JP2005257347A - Anticancer drug screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an excellent immune activating agent usable as an anticancer drug. <P>SOLUTION: In this anticancer drug screening method, an association inhibitor for inhibiting the associated state of a MIST molecule and an Fgr molecule is selected so as to select a substance for inhibiting the association of the Fgr molecule in the proline rich region on the C-terminal side of the MIST molecule or a substance for inhibiting the association of the MIST molecule in the SH3 domain of the Fgr molecule. The association inhibitor is preferably a proline rich region on C-terminal side of the MIST molecule and the selection thereof is preferably performed on the basis of the rise of the concentration of calcium in cell. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a screening method for an anticancer agent, and more particularly to a screening method for screening an anticancer agent having an immunostimulatory effect.

Many of the conventional anticancer agents have anticancer activity, but have strong side effects such as immunosuppression and hematopoietic disorder. On the other hand, immunostimulators used in combination with anticancer treatment have few side effects but weak effects. There was a problem. For this reason, there is still a strong social demand for an effective anticancer agent having strong anticancer activity and few side effects. In developing such an anticancer agent, there is a problem of the cancer cell selectivity of the anticancer agent, and the action mechanism of the immunostimulant is urgently clarified. In particular, there is an urgent need to develop an immunostimulatory drug that has few side-effect problems and restores self-healing ability.
On the other hand, it is known that various cell activities are caused by various signal transduction molecules present on the surface or inside of cells. In particular, elucidation of the mechanism of action of complex immune responses has identified several specific molecules involved in immune responses.
Examples of such a molecule include an adapter molecule specifically expressed in the B cell lineage, BASH (Non-patent Document 1). This BASH is similar in molecular structure to SLP-76 (Non-patent Document 2) expressed in T cells, and through its structural and functional analysis, a signal transduction molecule family specific to blood cell lineage and immune receptors exists. It shows that
In addition, elucidation of the molecular mechanism related to type I allergic reaction reveals a signal transduction mechanism involved in degranulation of mast cells via high affinity IgE receptor, in which a MIST molecule was identified ( Patent Document 1).
J. Immunol., (1998) 161: 5804-5808 J. Biol. Chem., (1995) 270: 7029-7032 JP 2001-78780 A

In spite of the progress of molecular level studies in this way, the identification of signaling-related molecules that can be used as anticancer agents is not sufficient, and no effective molecules that can lead to the development of immunostimulatory drugs have been identified.
Therefore, an object of the present invention is to obtain an excellent immunostimulatory substance that can be used as an anticancer agent.

The screening method of the present invention is a screening method for an anticancer substance, and is characterized by selecting an association inhibitor that inhibits the association state between the MIST molecule and the Fgr molecule.
Here, the selection is to select a substance that inhibits the association of the Fgr molecule in the C-terminal proline-rich region of the MIST molecule or a substance that inhibits the association of the MIST molecule in the SH3 domain of the Fgr molecule. It is preferable. The association inhibitor is preferably an amino acid that binds to the C-terminal proline-rich region of the MIST molecule.

  According to the present invention, since an association inhibitor that inhibits association between the MIST molecule and the Fgr molecule is selected, an excellent immunostimulatory substance that can be used as an anticancer agent can be obtained.

The screening method of the present invention is characterized by selecting an association inhibitor that inhibits the association state between the MIST molecule and the Fgr molecule.
MIST molecules are known to be highly expressed not only in mast cells but also in IL-2 stimulated NK cells. It has been found by the present invention that such a MIST molecule suppresses the function of NK (natural killer) cells, particularly the cytotoxic function and the IFN-γ production function, by association with the Fgr molecule. It was done. This means that if the association state between the MIST molecule and the Fgr molecule in the NK cell is inhibited, the function of the MIST molecule is inhibited, and the immunostimulatory antitumor activity of the NK cell can be enhanced. . Therefore, an excellent immunostimulatory substance can be obtained by selecting a substance that inhibits the association state between the MIST molecule and the Fgr molecule.

