JP2009108013A - Functional antibody against g protein conjugate receptor and its application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody that is specifically bound with a G protein conjugate receptor (GPCR) and has an interaction with the receptor and to provide a method for using the antibody. <P>SOLUTION: The antibody is specifically bound with an extracellular domain of GPCR or has an interaction with the domain and for example, a functional antibody having an agonist function of GPCR is prepared. More in detail, an anti-GPR56 extracellular domain antibody, in which GPCR is GPR56, is prepared. An agonist and/or antagonist of GPCR can be screened by using the functional antibody. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a functional antibody against a G protein-coupled receptor that is a neural stem cell marker protein and use thereof. Specifically, the present invention relates to an antibody specific for the extracellular domain of a G protein-coupled receptor and its application.

  G protein-coupled receptor (GPCR) is a biosensor that recognizes neurotransmitters and hormones on the cell membrane and acts as an antenna molecule when cells receive extracellular signals. It is essential in various biological systems such as the nervous system, endocrine system, circulatory system, and immune system for maintaining the system. Human genome analysis revealed the existence of over 800 types of GPCRs. These are characterized by having seven transmembrane sites. Since it is said that about half of the drugs currently used clinically work on GPCRs, it can be said to be a major target for drug discovery. The ligand-bound GPCR acts on a trimeric G protein (αGDPβγ) inside the cell membrane and promotes the release of GDP. After GDP is dissociated, GTP binds to the α subunit, and the trimeric G protein dissociates into αGTP and βγ. αGTP and βγ each act on an enzyme and an ion channel to transmit information in the cell.

  Numerous unknown genes have been found that appear to encode GPCRs from gene sequences revealed by the Human Genome Project, which are agonists or ligands (eg, hormones present in the body) to which the receptor binds. Because it is unknown, it is called “Orphan GPCR”. GPCRs are divided into several families based on amino acid sequence homology (Non-Patent Document 2), but most of the receptors belonging to the adhesive GPCR family having a long extracellular region are, for example, GPR56. An orphan receptor whose agonist is unknown.

  There is a disclosure of a novel receptor called TSR32 in the GPCR family (Patent Document 1). Here, there is disclosure of an isolated polynucleotide molecule that encodes a TSR32 receptor, as well as an antibody or antibody fragment that specifically binds to the TSR32 receptor. Also disclosed herein are methods for detecting TSR32 receptor agonist and / or antagonist compounds. However, there is no disclosure at all about the use of an antibody in performing the detection or that the antibody itself has an agonist function.

  Regarding the receptor called GPR56 in the GPCR family, there is a report on the relationship between abnormalities in the gene of the extracellular domain and BFPP disease of the brain (Non-patent Document 1). Here, it has been suggested that mutations in the extracellular domain of the GPR56 gene are associated with BFPP disease. In addition, it has been reported that when a GPR56 gene having a mutation found in a BFPP patient is expressed in a cell, the expression of the mutant GPR56 on the cell membrane surface is reduced (Non-patent Document 3).

  There is a disclosure regarding identification of a ligand that interacts with GPR56 and provision of a therapeutic drug (Patent Document 2). Here, GPR56 is shown to be expressed in specific regions, including the hippocampus, thalamus, and paraventricular nucleus of the hypothalamus. Furthermore, it has been confirmed here that mRNA expressed by a gene encoding GPR56 is expressed with a difference from the wild type in an appetite / obesity model of rodents. Therefore, here, it is an object to identify a ligand that interacts with GPR56 and to provide a therapeutic agent, and there is disclosure about screening an agonist and / or antagonist of GPR56. However, there is no disclosure about the relationship between agonists and / or antagonists and anti-GPR56 antibodies.

Furthermore, there is a disclosure of a gene encoding a polypeptide having the antigenicity of GPR56 (Patent Document 3). Here, a method for evaluating cell canceration using GPR56 expression as an index is described, and although anti-GPR56 antibody is disclosed as a GPR56 expression detection method, anti-GPR56 antibody has an agonistic function. There is no disclosure.
Special table 2001-513653 publication Special table 2003-531599 publication US application specification US20060137029 Science, 303, 2033 (2004) Mol. Pharmacol. 63, 1256 (2003) Hum. Mol. Genet., 16, 1972 (2007)

  It is an object of the present invention to provide a substance that can specifically bind to or interact with a G protein-coupled receptor (hereinafter sometimes simply referred to as “GPCR”). It is an object to provide a usage method.

  In order to solve the above problems, the present inventors have focused attention on an antibody that specifically binds or interacts with the extracellular domain of GPCR, and as a result of intensive research, for example, obtain a functional antibody having an agonist function of GPCR. The present invention was completed successfully.

