WO2019054460A1 - Anti-ramp2 antibody - Google Patents

Anti-ramp2 antibody Download PDF

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WO2019054460A1
WO2019054460A1 PCT/JP2018/034076 JP2018034076W WO2019054460A1 WO 2019054460 A1 WO2019054460 A1 WO 2019054460A1 JP 2018034076 W JP2018034076 W JP 2018034076W WO 2019054460 A1 WO2019054460 A1 WO 2019054460A1
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seq
amino acid
acid sequence
antibody
set forth
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PCT/JP2018/034076
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French (fr)
Japanese (ja)
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隆行 新藤
中村 哲朗
龍 前田
洋佑 中山
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株式会社ジーンテクノサイエンス
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Priority to CA3075867A priority Critical patent/CA3075867A1/en
Priority to JP2019542293A priority patent/JP7202011B2/en
Publication of WO2019054460A1 publication Critical patent/WO2019054460A1/en
Priority to JP2022136364A priority patent/JP2022169737A/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • A61P27/06Antiglaucoma agents or miotics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material

Definitions

  • the present invention provides an anti-angiogenic activity using an antibody against receptor activity regulatory protein 2 (RAMP2) having anti-angiogenic activity or an immunoreactive fragment thereof, and an antibody against such RAMP2 or an immunoreactive fragment thereof as an active ingredient It relates to the agent.
  • RAMP2 receptor activity regulatory protein 2
  • Adrenomedullin (hereinafter referred to as "AM") is a 52 amino acid peptide identified from human pheochromocytoma and is known as a vasoactive substance mainly secreted from blood vessels and having a function of dilating blood vessels. In addition to this, various actions such as organ protecting action, tissue regenerating action and anti-inflammatory action have been reported.
  • Receptor activity regulatory protein 2 (RAMP2) is a single-pass protein and forms a complex with the seven-pass protein calcitonin receptor-like receptor (CRLR) as a co-receptor for AM It is functioning. Its expression is found in vascular endothelial cells, and AM in autocrine and paracrine causes intracellular cAMP to rise and transmit signals to maintain vascular homeostasis.
  • AM37-52 which is an AM partial sequence peptide (antagonist) has been reported (Patent Document 1).
  • AM37-52 has a weak antagonist activity and a small molecular weight of 5 kDa or less, so its kinetics in blood are poor and development as a drug is difficult.
  • the present inventors have obtained an anti-RAMP2 antibody that inhibits AM-dependent activation of RAMP2 and CRLR complex, in particular, an anti-RAMP2 antibody having the ability to inhibit the growth of HUVEC (human umbilical vein endothelial cells).
  • a polyclonal antibody was prepared by immunizing a peptide fragment encoding the RAMP2 protein.
  • the antiserum-derived polyclonal antibody did not show the ability to suppress cAMP. Therefore, we attempted to obtain an anti-RAMP2 antibody by immunizing a protein in which the extracellular domain of RAMP2 was cell-freely synthesized.
  • the present inventors immunized a transgenic overexpressing cell as an antigen. Since human RAMP2 gene alone does not function as a receptor for AM peptide, fibroblast cell line NIH-3T3 cells in which RAMP2 gene was coexpressed with CRLR gene were prepared.
  • the RAMP2 protein is a small molecule with a total length of 175 amino acids (20 kDa), and in particular the extracellular domain is a small molecule of 102 amino acids (12 kDa), 62% of the extracellular domain is highly homologous with human RAMP2 and mouse RAMP2, and the same amino acid is encoded Therefore, it is difficult to obtain immunity to the functional domain of human RAMP2, and high quality antibodies can not be obtained by immunization to normal rats, hamsters or mice. Therefore, by immunizing MRL / lpr mice that generate an immune response (autoantibodies) against self by hyperimmunization, we succeeded in obtaining an anti-RAMP2 antibody containing an anti-RAMP2 antibody with excellent functionality.
  • autoantibodies autoantibodies
  • hCR2-CHOK1 cells were used to screen for signal-inhibiting antibodies using as an indicator whether or not to suppress an increase in intracellular cAMP due to AM addition. Furthermore, an antibody capable of inhibiting the growth of HUVEC by AM addition was screened as a test for inhibiting the biological activity of selected signal-inhibiting antibodies, and an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action was successfully obtained.
  • the present invention relates to an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action, and also relates to an angiogenesis inhibitor containing the antibody as an active ingredient.
  • RAMP2 receptor activity regulatory protein 2
  • CDRH1 consisting of amino acid sequence of GYFMN (SEQ ID NO: 5)
  • CDRH2 consisting of amino acid sequence of RX 8 NPYNGDX 9 X 10 YNQ KFKG (SEQ ID NO: 23)
  • X 1 is G or M
  • X 2 is F or Y
  • X 3 is A or V
  • X 4 is absent or H
  • X 5 is A or V
  • X 6 is D or E
  • X 7 is Y or N
  • X 8 is N or I
  • X 9 is S or T
  • X 10 is I, F or L.
  • a light chain complementarity determining region consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8), a CDRL 2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and QQDSKX 11 PWT (SEQ ID NO: 25)
  • X 11 is H or N.
  • CDRL1 comprising the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8), A CDRL2 consisting of an amino acid sequence of YTSRLHS (SEQ ID NO: 9) and a CDRL3 consisting of an amino acid sequence of QQDSKHPWT (SEQ ID NO: 10)
  • CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8)
  • a CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9)
  • CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17
  • CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8)
  • An antibody or an immunoreactive fragment thereof according to (10) having CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9) and
  • a VH has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 2 and a VL is set forth in SEQ ID NO: 4
  • a VH has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 12 and a VL is set forth in SEQ ID NO: 14
  • a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 11, and a polynucleotide encoding the VL having the nucleotide sequence set forth in SEQ ID NO: 13; or a VH encoding the nucleotide sequence set forth in SEQ ID NO: 18 A nucleic acid molecule according to (14), comprising a polynucleotide and a polynucleotide encoding a VL having the nucleotide sequence as set forth in SEQ ID NO: 20.
  • a vector comprising the nucleic acid molecule of (14) or (15).
  • a host cell comprising the vector according to (16).
  • a pharmaceutical composition comprising the antibody according to any one of (1) to (13) or an immunoreactive fragment thereof as an active ingredient.
  • the present invention provides a therapeutic agent or a preventive agent for a disease or disorder in which
  • FIG. 7 is a graph showing the inhibitory effect of various anti-RAMP2 antibodies (10 nM) on cAMP production upon addition of AM using hCR2-CHOK1 cells.
  • the vertical axis shows the ratio (%) of the amount of cAMP in each group to the amount (100%) of the amount of AMP addition / non-administration (control).
  • the horizontal axis shows the presence or absence of addition of AM and antibody, and the added antibody. It is a graph showing the result of having measured the inhibitory power of cAMP production by AM addition of various concentration 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody using hCR2-CHOK1 cells.
  • the vertical axis shows the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition.
  • the horizontal axis shows antibody concentration (nM). It is a graph which shows the inhibitory effect of various anti-RAMP2 antibodies (10 nM) on HUVEC proliferation by AM addition.
  • the vertical axis shows the percentage (%) of the HUVEC cell number of each group to the AM-added / non-antibody-administered (control) HUVEC cell number (100%).
  • the horizontal axis shows the presence or absence of addition of AM and antibody, and the added antibody.
  • the vertical axis shows the ratio (%) of HUVEC cell count at each antibody administration to the HUVEC cell count (100%) at the time of no antibody addition.
  • the horizontal axis shows antibody concentration (nM).
  • VH (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 4) of the 25H4-4F9 antibody, the VH (SEQ ID NO: 12) and light chain variable region (SEQ ID NO: 14) of the 85H7-2B11 antibody, and the 33H4-1G3 antibody The amino acid sequences of VH (SEQ ID NO: 19) and light chain variable region (SEQ ID NO: 21), and the CDR portions contained therein are shown. It is a graph which shows the result of having confirmed that the acquired antibody is anti-RAMP2 specific by Cell ELISA. The horizontal axis shows the antibody used.
  • the open bars in the dots indicate the results using cells expressing human CRLR alone, and the black bars indicate cells in which human CRLR and human RAMP2 are coexpressed (hCR2-CHOK1).
  • the vertical axis shows the binding amount (OD450) of each antibody to these expressing cells.
  • 25H4-4F9 black squares
  • 85H7-2B11 black circles
  • 33H4-1G3 black triangles
  • 4E5 white circles
  • 2F5 white squares
  • B5 open triangles
  • the ordinate represents the binding ratio (%), and the abscissa represents the log value of the antibody concentration (nM).
  • the antibody concentration at which the binding ratio is 50% was determined from the graph and used as the EC50 value.
  • 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and 4E5, 2F5, and B5, which are commercially available anti-RAMP2 antibodies, as test antibodies binding to the antigen on the cell membrane surface was carried out. It is a graph showing the result analyzed by FACS. Each test antibody name used at the top of each graph is shown.
  • each graph represents the number of cells ( ⁇ 10 3 cells), and the horizontal axis represents the fluorescence intensity of R-Phycoerythrin-labeled secondary antibody bound to the cells.
  • the peak located on the left indicates the control (in the absence of each test antibody), and the peak located on the right indicates the presence of each test antibody.
  • the ordinate represents the mean value (MFI) of the fluorescence intensity of the fluorescent label bound to the biotinylated 85H7-2B11 antibody bound to the cells, and the abscissa represents the concentration ( ⁇ g / mL) of each antibody used.
  • MFI mean value
  • the results of 85H7-2B11 (filled circles) competed with the labeled antibody for the same antibody show that the binding is in competition.
  • the sequences of the variable regions of the heavy chain (Heavy Chain) and light chain (Light Chain) of a humanized antibody designed based on the sequence of the 25H4-4F9 antibody are shown.
  • H25H4-4F9vh1 (SEQ ID NO: 27), h25H4-4F9vh2 (SEQ ID NO: 29), h25H4-4F9vh3 (SEQ ID NO: 31), h25H4-4F9vh4 based on the sequence h25H4-4F9vh (SEQ ID NO: 2) of the 25H4-4F9 antibody VH (SEQ ID NO: 33) and h25H4-4F9vh5 (SEQ ID NO: 35) were designed.
  • H25H4-4F9vk1 (SEQ ID NO: 37), h25H4-4F9vk2 (SEQ ID NO: 39), h25H4-4F9 vk3 (SEQ ID NO: 41), and h25H4-4F9vk1 (SEQ ID NO: 37) based on the sequence h25H4-4F9vk (SEQ ID NO: 4) of the 25H4-4F9 antibody VL.
  • h25H4-4F9vk4 (SEQ ID NO: 43) was designed. Underlining indicates each CDR sequence. The sequences of the variable regions of the heavy chain (Heavy Chain) and light chain (Light Chain) of a humanized antibody designed based on the sequence of the 85H7-2B11 antibody are shown.
  • H85H7-2B11vh1 (SEQ ID NO: 45), h85H7-2B11vh2 (SEQ ID NO: 47), h85H7-2B11vh3 (SEQ ID NO: 49), h85H7-2B11vh4 based on the sequence h85H7-2B11vh (SEQ ID NO: 12) of the VH of the 85H7-2B11 antibody (SEQ ID NO: 51) and h85H7-2B11 vh5 (SEQ ID NO: 53) were designed.
  • H85H7-2B11vk1 (SEQ ID NO: 55), h85H7-2B11vk2 (SEQ ID NO: 57), h85H7-2B11vk3 (SEQ ID NO: 59), and h85H7-2B11vk1 (SEQ ID NO: 55) based on the sequence h85H7-2B11vk (SEQ ID NO: 14) of the VL of the 85H7-2B11 antibody
  • the h85H7-2B11 vk4 (SEQ ID NO: 61) was designed. Underlining indicates each CDR sequence.
  • AM of the 16.7 nM humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) using hCR2-CHOK1 cells
  • the vertical axis shows the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition.
  • the horizontal axis shows the presence or absence of AM addition and the antibody used.
  • the vertical axis indicates the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition.
  • the horizontal axis shows the log value of the antibody concentration (nM).
  • the IC50 value (concentration for 50% inhibition of cAMP in the graph) of each antibody is shown together with the legend.
  • Humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (upper figure), and humanized 85H7-2B11 antibodies (85H7) Perform cell ELISA using 6.7 nM of -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8 (shown below), and perform cell membrane surface protein using It is a graph which shows the result of having analyzed the binding affinity with respect to.
  • the ordinate represents the binding ratio (OD 450%), and the abscissa represents the antibody used.
  • Humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (upper figure), and humanized 85H7-2B11 antibodies (85H7) Binding affinity to cell membrane surface protein by Cell ELISA using -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (lower figure) It is a graph which shows the analyzed result.
  • the ordinate represents the binding ratio (%), and the abscissa represents the log value of the antibody concentration (nM).
  • the antibody concentration at which the binding ratio is 50% was determined from the graph and used as the EC50 value.
  • the EC50 value (nM) of each antibody is shown together with the legend. It is a graph showing the result of having analyzed the epitope of an antibody.
  • the vertical axis represents the amount of bound antibody as OD450 (color intensity of secondary antibody).
  • the horizontal axis shows each peptide derived from the extracellular domain of RAMP2 used for binding.
  • the antibody of the present invention specifically binds to RAMP2.
  • the antibodies of the invention specifically bind to RAMP2 expressed on cell membranes or cell surfaces.
  • the phrase “specifically” (specifically) that an antibody or its immunoreactive fragment recognizes (binds) means that the antibody or its immunoreactive fragment has higher affinity for RAMP2 than the affinity for other proteins or peptides. It means binding with substantially high affinity.
  • "to bind with substantially high affinity” means that the specific protein or peptide of interest can be distinguished from other proteins or peptides and detected by the desired measuring device or method. It means high affinity.
  • substantially high affinity is 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, as the intensity (for example, fluorescence intensity) detected by ELISA or EIA. It may mean that it is 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 100 times or more.
  • binding rate constant (Ka1) in binding between the antibody of the present invention and RAMP2 for example, 1 ⁇ 10 4 Ms ⁇ 1 or more, 1 ⁇ 10 5 Ms ⁇ 1 or more, 5 ⁇ 10 5 Ms ⁇ 1 or more can be mentioned.
  • Kd1 in binding of the antibody of the present invention to RAMP2 for example, 1 ⁇ 10 ⁇ 3 or less and 1 ⁇ 10 4 or less can be mentioned.
  • the binding constant (KD) for binding between the antibody of the present invention and RAMP2 is, for example, 1 ⁇ 10 ⁇ 8 (M) or less, 5 ⁇ 10 ⁇ 8 (M) or less, 1 ⁇ 10 ⁇ 9 (M) or less, It may be 5 ⁇ 10 ⁇ 9 (M) or less.
  • the binding rate constant (Ka1), dissociation rate constant (Kd1), and binding constant (KD) of the antibody in the present specification are the manual provided by the manufacturer using BIACORE (GE Healthcare Biosciences Ltd., BIACORE-X100).
  • the test antibody is allowed to flow, the binding rate constant Ka1 and the dissociation rate constant Kd1 can be measured, and the binding constant KD value can be determined using bivalent fitting.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
  • a "monoclonal antibody” is an antibody that is substantially homogeneous in structure that reacts with a single antigenic determinant.
  • the antibody of the present invention includes non-human animal antibodies, antibodies having the amino acid sequences of non-human animal antibodies and human-derived antibodies, and human antibodies. Examples of non-human animal antibodies include mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, camels, chickens, ducks, etc., preferably hybridomas.
  • the antibody is preferably an animal antibody, more preferably a mouse, rat or rabbit antibody.
  • Examples of the antibody having the amino acid sequence of the non-human animal antibody and the amino acid sequence of the human-derived antibody include human chimeric antibodies and humanized antibodies.
  • chimeric antibody refers to an antibody that is genetically engineered to have the same constant region as a human antibody, which is a constant region of an antibody derived from non-human animals and specifically binding to RAMP2.
  • it is a human-mouse-chimeric antibody (see European Patent Publication EP0125023).
  • the “humanized antibody” refers to a primary structure other than the complementary recognition region (CDR) of the H chain and L chain of an antibody derived from non-human animals and specifically binding to RAMP2 as a primary structure corresponding to a human antibody.
  • CDR complementary recognition region
  • CDR means Kabat et al. ("Sequences of Proteins of Immunological Interest", Kabat, E. et al., U. S. Department of Health and Human Services, 1983) or Chothia et al. (Chothia & Lesk (1987) J. Mol. Biol., 196: 901-917).
  • the “human antibody” refers to a human antibody that is an expression product of a completely human-derived antibody gene, and for example, a monoclonal antibody (a human antibody produced by using a transgenic animal into which a gene involved in antibody production has been introduced) European Patent Publication No. EP0546073) and the like can be mentioned.
  • the antibody of the present invention when used for treatment, prevention, or diagnosis for use by administration into the body, is preferably a human / non-human animal chimeric antibody, a humanized antibody, or a human It is an antibody. Also preferably, the antibody of the present invention is a non-naturally occurring antibody.
  • the immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD or IgY, preferably IgG.
  • IgG immunoglobulin class
  • the antibody of the present invention may be any subclass (IgG1, IgG2, IgG3, or IgG4).
  • the antibody of the present invention is monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (for example, WO 1991/003493), or multispecific (multispecific antibody), It is also good.
  • immunosorbent fragment refers to a protein or peptide containing a portion (partial fragment) of an antibody, wherein the protein or peptide retains the action (immunoreactivity / binding) of the antibody to an antigen.
  • an immunoreactive fragment for example, F (ab ') 2 , Fab', Fab, Fab 3 , single chain Fv (hereinafter referred to as "scFv"), (tandem) bispecific single chain Fv ( sc (Fv) 2 ), single stranded triple body, nanobody, diversity VHH, pentavalent VHH, minibody, (double strand) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity Targeting molecule (DART), triabody (or tribody), tetrabody (or [sc (Fv) 2 ] 2 ), or (scFv-SA) 4 ) disulfide bond Fv (hereinafter referred to as “dsFv”), compact IgG, Heavy chain antibodies, or polymers thereof (Na Maneesh Jain et al., TRENDS in Biotechnology, 25 (7) (2007): 307-316; and Christoph stein et al., Anti
  • an immunoreactive fragment may be any of monospecific, bispecific (bispecific), trispecific (trispecific), and multispecific (multispecific).
  • an antibody is intended to also include immunoreactive fragments of the antibody.
  • the present invention provides 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7-
  • the present invention relates to 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody.
  • Each of these antibodies has a heavy chain (HC) and a light chain (LC) consisting of the amino acid sequences set forth in SEQ ID NOs shown in Table 2.
  • the antibody of the present invention specifically recognizes the conformation of the extracellular domain in RAMP2 on the cell surface.
  • the antibodies of the present invention can be 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7- 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody bound Specifically recognize an epitope that may be present in RAMP2 on the cell surface.
  • the antibody of the present invention does not recognize the primary structure of the amino acid sequence constituting the extracellular region of RAMP2 as an epitope.
  • not recognizing the primary structure of the amino acid sequence as an epitope means that it does not bind to an unfolded (not forming a three-dimensional structure) linear peptide or protein consisting of the amino acid sequence.
  • the antibody of the present invention does not bind to any of the peptides having the amino acid sequences set forth in SEQ ID NOS: 71 to 101, which are amino acid sequences constituting the extracellular region of RAMP2.
  • test antibody whether or not the test antibody "specifically binds (recognizes)" an epitope of interest (for example, an epitope to which the specific antibody described above binds), an antigen, or a peptide depends on the epitope. It can be determined by examining the binding of the antibody to the peptide or protein having the antigen, the antigen, or the peptide, and the binding of the antibody to another peptide, protein, or other antigen. In particular, since the antibody of the present invention specifically recognizes the three-dimensional structure of the extracellular domain in RAMP2 on the cell surface, RAMP2 expressed on the cell surface is used as the protein having the epitope in the method. Thus, the binding specificity can be determined.
  • the test antibody is the epitope, antigen, or peptide of interest It is determined that they specifically bind (recognize).
  • test antibody does not bind to a specific peptide or protein can be determined by examining the binding of the antibody to the peptide or protein.
  • the antibody of the present invention is a 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody , 85H7-2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody or 33H4-1G3 It competes with the antibody (referred to in this paragraph as "25H4-4F9 antibody etc") in binding to RAMP2 (especially RAMP2 on the cell surface).
  • test antibody competes with the 25H4-4F9 antibody etc. for binding to the RAMP2 or not is determined by contacting the labeled 25H4-4F9 antibody etc. with the test antibody to the antigen (RAMP2) Can.
  • RAMP2 antigen
  • a control one in which a labeled 25H4-4F9 antibody alone is brought into contact with an antigen (RAMP2) in the absence of a test antibody can be used.
  • the amount of binding to the antigen such as the labeled 25H4-4F9 antibody in the case of contacting the antigen simultaneously with the test antibody by comparing the amount of labeling such as the labeled 25H4-4F9 antibody bound to the antigen is the absence of the test antibody If the amount of binding of the labeled 25H4-4F9 antibody or the like to the antigen is lower than when it is contacted with the antigen below, it can be determined that the test antibody competes with the 25H4-4F9 antibody or the like used.
  • the method for measuring the binding of an antibody to a protein or peptide is, for example, EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, far western blotting method, homobifunctional crosslinker or heterobifunctional cross Linker-based cross-linking method, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (fluorescence resonance energy transfer) method, BIACORE method, AlphaScreen (registered trademark), AlphaPPI assay such as AlphaLISA (registered trademark)
  • EIA method EIA method
  • ELISA method ELISA method
  • FACS method co-immunoprecipitation method
  • pull-down assay method far western blotting method
  • homobifunctional crosslinker or heterobifunctional cross Linker-based cross-linking method label transfer reaction method
  • interaction mapping method surface plasmon resonance method
  • FRET fluorescence resonance energy transfer
  • BIACORE method BIAC
  • the invention is set forth in any one of VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. And has an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence encoding the amino acid sequence under stringent conditions, and wherein VL is SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, An antibody having an amino acid sequence encoded by a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence encoding an amino acid sequence described in any one of 55, 57, 59, and 61, or an antibody thereof It relates to an immunoreactive fragment.
  • hybridizing under stringent conditions means hybridizing under hybridization conditions commonly used by those skilled in the art. For example, it can be determined by the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).
