JP6730708B2 - Method for evaluating selective IL-4 production-inducing activity associated with NKT cell activation - Google Patents

Description

The present invention relates to a method for evaluating selective IL-4 production inducing activity associated with activation of natural killer T (NKT) cells.

NKT cells are cells having intermediate properties between T cells and natural killer (NK) cells. NKT cells have T cell antigen receptors (TCRs) and express NK cell markers (NK1.1 in mouse, CD56 in human). In particular, an NKT cell expressing an invariant TCR with almost no diversity in the Vα chain of the T cell antigen receptor (gene fragment Vα14-Jα28 in mouse, Vα24-Jα18 in human) is called invariant NKT cell. In 1997, it was reported that this invariant NKT cell recognizes the glycolipid α-galactosylceramide (α-GalCer) bound to CD1d which is an antigen-presenting molecule on the surface of dendritic cells (Non-Patent Document 1). ). Currently, cells that recognize and bind α-GalCer bound to CD1d and that express invariant TCR are often called NKT cells.

According to the results of experiments in humans and mice, CD1d-restricted NKT cells are activated by α-GalCer bound to CD1d, and interleukin 4 (IL-4), interferon γ (IFN-γ), interleukin. It is known to produce a large amount of cytokines such as 13 (IL-13) (Non-Patent Document 2). In addition, although NKT cells are a small number of cell populations, crosstalk through cytocan production as described above influences the functions of major lymphocytes including NK cells and is antigen-specific. It is known to regulate the immune response (Non-Patent Documents 3 and 4).

Α-GalCer is conventionally known as a substance capable of activating NKT cells (for example, Non-Patent Document 2). Patent Document 1 discloses a glycolipid capable of selectively inducing IL-4 production by NKT cells.

Japanese Patent No. 4064346

Kawano T, et al. , Science, 278, pp. 1626-1629. Bendelac A, et al. , 2007, Annu. Rev. Immunol. 25 volumes, pp. 297-336. Carnaud et al. , 1999, J. Immunol. , 163, pp. 4647. Berzins SP, et al. , 2011, Nat. Rev. Immunol. , Vol. 11, pp. 131-142.

The glycolipid described in Patent Document 1 (hereinafter, also referred to as “synthetic glycolipid compound”) can selectively induce IL-4 production by NKT cells (hereinafter, “selection accompanying activation of NKT cells”). IL-4 production-inducing activity"). Thereby, for example, it is possible to regulate the Th1/Th2 immune balance in the direction in which Th2 increases, reduce IL-17 production, and reduce GM-CSF production. Such a substance having a selective IL-4 production inducing activity associated with NKT cell activation is, for example, treatment of a disease in which the Th1/Th2 immune balance is biased to Th1 or a disease in which Th1 cells exacerbate the pathological condition, autoimmune disease. It is extremely effective for the treatment of the above, the treatment of the disease caused by the increase of the GM-CSF concentration in the living body, and the treatment of the disease caused by the increase of the IL-17-producing T cells.

By the way, in the case of evaluating whether or not a substance has a selective IL-4 production-inducing activity associated with NKT cell activation for the purpose of developing a new drug, in vitro (in vitro) evaluation alone is not sufficient. It is desirable to evaluate the immunomodulating ability when administered to humans. However, in mammals including human beings, NKT cells are scarcely present in peripheral blood, and it is very difficult to directly detect the activation state of NKT cells by the conventional technique.

In addition, since clinical trials for humans generally take time and cost, a method capable of evaluating drug efficacy at an early stage is required.

Therefore, an object of the present invention is to provide an evaluation method capable of rapidly evaluating the selective IL-4 production-inducing activity associated with NKT cell activation of a test substance in vivo.

The present invention relates to the following (1) to (17), for example.
(1) A method for evaluating a selective IL-4 production-inducing activity associated with in vivo NKT cell activation of a test substance, which is expressed in blood cells collected from a human subject to which the test substance is administered. Quantification step for measuring the amount of transcripts present, CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene , The amount of transcript measured for one or more genes selected from the group consisting of GSG1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene. , If more than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject not administered the test substance, or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2 gene. , AGPAT4-IT1 gene, FOS gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene. , FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4 gene, KIR2DL5A gene, KIR3DL2 gene, FOS gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222C gene, LOC10065 gene, LOC1002C, LOC400655 gene. Gene, XLOC_01024 The amount of transcripts measured for one or more genes selected from the group consisting of 5 genes and LOC286087 gene is the amount of transcripts expressed in blood cells collected from a human subject not administered with the test substance. A determination step of determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the amount is less than a threshold value determined based on the above.

(2) The quantification step includes measuring the amount of a transcript expressed in blood cells collected from a human subject administered the test substance 5 to 7 hours after the administration, and the determination step comprises CHRD. Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, XLOC_00270058 gene, XLOC_00270058 gene, XLOC_002727 gene. The amount of transcripts measured in blood cells 5 to 7 hours after administration of one or more genes selected from the group consisting of XLOC_I2_012046 gene is blood collected from a human subject not administered with the test substance. When it is more than a threshold value determined based on the amount of transcript expressed in cells, or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 Gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, FOSB gene, NR4A2 gene, FOS. Gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, and XLOC_I2_000092 gene, to the blood cells 5 to 7 hours after administration. When the measured amount of transcript is less than the threshold value determined based on the amount of transcript expressed in blood cells collected from a human subject not administered with the test substance, the test substance is The method according to (1), which comprises determining that it exhibited a selective IL-4 production-inducing activity associated with NKT cell activation in vivo.

(3) The quantification step includes measuring the amount of a transcript expressed in blood cells collected from a human subject administered the test substance 23 to 25 hours after administration, and the determination step comprises CHRD. Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene, CD180 gene, EGR2 gene, EGR2 gene. The transcript amount measured for blood cells 23 to 25 hours after administration for one or more genes selected from the group consisting of OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene is When the amount is greater than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject to which the test substance is not administered, or DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 Gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GOMY2 gene. Gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene. Transcripts measured in blood cells 23 to 25 hours after administration for one or more genes selected from the group consisting of KIR2DL5A gene, KIR3DL2 gene, FOS gene, XLOC_007314 gene, XLOC_I2_000092 gene, XLOC_010245 gene and LOC286087 gene If the amount is less than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject not receiving the test substance, the test substance is The method according to (1) or (2), comprising determining in vivo that a selective IL-4 production-inducing activity associated with NKT cell activation is exhibited.

