WO2018181656A1 - 抗gpr20抗体 - Google Patents
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- WO2018181656A1 WO2018181656A1 PCT/JP2018/013106 JP2018013106W WO2018181656A1 WO 2018181656 A1 WO2018181656 A1 WO 2018181656A1 JP 2018013106 W JP2018013106 W JP 2018013106W WO 2018181656 A1 WO2018181656 A1 WO 2018181656A1
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
Definitions
- the present invention relates to a novel anti-GPR20 antibody, a functional fragment of the antibody, a modified form of the antibody, a nucleotide comprising a base sequence encoding the amino acid sequence of the antibody, a vector into which the nucleotide is inserted, the nucleotide or the vector
- the present invention relates to an introduced cell, a method for producing the antibody including a step of culturing the cell, a pharmaceutical composition, a diagnostic or test composition, and the like.
- GPR20 G Protein-coupled receptor 20
- GPCR G protein-coupled receptor
- the N-terminal side is extracellular and the C-terminal side.
- GPR20 has an amino acid sequence similar to GPCR that recognizes nucleotides or lipids, but its physiological function and in vivo ligand have not been identified. From an experiment in which GPR20 is expressed in HEK293 cells, it has been reported that GPR20 constitutively activates a Gi-type trimeric G protein under conditions without stimulation by a ligand (Non-patent Document 1). .
- GPR20 has been confirmed to express messenger RNA (mRNA) in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, rectum, and white blood cell, especially in the small intestine. High expression was reported (Non-patent Document 1). In the brain, expression in the thalamus, putamen and caudate nucleus has been reported (Non-patent Document 2).
- GPR20 is present in mRNA in a part of gastrointestinal stromal tumors (GIST) (Non-patent Document 3) and in Cajal intervening cells (ICCs) that exist in the plexus of the gastrointestinal muscular layer and are involved in peristaltic movement of the intestinal tract Expression was reported.
- ICCs are the origin cells of GIST, and it has been reported that the expression of GPR20 is regulated in these cells by ets variant 1 (ETV1), which is a major transcription factor of GIST (Non-patent Document 4).
- GPR20-deficient mice show a hyperactivity disorder phenotype characterized by an increase in total distance traveled in the open field test, suggesting that GPR20 is associated with spontaneous activity in the central nervous system ( Patent Document 1).
- the provision of a method capable of detecting the expression of GPR20 is achieved by detecting tumor cells such as GIST, normal Cajal intervening cells present in the digestive tract, cells existing in the brain, etc. as GPR20 positive cells. It is useful for possible testing or diagnosis.
- One object of the present invention is to provide an antibody that specifically binds to GPR20.
- Another object of the present invention is to provide a GPR20 detection reagent comprising an anti-GPR20 antibody.
- Another object of the present invention is to provide a diagnostic reagent or test composition for a disease associated with expression of GPR20, comprising an anti-GPR20 antibody.
- the subject of the present invention also includes a nucleotide encoding the amino acid sequence of the antibody, a vector into which the nucleotide has been inserted, a cell into which the nucleotide or vector has been introduced, and a step of culturing the cell. Methods etc. are included.
- Another object of the present invention is to provide a pharmaceutical composition containing the anti-GPR20 antibody and a method for treating a disease involving the expression of GPR20 using the pharmaceutical composition.
- the present invention includes the following inventions. (1) an antibody that specifically binds to a peptide comprising the amino acid sequence represented by amino acid numbers 1 to 48 in SEQ ID NO: 1 or an antigen-binding fragment of the antibody, (2) For binding to GPR20, it has a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 466 of SEQ ID NO: 3 and a light chain consisting of the amino acid sequence shown in amino acid numbers 20 to 232 of SEQ ID NO: 11 An antibody having competitive inhibitory activity with an antibody or an antigen-binding fragment of the antibody, (3) The antibody or the functional fragment of the antibody according to (1) or (2), which binds to an epitope consisting of an amino acid sequence represented by LEVPLFHLFALD (SEQ ID NO: 31), (4) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 5
- the antibody according to any one of (3) or an antigen-binding fragment of the antibody (5) The heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4 and the light chain variable region shown in SEQ ID NO: 12 according to any one of (1) to (4) An antibody or an antigen-binding fragment of the antibody, (6) consisting of a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 466 of SEQ ID NO: 3 and a light chain comprising the amino acid sequence represented by amino acid numbers 20 to 232 of SEQ ID NO: 11 (1) to the antibody according to any one of (5) or an antigen-binding fragment of the antibody, (7) The antibody or the antigen-binding fragment of the antibody according to any one of (1) to (5), which is a chimeric antibody, (8) The chimeric antibody according to (7), wherein the constant region is derived from a rabbit antibody, (9) comprising a heavy chain consisting of the amino acid sequence represented by amino acid numbers 20 to 456 of SEQ ID NO: 19 and
- the antibody or the functional fragment of the antibody according to any one of (1) to (5), which is humanized (12) The antibody according to any one of (1) to (5) and (10), comprising the heavy chain according to (a) or (b) below and the light chain according to (c): Or an antigen-binding fragment of the antibody: (A) a heavy chain comprising the amino acid sequence of amino acid numbers 20 to 456 in SEQ ID NO: 23, (B) a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 456 in SEQ ID NO: 25, (C) a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 230 in SEQ ID NO: 27, (13) consisting of a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 456 in SEQ ID NO: 23 and a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 230 in SEQ ID NO: 27; (1) to (5) and (10) any one of the antibodies or antigen-binding fragments of the antibodies, (14) consisting of a
- composition according to (17) or (18), (20) When the GPR20 detection or measurement method detects or measures GPR20 in the test sample, or the expression level or expression level of GPR20 in the test sample is equal to or higher than a predetermined criterion
- the test specimen is determined to be positive, and GPR20 is not detected or measured in the test specimen, or the expression level or expression level of GPR20 in the test specimen is equal to or lower than a predetermined criterion
- Subjects determined to be A method for treating a GPR20 positive disease comprising the following steps (a) and (b): (A) GPR20 of a specimen using the antibody according to any one of (1) to (16) or an antigen-binding fragment of the antibody, and the composition according to (17) to (19), (21) Detecting or measuring (B) administering an anti-GPR20 antibody or an antigen-binding fragment of the antibody to a subject derived from the specimen in which the expression of GPR20 is detected or measured in (a), (27) a polynucleotide encoding the antibody or the antigen-binding fragment of the antibody according to any one of (1) to (16), (28) A vector comprising the polynucleotide according to (27), (29) A cell comprising the polynucleotide according to (27) or the vector according to (28), (30) The method of producing the antibody or antigen-binding fragment of the antibody according to any one of (1) to (16), comprising the following steps (a) and (b): (A) GPR
- the amino acid sequence (SEQ ID NO: 3) and nucleotide sequence (SEQ ID NO: 2) of the heavy chain of rat anti-GPR20 antibody 04-093 are shown.
- 1 shows the amino acid sequence (SEQ ID NO: 11) and nucleotide sequence (SEQ ID NO: 10) of the light chain of rat anti-GPR20 antibody 04-093.
- the CDRH1-3 (SEQ ID NOs: 5-9) and CDRRL1-3 amino acid sequences (SEQ ID NOs: 13-15) of rat anti-GPR20 antibody 04-093 are shown.
- 1 shows the amino acid sequence of the rabbit chimerized antibody heavy chain OcHch (SEQ ID NO: 19).
- FIG. 3 shows the amino acid sequence of the rabbit chimerized antibody light chain OcLch (SEQ ID NO: 21).
- the amino acid sequence of the rabbitized antibody heavy chain OcH01 (SEQ ID NO: 23) is shown.
- 1 shows the amino acid sequence of the rabbitized antibody light chain OcL01 (SEQ ID NO: 27).
- the flow cytometry analysis result at the time of making the culture supernatant of the hybridoma which produces a human GPR20 transient expression cell strain and anti-human GPR20 antibody react is shown.
- shaft shows the relative value of the average fluorescence intensity (MFI) measured by the flow cytometry method.
- the binding property of anti-human GPR20 antibody to a synthetic peptide consisting of 48 amino acids at the N-terminal of human GPR20 is shown.
- FIG. 11-1 shows a stained image of the rat anti-GPR20 monoclonal antibody.
- the immuno-staining image of the anti- GPR20 antibody with respect to a human GPR20 transient expression cell is shown.
- FIG. 11-2 shows a stained image of a commercially available rabbit anti-GPR20 polyclonal antibody.
- the immuno-staining image of the anti- GPR20 antibody with respect to a human GPR20 transient expression cell is shown.
- FIG. 11-3 shows a stained image of a commercially available rabbit anti-GPR20 polyclonal antibody.
- FIG. 12-1 shows a stained image of stomach-derived GIST.
- the staining image by the rat anti-human GPR20 antibody of the gastrointestinal stromal tumor GIST and the commercially available rabbit anti-GPR20 antibody is shown.
- FIG. 12-2 shows a stained image of GIST derived from the small intestine.
- the staining image by the rat anti-human GPR20 antibody of the gastrointestinal stromal tumor GIST and the commercially available rabbit anti-GPR20 antibody is shown.
- FIG. 12-3 shows a stained image of large intestine-derived GIST.
- FIG. 14-1 shows a stained image of stomach-derived GIST. Staining images of gastrointestinal stromal tumor GIST with rat anti-GPR20 antibody 04-093, rabbit chimerized anti-GPR20 antibody and rabbit anti-GPR20 antibody are shown.
- FIG. 14-2 shows a stained image of GIST derived from the small intestine. Staining images of gastrointestinal stromal tumor GIST with rat anti-GPR20 antibody 04-093, rabbit chimerized anti-GPR20 antibody and rabbit anti-GPR20 antibody are shown.
- FIG. 14-3 shows a stained image of large intestine-derived GIST. 2 shows the binding activity of a rat anti-GPR20 monoclonal antibody to a synthetic peptide consisting of N-terminal 1 to 48 amino acids of human GPR20 and a synthetic peptide consisting of 30 to 48 amino acids.
- the horizontal axis represents the clone number, and the vertical axis represents the amount of antibody bound by chemiluminescence (CPS).
- CPS chemiluminescence
- gene means a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof, for example, a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof.
- Certain polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA and the like are included in the meaning of “gene”.
- Such a gene is a single-stranded, double-stranded, or triple-stranded nucleotide, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) are mixed on a single nucleotide strand.
- RNA ribonucleotide
- DNA deoxyribonucleotide
- GPR20 gene include DNA, mRNA, cDNA, cRNA and the like containing a base sequence encoding the amino acid sequence of GPR20 protein.
- nucleotide and “nucleic acid” are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of “nucleotide”.
- a nucleotide is a nucleotide composed of a single strand, a double strand, or three or more strands, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) on a single nucleotide strand.
- RNA ribonucleotide
- DNA deoxyribonucleotide
- nucleotide also included within the meaning of “nucleotide” are those that are intermingled and aggregates of two or more strands containing such nucleotide strands.
- polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
- antigen is sometimes used to mean “immunogen”.
- cell includes various cells derived from individual animals, subculture cells, primary culture cells, cell lines, recombinant cells, microorganisms, and the like.
- antibodies that recognize GPR20 may be referred to as “anti-GPR20 antibodies”, respectively.
- Such antibodies include polyclonal antibodies, monoclonal antibodies, chimerized antibodies, rabbitized antibodies, humanized antibodies, human antibodies and the like.
- the “functional fragment of an antibody” means an antibody fragment that exhibits at least a part of the function exhibited by the original antibody.
- the “functional fragment of an antibody” include, but are not limited to, Fab, F (ab ′) 2, scFv, Fab ′, single chain immunoglobulin and the like.
- Such functional fragments of antibodies are recombinant proteins produced in appropriate host cells using recombinant genes in addition to those obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin. May be.
- the “site” to which the antibody binds that is, the “site” recognized by the antibody means a partial peptide or a partial higher order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site.
- bonds or recognizes the partial peptide on GPR20 protein, a partial higher-order structure, etc. can be illustrated.
- CDRs complementarity determining regions
- the complementarity-determining region is also called a hypervariable domain, and is located in the variable region of the heavy and light chains of an antibody and has a particularly high primary structure variability. In the primary structure of the polypeptide chain, it is usually separated at three points.
- the complementarity determining region of an antibody the complementarity determining region of an antibody
- the complementarity determining region of the heavy chain is denoted as CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence
- the complementarity determining region of the light chain is defined as the light chain amino acid.
- CDRL1, CDRL2, and CDRL3 are represented from the amino terminal side of the sequence. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to be bound.
- antibody variant has an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted (hereinafter collectively referred to as “mutation”) in the amino acid sequence of the original antibody. And a polypeptide that binds to the GPR20 protein of the present invention.
- the number of variant amino acids in such antibody variants is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50.
- Such antibody variants are also included in the “antibody” of the present invention.
- “several” in “1 to several” refers to 3 to 10.
- Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical properties, and the like. Specifically, various biological activities, binding activity to antigens and epitopes, stability during production and storage And thermal stability.
- hybridize under stringent conditions means hybridization at 65 ° C. in a solution containing 5 ⁇ SSC, and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS. 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS for 20 minutes at 65 ° C. and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS Means to hybridize under conditions of washing for 20 minutes or under equivalent conditions.
- SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and nx SSC means n-fold concentration of SSC.
- cytotoxicity refers to causing a pathological change in a cell in some form, and is not limited to direct trauma, but also includes DNA breakage, base dimer formation, chromosome breakage, It means any structural or functional damage of cells, such as damage to cell division apparatus or reduction of various enzyme activities.
- cytotoxic activity means causing the above cytotoxicity.
- antibody-dependent cytotoxic activity refers to “antibody dependent cellular cytotoxicity (ADCC) activity”, and means an activity in which NK cells damage target cells such as tumor cells via antibodies.
- cancer and “tumor” are used interchangeably.
- immunohistochemistry means a histological (histochemical) method for detecting an antigen in a tissue specimen, and is synonymous with “immunoantibody method”. “immunostaining”) is also used interchangeably.
- denatured GPR20 means a GPR20 molecule in a specimen fixed with formalin.
- the GPR20 molecule in the specimen fixed with formalin and then paraffin-treated and deparaffinized is also referred to as “denatured GPR20”.
- non-denatured GPR20 means GPR20 in a sample not fixed with formalin.
- the GPR20 molecule in the specimen not fixed with formalin is also referred to as “non-denatured GPR20”.
- GPR20 Is GPR20 used in the present invention directly purified from human, non-human mammals (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) or chicken T cells or mast cells?
- the cell membrane fraction of the above-mentioned cells can be prepared and used, or can be obtained by synthesizing GPR20 in vitro or by producing it in a host cell by genetic manipulation.