That is, the present invention is as follows.
1. An anti-GPCR extracellular domain antibody having a function of specifically binding to or interacting with the extracellular domain of GPCR and signaling inside the cell.
2. 2. The antibody according to item 1 above, wherein the antibody functions as a signal transducing into a cell by an agonist function of GPCR.
3. 3. The antibody according to item 1 or 2, wherein the GPCR is GPR56.
4). The antibody according to any one of the preceding items 1 to 3, wherein the antibody can recognize any part selected from the following regions as an antigen epitope:
1) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and containing at least 3 amino acid residues;
2) In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more of
3) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing and containing at least 3 amino acid residues;
4) In the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more.
5). A method for selecting an agonist and / or antagonist of a GPCR, wherein the GPCR interacts with the antibody according to any one of 1 to 4 above, and further a candidate compound is allowed to act to measure the function of the GPCR, A method for screening for an agonist and / or antagonist of GPCR, comprising selecting an alternative to an antibody or a substance that enhances or inhibits the interaction between the GPCR and the antibody.
6). A cell transplantation drug used for transplanting cells or tissues containing the antibody or the GPCR agonist and / or antagonist obtained by the screening method of the previous item 5 as an active ingredient for therapeutic purposes, And / or a regenerative medicine used to activate or protect a patient's cells and tissues.
7). Inhibition of proliferation, migration, invasion, metastasis, and engraftment of cancer cells containing the antibody according to any one of items 1 to 4 or the GPCR agonist and / or antagonist obtained by the screening method according to item 5 as an active ingredient Drug.
8). 5. A reagent for examining neural stem cells, comprising the antibody according to any one of 1 to 4 above or the GPCR agonist and / or antagonist obtained by the screening method according to 5 above.
9. Reagent for examination of proliferation, differentiation, migration and / or adhesion, engraftment of cancer cells containing the antibody according to any one of 1 to 4 above or the GPCR agonist and / or antagonist obtained by the screening method according to 5 above .
10. 5. A method for examining neural stem cells, comprising: interacting a GPCR with the antibody according to any one of 1 to 4 above; and causing a biological sample collected from a subject to act to measure GPCR receptor function.
11. A method for examining invasion and / or metastasis of cancer, which comprises measuring a GPCR function by allowing a GPCR to interact with the antibody according to any one of 1 to 4 above and causing a biological sample collected from a subject to act.

  The anti-GPCR extracellular domain antibody of the present invention specifically binds to or interacts with the extracellular domain of GPCR and has a function of signal transduction into the cell. The function of performing signal transmission into cells is, for example, an agonist function of GPCR.

  With the antibody of the present invention, it is possible to analyze the function of an orphan receptor such as GPR56 among GPCRs. In addition, by using the antibody of the present invention, a screening method for selecting an agonist and / or antagonist of GPCR can be provided. Furthermore, a reagent for examining proliferation, differentiation, migration, and / or adhesion of neural stem cells and cancer cells, which contains the antibody of the present invention and the GPCR agonist and / or antagonist selected by the screening method as an active ingredient, for regenerative medicine Drugs or cancer invasion and / or metastasis inhibitors can be provided.

  In the present invention, the GPCR is not particularly limited as long as it belongs to the GPCR family, and examples thereof include an adhesive GPCR, such as GPR56, GPR113, GPR115, GPR97, GPR114, GPR112 and GPR116. An example is GPR56. Adhesive GPCRs are distributed in various tissues and are thought to play an important role in physiological functions in the immune system, central nervous system, regeneration system, and the like. These are characterized by having a 7-transmembrane site, and each has a long N-terminus, a GPS (GPCR proteolytic site) domain, and each domain (Fredriksson R. et al., FEBSLetters 531, 407-414). (2004)). The N-terminus of the adhesive GPCR is considered to be functionally important for the recognition of ligands and the like, but most receptors are considered to be orphan receptors, and their functions are often unknown.

  FIG. 1 shows the distribution of GPR56 and GPR56 extracellular domain (hereinafter sometimes referred to as “GPR56ECD”) in adult mouse tissues and the expression in neural progenitor cells (NPCs) prepared from mouse fetuses. . In adult mice, GPR56ECD is expressed in the liver, kidney and placenta. Further, FIG. 2 shows the state of mouse fetal brain development in the central nervous system of GPR56, and FIG. 3 shows the results of examining SDS-PAGE for the expression of GPR56 and GPR56ECD at the stage of mouse fetal brain development.

