference value, the chemical substance is determined to have cell proliferation inhibition and metabolic activity inhibition. Furthermore, if at least one of the above two items is above the reference value, the chemical substance is determined not to have cell proliferation inhibition and metabolic activity inhibition. The reference value for (A-1) % suppression is preferably set in the range of -50% to -20%, more preferably -40% to -30%, and particularly preferably -35%. The reference value for (B-1) Inh-GAPLA is preferably set in the range of 0.5 to 0.9, more preferably 0.6 to 0.8, and particularly preferably 0.7.

[0048] Furthermore, in the above evaluation method, chemical substances for which the (A-1) % suppression value is within the range of -35% to 35% and the (B-1) Inh-GAPLA value is within the range of 0.7 to 1.0 may be water-insoluble chemical substances and can therefore be judged as "unevaluable."

[0049] Furthermore, the reliability of the evaluation method can be confirmed using the SLO luciferase activity (IFNLA) of chemical-untreated 2H4 cells.

[0050] In a preferred embodiment, in addition to the evaluations (A) and (B) (preferably (A-1) and (B-1)) above, the following evaluation (C) is also performed, allowing for more accurate evaluation of chemical substances. When mammalian cells for immunotoxicity evaluation are contacted with a chemical substance, the evaluation is performed based on whether or not there is an increase in the following item (C) compared to when the chemical substance is not contacted. Evaluation (C) is sometimes referred to as the IL-2 Luc assay. (C) A step of comparing and evaluating the increase or decrease in expression of the second reporter gene in the presence and absence of the chemical substance.

[0051] If the above item (C) increases (especially increases significantly), it can be determined that the chemical substance is highly likely to be immunotoxic. If it decreases or remains constant, it can be determined that the chemical substance is less likely to be immunotoxic.

[0052] When using the above-mentioned 2H4 cells, evaluation is performed by treating the cells with a chemical at a concentration ranging from 0.1 ng / mL to 2 mg / mL, followed by stimulation with PMA and ionomycin. After 6 to 12 hours of incubation, the luciferase activity of the three colors SLO, SLR, and SLG is quantified and compared with that measured without chemical exposure. More preferably, evaluation can be performed using the (A-1)% suppression value, and immunosuppression is determined to be present if the value is equal to or exceeds a separately established standard value. The standard value in this case is preferably set in the range of 20% to 50%, more preferably in the range of 30% to 40%, and particularly preferably 35%.

[0053] In the above method, if the value is equal to or less than a separately set reference value, it can be determined that the compound has an immunopotentiating effect. The reference value in this case is preferably set in the range of -20% to -50%, more preferably in the range of -30% to -40%, and particularly preferably -35%.

[0054] According to another aspect of the present invention, there is provided a method for screening an immunosuppressant, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method is provided, comprising:

[0055] The mammalian cells for immunotoxicity evaluation, two or more reporter genes, constitutive expression promoters, and promoters for immunotoxicity evaluation are as explained above in relation to the method for evaluating the immunotoxicity of chemical substances to mammals.

[0056] In step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is increased compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance, and the expression level of the first reporter gene in the presence of the chemical substance is decreased compared to the expression level of the first reporter gene in the absence of the chemical substance, this indicates that the chemical substance has an inhibitory effect on cell proliferation and / or metabolic activity, and therefore it can be determined that the chemical substance is likely to be an effective immunosuppressant.

[0057] In step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical, and / or the expression level of the first reporter gene in the presence of the chemical is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical, it can be determined that the chemical is unlikely to be effective as an immunosuppressant mediated by cell proliferation inhibitory effects and / or metabolic activity inhibitory effects.

[0058] According to another aspect of the present invention, there is provided a method for screening an anticancer agent, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; and Step 2: evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; A method is provided, comprising:

[0059] The mammalian cells for immunotoxicity evaluation, two or more reporter genes, constitutive expression promoters, and promoters for immunotoxicity evaluation are as explained above in relation to the method for evaluating the immunotoxicity of chemical substances to mammals.

[0060] In step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is increased compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance, and the expression level of the first reporter gene in the presence of the chemical substance is decreased compared to the expression level of the first reporter gene in the absence of the chemical substance, this indicates that the chemical substance has an inhibitory effect on cell proliferation and / or metabolic activity, and therefore it can be determined that the chemical substance is highly likely to be an effective anticancer drug.

[0061] In step 2, if the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance, and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance, it can be determined that the compound is unlikely to be an effective anticancer drug mediated by an inhibitory effect on cell proliferation and / or metabolic activity.

[0062] Another aspect of the present invention provides a kit for evaluating the immunotoxicity of chemicals in mammals. The kit of the present invention includes mammalian cells for immunotoxicity evaluation, which are prepared by transiently or stably expressing a first reporter gene under the control of a constitutive expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation. The kit may further include PMA (phorbol 12-myristate 13-acetate) and ionomycin. PMA and ionomycin are preferably included in the kit because they have the effect of activating cells. The kit may also include other reagents necessary for immunotoxicity evaluation, and tools such as well-equipped plates and tubes. Such a kit can be used in the method for evaluating the immunotoxicity of chemicals in mammals according to an embodiment of the present invention. [Example]

[0063] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

[0064] <List of chemical substances used in the study> Table 1 shows a list of chemical substances examined in the examples.