- GPR20 cDNA is incorporated into an expressible vector and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic or eukaryotic organisms.
- the protein can be obtained by expressing GPR20 by transforming a host cell of an organism.
- the nucleotide sequence of cDNA of human GPR20 is registered in GenBank with accession number: NM_005293. Further, the amino acid sequence of human GPR20 is registered in GenBank with an accession number: NP_005284.
- the GPR20 cDNA can be obtained by, for example, polymerase chain reaction (hereinafter referred to as “hereinafter referred to as“ polymerization chain reaction ”) using primers that specifically amplify the GPR20 cDNA using a cDNA library of an organ expressing GPR20 mRNA or genomic DNA extracted from human cells as a template. (Referred to as “PCR”) (Saiki, R. K., et al., Science, (1988) 239, 487-49).
- a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding human GPR20 under stringent conditions and encodes a protein having biological activity equivalent to that of GPR20 is also included in the GPR20 cDNA. included.
- a polynucleotide that is a splicing variant transcribed from the human GPR20 locus or a polynucleotide that hybridizes to this under stringent conditions and that encodes a protein having biological activity equivalent to that of GPR20 is also included in the GPR20 cDNA. included.
- a protein having a biological activity equivalent to that of GPR20 consisting of an amino acid sequence in which one, two, three, or four or five amino acids are substituted, deleted, or added in the amino acid sequence of human GPR20. included. Furthermore, an amino acid sequence encoded by a splicing variant transcribed from the human GPR20 locus, or an amino acid sequence in which one, two or three, or four or five amino acids are substituted, deleted, or added in the amino acid sequence A protein having a biological activity equivalent to that of GPR20 is also included in GPR20.
- amino acid sequence of human GPR20 used herein is described in SEQ ID NO: 1 in the sequence listing.
- the antibody against GPR20 of the present invention is obtained by immunizing an animal with GPR20 or any polypeptide selected from the amino acid sequence of GPR20 using a conventional method, and collecting and purifying the antibody produced in vivo. Can be obtained.
- the species of GPR20 serving as an antigen is not limited to humans, and GPR20 derived from animals other than humans such as monkeys, mice and rats can also be used to immunize animals.
- antibodies that can be applied to human diseases can be selected by examining the cross-reactivity between the obtained antibody that binds to the heterologous GPR20 and human GPR20.
- GPR20 serving as an antigen can be obtained by causing a host cell to produce the GPR20 gene by genetic manipulation. Specifically, a vector capable of expressing the GPR20 gene may be prepared, introduced into a host cell to express the gene, and the expressed GPR20 may be purified.
- the antibody against GPR20 of the present invention can also be obtained using a DNA immunization method.
- the DNA immunization method is a technique for inducing immunity to an antigen by introducing an antigen expression plasmid into an individual animal such as a mouse or a rat and expressing the antigen in the individual.
- Gene transfer methods include direct injection of plasmids into muscle, intravenous injection of liposomes and polyethyleneimine and other introduction reagents, viral vector methods, and gold particles with plasmids attached to them by Gene Gun.
- the amount of expression is further improved by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug; 8 (16) : 1264-70).
- Hybridomas can be established by fusing antibody-producing cells that produce antibodies against GPR20 and myeloma cells to obtain monoclonal antibodies. Specific examples of such a method are described in International Publication No. WO 09/48072 (published on April 16, 2009) and WO 10/11711 (published on October 14, 2010).
- the 04-093 antibody can be mentioned.
- the amino acid sequence of the 04-093 antibody heavy chain is shown in SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 2 in the sequence listing.
- the amino acid sequence of the 04-093 antibody light chain is shown in SEQ ID NO: 11 of the Sequence Listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 10 of the Sequence Listing.
- the 04-093 antibody specifically binds to a peptide comprising the amino acid sequence shown in amino acid numbers 1 to 48 in SEQ ID NO: 1.
- the 04-093 antibody binds to an epitope consisting of the amino acid sequence shown in LEVPLFHLFALD (SEQ ID NO: 31) in the peptide consisting of the amino acid sequence shown in amino acid numbers 1 to 48 in SEQ ID NO: 1.
- the antibody of the present invention may be any antibody that retains all six CDR sequences derived from the antibody 04-093 and has an activity of binding to GPR20. That is, the heavy chain variable region of the antibody of the present invention has CDRH1 (GFTFNYWMT (according to the definition of Abm) or NYWMT (according to the definition of Kabat)), SEQ ID NO: 6 or 9 consisting of the amino acid sequence shown in SEQ ID NO: 5 or 8.
- CDRH1 GFTFNYWMT (according to the definition of Abm) or NYWMT (according to the definition of Kabat)
- SEQ ID NO: 6 or 9 consisting of the amino acid sequence shown in SEQ ID NO: 5 or 8.
- CDRH2 (SITNIDGSSY (as defined by Abm) or SITNIDGSSYYPDSVKG (as defined by Kabat)) consisting of the amino acid sequence shown in the figure, and CDRH3 (GSFDY) consisting of the amino acid sequence shown as SEQ ID NO: 7 are possessed.
- the light chain variable region of the antibody comprises CDRL1 (KASQNVNKYLN) consisting of the amino acid sequence shown in SEQ ID NO: 13, CDRL2 (NTNNLQT) consisting of the amino acid sequence shown in SEQ ID NO: 14, and amino acids shown in SEQ ID NO: 15. It has CDRL3 (FQHVSWLT) consisting of the sequence.
- the amino acid sequences of these CDRs are also shown in FIG.
- the antibody of the present invention specifically recognizes the GPR20 protein.
- preferred antibodies of the present invention specifically bind to GPR20 protein.
- a suitable antibody specifically binds to both undenatured human GPR20 and denatured human GPR20 in formalin-fixed specimens.
- More preferable antibodies include an antibody that specifically binds to both non-denatured human GPR20 and denatured human GPR20 in a formalin-fixed specimen and does not specifically bind to other GPR families. However, it is not limited to them.
- the antibodies of the present invention include genetically engineered antibodies that have been artificially modified for the purpose of reducing xenoantigenicity, such as chimeric antibodies, humanized (humanized). ) Antibodies, or Rabbit type antibodies, murine antibodies, and the like are also included. These antibodies can be produced using known methods.
- chimeric antibody examples include antibodies in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad). Sci.U.S.A., 81, 6851-6855, (1984)).
- Another example includes a chimeric antibody in which a variable region of a mouse or rat-derived antibody is conjugated to a rabbit-derived constant region.
- the rabbit chimerized antibody include a heavy chain variable region derived from the antibody 04-093 and a heavy chain (OcHch) comprising a rabbit heavy chain constant region, and a light chain derived from the antibody 04-093.
- An antibody comprising a light chain (OcLch) comprising a variable region and a rabbit light chain constant region can be mentioned.
- the amino acid sequence of OcHch is represented by amino acid numbers 20 to 456 of SEQ ID NO: 19 in the sequence listing.
- the amino acid sequence of OcLch is shown in amino acid numbers 21 to 232 of SEQ ID NO: 21 in the sequence listing.
- the antibody of the present invention includes an antibody obtained by modifying CDRs of the humanized antibody or rabbitized antibody. These antibodies can be produced using known methods.
- humanized antibodies antibodies (see Nature (1986) 321, p.522-525) in which only complementarity determining regions (CDRs) are incorporated into human-derived antibodies (see Nature (1986) 321, p.522-525), some sequences in addition to CDR sequences
- the amino acid residues of the framework include an antibody grafted on a human antibody (International Publication No. WO90 / 07861 pamphlet).
- Rabbitized antibodies include antibodies in which only complementarity determining regions (CDRs) are incorporated into rabbit-derived antibodies, and antibodies in which some framework amino acid residues are transplanted into rabbit antibodies in addition to the CDR sequences.
- the amino acid sequence of the CDR can be determined by a known method such as the definition of Kabat, the definition of Chothia, the definition of Abm, etc.
- the CDR in the present invention may be defined by any method.
- a rabbitized antibody of the 04-093 antibody can be mentioned, and a more specific example is shown by amino acid numbers 20 to 456 of SEQ ID NO: 23 in the sequence listing. It consists of a heavy chain consisting of an amino acid sequence (OcH01), a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 456 of SEQ ID NO: 25 (OcH02), and an amino acid sequence shown by amino acid numbers 21 to 230 of SEQ ID NO: 27 Rabbitized antibodies comprising a light chain (OcL01) can be mentioned.
- the present invention also includes antibodies having such modifications, including deletions in which one or two amino acids have been deleted at the heavy chain carboxyl terminus, and such a deletions that have been amidated (eg, at the carboxyl terminus site).
- Heavy chain in which a proline residue is amidated e.g., at the carboxyl terminus site.
- the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention is not limited to the above type.
- the two heavy chains constituting the antibody according to the present invention may be either one of the full length and the heavy chain selected from the group consisting of the above-mentioned deletion forms, or a combination of any two of them. It may be a thing.
- the amount ratio of each deletion can be influenced by the type and culture conditions of the cultured mammalian cells that produce the antibody according to the present invention, but the main component of the antibody according to the present invention is a carboxyl in both two heavy chains. A case where one terminal amino acid residue is deleted can be mentioned.
- the antibody obtained by the above method can be evaluated for binding to an antigen and a suitable antibody can be selected.
- An example of another index for comparing antibody properties is antibody stability.
- Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is an indicator of good relative structural stability of proteins. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It is known that the storage stability of an antibody shows a certain degree of correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p.265-273), and heat.
- a suitable antibody can be selected using stability as an index.
- Other indicators for selecting antibodies include high yields in appropriate host cells and low aggregation in aqueous solutions. For example, since the antibody with the highest yield does not always exhibit the highest thermal stability, it is necessary to select the most suitable antibody based on a comprehensive judgment based on the above-described indicators.
- the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence.
- Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and are actually observed in camels or llamas and reported to retain antigen-binding ability.
- sdAbs single domain antibodies
- nanobodies are actually observed in camels or llamas and reported to retain antigen-binding ability.
- the above-described antibody can also be interpreted as a kind of antigen-binding fragment of the antibody in the present invention.
- an antibody gene When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
- Specific examples of the antibody gene include a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence.
- the heavy chain sequence gene and the light chain sequence gene When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
- eukaryotic cells When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.
- ATCC CRL-1650 which are monkey cells, mouse fibroblasts NIH3T3 (ATCC). No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strains (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220).
- Escherichia coli and Bacillus subtilis can be mentioned, for example.
- An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. In the above culture method, the yield may vary depending on the sequence of the antibody, and it is possible to select an antibody having an equivalent binding activity that can be easily produced as a drug using the yield as an index.
- IgG IgG1, IgG2, IgG3, IgG4
- IgM IgA (IgA1, IgA2)
- IgD or IgE preferably IgG or IgM
- IgG1 or IgG2 More preferably, IgG1 or IgG2 can be mentioned.
- the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof.
- a fragment of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or modifying the antibody gene by a genetic engineering technique and expressing it in an appropriate cultured cell.
- a fragment that retains all or part of the functions of the full-length antibody molecule can be called an antigen-binding fragment of an antibody.
- Antibody functions generally include antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and complement-dependence Mention may be made of cellular cytotoxic activity.
- the function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to GPR20.
- antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv), diabodies (diabodies), linear antibody in which heavy and light chain Fvs are linked by an appropriate linker.
- Fab ' which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antibody fragment.
- the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens.
- a molecule binds to two types of antigens (ie, bispecific antibodies), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.
- the multispecific antibody of the present invention may be a full-length antibody or a fragment of such an antibody (for example, F (ab ') 2 bispecific antibody).
- Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by hybridizing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).
- the antibody of the present invention may be a single chain antibody (also referred to as scFv).
- a single chain antibody is obtained by linking an antibody heavy chain variable region and a light chain variable region with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, edited by Springer Verlag, New). York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136)
- a BiscFv fragment produced by linking two scFvs with a polypeptide linker is used as a bispecific antibody. It can also be used.
- the heavy chain variable region and the light chain variable region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci.U.S.A. (1988), 85, p.5879-5883).
- the heavy chain variable region and the light chain variable region in scFv may be derived from the same antibody or different antibodies.
- the polypeptide linker that links the variable regions for example, any single chain peptide consisting of 12 to 19 residues is used.
- the DNA encoding the scFv is the DNA encoding the heavy chain or heavy chain variable region of the antibody, and the DNA encoding the light chain or light chain variable region.
- Amplification is performed by PCR using a coding DNA portion as a template and a primer pair defining both ends thereof, and then the DNA encoding the polypeptide linker portion and both ends thereof are connected to the heavy chain and light chain, respectively. Obtained by combining and amplifying the primer pairs defined in 1.
- an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.
- These antibody fragments can be produced by a host after obtaining and expressing the gene in the same manner as described above.
- the antibody of the present invention may be one that has been increased in quantity and has increased affinity for the antigen.
- the antibody that multiplies may be one type of antibody or a plurality of antibodies that recognize multiple epitopes of the same antigen. Examples of the method for increasing the amount of antibody include binding of IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of helix-turn-helix motif.
- the antibody of the present invention may be a polyclonal antibody that is a mixture of a plurality of types of anti-GPR20 antibodies having different amino acid sequences.
- a polyclonal antibody a mixture of plural kinds of antibodies having different CDRs can be mentioned.
- a polyclonal antibody a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO 2004/061104).
- an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
- PEG polyethylene glycol
- the antibody of the present invention may be one in which these antibody and another drug form a conjugate (Immunoconjugate).
- conjugate examples include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).
- the obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies) for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid chromatography, such as HPLC and FPLC.
- column used for affinity chromatography include a protein A column and a protein G column.
- a column using a protein A column Hyper D, POROS, Sepharose F.R. F. (Pharmacia) and the like. It is also possible to purify an antibody using a carrier on which an antigen is immobilized, utilizing the binding property to the antigen.
- composition comprising an anti-GPR20 antibody or a functional fragment thereof or a modified form thereof.
- the pharmaceutical composition of the present invention can be used in various diseases (hereinafter referred to as “GPR20 signal abnormalities or enhancements due to overexpression of GPR20 or its ligands, GPR20 mutation or gene amplification, or switching of isoforms of GPR20”). , "The disease relating to GPR20”), particularly useful for the treatment or prevention of various cancers.
- SNP single base substitution
- GIST gastrointestinal stromal tumor
- GIST gastrointestinal stromal tumor
- GIST gastrointestinal stromal tumor
- prevention of the onset of such a disease preferably the development of such a disease in an individual expressing GPR20 protein, suppression or inhibition of progression or progression
- Examples include, but are not limited to, alleviation of one or more symptoms present, suppression or remission of progression or progression, treatment or prevention of secondary diseases, and the like.