MIST (mast cell immunoreceptor signal transducer) molecule has a tyrosine residue that is phosphorylated at the N-terminus, a proline-rich region at the center (proline-rich region), and an SH2 domain at the C-terminus, and has kinase activity and enzyme activity. It is known to be a typical adapter molecule without it, and the sequences of mouse MIST and human MIST have already been identified (SEQ ID NO: 1 and 2 for mouse, SEQ ID NO: 3 and 4 for human).
The Fgr molecule is an Src-type tyrosine kinase expressed in blood cells, having an N-terminal to SH2 domain, an SH3 domain, and a C-terminal tyrosine kinase domain (DDBJ / GenBank / EMBL accession number; P14243).

The “association” in the present invention means that another molecule contacts or binds to the MIST molecule so that signaling for the functional expression of the MIST molecule occurs in the MIST molecule. For this reason, it includes not only irreversible chemical bonds and physical bonds, but also bonds that can be bonded and detached under conditions such as pH and temperature.
In addition, “inhibiting the association state” means not only blocking the association between the MIST molecule and the Fgr molecule with another substance interposed, but also missing or destroying at least one of at least one part, and at least It also includes preventing the overall expression of either one to prevent association of these molecules.
Therefore, inhibition of the association state of MIST molecules and Fgr molecules may be at the protein level of each molecule or at the gene level.

  The screening method of the present invention may select a substance that inhibits the association of Fgr molecules in the C-terminal proline-rich region of the MIST molecule. In such a selection, a molecule that can specifically bind to the C-terminal proline-rich region of the MIST molecule, such as a DNA fragment or amino acid having a sequence complementary to the base sequence or amino acid sequence of this region, A molecule can be selected. Similarly, a substance that inhibits the association of the MIST molecule in the SH3 domain of the Fgr molecule may be selected, or may have any property. Since the C-terminal proline-rich region of the MIST molecule and the SH3 region of the Fgr molecule are regions necessary for the functional expression of the MIST molecule, inhibition of the association state of the molecule is used as an index for at least one of these. By doing so, an anticancer substance can be effectively screened.

  Screening can be performed based on known screening techniques. Such screening methods include: 1. Method using surface plasmon sensor 2. a method using the yeast two-hybrid method; A method of measuring an indicator substance based on cell activation is included.

  For example, when a surface plasmon sensor is used, a MIST protein is immobilized on a sensor chip, and then Fgr protein is injected as an analyte, so that a MIST and Fgr complex is formed in advance, and various candidate substances are formed there. And the dissociation reaction of the MIST-Fgr complex on the sensor chip can be measured.

  When the yeast two-hybrid method is used, a fusion protein of the transcriptional activation domain of the Gal4 transcription factor and MIST and a fusion protein of the DNA binding domain and Fgr are expressed in yeast, whereby the association of MIST and Fgr. Dissociation can be evaluated by activation of a reporter gene that is a target of the Gal4 transcription factor, such as auxotrophy or color development by LacZ. By adding a specific substance to this system, inhibition of Gal4 transcription factor activation can be used as an indicator to screen for a binding inhibitor of MIST and Fgr. Further, by replacing the transcription factor from Gal4 to VP-16 and the reporter gene to the EGFP gene, it is possible to search for an association inhibitor by a two-hybrid method using mammalian cells in addition to yeast.

Examples of a method for measuring an indicator substance based on cell activation by receptor stimulation include measurement of intracellular calcium concentration and IFN-γ concentration. Measurement of cell activation such as intracellular calcium concentration and IFN-γ measurement is performed by adding specific substances to cells expressing either MIST molecule or Fgr molecule and cells expressing both. However, it can be performed by measuring cell activation by receptor stimulation according to a conventional method. At this time, there is no effect in cells expressing MIST molecule or Fgr molecule alone, and in cells expressing both, the increase in intracellular calcium concentration or the amount of IFN-γ produced changes. Since the association is inhibited, a substance having such an action can be recognized as an association inhibitor.
Examples of cells expressing MIST molecules or Fgr molecules alone include NK cells derived from MIST-deficient mice, and examples of cells expressing MIST molecules and Fgr molecules include wild-type mouse NK cells. In addition, the measurement of intracellular calcium concentration and the measurement of IFN-γ can use methods well known in this industry including the use of calcium fluorescent indicators and anti-IFN-γ antibodies.