  • the hybridization conditions are 6 ⁇ SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 ⁇ SSPE (3 M NaCl, 0.2 M NaH 2 PO 4 , 20 mM EDTA ⁇ 2 Na, pH 7.4)
  • the conditions may be such that hybridization is carried out at 42 ° C. and then washing with 0.5 ⁇ SSC at 42 ° C.
  • the present invention relates to a nucleic acid sequence wherein the VH encodes an amino acid sequence as set forth in SEQ ID NO: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, or 53.
  • the present invention 80% of the amino acid sequence of any one of SEQ ID NOs: 2, 12, 19, 27, 29, 31, 31, 33, 35, 45, 47, 49, 51, and 53 Any one amino acid having an amino acid sequence having the above identity and in which VL is selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61
  • the present invention relates to an antibody having an amino acid sequence having 80% or more identity to the sequence, or an immunoreactive fragment thereof.
  • the identity of the amino acid sequence means the percentage (%) of the number of identical amino acids in the amino acid sequence to be compared between the two types of proteins, for example, using a known program such as BLAST or FASTA. It can be decided.
  • any one amino acid sequence wherein VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And an amino acid sequence having an identity of 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, and VL has a sequence of SEQ ID NO: 4, 14, 21, 37, 39, 41 43, 55, 57, 59, and 61, and an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity with any one amino acid sequence selected from It may be an antibody or an immunoreactive fragment thereof.
  • any one amino acid sequence wherein VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And an amino acid sequence having an identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, and in each of which, VL has SEQ ID NO: 4, 14, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or any one amino acid sequence selected from 21, 37, 39, 41, 43, 55, 57, 59, and 61 An antibody having an amino acid sequence having an identity of 99% or more or an immunoreactive fragment thereof.
  • the present invention relates to the amino acid sequences of heavy chain (VC) and light chain (LC) described in SEQ ID NO: listed in Table 2 herein, or the amino acid sequences of VH and VL, or CDRH1-3. It is an antibody having a combination of amino acid sequences of CDRL1-3.
  • An antibody having an amino acid sequence having 80% or more identity with a specific amino acid sequence, or an immunoreactive fragment thereof preferably has CDRHs and CDRLs described below.
  • the invention provides an antibody or heavy chain comprising a heavy chain complementarity determining region (CDRH) 3 consisting of the amino acid sequence of LX 1 IX 2 DX 3 YX 4 YX 5 MX 6 X 7 (SEQ ID NO: 24) It relates to the immunoreactive fragment.
  • CDRH heavy chain complementarity determining region
  • X 1 is G or M
  • X 2 is F or Y
  • X 3 is A or V
  • X 4 is absent or H
  • X 5 is A or V
  • X 6 is D or E
  • X 7 is Y or N.
  • the present invention is an antibody or immunoreactive fragment thereof comprising a heavy chain having a CDRH3 consisting of the amino acid sequence of LMIFDAYYAMD (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16).
  • CDRH3 is important in determining its specificity (Xu JL et al., Immunity. 2000 Jul; 13 (1): 37-45.).
  • CDRH 3 preferably has the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16).
  • the antibody or immunoreactive fragment thereof of the present invention has a CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5).
  • the antibody of the present invention or the immunoreactive fragment thereof comprises the amino acid sequence of RX 8 NPYNGDX 9 X 10 YX 11 QKFX 12 G (SEQ ID NO: 66) or RX 8 NPYNGDX 9 X 10 YNQ KFK (SEQ ID NO: 23) And CDRH2.
  • X 8 is N or I
  • X 9 is S or T
  • X 10 is I, F or L
  • X 11 is A or N
  • X 12 is Q or K.
  • the CDRRH2 is RNNPYNGDSIYNQKFKG (SEQ ID NO: 6), RINPYNGDTFYNQKFKG (SEQ ID NO: 15), RINPYNGDTLYNQKFKG (SEQ ID NO: 22), RNNPYNGDSIYNEKF QG (SEQ ID NO: 62), RNNPYNGDSIYAEKF QG (SEQ ID NO: 63), It has the amino acid sequence of (SEQ ID NO: 65).
  • the antibody of the present invention or an immunoreactive fragment thereof has a light chain complementarity determining region (CDRL) 1 consisting of the amino acid sequence of X 13 ASQDIRNYLN (SEQ ID NO: 69).
  • X 13 is Q or R.
  • CDRL1 preferably has the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO: 67).
  • the antibody of the present invention or the immunoreactive fragment thereof has a CDRL2 consisting of the amino acid sequence of YTSRLX 14 X 15 (SEQ ID NO: 70).
  • X 14 is E or H
  • X 15 is S or T.
  • CDRL2 preferably has the amino acid sequence of YTSRLHS (SEQ ID NO: 9) or YTSRLET (SEQ ID NO: 68).
  • the antibody of the present invention or the immunoreactive fragment thereof has a CDRL3 consisting of the amino acid sequence of QQDSKX 16 PWT (SEQ ID NO: 25).
  • X 16 is H or N.
  • CDRL 3 preferably has the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT (SEQ ID NO: 17).
  • the antibody of the present invention has a combination of heavy chain CDRs of any one of the following (i) to (vii): (I) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7; (Ii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 62, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7; (Iii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 63, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7; (Iv) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 5
  • the antibody of the present invention may have a combination of light chain CDRs of any one of the following (a) to (f):
  • the invention includes antibodies or immunoreactive fragments thereof having the following combinations of CDRs:
  • amino acids are represented by a single letter code.
  • A is alanine
  • L leucine
  • R is arginine
  • K is lysine
  • N is asparagine
  • M is methionine
  • D is aspartic acid
  • F is phenylalanine
  • C cysteine
  • P proline
  • Q is glutamine
  • S represents serine
  • E represents glutamic acid
  • T represents threonine
  • G represents glycine
  • W represents tryptophan
  • H represents histidine
  • Y tyrosine
  • I isoleucine
  • V valine.
  • the antibody of the present invention and its immunoreactive fragment have a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. Whether or not the test antibody or its immunoreactive fragment has a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro was precultured for several hours in an appropriate medium By adding a test antibody or its immunoreactive fragment to vascular endothelial cells, adding AM to a final concentration of 50 to 200 nM, and observing proliferation of vascular endothelial cells after culturing for 2 to 7 days It can be carried out.
  • the proliferation of vascular endothelial cells may be performed by counting the number of cells, or by measuring OD450 as an indicator of the number of cells.
  • the test antibody or the immunoreactive fragment thereof is determined to have a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro.
  • the number of vascular endothelial cells cultured without adding the test antibody or its immunoreactive fragment is 100, and the number of vascular endothelial cells cultured in the presence of the test antibody or its immunoreactive fragment is 50.
  • the concentration of the test antibody or immunoreactive fragment thereof may be determined as an IC50 value (M).
  • the antibody of the present invention may have an IC50 value of 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1.6 nM or less Good.
  • the lower limit value of the IC 50 value need not be particularly defined, for example, 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, 0.5 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more Or 0.8 nM or more.
  • the IC50 value may be in the range of any combination of these upper and lower limits, and for example, 0.1 to 35 nM, 0.8 to 32.7 nM, 0.2 to 30 nM, 0.1.
  • test antibody or the immunoreactive fragment thereof may have a growth inhibitory effect on vascular endothelial cells in the vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. Good.
  • the antibody of the present invention or an immunoreactive fragment thereof according to the present invention has cAMP in animal cells by addition of adrenomedullin in addition to the growth inhibitory action of vascular endothelial cells in the vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. It has the effect of suppressing production.
  • test antibody or the immunoreactive fragment thereof has the effect of suppressing cAMP production by animal cells by addition of adrenomedullin depends on the animal cells producing cAMP by addition of adrenomedullin (for example, hCR2-CHOK1 cells) Is cultured overnight in an appropriate medium, the test antibody or its immunoreactive fragment is added, and a final concentration of 50 to 300 pM of AM is added, and this can be determined by measuring cAMP.
  • cAMP can be measured using a commercially available kit (eg, LANCE Ultra cAMP kit (Perkinelmer) and the like).
  • the antibody or the immunoreactive fragment thereof When the amount of cAMP produced by cells containing the test antibody or its immunoreactive fragment is low as compared to cAMP produced by cells without added antibody, the antibody or the immunoreactive fragment thereof is treated with animal cells by addition of adrenomedullin. It is determined to have the effect of suppressing cAMP production. In addition, when the cAMP produced by cells cultured without the addition of the test antibody or its immunoreactive fragment is 100, the cAMP produced by the cells cultured in the presence of the test antibody or its immunoreactive fragment is 50 The concentration of the test antibody or the immunoreactive fragment thereof may be determined as an IC 50 value (M).
  • the antibody of the present invention has an IC50 value of 2 nM or less, 1.5 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.15 nM or less May be
  • the lower limit value of the IC50 value need not be particularly defined, but may be, for example, 0.01 nM or more, 0.02 nM or more, or 0.03 nM or more.
  • the IC50 value may be in the range of any combination of these upper and lower limit values, and, for example, 0.01 to 2 nM, 0.01 to 1.5 nM, 0.01 to 1 nM, 0.1.
  • test antibody or the immunoreactive fragment thereof may be 01 to 0.5 nM, 0.03 to 1.35 nM, or 0.03 to 0.15 nM. Moreover, in the case of these IC50 values, it may be determined that the test antibody or the immunoreactive fragment thereof has an effect of suppressing cAMP production by animal cells by addition of adrenomedullin.
  • the antibody or immunoreactive fragment thereof of the present invention binds in vitro to cells co-expressing human CRLR and human RAMP2.
  • a cell co-expressing human CRLR and human RAMP2 is a stably expressing cell obtained by introducing human CRLR and human RAMP2 into a cell gene, and a cell co-expressing human CRLR and human RAMP2
  • a stably expressing cell line hCR2-CHOK1 or hCR2-
  • CHO-K1 or hCR2- obtained by gene transfer of human CRLR and human RAMP2 into hamster ovary cells (CHO-K1) or NIH3T3 cells. NIH 3 T 3).
  • a cell co-expressing human CRLR and human RAMP2 can be obtained by integrating human CRLR (NM_005795) and human RAMP2 (NM_005854) into a vector for gene transfer in tandem, and transferring the gene into a target cell.
  • a stable expression strain can be obtained by selective culture in the presence of a drug corresponding to a drug resistance gene previously inserted into a vector.
  • the antibody of the present invention may have a binding affinity (EC50) of 3.5 nM or less in a Cell ELISA test using cells co-expressing human CRLR and human RAMP2.
  • EC50 binding affinity
  • the Cell ELISA test can be performed by the following method. Cells co-expressing human CRLR and human RAMP2 in an appropriate medium are added to a 96-well culture plate at 1 ⁇ 10 3 to 1 ⁇ 10 5 cells / well and cultured overnight, and then 4% paraformaldehyde solution is added. The cells are fixed by standing at 4 ° C. for several dozen minutes, and after washing, blocking treatment with 1% BSA-PBS is performed for 1 hour or more.
  • each test antibody is added in appropriate serial dilutions, reacted with a labeled anti-IgG antibody as a secondary antibody, reacted with a reagent that matches the label, and measure the luminescence intensity and coloring intensity by the label.
  • the amount of binding can be determined by EC50 values can be determined as the antibody concentration that binds 50% of the maximal binding.
  • the antibody of the present invention has an EC50 value of 4 nM or less, 3.5 nM or less, 3.19 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, 1 nM or less, or 0.5 nM or less It may be.
  • the lower limit value of the EC50 value need not be particularly defined, but may be, for example, 0.01 nM or more, 0.03 nM or more, 0.05 nM or more, or 0.14 nM or more.
  • the EC50 value may be in the range of any combination of these upper and lower limit values, for example, 0.01 to 4 nM, 0.03 to 3 nM, 0.05 to 2 nM, or 0.14. It may be ⁇ 3.19 nM.
  • it may be determined that the test antibody or the immunoreactive fragment thereof binds to cells co-expressing human CRLR and human RAMP2 in vitro.
  • nucleic acid molecule in another aspect, relates to a nucleic acid molecule comprising a polynucleotide encoding an antibody of the invention as described above or an immunoreactive fragment thereof.
  • the nucleic acid molecule of the present invention is any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 as VH.
  • nucleic acid molecule may have a polynucleotide encoding the amino acid sequence described in
  • the nucleic acid molecule of the present invention is a nucleic acid molecule having a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as VH and SEQ ID NO: 4 as VL; amino acids of SEQ ID NO: 27 as VH and SEQ ID NO: 37 as VL
  • nucleic acid molecule having a polynucleotide encoding the sequence a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 29 as VH and SEQ ID NO: 37 as VL; described in SEQ ID NO: 39 as VH
  • amino acids of SEQ ID NO: 27 as VH and SEQ ID NO
  • the nucleic acid molecule of the present invention may comprise a polynucleotide having the following sequence: the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO: 3; the nucleotide sequence set forth in SEQ ID NO: 26 And the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28 and the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28; and the nucleotide sequence set forth in SEQ ID NO: 38; The nucleotide sequence set forth in SEQ ID NO: 30 and the nucleotide sequence set forth in SEQ ID NO: 38; the nucleotide sequence set forth in SEQ ID NO: 30 and the nucleotide sequence set forth in SEQ ID NO: 40; the nucleotide sequence set forth in SEQ ID NO: 32; Nucleotide
  • the invention includes a vector comprising the nucleic acid molecule.
  • a vector is not particularly limited as long as it can be used to express an antibody, and can be selected according to the host using an appropriate viral vector or plasmid vector.
  • the invention relates to a host cell containing said vector.
  • the host cell is not particularly limited as long as it can be used for antibody expression, and mammalian cells (mouse cells, rat cells, rabbit cells, human cells, etc.), yeast, microorganisms (E. coli etc.) It can be mentioned.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described antibody of the present invention or an immunoreactive fragment thereof as an active ingredient.
  • the target disease of the pharmaceutical composition of the present invention is a disease or disorder in which angiogenesis contributes to onset or exacerbation, for example, cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, Diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis can be mentioned.
  • the pharmaceutical composition of the present invention can be used as a preventive, a therapeutic, a progression inhibitor or a remedy for these diseases or disorders.
  • the present invention relates to the use of the antibody of the present invention or an immunoreactive fragment thereof for producing the above-mentioned pharmaceutical composition.
  • the pharmaceutical composition of the present invention may be any oral or parenteral preparation that can be administered to patients.
  • compositions for parenteral administration include injections, nasal drops, suppositories, patches, ointments and the like. Preferably, it is an injection.
  • the dosage form of the pharmaceutical composition of the present invention can include, for example, a solution or a lyophilized preparation.
  • a solubilizing agent such as propylene glycol or ethylene diamine, a buffer such as phosphate, a tonicity agent such as sodium chloride or glycerin, sulfite Additives such as stabilizers such as phenol, preservatives such as phenol, soothing agents such as lidocaine (see “Handbook of Pharmaceutical Supplements” Pharmaceutical Daily, Inc., “Handbook of Pharmaceutical Excipients Fifth Edition” APhA Publications) .
  • examples of the storage container include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and infusion bags.
  • the antibody of the present invention can be used as an immunogen with cells expressing human CRLR and human RAMP2 as an immunogen, if necessary, an immunostimulant (eg, mineral oil or aluminum precipitate, heat-killed bacteria or lipopolysaccharide, complete adjuvant with Freund's Or with Freund's incomplete adjuvant etc.) by immunizing a non-human animal that produces an immune response.
  • an immunostimulant eg, mineral oil or aluminum precipitate, heat-killed bacteria or lipopolysaccharide, complete adjuvant with Freund's Or with Freund's incomplete adjuvant etc.
  • the animal to be immunized is not particularly limited as long as it is an animal capable of producing a hybridoma such as mouse, rat, hamster, guinea pig, rabbit, dog, monkey, sheep, goat, chicken, duck, etc.
  • the cells to be the immunogen are desirably cells of the same species as the immunized animal in order to suppress antibody production to other than the target antigen.
  • the administration of the immunogen to an animal may be, for example, 1 ⁇ 10 6 cells once or several times at appropriate intervals (usually, 1 to 6 immunizations in total once every 1 to 6 weeks), subcutaneously It can be performed by injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or footpad injection.
  • the antibody of the present invention can be obtained by purification from serum of an animal exhibiting a sufficient antibody titer.
  • the monoclonal antibody can be obtained by culturing a hybridoma obtained by fusing a myeloma cell (myeloma cell) with an antibody-producing cell obtained from the immunized animal immunized by the above method.
  • Examples of the fusion method include the method of Milstein et al. (Galfre, G. & Milstein, C. (1981) Methods Enzymol., 73: 3-46).
  • the antibody-producing cells to be used can be collected from the spleen, pancreas, lymph nodes, and peripheral blood of mice or rats which have been immunized according to the above-mentioned method and their sera show a sufficient antibody titer.
  • the myeloma cells used are, for example, cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans, and are not particularly limited as long as they can be proliferated in vitro.
  • cells for example, P3-X63Ag8 (X63) (Nature, 256, 495, 1975), P3 / NS1 / 1-Ag4-1 (NS1) (Eur. J. Immunol., 6, 292, 1976).
  • P3X63Ag8U1 (P3U1) (Curr.Top.Microbiol.Immunol., 81,1,1978), P3X63Ag8.653 (653) (J.
  • clones that bind to RAMP2 are selected by Cell ELISA using cells coexpressing human CRLR and human RAMP2 (for example, hCR2-CHOK1 cells).
  • the selected clones are subjected to single-cellification by repeating limiting dilution 1 to 5 times, and among the antibodies produced by the single-cellified clones, candidate antibodies selected by Cell ELISA are further selected.
  • signal inhibitory antibodies are screened using as an index whether or not the increase in intracellular cAMP of cells (for example, hCR2-CHOK1 cells) co-expressing human CRLR and human RAMP2 by addition of AM is suppressed.
  • An antibody capable of inhibiting the growth of HUVEC by AM addition can be screened as a test for inhibiting the biological activity of the selected signal-inhibiting antibody, and an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action can be obtained.
  • the resulting antibodies can be purified to homogeneity.
  • separation and purification methods used in conventional proteins can be used.
  • antibodies can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988).
  • columns used for affinity chromatography include a protein A column and a protein G column.
  • RAMP2 or human CRLR and human RAMP2
  • immobilized columns ion exchange chromatography, hydrophobic interaction chromatography and the like can also be used.
  • antibodies that bind to RAMP2 can be obtained by utilizing an antibody phage library (Tomizuka et al., Nature Genet., 15, 146-156 (1997)).
  • an antibody phage library for example, cells expressing human CRLR and human RAMP2 were immobilized on a solid phase, reacted with a phage antibody library, washed to remove non-bound phage, and then bound. By recovering the phage, the desired clone can be obtained (panning).
  • the amino acid sequence of the antibody of interest or its immunoreactive fragment is designed with reference to the amino acid sequence described herein, and a DNA encoding the designed amino acid sequence is prepared and incorporated into an expression vector,
  • An antibody obtained by introducing and expressing the vector in a suitable host cell can be obtained by screening an antibody with high specificity to RAMP2 according to the method described above.
  • the antibody of the present invention is a human chimeric antibody
  • DNAs encoding VH and VL of non-human animal monoclonal antibodies that specifically recognize RAMP2 are prepared, and they are expressed by binding to human immunoglobulin constant region cDNA. It can be obtained by incorporating it into a vector and introducing the vector into an appropriate host cell and expressing it (Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984). .
  • the amino acid sequences encoding the CDRs of VH and VL of a non-human animal monoclonal antibody that specifically recognizes RAMP2 are transplanted into the framework regions (FR) of VH and VL of human antibody Obtained by constructing a DNA encoding the isolated V region, combining the constructed DNA with a constant region cDNA of a human-derived immunoglobulin, incorporating it into an expression vector, and introducing the vector into an appropriate host cell for expression (L. Rieohmann et al., Nature, 332, 323, 1988: Kettleborough, CA. et al., Protein Eng., 4, 773-783, 1991; Clark M., Immunol.
  • the CDRs of the non-human animal monoclonal antibody are the amino acid sequences predicted from the DNA sequences encoding the VH and VL of the non-human animal monoclonal antibody obtained by the above-mentioned method, and the entire amino acid sequences of VH and VL of the known antibody Can be obtained by comparing.
  • the amino acid sequences of known antibodies can be obtained, for example, from the amino acid sequences of antibodies registered in databases such as Protein Data Bank.
  • the FR of the humanized antibody is not particularly limited as long as the antibody after transplantation exerts the effect of the present invention, but preferably, the variable region of the humanized antibody (hereinafter referred to as "V region") is It is a human antibody FR having a steric structure similar to the V region of the non-human animal monoclonal antibody from which the CDRs are derived, or a human antibody FR having high identity to the amino acid sequence of the FR of the non-human animal monoclonal antibody used.
  • V region the variable region of the humanized antibody
  • a part of the amino acids constituting the FR derived from the human antibody are optionally non-human animal monoclonal antibodies from which the CDRs are derived (SEQ ID NO: 5) may be substituted by the FR sequence of (See, eg, Queen et al., US Pat. No. 5,855,089).
  • the DNA sequence encoding the V region of the humanized antibody to be used is designed as a DNA sequence corresponding to the amino acid sequence obtained by combining the amino acid sequence of CDR of non-human animal monoclonal antibody and the amino acid sequence of FR of human antibody.
  • the DNA encoding the V region of a humanized antibody can be prepared based on the designed DNA sequence by methods known to those skilled in the art.
  • Human antibodies can be obtained, for example, by using a human antibody phage library or a human antibody-producing transgenic mouse (Tomizuka et al., Nature Genet., 15, 146-156 (1997)).
  • a human antibody phage library for example, cells expressing human CRLR and human RAMP2 are immobilized on a solid phase, reacted with a phage antibody library, and washed to remove non-bound phage, and then bound.
  • the desired clones can be obtained (panning) by recovering the isolated phages.
  • a human antibody-producing transgenic mouse is a mouse in which an endogenous immunoglobulin (Ig) gene has been knocked out, into which an Ig gene of a human antibody has been introduced.
  • RAMP2 is specifically expressed by immunizing an antigen (preferably, a cell expressing human CRLR and human RAMP2) according to the above-described antibody production method of the present invention, using a human antibody-producing transgenic mouse as an immunized animal. Human antibodies that recognize can be obtained.
  • the nucleic acid of the present invention may be cloned from a hybridoma that produces the antibody obtained in the above, or, by appropriately designing a nucleic acid sequence based on the amino acid sequence of the antibody or its immunoreactive fragment obtained in the above. You can get it.