- the pharmaceutical composition of the present invention comprises a therapeutically or prophylactically effective amount of an anti-GPR20 antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Can be contained.
- “Therapeutically or prophylactically effective amount” means an amount having a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is synonymous with “pharmacologically effective amount”.
- the pharmaceutical composition of the present invention has pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or antibody contained therein, solubility, sustained release, absorbability, penetration.
- Substances for changing, maintaining, and maintaining properties, dosage forms, strength, properties, shapes, etc. can be included.
- the substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance.
- non-toxicity or low toxicity is a property that a substance for preparation preferably comprises.
- Substances for formulation include, for example, amino acids, antibacterial agents, antioxidants, buffers, fillers, chelating agents, complexing agents, bulking agents, monosaccharides, disaccharides, carbohydrates, coloring agents, flavoring agents, diluents , Emulsifier, hydrophilic polymer, preservative, solvent, sugar alcohol, suspending agent, surfactant, stabilization enhancer, elasticity enhancer, transport agent, diluent, excipient, and / or pharmaceutical adjuvant
- the amount of these substances to be added is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably the weight of the anti-GPR20 antibody or functional fragment thereof or a modified product thereof. Is 0.1 to 10 times.
- An immunoliposome containing an anti-GPR20 antibody or a functional fragment thereof or a modified product thereof in the liposome, or a pharmaceutical composition containing an antibody modified product (US Pat. No. 6,214,388, etc.) formed by binding the antibody and the liposome is also present in the present invention. Included in the pharmaceutical composition of the invention.
- the excipient or carrier is usually liquid or solid, and is not particularly limited as long as it is a substance used for water for injection, physiological saline, artificial cerebrospinal fluid, and other preparations for oral administration or parenteral administration.
- physiological saline include neutral ones and those containing serum albumin.
- the buffer examples include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetate buffer prepared so as to be 4.0 to 5.5, and 5. Examples thereof include a citrate buffer prepared to be 0 to 8.0, a histidine buffer prepared to be 5.0 to 8.0, and the like.
- the pharmaceutical composition of the present invention is a solid, liquid, suspension or the like. Mention may be made of freeze-dried preparations. An excipient such as sucrose can be used to mold the lyophilized preparation.
- the administration route of the pharmaceutical composition of the present invention may be enteral administration, topical administration or parenteral administration.
- intravenous administration intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration Administration, transdermal administration, intraosseous administration, intraarticular administration and the like.
- composition of such a pharmaceutical composition can be determined according to the administration method, the GPR20 protein binding affinity of the antibody, and the like.
- the dosage of the anti-GPR20 antibody of the present invention is not limited as long as it is a pharmacologically effective amount, and the species of the individual, the type of disease, the symptom, sex, age, chronic disease, GPR20 protein binding affinity of the antibody or Although it can be appropriately determined depending on the biological activity and other factors, it is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg once every 1 to 180 days, or 1 It can be administered twice or more times a day.
- the form of the pharmaceutical composition includes injections (including lyophilized preparations and infusions), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols. Examples thereof include pills, pills, powders, suspensions, emulsions, eye drops, and implantable preparations.
- a pharmaceutical composition containing an anti-GPR20 antibody or a functional fragment thereof or a modified form thereof as an active ingredient can be administered simultaneously with or separately from other drugs.
- a pharmaceutical composition comprising an anti-GPR20 antibody or a functional fragment of the antibody as an active ingredient is administered, or after administering such a pharmaceutical composition, another pharmaceutical is administered, or You may administer the said pharmaceutical composition and another pharmaceutical simultaneously.
- other medicaments include various anticancer agents such as chemotherapeutic agents and radiotherapy. These are collectively referred to as “the combined use of the antibody of the present invention with another drug”, and a pharmaceutical composition containing an additional drug in addition to the antibody of the present invention, a functional fragment thereof or a modified form thereof is also included in the present invention.
- the present invention relates to a method for treating or preventing a disease associated with GPR20 such as cancer, use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, and the present invention for treating or preventing the disease.
- a disease associated with GPR20 such as cancer
- use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease and the present invention for treating or preventing the disease.
- a therapeutic or prophylactic kit containing the antibody of the present invention is also included in the present invention.
- Diagnostic Composition The present invention also provides a test or diagnostic composition (hereinafter collectively referred to as “diagnostic composition”) comprising an anti-GPR20 antibody or a functional fragment thereof or a modified form thereof.
- diagnostic composition comprising an anti-GPR20 antibody or a functional fragment thereof or a modified form thereof.
- the diagnostic composition of the present invention is useful for examination or diagnosis of GPR20 expression, diseases relating to GPR20 such as cancer, gastrointestinal stromal tumor (GIST), and the like.
- examination or diagnosis includes, for example, determination or measurement of morbidity risk, determination of the presence or absence of illness, measurement of the degree of progression or deterioration, measurement or determination of the effect of drug treatment with a pharmaceutical composition such as an anti-GPR20 antibody.
- measurement or determination of the effect of treatment other than drug treatment, measurement of recurrence risk, determination of the presence or absence of recurrence, and the like are included, but the test or diagnosis is not limited thereto.
- the diagnostic composition of the present invention is useful for identifying an individual to which an anti-GPR20 antibody antibody or a functional fragment thereof or a modified product thereof, a composition containing them, or a pharmaceutical composition containing them is administered.
- Such a diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a developer, a sensitizer, an aggregation inhibitor, and the like.
- the present invention relates to a method for examining or diagnosing a disease associated with GPR20 such as cancer, use of the antibody of the present invention for preparing a diagnostic composition for the disease, use of the antibody of the present invention for examining or diagnosing the disease , Also provide.
- a test or diagnostic kit containing the antibody of the present invention is also included in the present invention.
- a sandwich ELISA is desirable as a test or diagnosis method using the diagnostic composition of the present invention, but a normal ELISA method, RIA method, ELISPOT (Enzyme-Linked ImmunoSpot) method, dot blot method, octalony method, CIE (Counterimmunoelectrophoresis). ), Detection methods using antibodies such as CLIA (Chemiluminescent immunoassay) and FCM (Flow Cytometry) can be used.
- CLIA Click-Linked ImmunoSpot
- FCM Flow Cytometry
- labeling methods in addition to biotin, labeling methods that can be used for biochemical analysis such as labels for fluorophores such as HRP, alkaline phosphatase, FITC and ALEXA, and radioisotopes can be used.
- TMB (3,3 ′, 5,5′-tetramethylbenzidine), BCIP (5-brom-4-chloro-3-indolyl phosphate), ⁇ -NPP ( ⁇ -nitrophenyl phosphate), OPD (O-Phenylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), SuperSignal ELISA Pico Chemiminent Sentrate (registered as Thermo-scientific scientist) In addition to fluorescent substrates, chemiluminescent substrates can be used.
- test samples derived from living organisms include, but are not limited to, blood, joint fluid, ascites, lymph fluid, cerebrospinal fluid, tissue homogenate supernatant, tissue section, and the like.
- the sandwich ELISA kit for testing or diagnosis containing the antibody of the present invention includes a control (standard solution of GPR20-derived peptide), a coloring reagent, a dilution buffer, a solid phase antibody, a detection antibody, a washing solution, and the like. It's okay.
- a control standard solution of GPR20-derived peptide
- a coloring reagent e.g., a coloring reagent
- a dilution buffer e.g., a coloring reagent for coloring a coloring reagent
- a dilution buffer e.g., a coloring reagent for coloring or diagnosis containing the antibody of the present invention. It's okay.
- an absorption method, a fluorescence method, a luminescence method, an RI (Radioisotope) method or the like is suitably applied.
- solubilized protein from a cell, tissue, organ or part thereof in a sample according to a conventional method and react the labeled protein with the solubilized protein. It can also be used in Western blotting or dot blotting to confirm the presence or absence of GPR20 in the solubilized protein.
- Test samples of interest include solubilized proteins prepared from exosomes secreted by various cells, including cells, circulating tumor cells, and cancer cells, contained in body fluids such as blood, It is not limited to them.
- the present invention provides antibodies useful for immunohistochemistry (IHC) analysis, functional fragments thereof and modifications thereof, and compositions containing them. Such a composition is also included in the “diagnostic composition” of the present invention.
- IHC immunohistochemistry
- the immunohistochemistry is not particularly limited as long as it is a technique for detecting a primary antibody bound to an antigen by reacting a tissue section with an antibody (primary antibody) that binds to the antigen.
- Tissue sections are preferably paraffin-embedded after formalin fixation. After embedding in paraffin, the sliced tissue section is deparaffinized and then subjected to antigen activation treatment and nonspecific reaction suppression treatment.
- antigen activation treatment method include heat treatment, enzyme treatment with protease, etc., and heat treatment is preferred.
- As conditions for the heat treatment a temperature range of 90 to 110 ° C., a pH of 8 to 10 and a treatment time of 20 to 60 minutes are usually preferable.
- Tris-EDTA buffer for example, 10 mM Tris buffer containing 1 mM EDTA
- a method of inactivating an endogenous enzyme having a catalytic activity similar to or similar to the enzyme used for color development is usually used.
- H 2 O 2 As the H 2 O 2 solvent, water, methanol or the like can be used, and the concentration of H 2 O 2 is 0.1 to 3%, preferably 0.3 to 3%.
- Sodium azide can be added to the H 2 O 2 solution.
- a method of blocking with serum or casein can also be used as a nonspecific reaction suppression treatment. Serum and casein can treat tissues before the primary antibody reaction, but can also be included in a solvent that dilutes the primary antibody.
- the reaction conditions for the primary antibody are not particularly limited, but the temperature is 4 to 50 ° C, preferably 20 to 37 ° C, more preferably 24 ° C.
- the reaction time is 5 minutes to 1 day, preferably 10 minutes to 4 hours, more preferably 30 minutes to 1 hour.
- an antibody that can be visualized and binds to the primary antibody can be preferably used.
- the reaction is performed three or more times using an antibody (tertiary antibody) that binds to the secondary antibody itself.
- the secondary or tertiary antibody can be visualized by binding an enzyme such as peroxidase or alkaline phosphatase to these antibodies, or by adding biotin or the like to these antibodies and binding to the aforementioned enzymes such as streptavidin.
- a method of reacting a chromogenic substrate corresponding to those enzymes can be preferably used.
- Examples of a method for binding an enzyme to a secondary antibody or a tertiary antibody include a method using a reagent in which a large number of the enzyme and secondary antibody are bound to a dextrin polymer or an amino acid polymer (polymer method).
- polymer method a method of reacting a biotinylated secondary antibody and peroxidase-labeled streptavidin (LSAB method), DAB or the like can be used as a chromogenic substrate.
- a secondary antibody labeled with a fluorescent dye or the like can also be used. When treated with a fluorescently labeled secondary antibody, positive cells are detected using a fluorescence microscope after the treatment.
- the isolated cells are applied to glass or separated by a centrifugal separator, divided into cell components and liquid components, and immunostaining is performed on the cell components. That is, cell components can be applied on a slide glass and fixed with an ethanol solution or a 10% formalin solution, and then immunostaining similar to a tissue section can be performed.
- the excised tissue is rapidly frozen with liquid nitrogen after embedding with an OCT compound and sliced with a cryostat to prepare a slide specimen. After fixing this specimen with 10% formalin or ethanol solution, immunostaining similar to the tissue section can be performed.
- the operation related to immunohistochemistry can be performed automatically by programming the reaction solution, reaction conditions, number of washings, etc., and incorporating it into the immune device.
- the antibody is labeled with a pharmaceutically acceptable radionuclide or illuminant, the antibody is administered to a subject, an image is taken using diagnostic imaging techniques such as PET / CT, and the presence of GPR20 Can be determined or inspected.
- the antibody, functional fragment thereof or modified product thereof contained in the diagnostic composition of the present invention is preferably an antibody that binds to GPR20, that is, an antibody having GPR20 selectivity, a functional fragment thereof, or a modified product thereof.
- Antibodies having human GPR20 selectivity include antibodies comprising the heavy chain CDRH1, CDRH2 and CDRH3 of rat 04-093 antibody, and the light chain comprising light chain CDRL1, CDRL2 and CDRL3, Examples thereof include an antibody comprising the heavy chain variable region and the light chain variable region of rat 04-093 antibody, an antibody comprising the heavy chain and light chain of rat 04-093 antibody, and the like.
- Such antibodies can include, but are not limited to, rat 04-093 antibody, chimeric antibody derived from 04-093 antibody, rabbitized antibody derived from 04-093 antibody, humanized antibody derived from 04-093 antibody, and the like. Is not to be done.
- the diagnostic composition is for GPR20 detection or measurement.
- the present invention provides a method for detecting or measuring human GPR20 in a test sample.
- the diagnostic composition of the present invention can be used for these detection or measurement methods. Such a measurement method and diagnostic composition are also included in the present invention for diagnosis or examination of human GPR20 positive cancer, preferably gastrointestinal stromal tumor.
- the present invention also includes a method for identifying an individual (patient) to whom a pharmaceutical composition targeting GPR20 is administered.
- a method for identifying an individual (patient) to whom a pharmaceutical composition targeting GPR20 is administered In such an identification method, human GPR20 in the sample derived from the individual is measured, and human GPR20 is detected in the sample or compared with the amount of human GPR20 detected in the sample derived from a healthy individual. When many human GPR20s are detected, the individual can be determined as positive and can be identified as an individual to which a pharmaceutical composition targeting GPR20 is administered.
- the expression level, the staining ratio by immunostaining in addition to the case where the expression of GPR20 is confirmed in a sample derived from a subject, the expression level, the staining ratio by immunostaining, The intensity of staining can be used as an index.
- a score of 0 to 3 shown in Table 1 is set according to the degree of staining when immunostaining with an anti-GPR20 antibody, and a subject whose score is 1 or more, 2 or more, or 3 or more is targeted to GPR20 Mention may be made of the method of identifying the patient to whom the composition is administered.
- the diagnostic composition of the present invention can be used.
- the individual can be used to determine whether or not the individual suffers from or is at risk of a cancer, preferably a gastrointestinal stromal tumor.
- the pharmaceutical composition of the present invention can be administered to an individual who has been determined to be positive by such an identification method.
- Reagent The antibody of the present invention or an antigen-binding fragment thereof or a modified form thereof is also useful as a reagent. Such reagents are used in the above-described examination or diagnostic, research and other applications.
- Example 1 Preparation of rat anti-human GPR20 antibody 1
- the cDNA encoding the human GPR20 protein (NP_005284) is a PCR method using human brain-derived cDNA as a template according to methods known to those skilled in the art.