  These screening methods may be performed alone, but are preferably performed by appropriately combining them. In this case, the order of combination of the screening methods is not particularly limited, but the last use of the screening method including measurement of an indicator substance based on cell activation is to inhibit the association of MIST and Fgr and change the cell function. This is preferable because sorting can be performed directly based on this.

Since the association-inhibiting substance obtained by the screening method of the present invention can be expected to have a function as an anticancer agent, the substance obtained by screening can be used as it is or in combination with known excipients. Can be generated.
Further, by analyzing the association state between the MIST molecule and the Fgr molecule on a molecular model and designing a molecule that inhibits the association state, a substance having an anticancer action can be obtained. Alternatively, the activity of known anticancer agents can be evaluated using the present invention.

[Example 1]
Preparation of MIST-deficient mice A fragment containing the second exon of the mouse MIST gene (including the translation initiation codon) was obtained by PCR using genomic DNA of E14ES cells.
The homologous region of the final vector is composed of a 5.75 kb genomic fragment immediately before (upstream side) the MIST translation initiation codon and a 0.85 kb EcoRI-BamHI intron DNA fragment immediately after the second exon (downstream side). It was. The 5 ′ portion of the second intron and the 50 bp second exon encoding the first 6 amino acids were replaced with the EFGP-neomycin resistance gene cassette. This cassette is constructed using an enhanced green fluorescent protein (EGFP) cDNA and SV40 poly A signal sequence (pEGFP-Cl, Clontech) and a neomycin resistance gene surrounded by pGKneo-derived loxP sequence. (See FIG. 1).

  All these fragments were incorporated into the pKSTKLokPNeo targeting vector containing the HSV thymidine kinase gene (HSV-TK). The obtained MIST targeting construct was introduced into E14ES cells by electroporation, and the introduced cells were screened using G418 (0.3 mg / ml) and gancyclovir (0.2 μM). Drug resistant colonies were screened for homologous recombination events by PCR and confirmed by Southern blot analysis. Target clones were introduced into C57BL / 6 blastocysts.

Chimeric mice were obtained, and Southern blot analysis was performed on the offspring to screen mutant offspring. A heterozygote (MIST ^{+ / neo} mouse) was crossed with a BALB / c or C57BL / 6 mouse to produce a control animal. EGFP fluorescence was not detected in MIST positive cells from MIST ^{+ / neo} mice due to translation silencing by the inserted neomycin resistance gene cassette. Therefore, in order to delete the neomycin selection cassette from the mutant homologous chromosome, the pCre-pac plasmid was temporarily introduced into a blastocyst derived from a female C57BL / 6 mouse crossed with a male MIST ^{+ / neo} mouse, MIST ^{+ / Dneo} mice were generated. Homozygous mice were obtained using MIST ^{+ / neo} mice hanged 8 times for BALB / c control animals and MIST ^{+ / Dneo} mice hanged 6 times for C57BL / 6 control animals.

[Example 2]
Cell distribution in MIST-deficient mice In order to examine various cell distributions in MIST-deficient mice, analysis by flow cytometry was performed using various antibodies (see FIG. 2).
Cell suspensions were prepared from thymus (FIG. 2a), bone marrow (FIG. 2b), liver (FIG. 2c) and spleen (FIG. 2d) of 4-6 week old MIST-deficient mice, and various cells were subjected to flow cytometry (Becton). -It investigated using the Dickinson company make; CELLQuest software. As primary antibodies, anti-CD18 (2.4G2), anti-TCRαβ (H57-597), anti-NK1.1 (PK136), anti-CD3ε (145-2C11), anti-B220 (RA3-6B2), DX-5, anti-c- kit, anti-CD4, anti-CD8α, anti-CD62L, anti-Ly49A (A1), anti-Ly49D (4E5), anti-Ly49C / I (5E6), anti-Ly49G2 (4D11), anti-CD94 and 2B4 (respectively, manufactured by BD Farmingen, San Jose , USA; eBioscience, San Diego, USA) and FITC-conjugated antibody, PE-conjugated antibody, biotin-conjugated antibody, streptavidin PerCP-Cy5.5 (BD Farmingen, San Jose, USA) as secondary antibodies did.