  The extracellular domain antibody of the GPCR of the present invention is specifically an antibody that can recognize any part selected from the following regions as an antigen epitope. The antibody can be obtained by selecting one of the following polypeptides as an antigen, inoculating it into an animal for obtaining an antibody such as a rabbit or rat, and selecting it. Furthermore, a mouse can be inoculated with any one of the following polypeptides as an antigen, and a monoclonal antibody can be prepared according to an ordinary method.

1) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and containing at least 3 amino acid residues;
2) In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more of
3) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing and containing at least 3 amino acid residues;
4) In the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more.

  In the above, the sequence described in SEQ ID NO: 1 in the sequence listing shows the amino acid sequence of human GPR56 registered in GenBank ACCESSION No. BC008770, and the sequence described in SEQ ID NO: 2 is registered in GenBank ACCESSION No. BC034678. The amino acid sequence of mouse GPR56 is shown.

  In any one of the above 1) to 4), the polypeptide containing at least 3 amino acid residues indicates a minimum number of amino acid residues having antigenicity. Furthermore, it may be a polypeptide that can specifically bind to the extracellular domain of GPR56 or can serve as an antigen for obtaining an antibody having an interaction. Such a polypeptide is a polypeptide selected from a mutated sequence in which one or more amino acids are substituted, deleted, added or derived from the amino acid sequence shown in SEQ ID NO: 1 or 2. Also good.

  The antibody of the present invention is not particularly limited as long as it specifically binds to or interacts with the extracellular domain of the GPCR and has a signal transduction function into the cell. Signal transduction into the cell is particularly suitable if it is a signal having an agonist function of GPCR. The agonist function of GPCR can be confirmed by measuring GPCR function. As a method for measuring the GPCR function, a new method known per se or developed in the future can be applied. For example, the following method of confirming gene expression by GPCR or confirming the effect of GPCR on cell migration ability can be mentioned.

  Measurement of gene expression by GPCR as a GPCR function can be performed by expressing GPCR together with a plasmid in which a reporter protein gene is incorporated downstream of an arbitrary transcriptional regulatory sequence and confirming the effect on expression of the reporter protein gene. . As the reporter protein, those known per se can be used, for example, Luc (luciferase), CAT (chloramphenicol acetyltransferase), DsRed (Discosoma sp. Red Fluorescent Protein), GFP (green fluorescent protein), GUZ (beta glucuronidase) and the like can be used.

  Among GPCRs, GPR56 is expressed in neural stem cells and neural progenitor cells (NPCs), and has been reported as a causative gene of human cerebral cortex dysplasia. GPR56 regulates the migration of neural progenitor cells from the ventricular zone (VZ) to the intermediate zone (IZ) and further to the cortical plate (CP) that becomes the cerebral cortex. Is estimated (see FIG. 4). Therefore, the function of GPCR can be measured by examining the migration ability of neural progenitor cells. Specifically, when forming the cerebral cortex, it can be measured by examining the influence of neural progenitor cells on migration from VZ of brain tissue to IZ and further to CP.

  The function of the GPCR extracellular domain antibody of the present invention can be confirmed by examining the influence of the GPCR function on the measurement.

In addition, screening for selecting an agonist and / or antagonist of GPCR can be performed using the antibody of the present invention. GPCR agonists and / or antagonists are selected by selecting a substitute for the antibody or a substance that enhances or inhibits the interaction between the GPCR and the antibody. Selection of substances that enhance or inhibit the interaction between the GPCR and the antibody is based on measuring the function of the GPCR. Here, the function of GPCR can use gene expression, cell migration, or dynamics of intracellular second messenger cyclic AMP and calcium. Specifically, by a method comprising the following steps:
1) The GPCR, the antibody and the candidate compound interact with each other simultaneously or at different times,
2) a step of measuring the function of GPCR by any one of the above indicators;
3) A step of selecting a substitute for the antibody or a substance that enhances or inhibits the interaction between the GPCR and the antibody from the measurement result of the GPCR function.

  By using the antibody of the present invention, screening for selecting an agonist and / or antagonist of GPCR can be performed. Candidate compounds may be proteins, peptides, oligonucleotides, etc. in addition to low molecular weight compounds. It may also be a substitute for the antibody of the present invention.

  The antibodies of the present invention and GPCR agonists and / or antagonists selected by the above screening can be used for the analysis of GPCR ligands and agonists that have not yet been clarified as orphan receptors.