- the human GPR20 expression vector pcDNA3.1-hGPR20 was prepared by amplification into a mammalian expression vector.
- the amino acid sequence of human GPR20 is shown in SEQ ID NO: 1 in the sequence listing. Large-scale preparation of pcDNA3.1-hGPR20 plasmid DNA was performed using EndoFree Plasmid Giga Kit (QIAGEN).
- Hybridoma preparation After lymph node cells and mouse myeloma SP2 / 0-ag14 cells were electro-fused using a Hybridum Hybridoma Production System (Cyto Pulse Sciences), ClonCell-HY SelemDelC Suspended at 37 ° C., diluted and cultured under conditions of 37 ° C. and 5% CO 2 . Each appearing hybridoma colony was recovered as a monoclone, suspended in ClonCell-HY Selection Medium E (StemCell Technologies), and cultured under conditions of 37 ° C. and 5% CO 2 . After moderate cell growth, a frozen stock of each hybridoma cell was prepared and the culture supernatant was collected and used for screening of hybridomas producing anti-GPR20 antibodies.
- a Hybridoma preparation After lymph node cells and mouse myeloma SP2 / 0-ag14 cells were electro-fused using a Hybridum Hybridoma Production System (Cyto Pulse Sciences),
- OPD coloring solution 0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate.12 water, pH 4. 5
- OPD coloring solution 0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate.12 water, pH 4. 5
- o-phenylenediamine dihydrochloride o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H 2 O 2 dissolved in 0.4 mg / mL and 0.6% (v / v), respectively, were added at 100 ⁇ L / well. did.
- the color development reaction was carried out with occasional stirring, 1M HCl was added at 100 ⁇ L / well to stop the color development reaction, and then the absorbance at 490 nm was measured with a plate reader (ENVISION: PerkinElmer).
- pcDNA3.1-hGPR20 expression vector-introduced 293 ⁇ cells Hybridomas producing culture supernatants showing high absorbance were selected as positive for anti-human GPR20 antibody production.
- the expression vector-introduced 293T cells were treated with TryLE Express (manufactured by Life Technologies), washed with DMEM containing 10% FBS, and then suspended in PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.
- Example 1 The suspension of transiently expressed 293T cells prepared in -5-1 was centrifuged, and the supernatant was removed. Then, the hybridoma culture supernatant was added to each suspension, and suspended at 4 ° C. for 1 hour. Left to stand. After washing twice with PBS containing 5% FBS, Anti-Rat IgG FITC conjugate (manufactured by SIGMA) diluted 500 times with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4 ° C. for 1 hour.
- the hybridoma producing an antibody in which the histogram of pcDNA3.1-hGPR20-introduced 293T cells is shifted to the strong fluorescence intensity side with respect to the fluorescence intensity histogram of pcDNA3.1-introducing 293T cells, which is a control, is used as an antibody-producing hybridoma that binds to human GPR20.
- 178 clones were selected.
- clones 04-093, 13-001, 13-006, 13-010, 13-040 and 13-046, and the primary antibody are not added as examples of antibodies specifically bound to human GPR20.
- the result of negative control (w / o 1stAb) is shown.
- the horizontal axis represents the clone number
- the vertical axis represents the amount of antibody binding by MFI (mean fluorescence intensity).
- 1% BSA-containing PBS was added at 100 ⁇ L / well and allowed to stand overnight at room temperature.
- the culture supernatant of the anti-human GPR20 antibody-producing hybridoma diluted 2-fold with PBS containing 1% BSA was added at 100 ⁇ L / well and allowed to stand at room temperature for 1 hour.
- Anti-Rat IgG-Peroxidase antibody produced in rabbit (SIGMA) diluted 500-fold with PBS was added at 100 ⁇ L / well and allowed to stand at room temperature for 1 hour.
- FIG. 10 shows typical reaction examples of six antibodies that showed specific binding to the N-terminal 1-48 amino acids of human GPR20.
- the horizontal axis represents the clone number
- the vertical axis represents the amount of antibody binding by chemiluminescence (CPS).
- each anti-GPR20 antibody-producing hybridoma was grown to a sufficient amount with ClonCell-HY Selection Medium E, and then Hyperoma SFM (LifeTechnol Exchange Medium) supplemented with 20% Ultra Low IgG FBS (Life Technologies). And cultured for 4-5 days. The main culture supernatant was collected, passed through a 0.8 ⁇ m filter, and then passed through a 0.2 ⁇ m filter to remove insoluble matters.
- the antibody was purified from the above hybridoma supernatant by Protein G affinity chromatography.
- the antibody was adsorbed onto a Protein G column (GE Healthcare Bioscience), and the column was washed with PBS and then eluted with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7).
- 1M Tris-HCl pH 9.0 was added to the eluate to adjust the pH to 7.0 to 7.5, and then HBSor (25mM histidine / 5%) with Centrifugal UF Filter Device VIASPIN20 (fractionated molecular weight UF30K, Sartorius).
- the buffer was replaced with sorbitol (pH 6.0) and the antibody was concentrated to prepare an antibody concentration of 0.7 mg / mL or more. Finally, it was filtered through a Minisart-Plus filter (Sartorius) to obtain a purified sample.
- Example 2 Evaluation of IHC suitability of rat anti-human GPR20 antibody 2) -1 GPR20 staining evaluation using GPR20 transiently expressed 293T cells 2) -1-1 Preparation of cell block 293T cells were prepared using Lipofectamine 2000 (Life Technologies). Was transfected with pcDNA3.1-hGPR20 or pcDNA3.1 (empty vector) (GPR20-expressing 293T cells). The pellet of GPR20-expressing 293T cells was fixed in formalin and used as a paraffin-embedded block. Similarly, 293T cells (control 293T cells) transfected with pcDNA3.1 (empty vector) were used as paraffin-embedded blocks after formalin fixation.
- LS-A104 C-terminal
- LS-B7724 291-340 amino acids
- ab75559 manufactured by abcam
- NLS101 C-terminal
- sc-87141 N-terminal
- Deparaffinization and antigen activation were performed at 97 ° C. for 20 minutes using an antigen activating solution (Target Retrieval Solution High pH: DAKO) using an autostainer link pretreatment system (PT Link: manufactured by DAKO). Subsequent staining was performed at room temperature using an automatic staining apparatus (Dako Autostainer Link48: manufactured by DAKO). After washing once with EnVision FLEX WASH BUFFER (manufactured by DAKO), Peroxidase Block 3% H2O2 (manufactured by DAKO) was added, incubated for 5 minutes, and washed once with EnVision FLEX WASH BUFFER.
- EnVision FLEX WASH BUFFER manufactured by DAKO
- Peroxidase Block 3% H2O2 manufactured by DAKO
- Protein Block serum free (manufactured by DAKO) was added, incubated for 15 minutes, and the solution was removed with Air blow.
- the anti-GPR20 antibody was diluted with Signal stain Antibody diluent (manufactured by Cell Signaling Technology) to the concentrations shown in Table 2-1 and Table 2-2, and reacted for 30 minutes.
- Histofine simplestein mouse MAX PO (Rat) # 4141431 (manufactured by Nichirei Biosciences) is used for the rat antibody
- EnVision + Systemyl-HRPL-Lolmel-HRPL is used for the rabbit antibody.
- Anti-Rabbit # K4003 (manufactured by DAKO) was added and incubated for 30 minutes, and then washed twice with EnVision FLEX WASH BUFFER.
- DAKO Liquid DAB + Substrate Chromogen System was added and incubated for a total of 10 minutes, and then washed once with EnVision FLEX WASH BUFFER. After adding EnVision FLEX Hematoxylin and incubating for 5 minutes, it was washed 3 times with EnVision FLEX WASH BUFFER and ion-exchanged water.
- FIGS. 11-1, 11-2, and 11-3 Typical staining results for each antibody are shown in FIGS. 11-1, 11-2, and 11-3.
- Table 2-1 and Table 2-2 show the results of scoring the staining intensity of each antibody in FIGS. 11-1, 11-2, and 11-3.
- ++ indicates strong positive
- ++ indicates positive
- + indicates weak positive
- Rat monoclonal anti-GPR20 antibodies 04-093, 13-001, and 13-046 showed stronger staining on GPR20-expressing 293T cells (293-GPR20) than rabbit polyclonal anti-GPR20 antibodies.
- the rat monoclonal anti-GPR20 antibody did not show staining for negative control 293T cells (293-EV), GPR20-specific staining was confirmed.
- Deparaffinization and antigen activation were performed at 97 ° C. for 20 minutes using an antigen activating solution (Target Retrieval Solution High pH: DAKO) using an autostainer link pretreatment system (PT Link: manufactured by DAKO). Subsequent staining was performed at room temperature using an automatic staining apparatus (Dako Autostainer Link48: manufactured by DAKO). After washing once with EnVision FLEX WASH BUFFER (manufactured by DAKO), Peroxidase Block 3% H2O2 (manufactured by DAKO) was added, incubated for 5 minutes, and washed once with EnVision FLEX WASH BUFFER.
- EnVision FLEX WASH BUFFER manufactured by DAKO
- Peroxidase Block 3% H2O2 manufactured by DAKO
- Protein Block serum free (manufactured by DAKO) was added, incubated for 15 minutes, and the solution was removed with Air blow.
- Rat monoclonal anti-GPR20 antibody or rabbit polyclonal anti-GPR20 antibody was diluted to 10 ⁇ g / mL or 5 ⁇ g / mL with Signal stain Antibody diluent (manufactured by Cell Signaling Technology) and reacted for 30 minutes. After washing 3 times with EnVision FLEX WASH BUFFER, Histofine simple stain mouse PO (Rat) # 414143 (manufactured by Nichirei Biosciences) was added and incubated for 30 minutes, followed by washing with EnVision FLEX WASH BUFFER twice.
- DAKO Liquid DAB + Substrate Chromogen System was added and incubated for a total of 10 minutes, and then washed once with EnVision FLEX WASH BUFFER. After adding EnVision FLEX Hematoxylin and incubating for 5 minutes, it was washed 3 times with EnVision FLEX WASH BUFFER and ion-exchanged water.
- FIG. 12 comparison of GPR20 staining of each antibody against (FIG. 12-1) gastric GIST, (FIG. 12-2) small intestine GIST, and (FIG. 12-3) large intestine GIST showed that rat anti-GPR20 antibody 04-093 was the most High sensitivity.
- Example 3 Sequence analysis of rat anti-human GPR20 antibody 04-093 3) -1 Preparation of total RNA from 04-093 producing hybridoma cDNA encoding heavy chain and light chain signal sequences of 04-093 and variable region To amplify, total RNA was prepared from 04-093 producing hybridoma using TRIzol Reagent (Ambion).
- the cDNA encoding the heavy chain signal sequence and variable region amplified by 5'-RACE PCR was cloned into a plasmid, and then the sequence analysis of the heavy chain signal sequence and the nucleotide sequence of the variable region cDNA was performed.
- the full-length sequences of the heavy and light chains of the 04-093 antibody were determined by joining them with known constant region sequences.
- base sequence and amino acid sequence of the rat heavy chain IgG2b reference was made to the base sequence and amino acid sequence of AABR03048905 (IGHG2B * 01) published in IMGT, the international ImMgeneTics information system (registered trademark).
- base sequence and amino acid sequence of the constant region of rat light chain IgK were referred to the base sequence and amino acid sequence of V01241 (IGKC * 01) published in the same system.
- the heavy chain of the 04-093 antibody has the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 133 is a variable region.
- the amino acid sequence consisting of the 134th to 466th amino acid residues is a constant region.
- variable region is represented by CDRH1, a 69th to 78th amino acid sequence (SITNIDGSSY) or a 69th amino acid sequence of 45 to 54th amino acid sequence (GFTFNNYWMT) or a 50th to 54th amino acid sequence (NYWMT), or 69 To CDRH2 consisting of the 85th amino acid sequence (SITNIDGSSYYPDSVKG), and CDRH3 consisting of the amino acid sequence (GSFDY) of 118 to 122.
- the heavy chain variable region of the 04-093 antibody has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
- the CDRH1 of the 04-093 antibody has the amino acid sequence shown in SEQ ID NO: 5 or 8 in the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 6 or 9 in the sequence listing
- the amino acid sequence of CDRH3 Has the amino acid sequence shown in SEQ ID NO: 7 in the Sequence Listing.
- the amino acid sequence of the heavy chain of the 04-093 antibody is shown in FIG.
- the light chain of the 04-093 antibody has the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 126 is a variable region.
- the amino acid sequence consisting of the 127th to 232nd amino acid residues is a constant region.
- the variable region has a CDRL1 consisting of the amino acid sequence described in 43 to 53, a CDRL2 consisting of the amino acid sequence described in 69 to 75, and a CDRL3 consisting of the amino acid sequence described in 108 to 115 in SEQ ID NO: 11 in the sequence listing. .
- the light chain variable region of the 04-093 antibody has the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing.
- the CDRL1 of the 04-093 antibody has the amino acid sequence (KASQNVNKYLN) shown in SEQ ID NO: 13 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence (NTNNLQT) shown in SEQ ID NO: 14 in the sequence listing
- the amino acid sequence has the amino acid sequence (FQHVSWLT) shown in SEQ ID NO: 15 in the sequence listing.
- the amino acid sequence of the light chain of the 04-093 antibody is shown in FIG.
- the heavy chain amino acid sequence of the 04-093 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
- the nucleotide sequence consisting of nucleotides 1 to 57 in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing is a signal sequence.
- the nucleotide sequence consisting of nucleotides 58 to 399 of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing encodes the heavy chain variable region of antibody 04-093, and the nucleotide sequence consisting of nucleotides 400 to 1398 Encodes the heavy chain constant region of the 04-093 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence of nucleotide numbers 133 to 162 or CDR numbers 118 to 162 encoding CDRH1, nucleotide numbers 205 to 234 encoding CDRH2 in SEQ ID NO: 2, or A polynucleotide comprising the nucleotide sequence of 205 to 183 and a polynucleotide comprising the nucleotide sequence of nucleotide numbers 352 to 366 encoding CDRH3.
- the signal sequence of the heavy chain of the 04-093 antibody and the nucleotide sequence of the variable region are also shown in SEQ ID NO: 16 in the Sequence Listing.
- nucleotide sequence consisting of the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO: 16 of the Sequence Listing shows the signal sequence, and the nucleotide sequence consisting of the 58th to 399th nucleotides encodes the heavy chain variable region.
- sequence of SEQ ID NO: 2 is also described in FIG.
- the light chain amino acid sequence of the 04-093 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing.
- a nucleotide sequence consisting of nucleotides 1 to 57 in the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing is a signal sequence.