  As shown in FIG. 2, no abnormalities were observed in the differentiation of hematopoietic cells including B cells, T cells, NK cells, and NKT cells in each organ of MIST-deficient mice. The ratio of the NK cell population expressing the inhibitory receptors such as Ly49G2 and CD94 and the activating receptor such as Ly49D and the expression level of each receptor were not changed compared to the wild type mice.

[Example 3]
NK cytotoxicity assay Next, MIST-deficient mouse cells were examined for cytotoxicity against various tumor cells.
Cytotoxicity was examined by a standard ⁵¹ Cr release assay (normal 4 hours) (J. Exp. Med., (1997) 186; 1957-1963). Briefly, 1 × 10 ⁶ target cells were incubated with 100 μCi Na ₂ ⁵¹ CrO ₄ (Amersham) for 1 hour and then washed thoroughly. FACS-sorted NK cells (NK1.1 ⁺ TCRαβ ⁻ ) cultured in the presence of IL-2 (2000 U / ml) for 5 to 7 days in a final volume of 200 μL in two wells in a U-bottom microtiter plate, target cells (4 × 10 ³ pieces / well). Target cells include YAC-1, B16 melanoma cells (provided by Dr. Takabe of Juntendo University) (a), Ba / F3 cells, and MHCV m157 antigen or RAE1-γ expressed in these cells. Tanto (provided by Dr. Arase of Chiba University) (b) and P815 of FcγR ⁺ in the presence of anti-NK1.1 antibody, anti-CD16 antibody, anti-2B4 antibody and anti-Ly49D antibody (provided by Dr. Takabe of Juntendo University) And P815 only (c).

As shown in FIG. 3, when IL-2 activated spleen NK cells (NK1.1 ⁺ TCRαβ ⁻ ) were compared with wild type (black circles) and MIST-deficient mice (white circles), YAC-1 cells and B16 Cytotoxic activity against cell cytotoxicity (FIG. 3a) and against Ba / F3 cells in which m157 antigen of mouse cytomegalovirus which is a ligand of Ly49H receptor and RAE1-γ which is a ligand of NKG2D receptor are expressed. All of the cytotoxic activities (FIG. 3a) were enhanced compared to wild-type NK cells. Furthermore, P815 expressing Fc receptors and various NK cell receptor-specific antibodies were used to analyze the cytotoxic activity via each receptor. MIST-deficient NK cells were mediated by any receptor. Cytotoxic activity was also enhanced compared to wild type NK cells.

[Example 4]
Enhancement of INF-γ production in MIST-deficient NK cells Wild-type NK cells (+ / +) and MIST-deficient NK cells (− / −) stimulated with 1 × 10 ⁵ IL-2, respectively, were treated with anti-NK1.1 antibody. Incubated for 6 hours in the presence of 2 μM monensin (Sigma-Aldrich) or in the presence of PMA (50 ng / ml) + ionomycin (1 μM) on microtiter plates pre-coated with anti-Ly49D antibody. After harvesting, the cells were fixed with 4% paraformaldehyde and permeabilized in a solution containing 50 mM NaCl, 5 mM EDTA, 0.02% NaN ₃ and 0.5% Triton X-100. After blocking with PBS containing 3% bovine serum albumin, the cells were stained with an anti-INF-γ-PE antibody (XMG1.2; manufactured by BDPharMingen) and examined for intracellular INF-γ using flow cytometry. The results are shown in FIG.