  • the vector of the present invention can be obtained by incorporating the obtained nucleic acid into a vector suitable for expression as appropriate.
  • the vector of the present invention may contain, in addition to the nucleic acid of the present invention, regions necessary for expression (promoter, enhancer, terminator, etc.).
  • the host cell of the present invention can be obtained by introducing the vector of the present invention into an appropriate cell line (for example, animal cells, insect cells, plant cells, microorganisms such as yeast and E. coli).
  • the antibody of the present invention or the immunoreactive fragment thereof can be used as a pharmaceutical composition by purifying it as necessary and then formulating it according to a conventional method.
  • the present invention also includes the use of the antibody of the present invention or an immunoreactive fragment thereof for producing a pharmaceutical composition.
  • the present invention includes the use of the antibody of the present invention or an immunoreactive fragment thereof for the treatment or prevention of a disease or disorder in which angiogenesis contributes to the onset or exacerbation.
  • the present invention provides a method for treating a disease or disorder in which angiogenesis contributes to the onset or exacerbation, comprising administering to a patient in need thereof an effective amount of the antibody of the present invention or an immunoreactive fragment thereof It relates to the prevention method.
  • the pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention can be used as an injection, and intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravitreal injection, drip injection Including dosage forms such as agents.
  • Such injections can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody etc. in a sterile aqueous or oily liquid usually used for injections, according to a known method.
  • aqueous solution for injection for example, physiological saline, glucose, sucrose, mannitol, isotonic solution containing other adjuvants, etc.
  • solubilizers such as alcohol (eg, ethanol, etc.) ), Polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc.
  • suitable solubilizers such as alcohol (eg, ethanol, etc.) ), Polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc.
  • suitable solubilizers such as alcohol (eg, ethanol, etc.)
  • Polyalcohols eg, propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) ad
  • a freeze-dried preparation is prepared by adding an appropriate excipient to the antibody of the present invention or the immunoreactive fragment thereof, and when used, it is dissolved in water for injection, physiological saline or the like to prepare an injection solution.
  • an appropriate excipient to the antibody of the present invention or the immunoreactive fragment thereof, and when used, it is dissolved in water for injection, physiological saline or the like to prepare an injection solution.
  • oral administration of proteins such as antibodies is considered to be difficult because it is degraded by the digestive tract, but there is also the possibility of oral administration due to the inventive idea of antibody fragments and modified antibody fragments and dosage forms.
  • preparations for oral administration include capsules, tablets, syrups, granules and the like.
  • compositions of the present invention are preferably formulated in dosage unit forms that are compatible with the dose of the active ingredient.
  • dosage forms of such dosage units include injections (ampoules, vials, prefilled syringes), and generally 5 to 500 mg, 5 to 100 mg, 10 to 250 mg of the antibody of the present invention or its immunity per dosage unit dosage form. It may contain a reactive fragment.
  • Administration of the antibody or pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention may be local or systemic. There is no particular limitation on the method of administration, and as described above, it is administered parenterally or orally. Parenteral administration routes include injection or infusion into the eye, subcutaneously, intraperitoneally, in the blood (intravenous or intraarterial) or spinal fluid, and preferably in the intraocular or blood. It is.
  • the pharmaceutical composition (therapeutic or prophylactic agent) of the present invention may be administered temporarily, or may be administered continuously or intermittently. For example, administration can be continuously performed for 1 minute to 2 weeks.
  • the antibodies of the present invention may be administered alone or in combination with other agents.
  • the antibody of the present invention may be an antibody-drug conjugate (ADC) bound to the other agent.
  • ADC antibody-drug conjugate
  • the other drug a drug or the like which is known to have a therapeutic effect on a disease or disorder in which angiogenesis contributes to onset or exacerbation can be used.
  • the dose of the pharmaceutical composition of the present invention is not particularly limited as long as a desired therapeutic effect or prophylactic effect can be obtained, and can be appropriately determined depending on symptoms, sex, age, and the like.
  • the dose of the pharmaceutical composition of the present invention can be determined, for example, using the therapeutic or prophylactic effect of a disease or disorder in which angiogenesis contributes to the onset or deterioration.
  • the amount of the active ingredient of the pharmaceutical composition of the present invention is usually 0.
  • similar amounts can be administered. If the condition is particularly severe, the dose may be increased or the number of administrations may be increased depending on the condition.
  • cells used as an antigen were prepared by the following method.
  • Human CRLR (NM — 005795) and human RAMP 2 (NM — 005854) were tandemly incorporated into pCHO 1.0 vector of Thermo, and transfected into NIH-3T3 cells (ATCC: CRL-1658) using Lipofectamine 2000 (Thermo) (hCR2) -NIH 3T3).
  • D-MEM medium Wi-MEM medium (Wako Pure Chemical Industries, Japan) containing 10 ⁇ g / mL Puromycin and 100 nM Methotrexate.
  • the resulting hCR2-NIH3T3 cells were prepared to 1 ⁇ 10 6 cells / MRL / lpr mouse, and intraperitoneally administered once a week for 5 weeks to immunize with antigen.
  • the sera before and after the immunization were collected, and an increase in antibody-specific antibody titer was confirmed by Cell ELISA. After confirming the increase in antibody titer, 1 ⁇ 10 6 cells of hCR2-NIH3T3 cells were again immunized as an antigen as a final immunization.
  • SP2ab cell line was used as mouse myeloma cells, fused with cells derived from spleen and lymph node by PEG method, and seeded in 96 well culture plate. The day after fusion, HAT-containing 10% FBS-TIL medium was added, and the medium was changed every three days to prepare a hybridoma.
  • hCRLR / hRAMP2-expressing CHO-K1 cells prepared by the same method as NIH-3T3 cells were seeded on a 96-well culture plate. After immobilization with 4% PFA and blocking with 1% BSA-PBS, the culture supernatant was added to react with Goat Anti-mouse-IgG-HRP (SouthernBiotech) as a secondary antibody. The positive wells were picked up by coloring with TMB solution (eBioscience) and used as candidate hybridomas.
  • Example 2 cAMP Suppression Test
  • the hCR2-CHOK1 cells were used to conduct a cAMP suppression test of the obtained anti-RAMP2 antibody.
  • As a control antibody a known commercially available antibody (Novus bio, 4E5, Cat No. NBP2-01853; Abnova, 2F5, Cat No. H00010266-M05; and Santacruz, B5, Cat No. sc-365240) was used. The half area 96 well plate was used for the measurement.
  • FIG. 1 shows the ratio of the cAMP concentration in each antibody-added group when the cAMP concentration in the case of no antibody addition is 100.
  • the control antibody hardly suppressed cAMP production, whereas the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody suppressed cAMP production to the same level as that in the group without AM.
  • the relationship between the concentration of each antibody and the cAMP suppression rate is shown in FIG.
  • the IC50 values for cAMP inhibition of the 25H4-4F9, 85H7-2B11 and 33H4-1G3 antibodies were 0.15 nM, 0.03 nM and 0.045 nM, respectively.
  • Example 3 Growth inhibition test of HUVEC HUVEC (KURABO) in Humedia-EG2 medium containing 2% FBS excluding growth factor was seeded at 2500 cells per well on 96 well culture plate and adhered by pre-incubation for 3 hours The Each antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5 so that the final concentration is 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM. And B5) were added, and a final concentration of 100 nM of AM was added to observe HUVEC proliferation after 96 hours, thereby conducting a HUVEC proliferation inhibition test.
  • Each antibody 25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5 so that the final concentration is 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM
  • FIG. 3 shows the percentage of OD450 (number of HUVEC cells) in each antibody-added group, where OD450 (number of HUVEC cells) in the case of no antibody addition is 100.
  • the control antibody hardly inhibited the proliferation of HUVEC, whereas the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody inhibited the proliferation to about 30%.
  • the relationship between the concentration of each antibody and the HUVEC growth inhibition rate is shown in FIG.
  • the IC50 values for inhibition of HUVEC proliferation of the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were 1.2 nM, 0.8 nM, and 1.6 nM, respectively.
  • RNA is extracted from cell pellets of each antibody-producing hybridoma using SV-Total RNA Isolation kit (Promega), and cDNA is synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche), The antibody sequences of the hybridomas were analyzed.
  • Wobble primers degenerate primers
  • the amino acid sequence was determined from the obtained base sequence, and the CDR region was specified by the KABAT method.
  • VH The determined VH (SEQ ID NO: 2) and VL (SEQ ID NO: 4) of 25H4-4F9 antibody, VH (SEQ ID NO: 12) and VL (SEQ ID NO: 14) of 85H7-2B11 antibody, and VH (sequence of 33H4-1G3 antibody)
  • VH sequence of 33H4-1G3 antibody
  • the sequences of No. 19) and VL (SEQ ID No. 21) are shown in FIG.
  • each determined CDR sequence is shown below.
  • 25H4-4F9 antibody heavy chain CDR sequences CDRH1: GYFMN (SEQ ID NO: 5)
  • CDRH2 RNNPYNGDSIYNQKFKG (SEQ ID NO: 6)
  • CDRH3 LMIFDAYYAMDY (SEQ ID NO: 7)
  • CDRH2 RINPYNGDTFYNQKFKG (SEQ ID NO: 15)
  • CDRH3 LGIYDV YHY VMEN (SEQ ID NO: 16) 85H7-2B11 Antibody Light Chain CDR Sequences: CDRL1:
  • Example 5 Confirmation of anti-RAMP2 specific antibody It was confirmed by Cell ELISA that the obtained antibody was anti-RAMP2 specific.
  • human CRLR and human RAMP2 were transfected into hamster ovary cells (CHO-K1). The binding properties of the antibodies were compared between cells transfected with human CRLR and human RAMP2 and cells transfected with human CRLR alone.
  • Example 6 Binding affinity analysis for cell membrane surface protein by Cell ELISA 96-well culture plate (BD-FALCON) with 1 ⁇ 10 4 cells / well of hCR2-CHOK1 cells in DMEM / F-12 medium containing 10% FCS And cultured overnight. Equal volume of 4% paraformaldehyde solution (MUTO Chemical) was added, cells were fixed by standing at 4 ° C for 20 minutes, and after washing with PBST, blocking treatment was performed for 1 hour or more with 1% BSA-PBS .
  • 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1, and 4E5, 2F5, and B5, which are commercially available anti-RAMP2 antibodies, were used.
  • 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 clearly showed superior binding activity as compared with the commercially available antibodies 4E5, 2F5, and B5. .
  • the vertical axis is calculated as a ratio to the maximum binding amount, where the maximum binding amount (OD value) of the antibody obtained in the same test is 100%.
  • the EC 50 representing binding affinity of each antibody was 0.40 nM for 25H4-4F9, 0.14 nM for 85H7-2B11, and 0.30 nM for 33H4-1G3. None of the commercially available antibodies can draw a normal binding curve due to their low binding ability, and EC50 values are 37.7 nM or more for 4E5, and 66.7 nM or more for 2F5 and B5.
  • Example 7 Binding analysis to cell membrane surface proteins by FACS Each antibody (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1, and 4E5, 2F5, which are commercially available anti-RAMP2 antibodies, And B5) hCR2-CHOK1 cells' binding ability to membrane surface RAMP2 protein was measured with a flow cytometer (ACEA). 3 ⁇ 10 5 cells of exfoliated hCR2-CHOK1 cells and 50 ⁇ L of 3 ⁇ g / mL antibody solution were added to a 96-well V-bottom plate (Thermo Fishcer Scientific) and allowed to stand on ice for 1 hour to allow antibody to bind.
  • ACEA flow cytometer
  • Example 1 As shown in FIG. 8, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 were all bound to cell membrane surface proteins. Among the commercially available antibodies, 4E5 was found to bind to cell membrane surface proteins, but the commercially available antibodies 2F5 and B5 were not observed to bind.
  • Example 8 Competitive analysis of epitope by FACS Biotinylated 85H7-2B11 and non-biotinylated antibodies (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and commercial antibody 4E5)
  • the competition test for the antigen was performed by simultaneously reacting hCR2-CHOK1 with hCR2-CHOK1.
  • the 85H7-2B11 antibody was biotinylated using Biotin Labeling kit-NH 2 (Dojindo) according to the manufacturer's manual.
  • Example 9 Design of Humanized Antibody A humanized antibody (humanized 25H4-4F9, humanized 85H7-2B11 antibody) was designed for the 25H4-4F9 and 85H7-2B11 antibodies.
  • the design of the amino acid sequence was performed according to the method (US Patent 5,766, 886; MODIFIED ANTIBODY VARIABLE DOMAINS, expiration of patent term) devised by Xoma corporation.
  • Amino acid sequences of 25H4-4F9 mouse antibody heavy chain and VL or amino acids of 85H7-2B11 mouse antibody heavy chain and VL were used for homology search with human immunoglobulin GERMLINE database (NCBI: IGBLAST) and amino acids showing the highest homology Sequence (25H4-4F9 heavy chain: IGHV1-69-2 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 25H4-4F9 light chain: IGKV1-33 * 01 and IGKJ2 * 01, 85H7-2B11 heavy chain: IGHV1- 46 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 85H7-2B11 light chains: IGKV1-33 * 01 and IGKJ2 * 01, except that the amino acid sequences of the 25H4-4F9 and 85H7-2B11 VL are excluded from one place Of the same sequence, It was selected as a work) and the humanized antibody framework.
  • Sequences of the designed 25H4-4F9 VH and VL are shown in FIG. 10, and sequences of the designed 85H7-2B11 VH and VL are shown in FIG.
  • combinations of heavy chain and light chain and VH, VL and CDR sequences in the produced humanized antibody are shown in Table 2 below.
  • Example 10 Preparation of Recombinant Humanized Antibody Gene sequence encoding heavy chain for preparation of recombinant antibody (SEQ ID NOs: 102, 104, 106, 108, 110, 120, 122, 124, 126, and 128) and The gene sequence encoding the light chain (SEQ ID NOS: 112, 114, 116, 118, 130, 132, 134 and 136) was cloned into the mammalian expression vector pcDNA3.4 (Thermo). The heavy and light chain cloned vectors were mixed at a weight ratio of 1: 1, and 10 ⁇ g to 30 ⁇ g of vector DNA was used to transfect mammalian cell CHO-S (Thermo).
  • the resin-bound recombinant antibody was eluted with 0.1 M Glycine-HCl pH 3.2 Elution buffer and neutralized with 1/50 volume of 1 M Tris-HCl pH 9.0. After neutralization, Buffer was replaced with phosphate buffer using a 30 kDa ultrafiltration membrane (Amicon Ultra: Millipore), and the concentration of recombinant antibody was determined by measuring the absorbance at 280 nm.
  • Example 11 cAMP Suppression Test of Humanized Antibody
  • the hCR2-CHOK1 cells were used to conduct a suppression test of cAMP of humanized antibody.
  • For measurement as in the measurement of mouse antibody activity, use of Perkinelmer's Half area 96 well plate, and using 2500 cells of hCR2-CHOK1 cells in D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd., 10% FBS) After overnight culture in Japan, each antibody (humanized 25H4-4F9, humanized 85H7-2B11, 4E5, 2F5, and B5) was added, and human AM with a final concentration of 150 pM (Peptide Research Laboratories, Japan) was added.
  • cAMP was measured using the LANCE Ultra cAMP kit (Perkinelmer) according to the manufacturer's manual. As a control, cAMP production by hCR2-CHOK1 cells without antibody addition / AM addition and without antibody addition / AM addition was similarly measured.
  • FIG. 12 and FIG. 13 show the cAMP inhibition rate when 16.7 nM of humanized 25H4-4F9 antibody and humanized 85H7-2B11 antibody were added, respectively.
  • the cAMP inhibition rate was shown as a ratio of the cAMP concentration of each antibody-added group when the cAMP concentration in the case of no antibody addition was 100.
  • the commercially available antibodies (4E5, 2F5, and B5) used as controls hardly suppressed cAMP production, whereas the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were equivalent to the non-AM added group. It suppressed cAMP production.
  • the IC50 values for cAMP inhibition of the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were 0.17 nM for h25H4-4F9v1, 0.14 nM for h25H4-4F9v2, and 0.26 nM for h25H4-4F9v3, respectively.
  • 4F9v4 is 0.17 nM
  • h25H4-4F9v5 is 0.50 nM
  • h25H4-4F9v6 is 1.35 nM
  • h25H4-4F9v8 is 1.07 nM
  • h85H7-2B11v1 is 0.07 nM
  • h85H7-2B11v2 is 0.11 nM
  • h85H7- 2B11v3 is 0.08 nM
  • h85H7-2B11v4 is 0.13 nM
  • h85H7-2B11v5 is 0.14 nM
  • h85H7-2B11v6 is 0.18 nM
  • h85H7-2B11v7 is 0.12 nM
  • h85 7-2B11v8 was 0.18nM.
  • Example 12 Binding Affinity Analysis of Humanized Antibody by Cell ELISA Binding to 96-well plate (BD-FALCON) hCR2-CHOK1 cells in DMEM / F-12 medium (Wako Pure Chemical Industries) containing 10% FCS 1 ⁇ 10 The cells were added at 4 cells / well and cultured overnight.
  • binding activity (EC50) of each antibody is 0.73 nM for h25H4-4F9v1, 1.05 nM for h25H4-4F9v2, 0.83 nM for h25H4-4F9v3, 1.83 nM for h25H4-4F9v4, and 2.32 nM for h25H4-4F9v5.
  • H25H4-4F9v6 3.19 nM h25H4-4F9v8 3.03 nM h85H7-2B11 v1 0.18 nM h85 H7-2 B11 v2 0.24 nM h85 H7-2 B11 v3 0.23 nM h85 H7-2 B11 v4 0.22 nM H85H7-2B11v5 was 0.25 nM, h85H7-2B11v6 was 0.24 nM, h85H7-2B11v7 was 0.23 nM, and h85H7-2B11v8 was 0.20 nM.
  • Example 13 Growth Inhibition Test of HUVEC of Humanized Antibody The HUVEC growth inhibitory ability of the humanized antibody was examined in the same manner as in Example 3. HUVECs were seeded at 2,500 cells / well in 96-well culture plates and adhered by pre-incubation for 3 hours. Thereafter, each concentration of humanized 25H4-4F9 and humanized 85H7-2B11 was added, and a final concentration of 100 nM of AM was added, and the proliferation of HUVEC after 96 hours was observed to confirm the proliferation inhibitory ability of HUVEC. . Cell Counting Kit-8 (DOJINDO) was used to measure the number of cells. 100 ⁇ L of a 10-fold diluted color solution was added to Humedia-EG2 medium containing 2% FBS excluding growth factors, and coloring was performed for 4 hours in a CO 2 incubator to measure OD450.
  • DOJINDO Cell Counting Kit-8
  • the HUVEC growth inhibition rate (IC50) was calculated from the ratio of the concentration of each antibody.
  • the IC50 values of the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were 16.0 nM for h25H4-4F9v1, 10.9 nM for h25H4-4F9v2, and 5.1 nM for h85H7-2B11v1, respectively.
  • -2B11v2 is 9.5 nM
  • h85H7-2B11v3 is 1.5 nM
  • h85H7-2B11v4 is 1.4 nM
  • h85H7-2B11v5 is 21.4 nM
  • h85H7-2B11v6 is 14.8 nM
  • h85H7-2B11v7 is 32.7 nM
  • h85H7-2B11v8 Was 22.6 nM.
  • Example 14 Epitope analysis of antibody
  • 12 amino acids are overlapped for the extracellular region of human RAMP2 protein 3
  • the following 31 13-15 amino acid peptide fragments (SEQ ID NOS: 71-101) shifted by amino acid were prepared, and the binding ability of the antibodies to these peptides was examined by ELISA.
  • a sufficient amount of 1.0 ⁇ g / mL was used for the solid phase of peptide and protein, and 85H7-2B11 antibody was reacted at 20 ng / mL for binding.
  • Goat Anti-mouse-IgG-HRP was reacted at 50.0 ng / mL, the bound antibody was developed with TMB solution, and after stopping the reaction with 2N H 2 SO 4 solution, OD450 was measured.
  • QPLPTTGTPGSEGGT (SEQ ID NO: 71) PTTGTPGSEGGTVKN (SEQ ID NO: 72) GTPGSEGGTVKNYET (SEQ ID NO: 73) GSEGGTVKNYETAVQ (SEQ ID NO: 74) GGTVKNYETAVQFSW (SEQ ID NO: 75) VKNYETAVQFSWNHY (SEQ ID NO: 76) YETAVQFSWNHYKDQ (SEQ ID NO: 77) AVQFSWNHYKDQMDP (SEQ ID NO: 78) FSWNHYKDQMDPIEK (SEQ ID NO: 79) NHYKDQMDPIEKDWS (SEQ ID NO: 80) KDQMDPIEKDWSDWA (SEQ ID NO: 81) MDPIEKDWSDWAMIS (SEQ ID NO: 82) IEKDWS DWAMISRPY (SEQ ID NO: 83) DWSDWAMISRPYSTL (SEQ ID NO:
  • the present antibody can specifically suppress the function of human RAMP2 which is a human adrenomedullin receptor, provision of a method for preventing, diagnosing or treating a disease associated with RAMP2, a preventive agent, a diagnostic agent or a treatment for the disease It is expected to play an important role in drug development.

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Abstract

With regard to an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC antiproliferative effect, the present invention provides an angiogenesis inhibitor containing said antibody as an active ingredient. Specifically, the present invention relates to, in an in vitro proliferation test: an antibody against a receptor activity modifying protein2 (RAMP2) having an antiproliferative effect of vascular endothelial cells, or immunoreactive fragments thereof; or an antibody having an effect of inhibiting cAMP production caused by animal cells through the addition of adrenomedullin, or immunoreactive fragments thereof.

Description

抗RAMP2抗体Anti-RAMP2 antibody
 本発明は、血管新生抑制作用を有する受容体活性調節タンパク質2(RAMP2)に対する抗体又はその免疫反応性断片、および、そのようなRAMP2に対する抗体又はその免疫反応性断片を有効成分とする血管新生抑制剤に関する。 The present invention provides an anti-angiogenic activity using an antibody against receptor activity regulatory protein 2 (RAMP2) having anti-angiogenic activity or an immunoreactive fragment thereof, and an antibody against such RAMP2 or an immunoreactive fragment thereof as an active ingredient It relates to the agent.