- the nucleotide sequence consisting of nucleotides 58 to 378 of the nucleotide sequence shown in SEQ ID NO: 10 of the sequence listing encodes the light chain variable region of the antibody 04-093, and the nucleotide sequence consisting of nucleotides 379 to 696 Encodes the light chain constant region of the 04-093 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 10, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 345 encoding CDRL3.
- the signal sequence of the light chain of the 04-093 antibody and the nucleotide sequence of the variable region are also shown in SEQ ID NO: 17 of the Sequence Listing.
- the nucleotide sequence consisting of the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO: 17 of the Sequence Listing shows a signal sequence, and the nucleotide sequence consisting of the 58th to 378th nucleotides encodes the light chain variable region.
- the sequence of SEQ ID NO: 10 is shown in FIG.
- the rabbit chimerized antibody heavy chain was named OcHch, and its amino acid sequence was shown in SEQ ID NO: 19.
- SEQ ID NO: 19 the amino acid sequence from amino acid numbers 1 to 19 represents a signal sequence
- the amino acid sequence from 20th to 133th represents a heavy chain variable region
- the amino acid sequence from 134th to 456th represents an amino acid sequence of a heavy chain constant region. Show.
- the rabbit chimerized antibody light chain was named OcLch, and its amino acid sequence was shown in SEQ ID NO: 21.
- SEQ ID NO: 21 the amino acid sequence from amino acid numbers 1 to 20 represents a signal sequence
- the 21st to 127th amino acid sequences represent the light chain variable region
- the 128th to 232nd amino acid sequences represent the amino acid sequence of the light chain constant region. Show.
- the heavy chain constant region was selected as rabbit IGHG * 02, and the light chain constant region was selected as rabbit IGKC1 * 01.
- SEQ ID NOs: 23 and 25 Two types of rabbitized antibody heavy chains, OcH01 and OcH02, were designed, and their amino acid sequences are shown in SEQ ID NOs: 23 and 25, respectively.
- the 1st to 19th amino acid sequences represent signal sequences
- the 20th to 133rd amino acid sequences represent variable regions
- the 134th to 456th amino acid sequences represent constant regions.
- the amino acid sequence thereof is shown in SEQ ID NO: 27.
- the 1st to 20th amino acid sequence represents a signal sequence
- the 21st to 127th amino acid sequence represents a variable region
- the 128th to 230th amino acid sequence represents a constant region.
- PCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.
- the synthesized DNA fragment and pCMA-LK are digested with XbaI and PmeI to bind the DNA fragment from which the light chain signal sequence and human ⁇ chain constant region have been removed.
- CLONTECH In-Fusion HD PCR cloning kit
- the OcHch and OcLch expression vectors were combined to produce OcChimera, the OcH01 and OcL01 expression vectors were combined, and the OcH1L1, OcH02 and OcL01 expression vectors were combined to produce the OcH2L1 antibody.
- Example 4 -2-5-2 Purification of Recombinant Antibody
- the culture supernatant obtained in Example 4) -2-5-1 was purified in one step of rProtein A affinity chromatography.
- the culture supernatant was applied to a column filled with MabSelectSuRe equilibrated with PBS (manufactured by GE Healthcare Bioscience), and then the column was washed with PBS twice the column volume.
- elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected.
- the fraction was subjected to buffer replacement with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
- the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractionated molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 2 mg / mL. Finally, it was filtered through a Minisart-Plus filter (Sartorius) to obtain a purified sample.
- VIVASPIN 20 fractionated molecular weight UF10K, Sartorius
- Example 5 Immunostaining using rabbit chimerized anti-GPR20 antibody and rabbit anti-GPR20 antibody 5) -1 Immunostaining of GIST cell line
- Example 4 Rabbit chimerized anti-GPR20 antibody and rabbit prepared in 2-5 The staining property of the conjugated anti-GPR20 antibody was examined using paraffin-embedded specimens of GIST cell lines (GIST430, GIST430 / 654) and prostate cancer cell line (PC-3) as a negative control. Deparaffinization and antigen activation were carried out at 97 ° C. for 40 minutes using an autostainer link pretreatment system (PT Link: manufactured by DAKO) and an antigen activation solution (Target Retrieval Solution High pH: manufactured by DAKO).
- PT Link manufactured by DAKO
- Target Retrieval Solution High pH: manufactured by DAKO an autostainer link pretreatment system
- Rat monoclonal anti-GPR20 antibody or rabbit chimerized anti-GPR20 antibody and rabbit anti-GPR20 antibody were diluted to 1.0 ⁇ g / mL to 10 ⁇ g / mL with REAL Antibody Diluent (manufactured by DAKO) and allowed to react for 30 minutes. After washing 3 times with EnVision FLEX WASH BUFFER, Hisfinine simplestein mouse MAX PO (Rat) # 4141431 (manufactured by Nichirei Biosciences) is used for rat antibodies, and EnVision + System-HolLabLabed-RRP is a rabbit antibody. # K4003 (manufactured by DAKO) was added, and each was incubated for 30 minutes, and then washed twice with EnVision FLEX WASH BUFFER.
- DAKO Liquid DAB + Substrate Chromogen System was added and incubated for a total of 10 minutes, and then washed once with EnVision FLEX WASH BUFFER. After adding EnVision FLEX Hematoxylin and incubating for 5 minutes, it was washed 3 times with EnVision FLEX WASH BUFFER and ion-exchanged water.
- FIG. 13 shows a typical stained image.
- the rabbit chimerized antibody OcChimera and the rabbitized antibodies OcH1L1 and OcH2L1 showed higher sensitivity staining than the rat antibody 04-093 against the GIST cell line. It did not show staining for a prostate cancer cell line not expressing GPR20.
- FIG. 14 (FIG. 14-1) Gastric GIST, (FIG. 14-2) Small intestine GIST, (FIG. 14-3) GPR20 staining properties of each antibody against large intestine GIST were compared. All of the anti-GPR20 antibodies were more sensitive than the rat anti-GPR20 antibody 04-093.
- Example 6 Identification of epitope 6) -1 Binding ability evaluation by peptide ELISA Binding of rat anti-human GPR20 antibody 04-093 to the following synthetic peptides was evaluated by peptide-ELISA method.
- NeutrAvidin (Pierce) diluted to 1 ⁇ g / mL with PBS was added to 96-well Maxisorp plate (Nunc) at 100 ⁇ L / well and allowed to stand overnight at 4 ° C. After removing the solution and washing three times with 300 ⁇ L / well of 0.05% Tween 20-containing PBS (hereinafter PBST), the C-terminal was biotinylated from amino acid Nos. 1 to 48 (SEQ ID No.
- FIG. 15 shows reaction examples of various antibodies.
- the horizontal axis represents the clone number, and the vertical axis represents the amount of antibody binding by chemiluminescence (CPS).
- CPS chemiluminescence
- the 04-093 antibody binds to an amino acid sequence consisting of amino acid numbers 30 to 48 of GPR20 (SEQ ID NO: 30: LEVPLFLFFALDDEELHGT). 6) -2 Binding ability evaluation using GPR20 fragment peptide
- the peptide consisting of amino acid numbers 29 to 48 of GPR20 and the terminal amino acid of this peptide were successively shortened one by one. Peptides were synthesized, and the N-terminal biotinylated and C-terminal amidated were prepared as a GPR20 fragment peptide library (Sigma Aldrich Japan).
- the dissociation constant between the prepared GPR20 fragment peptide and the 04-093 antibody was obtained by using the Octet RED384 system (Pall ForteBio) to capture the GPR20 fragment peptide as a ligand on a sensor chip and using the 04-093 antibody as an analyte. It was measured. PBS-T was used as a buffer solution, and a streptavidin sensor chip (Pall ForteBio) was used as a sensor chip.
- the sensor chip was immersed in a 1 ⁇ g / mL peptide solution for 300 seconds, and then immersed in a dilution series solution of 04-093 antibody (0.247-20 nM 3-fold dilution series, 5 concentrations) for 90 seconds.
- the binding phase was monitored and subsequently immersed in buffer to monitor the dissociation phase for 270 seconds.
- After measuring each concentration of 04-093 antibody it was immersed in citrate buffer pH 2.2 for 20 seconds to regenerate the sensor chip, and further immersed in buffer for 20 seconds for equilibration.
- the GPR20 fragment peptide (GLEVPLFHLFARLD: amino acid numbers 29 to 42 of SEQ ID NO: 29) showed strong binding to the 04-093 antibody, but (GLEVPLFHLFARL: amino acid numbers 29 to 41 of SEQ ID NO: 29) showed decreased binding. .
- the GPR20 fragment peptide (LEVPLFHLFARLDEEL: amino acid numbers 30 to 45 of SEQ ID NO: 29) showed strong binding to the 04-093 antibody, but (EVPLFHLFARLDEEL: amino acid numbers 31 to 45 of SEQ ID NO: 29) showed a decrease in binding. It was. From these results, amino acid numbers 30 to 42 (LEVPLFLFLARLD: SEQ ID NO: 31) of SEQ ID NO: 29 were identified as epitopes of the 04-093 antibody.
- Example 7 Detection of GPR20 protein by Western blotting method
- GPR20 protein transiently expressed in 293T cells was detected by Western blotting method.
- a protein lysate of the membrane fraction was prepared from each cell using Mem-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific) and BCA protein assay (ThermoFisher Scientific) was used.
- NuPAGE TM LDS Sample Buffer (4X), NuPAGE TM Sample Reducing Agent (10X) (Thermo Fisher Scientific) was added to the protein solution adjusted in concentration so as to have a concentration of 1X, followed by heating at 70 ° C. for 10 minutes. 20 ⁇ g / lane of the protein was electrophoresed by SDS-PAGE using Tris / Glysin / SDS buffer. Proteins were transferred from the gel after electrophoresis to an Immobilon-FL membrane (Millipore).
- This Immobilon-FL membrane was blocked with Odyssey Blocking Buffer (LI-COR) for 1 hour, and then diluted with 0.1% Tween20 / Odyssey Blocking Buffer, a rabbit chimeric anti-GPR20 antibody or a rabbit anti-GPR20 primary antibody The reaction was shaken overnight at 4 ° C. As controls, rabbit IgG, mouse anti-FLAG antibody, and mouse anti- ⁇ actin antibody were similarly used as the primary antibody reaction solution. SNAP i. d.
- the rabbit anti-GPR20 antibodies OcH1L1, OcH2L1, and the rabbit chimerized anti-GPR20 antibody OcChimera are expressed as 293T-pcDNA3.1-GPR20 cells expressing human GPR20 or 293T expressing human GPR20 with a FLAG tag added to the N-terminus. -Reactive to pFLAG-GPR20 cells, multiple bands detected. On the other hand, no band was detected in the negative control 293T-pcDNA3.1 cells. The mouse anti-FLAG antibody was reactive only with 293T-pFLAG-GPR20, and multiple bands were detected. From the above results, it was shown that the rabbit chimerized anti-GPR20 antibody and the rabbit anti-GPR20 antibody are useful for the detection of GPR20 by the Western blotting method.
- the anti-GPR20 antibody provided by the present invention it becomes possible to test or diagnose various cancers expressing GPR20, and a kit for testing or diagnosing various cancers expressing GPR20 containing the anti-GPR20 antibody of the present invention. Etc. can be provided. Moreover, the pharmaceutical composition etc. which contain the anti- GPR20 antibody of this invention as an active ingredient can be provided.