(4) The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, Blood collected from a human subject to which the amount of transcripts measured for IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene was not administered to the test substance. When it is more than a threshold value determined based on the amount of transcript expressed in cells, and/or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene , RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, FOS gene, KLF10 gene, SLC1A7 gene. , MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene Gene, KIR2DS4 gene, KIR2DL5A gene, KIR3DL2 gene, FOS gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, XLOC_I2_28_0102 gene, 60 gene, and XLOC_I2_00_90 gene, XLO_002_0102 gene, XLO_000092 gene, XLO_000092 gene, XLO_00002 gene, and XLO_00002 gene. Is less than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject not administered the test substance, the test substance is The method according to any one of (1) to (3), which comprises determining that it exhibited a selective IL-4 production-inducing activity associated with NKT cell activation in vivo.

(5) The quantification step includes measuring the amount of a transcript expressed in blood cells collected from a human subject administered the test substance 5 to 7 hours after the administration, and the determination step comprises CHRD. Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, XLOC_00270058 gene, XLOC_00270058 gene, XLOC_002727 gene. The amount of transcripts measured for blood cells 5 to 7 hours after administration of the XLOC_I2_012046 gene is based on the amount of transcripts expressed in blood cells collected from a human subject not administered with the above-mentioned test substance. When it is more than the defined threshold value and/or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene , MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, FOSB gene, NR4A2 gene, FOS gene, XLOC_000357 gene, XLOC_0066880061 gene, XLOC_006601 gene. , LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, and XLOC_I2_000092 gene, the amount of transcripts measured in blood cells 5 to 7 hours after administration was collected from a human subject not administered with the test substance. It is determined that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the amount is less than a threshold value determined based on the amount of transcript expressed in blood cells. The method according to any one of (1) to (4), which comprises:

(6) The quantification step includes measuring the amount of transcript expressed in blood cells collected from a human subject administered the test substance 23 to 25 hours after administration, and the determination step comprises CHRD. Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene, CD180 gene, EGR gene, EGR2 gene. The transcript amount of OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene measured in blood cells 23 to 25 hours after administration was collected from a human subject not administered with the test substance. When it is more than a threshold value determined based on the amount of transcript expressed in the blood cells, and/or DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene , MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP21 gene, C1or2. , COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4 gene, KIR2DL5X gene, CIR2DL5A2 gene, CIR2DL5A14 gene, KIR2DL5A2 gene, KIR2DL5A gene, KIR2DL5A gene, KIR2DL5A gene, KIR2DL5A gene, KIR3DL3A gene Gene, XLOC_I2_000092 gene, XLOC_010245 gene and LOC286087 gene, the amount of transcripts measured in blood cells 23 to 25 hours after administration was expressed in blood cells collected from a human subject not administered with the above-mentioned test substance. It was determined that the test substance exhibited selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the amount was less than a threshold value determined based on the amount of transcripts The method according to any one of (1) to (5), which comprises:

(7) The threshold value is the transcript amount of each gene expressed in blood cells collected from the human subject before administration of the test substance, according to any one of (1) to (6) the method of.

(8) The selective IL-4 production-inducing activity associated with the activation of NKT cells is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and that induces IL-4 production by NKT cells. The method according to any one of (1) to (7).

(9) In the quantification step, the amount of the transcript is measured using at least one selected from the group consisting of probes containing the nucleotide sequences shown in any of SEQ ID NOS: 1 to 78, (1) to (8) ).

(10) A method for screening a substance having a selective IL-4 production-inducing activity associated with NKT cell activation in vivo, wherein the candidate substance is described in any one of (1) to (7). A method of screening comprising performing the method.

(11) The screening method according to (10), wherein the candidate substance is a substance that binds to an NKT cell antigen receptor and CD1d.

(12) The selective IL-4 production-inducing activity associated with the activation of NKT cells is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and that induces IL-4 production by NKT cells. The screening method according to (10) or (11).

(13) A detection agent for a selective IL-4 production inducing activity associated with activation of a natural killer T (NKT) cell of a test substance in vivo, comprising the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 78. A detection agent comprising at least one selected from the group consisting of probes.

(14) The selective IL-4 production-inducing activity associated with the activation of NKT cells is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and that induces IL-4 production by NKT cells. The detection agent according to (13).

According to the present invention, based on a change in the transcript amount of a specific gene contained in a blood sample collected from a human subject, in vitro induction of selective IL-4 production of a test substance with activation of NKT cells is performed. Activity can be assessed rapidly (ie, early after administration). Therefore, it is suitable for evaluation of drug efficacy in clinical tests in humans.

Further, according to the present invention, for example, it is possible to evaluate the in vivo drug efficacy of the synthetic glycolipid compound described in Patent Document 1, and further has a selective IL-4 production inducing activity associated with NKT cell activation. It is also possible to screen for new substances (new drug candidates).

Furthermore, since the detection agent of the present invention comprises at least one probe containing the nucleotide sequence shown in any of SEQ ID NOS: 1 to 78, it is a transcription product of a specific gene contained in a blood sample collected from a human subject. The amount can be measured reproducibly and accurately. This makes it possible to detect with high accuracy whether or not the test substance exhibited in vivo selective IL-4 production-inducing activity associated with NKT cell activation.

3 is a scatter plot 6 hours after administration in the A cohort of Example 1. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization. 2 is a scatter plot 24 hours after administration in the A cohort of Example 1. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization. 6 is a scatter plot 6 hours after administration in the C cohort of Example 1. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization. 2 is a scatter plot 24 hours after administration in the C cohort of Example 1. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31. 3 is a graph showing changes over time in the amount of transcripts in peripheral blood in a subject administered with Compound 31.

Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

(Transcript)
As used herein, "transcript" includes messenger RNA (mRNA) as well as non-coding RNA that is not translated into protein.