 アドレノメジュリン(以下、「AM」という)はヒト褐色細胞腫から同定された52個のアミノ酸からなるペプチドで主として血管から分泌され血管を拡張させる働きをもつ血管作動性物質として知られている。また、この他にも臓器保護作用、組織再生作用、抗炎症作用などの多彩な作用が報告されている。受容体活性調節タンパク質2(RAMP2)は、1回膜貫通型タンパク質で、7回膜貫通型タンパク質であるカルシトニン受容体様受容体(CRLR)と複合体を形成することでAMの共受容体として機能している。その発現は血管内皮細胞に見られ、オートクライン、パラクラインでAMを受けることで細胞内cAMPが上昇しシグナルを伝達し、血管の恒常性を維持する。 Adrenomedullin (hereinafter referred to as "AM") is a 52 amino acid peptide identified from human pheochromocytoma and is known as a vasoactive substance mainly secreted from blood vessels and having a function of dilating blood vessels. In addition to this, various actions such as organ protecting action, tissue regenerating action and anti-inflammatory action have been reported. Receptor activity regulatory protein 2 (RAMP2) is a single-pass protein and forms a complex with the seven-pass protein calcitonin receptor-like receptor (CRLR) as a co-receptor for AM It is functioning. Its expression is found in vascular endothelial cells, and AM in autocrine and paracrine causes intracellular cAMP to rise and transmit signals to maintain vascular homeostasis.
 AM-RAMP2/CRLRシグナルを阻害することにより、血圧調節などの循環器疾患、さらには血管透過性の調節により炎症や浮腫、血管新生の関わる癌・網膜疾患、臓器移植などの拒絶免疫など、様々な疾患に対して医薬品の可能性が考えられる。このような阻害剤としては、AM部分配列ペプチド(アンタゴニスト)であるAM37-52が報告されている(特許文献1)。AM37-52は、アンタゴニスト活性が弱く、分子量が5kDa以下と小さいため血中動態が悪く医薬品として開発が困難である。 By inhibiting AM-RAMP2 / CRLR signal, cardiovascular diseases such as blood pressure regulation, and further, various factors such as inflammation and edema by regulation of vascular permeability, cancer / retinal diseases associated with angiogenesis, rejection immunity such as organ transplantation, etc. Potential for medicines for various diseases. As such an inhibitor, AM37-52 which is an AM partial sequence peptide (antagonist) has been reported (Patent Document 1). AM37-52 has a weak antagonist activity and a small molecular weight of 5 kDa or less, so its kinetics in blood are poor and development as a drug is difficult.
 その他、RAMP2に対するポリクローナル抗体やその混合物がこれまでに報告されている(特許文献2、3)が、RAMP2抗体(特にはモノクローナル抗体)単独で血管新生を効果的に抑制するには至っていない。 In addition, although a polyclonal antibody against RAMP2 and a mixture thereof have been reported so far (Patent Documents 2 and 3), the RAMP2 antibody (particularly, a monoclonal antibody) alone has not been effective in suppressing angiogenesis.
国際公開WO2016/183479号International Publication WO2016 / 183479 国際公開WO2010/012911号International Publication WO 2010/012911 国際公開WO2012/029023号International Publication WO2012 / 029023
 本発明者らは、AM依存的なRAMP2とCRLR複合体の活性化を阻害する抗RAMP2抗体、特にはHUVEC(ヒト臍帯静脈内皮細胞)の増殖抑制能を有する抗RAMP2抗体を取得するため、ヒトRAMP2タンパクをコードするペプチド断片を免疫しポリクローナル抗体を作製した。しかしながら、抗血清由来のポリクローナル抗体にはcAMP抑制能は見られなかった。そこで、RAMP2の細胞外ドメインを無細胞合成したタンパクを免疫することにより、抗RAMP2抗体の取得を試みた。抗原として、RAMP2タンパク単体の他、共受容体であるCRLRとの複合タンパク質を使用した結果、優れたcAMP抑制能を有する抗RAMP2抗体が得られた。しかし、これらの抗体はHUVECの増殖を抑制する作用は示さなかった。 The present inventors have obtained an anti-RAMP2 antibody that inhibits AM-dependent activation of RAMP2 and CRLR complex, in particular, an anti-RAMP2 antibody having the ability to inhibit the growth of HUVEC (human umbilical vein endothelial cells). A polyclonal antibody was prepared by immunizing a peptide fragment encoding the RAMP2 protein. However, the antiserum-derived polyclonal antibody did not show the ability to suppress cAMP. Therefore, we attempted to obtain an anti-RAMP2 antibody by immunizing a protein in which the extracellular domain of RAMP2 was cell-freely synthesized. As a result of using a complex protein with CRLR which is a co-receptor in addition to RAMP2 protein alone as an antigen, an anti-RAMP2 antibody having excellent cAMP inhibitory ability was obtained. However, these antibodies did not show the effect of suppressing HUVEC proliferation.
 そこで、本発明者らは、遺伝子導入した過剰発現細胞を抗原として免疫した。ヒトRAMP2遺伝子単独ではAMペプチドの受容体としては機能しないため、RAMP2遺伝子をCRLR遺伝子とともに共発現させた線維芽細胞株NIH-3T3細胞を作製した。 Therefore, the present inventors immunized a transgenic overexpressing cell as an antigen. Since human RAMP2 gene alone does not function as a receptor for AM peptide, fibroblast cell line NIH-3T3 cells in which RAMP2 gene was coexpressed with CRLR gene were prepared.
 しかし、RAMP2タンパクは全長175アミノ酸(20kDa)で、特に細胞外ドメインは102アミノ酸(12kDa)の小さい分子であり、ヒトRAMP2とマウスRAMP2の相同性が高く細胞外ドメインの62%が同一アミノ酸をコードするため、ヒトRAMP2の機能ドメインに対して免疫が成立しにくく、通常のラット、ハムスター、又はマウスへの免疫では良質な抗体が取得できなかった。そこで、過剰免疫により自己に対して免疫反応(自己抗体)を生じるMRL/lprマウスを免疫することにより、機能性に優れた抗RAMP2抗体を含む抗RAMP2抗体の取得に成功した。 However, the RAMP2 protein is a small molecule with a total length of 175 amino acids (20 kDa), and in particular the extracellular domain is a small molecule of 102 amino acids (12 kDa), 62% of the extracellular domain is highly homologous with human RAMP2 and mouse RAMP2, and the same amino acid is encoded Therefore, it is difficult to obtain immunity to the functional domain of human RAMP2, and high quality antibodies can not be obtained by immunization to normal rats, hamsters or mice. Therefore, by immunizing MRL / lpr mice that generate an immune response (autoantibodies) against self by hyperimmunization, we succeeded in obtaining an anti-RAMP2 antibody containing an anti-RAMP2 antibody with excellent functionality.
 得られたハイブリドーマから機能性に優れた抗RAMP2抗体をスクリーニングするため、遺伝子導入した過剰発現細胞のCell ELISAを実施した。抗原に使用したマウスNIH-3T3細胞では細胞自身に対する抗体の結合のため、擬陽性が見られる事から、ハムスター卵巣細胞(CHO-K1)にヒトRAMP2-CRLRを遺伝子導入して得られた安定発現細胞(hCR2-CHOK1)をCell ELISAに用いた。 In order to screen the highly functional anti-RAMP2 antibody from the obtained hybridoma, Cell ELISA of the overexpressing cells into which the gene was introduced was performed. Since false positives are observed in mouse NIH-3T3 cells used as antigens because of the binding of the antibody to the cells themselves, stable expression cells obtained by gene transfer of human RAMP2-CRLR into hamster ovary cells (CHO-K1) (HCR2-CHOK1) was used for Cell ELISA.
 Cell ELISAで選択された候補抗体について、hCR2-CHOK1細胞を用いAM添加による細胞内cAMPの上昇を抑制するか否かを指標としてシグナル阻害抗体をスクリーニングした。さらに、選択されたシグナル阻害抗体について生物活性の阻害試験としてAM添加によるHUVECの増殖を阻害できる抗体をスクリーニングし、cAMP阻害作用及びHUVEC増殖抑制作用を有する抗RAMP2抗体を取得することに成功した。 With regard to candidate antibodies selected by Cell ELISA, hCR2-CHOK1 cells were used to screen for signal-inhibiting antibodies using as an indicator whether or not to suppress an increase in intracellular cAMP due to AM addition. Furthermore, an antibody capable of inhibiting the growth of HUVEC by AM addition was screened as a test for inhibiting the biological activity of selected signal-inhibiting antibodies, and an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action was successfully obtained.
 よって、本発明は、cAMP阻害作用及びHUVEC増殖抑制作用を有する抗RAMP2抗体に関し、また当該抗体を有効成分として含有する血管新生抑制剤に関する。
(1) in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有する、受容体活性調節タンパク質2(RAMP2)に特異的に結合する抗体又はその免疫反応性断片。
(2) 前記血管内皮細胞の増殖抑制作用が、0.5~5nMのIC50値である、(1)に記載の抗体又はその免疫反応性断片。
(3) アドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有する、(1)又は(2)に記載の抗体又はその免疫反応性断片。
(4) in vitroのアドレノメジュリン添加による動物細胞によるcAMP産生試験において、cAMP産生抑制作用が、0.03~0.5nMのIC50値である、(3)に記載の抗体又はその免疫反応性断片。
(5) 配列番号2に記載のアミノ酸配列を有する重鎖可変領域(VH)及び配列番号4に記載のアミノ酸配列を有する軽鎖可変領域(VL)を含む抗体、配列番号12に記載のアミノ酸配列を有するVH及び配列番号14に記載のアミノ酸配列を有するVLを含む抗体、及び配列番号19に記載のアミノ酸配列を有するVH及び配列番号21に記載のアミノ酸配列を有するVLを含む抗体から選択されるいずれか1種類の抗体とRAMP2への結合において競合する抗体又はその免疫反応性断片、あるいは、
 配列番号2に記載のアミノ酸配列を有するVH及び配列番号4に記載のアミノ酸配列を有するVLを含む抗体、配列番号12に記載のアミノ酸配列を有するVH及び配列番号14に記載のアミノ酸配列を有するVLを含む抗体、及び配列番号19に記載のアミノ酸配列を有するVH及び配列番号21に記載のアミノ酸配列を有するVLを含む抗体から選択されるいずれか1種類の抗体と同じエピトープに結合する抗体又はその免疫反応性断片。
(6) LXIXDXYXYXMX(配列番号24)のアミノ酸配列であって、ここで、XはG又はMであり、XはF又はYであり、XはA又はVであり、Xは存在しないかHであり、XはAかVであり、XはDかEであり、かつ、XはYかNであるアミノ酸配列からなる重鎖相補性決定領域(CDRH)3を有する重鎖を含む抗体又はその免疫反応性断片。
(7) LMIFDAYYAMDY(配列番号7)又はLGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有する重鎖を含む、(6)に記載の抗体又はその免疫反応性断片。
(8) GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、RXNPYNGDX10YNQKFKG(配列番号23)のアミノ酸配列からなるCDRH2、及び、LXIXDXYXYXMX(配列番号24)のアミノ酸配列からなるCDRH3を有する重鎖を含む、(6)に記載の抗体又はその免疫反応性断片、
 ここで、XはG又はMであり、XはF又はYであり、XはA又はVであり、Xは存在しないかHであり、XはAかVであり、XはDかEであり、XはYかNであり、XはN又はIであり、XはS又はTであり、X10はI、F又はLである。
(9) 更に、RASQDIRNYLN(配列番号8)のアミノ酸配列からなる軽鎖相補性決定領域(CDRL)1、YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及びQQDSKX11PWT(配列番号25)のアミノ酸配列からなるCDRL3を有する軽鎖を含む、(8)に記載の抗体又はその免疫反応性断片、
 ここで、X11はH又はNである。
(10) (i)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
RNNPYNGDSIYNQKFKG(配列番号6)のアミノ酸配列からなるCDRH2、及び
LMIFDAYYAMDY(配列番号7)のアミノ酸配列からなるCDRH3を有するか、
 (ii)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
RINPYNGDTFYNQKFKG(配列番号15)のアミノ酸配列からなるCDRH2、及び
LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有するか、又は
 (iii)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
RINPYNGDTLYNQKFKG(配列番号22)のアミノ酸配列からなるCDRH2、及び
LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有する、(6)に記載の抗体又はその免疫反応性断片。
(11) 更に、(i)の場合、RASQDIRNYLN(配列番号8)のアミノ酸配列からなるCDRL1、
YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
QQDSKHPWT(配列番号10)のアミノ酸配列からなるCDRL3を有し、
 (ii)の場合、RASQDIRNYLN(配列番号8)のアミノ酸配列からなるCDRL1、
YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
QQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3を有し、
 (iii)の場合、RASQDIRNYLN(配列番号8)のアミノ酸配列からなるCDRL1、
YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
QQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3を有する、(10)に記載の抗体又はその免疫反応性断片。
(12) 以下の群から選択されるいずれか1つの抗体又はその免疫反応性断片である、(1)~(11)のいずれか1項に記載の抗体:
 VHが、配列番号2に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、VLが配列番号4に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片;
 VHが、配列番号12に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、VLが配列番号14に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片;
 VHが、配列番号19に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、VLが配列番号21に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片;
 VHが、配列番号2のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号4のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片;
 VHが、配列番号12のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号14のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片;及び
 VHが、配列番号19のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号21のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片。
(13) 以下の群から選択されるいずれか1つの抗体又はその免疫反応性断片である(11)に記載の抗体又はその免疫反応性断片:
 配列番号2に記載のアミノ酸配列を有するVH及び配列番号4に記載のアミノ酸配列を有するVLを含む抗体又はその免疫反応性断片;
 配列番号12に記載のアミノ酸配列を有するVH及び配列番号14に記載のアミノ酸配列を有するVLを含む抗体又はその免疫反応性断片;及び
 配列番号19に記載のアミノ酸配列を有するVH及び配列番号21に記載のアミノ酸配列を有するVLを含む抗体又はその免疫反応性断片。
(14) (1)~(13)のいずれか1項に記載の抗体又はその免疫反応性断片のアミノ酸配列をコードする核酸分子。
(15) 配列番号1に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号3に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
 配列番号11に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号13に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;又は
 配列番号18に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号20に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチドを含有する、(14)に記載の核酸分子。
(16) (14)又は(15)に記載の核酸分子を有するベクター。
(17) (16)に記載のベクターを有する宿主細胞。
(18) (17)の宿主細胞を培養することを含む、(1)~(13)のいずれか1項に記載の抗体又はその免疫反応性断片の製造方法。
(19) (1)~(13)のいずれか1項に記載の抗体又はその免疫反応性断片を有効成分として含有する、医薬組成物。
(20) 血管新生抑制剤である、(19)に記載の医薬組成物。
(21) 癌、癌転移、糖尿病性網膜症、未熟児網膜症、加齢黄斑変性症、糖尿病網膜症、網膜静脈閉塞症、血管新生緑内障、炎症性皮膚疾患、関節リウマチ、又は変形性関節症の治療薬又は予防薬である、(19)に記載の医薬組成物。
Therefore, the present invention relates to an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action, and also relates to an angiogenesis inhibitor containing the antibody as an active ingredient.
(1) An antibody or immunoreactive fragment thereof specifically binding to receptor activity regulatory protein 2 (RAMP2), which has a proliferation inhibitory effect on vascular endothelial cells in vascular endothelial cell proliferation test by addition of adrenomedullin in vitro .
(2) The antibody or immunoreactive fragment thereof according to (1), wherein the growth inhibitory effect of the vascular endothelial cell is an IC 50 value of 0.5 to 5 nM.
(3) The antibody or immunoreactive fragment thereof according to (1) or (2), which has the effect of suppressing cAMP production by animal cells by addition of adrenomedullin.
(4) The antibody according to (3) or its immunoreactivity according to (3), wherein the cAMP production inhibitory action is an IC50 value of 0.03 to 0.5 nM in a test of cAMP production by animal cells by addition of adrenomedullin in vitro. fragment.
(5) An antibody comprising a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 4, an amino acid sequence set forth in SEQ ID NO: 12 Selected from antibodies comprising a VH having the amino acid sequence set forth in SEQ ID NO: 14 and an antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 19 and a VL having the amino acid sequence set forth in SEQ ID NO: 21 An antibody or an immunoreactive fragment thereof that competes with any one type of antibody for binding to RAMP2, or
A VH having the amino acid sequence set forth in SEQ ID NO: 2 and an antibody comprising the VL having the amino acid sequence set forth in SEQ ID NO: 4, a VH having the amino acid sequence set forth in SEQ ID NO: 12 and a VL having the amino acid sequence set forth in SEQ ID NO: 14 An antibody that binds to the same epitope as any one type of antibody selected from an antibody comprising: a VH comprising an amino acid sequence as set forth in SEQ ID NO: 19 and an antibody comprising a VL having an amino acid sequence as set forth in SEQ ID NO: 21 Immunoreactive fragment.
(6) An amino acid sequence of LX 1 IX 2 DX 3 YX 4 YX 5 MX 6 X 7 (SEQ ID NO: 24), wherein X 1 is G or M and X 2 is F or Y, From the amino acid sequence, X 3 is A or V, X 4 is absent or H, X 5 is A or V, X 6 is D or E, and X 7 is Y or N An antibody or an immunoreactive fragment thereof comprising a heavy chain having a heavy chain complementarity determining region (CDRH) 3
(7) The antibody or immunoreactive fragment thereof according to (6), which comprises a heavy chain having a CDRH3 consisting of the amino acid sequence of LMIFDAYYAMD (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16).
(8) CDRH1 consisting of amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2 consisting of amino acid sequence of RX 8 NPYNGDX 9 X 10 YNQ KFKG (SEQ ID NO: 23), and LX 1 IX 2 DX 3 YX 4 YX 5 MX 6 X The antibody according to (6) or an immunoreactive fragment thereof, comprising a heavy chain having a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7 (SEQ ID NO: 24),
Here, X 1 is G or M, X 2 is F or Y, X 3 is A or V, X 4 is absent or H, and X 5 is A or V, X 6 is D or E, X 7 is Y or N, X 8 is N or I, X 9 is S or T, and X 10 is I, F or L.
(9) Furthermore, a light chain complementarity determining region (CDRL) 1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8), a CDRL 2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and QQDSKX 11 PWT (SEQ ID NO: 25) The antibody according to (8) or an immunoreactive fragment thereof, comprising a light chain having a CDRL3 consisting of an amino acid sequence.
Here, X 11 is H or N.
(10) (i) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
Whether there is CDRH2 consisting of the amino acid sequence of RNNP YNGDSIYNQ KF KF (SEQ ID NO: 6) and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7),
(Ii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
CDRH2 consisting of the amino acid sequence of RINPYNGDTFYNQKFKG (SEQ ID NO: 15) and CDRH3 consisting of the amino acid sequence of LGIYDV YHYVMEN (SEQ ID NO: 16), or (iii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5)
An antibody or an immunoreactive fragment thereof according to (6), comprising CDRH2 consisting of the amino acid sequence of RINPYNGDTLYNQKFKG (SEQ ID NO: 22) and CDRH3 consisting of the amino acid sequence of LGIYDV YHYVMEN (SEQ ID NO: 16).
(11) Furthermore, in the case of (i), CDRL1 comprising the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
A CDRL2 consisting of an amino acid sequence of YTSRLHS (SEQ ID NO: 9) and a CDRL3 consisting of an amino acid sequence of QQDSKHPWT (SEQ ID NO: 10),
In the case of (ii), CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
A CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and a CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17),
In the case of (iii), CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
An antibody or an immunoreactive fragment thereof according to (10), having CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9) and CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17).
(12) The antibody according to any one of (1) to (11), which is any one antibody selected from the following group or an immunoreactive fragment thereof:
A VH has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 2 and a VL is set forth in SEQ ID NO: 4 An antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence under stringent conditions; or an immunoreactive fragment thereof;
A VH has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 12 and a VL is set forth in SEQ ID NO: 14 An antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence under stringent conditions; or an immunoreactive fragment thereof;
A VH has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 19 and a VL is set forth in SEQ ID NO: 21 An antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence under stringent conditions; or an immunoreactive fragment thereof;
An antibody having an amino acid sequence having an identity of 80% or more with the amino acid sequence of SEQ ID NO: 2 and an antibody having an amino acid sequence having an identity of 80% or more with VL; Its immunoreactive fragment;
An antibody having an amino acid sequence having an identity of 80% or more with the amino acid sequence of SEQ ID NO: 12 and an antibody having an amino acid sequence having an identity of 80% or more with VL The immunoreactive fragment; and VH have an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 19 and VL has 80% or more identity with the amino acid sequence of SEQ ID NO: 21 An antibody having an amino acid sequence or an immunoreactive fragment thereof.
(13) The antibody or immunoreactive fragment thereof according to (11), which is any one antibody or immunoreactive fragment thereof selected from the following group:
An antibody or immunoreactive fragment thereof comprising a VH having the amino acid sequence set forth in SEQ ID NO: 2 and a VL having the amino acid sequence set forth in SEQ ID NO: 4;
An antibody comprising the amino acid sequence set forth in SEQ ID NO: 12 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 14 or an immunoreactive fragment thereof; and VH having the amino acid sequence set forth in SEQ ID NO 19 and SEQ ID NO: 21 An antibody or an immunoreactive fragment thereof comprising a VL having the described amino acid sequence.
(14) A nucleic acid molecule encoding an amino acid sequence of the antibody according to any one of (1) to (13) or an immunoreactive fragment thereof.
(15) A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 1, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 3;
A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 11, and a polynucleotide encoding the VL having the nucleotide sequence set forth in SEQ ID NO: 13; or a VH encoding the nucleotide sequence set forth in SEQ ID NO: 18 A nucleic acid molecule according to (14), comprising a polynucleotide and a polynucleotide encoding a VL having the nucleotide sequence as set forth in SEQ ID NO: 20.
(16) A vector comprising the nucleic acid molecule of (14) or (15).
(17) A host cell comprising the vector according to (16).
(18) A method for producing the antibody or the immunoreactive fragment thereof according to any one of (1) to (13), which comprises culturing the host cell of (17).
(19) A pharmaceutical composition comprising the antibody according to any one of (1) to (13) or an immunoreactive fragment thereof as an active ingredient.
(20) The pharmaceutical composition according to (19), which is an angiogenesis inhibitor.