[0065] [Table 1]

[0066] Example 1. Establishment of 2H4 cells 2H4 cells were established from Jurkat cells according to the method described in Patent Document 1. To generate 2H4 cells, we first constructed a plasmid carrying SLG downstream of the IL-2 promoter as an internal control, a plasmid carrying SLR downstream of the G3PDH promoter, and a plasmid carrying SLO downstream of the INF-γ promoter. These plasmids contained hygromycin B, puromycin, and neomycin resistance genes downstream of SLG, SLR, and SLO, respectively. After transfection into the host Jurkat cells, drug selection was performed for one week in the presence of drug. Surviving the primary selection were seeded at 1 cell / well or 200 cells / well in 96-well plates and cultured under the same drug conditions for two weeks. After the secondary selection, clones were selected based on signal intensity and signal induction upon drug treatment. First, we transfected SLR into the G3PDH promoter, establishing a single-color stable Jurkat:G3PDH-SLR #14D1 cell line. We then used this parent line to establish the two-color stable Jurkat:INFγ-SLO / G3PDH-SLR #2B12 cells by introducing the INF-γ promoter SLO, and then to establish the three-color stable Jurkat:IL-2-SLG / INFγ-SLO / G3PDH-SLR #2H4 cells by introducing the IL-2 promoter SLG.

[0067] Example 2. Chemical Evaluation in 2H4 Cells (IL-2 Luc LTT) The chemicals listed in Table 1 were evaluated using 2H4 cells. 2H4 cells were cultured at 1 × 10 4 The cells were seeded at 1 / well in a 96-well plate, treated with chemicals for 1 hour, and then incubated with 25 nM PMA and 1 μM ionomycin for 24 hours. After 24 hours of PMA / ionomycin treatment, Tripluc (R)Assay Reagent (Toyobo Co., Ltd.) was added, and luminescence was measured using a Phelios microplate luminometer (ATTO Corporation). As a control, 2H4 cells untreated with chemicals were also examined in the same manner. The luminescence intensity of SLG luciferase activity (IL2LA), SLR luciferase activity (GAPLA), and SLO luciferase activity (IFNLA) was measured, and IL-2LA and IFNLA were normalized to GAPLA to obtain nIL2LA and nIFNLA, respectively. Then, for each concentration of chemical, % suppression and Inh-GAPLA were calculated using the following formulas (1) and (2), and the minimum values ​​(Min % suppression, Min Inh-GAPLA) were used to evaluate the chemicals.

[0068] During the evaluation, nIL2LA was also measured for PMA / Ionomycin-treated 2H4 cells and PMA / Ionomycin-untreated 2H4 cells as controls, and if the ratio of nIL2LA in PMA / Ionomycin-treated 2H4 cells to nIL2LA in PMA / Ionomycin-untreated 2H4 cells was less than 3.0, the results were not used.

[0069] % suppression = {(nIL2LA in chemical-untreated 2H4 cells) - (nIL2LA in chemical-treated 2H4 cells)} × 100 / (nIL2LA in chemical-untreated 2H4 cells) (1) Inh-GAPLA = (GAPLA in chemical-treated 2H4 cells) / (GAPLA in chemical-untreated 2H4 cells) (2)

[0070] Example 3. Evaluation of chemicals in 2H4 cells (IL-2 Luc assay) The chemicals listed in Table 1 were evaluated using 2H4 cells. 2 × 10 5Seeded in a 96-well plate at 50 μl / well, treated with the chemical substance for 1 hour, and then incubated with 25 nM PMA and 1 μM ionomycin for 24 hours. Six hours after PMA / Ionomycin treatment, Tripluc (R) Assay Reagent (manufactured by Toyobo Co., Ltd.) was added, and the luminescence was measured using a microplate type luminometer Phelios (manufactured by ATTO Corporation). Also, as a control, 2H4 cells not treated with the chemical substance were examined in the same manner. The luminescence of each of the SLG luciferase activity (IL2LA), SLR luciferase activity (GAPLA), and SLO luciferase activity (IFNLA) was measured, and IL-2LA and IFNLA were normalized with GAPLA, respectively, to obtain nIL2LA and nIFNLA. Then, for each concentration of the chemical substance, the numerical value of % suppression was calculated using the above formula (1), and the chemical substance was evaluated using the minimum value (Min % suppression).