[Evaluation method of selective IL-4 production inducing activity associated with activation of NKT cells in vivo of test substance]
The method for evaluating the selective IL-4 production inducing activity associated with in vivo NKT cell activation of a test substance according to the present invention (hereinafter, also simply referred to as “evaluation method”) is a human to which the test substance is administered. A quantification step for measuring the transcript amount of a predetermined gene expressed in blood cells collected from a subject (hereinafter, also simply referred to as “quantification step”), and the measured transcript amount and each gene Based on the comparison with the set threshold value, the determination step of determining that the test substance exhibited the selective IL-4 production-inducing activity associated with NKT cell activation in vivo (hereinafter, also simply referred to as “determination step”). ), and. The present invention can also be regarded as a method of collecting data for evaluating a selective IL-4 production inducing activity associated with in vivo NKT cell activation of a test substance, which includes the above-mentioned quantification step.

(Predetermined gene)
The “predetermined gene” in the evaluation method according to the present invention is one of the synthetic glycolipid compounds described in Patent Document 1 (2S,3S,4R)-1-O-(α-D-galactosyl)-2. When (-N-tetracosanoylamino)-1,3,4-nonanetriol (hereinafter, also referred to as "Compound 31") is administered to a human subject (healthy subject), blood is compared with that before administration. A gene in which the amount of transcript in a sample (blood cell) is statistically significantly increased or decreased. That is, since the increase or decrease in the amount of the transcript of the “predetermined gene” is correlated with the selective IL-4 production-inducing activity associated with NKT cell activation, the evaluation method according to the present invention Evaluation is possible.

In the first embodiment, the “predetermined gene” is a CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene. , GSG1 gene, CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAG. Gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKRKR2 gene, AKRKR2 gene, AKRKR2 gene. Gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene, LOC645195 gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_01LO gene, LINC00222 gene, LOC10025 gene, LOC41005 gene, LOC40065 gene. XLOC — 010245 gene and LOC286087 gene (hereinafter, also referred to as “gene group 1”). Details of each gene are shown in Tables 1 to 9.

In the first embodiment, the quantification step and the determination step may be carried out for one kind of gene selected from the gene group 1, and also for two or more kinds of genes selected from the gene group 1, or the gene group. The quantification step and the determination step may be performed for all the genes of 1. By performing the quantification step and the determination step for a plurality of genes, the selective IL-4 production-inducing activity associated with the in vivo NKT cell activation of the test substance can be evaluated more accurately.

In the second embodiment, the “predetermined gene” is each gene of gene group 1, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4. A gene, a KIR2DL5A gene, a KIR3DL2 gene and a FOS gene (hereinafter, also referred to as "gene group 2"). Details of each gene are shown in Tables 1 to 9.

In the second embodiment, the quantification step and the determination step may be performed on one or more genes selected from the gene group 2, and the quantification step and the determination step may be performed on two or more genes selected from the gene group 2. The steps may be performed. In addition, in the second embodiment, it is preferable to perform the quantification step and the determination step for all the genes of the gene group 2 from the viewpoint of further improving the evaluation accuracy.

Each gene included in the gene group 1 or 2 has a gene group A, a gene group B, a gene group C, a gene group D, and a gene group E in accordance with the change pattern of the transcript amount with the lapse of time after administration of the test substance. Can be divided into

In the gene group A, the transcript amount is statistically significantly increased 6 hours after administration and 24 hours after administration, as compared with before administration. Genes included in the gene group A include CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, IL4I1 gene, CD180 gene, EGR2 gene, RCC2 gene, OAS2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene. , POT1 gene, XLOC_002727 gene, XLOC_005851 gene and XLOC_I2_012046 gene.

The gene group B has a statistically significant increase in the amount of transcripts 24 hours after administration, as compared with that before administration. There is no statistically significant difference between the transcript amount 6 hours after administration and the transcript amount before administration. The genes included in the gene group B are OAS3 gene, RUFY4 gene, GPER gene, GSG1 gene and LOC645195 gene.

In the gene group C, the transcript amount is statistically significantly reduced 6 hours after the administration, as compared with that before the administration. There is no statistically significant difference between the transcript amount 24 hours after administration and the transcript amount before administration. The genes included in the gene group C are CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene and LOC100130581 gene.

In the gene group D, the transcript amount is statistically significantly decreased 6 hours after administration and 24 hours after administration, as compared with before administration. The genes included in the gene group D are DRC1 gene, FOSB gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, NR4A2 gene, GATA2 gene, TAGAP gene, F12 gene. , KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, FOS gene, KLF10 gene, XLOC_007314 gene, and XLOC_I2_000092 gene.

In the gene group E, the amount of transcripts is statistically significantly reduced 24 hours after administration, as compared with that before administration. There is no statistically significant difference between the transcript amount 6 hours after administration and the transcript amount before administration. Genes included in the gene group E include SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, KIR2DL. , KIR2DL4 gene, KIR2DL5A gene, KIR2DS2 gene, KIR2DS4 gene, KIR3DL2 gene, XLOC_010245 gene and LOC286087 gene.

Therefore, in the evaluation method according to the present invention, the quantification step includes measuring the amount of a transcript expressed in blood cells collected from a human subject administered with the test substance 5 to 7 hours after the administration. In the step, when the transcript amount of the gene included in the gene group A is larger than the threshold value set for each gene, or when the transcript amount of the gene included in the gene group C or the gene group D is It may include determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the amount is less than the threshold value set for , For convenience, also referred to as "6 hour evaluation".) The blood cells are preferably collected 5.5 to 6.5 hours after the administration of the test substance, more preferably 5.75 to 6.25 hours after the administration of the test substance, and 6 hours after the administration of the test substance. It is more preferable to carry out.

In the first embodiment, in the case of 6-hour evaluation, the genes included in the gene group A are CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene. , PPARGC1B gene, POT1 gene, XLOC_002727 gene, XLOC_005851 gene and XLOC_I2_012046 gene, and the genes contained in gene group C or gene group D are CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4. Gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLFF10 gene, KLPF10 gene. Genes, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, and XLOC_I2_000092 gene.

In the second embodiment, in the case of 6-hour evaluation, the genes included in the gene group A are the gene of the first embodiment, the IL4I1 gene, the CD180 gene, the EGR2 gene and the OAS2 gene, and the gene group C or the gene group D. The genes included in are the gene of the first embodiment, the FOSB gene, the NR4A2 gene and the FOS gene.

