JP2012111706A - Monoclonal antibody and hybridoma - Google Patents

Abstract

A novel monoclonal antibody against human-derived endothelin receptor type B (hETBR) is provided.
A monoclonal antibody against human-derived endothelin receptor type B, which specifically reacts with the extracellular domain of human-derived endothelin receptor type B, is provided. It is preferable that it can inhibit the binding between the endothelin receptor type B and the natural ligand, and can inhibit the natural ligand-specific signal transduction of the endothelin receptor type B. As a hybridoma producing the monoclonal antibody, a hybridoma whose accession number is FERM P-21196 is also provided.
[Selection] Figure 1

Description

  The present invention relates to a monoclonal antibody and a hybridoma, and more particularly to a novel monoclonal antibody against human-derived endothelin receptor type B and a hybridoma producing the monoclonal antibody.

  Membrane receptor proteins on the cell surface play an extremely important role in transmitting extracellular information into the cell. Therefore, a substance that binds to a membrane receptor protein becomes a candidate substance as an agonist-type drug or an antagonist-type drug. Among membrane receptor proteins, the G protein-coupled receptor (GPCR) is particularly focused on drug development because about half of currently marketed small molecule drugs target this receptor. Collecting. In addition, about half of the GPCRs are orphan GPCRs whose natural ligands functioning in vivo have not been identified, and natural ligands and related compounds are expected as candidates for new drugs. Ligand screening for GPCRs is extremely intense.

  The GPCR penetrates the cell membrane seven times, and exists with its N-terminus facing out of the cell and its C-terminus facing into the cell. That is, the extracellular domain of GPCR is composed of an N-terminal domain, an extracellular first loop, an extracellular second loop, and an extracellular third loop. Similarly, the intracellular domain of GPCR is composed of an intracellular first loop, an intracellular second loop, an intracellular third loop, and a C-terminal domain.

  Endothelin (ET) is a peptide consisting of 21 amino acid residues and is known to have a strong vasoconstrictive action. Endothelin has been found in three isoforms, ET-1, ET-2 and ET-3. On the other hand, a receptor that specifically binds to endothelin is an endothelin receptor. Two types of endothelin receptors, endothelin receptor type A (ETAR) and endothelin receptor type B (ETBR), have been found, ETAR shows strong binding to ET-1 and ET-2, ETBR is It shows the same degree of connectivity with three types of ET.

  The endothelin receptor is a kind of GPCR and has a structure that penetrates the cell membrane seven times. The primary structure of human-derived endothelin receptor type B (hereinafter sometimes abbreviated as “hETBR”) has already been elucidated, and its N-terminal domain, each extracellular domain, each intracellular domain, and the C-terminal A site corresponding to the domain has also been identified.

  Endothelin receptor antagonists may be therapeutic agents for various diseases involving endothelin, and new antagonists are actively searched for (for example, Non-Patent Documents 1 and 2). For example, the antagonist is expected to have an action of suppressing vascular relaxation and may be a pressor. In addition, it may be an anticancer agent that suppresses the growth and invasion of various cancers. On the other hand, it has been generally attempted to apply an antibody against a receptor as an antagonist (antagonist antibody), and an antibody against an endothelin receptor type B may become a pressor or an anticancer agent.

  Currently, polyclonal antibodies have already been obtained as antibodies against human-derived endothelin receptor type B, and some of them are commercially available. On the other hand, monoclonal antibodies that specifically recognize only the primary structure of hETBR have been obtained (Non-patent Document 3). However, neither a polyclonal antibody nor a monoclonal antibody has been obtained that specifically recognizes the extracellular domain of hETBR, for example, that specifically recognizes the three-dimensional structure of the extracellular domain. Therefore, an antibody capable of inhibiting the binding and signal transduction between hETBR and a natural ligand (ET-1, ET-2, ET-3, etc.) has not been obtained.

Endocrine-Related Cancer, 2007, Vol. 14, p. 233-244 Journal of translational Medicine, 2004, Vol. 2, No. 16, 1-9 PloS ONE, 2010, Vol. 5, No. 6, e11241,

  An object of the present invention is to provide a novel monoclonal antibody against human-derived endothelin receptor type B useful for new drug development and the like, and a hybridoma that produces the monoclonal antibody.

  The present inventors use a fusion gene of a gene encoding chaperonin, which is a kind of molecular chaperone, and a gene encoding human-derived endothelin receptor type B, to immunize mice with human-derived endothelin receptor type. An immune response against B was induced. Then, spleen cells of the mouse and myeloma were fused to prepare a hybridoma population, and a hybridoma producing a monoclonal antibody that specifically recognizes the extracellular domain of hETBR was searched. As a result, one type of clone was successfully selected. And it succeeded in acquiring a desired monoclonal antibody from the said hybridoma, and completed this invention. The gist of the present invention is as follows.