  Among GPCRs, for example, GPR56 is one of the orphan receptors, but as described above, it is known as a causative gene of, for example, human cerebral cortex dysplasia. It has also been suggested to be associated with cancer cell adhesion, metastasis, and malignancy (PNAS, 103 (24), 9023-9028 (2006)). Therefore, the antibodies of the present invention and GPCR agonists and / or antagonists selected by the above screening, in particular GPR56 agonists and / or antagonists, are used as test reagents for neural stem cells, neural progenitor cells, proliferation, differentiation, and migration of cancer cells. , And / or can be used as a test reagent for adhesion. The antibody of the present invention or the GPCR agonist and / or antagonist selected by the above screening, particularly the GPR56 agonist and / or antagonist, is a cell transplant drug and / or patient used for transplanting cells or tissues for therapeutic purposes. It can be used as a drug for regenerative medicine used for activating or protecting the cells and tissues, and a drug for suppressing proliferation, migration, invasion, metastasis, and engraftment of cancer cells. Furthermore, (a) a GPCR, (b) an agonist and / or antagonist of the GPCR selected by the antibody of the present invention or the above screening, particularly an agonist and / or antagonist of GPR56, and (c) a biological sample are allowed to act on By measuring the GPCR function, it is possible to examine neural stem cells and proliferation, differentiation, migration, and / or adhesion and engraftment of cancer cells. Further, an appropriate excipient or stabilizer can be added to the test reagent of the present invention.

  The present invention includes (1) a reagent for examination of neural stem cells, and (2) proliferation, differentiation, and migration of cancer cells, containing as an active ingredient the antibody of the present invention or the GPCR agonist and / or antagonist selected by the above screening. And / or a range of reagents for adhesion and engraftment. Further, the present invention extends to (1) cell transplantation agents and / or regenerative medicine agents and (2) cancer invasion and / or metastasis inhibitors containing the above substances as active ingredients. Furthermore, the present invention extends to (a) a GPCR, (b) a method for examining neural stem cells and a method for examining cancer invasion and / or metastasis by measuring the GPCR function by allowing the above substance and (c) biological sample to act. .

  The agent for cell transplantation and / or regenerative medicine of the present invention and the agent for suppressing cancer cell proliferation, migration, invasion, metastasis, and engraftment may further contain a pharmaceutically acceptable carrier. Such compositions can be administered orally or parenterally. In the case of oral administration, the drug of the present invention is any of ordinary preparations, for example, solid preparations such as tablets, powders, granules, capsules, etc .; liquids; oily suspensions; It can also be used as a dosage form. In the case of parenteral administration, the agent of the present invention can be used as an aqueous or oily suspension injection or nasal solution. In the preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be arbitrarily used. About the dosage of a chemical | medical agent, it can determine suitably according to the intended purpose.

  The present invention will be described below with reference to examples, but it is needless to say that the present examples are for the purpose of better understanding the contents of the invention, and the present invention is not limited to these examples.

Example 1 Preparation of Antigen for Production of Anti-GPR56 Extracellular Domain (GPR56ECD) Antibody His-GPR56 cells were prepared by genetic recombination using baculovirus to prepare an antigen for production of anti-GPR56ECD antibody. The outer domain was expressed.
GPR56 is represented by human GPR56 (GenBank ACCESSION No. BC008770) shown in SEQ ID NO: 1 in the sequence listing and mouse GPR56 (GenBank ACCESSION No. BC034678) shown in SEQ ID NO: 2.

1) Construction of His-GPR56 extracellular domain construct In order to produce a recombinant protein in which 6x His tag is added to the C-terminus of the polypeptide consisting of Met1-Leu403 obtained from GPR56 shown in SEQ ID NOs: 1 and 2, from ATCC. Using the obtained full-length cDNA of GPR56 as a template, PCR reaction was performed using the following primers to amplify the gene fragment.

A. Primer for preparing human GPR56ECD
5 '-GAAGATCTATGACTCCCCAGTCGCTGC-3' (SEQ ID NO: 3)
3 '-GAAGATCTCTAGTGATGGTGATGGTGATGCAGGTAGTGCTTGTGCACGG-5' (SEQ ID NO: 4)
B. Primer for making mouse GPR56ECD
5'-GAAGATCTATGGCTGTCCAGGTGCTGC-3 '(SEQ ID NO: 5)
3 '-GAAGATCTCTAGTGATGGTGATGGTGATGGAGGTAGTGTTTGTGAGTGG-5' (SEQ ID NO: 6)

2) Transposition The human or mouse GPR56ECD cDNA obtained by the PCR reaction is incorporated into a pFastBac_1 (Invitrogen) vector and introduced into Escherichia coli DH10Bac ^™ (Invitrogen) to produce recombinant Bacmid DNA, and the alkali miniprep method Purified by

3) Sf9 cells of recombinant Bacmid DNA (recombinant Bacmid DNA introduced purified into insect cells) cellfectin ^(R) reagent (Cellfectin ^(R) is introduced into Sf9 cells attached incubated with Reagent (Invitrogen, Inc.) Then, the cells were cultured at 27 ° C. for 72 hours using TC-100 medium.