In the evaluation method according to the present invention, the quantification step further includes the step of determining the amount of transcript expressed in blood cells collected from a human subject administered with the test substance 23 to 25 hours after administration, and the determination step However, when the transcript amount of the gene contained in the gene group A or gene group B is larger than the threshold value set for each gene, or the transcript amount of the gene contained in the gene group D or gene group E is , Which includes determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the amount is less than the threshold value set for each gene. Good (hereinafter, also referred to as “24-hour evaluation” for convenience). Blood cells are preferably collected 23.5 to 24.5 hours after administration of the test substance, more preferably 23.75 to 24.25 hours after administration of the test substance, and more preferably 24 hours after administration of the test substance. It is more preferable to carry out.

In the first embodiment, in the case of 24-hour evaluation, the genes included in the gene group A or the gene group B are CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1. Gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene, and the genes included in gene group D or gene group E are DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene. KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, XLO_0_C1_XLO_CII_XLO_CII_XLOC_007314 gene, XLOC_007314 gene, XLOC_007314 gene. And LOC286087 gene.

In the second embodiment, in the case of 24-hour evaluation, the genes included in the gene group A or the gene group B are the gene of the first embodiment, the IL4I1 gene, the CD180 gene, the EGR2 gene, the OAS2 gene, and the OAS3 gene, The genes included in the gene group D or the gene group E are the gene of the first embodiment, the FOSB gene, the NR4A2 gene, the KIR2DL2 gene, the KIR2DL4 gene, the KIR2DS2 gene, the KIR2DS4 gene, the KIR2DL5A gene, the KIR3DL2 gene and the FOS gene.

In any of the first embodiment and the second embodiment, when carrying out the 6-hour evaluation or the 24-hour evaluation, either the 6-hour evaluation or the 24-hour evaluation may be carried out. You may carry out combining evaluation.

(Test substance)
The test substance may be a low molecular weight compound or a high molecular weight compound. The low molecular weight compound is arbitrarily selected from, for example, peptides, siRNA, miRNA, glycolipids and the like. The polymer compound is arbitrarily selected from, for example, proteins and antibodies.

In one embodiment, the test substance is a glycolipid described in Patent Document 1 (eg, represented by the following formula (I)), which has been confirmed to have selective IL-4 production-inducing activity associated with NKT cell activation. A derivative of α-galactosylceramide) may be selected. In this case, for example, it is possible to evaluate the correlation with the selective IL-4 production-inducing activity associated with activation of NKT cells in vivo when the dosage, administration method, etc. of the glycolipid are changed.

[In general formula (I), R ¹ represents an aldopyranose residue, R ² represents a hydrogen atom or a hydroxyl group, and R ³ represents —CH ₂ —, —CH(OH)—CH ₂ —, or —CH═. represents CH-, ^{R 4} represents a hydrogen atom or CH _3, x is 0 to 35, y and z represent integers satisfying y + z = 0 to 3. ]

In addition, as a test substance, a substance which is unknown whether or not it has a selective IL-4 production inducing activity associated with NKT cell activation in vivo may be selected. The substance determined to exhibit the selective IL-4 production-inducing activity associated with NKT cell activation in vivo by the evaluation method of the present invention includes, for example, a disease in which the Th1/Th2 immune balance is biased to Th1 or Th1 cells. It is extremely effective for the treatment of diseases that worsen the pathological condition, the treatment of autoimmune diseases, the treatment of diseases caused by an increase in GM-CSF concentration in vivo, and the treatment of diseases caused by an increase in IL-17-producing T cells. .. The test substance is preferably a substance which has achieved the safety standard set in the non-clinical test, and more preferably a substance which is judged to be safe when administered to a human subject. “Has safety when administered to a human subject” means, for example, that safety was confirmed in Phase I of a clinical trial, and more specifically, it is 10 times the effective dose of the substance or It can be said that no serious side effect is exhibited even at a blood concentration of 100 times.

(Blood cells)
Blood cells were taken before administration of the test substance or from a human subject administered with the test substance. The time from administration of the test substance to collection of blood cells can be set, for example, between 1 hour after administration and 72 hours after administration. Blood cells can be collected by a known method such as collecting blood cells by a vacuum blood collection tube and collecting blood cells by centrifugation. The sex, age, height, weight, health condition, etc. of the human subject are not particularly limited, and may be appropriately set according to the purpose of evaluation. The administration method of the test substance may be set according to the purpose, and may be oral administration or parenteral administration. Parenteral administration may be systemic administration (for example, intravascular injection) or local administration (for example, subcutaneous administration, transdermal administration, transmucosal administration, transrectal administration, etc.). The dose of the test substance may be set according to the type of the test substance and the purpose.

(Quantitative process)
As a method for measuring the amount of transcript expressed in blood cells, any method known to those skilled in the art may be adopted. The quantification step can be performed, for example, according to the following procedure.
(1) A blood sample collected from a human subject administered with a test substance is collected 1 day before administration, 6 hours after administration, and 24 hours after administration. A blood sample one day before administration is used as a standard for the amount of transcript (for example, used to determine a threshold value).
(2) The blood sample is centrifuged to collect blood cells as a precipitate.
(3) Purify total RNA from the collected blood cells according to a conventional method. A commercially available total RNA extraction kit or purification kit may be used. Transcripts may be purified (or enriched) from total RNA samples.
(4) Using the purified total RNA as a template, the transcription amount of a predetermined gene is measured by a conventional method. The amount of transcripts using total RNA as a template can be measured by, for example, microarray analysis, (real-time) quantitative PCR analysis, nucleic acid digital counting system (ncounter), RNA-Seq, or the like.

In the quantification step, in order to correct the variation in the amount of the blood sample (blood cells), in addition to the predetermined gene, the amount of the transcript of the housekeeping gene (eg, GAPDH) as an internal standard may also be measured. This allows a more accurate comparison of transcript amounts.

(Judgment process)
In the determination step, the test substance was tested for in vivo NKT cell activity based on the comparison between the transcript amount (or corrected transcript amount) measured in the quantification step and the threshold value set for each gene. It is determined whether or not the selective IL-4 production-inducing activity associated with the activation was exhibited.