(21) cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis The pharmaceutical composition according to (19), which is a therapeutic agent or prophylactic agent for
 本発明の抗体又はその免疫反応性断片は、細胞表面に発現したRAMP2に結合して単独で血管内皮細胞の増殖を抑制することができることから、新規なメカニズムに基づく血管新生抑制剤、及び血管新生が発症又は増悪化に寄与する疾患又は障害の治療薬又は予防薬を提供するものである。 Since the antibody of the present invention or the immunoreactive fragment thereof can bind to RAMP2 expressed on the cell surface and can suppress the proliferation of vascular endothelial cells alone, an angiogenesis inhibitor based on a novel mechanism, and angiogenesis The present invention provides a therapeutic agent or a preventive agent for a disease or disorder in which
hCR2-CHOK1細胞を用いて、AM添加時のcAMP産生に対する各種抗RAMP2抗体(10nM)の抑制効果を表すグラフである。縦軸は、AM添加/抗体非投与(コントロール)のcAMP量(100%)に対する、各群のcAMP量の割合(%)を示す。横軸は、AM及び抗体の添加の有無、及び添加した抗体を示す。7 is a graph showing the inhibitory effect of various anti-RAMP2 antibodies (10 nM) on cAMP production upon addition of AM using hCR2-CHOK1 cells. The vertical axis shows the ratio (%) of the amount of cAMP in each group to the amount (100%) of the amount of AMP addition / non-administration (control). The horizontal axis shows the presence or absence of addition of AM and antibody, and the added antibody. hCR2-CHOK1細胞を用いて、各種濃度の25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体のAM添加によるcAMP産生の抑制能を測定した結果を表すグラフである。縦軸は、抗体非添加時のcAMP量(100%)に対する、各抗体投与時のcAMP量の割合(%)を示す。横軸は、抗体濃度(nM)を示す。It is a graph showing the result of having measured the inhibitory power of cAMP production by AM addition of various concentration 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody using hCR2-CHOK1 cells. The vertical axis shows the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition. The horizontal axis shows antibody concentration (nM). AM添加によるHUVEC増殖に対する、各種抗RAMP2抗体(10nM)の抑制作用を示すグラフである。縦軸は、AM添加/抗体非投与(コントロール)のHUVEC細胞数(100%)に対する、各群のHUVEC細胞数の割合(%)を示す。横軸は、AM及び抗体の添加の有無、及び添加した抗体を示す。It is a graph which shows the inhibitory effect of various anti-RAMP2 antibodies (10 nM) on HUVEC proliferation by AM addition. The vertical axis shows the percentage (%) of the HUVEC cell number of each group to the AM-added / non-antibody-administered (control) HUVEC cell number (100%). The horizontal axis shows the presence or absence of addition of AM and antibody, and the added antibody. HUVECを用いて、各種濃度の25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体のAM添加による増殖の抑制能を測定した結果を表すグラフである。縦軸は、抗体非添加時のHUVEC細胞数(100%)に対する、各抗体投与時のHUVEC細胞数の割合(%)を示す。横軸は、抗体濃度(nM)を示す。It is a graph showing the result of having measured the ability to suppress the proliferation by AM addition of various concentrations of 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody using HUVEC. The vertical axis shows the ratio (%) of HUVEC cell count at each antibody administration to the HUVEC cell count (100%) at the time of no antibody addition. The horizontal axis shows antibody concentration (nM). 25H4-4F9抗体のVH(配列番号2)及び軽鎖可変領域(配列番号4)、85H7-2B11抗体のVH(配列番号12)及び軽鎖可変領域(配列番号14)、及び33H4-1G3抗体のVH(配列番号19)及び軽鎖可変領域(配列番号21)のアミノ酸配列、及びそれらに含まれるCDR部分を示す。The VH (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 4) of the 25H4-4F9 antibody, the VH (SEQ ID NO: 12) and light chain variable region (SEQ ID NO: 14) of the 85H7-2B11 antibody, and the 33H4-1G3 antibody The amino acid sequences of VH (SEQ ID NO: 19) and light chain variable region (SEQ ID NO: 21), and the CDR portions contained therein are shown. 取得した抗体が抗RAMP2特異的である事をCell ELISAによって確認した結果を示すグラフである。横軸は、使用した抗体を示す。ドットの白抜きバーはヒトCRLR単独の発現細胞を用いた結果を示し、黒色のバーはヒトCRLRとヒトRAMP2を共発現させた細胞(hCR2-CHOK1)を示す。縦軸は、これら発現細胞への各抗体の結合量(OD450)を示す。It is a graph which shows the result of having confirmed that the acquired antibody is anti-RAMP2 specific by Cell ELISA. The horizontal axis shows the antibody used. The open bars in the dots indicate the results using cells expressing human CRLR alone, and the black bars indicate cells in which human CRLR and human RAMP2 are coexpressed (hCR2-CHOK1). The vertical axis shows the binding amount (OD450) of each antibody to these expressing cells. 実施例1で得られた25H4-4F9(黒四角)、85H7-2B11(黒丸)、及び33H4-1G3(黒三角)、並びに、市販の抗RAMP2抗体である、4E5(白丸)、2F5(白四角)、及びB5(白三角)を用いて、Cell ELISAによる細胞膜表面タンパクに対する結合親和性を解析した結果を示すグラフである。縦軸は結合割合(%)、横軸は抗体濃度(nM)のlog値を表す。各抗体について、結合割合が50%となる抗体濃度をグラフから求めてEC50値とした。25H4-4F9 (black squares), 85H7-2B11 (black circles), and 33H4-1G3 (black triangles) obtained in Example 1, and 4E5 (white circles), 2F5 (white squares) which are commercially available anti-RAMP2 antibodies And B5 (open triangles) are graphs showing the results of analysis of binding affinity to cell membrane surface proteins by Cell ELISA. The ordinate represents the binding ratio (%), and the abscissa represents the log value of the antibody concentration (nM). For each antibody, the antibody concentration at which the binding ratio is 50% was determined from the graph and used as the EC50 value. 実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3、並びに、市販の抗RAMP2抗体である、4E5、2F5、及びB5を被検抗体として、細胞膜表面上の抗原に対する結合をFACSで解析した結果を表すグラフである。各グラフの上部に使用した各被検抗体名を示す。各グラフの縦軸は細胞数(×10細胞)、横軸は細胞に結合したR-Phycoerythrin標識二次抗体の蛍光強度を示す。グラフ中、左側に位置するピークはコントロール(各被検抗体非存在下)を示し、右側に位置するピークは各被検抗体存在下を表す。Using 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and 4E5, 2F5, and B5, which are commercially available anti-RAMP2 antibodies, as test antibodies, binding to the antigen on the cell membrane surface was carried out. It is a graph showing the result analyzed by FACS. Each test antibody name used at the top of each graph is shown. The vertical axis of each graph represents the number of cells (× 10 3 cells), and the horizontal axis represents the fluorescence intensity of R-Phycoerythrin-labeled secondary antibody bound to the cells. In the graph, the peak located on the left indicates the control (in the absence of each test antibody), and the peak located on the right indicates the presence of each test antibody. 実施例1で得られた25H4-4F9(黒三角)、85H7-2B11(黒丸)、及び33H4-1G3(黒四角)、並びに市販抗体4E5(白丸)の細胞表面上の抗原への結合がビオチン化85H7-2B11抗体と競合するか否かをFACSにより解析した結果を示すグラフである。縦軸は細胞に結合したビオチン化85H7-2B11抗体と結合した蛍光標識の蛍光強度の平均値(MFI)、横軸は使用した各抗体の濃度(μg/mL)を表す。標識化抗体と同じ抗体で競合させた85H7-2B11(黒丸)の結果は、結合が競合していることを示す。Binding to the antigen on the cell surface of 25H4-4F9 (black triangle), 85H7-2B11 (black circle), and 33H4-1G3 (black square) obtained in Example 1 and the commercially available antibody 4E5 (white circle) was biotinylated It is a graph which shows the result of having analyzed by FACS whether it competes with the 85H7-2B11 antibody. The ordinate represents the mean value (MFI) of the fluorescence intensity of the fluorescent label bound to the biotinylated 85H7-2B11 antibody bound to the cells, and the abscissa represents the concentration (μg / mL) of each antibody used. The results of 85H7-2B11 (filled circles) competed with the labeled antibody for the same antibody show that the binding is in competition. 25H4-4F9抗体の配列を基にデザインされたヒト化抗体の重鎖(Heavy Chain)及び軽鎖(Light Chain)の可変領域の配列を示す。25H4-4F9抗体のVHの配列h25H4-4F9vh(配列番号2)を基に、h25H4-4F9vh1(配列番号27)、h25H4-4F9vh2(配列番号29)、h25H4-4F9vh3(配列番号31)、h25H4-4F9vh4(配列番号33)、及びh25H4-4F9vh5(配列番号35)をデザインした。また、25H4-4F9抗体のVLの配列h25H4-4F9vk(配列番号4)を基に、h25H4-4F9vk1(配列番号37)、h25H4-4F9vk2(配列番号39)、h25H4-4F9vk3(配列番号41)、及びh25H4-4F9vk4(配列番号43)をデザインした。下線部は各CDR配列を示す。The sequences of the variable regions of the heavy chain (Heavy Chain) and light chain (Light Chain) of a humanized antibody designed based on the sequence of the 25H4-4F9 antibody are shown. H25H4-4F9vh1 (SEQ ID NO: 27), h25H4-4F9vh2 (SEQ ID NO: 29), h25H4-4F9vh3 (SEQ ID NO: 31), h25H4-4F9vh4 based on the sequence h25H4-4F9vh (SEQ ID NO: 2) of the 25H4-4F9 antibody VH (SEQ ID NO: 33) and h25H4-4F9vh5 (SEQ ID NO: 35) were designed. H25H4-4F9vk1 (SEQ ID NO: 37), h25H4-4F9vk2 (SEQ ID NO: 39), h25H4-4F9 vk3 (SEQ ID NO: 41), and h25H4-4F9vk1 (SEQ ID NO: 37) based on the sequence h25H4-4F9vk (SEQ ID NO: 4) of the 25H4-4F9 antibody VL. h25H4-4F9vk4 (SEQ ID NO: 43) was designed. Underlining indicates each CDR sequence. 85H7-2B11抗体の配列を基にデザインされたヒト化抗体の重鎖(Heavy Chain)及び軽鎖(Light Chain)の可変領域の配列を示す。85H7-2B11抗体のVHの配列h85H7-2B11vh(配列番号12)を基に、h85H7-2B11vh1(配列番号45)、h85H7-2B11vh2(配列番号47)、h85H7-2B11vh3(配列番号49)、h85H7-2B11vh4(配列番号51)、及びh85H7-2B11vh5(配列番号53)をデザインした。また、85H7-2B11抗体のVLの配列h85H7-2B11vk(配列番号14)を基に、h85H7-2B11vk1(配列番号55)、h85H7-2B11vk2(配列番号57)、h85H7-2B11vk3(配列番号59)、及びh85H7-2B11vk4(配列番号61)をデザインした。下線部は各CDR配列を示す。The sequences of the variable regions of the heavy chain (Heavy Chain) and light chain (Light Chain) of a humanized antibody designed based on the sequence of the 85H7-2B11 antibody are shown. H85H7-2B11vh1 (SEQ ID NO: 45), h85H7-2B11vh2 (SEQ ID NO: 47), h85H7-2B11vh3 (SEQ ID NO: 49), h85H7-2B11vh4 based on the sequence h85H7-2B11vh (SEQ ID NO: 12) of the VH of the 85H7-2B11 antibody (SEQ ID NO: 51) and h85H7-2B11 vh5 (SEQ ID NO: 53) were designed. H85H7-2B11vk1 (SEQ ID NO: 55), h85H7-2B11vk2 (SEQ ID NO: 57), h85H7-2B11vk3 (SEQ ID NO: 59), and h85H7-2B11vk1 (SEQ ID NO: 55) based on the sequence h85H7-2B11vk (SEQ ID NO: 14) of the VL of the 85H7-2B11 antibody The h85H7-2B11 vk4 (SEQ ID NO: 61) was designed. Underlining indicates each CDR sequence. hCR2-CHOK1細胞を用いて、16.7nMのヒト化25H4-4F9抗体(25H4-4F9v1、25H4-4F9v2、25H4-4F9v3、25H4-4F9v4、25H4-4F9v5、25H4-4F9v6、及び25H4-4F9v8)のAM添加によるcAMP産生の抑制能を測定した結果を表すグラフである。縦軸は、抗体非添加時のcAMP量(100%)に対する、各抗体投与時のcAMP量の割合(%)を示す。横軸は、AM添加の有無及び使用した抗体を示す。AM of the 16.7 nM humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) using hCR2-CHOK1 cells It is a graph showing the result of having measured the ability to suppress cAMP production by addition. The vertical axis shows the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition. The horizontal axis shows the presence or absence of AM addition and the antibody used. hCR2-CHOK1細胞を用いて、16.7nMのヒト化85H7-2B11抗体(85H7-2B11v1、85H7-2B11v2、85H7-2B11v3、85H7-2B11v4、85H7-2B11v5、85H7-2B11v6、85H7-2B11v7、及び85H7-2B11v8)のAM添加によるcAMP産生の抑制能を測定した結果を表すグラフである。縦軸は、抗体非添加時のcAMP量(100%)に対する、各抗体投与時のcAMP量の割合(%)を示す。横軸は、AM添加の有無及び使用した抗体を示す。Using the hCR2-CHOK1 cells, 16.7 nM of the humanized 85H7-2B11 antibody (85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7- It is a graph showing the result of having measured the inhibitory capacity of cAMP production by AM addition of 2B11v8). The vertical axis shows the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition. The horizontal axis shows the presence or absence of AM addition and the antibody used. hCR2-CHOK1細胞を用いて、各種濃度のヒト化25H4-4F9抗体(25H4-4F9v1、25H4-4F9v2、25H4-4F9v3、25H4-4F9v4、25H4-4F9v5、25H4-4F9v6、及び25H4-4F9v8)(上図)、及びヒト化85H7-2B11抗体(85H7-2B11v1、85H7-2B11v2、85H7-2B11v3、85H7-2B11v4、85H7-2B11v5、85H7-2B11v6、85H7-2B11v7、及び85H7-2B11v8)(下図)のAM添加によるcAMP産生の抑制能を測定した結果を表すグラフである。各グラフにおいて縦軸は、抗体非添加時のcAMP量(100%)に対する、各抗体投与時のcAMP量の割合(%)を示す。横軸は、抗体濃度(nM)のlog値を示す。凡例と共に、各抗体のIC50値(グラフにおいてcAMPを50%阻害する濃度)を示す。Various concentrations of humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (upper figure) using hCR2-CHOK1 cells. And AM by addition of humanized 85H7-2B11 antibody (85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (shown below) It is a graph showing the result of having measured the ability to suppress cAMP production. In each graph, the vertical axis indicates the ratio (%) of the amount of cAMP at the time of administration of each antibody to the amount of cAMP (100%) at the time of no antibody addition. The horizontal axis shows the log value of the antibody concentration (nM). The IC50 value (concentration for 50% inhibition of cAMP in the graph) of each antibody is shown together with the legend. ヒト化25H4-4F9抗体(25H4-4F9v1、25H4-4F9v2、25H4-4F9v3、25H4-4F9v4、25H4-4F9v5、25H4-4F9v6、及び25H4-4F9v8)(上図)、及びヒト化85H7-2B11抗体(85H7-2B11v1、85H7-2B11v2、85H7-2B11v3、85H7-2B11v4、85H7-2B11v5、85H7-2B11v6、85H7-2B11v7、及び85H7-2B11v8)(下図)を6.7nM用いて、Cell ELISAを行い、細胞膜表面タンパクに対する結合親和性を解析した結果を示すグラフである。縦軸は結合割合(OD450%)、横軸は用いた抗体を表す。Humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (upper figure), and humanized 85H7-2B11 antibodies (85H7) Perform cell ELISA using 6.7 nM of -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8 (shown below), and perform cell membrane surface protein using It is a graph which shows the result of having analyzed the binding affinity with respect to. The ordinate represents the binding ratio (OD 450%), and the abscissa represents the antibody used. ヒト化25H4-4F9抗体(25H4-4F9v1、25H4-4F9v2、25H4-4F9v3、25H4-4F9v4、25H4-4F9v5、25H4-4F9v6、及び25H4-4F9v8)(上図)、及びヒト化85H7-2B11抗体(85H7-2B11v1、85H7-2B11v2、85H7-2B11v3、85H7-2B11v4、85H7-2B11v5、85H7-2B11v6、85H7-2B11v7、及び85H7-2B11v8)(下図)を用いて、Cell ELISAによる細胞膜表面タンパクに対する結合親和性を解析した結果を示すグラフである。縦軸は結合割合(%)、横軸は抗体濃度(nM)のlog値を表す。各抗体について、結合割合が50%となる抗体濃度をグラフから求めてEC50値とした。凡例と共に、各抗体のEC50値(nM)を示す。Humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (upper figure), and humanized 85H7-2B11 antibodies (85H7) Binding affinity to cell membrane surface protein by Cell ELISA using -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (lower figure) It is a graph which shows the analyzed result. The ordinate represents the binding ratio (%), and the abscissa represents the log value of the antibody concentration (nM). For each antibody, the antibody concentration at which the binding ratio is 50% was determined from the graph and used as the EC50 value. The EC50 value (nM) of each antibody is shown together with the legend. 抗体のエピトープを解析した結果を表すグラフである。縦軸は結合した抗体量をOD450(二次抗体の発色強度)で表す。横軸は結合に用いたRAMP2の細胞外領域に由来する各ペプチドを示す。It is a graph showing the result of having analyzed the epitope of an antibody. The vertical axis represents the amount of bound antibody as OD450 (color intensity of secondary antibody). The horizontal axis shows each peptide derived from the extracellular domain of RAMP2 used for binding.
(抗体又はその免疫反応性断片)
 本発明の抗体は、RAMP2と特異的に結合する。特には、本発明の抗体は細胞膜又は細胞表面上に発現したRAMP2と特異的に結合する。本明細書において、抗体又はその免疫反応性断片が「特異的に」認識する(結合する)とは、その抗体又はその免疫反応性断片が他のタンパク質やペプチドに対する親和性よりも、RAMP2に対して実質的に高い親和性で結合することを意味する。ここで、「実質的に高い親和性で結合する」とは、所望の測定装置または方法によって、目的とする特定のタンパク質やペプチドを他のタンパク質やペプチドから区別して検出することが可能な程度に高い親和性を意味する。例えば、実質的に高い親和性は、ELISA又はEIAにより検出された強度(例えば、蛍光強度)として、3倍以上、4倍以上、5倍以上、6倍以上、7倍以上、8倍以上、9倍以上、10倍以上、20倍以上、30倍以上、40倍以上、50倍以上、100倍以上であることを意味していてもよい。
(Antibody or immunoreactive fragment thereof)
The antibody of the present invention specifically binds to RAMP2. In particular, the antibodies of the invention specifically bind to RAMP2 expressed on cell membranes or cell surfaces. In the present specification, the phrase “specifically” (specifically) that an antibody or its immunoreactive fragment recognizes (binds) means that the antibody or its immunoreactive fragment has higher affinity for RAMP2 than the affinity for other proteins or peptides. It means binding with substantially high affinity. Here, "to bind with substantially high affinity" means that the specific protein or peptide of interest can be distinguished from other proteins or peptides and detected by the desired measuring device or method. It means high affinity. For example, substantially high affinity is 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, as the intensity (for example, fluorescence intensity) detected by ELISA or EIA. It may mean that it is 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 100 times or more.
 本発明の抗体とRAMP2との結合における結合速度定数(Ka1)としては、例えば、1×10Ms-1以上、1×10Ms-1以上、5×10Ms-1以上を挙げることができる。また、本発明の抗体とRAMP2との結合における解離速度定数(Kd1)としては、例えば、1×10-3以下、1×10-4以下を挙げられる。本発明の抗体とRAMP2との結合における結合定数(KD)としては、例えば、1×10-8(M)以下、5×10-8(M)以下、1×10-9(M)以下、5×10-9(M)以下であり得る。本明細書における抗体の結合速度定数(Ka1)、解離速度定数(Kd1)、及び結合定数(KD)はBIACORE(GEヘルスケアバイオサイエンス株式会社,BIACORE-X100)を用いて、製造者提供のマニュアルに従い、SAチップにビオチン化RAMP2を固定後、被検抗体を流し、結合速度定数Ka1、解離速度定数Kd1を測定し、bivalentフィッティングを用いて結合定数KD値を決定することができる。 As a binding rate constant (Ka1) in binding between the antibody of the present invention and RAMP2, for example, 1 × 10 4 Ms −1 or more, 1 × 10 5 Ms −1 or more, 5 × 10 5 Ms −1 or more can be mentioned. Can. Moreover, as a dissociation rate constant (Kd1) in binding of the antibody of the present invention to RAMP2, for example, 1 × 10 −3 or less and 1 × 10 4 or less can be mentioned. The binding constant (KD) for binding between the antibody of the present invention and RAMP2 is, for example, 1 × 10 −8 (M) or less, 5 × 10 −8 (M) or less, 1 × 10 −9 (M) or less, It may be 5 × 10 −9 (M) or less. The binding rate constant (Ka1), dissociation rate constant (Kd1), and binding constant (KD) of the antibody in the present specification are the manual provided by the manufacturer using BIACORE (GE Healthcare Biosciences Ltd., BIACORE-X100). According to the above, after immobilizing biotinylated RAMP2 on the SA chip, the test antibody is allowed to flow, the binding rate constant Ka1 and the dissociation rate constant Kd1 can be measured, and the binding constant KD value can be determined using bivalent fitting.