[0071] <Evaluation method> IL-2 Luc LTT Min % suppression ≤ -35 and Min Inh-GAPLA < 0.7: Leukocyte toxic (abbreviated as LCT; determined to have cell growth inhibition and / or metabolic activity inhibition) -35 < Min % suppression < 35 and 0.7 ≤ Min Inh-GAPLA: Inconclusive (abbreviated as I; may be unmeasurable due to low water solubility) Otherwise: Non-leukocyte toxic (abbreviated as NLCT; determined to have no cell growth inhibition and metabolic activity inhibition)

[0072] IL-2 Luc assay Min % suppression ≥ 35: Suppression (abbreviated as S; determined to have an immunosuppressive effect) Min % suppression ≤ -35: Augmentation (abbreviated as A; determined to have an immunostimulatory effect) -35 < Min % suppression < 35: No effect (denoted as N; determined not to affect immunity)

[0073] Combination Evaluation Positive (Pos): Determined to be an immunosuppressive substance if it meets either or both of the conditions of being determined as LCT in the IL-2 Luc LTT or being determined as S in the IL-2 Luc assay Negative (Neg): Determined to be a non-immunosuppressive substance if it does not meet the above conditions

[0074] <Evaluation Results> The results are shown in Table 2. Mzoribine, Mycophenolic acid, and Rapamycin, which could not be evaluated by the IL-2 Luc assay alone (not determined to have an immunosuppressive effect), were correctly determined to be chemicals with cell growth inhibition and / or metabolic activity inhibition in the IL-2 Luc LTT. Looking at the evaluation results for the anticancer agents or chemicals No. 1 - 19, which are immunosuppressive agents with cell growth inhibition and / or metabolic activity inhibition, there were 12 chemicals correctly determined as LCT in the IL-2 Luc LTT, which is more accurate than the results of the IL-2 Luc assay (5 chemicals). Also, when combining the IL-2 Luc LTT and the IL-2 Luc assay, for 28 chemicals No. 1 - 28, 24 were determined to have immunotoxicity, enabling evaluation with higher accuracy than the 14 chemicals with the IL-2 Luc LTT alone and the 15 chemicals with the IL-2 Luc assay alone.

[0075]

Table 2

Claims

1. A method for evaluating the cell proliferation inhibitory effect and / or metabolic activity inhibitory effect of a chemical substance on mammalian cells, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is highly likely to be a chemical substance having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect when the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance; wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

2. A method for evaluating the cell proliferation inhibitory effect and / or metabolic activity inhibitory effect of a chemical substance on mammalian cells, comprising the steps of: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is unlikely to be a chemical substance having cell proliferation inhibitory activity and / or metabolic activity inhibitory activity when the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance. wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

3. A method for screening an immunosuppressant having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect on mammalian cells, comprising the following steps: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is highly likely to be a chemical substance having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect when the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance; wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

4. A method for screening an immunosuppressant having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect on mammalian cells, comprising the following steps: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is unlikely to be an effective immunosuppressant via cell proliferation inhibitory action and / or metabolic activity inhibitory action, when the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance. wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

5. A method for screening an anticancer agent having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect on mammalian cells, comprising the following steps: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is highly likely to be a chemical substance having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect when the expression level of the second reporter gene relative to the expression level of the first reporter gene in the presence of the chemical substance is increased relative to the expression level of the second reporter gene relative to the expression level of the first reporter gene in the absence of the chemical substance and the expression level of the first reporter gene in the presence of the chemical substance is decreased relative to the expression level of the first reporter gene in the absence of the chemical substance; wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

6. A method for screening an anticancer agent having a cell proliferation inhibitory effect and / or a metabolic activity inhibitory effect on mammalian cells, comprising the following steps: Step 1: contacting a chemical substance with mammalian cells for immunotoxicity evaluation, which have been introduced with a first reporter gene under the control of a constant expression promoter and a second reporter gene under the control of a promoter for immunotoxicity evaluation so as to be transiently or stably expressible; Step 2: Evaluating the increase or decrease in expression of the second reporter gene relative to the first reporter gene, and the increase or decrease in expression of the first reporter gene, in the presence and absence of the chemical substance; and Step 3: determining that the chemical substance is unlikely to be an effective anticancer agent mediated by an inhibitory effect on cell proliferation and / or metabolic activity, when the expression level of the second reporter gene relative to the first reporter gene in the presence of the chemical substance is decreased or constant compared to the expression level of the second reporter gene relative to the first reporter gene in the absence of the chemical substance and / or the expression level of the first reporter gene in the presence of the chemical substance is increased or constant compared to the expression level of the first reporter gene in the absence of the chemical substance. wherein the mammalian cells for immunotoxicity evaluation are Jurkat cells.

7. Step 4: comparing and evaluating the increase or decrease in expression of the second reporter gene in the presence and absence of the chemical substance; The method of any one of claims 1 to 6, further comprising:

8. The method according to any one of claims 1 to 7, wherein the first reporter gene and the second reporter gene are genes encoding different luciferases, and the maximum emission wavelengths of the different luciferases differ from each other by 20 nm or more.

9. The method according to any one of claims 1 to 8, wherein the chemical substance is at least one chemical substance selected from the group consisting of an anticancer drug, a drug that induces immunosuppression by inhibiting cell proliferation or metabolic activity, an immunosuppressant, and a non-immunosuppressant.

Images