(Threshold)
The threshold is established based on the amount of transcript expressed in blood cells collected from a human subject that has not received the test substance. The threshold value is preferably the transcript amount of each gene expressed in the blood cell for each gene. This makes it possible to more sensitively determine the change in the transcript amount of a given gene due to the administration of the test substance. The threshold value is, for example, the amount of a transcript of a predetermined gene expressed in blood cells collected from a human subject (one or more) who has not been administered the test substance, and the amount of the transcript is measured in advance. (For a plurality of people, for example, an average value) may be set in advance as a threshold value. In addition, the threshold is measured by measuring the transcript amount of a predetermined gene expressed in blood cells collected before administration of the test substance from a human subject who is scheduled to administer the test substance, and measuring the transcript amount. It may be a threshold value. This makes it possible to make a determination with even higher sensitivity.

(Selective IL-4 production-inducing activity associated with NKT cell activation)
In the present specification, the selective IL-4 production-inducing activity associated with in vivo NKT cell activation of a test substance refers to the activity of activating NKT cells and selectively inducing IL-4 production by NKT cells. means. More preferably, the selective IL-4 production-inducing activity associated with NKT cell activation is an activity that does not induce IFN-γ production by NKT cells and induces IL-4 production by NKT cells.

[Method of screening for substance having selective IL-4 production inducing activity associated with activation of NKT cells in vivo]
Since the evaluation method according to the present invention employs the above-mentioned constitution, it can be used also as a screening method for a substance having a selective IL-4 production inducing activity associated with NKT cell activation in vivo. .. That is, by carrying out the evaluation method according to the present invention using one or more candidate substances as test substances, it is possible to screen a substance having a selective IL-4 production inducing activity associated with NKT cell activation in vivo. become.

The substance selected by the screening method according to the present invention has a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. Therefore, the substance can be used as a therapeutic or prophylactic agent for a disease in which the Th1/Th2 immune balance is biased to Th1 or a disease in which Th1 cells exacerbate the pathological condition. In addition, the substance can also be used as a therapeutic or prophylactic agent for autoimmune diseases. Furthermore, the substance can also be used as a therapeutic or prophylactic agent for diseases caused by an increase in GM-CSF concentration in the body. Furthermore, the substance can also be used as a therapeutic or prophylactic agent for diseases caused by an increase in IL-17-producing T cells.

Diseases in which Th1/Th2 immune balance is biased to Th1 include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, uveitis, and Sjogren's syndrome, as well as fulminant hepatitis, graft rejection, It mainly means diseases caused by cell-mediated immunity such as infectious diseases caused by intracellular infectious agents. Autoimmune diseases include multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, vitiligo vulgaris, Behcet's disease, collagen disease, type I diabetes, uveitis, Sjogren's syndrome, autoimmune myocarditis, autoimmune liver. It means diseases such as diseases, autoimmune gastritis, pemphigus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuritis, HTLV-1 associated myelopathy and the like. The disease caused by the increase in GM-CSF concentration in the living body means a disease such as chronic organ inflammation, chronic inflammatory disease such as rheumatoid arthritis and the like. The disease caused by an increase in IL-17-producing T cells means diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis, neuromyelitis optica and inflammatory bowel disease.

[Detective agent for selective IL-4 production inducing activity associated with in vivo NKT cell activation of test substance]
The agent for detecting the selective IL-4 production inducing activity associated with in vivo NKT cell activation of the test substance according to the present invention is selected from the group consisting of probes including the nucleotide sequences shown in SEQ ID NOs: 1 to 78. It consists of at least one selected. Each probe may consist of the base sequence shown in any of SEQ ID NOs: 1 to 78.

As shown in Tables 1 to 9, the nucleotide sequences shown in SEQ ID NOs: 1 to 78 hybridize with cRNAs (or transcripts) of the genes included in gene group 2. Therefore, the above-mentioned detection agent can quantitatively hybridize with cRNA (or transcript) of a predetermined gene, and thus, for example, by a fluorescent signal or the like reflecting the amount of transcript expressed in blood cells. A signal can be taken out, and the selective IL-4 production inducing activity associated with activation of NKT cells in vivo of the test substance can be detected.

Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to the following examples.

<Example 1: Gene identification>
Compound 31 was orally administered once to human subjects, and changes in gene expression in peripheral blood were analyzed by microarray. In Example 1, a healthy adult (hereinafter referred to as “subject”) was adopted as a human subject, and the test was conducted according to the following procedure.

(Method)
(1) Administration of Synthetic Glycolipid Compound to Subjects Six subjects who obtained informed consent were classified into two cohorts (A and C cohorts), and each subject was represented by compound 31, that is, the following formula (II). (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-nonanetriol was orally administered once as a single dose. Subjects in the A cohort received Compound 31 at a dose of 0.3 mg/person, and subjects in the C cohort received Compound 31 at a dose of 3 mg/person.

(2) Extraction of Total RNA from Peripheral Blood First, using a vacuum blood collection tube (PAXgene Blood miRNA Kit; QIAGEN), 6 hours and 24 hours (1 day) after administration of Compound 31, 2.5 mL of blood was collected from each subject's vein. On the day before the administration of Compound 31, 2.5 mL of blood was similarly collected from the vein of each subject and used as a control.

Then, the vacuum blood collection tube containing the collected blood was centrifuged at 2,500 rpm for 10 minutes to precipitate blood cells and remove the supernatant. The obtained precipitate was washed with RNase Free water and suspended in BM1 (resuspension buffer), and then BM2 (binding buffer) and proteinase K were added and dissolved at 55° C. and 900 rpm for 10 minutes. .. The resulting lysate was crushed by passing through a PAXgene Shredder spin column attached to the above kit, and then unnecessary pellets were removed by centrifugation to obtain a supernatant. 700 μL of isopropanol was added to the obtained supernatant to precipitate total RNA. Then, total RNA was purified using PAXgene miRNA Spin Column attached to the kit, and eluted with 20 μL or 30 μL of BM5 (elution buffer).