 本発明の抗体は、ポリクローナル抗体であっても、モノクローナル抗体であってもよいが、好ましくは、モノクローナル抗体である。本発明において、「モノクローナル抗体」は、単一な抗原決定基と反応する、構造がほぼ均一な抗体である。更に、本発明の抗体は、非ヒト動物の抗体、非ヒト動物の抗体のアミノ酸配列とヒト由来の抗体のアミノ酸配列を有する抗体、及び、ヒト抗体を包含する。非ヒト動物の抗体としては、例えば、マウス、ラット、ハムスター、モルモット、ラビット、イヌ、サル、ヒツジ、ヤギ、ラクダ、ニワトリ、アヒル等の抗体を挙げることができ、好ましくは、ハイブリドーマを作製することができる動物の抗体であり、より好ましくはマウス、ラット又はウサギの抗体である。非ヒト動物の抗体のアミノ酸配列とヒト由来の抗体のアミノ酸配列を有する抗体としては、ヒト型キメラ抗体、ヒト化抗体を挙げることができる。上記において、「キメラ抗体」とは、非ヒト動物由来であってRAMP2と特異的に結合する抗体の定常領域をヒトの抗体と同じ定常領域を有するように遺伝子工学的に改変した抗体のことであり、好ましくは、ヒト・マウス・キメラ抗体(欧州特許公開公報EP0125023参照)である。「ヒト化抗体」とは、非ヒト動物由来であってRAMP2と特異的に結合する抗体のH鎖とL鎖の相補認識領域(CDR)以外の一次構造をヒトの抗体に対応する一次構造に遺伝子工学的に改変した抗体のことである。ここで、CDRとは、Kabatら(“Sequences of Proteins of Immunological Interest”,Kabat,E.ら,U.S.Department of Health and Human Services,1983)またはChothiaら(Chothia&Lesk(1987)J.Mol.Biol.,196:901-917)のいずれの定義によるものであってもよい。「ヒト抗体」とは、完全にヒト由来の抗体遺伝子の発現産物であるヒト抗体のことであり、例えば、ヒトの抗体産生に関与する遺伝子を導入したトランスジェニック動物を用いて作製したモノクローナル抗体(欧州特許公開公報EP0546073参照)等を挙げることができる。例えば、本発明の抗体を治療、予防、又は体内に投与することにより用いる診断に使用する場合には、本発明の抗体として好ましくは、ヒト・非ヒト動物のキメラ抗体、ヒト化抗体、又はヒト抗体である。また、好ましくは本発明の抗体は天然に存在しない抗体である。 The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. In the present invention, a "monoclonal antibody" is an antibody that is substantially homogeneous in structure that reacts with a single antigenic determinant. Furthermore, the antibody of the present invention includes non-human animal antibodies, antibodies having the amino acid sequences of non-human animal antibodies and human-derived antibodies, and human antibodies. Examples of non-human animal antibodies include mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, camels, chickens, ducks, etc., preferably hybridomas. The antibody is preferably an animal antibody, more preferably a mouse, rat or rabbit antibody. Examples of the antibody having the amino acid sequence of the non-human animal antibody and the amino acid sequence of the human-derived antibody include human chimeric antibodies and humanized antibodies. In the above, “chimeric antibody” refers to an antibody that is genetically engineered to have the same constant region as a human antibody, which is a constant region of an antibody derived from non-human animals and specifically binding to RAMP2. Preferably, it is a human-mouse-chimeric antibody (see European Patent Publication EP0125023). The “humanized antibody” refers to a primary structure other than the complementary recognition region (CDR) of the H chain and L chain of an antibody derived from non-human animals and specifically binding to RAMP2 as a primary structure corresponding to a human antibody. It refers to a genetically engineered antibody. Here, CDR means Kabat et al. ("Sequences of Proteins of Immunological Interest", Kabat, E. et al., U. S. Department of Health and Human Services, 1983) or Chothia et al. (Chothia & Lesk (1987) J. Mol. Biol., 196: 901-917). The “human antibody” refers to a human antibody that is an expression product of a completely human-derived antibody gene, and for example, a monoclonal antibody (a human antibody produced by using a transgenic animal into which a gene involved in antibody production has been introduced) European Patent Publication No. EP0546073) and the like can be mentioned. For example, when the antibody of the present invention is used for treatment, prevention, or diagnosis for use by administration into the body, the antibody of the present invention is preferably a human / non-human animal chimeric antibody, a humanized antibody, or a human It is an antibody. Also preferably, the antibody of the present invention is a non-naturally occurring antibody.
 本発明の抗体のイムノグロブリンクラスは特に限定されるものではなく、IgG、IgM、IgA、IgE、IgD、又はIgYのいずれのイムノグロブリンクラス(アイソタイプ)であってもよく、好ましくはIgGである。また、本発明の抗体がIgGの場合、いずれのサブクラス(IgG1、IgG2、IgG3、又はIgG4)であってもよい。また、本発明の抗体は、モノスペシフィック、バイスペシフィック(二重特異性抗体)、トリスペシフィック(三重特異性抗体)(例えば、WO1991/003493号)、又はマルチスペシフィック(多重特異性抗体)であってもよい。 The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD or IgY, preferably IgG. When the antibody of the present invention is IgG, it may be any subclass (IgG1, IgG2, IgG3, or IgG4). Furthermore, the antibody of the present invention is monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (for example, WO 1991/003493), or multispecific (multispecific antibody), It is also good.
 抗体はその可変領域(特には、CDRs)が結合特性を付与していることが知られており、完全抗体でない抗体断片であってもその結合特性を利用可能であることが当業者に広く知られている。本明細書において、「免疫反応性断片」とは、抗体の一部分(部分断片)を含むタンパク質又はペプチドであって、抗体の抗原への作用(免疫反応性・結合性)を保持するタンパク質又はペプチドを意味する。このような免疫反応性断片としては、例えば、F(ab’)、Fab’、Fab、Fab、一本鎖Fv(以下、「scFv」という)、(タンデム)バイスペシフィック一本鎖Fv(sc(Fv))、一本鎖トリプルボディ、ナノボディ、ダイバレントVHH、ペンタバレントVHH、ミニボディ、(二本鎖)ダイアボディ、タンデムダイアボディ、バイスペシフィックトリボディ、バイスペシフィックバイボディ、デュアルアフィニティリターゲティング分子(DART)、トリアボディ(又はトリボディ)、テトラボディ(又は[sc(Fv))、若しくは(scFv-SA))ジスルフィド結合Fv(以下、「dsFv」という)、コンパクトIgG、重鎖抗体、又はそれらの重合体を挙げることができる(Nature Biotechnology, 29(1):5-6 (2011);Maneesh Jain et al., TRENDS in Biotechnology, 25(7)(2007):307-316;及び、Christoph steinら、Antibodies(1):88-123(2012)参照)。本明細書において、免疫反応性断片は、モノスペシフィック、バイスペシフィック(二重特異性)、トリスペシフィック(三重特異性)、及びマルチスペシフィック(多重特異性)のいずれであってもよい。本明細書において、特にそのように解釈することが不整合である場合を除き、「抗体」の語は抗体の免疫反応性断片をも含むことを意図している。 It is widely known to those skilled in the art that antibodies are known to have binding characteristics in their variable regions (in particular, CDRs), and even non-complete antibody fragments are able to utilize their binding characteristics. It is done. As used herein, the term "immunoreactive fragment" refers to a protein or peptide containing a portion (partial fragment) of an antibody, wherein the protein or peptide retains the action (immunoreactivity / binding) of the antibody to an antigen. Means As such an immunoreactive fragment, for example, F (ab ') 2 , Fab', Fab, Fab 3 , single chain Fv (hereinafter referred to as "scFv"), (tandem) bispecific single chain Fv ( sc (Fv) 2 ), single stranded triple body, nanobody, diversity VHH, pentavalent VHH, minibody, (double strand) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity Targeting molecule (DART), triabody (or tribody), tetrabody (or [sc (Fv) 2 ] 2 ), or (scFv-SA) 4 ) disulfide bond Fv (hereinafter referred to as “dsFv”), compact IgG, Heavy chain antibodies, or polymers thereof (Na Maneesh Jain et al., TRENDS in Biotechnology, 25 (7) (2007): 307-316; and Christoph stein et al., Antibodies (1): 88-. 123 (2012)). As used herein, an immunoreactive fragment may be any of monospecific, bispecific (bispecific), trispecific (trispecific), and multispecific (multispecific). As used herein, unless the context is so inconsistent, the term "antibody" is intended to also include immunoreactive fragments of the antibody.
 一態様において、本発明は、25H4-4F9抗体、25H4-4F9v1抗体、25H4-4F9v2抗体、25H4-4F9v3抗体、25H4-4F9v4抗体、25H4-4F9v5抗体、25H4-4F9v6抗体、25H4-4F9v8抗体、85H7-2B11抗体、85H7-2B11v1抗体、85H7-2B11v2抗体、85H7-2B11v3抗体、85H7-2B11v4抗体、85H7-2B11v5抗体、85H7-2B11v6抗体、85H7-2B11v7抗体、85H7-2B11v8抗体、又は33H4-1G3抗体に関する。これらの抗体はそれぞれ、表2に示された配列番号に記載のアミノ酸配列からなる重鎖(HC)及び軽鎖(LC)を有する。 In one embodiment, the present invention provides 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7- The present invention relates to 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody. Each of these antibodies has a heavy chain (HC) and a light chain (LC) consisting of the amino acid sequences set forth in SEQ ID NOs shown in Table 2.
 一態様において、本発明の抗体は、細胞表面上のRAMP2内の細胞外領域の立体構造を特異的に認識する。例えば、本発明の抗体は、25H4-4F9抗体、25H4-4F9v1抗体、25H4-4F9v2抗体、25H4-4F9v3抗体、25H4-4F9v4抗体、25H4-4F9v5抗体、25H4-4F9v6抗体、25H4-4F9v8抗体、85H7-2B11抗体、85H7-2B11v1抗体、85H7-2B11v2抗体、85H7-2B11v3抗体、85H7-2B11v4抗体、85H7-2B11v5抗体、85H7-2B11v6抗体、85H7-2B11v7抗体、85H7-2B11v8抗体、又は33H4-1G3抗体が結合する、細胞表面上のRAMP2内に存在し得るエピトープを特異的に認識する。 In one embodiment, the antibody of the present invention specifically recognizes the conformation of the extracellular domain in RAMP2 on the cell surface. For example, the antibodies of the present invention can be 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7- 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody bound Specifically recognize an epitope that may be present in RAMP2 on the cell surface.
 また、本発明の抗体は、RAMP2の細胞外領域を構成するアミノ酸配列の一次構造をエピトープとして認識しない。ここで、アミノ酸配列の一次構造をエピトープとして認識しないとは、ホールディングされていない(立体構造を形成していない)直鎖状の当該アミノ酸配列からなるペプチド又はタンパク質とは結合しないことを意味する。より具体的には、本発明の抗体は、RAMP2の細胞外領域を構成するアミノ酸配列である、配列番号71~101に記載の各アミノ酸配列を有するペプチドにいずれとも結合しない。 Furthermore, the antibody of the present invention does not recognize the primary structure of the amino acid sequence constituting the extracellular region of RAMP2 as an epitope. Here, not recognizing the primary structure of the amino acid sequence as an epitope means that it does not bind to an unfolded (not forming a three-dimensional structure) linear peptide or protein consisting of the amino acid sequence. More specifically, the antibody of the present invention does not bind to any of the peptides having the amino acid sequences set forth in SEQ ID NOS: 71 to 101, which are amino acid sequences constituting the extracellular region of RAMP2.
 本明細書において、被検抗体が、目的のエピトープ(例えば、上述の特定の抗体が結合するエピトープ)、抗原、又はペプチドと「特異的に結合する(認識する)」か否かは、当該エピトープを有するペプチド若しくはタンパク質、当該抗原、又は当該ペプチドへの当該抗体の結合と、その他のペプチド、タンパク質又はその他の抗原への当該抗体の結合を調べることにより判定することができる。特に、本発明の抗体は、細胞表面上のRAMP2内の細胞外領域の立体構造を特異的に認識することから、前記方法における当該エピトープを有するタンパク質として、細胞表面上に発現させたRAMP2を用いることにより結合特異性を判定することができる。目的のエピトープを有するペプチド若しくはタンパク質、目的の抗原、又は目的のペプチドと結合し、その他のペプチド、タンパク質、又はその他の抗原と結合しない場合、当該被検抗体は目的のエピトープ、抗原、又はペプチドと特異的に結合する(認識する)と判定される。 In the present specification, whether or not the test antibody "specifically binds (recognizes)" an epitope of interest (for example, an epitope to which the specific antibody described above binds), an antigen, or a peptide depends on the epitope. It can be determined by examining the binding of the antibody to the peptide or protein having the antigen, the antigen, or the peptide, and the binding of the antibody to another peptide, protein, or other antigen. In particular, since the antibody of the present invention specifically recognizes the three-dimensional structure of the extracellular domain in RAMP2 on the cell surface, RAMP2 expressed on the cell surface is used as the protein having the epitope in the method. Thus, the binding specificity can be determined. When the peptide or protein having the epitope of interest, the antigen of interest, or the peptide of interest binds to the peptide, but does not bind to other peptides, proteins, or other antigens, the test antibody is the epitope, antigen, or peptide of interest It is determined that they specifically bind (recognize).
 また、本明細書において、被検抗体が、特定のペプチド又はタンパク質と結合しないことは、当該ペプチド若しくはタンパク質への当該抗体の結合を調べることにより判定することができる。 In addition, in the present specification, that a test antibody does not bind to a specific peptide or protein can be determined by examining the binding of the antibody to the peptide or protein.
 別の態様において、本発明の抗体は、25H4-4F9抗体、25H4-4F9v1抗体、25H4-4F9v2抗体、25H4-4F9v3抗体、25H4-4F9v4抗体、25H4-4F9v5抗体、25H4-4F9v6抗体、25H4-4F9v8抗体、85H7-2B11抗体、85H7-2B11v1抗体、85H7-2B11v2抗体、85H7-2B11v3抗体、85H7-2B11v4抗体、85H7-2B11v5抗体、85H7-2B11v6抗体、85H7-2B11v7抗体、85H7-2B11v8抗体、又は33H4-1G3抗体(本段落において、「25H4-4F9抗体等」という)と、RAMP2(特には、細胞表面上のRAMP2)への結合において競合する。被検抗体が、25H4-4F9抗体等と、RAMP2への結合において競合するか否かは、標識化された25H4-4F9抗体等を被検抗体と共に抗原(RAMP2)に接触させることにより決定することができる。好ましくは、コントロールとして、被検抗体非存在下で標識化25H4-4F9抗体等を単独で抗原(RAMP2)に接触させたものを用いることができる。抗原に結合した標識化25H4-4F9抗体等の標識量を比較して、被検抗体と同時に抗原に接触させた場合の標識化25H4-4F9抗体等の抗原への結合量が被検抗体非存在下で抗原に接触させた場合の標識化25H4-4F9抗体等の抗原への結合量よりも少ない場合には、当該被検抗体は用いた25H4-4F9抗体等と競合すると決定することができる。 In another embodiment, the antibody of the present invention is a 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody , 85H7-2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody or 33H4-1G3 It competes with the antibody (referred to in this paragraph as "25H4-4F9 antibody etc") in binding to RAMP2 (especially RAMP2 on the cell surface). Whether the test antibody competes with the 25H4-4F9 antibody etc. for binding to the RAMP2 or not is determined by contacting the labeled 25H4-4F9 antibody etc. with the test antibody to the antigen (RAMP2) Can. Preferably, as a control, one in which a labeled 25H4-4F9 antibody alone is brought into contact with an antigen (RAMP2) in the absence of a test antibody can be used. The amount of binding to the antigen such as the labeled 25H4-4F9 antibody in the case of contacting the antigen simultaneously with the test antibody by comparing the amount of labeling such as the labeled 25H4-4F9 antibody bound to the antigen is the absence of the test antibody If the amount of binding of the labeled 25H4-4F9 antibody or the like to the antigen is lower than when it is contacted with the antigen below, it can be determined that the test antibody competes with the 25H4-4F9 antibody or the like used.
 抗体とタンパク質又はペプチドとの結合の測定方法は、例えば、EIA法、ELISA法、FACS法、共免疫沈降法、プルダウンアッセイ法、ファーウェスタンブロッティング法、ホモバイファンクショナルクロスリンカー若しくはヘテロバイファンクショナルクロスリンカーを利用するクロスリンク法、ラベル転移反応法、相互作用マッピング法、表面プラズモン共鳴法、FRET(Fluorescence resonance energy transfer)法、BIACORE法、AlphaScreen(登録商標)やAlphaLISA(登録商標)などのAlphaPPIアッセイ(PerkinElmer)など、多様な方法が本技術分野において周知であり、目的とする結合の検出に応じて適宜好ましい方法を採用することができる。 The method for measuring the binding of an antibody to a protein or peptide is, for example, EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, far western blotting method, homobifunctional crosslinker or heterobifunctional cross Linker-based cross-linking method, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (fluorescence resonance energy transfer) method, BIACORE method, AlphaScreen (registered trademark), AlphaPPI assay such as AlphaLISA (registered trademark) A variety of methods are known in the art, such as (PerkinElmer), and where appropriate a preferred method depending on the detection of the desired binding. It is possible to use.
 一態様において、本発明は、VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、VLが配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片に関する。本明細書において、ストリンジェントな条件下でハイブリダイズするとは、当業者に通常用いられるハイブリダイゼーション条件でハイブリダイズすることを意味する。例えば、Molecular Cloning,A Laboratory Mannual,Second Edition,Cold Spring Harbor Laboratory Press(1989)記載の方法によりハイブリダイズするか否かを決定することができる。例えば、ハイブリダイズの条件は、6×SSC(0.9M NaCl,0.09M クエン酸三ナトリウム)または6×SSPE(3M NaCl,0.2M NaHPO,20mM EDTA・2Na,pH7.4)中42℃でハイブリダイズさせ、その後42℃で0.5×SSCで洗浄する条件であってもよい。好ましくは、本発明は、VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,又は53に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、それぞれ順に、VLが配列番号4、14,21、37,39,41、43、55,57,59,又は61に記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片である。 In one aspect, the invention is set forth in any one of VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. And has an amino acid sequence encoded by a nucleic acid sequence that hybridizes with the nucleic acid sequence encoding the amino acid sequence under stringent conditions, and wherein VL is SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, An antibody having an amino acid sequence encoded by a nucleic acid sequence hybridizing under stringent conditions with a nucleic acid sequence encoding an amino acid sequence described in any one of 55, 57, 59, and 61, or an antibody thereof It relates to an immunoreactive fragment. As used herein, hybridizing under stringent conditions means hybridizing under hybridization conditions commonly used by those skilled in the art. For example, it can be determined by the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). For example, the hybridization conditions are 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0.2 M NaH 2 PO 4 , 20 mM EDTA · 2 Na, pH 7.4) The conditions may be such that hybridization is carried out at 42 ° C. and then washing with 0.5 × SSC at 42 ° C. Preferably, the present invention relates to a nucleic acid sequence wherein the VH encodes an amino acid sequence as set forth in SEQ ID NO: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, or 53. SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, each having an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions, and Or an antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence described in 61 under stringent conditions, or an immunoreactive fragment thereof.
 また、本発明は、VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つのアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つのアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片に関する。アミノ酸配列の同一性は、2種類のタンパク質間において,比較対象とするアミノ酸配列範囲における種類が同一なアミノ酸数の割合(%)を意味し、例えば、BLAST、FASTA等の公知のプログラムを用いて決定することができる。本発明の抗体としては、例えば、VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つのアミノ酸配列と85%以上、90%以上、95%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つのアミノ酸配列と85%以上、90%以上、95%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片であってもよい。好ましくは、本発明の抗体は、VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つのアミノ酸配列と80%以上、85%以上、90%以上、95%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を有し、かつ、それぞれ順に、VLが、配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つのアミノ酸配列と80%以上、85%以上、90%以上、95%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片である。 Furthermore, in the present invention, 80% of the amino acid sequence of any one of SEQ ID NOs: 2, 12, 19, 27, 29, 31, 31, 33, 35, 45, 47, 49, 51, and 53 Any one amino acid having an amino acid sequence having the above identity and in which VL is selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 The present invention relates to an antibody having an amino acid sequence having 80% or more identity to the sequence, or an immunoreactive fragment thereof. The identity of the amino acid sequence means the percentage (%) of the number of identical amino acids in the amino acid sequence to be compared between the two types of proteins, for example, using a known program such as BLAST or FASTA. It can be decided. In the antibody of the present invention, for example, any one amino acid sequence wherein VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And an amino acid sequence having an identity of 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, and VL has a sequence of SEQ ID NO: 4, 14, 21, 37, 39, 41 43, 55, 57, 59, and 61, and an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity with any one amino acid sequence selected from It may be an antibody or an immunoreactive fragment thereof. Preferably, in the antibody of the present invention, any one amino acid sequence wherein VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And an amino acid sequence having an identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, and in each of which, VL has SEQ ID NO: 4, 14, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or any one amino acid sequence selected from 21, 37, 39, 41, 43, 55, 57, 59, and 61 An antibody having an amino acid sequence having an identity of 99% or more or an immunoreactive fragment thereof.
 好ましくは、本発明は、本明細書の表2に記載の配列番号に記載された重鎖(VC)と軽鎖(LC)のアミノ酸配列、又はVHとVLのアミノ酸配列、又はCDRH1~3とCDRL1~3のアミノ酸配列の組み合わせを有する抗体である。 Preferably, the present invention relates to the amino acid sequences of heavy chain (VC) and light chain (LC) described in SEQ ID NO: listed in Table 2 herein, or the amino acid sequences of VH and VL, or CDRH1-3. It is an antibody having a combination of amino acid sequences of CDRL1-3.
 上述のVH及びVLが、特定のアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片、並びに、VH及びVLが、特定のアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片は、好ましくは、以下に記載されたCDRHs及びCDRLsを有する。 An antibody or an immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the specific amino acid sequence under the stringent conditions described above, and VH and VL An antibody having an amino acid sequence having 80% or more identity with a specific amino acid sequence, or an immunoreactive fragment thereof preferably has CDRHs and CDRLs described below.
 一態様において、本発明は、LXIXDXYXYXMX(配列番号24)のアミノ酸配列からなる重鎖相補性決定領域(CDRH)3を有する重鎖を含む抗体又はその免疫反応性断片に関する。ここで、XはG又はMであり、XはF又はYであり、XはA又はVであり、Xは存在しないかHであり、XはAかVであり、XはDかEであり、XはYかNである。好ましくは、本発明はLMIFDAYYAMDY(配列番号7)又はLGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有する重鎖を含む抗体又はその免疫反応性断片である。抗体において、CDRH3がその特異性決定において重要であることは既に知られている(Xu JLら、Immunity. 2000 Jul;13(1):37-45.)。CDRH3は、好ましくは、LMIFDAYYAMDY(配列番号7)又はLGIYDVYHYVMEN(配列番号16)のアミノ酸配列を有する。 In one aspect, the invention provides an antibody or heavy chain comprising a heavy chain complementarity determining region (CDRH) 3 consisting of the amino acid sequence of LX 1 IX 2 DX 3 YX 4 YX 5 MX 6 X 7 (SEQ ID NO: 24) It relates to the immunoreactive fragment. Here, X 1 is G or M, X 2 is F or Y, X 3 is A or V, X 4 is absent or H, and X 5 is A or V, X 6 is D or E, and X 7 is Y or N. Preferably, the present invention is an antibody or immunoreactive fragment thereof comprising a heavy chain having a CDRH3 consisting of the amino acid sequence of LMIFDAYYAMD (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16). In antibodies, it is already known that CDRH3 is important in determining its specificity (Xu JL et al., Immunity. 2000 Jul; 13 (1): 37-45.). CDRH 3 preferably has the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16).