  The invention according to claim 1 is a monoclonal antibody against human-derived endothelin receptor type B, which specifically reacts with the extracellular domain of human-derived endothelin receptor type B.

  The present invention relates to a monoclonal antibody against human-derived endothelin receptor type B (hETBR) (hereinafter sometimes abbreviated as “anti-hETBR monoclonal antibody”). The monoclonal antibody of the present invention specifically reacts with the extracellular domain of hETBR. In other words, it does not recognize only the primary structure of hETBR, nor does it react only with the intracellular domain of hETBR. The monoclonal antibody of the present invention is useful for developing therapeutic agents for diseases related to the extracellular domain of human-derived endothelin receptor type B. The monoclonal antibody of the present invention is also useful for diagnosis of diseases related to the extracellular domain of endothelin receptor type B and construction of immunoassay for endothelin receptor type B.

  The invention according to claim 2 is the monoclonal antibody according to claim 1, which recognizes the three-dimensional structure of the extracellular domain.

  The monoclonal antibody of the present invention recognizes the three-dimensional structure of the extracellular domain. In other words, it does not recognize only the primary structure of the extracellular domain. Such a configuration provides a monoclonal antibody particularly suitable as an antagonist antibody for hETBR. The extracellular domain that retains the three-dimensional structure corresponds to the extracellular domain of native hETBR (active hETBR), and exists on the surface of a living cell, for example.

  The monoclonal antibody according to claim 1 or 2, wherein the extracellular domain is preferably an N-terminal domain, an extracellular first loop, an extracellular second loop, or an extracellular third loop (claim 3). .

The monoclonal antibody according to any one of claims 1 to 3 preferably has the following properties (claim 4).
(A) Reacts with human-derived endothelin receptor type B and does not substantially react with human-derived endothelin receptor type A. (b) Reacts with human-derived endothelin receptor type B and mouse-derived endothelin receptor type B. It does not substantially respond to any of the rat-derived endothelin receptor type B.

  The monoclonal antibody according to any one of claims 1 to 4, wherein the subclass is mouse IgG2a and the light chain is a κ chain (claim 5).

  Invention of Claim 6 is the monoclonal antibody in any one of Claims 1-5 which can inhibit the coupling | bonding of endothelin receptor type B and a natural ligand.

  The invention according to claim 7 is the monoclonal antibody according to any one of claims 1 to 6 capable of inhibiting natural ligand-specific signal transduction possessed by endothelin receptor type B.

  Such a configuration provides a monoclonal antibody that can be used as an antagonist for human-derived endothelin receptor type B.

  The invention according to claim 8 is a monoclonal antibody against human-derived endothelin receptor type B, which is produced by a hybridoma whose accession number is FERM P-211961.

  The invention according to claim 9 is a monoclonal antibody that binds to the same epitope as the monoclonal antibody according to claim 8.

  The monoclonal antibody of the present invention specifically reacts with the extracellular domain of hETBR. In other words, it does not recognize only the primary structure of hETBR, nor does it react only with the intracellular domain of hETBR. The monoclonal antibody of the present invention is useful for developing therapeutic agents for diseases related to the extracellular domain of human-derived endothelin receptor type B. The monoclonal antibody of the present invention is also useful for diagnosis of diseases related to the extracellular domain of endothelin receptor type B and construction of immunoassay for endothelin receptor type B.

  The invention described in claim 10 is a hybridoma whose accession number is FERM P-211961.

  By using the hybridoma of the present invention, the above-described monoclonal antibody of the present invention can be produced. For example, the monoclonal antibody of the present invention can be obtained from the culture.

  The monoclonal antibody of the present invention is useful for developing therapeutic agents for diseases related to the extracellular domain of human-derived endothelin receptor type B. It is also useful for diagnosing diseases related to the extracellular domain of endothelin receptor type B and constructing an immunoassay for endothelin receptor type B.

  According to the hybridoma of the present invention, the monoclonal antibody of the present invention can be obtained from the culture.

It is a histogram showing the analysis result of interaction between hETBR gene introduction cell and hB07 antibody. It is a histogram showing the analysis result of interaction with a hETAR gene transfer cell and hB07 antibody. It is a histogram showing the analysis result of interaction with a control cell and hB07 antibody. It is a photograph showing the result of having observed the hETBR gene transfer cell incubated with the hB07 antibody solution with the fluorescence microscope. It is a photograph showing the result of having observed the hETAR gene introduction cell incubated with the hB07 antibody solution with the fluorescence microscope. It is a graph showing the relationship between intracellular Ca ^<2+ > density ^| concentration and the density ^| concentration of each additive. (A) Histogram showing analysis result of interaction between mouse-derived ETBR (mETBR) gene-introduced cell and hB07 antibody, (b) Analysis result of interaction between rat-derived ETBR (rETBR) gene-introduced cell and hB07 antibody It is a histogram showing.