(3) Gene Expression Analysis by Microarray Next, according to the protocol recommended by Agilent (One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling), ver 6.5, May 2010 gene expression). Analysis was performed. First, cDNA was synthesized using 100 ng of total RNA extracted in the above step as a template. Next, labeled cRNA was obtained from the synthesized cDNA using One Color Spike Mix Kit (manufactured by Agilent) and Quick-Amp Labeling Kit (for one color) (manufactured by Agilent). After the labeled cRNA was purified using RNeasy Mini kit (manufactured by QIAGEN), the concentration was measured using NanoDrop ND-1000 (Thermo Fisher Scientific). At this time, a sample in which the amplification efficiency of the purified labeled cRNA was 15 times or more and the label incorporation efficiency was 6 pmol/μg or more was regarded as a pass and used for the subsequent tests.

Subsequently, 600 ng of the labeled cRNA was collected, and the labeled cRNA and the microarray were hybridized using the Expression Hybridization kit (manufactured by Agilent). As the microarray, SurePrint G3 Human GE microarray (8×60K) ver 2.0 (manufactured by Agilent) was used. After applying the sample to the microarray surface, it was covered with an 8×60K gasket slide (manufactured by Agilent) and fixed by a hybridization chamber (manufactured by Agilent). The immobilized array was hybridized in a DNA Microarray Hybridization Oven (Agilent) under conditions of 17 hours (65° C., 10 rotations), and then the microarray was used as a Gene Expression Wash Buffer 1 (Agilent), Gene. After washing with Expression Wash Buffer 2 (manufactured by Agilent) and 10% Triton X-102 (manufactured by Agilent), the washed microarray was scanned using an Agilent DNA Microarray Scanner (manufactured by Agilent).

The obtained scanning data was quantified using the software Feature Extraction ver 10.7.1.1 (manufactured by Agilent) using the quantification protocol GE1_107_Sep09 (numericalized grid file: 039494_D_F_2012628). The digitized data was analyzed by the following method.
(A) Normalization between Arrays As a normalization method between arrays in the one-color method, GeneSpring GX 12.0 (manufactured by Agilent) was used to perform global normalization promoted by Agilent. After excluding probes whose numerical data (Raw Signal) was 1.0 or less, correction was carried out by the 75th percentile value of the measured value for each array, the variation between arrays was suppressed, and only biological variation was extracted.
Simultaneously with the normalization between arrays, based on the flag information of 5 items described in the digitized data (Raw Data), it is converted into three types (Detected, Compromised, Not Detected) of GeneSpring flags which are indicators of the reliability of the data. did). Table 1 shows the digitized data (Raw Data) and flag information in GeneSpring.

The selection criteria of the GeneSpring flag attached to each probe are as follows. The GeneSpring flag of "Not Detected" was attached to the probe determined to be "Not Detected" in any one item. Further, although there is no “Not Detected” determination, the probe determined to be “Compromised” in any of the items is given a GeneSpring flag of “Compromised”. GeneSpring flags of "Detected" were attached to the probes that had no determination of "Compromised" and "Not Detected". It is to be noted that the probe with GeneSpring flags of "Detected" is the most reliable probe. After applying any of the three GeneSpring flags to all the probes, the number of probes classified by the GeneSpring flag was counted for each data.

(B) Correlation analysis In order to investigate the correlation between the subjects in each group, a correlation analysis was performed between two subjects in the same group (3 ways/group). First, a probe with a GeneSpring flag flagged as “Detected” or “Compromised” is selected for each of the two subjects in the same group, and a scatter plot is generated based on their normalized signal value data. It was created and the coefficient of determination (r2) was calculated.

(C) Hierarchical clustering analysis In order to investigate the similarity and independence between gene expression profile data, “Detected” or “Detected” is commonly used for all data of a total of 18 samples of 6 subjects×3 samples. After selecting a probe indicating “Compromised”, hierarchical clustering was performed using GeneSpring GX 12.0 (Agilent) based on these normalized signal values. Among the analysis conditions, the Distance metric was set as "Pearson Uncentered" and the Linkage rule was set as "Ward's". Then, together with the results of the coefficient of determination (r2) obtained by the above correlation analysis, the similarity within the same group and the independence of each group were evaluated to determine whether or not a significant difference test could be performed.

(D) Calculation of expression ratio and extraction of variable gene group Based on the signal value after normalization, in which correlation was observed in each group, GeneSpring GX 12.0 (Agilent) was used for 0 hours (before administration). ) Group and a 6-hour group or a 24-hour group were subjected to a significant difference test between two groups (Welch's t-test) to calculate the p-value.

Moreover, after calculating the average signal value of each group and the expression ratio (log2 ratio) of the 6-hour group or the 24-hour group relative to the 0-hour group, for all the probes, the probe with the "Detected" flag or the "Compromised" flag was added. , Scatter plots of the treatment group (y-axis) versus the 0-hour group (x-axis) were prepared (FIGS. 1 to 4). In addition, the probe which attached the "Detected" flag or the "Compromised" flag in both groups was used for the analysis. 1-4 are scatter plots 6 hours after administration in the A cohort, 24 hours after administration in the A cohort, 6 hours after administration in the C cohort, and 24 hours after administration in the C cohort, respectively. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization. In addition, in FIGS. 1 to 4, a dotted line indicates a boundary of 1.5 times, a broken line indicates a boundary of 0.67 times, and a white square dot indicates a variable gene.