 好ましくは、本発明の抗体又はその免疫反応性断片は、GYFMN(配列番号5)のアミノ酸配列からなるCDRH1を有する。
 また、好ましくは、本発明の抗体又はその免疫反応性断片は、RXNPYNGDX10YX11QKFX12G(配列番号66)又はRXNPYNGDX10YNQKFKG(配列番号23)のアミノ酸配列からなるCDRH2を有する。ここで、XはN又はIであり、XはS又はTであり、X10はI、F又はLであり、X11はA又はNであり、X12はQ又はKである。CDRH2は、好ましくは、RNNPYNGDSIYNQKFKG(配列番号6)、RINPYNGDTFYNQKFKG (配列番号15)、RINPYNGDTLYNQKFKG(配列番号22)、RNNPYNGDSIYNEKFQG(配列番号62)、RNNPYNGDSIYAEKFQG(配列番号63)、RINPYNGDTFYNQKFQG(配列番号64)、又はRINPYNGDTFYAQKFQG(配列番号65)のアミノ酸配列を有する。
Preferably, the antibody or immunoreactive fragment thereof of the present invention has a CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5).
Also preferably, the antibody of the present invention or the immunoreactive fragment thereof comprises the amino acid sequence of RX 8 NPYNGDX 9 X 10 YX 11 QKFX 12 G (SEQ ID NO: 66) or RX 8 NPYNGDX 9 X 10 YNQ KFK (SEQ ID NO: 23) And CDRH2. Here, X 8 is N or I, X 9 is S or T, X 10 is I, F or L, X 11 is A or N, and X 12 is Q or K. Preferably, the CDRRH2 is RNNPYNGDSIYNQKFKG (SEQ ID NO: 6), RINPYNGDTFYNQKFKG (SEQ ID NO: 15), RINPYNGDTLYNQKFKG (SEQ ID NO: 22), RNNPYNGDSIYNEKF QG (SEQ ID NO: 62), RNNPYNGDSIYAEKF QG (SEQ ID NO: 63), It has the amino acid sequence of (SEQ ID NO: 65).
 更に好ましくは、本発明の抗体又はその免疫反応性断片は、X13ASQDIRNYLN(配列番号69)のアミノ酸配列からなる軽鎖相補性決定領域(CDRL)1を有する。ここで、X13はQ又はRである。CDRL1は、好ましくは、RASQDIRNYLN(配列番号8)又はQASQDIRNYLN(配列番号67)のアミノ酸配列を有する。 More preferably, the antibody of the present invention or an immunoreactive fragment thereof has a light chain complementarity determining region (CDRL) 1 consisting of the amino acid sequence of X 13 ASQDIRNYLN (SEQ ID NO: 69). Here, X 13 is Q or R. CDRL1 preferably has the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO: 67).
 また、本発明の抗体又はその免疫反応性断片は、YTSRLX1415(配列番号70)のアミノ酸配列からなるCDRL2を有する。ここで、X14はE又はHであり、X15はS又はTである。CDRL2は、好ましくは、YTSRLHS(配列番号9)又はYTSRLET(配列番号68)のアミノ酸配列を有する。 Furthermore, the antibody of the present invention or the immunoreactive fragment thereof has a CDRL2 consisting of the amino acid sequence of YTSRLX 14 X 15 (SEQ ID NO: 70). Here, X 14 is E or H, and X 15 is S or T. CDRL2 preferably has the amino acid sequence of YTSRLHS (SEQ ID NO: 9) or YTSRLET (SEQ ID NO: 68).
 また、本発明の抗体又はその免疫反応性断片は、及びQQDSKX16PWT(配列番号25)のアミノ酸配列からなるCDRL3を有する。ここで、X16はH又はNである。CDRL3は、好ましくはQQDSKHPWT(配列番号10)又はQQDSKNPWT(配列番号17)のアミノ酸配列を有する。 In addition, the antibody of the present invention or the immunoreactive fragment thereof has a CDRL3 consisting of the amino acid sequence of QQDSKX 16 PWT (SEQ ID NO: 25). Here, X 16 is H or N. CDRL 3 preferably has the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT (SEQ ID NO: 17).
 一例において、本発明の抗体は以下の(i)~(vii)のいずれか1つの重鎖CDRの組み合わせを有する:
(i)配列番号5のアミノ酸配列からなるCDRH1、配列番号6のアミノ酸配列からなるCDRH2、及び配列番号7のアミノ酸配列からなるCDRH3;
(ii)配列番号5のアミノ酸配列からなるCDRH1、配列番号62のアミノ酸配列からなるCDRH2、及び配列番号7のアミノ酸配列からなるCDRH3;
(iii)配列番号5のアミノ酸配列からなるCDRH1、配列番号63のアミノ酸配列からなるCDRH2、及び配列番号7のアミノ酸配列からなるCDRH3;
(iv)配列番号5のアミノ酸配列からなるCDRH1、配列番号15のアミノ酸配列からなるCDRH2、及び配列番号16のアミノ酸配列からなるCDRH3;
(v)配列番号5のアミノ酸配列からなるCDRH1、配列番号64のアミノ酸配列からなるCDRH2、及び配列番号16のアミノ酸配列からなるCDRH3;
(vi)配列番号5のアミノ酸配列からなるCDRH1、配列番号65のアミノ酸配列からなるCDRH2、及び配列番号16のアミノ酸配列からなるCDRH3;又は、
(vii)配列番号5のアミノ酸配列からなるCDRH1、RINPYNGDTLYNQKFKG(配列番号22)のアミノ酸配列からなるCDRH2、及び配列番号16のアミノ酸配列からなるCDRH3。
In one example, the antibody of the present invention has a combination of heavy chain CDRs of any one of the following (i) to (vii):
(I) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7;
(Ii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 62, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7;
(Iii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 63, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7;
(Iv) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 15, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16;
(V) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16;
(Vi) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 65, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16;
(Vii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of RINPYNGDTLYNQKFKG (SEQ ID NO: 22), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16.
 更に、本発明の抗体は、以下の(a)~(f)のいずれか1つの軽鎖CDRの組み合わせを有していてもよい:
(a)配列番号8のアミノ酸配列からなるCDRL1、配列番号9のアミノ酸配列からなるCDRL2、及び配列番号10のアミノ酸配列からなるCDRL3;
(b)QASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、配列番号9のアミノ酸配列からなるCDRL2、及び配列番号10のアミノ酸配列からなるCDRL3;
(c)配列番号67のアミノ酸配列からなるCDRL1、配列番号68のアミノ酸配列からなるCDRL2、及び配列番号10のアミノ酸配列からなるCDRL3;
(d)配列番号8のアミノ酸配列からなるCDRL1、配列番号9のアミノ酸配列からなるCDRL2、及び配列番号17のアミノ酸配列からなるCDRL3;
(e)配列番号67のアミノ酸配列からなるCDRL1、配列番号9のアミノ酸配列からなるCDRL2、及び配列番号17のアミノ酸配列からなるCDRL3;又は、
(f)配列番号67のアミノ酸配列からなるCDRL1、配列番号68のアミノ酸配列からなるCDRL2、及び配列番号17のアミノ酸配列からなるCDRL3。
Furthermore, the antibody of the present invention may have a combination of light chain CDRs of any one of the following (a) to (f):
(A) CDR L1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR L2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDR L3 consisting of the amino acid sequence of SEQ ID NO: 10;
(B) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67), CDRL2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 10;
(C) CDRL1 consisting of the amino acid sequence of SEQ ID NO: 67, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 68, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 10;
(D) CDR L1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR L2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDR L3 consisting of the amino acid sequence of SEQ ID NO: 17;
(E) CDRL1 consisting of the amino acid sequence of SEQ ID NO: 67, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 17;
(F) CDR L1 consisting of the amino acid sequence of SEQ ID NO: 67, CDR L2 consisting of the amino acid sequence of SEQ ID NO: 68, and CDR L3 consisting of the amino acid sequence of SEQ ID NO: 17.
 例えば、本発明は、以下のCDRの組み合わせを有する抗体又はその免疫反応性断片を含む: For example, the invention includes antibodies or immunoreactive fragments thereof having the following combinations of CDRs:
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
 本明細書において、アミノ酸は一文字表記で表される。具体的には、Aはアラニン、Lはロイシン、Rはアルギニン、Kはリシン、Nはアスパラギン、Mはメチオニン、Dはアスパラギン酸、Fはフェニルアラニン、Cはシステイン、Pはプロリン、Qはグルタミン、Sはセリン、Eはグルタミン酸、Tはトレオニン、Gはグリシン、Wはトリプトファン、Hはヒスチジン、Yはチロシン、Iはイソロイシン、Vはバリンを表す。 In the present specification, amino acids are represented by a single letter code. Specifically, A is alanine, L is leucine, R is arginine, K is lysine, N is asparagine, M is methionine, D is aspartic acid, F is phenylalanine, C is cysteine, P is proline, Q is glutamine, S represents serine, E represents glutamic acid, T represents threonine, G represents glycine, W represents tryptophan, H represents histidine, Y represents tyrosine, I represents isoleucine, and V represents valine.
 本発明の抗体及びその免疫反応性断片は、in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有する。被検抗体又はその免疫反応性断片がin vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有するか否かは、適切な培地中で数時間程度前培養した血管内皮細胞に被検抗体又はその免疫反応性断片を添加し、AMを終濃度50~200nMとなるように添加して、2~7日培養した後における血管内皮細胞の増殖を観察することにより行うことができる。血管内皮細胞の増殖は、細胞数をカウントしてもよいし、細胞数の指標となるOD450等を測定してもよい。被検抗体又はその免疫反応性断片を添加せずに培養した血管内皮細胞数と比較して、被検抗体又はその免疫反応性断片の存在下で培養した血管内皮細胞数が少ない場合、当該被検抗体又はその免疫反応性断片は、in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有すると判定される。また、被検抗体又はその免疫反応性断片を添加せずに培養した血管内皮細胞数を100として、被検抗体又はその免疫反応性断片の存在下で培養した血管内皮細胞数が50となる、被検抗体又はその免疫反応性断片の濃度をIC50値(M)として求めてもよい。例えば、本発明の抗体は、IC50値が35nM以下、30nM以下、25nM以下、20nM以下、15nM以下、10nM以下、5nM以下、4nM以下、3nM以下、2nM以下、若しくは1.6nM以下であってもよい。IC50値の下限値は特に定められる必要はないが、例えば、0.1nM以上、0.2nM以上、0.3nM以上、0.4nM以上、0.5nM以上、0.6nM以上、0.7nM以上、若しくは0.8nM以上であってもよい。また、IC50値としては、これらの上限値及び下限値の任意の組み合わせの範囲であってもよく、例えば、0.1~35nM、0.8~32.7nM、0.2~30nM、0.3~25nM、0.4~20nM、0.5~15nM、0.5~10nM、0.5~5nM、0.8~1.6nMであってもよい。また、これらのIC50値の場合、当該被検抗体又はその免疫反応性断片は、in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有すると判定してもよい。 The antibody of the present invention and its immunoreactive fragment have a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. Whether or not the test antibody or its immunoreactive fragment has a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro was precultured for several hours in an appropriate medium By adding a test antibody or its immunoreactive fragment to vascular endothelial cells, adding AM to a final concentration of 50 to 200 nM, and observing proliferation of vascular endothelial cells after culturing for 2 to 7 days It can be carried out. The proliferation of vascular endothelial cells may be performed by counting the number of cells, or by measuring OD450 as an indicator of the number of cells. When the number of vascular endothelial cells cultured in the presence of the test antibody or the immunoreactive fragment thereof is smaller than the number of vascular endothelial cells cultured without the addition of the test antibody or the immunoreactive fragment thereof, The test antibody or the immunoreactive fragment thereof is determined to have a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. In addition, the number of vascular endothelial cells cultured without adding the test antibody or its immunoreactive fragment is 100, and the number of vascular endothelial cells cultured in the presence of the test antibody or its immunoreactive fragment is 50. The concentration of the test antibody or immunoreactive fragment thereof may be determined as an IC50 value (M). For example, the antibody of the present invention may have an IC50 value of 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1.6 nM or less Good. Although the lower limit value of the IC 50 value need not be particularly defined, for example, 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, 0.5 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more Or 0.8 nM or more. In addition, the IC50 value may be in the range of any combination of these upper and lower limits, and for example, 0.1 to 35 nM, 0.8 to 32.7 nM, 0.2 to 30 nM, 0.1. It may be 3 to 25 nM, 0.4 to 20 nM, 0.5 to 15 nM, 0.5 to 10 nM, 0.5 to 5 nM, or 0.8 to 1.6 nM. In addition, in the case of these IC50 values, even if it is determined that the test antibody or the immunoreactive fragment thereof has a growth inhibitory effect on vascular endothelial cells in the vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. Good.
 好ましくは、本発明の抗体又はその免疫反応性断片は、in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用に加えて、アドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有する。被検抗体又はその免疫反応性断片がアドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有するか否かは、アドレノメジュリン添加によりcAMPを産生する動物細胞(例えば、hCR2-CHOK1細胞)を適切な培地で一晩培養し、被検抗体又はその免疫反応性断片を添加し、終濃度50~300pMのAMを添加し、cAMPを測定することにより決定することができる。cAMPは、市販のキット(例えば、LANCE Ultra cAMP kit(Perkinelmer社)など)を用いて測定することができる。抗体非添加の細胞によるcAMP産生と比較して、被検抗体又はその免疫反応性断片添加の細胞によるcAMP産生が少ない場合、当該抗体又はその免疫反応性断片は、アドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有すると判定される。また、被検抗体又はその免疫反応性断片を添加せずに培養した細胞が産生するcAMPを100として、被検抗体又はその免疫反応性断片の存在下で培養した細胞が産生するcAMPが50となる、被検抗体又はその免疫反応性断片の濃度をIC50値(M)として求めてもよい。例えば、本発明の抗体は、IC50値が、2nM以下、1.5nM以下、1nM以下、0.5nM以下、0.4nM以下、0.3nM以下、0.2nM以下、若しくは0.15nM以下であってもよい。IC50値の下限値は特に定められる必要はないが、例えば、0.01nM以上、0.02nM以上、若しくは0.03nM以上であってもよい。また、IC50値としては、これらの上限値及び下限値の任意の組み合わせの範囲であってもよく、例えば、0.01~2nM、0.01~1.5nM、0.01~1nM、0.01~0.5nM、0.03~1.35nM、又は0.03~0.15nMであってもよい。また、これらのIC50値の場合、当該被検抗体又はその免疫反応性断片は、アドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有すると判定してもよい。 Preferably, the antibody of the present invention or an immunoreactive fragment thereof according to the present invention has cAMP in animal cells by addition of adrenomedullin in addition to the growth inhibitory action of vascular endothelial cells in the vascular endothelial cell proliferation test by addition of adrenomedullin in vitro. It has the effect of suppressing production. Whether the test antibody or the immunoreactive fragment thereof has the effect of suppressing cAMP production by animal cells by addition of adrenomedullin depends on the animal cells producing cAMP by addition of adrenomedullin (for example, hCR2-CHOK1 cells) Is cultured overnight in an appropriate medium, the test antibody or its immunoreactive fragment is added, and a final concentration of 50 to 300 pM of AM is added, and this can be determined by measuring cAMP. cAMP can be measured using a commercially available kit (eg, LANCE Ultra cAMP kit (Perkinelmer) and the like). When the amount of cAMP produced by cells containing the test antibody or its immunoreactive fragment is low as compared to cAMP produced by cells without added antibody, the antibody or the immunoreactive fragment thereof is treated with animal cells by addition of adrenomedullin. It is determined to have the effect of suppressing cAMP production. In addition, when the cAMP produced by cells cultured without the addition of the test antibody or its immunoreactive fragment is 100, the cAMP produced by the cells cultured in the presence of the test antibody or its immunoreactive fragment is 50 The concentration of the test antibody or the immunoreactive fragment thereof may be determined as an IC 50 value (M). For example, the antibody of the present invention has an IC50 value of 2 nM or less, 1.5 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.15 nM or less May be The lower limit value of the IC50 value need not be particularly defined, but may be, for example, 0.01 nM or more, 0.02 nM or more, or 0.03 nM or more. In addition, the IC50 value may be in the range of any combination of these upper and lower limit values, and, for example, 0.01 to 2 nM, 0.01 to 1.5 nM, 0.01 to 1 nM, 0.1. It may be 01 to 0.5 nM, 0.03 to 1.35 nM, or 0.03 to 0.15 nM. Moreover, in the case of these IC50 values, it may be determined that the test antibody or the immunoreactive fragment thereof has an effect of suppressing cAMP production by animal cells by addition of adrenomedullin.
 好ましくは、本発明の抗体又はその免疫反応性断片は、in vitroでヒトCRLRとヒトRAMP2を共発現する細胞に結合する。本明細書において、「ヒトCRLRとヒトRAMP2を共発現する細胞」は、ヒトCRLRとヒトRAMP2を細胞遺伝子導入して得られた安定発現細胞であって、ヒトCRLRとヒトRAMP2を共発現する細胞あれば特に限定されるものではないが、好ましくは、ハムスター卵巣細胞(CHO-K1)又はNIH3T3細胞にヒトCRLRとヒトRAMP2を遺伝子導入して得られた安定発現細胞株(hCR2-CHOK1又はhCR2-NIH3T3)である。ヒトCRLRとヒトRAMP2を共発現する細胞は、ヒトCRLR(NM_005795)及びヒトRAMP2(NM_005854)を遺伝子導入用のベクターにタンデムに組み込み、目的の細胞に遺伝子導入することにより得ることができる。安定発現株は、予めベクターに挿入されていた薬剤耐性遺伝子に対応する薬剤存在下で選択培養することにより得ることができる。 Preferably, the antibody or immunoreactive fragment thereof of the present invention binds in vitro to cells co-expressing human CRLR and human RAMP2. As used herein, “a cell co-expressing human CRLR and human RAMP2” is a stably expressing cell obtained by introducing human CRLR and human RAMP2 into a cell gene, and a cell co-expressing human CRLR and human RAMP2 There is no particular limitation if it is present, but preferably, a stably expressing cell line (hCR2-CHOK1 or hCR2-) obtained by gene transfer of human CRLR and human RAMP2 into hamster ovary cells (CHO-K1) or NIH3T3 cells. NIH 3 T 3). A cell co-expressing human CRLR and human RAMP2 can be obtained by integrating human CRLR (NM_005795) and human RAMP2 (NM_005854) into a vector for gene transfer in tandem, and transferring the gene into a target cell. A stable expression strain can be obtained by selective culture in the presence of a drug corresponding to a drug resistance gene previously inserted into a vector.
 例えば、本発明の抗体は、ヒトCRLRとヒトRAMP2を共発現する細胞を用いたCell ELISA試験による結合親和性(EC50)が3.5nM以下であってもよい。例えば、被検抗体のイムノグロブリンクラスがIgGの場合、Cell ELISA試験は、以下の方法により行うことができる。適切な培地中のヒトCRLRとヒトRAMP2を共発現する細胞を1×10~1×10cells/wellで96well培養プレートに添加して一晩培養した後、4%パラホルムアルデヒド溶液を添加して4℃で数十分間静置することにより細胞を固定し、洗浄した後1%BSA-PBSで1時間以上のブロッキング処理を行う。精製した各被検抗体を適宜段階希釈して添加し、二次抗体として標識化抗IgG抗体を反応させ、当該標識に合致する試薬を反応させて、標識による発光強度や発色強度を測定することにより結合量を決定することができる。EC50値は最大結合量の50%と結合する抗体濃度として求めることができる。例えば、本発明の抗体は、EC50値が、4nM以下、3.5nM以下、3.19nM以下、3nM以下、2.5nM以下、2nM以下、1.5nM以下、1nM以下、若しくは0.5nM以下であってもよい。EC50値の下限値は特に定められる必要はないが、例えば、0.01nM以上、0.03nM以上、0.05nM以上、若しくは0.14nM以上であってもよい。また、EC50値としては、これらの上限値及び下限値の任意の組み合わせの範囲であってもよく、例えば、0.01~4nM、0.03~3nM、0.05~2nM、又は0.14~3.19nMであってもよい。また、これらのEC50値の場合、当該被検抗体又はその免疫反応性断片は、in vitroでヒトCRLRとヒトRAMP2を共発現する細胞に結合すると判定してもよい。 For example, the antibody of the present invention may have a binding affinity (EC50) of 3.5 nM or less in a Cell ELISA test using cells co-expressing human CRLR and human RAMP2. For example, when the immunoglobulin class of the test antibody is IgG, the Cell ELISA test can be performed by the following method. Cells co-expressing human CRLR and human RAMP2 in an appropriate medium are added to a 96-well culture plate at 1 × 10 3 to 1 × 10 5 cells / well and cultured overnight, and then 4% paraformaldehyde solution is added. The cells are fixed by standing at 4 ° C. for several dozen minutes, and after washing, blocking treatment with 1% BSA-PBS is performed for 1 hour or more. Purified each test antibody is added in appropriate serial dilutions, reacted with a labeled anti-IgG antibody as a secondary antibody, reacted with a reagent that matches the label, and measure the luminescence intensity and coloring intensity by the label. The amount of binding can be determined by EC50 values can be determined as the antibody concentration that binds 50% of the maximal binding. For example, the antibody of the present invention has an EC50 value of 4 nM or less, 3.5 nM or less, 3.19 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, 1 nM or less, or 0.5 nM or less It may be. The lower limit value of the EC50 value need not be particularly defined, but may be, for example, 0.01 nM or more, 0.03 nM or more, 0.05 nM or more, or 0.14 nM or more. In addition, the EC50 value may be in the range of any combination of these upper and lower limit values, for example, 0.01 to 4 nM, 0.03 to 3 nM, 0.05 to 2 nM, or 0.14. It may be ̃3.19 nM. In addition, in the case of these EC50 values, it may be determined that the test antibody or the immunoreactive fragment thereof binds to cells co-expressing human CRLR and human RAMP2 in vitro.