First, the structure of human-derived endothelin receptor type B (hETBR) will be described.
As described above, hETBR is a kind of G protein-coupled receptor (GPCR), and is present seven times through the cell membrane, with its N-terminus directed outside the cell and the C-terminus directed into the cell. The gene (cDNA) encoding hETBR has already been isolated, and the amino acid sequence of hETBR is also known. SEQ ID NO: 3 shows the base sequence of the hETBR gene and the amino acid sequence corresponding to the base sequence, and SEQ ID NO: 4 shows only the amino acid sequence.

  According to the structure generally considered by the hydrophobic model of hETBR, each domain of hETBR corresponds to the following portion in the amino acid sequence shown in SEQ ID NO: 4. The left side is the amino acid number, and the right side is each domain. When the amino acid sequences of the extracellular domains (N-terminal domain, extracellular first loop, extracellular second loop, and extracellular third loop) are compared between different animals, the homology of the N-terminal domain is low, and each loop The homology of is known to be high.

1-27: Membrane translocation signal peptide sequence (cleaved and removed after expression)
28-99: N-terminal domain 123-137: intracellular first loop 161-172: extracellular first loop 196-220: intracellular second loop 244-276: extracellular second loop 300-324: intracellular first loop 3 loops 348 to 357: extracellular third loop 381 to 442: C-terminal domain

  The monoclonal antibody of the present invention is a monoclonal antibody against human-derived endothelin receptor type B and specifically reacts with the extracellular domain of human-derived endothelin receptor type B. As a method for producing the monoclonal antibody of the present invention, a known method can be employed as it is, and for example, it can be produced based on the cell fusion method of Kohler and Milstein. In brief, hybridoma populations are prepared by fusing spleen cells of animals such as mice in which an immune response to hETBR has been induced and myeloma, and those producing a desired monoclonal antibody are selected from the hybridoma population. Then, the selected hybridoma can be cultured, and a desired monoclonal antibody can be isolated and purified from the culture.

  As a method for inducing an immune response to hETBR, a general protein immunization technique in which an animal is inoculated with hETBR (protein) may be used, but a genetic immunization technique in which an hETBR gene is administered to an animal is preferably used.

  That is, since hETBR is a membrane protein, it is generally difficult to use full-length hETBR as an immunogen. Therefore, when it is desired to induce an immune response against the extracellular domain of hETBR with protein immunity, a peptide of the extracellular domain is used as an immunogen. However, since the extracellular domain has high homology among different organisms and has a complicated structure, it may not be possible to induce a sufficient immune response even when the peptide is used as an immunogen. After all, it is difficult to sufficiently induce an immune response against the extracellular domain by protein immunization.

  On the other hand, according to gene immunity, by using a gene encoding the full length of hETBR, it is possible to create a situation similar to the case where full length hETBR is used as an immunogen. Furthermore, since the expressed hETBR can exist in a native state (active state) in the animal body, it is possible to induce an immune response specific to the three-dimensional structure of hETBR. In addition, according to the gene immunization technique, it can be carried out as long as the hETBR gene is available, and there is no need to prepare purified hETBR.

  However, even in this case, in hETBR, the homology between different animals remains relatively high, and it is difficult to sufficiently induce an immune response against the extracellular domain. Conventionally, only antibodies that recognize only the primary structure such as an antibody for Western blotting have been obtained for antibodies against hETBR, which is considered to be one of the causes. Therefore, a fusion gene of a gene encoding chaperonin and a gene encoding hETBR is used as a gene to be inoculated into animals. By using the fusion gene as an immunogen, an immune response against the extracellular domain can be induced more strongly than when the hETBR gene is used alone. Details of the gene immunization method using the fusion gene are described in International Publication No. 2006/041157.

  Chaperonins are a type of molecular chaperone and are present in all living organisms such as bacteria, archaea, and eukaryotes. In particular, it is abundant in bacterial cytoplasm, eukaryotic mitochondria, and chloroplasts. Chaperonin has an activity of promoting protein folding and an activity of preventing protein denaturation. Chaperonin is a large cylinder-like complex protein with a total molecular weight of about 800,000 to 1,000,000, in which two ring-shaped structures composed of 7 to 9 chaperonin subunits (also referred to as HSP60) having a molecular weight of about 60,000 overlap. . Chaperonin has a cavity inside the ring-shaped structure, and temporarily folds the protein in the middle of folding or denatured protein to form a complex. And it is known that the protein accommodated in the cavity is correctly folded, and then the correctly folded protein is released from the cavity.