Next, in order to extract a group of genes whose expression varied after administration of Compound 31, a probe matching any of the following three conditions was selected.
(Condition 1) In both the 6-hour group and the 24-hour group, the "Detected" flag or the "Compromised" flag was added, and the expression ratio was 1.5 times or more or 0.67 times or less, and a significant difference test In both the 6-hour group and the 24-hour group of the probe (condition 2) that showed p<0.05 in 1), the "Detected" flag or the "Compromised" flag was added, and the expression ratio was more than 1 time and 1.5 times. Probes that were less than or more than 0.67 times and less than 1 time and showed p<0.01 in the significance test (condition 3)
・Switch ON type:
One or more subjects were flagged as "Not Detected" in the 0-hour group, but all were flagged as "Detected" or "Compromised" in the treatment group, and the expression ratio was 4 times or more and a significant difference test Probe switch OFF type showing p<0.01 in:
In the treatment group, one or more subjects were flagged as “Not Detected”, but in the 0-hour group, all were flagged as “Detected” or “Compromised”, and the expression ratio was 0.25 times or less, and significant. However, a probe showing p<0.01 in the difference test However, in the correlation analysis and the hierarchical clustering analysis, when there was a large variation in the same treatment group, the subject that caused the variation was excluded from the analysis. In this case, since the statistical processing cannot be performed, the selection criterion based on the p-value is not applied, and since the accuracy of the average value is reduced due to the decrease in the number of subjects, (Condition 2) is also not applied. In this case, a probe that meets either of the following two conditions was selected.
(Condition 1') A probe having a "Detected" flag or a "Compromised" flag and an expression ratio of 1.5 times or more or 0.67 times or less in both the 6-hour group and the 24-hour group (condition 3')
・Switch ON type:
One or more subjects were flagged as "Not Detected" in the 0-hour group, but all were flagged as "Detected" or "Compromised" in the treatment group and the expression ratio was 4 times or more. Type:
In the treatment group, one or more subjects were flagged as "Not Detected", but in the 0-hour group, all were flagged as "Detected" or "Compromised" and the expression ratio was 0.25 times or less.

(E) Venn diagram analysis To investigate the number of probes in which the transcript amount varied in common between the A and C cohorts, the probe transcript amount was divided into increase and decrease in each of the 6-hour group and the 24-hour group. Venn diagram analysis was performed. Probes that showed overlapping probe groups and specific variations between the two combinations were extracted.

(result)
In both the A cohort and the C cohort, the time course of the transcript amount of the gene in which the transcript amount was significantly increased 6 hours and 24 hours after the administration of Compound 31 as compared with that before the administration is shown in FIG. 7 and FIGS. 15 to 16. 5, 6 and 15 show genes in which the transcript amount was significantly increased as compared with that before administration, both 6 hours and 24 hours after administration. FIG. 7 and FIG. 16 show genes in which the transcript amount was significantly increased as compared with that before the administration only after 24 hours from the administration.

That is, as genes in which the amount of transcripts was increased, CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS3 gene, RUFY4 gene, GPER gene, RCC2 gene, The OAS2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, GSG1 gene, XLOC_002727 gene, XLOC_005851 gene and XLOC_I2_012046 gene and LOC645195 gene were identified (see also Table 2, Table 3 and Table 8).

Common to the A cohort and the C cohort, the time course of the transcript amount of the gene in which the transcript amount was significantly reduced 6 hours and 24 hours after the administration of Compound 31 as compared with that before the administration is shown in FIGS. 14 and FIGS. 17 to 19. FIG. 8 and FIG. 17 show genes in which the transcript amount was significantly reduced as compared with that before the administration only 6 hours after the administration. FIGS. 9 to 11 and FIG. 18 show genes in which the transcript amount was significantly reduced as compared with that before administration, both 6 hours after administration and 24 hours after administration. FIGS. 12 to 14 and 19 show genes in which the transcript amount was significantly reduced only 24 hours after the administration as compared with that before the administration.

That is, as genes in which the amount of transcripts was decreased, DRC1 gene, FOSB gene, PDK4 gene, CNTNAP3 gene, CPT1A gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, NR4A2 gene, GATA2 gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21C gene, COLGALT2 gene. , HDC gene, F2R gene, ZNF683 gene, PRF1 gene, SH2D2A gene, DAAM2 gene, AGPAT4-IT1 gene, MYBL1 gene, FOS gene, KLF10 gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene. , XLOC_007314 gene, XLOC_I2_000092 gene, XLOC_010245 gene and LOC286087 gene were identified. Furthermore, as the genes whose expression was decreased, genes belonging to the KIR family such as KIR2DL2 gene, KIR2DL4 gene, KIR2DL5A gene, KIR2DS2 gene, KIR2DS4 gene and KIR3DL2 gene were identified (see Tables 4 to 6 and Table 8). reference).

Here, according to Miyamoto et al. (2001, Nature, 413, pp.531-534), administration of compound 31 activates NKT cells and temporarily enhances the secretion amount of IL-4 protein. However, it can be understood that the secreted amount of IFN-γ protein does not change significantly. IL4I1 protein is secreted from B cells and myeloid cells by stimulation with IL-4 and suppresses T cell proliferation (Boulland et al., 2007, Blood, 110, pp. 220-227. ), and Th17 cells, and is involved in the suppression of IL-2 production and cell growth (Santalarsci et al., 2012, Immunity, 36, pp. 201-214). Although these are reports at the protein level, it can be understood that the increase in the amount of IL4I1 transcript by administration of compound 31 in this example reflects the activation of NKT cells by compound 31. Therefore, it can be said that the change in the transcript amount of the above-mentioned gene group reflects that Compound 31 showed the selective IL-4 production-inducing activity associated with NKT cell activation in vivo.

In the present Example, the genes whose increase or decrease in the transcript amount were observed are summarized in Tables 2 to 6 and 8. The sequence numbers in Tables 2 to 6 and 8 correspond to the probe sequences on the microarray. The sequences of each probe are shown in Tables 7 and 9. The ensembl ID is an identification number of Ensemble Genome Browser (http://www.ensembl.org/index.html) which is a database of genome. The nucleotide sequences of the transcripts corresponding to the genes of SEQ ID NOs: 65 to 78 are shown in SEQ ID NOs: 79 to 103 of the sequence listing, and the correspondence is shown in Table 10.