Further, wild type NK cells (black) and MIST-deficient NK cells (white) cultured in the presence of IL-2 are each cultured at 5 × 10 ⁴ cells / 200 μL in a flat bottom microtiter plate in the presence of IL-2. Then, each of the anti-CD3 antibody, the anti-NK1.1 antibody, the CD16 antibody, the Ly49D antibody, or the 2B4 antibody was stimulated with each immobilized antibody or IL-12 (10 ng / mL). Target cells (YAC-1 and B16) were then co-cultured for 24 hours in U-bottom plates in a 1: 1 ratio with NK cells in the presence of IL-2. After the culture, the cell-free culture supernatant was collected, and the amounts of IFN-γ and IL-4 were measured using an ELISA kit (manufactured by BioSource). The results are shown in FIG. *; P <0.05, **; P <0.01.

  As shown in FIGS. 4 (A) and (B), IFN-γ production by NK cell receptor stimulation was analyzed by intracellular staining and ELISA. As a result, in any of NK1.1, Ly49D, CD16, and 2B4 stimulation However, MIST-deficient NK cells showed significantly higher production than the wild type. The above results revealed that MIST negatively regulates a wide range of activated NK receptor signals.

[Example 5]
Enhancement of NK receptor signaling in MIST-deficient NK cells In order to elucidate the mechanism of activation enhancement associated with NK receptor stimulation observed in MIST-deficient NK cells, biochemical analysis of signal transduction downstream of NK receptors was performed. .
NK1.1-mediated IFN-γ production was compared between wild-type NK cells and MIST-deficient NK cells in the presence of PP2 or picetanol, which are Src family PTK inhibitors. As a result, enhanced tyrosine phosphorylation of PLC-γ2 and activation of ERK2 and p38MAP kinase accompanying stimulation with NK1.1 were observed. In addition, as a result of analyzing a signal transduction pathway involving MIST with a specific inhibitor of Src type tyrosine kinase or Syk / ZAP-70 type tyrosine kinase using IFN-γ production as an index, MIST-deficient NK cells are wild type cells. Compared with SYK / ZAP-70 tyrosine kinase inhibitor, MIST-deficient NK cells have enhanced activation of Syk / ZAP-70 pathway associated with NK receptor stimulation. (Data not shown).

[Example 6]
Identification of functional domain of MIST involved in NK receptor signaling In order to clarify the functional domain of MIST involved in negative regulation of NK receptor signaling, various MIST functional domain mutants were introduced into MIST-deficient NK cells. It was introduced using a retroviral vector.
CDNAs of various mutants of mouse wild type MIST (WT), N-terminal proline-rich region deletion mutant (dPR1), and C-terminal proline-rich region deletion mutant (dPR2) were transformed into pMX-IRES-GFP retro. Inserted into viral vector. Since this retroviral vector expresses EGFP in addition to the inserted gene, the transfected cells can be analyzed by FACS using the fluorescence of EGFP as an indicator. Each of the obtained plasmids was introduced into the package cell line PLAT-E using FuGNEN6 (Roche) and incubated for 24 hours, and then the culture supernatant was collected and concentrated as a virus stock.

NK cells or NKT cells sorted using FACS were cultured for 24 hours in the presence of IL-2 and infected with retrovirus in the presence of 0.5 μg / ml polybrene. Cells were further cultured for 3 days in the presence of IL-2, and stimulated with anti-NK1.1 antibody or PMA + ionomycin for 6 hours on day 5 to perform intracellular IFN-γ staining.
The results are shown in FIG. In contrast to NK cells using the control virus (second row in the middle row), suppression of IFN-γ production was observed in the wild-type NK cells (third row in the middle row). In contrast, when the mutant MIST was introduced, a partial increase in IFN-γ production was observed when the N-terminal proline-rich region was deleted (middle row 4th row). In addition, when the C-terminal proline-rich region was deleted, production of IFN-γ was not suppressed (lower row in the middle row).
These facts revealed that the proline-rich region present at the C-terminus of MIST and the molecule associated therewith are essential for negatively controlling the NK receptor signal.
When a mutant in which tyrosine residues are substituted with phenylalanine and a non-functional SH2 domain mutant are introduced, as in the case of introducing wild-type MIST, IFN− of MIST-deficient NK cells accompanying NK receptor stimulation is introduced. Increased production of γ was reduced to the level of wild type NK cells (data not shown).