  Chaperonins are classified into group I type found in bacteria and eukaryotic organelles and group II type found in eukaryotes and archaea. In the gene immunization performed when the monoclonal antibody of the present invention is produced, when using the above-mentioned fusion gene, any group I type or group II type chaperonin gene may be employed. A representative example of group I chaperonins is GroEL derived from E. coli. A representative example of group II type chaperonins is TCP derived from archaea. The base sequence of the gene encoding the GroEL subunit is shown in SEQ ID NO: 8.

  Here, in the “gene encoding chaperonin (chaperonin gene)”, in addition to the gene encoding chaperonin subunit (chaperonin subunit gene), a “chaperonin subunit linked body” in which a plurality of chaperonin subunits are connected in tandem. Including the gene encoding. The gene encoding the chaperonin subunit conjugate is the same as a gene in which a plurality of chaperonin subunit genes are linked in tandem. Note that the chaperonin subunit linked body can form a ring-shaped structure similar to natural chaperonin.

  As a method for producing the fusion gene, for example, the hETBR gene and the chaperonin gene may be linked by a ligation reaction. In addition, when administering the fusion gene to an animal, it is preferable to administer the fusion gene in an expression vector and under the control of a promoter. Examples of expression vectors include pCI, pSI, pAdVantage, pTriEX, pKA1, pCDM8, and pS. Examples of the promoter on the expression vector include CMV promoter, AML promoter, SV40 promoter, SRα promoter, EF-1α promoter, and the like. Furthermore, an enhancer that enhances promoter activity may be included in the expression vector. Furthermore, the expression vector may contain a CpG motif.

  Examples of the method for administering the fusion gene to animals include subcutaneous injection, intramuscular injection, intravenous injection and the like. Administration by particle gun is also applicable. The dose may be appropriately determined according to the expression vector used, the type of promoter, etc., but is generally 1 to 3 mg / kg body weight per time, which is 25 to 100 μg / time for mice. The number of administrations may be one, but a higher immune response can be induced by performing multiple administrations at regular intervals.

  In addition, when an immune response is induced in an animal by protein immunization, a generally performed method can be employed as it is. For example, purified hETBR is prepared and a mixed solution with an adjuvant is prepared. This mixed solution is injected subcutaneously into a mouse or the like to induce an immune response against hETBR. If necessary, it may be administered multiple times at intervals, and boosted.

  The animal to be immunized is not particularly limited, but preferably a mouse is used. Thereby, a mouse-derived monoclonal antibody can be obtained.

  Hybridoma can be produced by the method of Kohler and Milstein. That is, the spleen is extracted from an animal that has been genetically or protein-immunized by the above-described procedure and induced an immune response against hETBR, and spleen cells are collected. Then, the spleen cells and myeloma are fused to produce a hybridoma population. Selection of a hybridoma can be performed using, for example, a HAT selection medium. In addition, the hybridoma can be cloned by, for example, a limiting dilution method. Thus, a hybridoma that produces an anti-hETBR monoclonal antibody that reacts with the extracellular domain of hETBR may be selected and cloned.

  One of the hybridomas producing the monoclonal antibody of the present invention is named “hB07” and is deposited with the Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology. Details of the deposit are as follows.

Display: hB07
Accession Number: FERM P-21196
Date of receipt: May 11, 2010

  By culturing the selected and cloned hybridoma, an anti-hETBR monoclonal antibody having the above-mentioned properties can be produced. Hybridoma culture may be performed in the peritoneal cavity of an animal or in vitro using a dish or the like. When culturing a hybridoma in the abdominal cavity of an animal, ascites can be collected and a monoclonal antibody can be isolated and purified from the ascites. When culturing in vitro, monoclonal antibodies can be isolated and purified from the culture solution.

  The purification of the monoclonal antibody can be performed by combining various known techniques such as chromatography, salting out, dialysis, and membrane separation. When the subclass of the monoclonal antibody is IgG, it can be simply purified by affinity chromatography using protein A.