4) Preparation of recombinant baculovirus The culture supernatant after 72 hours of culture was added to Sf9 cells at 1/10 volume of the medium and cultured at 27 ° C for 72 hours. Liquid. By repeating this at least three times, a stock solution with increased virus and high infection efficiency was prepared.

5) Extraction and purification of GPR56ECD recombinant protein To 4 L of the culture solution containing Sf9 cells (2 × 10 ⁶ cells / ml), 1/100 volume of the medium with the high-titer virus solution obtained above was added. The cells were cultured at 72 ° C. for 72 hours. Thereafter, the mixture was centrifuged at 5000 rpm for 10 minutes at 4 ° C., and the culture supernatant was collected. The collected culture supernatant is passed through an SP Sepharose ^TM Fast Flow column (GE Healthcare), and the column is washed with 50 mM phosphate buffer (Na-Pi buffer) (pH 6.0), and then 50 mM Na-Pi buffer. The solution (pH 8.0) was eluted with 500 mM NaCl and 10 mM imidazole. The eluate was passed through a Ni-NTA agarose column (QIAGEN), washed with 50 mM Na-Pi buffer (pH 8.0), 500 mM NaCl, 20 mM imidazole, then 50 mM Na-Pi buffer (pH 8.0), 500 mM NaCl. The eluate was replaced with 50 mM Na-Pi buffer (pH 6.0) by dialysis and passed through a Mono ^™ S (Pharmacia) column and 50 mM Na-Pi buffer (pH 8.0). Elution was performed to obtain purified GPR56ECD recombinant protein.

(Example 2) Production of anti-GPR56ECD antibody (production of polyclonal antibody)
1) Preparation of rabbit antiserum Rabbit anti-GPR56ECD polyclonal antibody was prepared using the purified GPR56ECD recombinant protein obtained in Example 1 as an antigen.
SPF Japanese white rabbits were inoculated 5 times with the above purified GPR56ECD recombinant protein of the mouse as an antigen, the antibody titer was checked by ELISA (Enzyme-Linked Immuno Sorbent Assay), and then whole blood was collected to obtain antiserum. Here, SPF means absence of a specific pathogen (Specific Pathogen Free).

2) Affinity Purification of Polyclonal Antibody The antibody was purified using an affinity column (HiTrap NHS-activated HP column ™, GE Healthcare) coupled with mouse GPR56ECD prepared from Sf9 prepared by the method of Example 1 from antiserum. did.

(Example 3) Production of anti-GPR56ECD antibody (production of monoclonal antibody)
1) Immunization to mice Purified GPR56ECD recombinant protein (2 mg / ml) of mouse obtained in Example 1 and Freund's complete adjuvant (SIGMA) were mixed at 1: 1, and 50 μl of the mixed solution was mixed for 6 weeks. The footpads of aged mice (strain Balb / c female) were inoculated. Thereafter, when the mice were inoculated every second week for the second and third time, the samples were inoculated with an incomplete mixture of Freund's incomplete adjuvant (SIGMA) and antigen. After a total of 3 immunizations, lymphocytes were removed from the lymph nodes of the mice.

2) Cell fusion The above-obtained lymph node cells and myeloma cells were subjected to cell fusion using 50% polyethylene glycol (PEG) 1500 (Roche) to prepare hybridomas. Thereafter, 100 μl / well was seeded in a 96-well plate so that the total number of cells was 1 × 10 ⁶ cells / ml, and RPMI-1640 (containing 10% FBS, penicillin, streptomycin) and HAT medium (Kohjin-Bio) ( 50-fold dilution) and BM Condimed H1 ^™ (Roche) (10-fold dilution) at 37 ° C. in the presence of 5% CO ₂ .

3) Hybridoma screening
To a 96-well plate for ELISA, an alkaline buffer (sodium carbonate buffer (pH 9.6)) was used as a solvent, a solution containing 5 μg / ml of mouse GPR56ECD was added at 50 μl / well, and allowed to stand at 4 ° C. overnight. The collected hybridoma culture supernatant was added to the antigen-coated plate and allowed to stand at room temperature for 1 hour. After washing with PBST (1 × PBS, 0.2% TritonX-100), 100 μl / well of HRP-labeled anti-mouse antibody IgM + IgG (Jackson Immuno Research LABORATORIES) diluted 5000 times with PBST as a secondary antibody reaction was added for 1 hour at room temperature. Left to stand. After washing again with PBST, a coloroma reaction and a color development reaction were stopped using a peroxidase (POD) chromogenic substrate TMB kit (Nacalai), and a hybridoma was screened by detecting absorbance at a wavelength of 450 nm.