[Example 7]
Identification of molecules associated with the C-terminal proline-rich region of MIST Using the cDNA library derived from human mast cells and spleen using the amino acid residues 1 to 213 of human MIST as a bait, We searched for binding molecules. As a result, Fgr, which is an Src tyrosine kinase, was identified as a molecule that associates with the MIST molecule.
Spleen NK cells (NK1.1 + TCRαβ−) and NKT (NK1.1 + CD4 +) selected by FACS were activated with IL-2 for 5 days and subjected to RT-PCR analysis. Total RNA was isolated using TRIZOL reagent (BRL) and then converted to single stranded cDNA using SuperScript II reverse transcriptase with oligo (dT) primer (Invitorogen). Using the following primers, the obtained cDNA was subjected to PCR on MIST, Fgr or β-actin mRNA.

For MIST
5'-CTTACAGAGTGCTCCAGGATGCGACCGTGG-3 '(SEQ ID NO: 5)
5'-GAGGGGTTGGTACTTCTTGGGGAGAGTGATAGC-3 '(SEQ ID NO: 6)
Against Fgr
5'-TGGTGGGGGAATACCTAATATGCAAGATCG-3 '(SEQ ID NO: 7)
5'-TGCCCAGTCAGGCTATGTCTGGTCTCCAGG-3 '(SEQ ID NO: 8)
against β-actin
5'-TACAATGAGCTGCGTGTGGC-3 '(SEQ ID NO: 9)
5'-TAGCTCTTCTCCAGGGAGG-3 '(SEQ ID NO: 10)

  This Fgr was also co-expressed in COS-7 cells with either or both of the wild type (WT) or proline rich region deletion mutants (dPR1 and dPR2). Cell lysate (WCL) was subjected to immunoprecipitation (IP) using an anti-MIST antibody and blotted with an anti-Fgr antibody (Santa Cruz Biotechnology Inc.) or anti-MIST antibody.

  As a result, as shown in FIGS. 6a and 6b, it was revealed that in NK cells, the MIST molecule and the Fgr molecule are expressed together at the RNA level and immunoprecipitated together with MIST. Furthermore, as shown in FIG. 6 c, Fgr cannot be associated when the C-terminal proline-rich region of MIST is deleted, suggesting that it binds to the C-terminal proline-rich region via the SH3 domain. .

[Example 8]
Suppression of NK cell activation depending on interaction between MIST and Fgr It was found that both MIST and Fgr are expressed in NK cells, but only MIST is expressed in NKT cells. Using this, the influence of the interaction between MIST and Fgr on the production of IFN-γ by NK cell receptor stimulation by ectopically expressing the Fgr molecule in NKT cells was analyzed.
NKT cells (wild type mouse cells) cultured in the presence of IL-2 were infected with a retrovirus expressing MIST and EGFP. On day 5, cells were stimulated with anti-NK1.1 antibody, anti-CD3 antibody or PMA + ionomycin, respectively, for 6 hours and analyzed for intracellular IFN-γ production.
As a result, as shown in FIG. 7, in NKT cells that do not express Fgr, IFN-γ production by NK1.1 stimulation is enhanced in MIST-deficient mice (− / −) as compared to wild-type mice (+ / +). In addition, when Fgr is ectopically expressed in wild-type NKT cells, the production of IFN-γ decreases, and in MIST-deficient NKT cells, the decrease in IFN-γ production by Fgr introduction is not observed. It was revealed that the interaction between MIST and Fgr is essential for the suppression of NK cell receptor signal.