  A monoclonal antibody that binds to the same epitope as the anti-hETBR monoclonal antibody produced by hB07 (a hybridoma whose accession number is FERM P-21196), in other words, “functionally equivalent to the anti-hETBR monoclonal antibody produced by hB07” Are also included in the present invention. As a method for obtaining such a monoclonal antibody, for example, an epitope of an anti-hETBR monoclonal antibody produced by hB07 is analyzed by an epitope mapping method using a partial peptide of hETBR. Then, using a synthetic peptide containing the identified epitope as an antigen, a monoclonal antibody that binds to the same epitope as the anti-hETBR monoclonal antibody can be obtained. A method for examining whether two antibodies have the same epitope includes a method based on a competition experiment. For example, an anti-hETBR monoclonal antibody produced by hB07 is used as the first antibody. When the binding between the first antibody and hETBR is subjected to competitive inhibition by the second antibody to be tested, it can be said that the second antibody binds to the same epitope as the first antibody. .

  The monoclonal antibody of the present invention can be used for various applications. For example, it can be applied to drug development as an antagonist to hETBR. That is, it can be applied to a vasopressor that suppresses vascular relaxation by inhibiting natural ligand-specific signal transduction possessed by hETBR. In addition, it may be an anticancer agent that suppresses the growth and invasion of various cancers. Moreover, if the structure of the complementarity determining region (CDR) of the monoclonal antibody of the present invention is specified and chimerized or humanized, it can be made a safer drug. In addition, the monoclonal antibody of the present invention comprises a reagent for screening a low molecular weight drug, a cardiomyopathy / hypertension / cancer test reagent, a research reagent for ETBR localization verification using tissue sections / cells, a test for preparing recombinant ETBR-expressing cells. It can also be used for reagents, research reagents for crystal structure analysis of ETBR, and the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(1) Isolation of human-derived endothelin receptor type B gene PCR was performed using a human placenta cDNA library (Takara Bio Inc.) as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 as primer pairs. A DNA fragment containing the human-derived endothelin receptor type B (hETBR) gene having the base sequence shown in No. 3 (hereinafter referred to as “DNA fragment A”) was amplified. In DNA fragment A, an NheI site was introduced at the 5 ′ end and a SalI site was introduced at the 3 ′ end from the primer. Similarly, PCR was performed using an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 and 5 as a primer pair, and a DNA fragment containing the hETBR gene having the nucleotide sequence shown in SEQ ID NO: 3 (hereinafter referred to as “DNA fragment B”). Amplified). In DNA fragment B, a sequence encoding NheI site at the 5 ′ end and a SalI site encoding two stop codons (TAATAG) at the 3 ′ end were introduced into the DNA fragment B.

(2) Isolation of GroEL subunit gene Genomic DNA was extracted and purified from Escherichia coli HMS174 (DE3) strain (Novagen). Next, PCR was performed using the purified genomic DNA as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 6 and 7 as primer pairs, and a DNA fragment containing the GroEL subunit gene having the nucleotide sequence shown in SEQ ID NO: 8 ( Hereinafter, it is referred to as “DNA fragment C”). In DNA fragment C, a sequence encoding a SalI site at the 5 ′ end and two stop codons (TAATAG) at the 3 ′ end and a NotI site were introduced from the primer.

(3) Construction of a vector for gene immunization expressing a fusion protein of hETBR and GroEL subunit A mammalian expression vector pCI Mammalian Expression Vector (Promega) was digested with restriction enzymes NheI and SalI, and bacterial alkaline phosphatase (BAP) After dephosphorylating the ends with, the DNA fragment A prepared in (1) above was inserted. Furthermore, this expression vector was digested with SalI and NotI, and the end was dephosphorylated with BAP, and then the DNA fragment C prepared in (2) above was inserted to construct the vector pCI-hETBR · GroEL. That is, the vector pCI-hETBR · GroEL has a fusion gene of a gene encoding hETBR and a gene encoding GroEL subunit. On the other hand, similarly, after digesting the mammalian expression vector pCI Mammalian Expression Vector with restriction enzymes NheI and SalI, and dephosphorylating the end with BAP, the DNA fragment B prepared in (1) above was inserted, Vector pCI-hETBR was constructed. That is, the vector pCI-hETBR has only the hETBR gene.

(4) Preparation of hETBR stable expression cells and hETAR stable expression cells PCR is performed using the pCI-hETBR · GroEL constructed in (3) as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 5 as primer pairs, A DNA fragment containing the hETBR gene was obtained. This DNA fragment was introduced into the NheI-XhoI site of pCIneo (Promega) to construct pCIneo-hETBR.
On the other hand, PCR was performed using a human lung cDNA library (Takara Bio Inc.) as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 9 and 10 as primer pairs, and a human-derived endothelin receptor type A (hETAR) gene (sequence) A DNA fragment containing No. 11) was obtained. This DNA fragment was introduced into the NheI-XhoI site of pCIneo to construct pCIneo-hETAR.