- SEQ ID NO: 18 Nucleotide sequence of a DNA fragment comprising a sequence encoding the amino acid sequence of rabbit chimerized antibody heavy chain OcHch
- SEQ ID NO: 19 Amino acid sequence of rabbit chimerized antibody heavy chain
- OcHch SEQ ID NO: 20: Rabbit chimerized antibody light chain
- SEQ ID NO: 22 DNA fragment comprising a sequence encoding the amino acid sequence of rabbitized antibody heavy chain OcH01
- Nucleotide sequence SEQ ID NO: 23 Amino acid sequence of rabbitized antibody heavy chain OcH01
- SEQ ID NO: 24 Nucleotide sequence of a DNA fragment comprising a sequence encoding the amino acid sequence of rabbitized antibody heavy chain OcH02
- SEQ ID NO: 25 Rabbitized antibody heavy chain H02
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Abstract
Description
(1)配列番号1においてアミノ酸番号1乃至48に示されるアミノ酸配列を含むことからなるペプチドに特異的に結合する抗体または当該抗体の抗原結合性断片、
(2)GPR20への結合に対して、配列番号3のアミノ酸番号20乃至466に示されるアミノ酸配列からなる重鎖及び配列番号11のアミノ酸番号20乃至232に示されるアミノ酸配列からなる軽鎖を有する抗体と競合阻害活性を有する抗体または当該抗体の抗原結合性断片、
(3)LEVPLFHLFARLD(配列番号31)に示されるアミノ酸配列からなるエピトープに結合することを特徴とする(1)又は(2)に記載の抗体又は当該抗体の機能性断片、
(4)重鎖配列が、配列番号5または8に示されるアミノ酸配列からなるCDRH1、配列番号6または9に示されるアミノ酸配列からなるCDRH2、並びに、配列番号7に示されるアミノ酸配列からなるCDRH3を有する可変領域を含み;
軽鎖配列が配列番号13に示されるアミノ酸配列からなるCDRL1、配列番号14に示されるアミノ酸配列からなるCDRL2、及び配列番号15に示されるアミノ酸配列からなるCDRL3を有する可変領域を含む、(1)乃至(3)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(5)配列番号4に示されるアミノ酸配列からなる重鎖可変領域及び、配列番号12に示される軽鎖可変領域を含むことからなる、(1)乃至(4)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(6)配列番号3のアミノ酸番号20乃至466に示されるアミノ酸配列を含むことからなる重鎖、及び、配列番号11のアミノ酸番号20乃至232に示されるアミノ酸配列を含むことからなる軽鎖からなる(1)乃至(5)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(7)キメラ抗体であることを特徴とする(1)乃至(5)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(8)定常領域がウサギ抗体由来であることを特徴とする、(7)に記載のキメラ抗体、
(9)配列番号19のアミノ酸番号20乃至456に示されるアミノ酸配列からなる重鎖及び配列番号21のアミノ酸番号21乃至232に示されるアミノ酸配列からなる軽鎖を含む(1)乃至(5)、(7)及び(8)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(10)ウサギ化されていることを特徴とする、(1)乃至(5)のいずれか1項に記載の抗体または当該抗体の機能性断片、
(12)以下の(a)または(b)に記載の重鎖、及び(c)に記載の軽鎖を含む、(1)乃至(5)及び(10)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片:
(a)配列番号23においてアミノ酸番号20乃至456に記載のアミノ酸配列からなる重鎖、
(b)配列番号25においてアミノ酸番号20乃至456に示されるアミノ酸配列からなる重鎖、
(c)配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列からなる軽鎖、
(13)配列番号23においてアミノ酸番号20乃至456に示されるアミノ酸配列を含むことからなる重鎖及び、配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列を含むことからなる軽鎖からなる、(1)乃至(5)及び(10)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(14)配列番号25においてアミノ酸番号20乃至456に示されるアミノ酸配列を含むことからなる重鎖及び、配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列を含むことからなる軽鎖からなる、(1)乃至(5)及び(10)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片、
(15)Fab、F(ab’)2、Fab’およびFvからなる群から選択される、(1)乃至(14)のいずれか1項に記載の抗体の抗原結合性断片、
(16)scFvであることを特徴とする、(1)乃至(14)のいずれか1項に記載の抗体、
(17)(1)乃至(16)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片を含む組成物、
(18)(1)乃至(16)のいずれか1項に記載の抗体または当該抗体の抗原結合性断片を含み、パラフィン包埋処理した後脱パラフィン処理した組織標本(以下、単に「標本」という。)中のGPR20の検出または測定方法に使用される、(17)に記載の組成物、
(19)(1)乃至(16)のいずれか1項に記載の抗体または該抗体の抗原結合性断片と被検標本を接触させる工程を含む、標本中のGPR20の検出または測定方法に使用される、(17)または(18)に記載の組成物、
(20)GPR20の検出または測定方法が、被検標本においてGPR20が検出もしくは測定されたか、または被検標本におけるGPR20の発現レベルもしくは発現量が事前に決定された基準と同等かまたはそれより高い場合、該被検標本を陽性と判定し、被検標本においてGPR20が検出もしくは測定されなかったか、または被検標本におけるGPR20の発現レベルもしくは発現量が事前に決定された基準と同等かまたはそれより低い場合、該被検標本を陰性と判定する工程を含む、(18)または(19)に記載の組成物、
(22)GPR20陽性疾患の検査または診断方法が、GPR20の検出または測定において、陽性と判定された被検標本が由来する被験者は、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防方法に適していると判定し、陰性と判定された被検標本が由来する被験者は、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防方法には適していないと判定することを含む、(17)乃至(21)のいずれか1項に記載の組成物、
(23)GPR20陽性疾患がGPR20陽性癌である、(21)または(22)に記載の組成物、
(24)GPR20陽性疾患が消化管間質腫瘍(GIST)である、(21)乃至(23)のいずれか1項に記載の組成物、
(25)下記(a)乃至(c)のいずれか一つに記載の被験者に投与される、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を含む医薬組成物:
(a)(17)乃至(19)及び(21)のいずれか1項に記載の組成物を用いてGPR20が検出または測定された被検標本の由来する被験者;
(b)(20)記載の組成物を用いたGPR20の検出または測定において陽性と判定された被検標本の由来する被験者;
(c)(22)または(24)記載の組成物を用いて、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防に適していると判定された被験者、
(26)以下の工程(a)及び(b)を含むGPR20陽性疾患の治療方法:
(a)(1)乃至(16)のいずれか1項に記載の抗体または該抗体の抗原結合性断片、(17)乃至(19)、(21)に記載の組成物を用いて検体のGPR20を検出または測定する工程;
(b)(a)においてGPR20の発現が検出または測定された検体に由来する被験者に抗GPR20抗体又は該抗体の抗原結合性断片を投与する工程、
(27)(1)乃至(16)のいずれか1項に記載の抗体または該抗体の抗原結合性断片をコードするポリヌクレオチド、
(28)(27)記載のポリヌクレオチドを含むベクター、
(29)(27)記載のポリヌクレオチドまたは(28)記載のベクターを含む細胞、
(30)下記の工程(a)および(b)を含む、(1)乃至(16)のいずれか1項に記載の抗体または該抗体の抗原結合性断片の製造方法:
(a)(29)記載の細胞を培養する工程;
(b)工程(a)の培養物からモノクローナル抗体または該抗体の抗原結合性断片を回収する工程。
本発明において、「遺伝子」とは、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチドまたはその相補鎖を意味し、例えば、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチドまたはその相補鎖であるポリヌクレオチド、オリゴヌクレオチド、DNA、mRNA、cDNA、cRNA等は「遺伝子」の意味に含まれる。かかる遺伝子は一本鎖、二本鎖または三本鎖以上のヌクレオチドであり、DNA鎖とRNA鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド(RNA)とデオキシリボヌクレオチド(DNA)が混在するものおよびそのようなヌクレオチド鎖を含む二本鎖または三本鎖以上のヌクレオチドも「遺伝子」の意味に含まれる。本発明の「GPR20遺伝子」としては、例えば、GPR20蛋白質のアミノ酸配列をコードする塩基配列が含まれるDNA、mRNA、cDNA、cRNA等を挙げることができる。
本発明で用いるGPR20は、ヒト、ヒト以外の哺乳動物(例えば、モルモット、ラット、マウス、ウサギ、ブタ、ヒツジ、ウシ、サルなど)あるいはニワトリのT細胞あるいは肥満細胞から直接精製して使用するか、あるいは上記の細胞の細胞膜画分を調製して使用することができ、また、GPR20をin vitroにて合成する、あるいは遺伝子操作により宿主細胞に産生させることによって得ることができる。遺伝子操作では、具体的には、GPR20 cDNAを発現可能なベクターに組み込んだ後、転写と翻訳に必要な酵素、基質およびエネルギー物質を含む溶液中で合成する、あるいは他の原核生物、または真核生物の宿主細胞を形質転換させることによってGPR20を発現させることにより、該蛋白質を得ることが出来る。
本発明のGPR20に対する抗体は、常法を用いて、GPR20またはGPR20のアミノ酸配列から選択される任意のポリペプチドを動物に免疫し、生体内に産生される抗体を採取、精製することによって得ることができる。抗原となるGPR20の生物種はヒトに限定されず、サル、マウス、ラット等のヒト以外の動物に由来するGPR20を動物に免疫することもできる。この場合には、取得された異種GPR20に結合する抗体とヒトGPR20との交差性を試験することによって、ヒトの疾患に適用可能な抗体を選別できる。なお、抗原となるGPR20はGPR20遺伝子を遺伝子操作により宿主細胞に産生させることによって得ることができる。具体的には、GPR20遺伝子を発現可能なベクターを作製し、これを宿主細胞に導入して該遺伝子を発現させ、発現したGPR20を精製すればよい。
ある態様において、好適な抗体は、非変性型のヒトGPR20、及び、ホルマリンで固定した標本中の変性型ヒトGPR20の両方に特異的に結合する。また、より好適な抗体は、非変性型のヒトGPR20、及び、ホルマリンで固定した標本中の変性型ヒトGPR20の両方に特異的に結合し、かつ他のGPRファミリーに特異的に結合しない抗体等を挙げることができるが、それらに限定されるものではない。
本発明は抗GPR20抗体もしくはその機能断片またはその修飾体を含む医薬組成物を提供する。
本発明は抗GPR20抗体もしくはその機能断片またはその修飾体を含む検査用または診断用組成物(以下、まとめて「診断用組成物」という)をも提供する。
組織切片は、好適にはホルマリン固定後にパラフィン包埋処理する。パラフィン包埋後、薄切した組織切片を脱パラフィン処理した後、抗原賦活処理および非特異的反応抑制処理を行う。抗原賦活処理の方法としては、加熱処理、プロテアーゼ等による酵素処理を例示することができ、好適には加熱処理である。加熱処理の条件としては、通常、温度90乃至110℃、pH8乃至10、処理時間20乃至60分の範囲が好適である。pHの調整にはTris-EDTA緩衝液(例えば、1mMのEDTAを含有する10mMトリス緩衝液)等を使用することができる。非特異的反応抑制処理としては、発色に使用する酵素と同様または類似の触媒活性を有する内因性の酵素を不活性化する方法が通常用いられる。ペルオキシダーゼ反応により発色させる場合、予め組織中に存在する内因性のペルオキシダーゼをH2O2等で阻害することが好ましい。H2O2の溶媒は水、メタノール等を使用することができ、H2O2の濃度は0.1乃至3%、好適には0.3乃至3%である。H2O2溶液にはアジ化ナトリウムを添加することができる。また、血清やカゼインによりブロッキングする方法も非特異的反応抑制処理として使用することができる。血清やカゼインは、一次抗体反応の前に組織を処理することができるが、1次抗体を希釈する溶媒に含有せしめることもできる。
本発明の抗体もしくはその抗原結合性断片またはその修飾体は、試薬としても有用である。かかる試薬は、上述の検査または診断用、研究用およびその他の用途で使用される。
1)-1 ヒトGPR20発現ベクターの構築
ヒトGPR20タンパク質(NP_005284)をコードするcDNAは、当業者に公知な方法に従いヒト脳由来cDNAを鋳型としたPCR法により増幅され、哺乳動物発現用ベクターに組み込むことによってヒトGPR20発現ベクターpcDNA3.1-hGPR20が作製された。ヒトGPR20のアミノ酸配列を配列表の配列番号1に示す。pcDNA3.1-hGPR20プラスミドDNAの大量調製は、EndoFree Plasmid Giga Kit(QIAGEN社)を用いて行った。
免疫には6週齢のWKY/Izmラットの雌(日本エスエルシー社)が使用された。まずラット両足下腿部をHyaluronidase(SIGMA-ALDRICH社)にて前処理後、同部位にヒトGPR20発現ベクターpcDNA3.1-hGPR20が筋肉内注射された。続けて、ECM830(BTX社)を使用し、2ニードル電極を用いて、同部位にインビボ エレクトロポレーションを実施した。二週間に一度、同様のインビボ エレクトロポレーションを繰り返した後、79日目にラットのリンパ節を採取しハイブリドーマ作製に用いた。
リンパ節細胞とマウスミエローマSP2/0-ag14細胞とをHybrimune Hybridoma Production System(Cyto Pulse Sciences社)を用いて電気細胞融合した後、ClonaCell-HY Selection Medium D(StemCell Technologies社)に懸濁し、希釈して37℃、5% CO2の条件下で培養した。出現した各々のハイブリドーマコロニーは、モノクローンとして回収され、ClonaCell-HY Selection Medium E(StemCell Technologies社)に懸濁して37℃、5% CO2の条件下で培養した。適度に細胞が増殖した後、各々のハイブリドーマ細胞の凍結ストックを作製すると共に、培養上清が回収され、抗GPR20抗体を産生するハイブリドーマのスクリーニングに用いられた。
1)-4-1 Cell-ELISA用抗原遺伝子発現細胞の調製
293α細胞(インテグリンαv及びインテグリンβ3を発現するHEK293由来の安定発現細胞株)を10% FBS含有DMEM培地中5x106細胞/10mLになるよう調製した。Lipofectamine 2000(インビトロジェン社)を用いた形質移入手順に従って、この293α細胞に対して、pcDNA3.1-hGPR20もしくは陰性コントロールとしてpcDNA3.1のDNAを導入し、96-well plate(Corning社)に100μLずつ分注後、10% FBS含有DMEM培地中で37℃、5% CO2の条件下で24から27時間培養した。得られた形質移入細胞を接着状態のまま、Cell-ELISAに使用した。
実施例1)-4-1で調製した発現ベクター導入293α細胞の培養上清を除去後、pcDNA3.1-hGPR20またはpcDNA3.1導入293α細胞のそれぞれに対しハイブリドーマ培養上清を添加し、4℃で1時間静置した。well中の細胞を5% FBS含有PBS(+)で1回洗浄後、5% FBS含有PBS(+)で500倍に希釈したAnti-Rat IgG-Peroxidase antibody produced in rabbit(SIGMA社)を加えて、4℃で1時間静置した。well中の細胞を5% FBS含有PBS(+)で3回洗浄した後、OPD発色液(OPD溶解液(0.05 M クエン酸3ナトリウム、0.1M リン酸水素2ナトリウム・12水 pH4.5)にo-フェニレンジアミン二塩酸塩(和光純薬社)、H2O2をそれぞれ0.4mg/mL、0.6%(v/v)になるように溶解)を100μL/wellで添加した。時々攪拌しながら発色反応を行い、1M HClを100μL/wellで添加して発色反応を停止させた後、プレートリーダー(ENVISION:PerkinElmer社)で490nmの吸光度を測定した。細胞膜表面上に発現するヒトGPR20に特異的に結合する抗体を産生するハイブリドーマを選択するため、コントロールのpcDNA3.1導入293α細胞と比較し、pcDNA3.1-hGPR20発現ベクター導入293α細胞の方でより高い吸光度を示す培養上清を産生するハイブリドーマを抗ヒトGPR20抗体産生陽性として選択した。