Claims (14)

A method for evaluating a selective IL-4 production inducing activity associated with activation of a natural killer T (NKT) cell of a test substance in vivo, comprising:
A quantification step of measuring the amount of transcripts expressed in blood cells collected from a human subject administered with the test substance,
CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1R gene, CD180 gene. , OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene, and LOC645195 gene, the amount of transcripts measured for one or more genes selected from the human subject not administered with the test substance When it is more than the threshold value determined based on the amount of transcripts expressed in the collected blood cells, or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, FOS gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4DL2 gene, KIR4DL2 gene, KIR2A2 gene, KIR4DL2 gene, KIR4DL2 gene, KIR4DL2 gene , KIR2DS2 gene, KIR2DS4 gene, KIR2DL5A gene, KIR3DL2 gene , XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, XLOC_45 gene and XLOC_ILO_CLO_I2_000092 gene. The amount of transcript measured for one or more genes is a threshold value determined based on the amount of transcript expressed in blood cells collected from a human subject not administered with the test substance. Less than
A determination step of determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo;
Comprising a method. The quantification step comprises measuring the amount of transcript expressed in blood cells collected from a human subject administered the test substance 5-7 hours after administration,
The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, A human in which the amount of transcript measured in blood cells 5 to 7 hours after administration of one or more genes selected from the group consisting of XLOC_002727 gene, XLOC_005851 gene and XLOC_I2_012046 gene is not administered with the test substance. When the amount is greater than a threshold value determined based on the amount of transcript expressed in blood cells collected from the subject, or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 Gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, FOSB. 5 to 7 hours after administration for at least one gene selected from the group consisting of the gene, the NR4A2 gene, the FOS gene, the XLOC_000357 gene, the XLOC_006688 gene, the XLOC_010061 gene, the LINC00222 gene, the LOC400655 gene, the LOC100130581 gene, the XLOC_007314 gene, and the XLOC_I2_000092 gene. When the amount of transcripts measured for blood cells of is less than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject not administered with the test substance. ,
The method according to claim 1, which comprises determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. The quantifying step comprises measuring the amount of transcript expressed in blood cells collected from a human subject administered the test substance 23-25 hours after administration,
The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene. One or more genes selected from the group consisting of CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene were measured in blood cells 23 to 25 hours after administration. The amount of transcripts is higher than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject not administered with the test substance, or DRC1 gene, PDK4 gene, CNTNAP3 Gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1 gene. Gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL2 gene, KIR2DL2 gene. One or more genes selected from the group consisting of KIR2DS2 gene, KIR2DS4 gene, KIR2DL5A gene, KIR3DL2 gene, FOS gene, XLOC_007314 gene, XLOC_I2_000092 gene, XLOC_010245 gene and LOC286087 gene to blood cells 23 to 25 hours after administration When the amount of transcript measured by the method is less than a threshold value determined based on the amount of transcript expressed in blood cells collected from a human subject not administered with the test substance,
The method according to claim 1 or 2, which comprises determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene. The amount of transcripts measured for CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene is expressed in blood cells collected from a human subject not administered with the test substance. When the amount is greater than a threshold value determined based on the amount of transcripts being produced, and/or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene , CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, FOS gene, KLF10 gene, SLC1A7 gene, MYOM2 gene. , GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DL4 gene, KIR2DL4 gene, KIR2DL4 gene. Gene, KIR2DL5A gene, KIR3DL2 gene, XLOC_000357 gene , XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, XLOC_I2_000092 gene, XLOC_010245 gene, the amount of the test substance and the LOC28608 gene measured, and LOC28608 gene of the tested substance and LOC28608. Less than a threshold determined based on the amount of transcripts expressed in blood cells collected from a human subject not receiving
The method according to any one of claims 1 to 3, which comprises determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. The quantification step comprises measuring the amount of transcript expressed in blood cells collected from a human subject administered the test substance 5-7 hours after administration,
The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, The transcript amount of XLOC_002727 gene, XLOC_005851 gene and XLOC_I2_012046 gene measured in blood cells 5 to 7 hours after administration is expressed in blood cells collected from a human subject not administered with the test substance. When it is more than a threshold value determined based on the amount of transcript, and/or CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene , DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, FOSB gene, NR4A2 gene, FOS gene, XLOC_000357 gene. , XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC400655 gene, LOC100130581 gene, XLOC_007314 gene, and XLOC_I2_000092 gene, the amount of transcript measured in blood cells 5 to 7 hours after administration was not administered with the test substance. When it is less than a threshold value determined based on the amount of transcript expressed in blood cells collected from a human subject,
The method according to any one of claims 1 to 4, which comprises determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. The quantifying step comprises measuring the amount of transcript expressed in blood cells collected from a human subject administered the test substance 23-25 hours after administration,
The determination step includes CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene. CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2_012046 gene and LOC645195 gene, the amount of transcript measured in blood cells 23 to 25 hours after the administration, the test substance was administered. When the amount is higher than a threshold value determined based on the amount of transcripts expressed in blood cells collected from a human subject that is not present, and/or DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene , DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBTBBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene. , FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4 gene, KIR2DS4KIR2DS4 gene Gene, FOS gene, XLOC_007314 gene, XLOC_I2_000092 gene, XLOC_010245 gene and LOC286087 gene, the amount of transcript measured in blood cells 23 to 25 hours after administration was collected from a human subject not administered with the test substance. When it is less than the threshold value determined based on the amount of transcript expressed in the blood cells,
The method according to any one of claims 1 to 5, which comprises determining that the test substance exhibits a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. 7. The method according to any one of claims 1 to 6, wherein the threshold value is a transcript amount of each gene expressed in blood cells collected from the human subject before administration of the test substance. The selective IL-4 production-inducing activity associated with NKT cell activation is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and induces IL-4 production by NKT cells. The method according to any one of 1 to 7. 9. In the quantification step, the amount of transcript is measured using at least one kind selected from the group consisting of probes containing the nucleotide sequences shown in any of SEQ ID NOS: 1 to 78. The method described in the section. A method for screening a substance having a selective IL-4 production-inducing activity associated with NKT cell activation in vivo, comprising:
A screening method comprising performing the method according to any one of claims 1 to 7 on a candidate substance. The screening method according to claim 10, wherein the candidate substance is a substance that binds to an NKT cell antigen receptor and CD1d. The selective IL-4 production-inducing activity associated with NKT cell activation is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and induces IL-4 production by NKT cells. The screening method according to 10 or 11. A detection agent for a selective IL-4 production inducing activity associated with activation of a natural killer T (NKT) cell of a test substance in vivo,
A detection agent comprising at least one selected from the group consisting of probes containing the nucleotide sequences shown in any of SEQ ID NOs: 1 to 78. The selective IL-4 production-inducing activity associated with NKT cell activation is an activity that does not induce interferon γ (IFN-γ) production by NKT cells and induces IL-4 production by NKT cells. 13. The detection agent according to 13.