37.5 μL of Lipofectamine solution, 625 μL of OPTI-MEMI medium, and 625 μL of OPTI-MEMI medium containing 20 μg of pCIneo-hETBR were mixed. Using this mixed solution, pCIneo-hETBR was introduced into 2 × 10 ⁵ CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.). In a similar procedure, pCIneo-hETAR was introduced into CHO-K1 cells. As a control, only pCIneo was introduced into CHO-K1 cells.
Each CHO-K1 cell into which the gene was introduced was cultured in Ham's F12K + 10% FBS medium (ICN) for 30 hours. Further, each cell was detached, suspended, seeded with 5 × 10 ⁵ cells in a 100 mm dish, and treated with Ham's F12K + 10% FBS medium containing antibiotic G418 (Promega) at a concentration of 0.8 mg / mL for 2 weeks. Was done. After drug treatment, antibiotic resistant cells were cloned by limiting dilution. Each cloned CHO-K1 cell was further cultured overnight using a 96-well microtiter plate at an initial cell concentration of 2 × 10 ⁴ cells / 100 μL. When the cells were stimulated with ET-1 (Peptide Institute) within the concentration range of 10 ⁻⁶ to 10 ⁻¹² M after the completion of the culture, the intracellular Ca ²⁺ concentration increased transiently. The Ca ²⁺ concentration was measured using a Ca ²⁺ signal analyzer (FLIPR; Molecular Devices) and an intracellular Ca ²⁺ staining kit (Ca3kit; Molecular Devices). From this, it was found that both active hETAR and active hETBR were normally stably expressed on the CHO-K1 cell membrane.

(5) Gene immunization The vector pCI-hETBR · GroEL was dissolved in physiological saline to a concentration of 250 μg / mL to prepare a composition for immunization. This immunizing composition was immunized by injecting 0.12 mL each into both thigh muscles of 8-week-old mouse BALB / c (female) (day 0). Thereby, 30 μg of pCI-hETBR · GroEL was administered to both legs, that is, 60 μg per animal. Thereafter, immunization was repeated repeatedly on the 7th, 21st, and 28th days. Then, blood was collected on days 0, 7, 14, 21, 28, 35, and 42 to prepare serum.

(6) Evaluation of antibody binding in serum to activated hETBR by flow cytometry CHO-K1 cells (hereinafter referred to as “hETBR gene-introduced cells”) into which pCIneo-hETBR has been introduced and stable expression has been confirmed, and pCIneo-hETAR are used. CHO-K1 cells (hereinafter referred to as “hETAR gene-introduced cells”) that were introduced and confirmed to have stable expression, and CHO-K1 cells (control cells) into which pCIneo had been introduced were washed with PBS. Serum on day 35 after immunization was diluted 500-fold and incubated with each cell. Further, each cell was washed with PBS, phycoerythrin-labeled anti-mouse IgG antibody (Beckman Coulter) was added as a secondary antibody, and each cell and serum were then used using a flow cytometer FACSCalibur (Becton Dickinson). The interaction with anti-hETBR antibody was analyzed.

As a result, when hETBR gene-introduced cells were used, phycoerythrin was hardly detected in the serum before gene immunization, but was detected in the serum after gene immunization. This indicated that anti-hETBR antibodies in the serum after immunization bind to hETBR gene-introduced cells. On the other hand, when hETAR gene-introduced cells were used, phycoerythrin was not detected in the serum after gene immunization. This indicated that anti-hETBR antibody in the serum after immunization did not bind to hETAR gene-introduced cells. Even when control cells were used, phycoerythrin was not detected in the serum after gene immunization. This indicated that the anti-hETBR antibody in the serum after immunization did not bind to the control cells.
From the above, gene immunization with the vector pCI-hETBR · GroEL was able to induce the production of antibodies that specifically recognize the extracellular domain of active hETBR in mouse serum.

(7) Preparation of anti-hETBR monoclonal antibody Booster immunization was carried out on 6 mice genetically immunized by the same procedure as in (5) above. Three days after the booster, the spleen was removed and spleen cells were prepared. By the PEG method, 1 × 10 ⁸ spleen cells and 1 × 10 ⁷ BALB / C mouse-derived HAT-selective myeloma SP2 / 0 cells were fused (cell fusion). A population of fused cells (hybridoma) was suspended in RPMI medium and seeded in each well of 14 96-well microplates. At this stage, about 990 hybridomas were obtained.

  The medium in the microplate was replaced with RPMI medium supplemented with HAT Media Supplement (50 ×) (Sigma, product number: H0262) at a frequency of once every 3 days for 2 weeks from the day after cell fusion.