1)-5-1 フローサイトメトリー解析用抗原遺伝子発現細胞の調製
293T細胞を5×104細胞/cm2になるよう225cm2フラスコ(住友ベークライト社製)に播種し、10% FBS含有DMEM培地中で37℃、5% CO2の条件下で一晩培養した。翌日、pcDNA3.1-hGPR20および陰性コントロールとしてpcDNA3.1をそれぞれ293T細胞にLipofectamine 2000を用いて導入し、37℃、5% CO2の条件下でさらに一晩培養した。翌日、発現ベクター導入293T細胞をTrypLE Express(Life Technologies社製)で処理し、10% FBS含有DMEMで細胞を洗浄した後、5% FBS含有PBSに懸濁した。得られた細胞懸濁液をフローサイトメトリー解析に使用した。
実施例1)-4-2のCell-ELISAで陽性と判定されたハイブリドーマが産生する抗体のヒトGPR20に対する結合特異性をフローサイトメトリー法によりさらに確認した。実施例1)-5-1で調製した一過性発現293T細胞の懸濁液を遠心し、上清を除去した後、各々に対しハイブリドーマ培養上清を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugate(SIGMA社製)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、2μg/ml 7-aminoactinomycin D(Molecular Probes社製)を含む5% FBS含有PBSに再懸濁し、フローサイトメーター(FC500:BeckmanCoulter社製)で検出を行った。データ解析はFlowjo(TreeStar社製)で行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成した。コントロールであるpcDNA3.1導入293T細胞の蛍光強度ヒストグラムに対しpcDNA3.1-hGPR20導入293T細胞のヒストグラムが強蛍光強度側にシフトしている抗体を産生するハイブリドーマをヒトGPR20に結合する抗体産生ハイブリドーマとして178クローン選択した。図9にヒトGPR20に特異的に結合した抗体の例としてクローン番号04-093、13-001、13-006、13-010、13-040及び13-046、並びに1次抗体を添加していない陰性コントロール(w/o 1stAb)の結果を示す。図9の横軸はクローン番号、縦軸はMFI(mean fluorescence intensity)による抗体結合量を示す。
ヒトGPR20のN末端側48アミノ酸に対する結合性をペプチド-ELISA法により評価した。96-well Maxisorp plate(Nunc社)にPBSで1μg/mLに希釈したNeutrAvidin(Pierce社)を100μL/wellで添加し、4℃で1晩静置した。溶液を除去し、300μL/wellの0.05%Tween20含有PBS(以下PBST)で3回洗浄した後、C末端がビオチン化されたヒトGPR20のN末端側1-48アミノ酸(配列番号29)からなる合成ペプチド1をPBSで10 nMに溶解した溶液を100μL/wellで添加し、室温で1時間静置した。同様にC末端がビオチン化された、GPR20のアミノ酸配列とは異なる配列を有する合成ペプチドを添加したプレートを陰性コントロールとして用意し、以下同様に処理した。プレートから溶液を除去し、PBSTで3回洗浄した後、1%BSA含有PBSを100μL/wellで添加し、室温で1晩静置した。溶液を除去し、PBSTで3回洗浄した後、1%BSA含有PBSで2倍希釈した抗ヒトGPR20抗体産生ハイブリドーマの培養上清を100μL/wellで添加し、室温で1時間静置した。溶液を除去し、PBSTで3回洗浄した後、PBSで500倍希釈したAnti-Rat IgG-Peroxidase antibody produced in rabbit(SIGMA社)を100μL/wellで添加し、室温で1時間静置した。溶液を除去し、PBSTで3回洗浄した後、SuperSignal ELISA Pico Chemiluminescent Substrateを100μl/wellで添加し、室温で10分間静置した後、プレートリーダー(ARVO,PerkinElmer社)で化学発光を測定した。図10にヒトGPR20のN末端側1-48アミノ酸に特異的な結合を示した6種の抗体の典型的な反応例を示す。図10の横軸はクローン番号、縦軸は化学発光量(CPS)による抗体結合量を示す。
ラット抗ヒトGPR20モノクローナル抗体の重鎖のサブクラス、軽鎖のタイプは、RAT MONOCLONAL ANTIBODY ISOTYPING TEST KIT(DSファーマバイオメディカル社)により決定された。その結果、04-093、13-001、13-006、13-010、13-040及び13-046は、いずれもIgG2b、κ鎖であることが確認された。また、実施例4に記載の方法と同様の方法により塩基配列を解析した結果、13-001、13-006、13-010、13-040の各抗体のアミノ酸配列は高い相同性を示す配列を有しており、同一のエピトープを認識することが推定された。
ラット抗ヒトGPR20モノクローナル抗体04-093、13-001及び13-046は、ハイブリドーマ培養上清から精製された。
2)-1 GPR20一過性発現293T細胞を用いたGPR20染色性評価
2)-1-1 セルブロックの作製
293T細胞は、Lipofectamine 2000(Life Technologies社)を用いて、pcDNA3.1-hGPR20またはpcDNA3.1(空ベクター)でトランスフェクションされた(GPR20発現293T細胞)。このGPR20発現293T細胞のペレットをホルマリン固定後にパラフィン包埋ブロックとした。同様にpcDNA3.1(空ベクター)をトランスフェクションした293T細胞(コントロール293T細胞)についてもホルマリン固定後にパラフィン包埋ブロックとした。
1)-8で調製したラットモノクローナル抗ヒトGPR20抗体04-093、13-001及び13-046と市販のウサギポリクローナル抗ヒトGPR20抗体のGPR20染色性を比較した。市販の抗体は、いずれもヒトGPR20に由来する合成ペプチドを抗原として作製されており、LS Bio社製のLS-A101(C末端)、LS-A102(N末端)、LS-A103(細胞質ドメイン)、LS-A104(C末端)、LS-B7724(291~340アミノ酸)、abcam社製のab75559、Novus Biologicals社製のNLS101(C末端)、Santa Cruz Biotechnology社製のsc-87141(N末端)が用いられた。なお、括弧内は各ウサギポリクローナル抗体作製時の免疫に使用された合成ペプチドのGPR20内の部位を示している。
2)-2-1 GIST組織アレイの染色
臨床検体における抗GPR20抗体の染色性をGIST481, Gastrointestinal stromal tumor tissue array, 24 cases/48 cores(US Biomax社製)を用いて検討した。
3)-1 04-093産生ハイブリドーマからのtotal RNAの調製
04-093の重鎖及び軽鎖のシグナル配列と可変領域をコードするcDNAを増幅するため、04-093産生ハイブリドーマよりTRIzol Reagent(Ambion社)を用いてtotal RNAを調製した。
04-093の重鎖のシグナル配列と可変領域をコードするcDNAの増幅は、実施例3)-1で調製したtotal RNAの約1μgとSMARTer RACE cDNA Amplification Kit(Clontech社)を用いて実施した。04-093の重鎖のシグナル配列と可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM (Universal Primer A Mix:SMARTer RACE cDNA Amplification Kitに付属)、及び公知のラット重鎖の定常領域の配列から設計したプライマーを用いた。
実施例3)-2と同様の方法で実施した。ただし、04-093の軽鎖のシグナル配列と可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM (Universal Primer A Mix:SMARTer RACE cDNA Amplification Kitに付属)、及び公知のラット軽鎖の定常領域の配列から設計したプライマーを用いた。
4)-1 抗GPR20抗体04-093のウサギキメラ化体のデザイン
ウサギキメラ化配列は、ウサギの重鎖定常領域IGHG*02とウサギの軽鎖定常領域IGKC2*01をIMGT(登録商標), the international ImMunoGeneTics information system(登録商標)から参照して、クローン04-093のそれぞれの可変領域につなげることで設計した。
ウサギ化抗体の可変領域のアミノ酸配列は、CDRグラフティング(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))によって設計した。可変領域のアミノ酸配列の同一性とウサギgermline配列における中庸さに基づき、重鎖アクセプター配列はIGHV1S7*01とIGHJ3*01を、軽鎖アクセプター配列はIGKV1S39*01とIGKJ1-2*01を選択した。タンパク質立体構造解析プログラムBioLuminate(Schrodinger社製)で構築されたクローン04-093のホモロジーモデルを分析し、アクセプター上にグラフティングされるべきドナー残基をQueen et al.(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))によって与えられる基準などを参考に選択した。重鎖定常領域はウサギのIGHG*02を、軽鎖定常領域はウサギのIGKC1*01を選択した。
4)-2-1 ウサギキメラ化抗GPR20抗体の重鎖発現ベクターの構築
4)-2-1-1 抗体発現ベクターpCMA-LKの構築
プラスミドpcDNA3.3-TOPO/LacZ(Invitrogen社)を制限酵素XbaI及びPmeIで消化して得られる約5.4kbのフラグメントと、配列番号28に示すヒト軽鎖シグナル配列及びヒトκ鎖定常領域のアミノ酸配列をコードするDNA配列を含むDNA断片をIn-Fusion Advantage PCRクローニングキット(CLONTECH社)を用いて結合して、pcDNA3.3/LKを作製した。
配列番号18に示すウサギキメラ化抗体重鎖OcHchのアミノ酸配列をコードするDNA配列を含むDNA断片を合成した(GENEART社)。配列表の配列番号18に示されるヌクレオチド配列の26乃至82番目のヌクレオチドからなるヌクレオチド配列はシグナル配列を示し、83乃至424番目のヌクレオチドからなるヌクレオチド配列は重鎖可変領域をコードし、425乃至1393番目のヌクレオチドからなるヌクレオチド配列は定常領域をコードしている。
4)-2-2-1 ウサギキメラ化抗体軽鎖OcLch発現ベクターの構築
配列番号20に示すウサギキメラ化抗体軽鎖OcLchのアミノ酸配列をコードするDNA配列を含むDNA断片を合成した(GENEART社)。配列表の配列番号20に示されるヌクレオチド配列の26乃至85番目のヌクレオチドからなるヌクレオチド配列はシグナル配列を示し、86乃至406番目のヌクレオチドからなるヌクレオチド配列は軽鎖可変領域をコードし、407乃至721番目のヌクレオチドからなるヌクレオチド配列は定常領域をコードしている。実施例4)-2-1-2と同様の方法でウサギキメラ化抗体軽鎖OcLchの発現ベクターを構築した。
4)-2-3-1 ウサギ化抗体重鎖OcH01の発現ベクターの構築
配列番号22に示すウサギ化抗体重鎖OcH01のアミノ酸配列をコードするDNA配列を含むDNA断片を合成した(GENEART社)。配列表の配列番号22に示されるヌクレオチド配列の26乃至82番目のヌクレオチドからなるヌクレオチド配列はシグナル配列を示し、83乃至424番目のヌクレオチドからなるヌクレオチド配列は重鎖可変領域をコードし、425乃至1393番目のヌクレオチドからなるヌクレオチド配列は定常領域をコードしている。実施例4)-2-1-2と同様の方法でウサギ化抗体重鎖OcH01発現ベクターを構築した。
配列番号24に示すウサギ化抗体重鎖OcH02のアミノ酸をコードするDNA配列を含むDNA断片を合成した(GENEART社)。配列表の配列番号24に示されるヌクレオチド配列の26乃至82番目のヌクレオチドからなるヌクレオチド配列はシグナル配列を示し、83乃至424番目のヌクレオチドからなるヌクレオチド配列は重鎖可変領域をコードし、425乃至1393番目のヌクレオチドからなるヌクレオチド配列は定常領域をコードしている。実施例4)-2-1-2と同様の方法でウサギ化抗体重鎖OcH02発現ベクターを構築した。
4)-2-4-1 ウサギ化抗体軽鎖OcL01の発現ベクターの構築
配列番号26に示すウサギ化抗体軽鎖OcL01のアミノ酸をコードするDNA配列を含むDNA断片を合成した(GENEART社)。配列表の配列番号26に示されるヌクレオチド配列の26乃至85番目のヌクレオチドからなるヌクレオチド配列はシグナル配列を示し、86乃至406番目のヌクレオチドからなるヌクレオチド配列は重鎖可変領域をコードし、407乃至721番目のヌクレオチドからなるヌクレオチド配列は定常領域をコードしている。実施例5)-2-1-2と同様の方法でウサギ化抗体軽鎖OcL01発現ベクターを構築した。
4)-2-5-1 リコンビナント抗体の生産
FreeStyle 293F細胞(Invitrogen社)はマニュアルに従い、継代、培養をおこなった。対数増殖期の1.2×109個のFreeStyle 293F細胞(Invitrogen社)を3L Fernbach Erlenmeyer Flask(CORNING社)に播種し、FreeStyle293 expression medium (Invitrogen社)で希釈して2.0×106細胞/mLに調製した。40mLのOpti-Pro SFM培地(Invitrogen社)に0.24mgの重鎖発現ベクターと0.36mgの軽鎖発現ベクターと1.8mgのPolyethyleneimine(Polyscience #24765)を加えて穏やかに攪拌し、さらに5分間放置した後にFreeStyle 293F細胞に添加した。37℃、8%CO2インキュベーターで4時間、90rpmで振とう培養後に600mLのEX-CELL VPRO培地(SAFC Biosciences社)、18mLのGlutaMAX I(GIBCO社)、及び30mLのYeastolate Ultrafiltrate(GIBCO社)を添加し、37℃、8%CO2インキュベーターで7日間、90rpmで振とう培養して得られた培養上清をDisposable Capsule Filter (Advantec #CCS-045-E1H)でろ過した。
実施例4)-2-5-1で得られた培養上清をrProtein Aアフィニティークロマトグラフィーの1段階工程で精製した。培養上清をPBSで平衡化したMabSelectSuReが充填されたカラム(GE Healthcare Bioscience社製)にアプライしたのちに、カラム容量の2倍以上のPBSでカラムを洗浄した。次に2Mアルギニン塩酸塩溶液(pH4.0)で溶出し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりPBSへのバッファー置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社)で抗体を濃縮し、IgG濃度を2mg/mLに調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
5)-1 GIST細胞株の免疫染色
実施例4)-2-5で作製したウサギキメラ化抗GPR20抗体及びウサギ化抗GPR20抗体の染色性を、GIST細胞株(GIST430、GIST430/654)および陰性対照として前立腺癌細胞株(PC-3)のパラフィン包埋標本を用いて検討した。脱パラフィンおよび抗原賦活はAutostainer Link用前処理システム(PT Link:DAKO社製)を用いて、抗原賦活液(Target Retrieval Solution High pH:DAKO社製)、97℃で40分間実施した。その後の染色作業は自動染色装置(ダコ Autostainer Link48:DAKO社製)を用いて室温で実施した。EnVision FLEX WASH BUFFER(DAKO社製)で1回洗浄した後、Peroxidase Block 3% H2O2(DAKO社製)を加え5分間インキュベートし、EnVision FLEX WASH BUFFERで1回洗浄した。Protein Block serum free(DAKO社製)を加え、30分間インキュベートし、Air blowで液を除去した。ラットモノクローナル抗GPR20抗体あるいはウサギキメラ化抗GPR20抗体及びウサギ化抗GPR20抗体をREAL Antibody Diluent(DAKO社製)で1.0μg/mL~10μg/mLに希釈し、30分間反応させた。EnVision FLEX WASH BUFFERで3回洗浄後、ラット抗体に対してはHistofine simplestain mouse MAX PO(Rat)#414311(ニチレイバイオサイエンス社製)を、ウサギ抗体に対してはEnVision+ System-HRP Labelled Polymer Anti-Rabbit #K4003(DAKO社製)を加え、いずれも30分インキュベートした後、EnVision FLEX WASH BUFFERで2回洗浄した。