[Example 9]
Enhancement of resistance to tumor lung metastasis in MIST-deficient mice In order to grasp the NK cell function in MIST-deficient mice at a living body level, examination was performed using a lung metastasis model of tumor cells.
Colon cancer cells Colon-26 cells (2.4 × 10 ⁴ cells) and black cell type B16-BL6 (2 × 10 ⁵ cells) were passed through BALS / c and C56BL / 6 strains of MIST-deficient mice and wild-type mice, respectively. It was administered intravenously. The number of nodules of tumor cells that had metastasized to the lung after 2 weeks was counted using a dissecting microscope.
As a result, as shown in FIG. 8a and FIG. 9, a significant decrease in the number of lung metastatic nodules was observed in the MIST-deficient mouse (black) compared to the wild-type mouse (white) in any tumor cell administration group. (*; P <0.01).
Then, in order to examine the effect of NK cell removal, anti-asialo GM1 antibody was administered intraperitoneally on the 2nd, 3rd and 8th days. NK cell depletion was confirmed by the absence of NK1.1 ⁺ CD3 ⁻ cells in the spleens of treated mice. As a result, by removing NK cells in this way, metastasis resistance in MIST-deficient mice disappeared (see FIG. 8b).
From these facts, it was clarified that NK cell function enhancement due to MIST deficiency exists at the living body level, leading to enhancement of antitumor activity.

  Thus, by inhibiting the association state of the MIST molecule and the Fgr molecule, it is clear that the suppressive function of the MIST molecule is inhibited and an immunostimulatory anticancer effect is produced. Therefore, a substance useful as an anticancer agent can be obtained by screening a substance that inhibits the association state between the MIST molecule and the Fgr molecule by various techniques. For example, by using a method using a surface plasmon sensor, a method using a yeast two-hybrid method, or a method of measuring intracellular calcium concentration or IFN-γ concentration, the association state between the MIST molecule and the Fgr molecule is inhibited. A useful anticancer substance can be easily obtained by using whether or not to do so as an index.

FIG. 1 is a configuration diagram of a targeting construct used for producing a MIST-deficient mouse according to this example. FIG. 2 is an analysis diagram of flow cytometry showing various cell distributions in the spleen (a), bone marrow (b), liver (c) and spleen (d) of MIST-deficient mice. FIG. 3 shows Ba / 3 cells (a) in MIST-deficient mouse cells, transfectants in which MHCV m157 antigen or RAE1-γ is expressed in Ba / 3 cells, and P815 expressing receptors for IgG. It is the graph which compared (c) cytotoxicity when the antibody specific to various NK receptors was couple | bonded with the cell. FIG. 4A is a flow cytometric analysis showing intracellular INF-γ expression in MIST-deficient NK cells. FIG. 4B shows wild-type NK cells (black) cultured in the presence of IL-2 and MIST. It is the graph which compared the production of INF-γ in a deficient NK cell (white). FIG. 5 is an analysis diagram of a flow cytometer comparing the production state of intracellular INF-γ. Gel electrophoresis photograph (a) showing the expression state of mRNA of MIST molecule and Fgr molecule in IL-2 activated NK cell or NKT cell, stained diagram after immunoprecipitation (b), specific site of MIST molecule and Fgr molecule It is the dyeing | staining figure after the immunoprecipitation explaining the association state with (c). It is an analysis figure of the flow cytometer which shows the production of INF-γ by the interaction between the MIST molecule and the Fgr molecule. It is a graph which shows the comparison of tumor lung metastasis in (a) and anti-asialo GM1 antibody presence with respect to various tumor cells (b), MIST deficient mouse (black), and wild type mouse (white). It is a photograph which shows the state of tumor lung metastasis in a MIST deficient mouse (black) and a wild type mouse (white).

Claims (6)

A screening method for anticancer substances,
A screening method comprising selecting an association inhibitor that inhibits the association state between a MIST molecule and an Fgr molecule. 2. The screening method according to claim 1, wherein the selection is to select a substance that inhibits association of Fgr molecules in the C-terminal proline-rich region of the MIST molecule. The screening method according to claim 1, wherein the selection is to select a substance that inhibits association of MIST molecules in the SH3 domain of the Fgr molecule. The screening method according to claim 1, wherein the association inhibitor is an amino acid that binds to a C-terminal proline-rich region of a MIST molecule. The screening method according to claim 1, wherein the selection includes measurement of an indicator substance based on cell activation. 6. The screening method according to claim 5, wherein the indicator substance is intracellular calcium or interferon γ.