  Antibody binding was evaluated by the same flow cytometry as in (6) above, and the binding between hETBR gene-introduced cells and antibodies in the hybridoma culture supernatant of each well was examined. As a result, binding to the antibody was confirmed in the 4 holes.

  Four types of hybridomas that were confirmed to be bound to antibodies were cloned by limiting dilution. That is, four types of hybridomas were seeded in a 96-well microplate so that there were 1 cell / hole or less and cultured. Two weeks later, the same flow cytometry was performed, and the binding of the antibody (anti-hETBR monoclonal antibody) in the cloned culture supernatant was confirmed. As a result, four types of hybridomas producing anti-hETBR monoclonal antibodies were cloned.

  Each hybridoma was cultured in 100 mL of RPMI medium for 2 weeks. Each culture supernatant was subjected to a protein G column (Amersham Bioscience) and purified and concentrated to obtain about 2 mg of each of the four types of purified anti-hETBR monoclonal antibodies.

  Each anti-hETBR monoclonal antibody was tested as follows. From these results, one type of anti-hETBR monoclonal antibody having the desired properties was selected (clone number hB07). This hybridoma was deposited with the Patent Organism Depositary (IPOD) of the National Institute of Advanced Industrial Science and Technology (Accession Number: FERM P-21196; Received Date: May 11, 2010).

  Hereinafter, the following evaluation was performed on an anti-hETBR monoclonal antibody (hereinafter referred to as “hB07 antibody”) purified from a hybridoma having an accession number of FERM P-211961.

(8) Evaluation of binding to hETBR by flow cytometry A PBS solution of hB07 antibody (hereinafter referred to as “hB07 antibody solution”) at a concentration of 10 μg / mL was prepared. On the other hand, hETBR gene-introduced cells, hETAR gene-introduced cells, and control cells were washed with PBS, respectively, and an hB07 antibody solution was incubated with each cell. Further, each cell was washed with PBS, phycoerythrin-labeled anti-mouse IgG antibody (Beckman Coulter) was added as a secondary antibody, and each cell and hB07 antibody were then used using a flow cytometer FACSCalibur (Becton Dickinson). The interaction with was analyzed. The results are shown in FIGS. FIG. 1 is a histogram showing the analysis results of the interaction between hETBR gene-introduced cells and hB07 antibody. FIG. 2 is a histogram showing the analysis results of the interaction between hETAR gene-introduced cells and hB07 antibody. FIG. 3 is a histogram showing the analysis result of the interaction between the control cells and the hB07 antibody. 1-3, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity derived from phycoerythrin (PE). Moreover, the cell which belongs to M2 (right area | region) among two areas (M1, M2) represents the cell couple | bonded with hB07 antibody.

  As a result, when hETBR gene-introduced cells were used, many cells were detected in the M2 area (FIG. 1). This indicated that hB07 antibody binds to hETBR gene-introduced cells. On the other hand, when hETAR gene-introduced cells were used (FIG. 2) and control cells were used (FIG. 3), almost no cells were detected in the M2 area. This indicated that the hB07 antibody did not bind to either hETAR transgenic cells or control cells. From the above, it was shown that the hB07 antibody specifically recognizes the extracellular domain of active hETBR.

(9) Evaluation of binding to hETBR by immunological cell staining The hETBR gene-introduced cells and hETAR gene-introduced cells were seeded in a 6-well plate with a cover glass and cultured. After confirming that the cells adhered and proliferated, the cover glass was immersed in a 4% paraformaldehyde / PBS solution to immobilize the cells, and subjected to cell membrane permeabilization with saponin. Thereafter, the cover glass was incubated with the hB07 antibody solution. Further, the cover glass was washed with PBS, phycoerythrin-labeled anti-mouse IgG antibody (Beckman Coulter, Inc.) was added as a secondary antibody, and then observed using a fluorescence microscope (Olympus). Each cell, hB07 antibody, The interaction of was analyzed. The results are shown in FIGS. FIG. 4 is a photograph showing the results of observing the hETBR gene-introduced cells incubated with the hB07 antibody solution with a fluorescence microscope, and FIG. 5 is a photograph showing the results of observing the hETAR gene-introduced cells incubated with the hB07 antibody solution with a fluorescence microscope. is there. 4 and 5, the location where fluorescence is detected is the location where the hB07 antibody is bound on the cell. The scale bar shown in FIGS. 4 and 5 indicates 100 μm.

  As shown in FIG. 4, when hETBR gene-introduced cells expressed on the cell membrane were used, fluorescence was detected and hB07 antibody was bound on the cell membrane. On the other hand, when hETAR gene-introduced cells were used, no fluorescence was detected and hB07 antibody was not bound on the cell membrane. From the above, immunological cell staining also showed that the hB07 antibody specifically binds to hETBR expressed on the cells (that is, the extracellular domain of hETBR).