(Experimental Example 1) Confirmation by Western Blotting A schematic diagram of the antibody of the present invention (anti-GPR56ECD antibody) obtained in Examples 2 and 3 is shown in FIG.

  0.1, 1.0, 10 ng of the extracellular domain of mouse GPR56 as an antigen prepared in Example 1 (GPR56ECD) and the neural progenitor cell extract were separated by SDS-PAGE, followed by Western blotting with an anti-mouse GPR antibody. went. From FIG. 6, a band of GPR56ECD was observed at a position of about 50 kDa. Further, from the neural progenitor cell extract, a band was observed at a position of about 62 kDa, and it was confirmed that GPR56 was expressed in the neural progenitor cells. The difference in mobility on electrophoresis, that is, the apparent molecular weight, is thought to be due to the difference between the sugar chain modification of proteins in insect cells Sf9 and the sugar chain modification in mammalian mice.

(Experimental example 2) Measurement of GPR56 function (reporter assay)
In order to measure the GPR56 function of the present invention, a reporter assay using luciferase as a reporter protein was performed. The reporter assay was performed using Dual-Luciferase Reporter Assay ^™ (Promega).

  Regarding the mouse GPR56-expressing cells and control cells in human 293T cells, the relative activity of the reporter protein when each concentration of anti-mouse GPR antibody was added was confirmed.

Human 293T cells were cultured in 48-well plates.
Upstream of the luciferase gene, luciferase gene (pSRE-Luc) with SRE (Serum responsive element) binding sequence of transcriptional regulatory factor, pEF-RL luciferase vector for internal control, and various expression vectors were introduced by the calcium phosphate method did. After culturing for 24 hours, each concentration of the anti-mouse GPR56ECD antibody prepared in Example 2 was added, and the culture was further continued for 6 hours. Thereafter, the cultured cells were collected and a cell extract was prepared.

For the preparation of the cell extract, a lysis reagent (Passive Lysis Buffer ^™ , PLB) included in Dual-Luciferase Reporter Assay ^™ (Promega) was used. Luminescence measurement was performed using a plate reader ARVO ^™ MX (Perkin Elmer). The difference in gene transfer efficiency between samples was corrected using the luminescence intensity of Renilla luciferase to evaluate firefly luciferase activity.

  As a result, it was confirmed that the transcriptional activity via SRE increases in GPR56-expressing cells depending on the antibody concentration (FIG. 7). Accordingly, it was confirmed that the anti-GPR56ECD antibody has a GPR56 agonist function that can activate GPR56 and increase the transcriptional activity via SRE working downstream.

(Experimental example 3) Measurement of GPR56 function (reporter assay) 2
Similar to Experimental Example 2, in order to measure the GPR56 function of the present invention, a reporter assay using luciferase as a reporter protein was performed. The reporter assay was performed using Dual-Luciferase Reporter Assay ^™ (Promega) in the same manner as in Experimental Example 2.

  In the same manner as in Experimental Example 2, a test for confirming the luminescence intensity by SRE luciferase was performed using human 293T cells.

  As a result, as shown in FIG. 8, it was confirmed that the transcriptional activity via SRE increases in the GPR56-expressing cells depending on the anti-GPR56ECD antibody concentration. On the other hand, in cells (mock) that do not express GPR56, the luminescence was low even in the presence of the antibody, and no increase in transcriptional activity via SRE was observed. In addition, even in the case of GPR56-expressing cells, when a control antibody was used instead of the anti-GPR56ECD antibody, an increase in transcriptional activity via SRE depending on the antibody concentration was not observed. Furthermore, when a mixture of an anti-GPR56ECD antibody and GPR56ECD was added to GPR56-expressing cells, the increase in transcriptional activity via SRE dependent on the antibody concentration was suppressed.

  As a control antibody, a rabbit polyclonal antibody prepared in the same manner using mouse Asef2 which is an intracellular protein as an antigen was used. The antibody was purified from the antiserum in the same manner as the anti-GPR56ECD antibody.

  From the above results, it was confirmed that the anti-GPR56ECD antibody has a GPR56 agonist function capable of increasing the transcriptional activity via SRE.