臨床検体におけるウサギキメラ化抗GPR20抗体及びウサギ化抗GPR20抗体の染色性をGIST801, Gastrointestinal stromal tumor tissue array, 80 cases/80 cores(US Biomax社製)を用いて検討した。染色は実施例5)-1と同様の方法で実施した。
6)-1 ペプチドELISAによる結合能評価
以下の合成ペプチドに対するラット抗ヒトGPR20抗体04-093の結合をペプチド-ELISA法により評価した。96-well Maxisorp plate(Nunc社)にPBSで1μg/mLに希釈したNeutrAvidin(Pierce社)を100μL/wellで添加し、4℃で1晩静置した。溶液を除去し、300μL/wellの0.05%Tween20含有PBS(以下PBST)で3回洗浄した後、C末端がビオチン化された、ヒトGPR20のアミノ酸番号1から48番目(配列番号29)からなる合成ペプチド1、ヒトGPR20のアミノ酸番号30から48番目(配列番号30)からなる合成ペプチド2、GPR20のアミノ酸配列とは異なる配列を有する陰性コントロールの合成ペプチドを各々PBSで10 nMに溶解した溶液を100μL/wellで添加し、室温で1時間静置した。プレートから溶液を除去し、PBSTで3回洗浄した後、Blocker Casein in PBS(Thermo Fisher Scientific社)を100μL/wellで添加し、室温で1晩静置した。溶液を除去し、PBSTで3回洗浄した後、Blocker Casein in PBSで1μg/mLに希釈した04-093抗体を100μL/wellで添加し、室温で1時間静置した。溶液を除去し、PBSTで3回洗浄した後、PBSで500倍希釈したAnti-Rat IgG-Peroxidase antibody produced in rabbit(Jackson ImmunoResearch Laboratories社)を100μL/wellで添加し、室温で1時間静置した。溶液を除去し、PBSTで3回洗浄した後、SuperSignal ELISA Pico Chemiluminescent Substrateを100μl/wellで添加し、室温で10分間静置した後、プレートリーダー(SpectraMax M3,Molecular Devices社)で化学発光を測定した。図15に各種抗体の反応例を示す。図15の横軸はクローン番号、縦軸は化学発光量(CPS)による抗体結合量を示す。04-093抗体は、合成ペプチド1及び合成ペプチド2に対して同等の結合活性を示した。一方、他の抗GPR20抗体は、合成ペプチド2に対して結合性を示さなかった。本結果から、04-093抗体はGPR20のアミノ酸番号30から48番目からなるアミノ酸配列(配列番号30:LEVPLFHLFARLDEELHGT)に結合することが示された。
6)-2 GPR20断片ペプチドを用いた結合能評価
エピトープを詳細に解析するため、GPR20のアミノ酸番号29から48番目からなるペプチド、並びに本ペプチドの末端のアミノ酸を一残基ずつ連続的に短縮したペプチドを合成し、N-末端をビオチン化、C-末端をアミド化したものをGPR20断片ペプチドライブラリーとして作製した(シグマ アルドリッチ ジャパン社)。作製したGPR20断片ペプチドと04-093抗体の解離定数は、Octet RED384システム(Pall ForteBio社製)を使用し、GPR20断片ペプチドをリガンドとしてセンサーチップに捕捉、04-093抗体をアナライトとすることで測定した。緩衝液としてPBS-T、センサーチップとしてストレプトアビジンセンサーチップ(Pall ForteBio社製)を用いた。各GPR20断片ペプチドについて、センサーチップを1μg/mLのペプチド溶液に300秒間浸した後、04-093抗体の希釈系列溶液(0.247~20nMの3倍希釈系列、5濃度)に90秒間浸して結合相をモニターし、引き続き緩衝液に浸して270秒間の解離相をモニターした。各濃度の04-093抗体を測定後は、センサーチップを再生するためにクエン酸緩衝液pH 2.2に20秒間浸し、さらに平衡化のため緩衝液に20秒間浸した。データの解析には1:1結合モデルを用いて、結合速度定数ka、解離速度定数kd及び解離定数(KD;KD=kd/ka)を算出した。
結果を表3に示す。
293T細胞に一過性に発現させたGPR20タンパク質をウエスタンブロッティング法により検出した。293T細胞にLipofectamine 2000(Life Technologies社)を用いて、ヒトGPR20発現ベクターpcDNA3.1-hGPR20、N末端にFLAGタグを付加したヒトGPR20の発現ベクターpFLAG-GPR20並びにpcDNA3.1(空ベクター)を各々導入した。各細胞よりMem-PER Plus Membrane Protein Extraction Kit(ThermoFisher Scientific社)を用いて膜画分のタンパク質溶解液を調製し、BCA protein assay(ThermoFisher Scientific社)を用いてタンパク質濃度を測定した。濃度調整したタンパク質溶液にNuPAGETM LDS Sample Buffer (4X)、NuPAGETM Sample Reducing Agent (10X) (Thermo Fisher Scientific社)を1X濃度となるように添加し、70℃で10分間加熱した後、定法に従いTris /Glysin/SDS bufferを用いたSDS-PAGEで20μg/laneのタンパク質を電気泳動した。泳動後のゲルからImmobilon-FL膜(Millipore社)にタンパク質を転写した。このImmobilon-FL膜をOdyssey Blocking Buffer(LI-COR社)で1時間ブロッキングした後、0.1% Tween20/ Odyssey Blocking Bufferで希釈したウサギキメラ化抗GPR20抗体またはウサギ化抗GPR20抗体の1次抗体反応液中で4℃、一晩振盪した。また、対照としてウサギIgG及びマウス抗FLAG抗体、マウス抗β actin抗体を同様に一次抗体反応液として用いた。SNAP i.d. システム(Millipore社)を用いてTBS-0.1%Tween20バッファーで3回洗浄した後、0.1% Tween20/ Odyssey Blocking Bufferで希釈した2次抗体液(1次抗体がウサギ抗体の場合はIRDye 680CW Goat anti-rabbit IgG、1次抗体がマウス抗体の場合はIRDye 800CW Goat anti-mouse IgGを使用した)で反応させた後、TBS-0.1%Tween20バッファーで2回、TBSで1回洗浄した。蛍光の検出にはOdyssey Infrared Imaging System(LI-COR社)を用いた。図17において、ウサギ化抗GPR20抗体OcH1L1、OcH2L1、ウサギキメラ化抗GPR20抗体OcChimeraは、ヒトGPR20を発現する293T-pcDNA3.1-GPR20細胞またはN末端にFLAGタグを付加したヒトGPR20を発現する293T-pFLAG-GPR20細胞に対して反応性を示し、複数のバンドが検出された。一方、陰性コントロールである293T-pcDNA3.1細胞ではバンドが検出されなかった。また、マウス抗FLAG抗体は293T-pFLAG-GPR20に対してのみ反応性を示し、複数のバンドが検出された。以上の結果より、ウサギキメラ化抗GPR20抗体及びウサギ化抗GPR20抗体はウエスタンブロッティング法によるGPR20の検出に有用であることが示された。
配列番号19: ウサギキメラ化抗体重鎖OcHchのアミノ酸配列
配列番号20:ウサギキメラ化抗体軽鎖OcLchのアミノ酸配列をコードする配列を含むDNA断片のヌクレオチド配列
配列番号21: ウサギキメラ化抗体軽鎖OcLchのアミノ酸配列
配列番号22:ウサギ化抗体重鎖OcH01のアミノ酸配列をコードする配列を含むDNA断片のヌクレオチド配列
配列番号23: ウサギ化抗体重鎖OcH01のアミノ酸配列
配列番号24:ウサギ化抗体重鎖OcH02のアミノ酸配列をコードする配列を含むDNA断片のヌクレオチド配列
配列番号25: ウサギ化抗体重鎖H02のアミノ酸配列
配列番号26:ウサギ化抗体軽鎖OcL01のアミノ酸配列をコードする配列を含むDNA断片のヌクレオチド配列
配列番号27:ウサギ化抗体軽鎖OcL01のアミノ酸配列
配列番号28:ヒト軽鎖シグナル配列及びヒトκ鎖定常領域のアミノ酸配列をコードするDNA配列を含むDNA断片
配列番号29:合成ペプチド1 ヒトGPR20の1から48番目のアミノ酸配列
Claims (30)
- 配列番号1においてアミノ酸番号1乃至48に示されるアミノ酸配列を含むことからなるペプチドに特異的に結合する抗体または当該抗体の抗原結合性断片。
- GPR20への結合に対して、配列番号3のアミノ酸番号20乃至466に示されるアミノ酸配列からなる重鎖及び配列番号11のアミノ酸番号20乃至232に示されるアミノ酸配列からなる軽鎖を有する抗体と競合阻害活性を有する抗体または当該抗体の抗原結合性断片。
- LEVPLFHLFARLD(配列番号31)に示されるアミノ酸配列からなるエピトープに結合することを特徴とする請求項1又は2に記載の抗体又は当該抗体の機能性断片。
- 重鎖配列が、配列番号5または8に示されるアミノ酸配列からなるCDRH1、配列番号6または9に示されるアミノ酸配列からなるCDRH2、並びに、配列番号7に示されるアミノ酸配列からなるCDRH3を有する可変領域を含み;
軽鎖配列が配列番号13に示されるアミノ酸配列からなるCDRL1、配列番号14に示されるアミノ酸配列からなるCDRL2、及び配列番号15に示されるアミノ酸配列からなるCDRL3を有する可変領域を含む、請求項1乃至3のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。 - 配列番号4に示されるアミノ酸配列からなる重鎖可変領域及び、配列番号12に示される軽鎖可変領域を含むことからなる、請求項1乃至4のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- 配列番号3のアミノ酸番号20乃至466に示されるアミノ酸配列を含むことからなる重鎖、及び、配列番号11のアミノ酸番号20乃至232に示されるアミノ酸配列を含むことからなる軽鎖からなる請求項1乃至5のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- キメラ抗体であることを特徴とする請求項1乃至5のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- 定常領域がウサギ抗体由来であることを特徴とする、請求項7に記載のキメラ抗体。
- 配列番号19のアミノ酸番号20乃至456に示されるアミノ酸配列からなる重鎖及び配列番号21のアミノ酸番号21乃至232に示されるアミノ酸配列からなる軽鎖を含む請求項1乃至5、7及び8のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- ウサギ化されていることを特徴とする、請求項1乃至5のいずれか1項に記載の抗体または当該抗体の機能性断片。
- ヒト化されていることを特徴とする、請求項1乃至5のいずれか1項に記載の抗体または当該抗体の機能性断片。
- 以下の(a)または(b)に記載の重鎖、及び(c)に記載の軽鎖を含む、請求項1乃至5及び10のいずれか1項に記載の抗体または当該抗体の抗原結合性断片:
(a)配列番号23においてアミノ酸番号20乃至456に記載のアミノ酸配列からなる重鎖、
(b)配列番号25においてアミノ酸番号20乃至456に示されるアミノ酸配列からなる重鎖、
(c)配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列からなる軽鎖。 - 配列番号23においてアミノ酸番号20乃至456に示されるアミノ酸配列を含むことからなる重鎖及び、配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列を含むことからなる軽鎖からなる、請求項1乃至5及び10のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- 配列番号25においてアミノ酸番号20乃至456に示されるアミノ酸配列を含むことからなる重鎖及び、配列番号27においてアミノ酸番号21乃至230に示されるアミノ酸配列を含むことからなる軽鎖からなる、請求項1乃至5及び10のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
- Fab、F(ab’)2、Fab’およびFvからなる群から選択される、請求項1乃至14のいずれか1項に記載の抗体の抗原結合性断片。
- scFvであることを特徴とする、請求項1乃至14のいずれか1項に記載の抗体。
- 請求項1乃至16のいずれか1項に記載の抗体または当該抗体の抗原結合性断片を含む組成物。
- 請求項1乃至16のいずれか1項に記載の抗体または当該抗体の抗原結合性断片を含み、パラフィン包埋処理した後脱パラフィン処理した組織標本(以下、単に「標本」という。)中のGPR20の検出または測定方法に使用される、請求項17に記載の組成物。
- 請求項1乃至16のいずれか1項に記載の抗体または該抗体の抗原結合性断片と被検標本を接触させる工程を含む、標本中のGPR20の検出または測定方法に使用される、請求項17または18に記載の組成物。
- GPR20の検出または測定方法が、被検標本においてGPR20が検出もしくは測定されたか、または被検標本におけるGPR20の発現レベルもしくは発現量が事前に決定された基準と同等かまたはそれより高い場合、該被検標本を陽性と判定し、被検標本においてGPR20が検出もしくは測定されなかったか、または被検標本におけるGPR20の発現レベルもしくは発現量が事前に決定された基準と同等かまたはそれより低い場合、該被検標本を陰性と判定する工程を含む、請求項18または19に記載の組成物。
- GPR20陽性疾患の検査または診断方法に使用される、請求項17乃至20のいずれか1項に記載の組成物。
- GPR20陽性疾患の検査または診断方法が、GPR20の検出または測定において、陽性と判定された被検標本が由来する被験者は、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防方法に適していると判定し、陰性と判定された被検標本が由来する被験者は、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防方法には適していないと判定することを含む、請求項17乃至21のいずれか1項に記載の組成物。
- GPR20陽性疾患がGPR20陽性癌である、請求項21または22に記載の組成物。
- GPR20陽性疾患が消化管間質腫瘍(GIST)である、請求項21乃至23のいずれか1項に記載の組成物。
- 下記(a)乃至(c)のいずれか一つに記載の被験者に投与される、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を含む医薬組成物:
(a)請求項17乃至19及び21のいずれか1項に記載の組成物を用いてGPR20が検出または測定された被検標本の由来する被験者;
(b)請求項20記載の組成物を用いたGPR20の検出または測定において陽性と判定された被検標本の由来する被験者;
(c)請求項22または24記載の組成物を用いて、GPR20に特異的に結合する抗体もしくは該抗体の抗原結合性断片を投与する工程を含むGPR20陽性疾患の治療または予防に適していると判定された被験者。 - 以下の工程(a)及び(b)を含むGPR20陽性疾患の治療方法:
(a)請求項1乃至16のいずれか1項に記載の抗体または該抗体の抗原結合性断片、請求項17乃至19、21に記載の組成物を用いて検体のGPR20を検出または測定する工程;
(b)(a)においてGPR20の発現が検出または測定された検体に由来する被験者に抗GPR20抗体又は該抗体の抗原結合性断片を投与する工程。 - 請求項1乃至16のいずれか1項に記載の抗体または該抗体の抗原結合性断片をコードするポリヌクレオチド。
- 請求項27記載のポリヌクレオチドを含むベクター。
- 請求項27記載のポリヌクレオチドまたは請求項28記載のベクターを含む細胞、
- 下記の工程(a)および(b)を含む、請求項1乃至16のいずれか1項に記載の抗体または該抗体の抗原結合性断片の製造方法:
(a)請求項29記載の細胞を培養する工程;
(b)工程(a)の培養物からモノクローナル抗体または該抗体の抗原結合性断片を回収する工程。
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Publication number | Publication date |
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EP3604518A1 (en) | 2020-02-05 |
JP7082112B2 (ja) | 2022-06-07 |
TW201840588A (zh) | 2018-11-16 |
US11261249B2 (en) | 2022-03-01 |
EP3604518A4 (en) | 2020-12-09 |
JPWO2018181656A1 (ja) | 2020-02-06 |
US20200017583A1 (en) | 2020-01-16 |
CA3058357A1 (en) | 2018-10-04 |
AU2018244047A1 (en) | 2019-10-17 |
CN110573618A (zh) | 2019-12-13 |
TWI782000B (zh) | 2022-11-01 |
KR20190135478A (ko) | 2019-12-06 |
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