(10) Evaluation of intracellular Ca ²⁺ signaling inhibition activity hETBR gene-introduced cells were cultured overnight at an initial cell concentration of 2 × 10 ⁴ cells / 100 μL using a 96-well microtiter plate. After completion of the culture, hB07 antibody within a concentration range of 10 ⁻⁶ to 10 ⁻¹² M was added to each well. As a control, mouse IgG (Pierce, negative control) was similarly added in a concentration range of 10 ⁻⁶ to 10 ⁻¹² M. One hour later, the degree of antibody concentration-dependent decrease in the transient increase in intracellular Ca ²⁺ concentration when each cell was stimulated with 1 × 10 ⁻⁸ M endothelin ET-1 (Peptide Institute) was measured. The Ca ²⁺ concentration was measured using a Ca ²⁺ signal analyzer (FLIPR; Molecular Devices) and an intracellular Ca staining kit (Ca3kit; Molecular Devices). The results are shown in FIG. FIG. 6 is a graph showing the relationship between the intracellular Ca ²⁺ concentration and the concentration of each additive (hB07 antibody or mouse IgG). As shown in FIG. 6, when hB07 antibody was added, a decrease in the ET-1-induced intracellular Ca ²⁺ concentration was observed. This indicated that hB07 antibody competitively inhibited the binding between endothelin ET-1 and hETBR, resulting in inhibition of signal transduction into the cell. The 50% inhibitory concentration (IC50) was calculated to be 1.708 × 10 ⁻⁷ M for the hB07 antibody.
From the above, it was shown that hB07 antibody can inhibit natural ligand-specific signal transduction possessed by hETBR.

(11) Isotype analysis The isotype of hB07 antibody was determined using a mouse monoclonal antibody isotyping kit (GE Healthcare). Detection was performed by sandwich ELISA using horseradish peroxidase-labeled mouse IgG antibody. As a result, the isotype of hB07 antibody was IgG2a, and the light chain was κ chain.

(12) Evaluation of binding ability to heterologous ETBR In the same manner as in (1) to (6) above, “mouse-derived ETBR (mETBR) gene-introduced cells” and “rat-derived ETBR (rETBR) gene-introduced cells” were prepared. Using these cells, the binding of hB07 antibody to mETBR and rETBR was evaluated by flow cytometry similar to (8) above. The results are shown in FIG. FIG. 7A is a histogram showing the analysis result of the interaction between the mETBR gene-introduced cell and the hB07 antibody, and FIG. 7B is a histogram showing the analysis result of the interaction between the rETBR gene-introduced cell and the hB07 antibody. 7A and 7B, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity derived from phycoerythrin (PE). Moreover, the cell which belongs to M2 (right area | region) among two areas (M1, M2) represents the cell couple | bonded with hB07 antibody. That is, in any case, almost no cells were detected in the M2 area. This indicated that the hB07 antibody did not bind to either the mETBR gene-introduced cell or the rETBR gene-introduced cell. From the above, it was shown that the hB07 antibody specifically reacts with hETBR, but does not substantially react with either mETBR or rETBR.

Claims (10)

  A monoclonal antibody against human endothelin receptor type B, which specifically reacts with the extracellular domain of human endothelin receptor type B.   The monoclonal antibody according to claim 1, which recognizes the three-dimensional structure of the extracellular domain.   The monoclonal antibody according to claim 1 or 2, wherein the extracellular domain is an N-terminal domain, an extracellular first loop, an extracellular second loop, or an extracellular third loop. The monoclonal antibody according to any one of claims 1 to 3, which has the following properties (a) and (b).
(A) Reacts with human-derived endothelin receptor type B and does not substantially react with human-derived endothelin receptor type A. (b) Reacts with human-derived endothelin receptor type B and mouse-derived endothelin receptor type B. It does not substantially respond to any of the rat-derived endothelin receptor type B.   The monoclonal antibody according to any one of claims 1 to 4, wherein the subclass is mouse IgG2a and the light chain is κ chain.   The monoclonal antibody according to any one of claims 1 to 5, which can inhibit the binding between endothelin receptor type B and a natural ligand.   The monoclonal antibody according to any one of claims 1 to 6, which can inhibit a natural ligand-specific signal transduction possessed by an endothelin receptor type B.   A monoclonal antibody against human-derived endothelin receptor type B produced by a hybridoma having the accession number FERM P-211961.   A monoclonal antibody that binds to the same epitope as the monoclonal antibody of claim 8.   A hybridoma whose accession number is FERM P-211961.