(Experimental example 4) Measurement of GPR56 function (migration assay)
1) Neurosphere culture Neurospheres are neural stem cell colonies. Neurospheres were prepared from the ICR mouse telencephalon on embryonic day 11.5 (E11.5).
Remove brain from mouse fetus, transfer to phosphate buffer on ice (PBS; 138 mM NaCl, 2.68 mM KCl, 8.10 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ ), then remove telencephalon under stereo microscope , Recovered in DF (DMEM: F12 = 1: 1) medium. Thereafter, it was incubated with trypsin at 37 ° C. for 15 minutes to separate into single cells. After suspended in DF culture medium were seeded so that 20μg / ml poly-2-hydroxyethyl methacrylate (poly-HEMA) 2 × 10 6 cells in 10cm dishes coated with 50-fold diluted B-27 supplement ^TM (Invitrogen In the presence of 20 ng / ml bFGF, 20 ng / ml EGF, 1 mg / ml BSA, and 2 μg / ml heparin, suspension culture was performed in DF medium containing 100 U / ml penicillin and 100 μg / ml streptomycin. Here, the B-27 supplement ^TM is an auxiliary reagent necessary for the proliferation and long-term survival of hippocampal neurons and is commercially available. Neurospheres were obtained after culturing at 37 ° C. in the presence of 5% CO ₂ for 3 days. The cell mass was further dispersed by trypsin treatment, seeded to 2 × 10 ⁶ cells, suspended in culture for 3 days to form neurospheres, and used for migration assay.

2) Migration assay DF medium containing 20-40 neurospheres per well in a 96-well plate coated with polyethyleneimine (PEI), B-27 supplement ^TM (Invitrogen), 1 mg / ml BSA The cells were allowed to adhere and cultured, and the morphology after 24 hours was observed. Centering on the attached neurosphere, neural progenitor cells move to the outside of the sphere and spread concentrically. The distance to the outer periphery of the cells that migrated on the concentric circles was measured, and when the diameter of the neurosphere was more than twice the diameter of the neurosphere, it was evaluated as “migrated”, and the ratio of the migrated sphere in the total neurosphere was calculated.

3) Tissue immunostaining using anti-GPR56ECD antibody Tissue immunostaining of embryonic day 16.5 mouse cerebral cortex was performed using anti-GPR56ECD antibody. Embryonic day 16.5 mouse brains were fixed overnight with 4% paraformaldehyde and then immersed in 30% sucrose for 3 days. Thereafter, a frozen section having a thickness of 30 μm was prepared and used for immunostaining. Along with the anti-GPR56ECD antibody, immunostaining was performed with an antibody of nestin which is a marker molecule of neural stem cells / neural progenitor cells or β3-tubulin which is a marker of differentiated nerve cells.

As a result, as shown in FIG. 9, among the ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP) shown in the phase contrast micrograph (phase) GPR56 was highly expressed in VZ where neural stem cells / neural progenitor cells recognized by nestin antibody were present. Further, as shown in FIG. 10, the expression of GPR56 in the CP in which cells differentiated into nerve cells recognized by βIII-tubulin antibody existed was slight.
From the above results, it was confirmed that GPR56 is highly expressed in neural stem cells / progenitor cells, and that the anti-GPR56ECD antibody is an antibody that recognizes neural stem cells / progenitor cells.

4) Effect of anti-mouse GPR56ECD antibody on migration of neural progenitor cells The effect of anti-mouse GPR56ECD antibody on migration of neural progenitor cells was examined.
Buffer (PBS), control antibody (15 μg / ml), anti-mouse GPR56ECD antibody (15 μg / ml), anti-mouse GPR56ECD antibody (15 μg / ml) and GPR56ECD (15 μg / ml) in the presence of the above 2) The migration activity was measured using the method shown in. As a result, as shown in FIG. 11, migration of neural progenitor cells was suppressed only in the presence of the anti-mouse GPR56ECD antibody (15 μg / ml). Further, neutralization of the anti-mouse GPR56ECD antibody with GPR56ECD, which is an antigen, no longer showed its migration inhibitory effect.
From the above results, it was shown that the anti-mouse GPR56ECD antibody has a function of suppressing migration of neural progenitor cells by acting on GPR56.

  As described above, the function of an orphan receptor such as GPR56 among GPCRs can be analyzed by the anti-GPCR extracellular domain antibody of the present invention. In addition, by using the antibody of the present invention, a screening method for selecting an agonist and / or antagonist of GPCR can be provided. Furthermore, a reagent for examining proliferation, differentiation, migration, and / or adhesion of neural stem cells and cancer cells, which contains the antibody of the present invention and the GPCR agonist and / or antagonist selected by the screening method as an active ingredient, for regenerative medicine Drugs or cancer invasion and / or metastasis inhibitors can be provided.

It is a figure which shows distribution in adult mouse | mouth tissue of GPR56ECD. GPR56FL indicates the full length GPR56 that is not cut in the GPS domain. It is a figure which shows the process of a mouse | mouth fetus brain development. It is a figure which shows the expression in the developmental stage of the mouse | mouth fetus brain of GPR56ECD. GPR56FL indicates the full length GPR56 that is not cut in the GPS domain. It is a figure which shows the mode of the asymmetric division and movement of the neural progenitor cell at the time of cerebral cortex formation. It is a figure which illustrates the antibody of this invention typically. It is a figure which confirms the peptide for antibody preparation of this invention by Western blotting. (Experimental example 1) FIG. 3 shows that the antibody of the present invention activates GPR56 and promotes transcriptional activity via SRE. (Experimental example 2) FIG. 3 shows that the antibody of the present invention activates GPR56 and promotes transcriptional activity via SRE. (Experimental example 3) It is the figure which investigated the expression of GPR56 and the neural stem cell / neural progenitor cell marker nestin in the embryonic day 16 mouse | mouth cerebrum using the antibody of this invention. (Experimental example 4) It is the figure which investigated the expression of (beta) III-tubulin which is GPR56 and the neuronal marker in the embryonic day 16 mouse | mouth cerebrum using the antibody of this invention. (Experimental example 4) It is the figure which investigated the influence which acts on the migration of the neural progenitor cell of the antibody of this invention. (Experimental example 5)

Explanation of symbols

In each drawing, “GPR56ECD” means “GPR56 extracellular domain”.
In each drawing, “anti-GPR56 antibody” means “anti-mouse GPR56 extracellular domain antibody”.

Claims (11)

An anti-G protein-coupled receptor extracellular domain antibody having a function of specifically binding to or interacting with the extracellular domain of a G protein-coupled receptor and performing signal transduction into the cell. The antibody according to claim 1, wherein the antibody functions as a signal transducing into a cell by an agonist function of a G protein-coupled receptor. The antibody according to claim 1 or 2, wherein the G protein-coupled receptor is GPR56. The antibody according to any one of claims 1 to 3, wherein the antibody can recognize any part selected from the following regions as an antigen epitope:
1) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and containing at least 3 amino acid residues;
2) In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more of
3) A polypeptide selected from a portion from amino acid sequence 1 to 403 of the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing and containing at least 3 amino acid residues;
4) In the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, in the portion from 1 to 403, one or more amino acids are selected from the substituted, deleted, added or derived sequences, and amino acid residues A polypeptide comprising at least 3 or more. A method for selecting an agonist and / or antagonist of a G protein-coupled receptor, wherein the G protein-coupled receptor and the antibody according to any one of claims 1 to 4 are allowed to interact with each other, and further a candidate compound is allowed to act. G protein-coupled receptor, wherein the function of G protein-coupled receptor is measured and a substitute for the antibody or a substance that enhances or inhibits the interaction between the G protein-coupled receptor and the antibody is selected. A method for screening for agonists and / or antagonists. When transplanting a cell or tissue containing an agonist and / or antagonist of the G protein-coupled receptor obtained by the antibody according to any one of claims 1 to 4 or the screening method according to claim 5 as an active ingredient for therapeutic purposes. Regenerative medicine used for activating or protecting cells and tissues of patients and / or cell transplantation drugs used in the field. Growth, migration, invasion, and metastasis of cancer cells containing as an active ingredient the agonist and / or antagonist of the G protein-coupled receptor obtained by the antibody according to any one of claims 1 to 4 or the screening method according to claim 5. A drug that suppresses engraftment. A reagent for testing neural stem cells, comprising the antibody according to any one of claims 1 to 4 or the agonist and / or antagonist of a G protein-coupled receptor obtained by the screening method according to claim 5. Proliferation, differentiation, migration, and / or adhesion of cancer cells containing the antibody according to any one of claims 1 to 4 or the G protein-coupled receptor agonist and / or antagonist obtained by the screening method according to claim 5, Test reagent for engraftment. A G protein-coupled receptor and the antibody according to any one of claims 1 to 4 are allowed to interact, and a biological sample collected from a subject is allowed to act to measure the G protein-coupled receptor receptor function. Neural stem cell testing method. A G protein-coupled receptor and an antibody according to any one of claims 1 to 4 are allowed to interact, and a biological sample collected from a subject is allowed to act to measure the G protein-coupled receptor function. Inspection method for infiltration and / or metastasis.