(核酸分子)
 別の態様において、本発明は、上述の本発明の抗体又はその免疫反応性断片をコードするポリヌクレオチドを有する核酸分子に関する。具体的には、本発明の核酸分子は、VHとして、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載のアミノ酸をコードするポリヌクレオチドを有し、かつ、VLとして配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子であってもよい。例えば、本発明の核酸分子は、VHとして配列番号2及びVLとして配列番号4に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号27及びVLとして配列番号37に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号29及びVLとして配列番号37に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号29及びVLとして配列番号39に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号31及びVLとして配列番号39に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号31及びVLとして配列番号41に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号33及びVLとして配列番号41に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号35及びVLとして配列番号43に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号12及びVLとして配列番号14に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号45及びVLとして配列番号55に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号12及びVLとして配列番号14に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号47及びVLとして配列番号55に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号47及びVLとして配列番号57に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号49及びVLとして配列番号57に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号49及びVLとして配列番号59に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号51及びVLとして配列番号59に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号51及びVLとして配列番号61に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;VHとして配列番号53及びVLとして配列番号61に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子;並びに、VHとして配列番号19及びVLとして配列番号21に記載のアミノ酸配列をコードするポリヌクレオチドを有する核酸分子であってもよい。
(Nucleic acid molecule)
In another aspect, the invention relates to a nucleic acid molecule comprising a polynucleotide encoding an antibody of the invention as described above or an immunoreactive fragment thereof. Specifically, the nucleic acid molecule of the present invention is any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 as VH. Any one selected from SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 as a VL; The nucleic acid molecule may have a polynucleotide encoding the amino acid sequence described in For example, the nucleic acid molecule of the present invention is a nucleic acid molecule having a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as VH and SEQ ID NO: 4 as VL; amino acids of SEQ ID NO: 27 as VH and SEQ ID NO: 37 as VL A nucleic acid molecule having a polynucleotide encoding the sequence; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 29 as VH and SEQ ID NO: 37 as VL; described in SEQ ID NO: 39 as VH A nucleic acid molecule having a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 as a VH, and a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 39 as a VL; The amino acid sequence described in A nucleic acid molecule having a polynucleotide which comprises the polynucleotide; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 33 as VH and SEQ ID NO: 41 as VL; an amino acid set forth in SEQ ID NO: 43 as VH A nucleic acid molecule having a polynucleotide encoding a sequence; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 12 as VH and SEQ ID NO: 14 as VL; described in SEQ ID NO: 55 as VH A nucleic acid molecule having a polynucleotide encoding the amino acid sequence of SEQ ID NO: 12 and a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 14 as the VH; Described in A nucleic acid molecule having a polynucleotide encoding an amino acid sequence; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 47 as VH and SEQ ID NO: 57 as SEQ ID NO: 49 as VH Nucleic acid molecule having a polynucleotide encoding the described amino acid sequence; nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 49 as VH and SEQ ID NO: 59 as VL; SEQ ID NO: 51 as VH A nucleic acid molecule having a polynucleotide encoding the amino acid sequence described in 59; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence described in SEQ ID NO: 61 as VH and SEQ ID NO: 61; Distribution A nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 61; and a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 21 as VH and SEQ ID NO: 21 .
 例えば、本発明の核酸分子は、以下の配列を有するポリヌクレオチドを含んでいてもよい:配列番号1に記載のヌクレオチド配列、及び配列番号3に記載のヌクレオチド配列;配列番号26に記載のヌクレオチド配列、及び配列番号36に記載のヌクレオチド配列;配列番号28に記載のヌクレオチド配列、及び配列番号36に記載のヌクレオチド配列;配列番号28に記載のヌクレオチド配列、及び配列番号38に記載のヌクレオチド配列;配列番号30に記載のヌクレオチド配列、及び配列番号38に記載のヌクレオチド配列;配列番号30に記載のヌクレオチド配列、及び配列番号40に記載のヌクレオチド配列;配列番号32に記載のヌクレオチド配列、及び配列番号40に記載のヌクレオチド配列;配列番号34に記載のヌクレオチド配列、及び配列番号42に記載のヌクレオチド配列;配列番号11に記載のヌクレオチド配列、及び配列番号13に記載のヌクレオチド配列;配列番号44に記載のヌクレオチド配列、及び配列番号54に記載のヌクレオチド配列;配列番号46に記載のヌクレオチド配列、及び配列番号54に記載のヌクレオチド配列;配列番号46に記載のヌクレオチド配列、及び配列番号56に記載のヌクレオチド配列;配列番号48に記載のヌクレオチド配列、及び配列番号56に記載のヌクレオチド配列;配列番号48に記載のヌクレオチド配列、及び配列番号58に記載のヌクレオチド配列;配列番号50に記載のヌクレオチド配列、及び配列番号58に記載のヌクレオチド配列;配列番号50に記載のヌクレオチド配列、及び配列番号60に記載のヌクレオチド配列;配列番号52に記載のヌクレオチド配列、及び配列番号60に記載のヌクレオチド配列;又は配列番号18に記載のヌクレオチド配列、及び配列番号20に記載のヌクレオチド配列。 For example, the nucleic acid molecule of the present invention may comprise a polynucleotide having the following sequence: the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO: 3; the nucleotide sequence set forth in SEQ ID NO: 26 And the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28 and the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28; and the nucleotide sequence set forth in SEQ ID NO: 38; The nucleotide sequence set forth in SEQ ID NO: 30 and the nucleotide sequence set forth in SEQ ID NO: 38; the nucleotide sequence set forth in SEQ ID NO: 30 and the nucleotide sequence set forth in SEQ ID NO: 40; the nucleotide sequence set forth in SEQ ID NO: 32; Nucleotide sequence as set forth in SEQ ID NO: 34; The nucleotide sequence set forth in SEQ ID NO: 42; the nucleotide sequence set forth in SEQ ID NO: 11 and the nucleotide sequence set forth in SEQ ID NO: 13; the nucleotide sequence set forth in SEQ ID NO: 44; and the nucleotide sequence set forth in SEQ ID NO: 54 The nucleotide sequence set forth in SEQ ID NO: 46, and the nucleotide sequence set forth in SEQ ID NO: 54; the nucleotide sequence set forth in SEQ ID NO: 46, and the nucleotide sequence set forth in SEQ ID NO: 56; the nucleotide sequence set forth in SEQ ID NO: 48; The nucleotide sequence set forth in SEQ ID NO: 56; the nucleotide sequence set forth in SEQ ID NO: 48, and the nucleotide sequence set forth in SEQ ID NO: 58; the nucleotide sequence set forth in SEQ ID NO: 50, and the nucleotide sequence set forth in SEQ ID NO: 58; The described nucleotide sequence, and SEQ ID NO: 6 Nucleotide sequence, and nucleotide sequence set forth in SEQ ID NO: 60 as set forth in SEQ ID NO: 52; nucleotide sequence, and nucleotide sequence set forth in SEQ ID NO: 20 set forth in or SEQ ID NO: 18 nucleotide sequence set forth in.
 更に、本発明は、前記核酸分子を有するベクターを包含する。このようなベクターとしては、抗体の発現に利用可能なベクターであれば特に制限されるものではなく、適切なウイルスベクター又はプラスミドベクターなどを使用する宿主に応じて選択することができる。別の態様において、本発明は、前記ベクターを含有する宿主細胞に関する。宿主細胞としては、抗体の発現に利用可能な宿主細胞であれば特に制限されるものではなく、哺乳類細胞(マウス細胞、ラット細胞、ウサギ細胞、ヒト細胞など)、酵母、微生物(大腸菌など)を挙げることができる。 Furthermore, the invention includes a vector comprising the nucleic acid molecule. Such a vector is not particularly limited as long as it can be used to express an antibody, and can be selected according to the host using an appropriate viral vector or plasmid vector. In another aspect, the invention relates to a host cell containing said vector. The host cell is not particularly limited as long as it can be used for antibody expression, and mammalian cells (mouse cells, rat cells, rabbit cells, human cells, etc.), yeast, microorganisms (E. coli etc.) It can be mentioned.
 また、別の態様において、本発明は、上述の本発明の抗体又はその免疫反応性断片を有効成分として含有する医薬組成物に関する。本発明の医薬組成物の対象疾患は、血管新生が発症又は増悪化に寄与する疾患又は障害であり、例えば、癌、癌転移、糖尿病性網膜症、未熟児網膜症、加齢黄斑変性症、糖尿病網膜症、網膜静脈閉塞症、血管新生緑内障、炎症性皮膚疾患、関節リウマチ、又は変形性関節症を挙げることができる。よって、本発明の医薬組成物は、これらの疾患又は障害の予防薬、治療薬、進行抑制剤又は改善薬とすることができる。あるいは、本発明は、前記医薬組成物を製造するための、本発明の抗体又はその免疫反応性断片の使用に関する。 In another aspect, the present invention relates to a pharmaceutical composition comprising the above-described antibody of the present invention or an immunoreactive fragment thereof as an active ingredient. The target disease of the pharmaceutical composition of the present invention is a disease or disorder in which angiogenesis contributes to onset or exacerbation, for example, cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, Diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis can be mentioned. Therefore, the pharmaceutical composition of the present invention can be used as a preventive, a therapeutic, a progression inhibitor or a remedy for these diseases or disorders. Alternatively, the present invention relates to the use of the antibody of the present invention or an immunoreactive fragment thereof for producing the above-mentioned pharmaceutical composition.
 本発明の医薬組成物は、患者に投与することができる製剤であれば経口又は非経口のいかなる製剤を採用してもよい。非経口投与のための組成物としては、例えば、注射剤、点鼻剤、坐剤、貼布剤、軟膏等を挙げることができる。好ましくは、注射剤である。本発明の医薬組成物の剤形は、例えば、液剤又は凍結乾燥製剤を挙げることができる。本発明の医薬組成物を注射剤として使用する場合、必要に応じて、プロピレングリコール、エチレンジアミン等の溶解補助剤、リン酸塩等の緩衝材、塩化ナトリウム、グリセリン等の等張化剤、亜硫酸塩等の安定剤、フェノール等の保存剤、リドカイン等の無痛化剤等の添加物(「医薬品添加物事典」薬事日報社、「Handbook of Pharmaceutical Excipients Fifth Edition」APhA Publications社参照)を加えることができる。また、本発明の医薬組成物を注射剤として使用する場合、保存容器としては、アンプル、バイアル、プレフィルドシリンジ、ペン型注射器用カートリッジ、及び、点滴用バッグ等を挙げることができる。 The pharmaceutical composition of the present invention may be any oral or parenteral preparation that can be administered to patients. Examples of compositions for parenteral administration include injections, nasal drops, suppositories, patches, ointments and the like. Preferably, it is an injection. The dosage form of the pharmaceutical composition of the present invention can include, for example, a solution or a lyophilized preparation. When the pharmaceutical composition of the present invention is used as an injection, if necessary, a solubilizing agent such as propylene glycol or ethylene diamine, a buffer such as phosphate, a tonicity agent such as sodium chloride or glycerin, sulfite Additives such as stabilizers such as phenol, preservatives such as phenol, soothing agents such as lidocaine (see “Handbook of Pharmaceutical Supplements” Pharmaceutical Daily, Inc., “Handbook of Pharmaceutical Excipients Fifth Edition” APhA Publications) . When the pharmaceutical composition of the present invention is used as an injection, examples of the storage container include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and infusion bags.
(抗体の取得)
 本発明の抗体は、ヒトCRLR及びヒトRAMP2を発現している細胞を免疫原として、必要に応じて免疫賦活剤(例えば、鉱油若しくはアルミニウム沈殿物と加熱死菌若しくはリポ多糖体、フロインドの完全アジュバント、または、フロインドの不完全アジュバント等)とともに、免疫反応を生じる非ヒト動物を免疫することにより作製することができる。免疫動物としては、マウス、ラット、ハムスター、モルモット、ラビット、イヌ、サル、ヒツジ、ヤギ、ニワトリ、アヒル等ハイブリドーマを作製することが可能な動物であれば特に限定はないが、好ましくは、マウスまたはラットであり、より好ましくはマウスであり、最も好ましくはMRL/lprマウスである。免疫原となる細胞は、目的とする抗原以外への抗体産生を抑制するため、免疫動物と同種の細胞であることが望ましい。動物への免疫原の投与は、例えば、1×10cellsを1回または適当な間隔をあけて数回(通常1~6週毎に1回の免疫を合計2~10回程度)、皮下注射、腹腔内注射、静脈内注射、皮内注射、筋肉内注射または足蹠注射により行うことができる。最後の免疫から1~2週間後に、免疫した動物の眼窩または尾静脈から採血を行い、その血清を用いて抗体価の測定を行う。本発明の抗体は、十分な抗体価を示す動物の血清から精製することにより得ることができる。
(Acquisition of antibody)
The antibody of the present invention can be used as an immunogen with cells expressing human CRLR and human RAMP2 as an immunogen, if necessary, an immunostimulant (eg, mineral oil or aluminum precipitate, heat-killed bacteria or lipopolysaccharide, complete adjuvant with Freund's Or with Freund's incomplete adjuvant etc.) by immunizing a non-human animal that produces an immune response. The animal to be immunized is not particularly limited as long as it is an animal capable of producing a hybridoma such as mouse, rat, hamster, guinea pig, rabbit, dog, monkey, sheep, goat, chicken, duck, etc. It is a rat, more preferably a mouse, most preferably a MRL / lpr mouse. The cells to be the immunogen are desirably cells of the same species as the immunized animal in order to suppress antibody production to other than the target antigen. The administration of the immunogen to an animal may be, for example, 1 × 10 6 cells once or several times at appropriate intervals (usually, 1 to 6 immunizations in total once every 1 to 6 weeks), subcutaneously It can be performed by injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or footpad injection. One to two weeks after the last immunization, blood is collected from the orbit or tail vein of the immunized animal, and the serum is used to measure antibody titer. The antibody of the present invention can be obtained by purification from serum of an animal exhibiting a sufficient antibody titer.
 モノクローナル抗体は、上記方法により免疫した免疫感作動物から得た抗体産生細胞と、骨髄腫系細胞(ミエローマ細胞)を融合することにより得られるハイブリドーマを培養することにより得ることができる。当該融合方法としては、例えば、ミルステインらの方法(Galfre,G.&Milstein,C.(1981)Methods Enzymol.,73:3-46)を挙げることができる。使用する抗体産生細胞は、上記方法により免疫し血清が十分な抗体価を示したマウスまたはラットの脾臓、膵臓、リンパ節、末梢血より採取することができる。使用する骨髄腫系細胞は、例えば、マウス、ラット、モルモット、ハムスター、ラビット又はヒト等の哺乳動物に由来する細胞であって、in vitroで増殖可能な細胞であれば特に限定はない。このような細胞としては、例えば、P3-X63Ag8(X63)(Nature,256,495,1975)、P3/NS1/1-Ag4-1(NS1)(Eur.J.Immunol.,6,292,1976)、P3X63Ag8U1(P3U1)(Curr.Top.Microbiol.Immunol.,81,1,1978)、P3X63Ag8.653(653)(J.Immunol.,123,1548,1979)、Sp2/0-Ag14(Sp2/O)(Nature,276,269,1978)、Sp2/O/FO-2(FO-2)(J.Immunol.Methods,35,1,1980)、SP2ab等を挙げることができ、好ましくは、抗体産生細胞と同種動物由来の細胞であり、より好ましくは、抗体産生細胞と同系統の動物由来の細胞である。 The monoclonal antibody can be obtained by culturing a hybridoma obtained by fusing a myeloma cell (myeloma cell) with an antibody-producing cell obtained from the immunized animal immunized by the above method. Examples of the fusion method include the method of Milstein et al. (Galfre, G. & Milstein, C. (1981) Methods Enzymol., 73: 3-46). The antibody-producing cells to be used can be collected from the spleen, pancreas, lymph nodes, and peripheral blood of mice or rats which have been immunized according to the above-mentioned method and their sera show a sufficient antibody titer. The myeloma cells used are, for example, cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans, and are not particularly limited as long as they can be proliferated in vitro. As such cells, for example, P3-X63Ag8 (X63) (Nature, 256, 495, 1975), P3 / NS1 / 1-Ag4-1 (NS1) (Eur. J. Immunol., 6, 292, 1976). P3X63Ag8U1 (P3U1) (Curr.Top.Microbiol.Immunol., 81,1,1978), P3X63Ag8.653 (653) (J. Immunol., 123, 1548, 1979), Sp2 / 0-Ag14 (Sp2 /) O) (Nature, 276, 269, 1978), Sp2 / O / FO-2 (FO-2) (J. Immunol. Methods, 35, 1, 1980), SP2ab and the like, preferably antibodies It is a cell derived from the same species as the producer cell, more preferably It is a cell of origin of the animal antibody-producing cells of the same lineage.
 培養後、培養上清を採取し、ヒトCRLRとヒトRAMP2を共発現する細胞(例えば、hCR2-CHOK1細胞)を用いたCell ELISAにより、RAMP2に結合するクローンを選択する。選択されたクローンについて、限界希釈法を1~5回繰り返すことにより単一細胞化を行い、当該単一細胞化されたクローンが産生する抗体のうち、更に、Cell ELISAで選択された候補抗体を、AM添加によるヒトCRLRとヒトRAMP2を共発現する細胞(例えば、hCR2-CHOK1細胞)の細胞内cAMPの上昇を抑制するか否かを指標としてシグナル阻害抗体をスクリーニングする。選択されたシグナル阻害抗体について生物活性の阻害試験としてAM添加によるHUVECの増殖を阻害できる抗体をスクリーニングし、cAMP阻害作用及びHUVEC増殖抑制作用を有する抗RAMP2抗体を取得することができる。 After culture, culture supernatants are collected, and clones that bind to RAMP2 are selected by Cell ELISA using cells coexpressing human CRLR and human RAMP2 (for example, hCR2-CHOK1 cells). The selected clones are subjected to single-cellification by repeating limiting dilution 1 to 5 times, and among the antibodies produced by the single-cellified clones, candidate antibodies selected by Cell ELISA are further selected. Then, signal inhibitory antibodies are screened using as an index whether or not the increase in intracellular cAMP of cells (for example, hCR2-CHOK1 cells) co-expressing human CRLR and human RAMP2 by addition of AM is suppressed. An antibody capable of inhibiting the growth of HUVEC by AM addition can be screened as a test for inhibiting the biological activity of the selected signal-inhibiting antibody, and an anti-RAMP2 antibody having a cAMP inhibitory action and a HUVEC growth inhibitory action can be obtained.
 得られた抗体は、均一にまで精製することができる。抗体の分離、精製は通常のタンパク質で使用されている分離、精製方法を使用することができる。例えば、アフィニティークロマトグラフィ等のカラムクロマトグラフィ、フィルター、限外濾過、塩析、透析、SDSポリアクリルアミドゲル電気泳動、等電点電気泳動等を適宜選択、組み合わせることにより、抗体を分離、精製することができる(Antibodies:A Laboratory Manual.Ed Harlow and David Lane,Cold Spring Harbor Laboratory,1988)。アフィニティークロマトグラフィに用いるカラムとしては、例えば、プロテインAカラム、プロテインGカラムを挙げることができる。更に、抗体のクラスによらず、RAMP2(又はヒトCRLR及びヒトRAMP2)固相化カラム、イオン交換クロマトグラフィ、疎水相互作用クロマトグラフィ等を用いることもできる。 The resulting antibodies can be purified to homogeneity. For separation and purification of antibodies, separation and purification methods used in conventional proteins can be used. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Examples of columns used for affinity chromatography include a protein A column and a protein G column. Furthermore, regardless of the antibody class, RAMP2 (or human CRLR and human RAMP2) immobilized columns, ion exchange chromatography, hydrophobic interaction chromatography and the like can also be used.
 あるいは、例えば、抗体ファージライブラリーを利用することによりRAMP2に結合する抗体を得ることができる(富塚ら,Nature Genet.,15,146-156(1997))。抗体ファージライブラリーを利用する場合、例えば、ヒトCRLR及びヒトRAMP2を発現している細胞を固相に固定化し、ファージ抗体ライブラリーを反応させて、非結合のファージを洗浄除去した後、結合したファージを回収することにより、所望のクローンを得ることができる(パンニング)。 Alternatively, for example, antibodies that bind to RAMP2 can be obtained by utilizing an antibody phage library (Tomizuka et al., Nature Genet., 15, 146-156 (1997)). In the case of using an antibody phage library, for example, cells expressing human CRLR and human RAMP2 were immobilized on a solid phase, reacted with a phage antibody library, washed to remove non-bound phage, and then bound. By recovering the phage, the desired clone can be obtained (panning).
 あるいは、本明細書記載のアミノ酸配列を参照して、目的の抗体又はその免疫反応性断片のアミノ酸配列をデザインし、当該デザインされたアミノ酸配列をコードするDNAを調製して、発現ベクターに組み込み、適当な宿主細胞に当該ベクターを導入して発現させることにより得た抗体から、上述の方法に従ってRAMP2への特異性が高い抗体をスクリーニングすることにより得ることができる。 Alternatively, the amino acid sequence of the antibody of interest or its immunoreactive fragment is designed with reference to the amino acid sequence described herein, and a DNA encoding the designed amino acid sequence is prepared and incorporated into an expression vector, An antibody obtained by introducing and expressing the vector in a suitable host cell can be obtained by screening an antibody with high specificity to RAMP2 according to the method described above.
(ヒト型キメラ抗体作製)
 本発明の抗体がヒト型キメラ抗体の場合、RAMP2を特異的に認識する非ヒト動物モノクローナル抗体のVH及びVLをコードするDNAを調製し、これをヒト免疫グロブリンの定常領域cDNAと結合して発現ベクターに組み込み、適当な宿主細胞に当該ベクターを導入して発現させることにより得ることができる(Morrison,S.L.ら,Proc.Natl.Acad.Sci.USA,81,6851-6855,1984)。
(Human type chimeric antibody production)
When the antibody of the present invention is a human chimeric antibody, DNAs encoding VH and VL of non-human animal monoclonal antibodies that specifically recognize RAMP2 are prepared, and they are expressed by binding to human immunoglobulin constant region cDNA. It can be obtained by incorporating it into a vector and introducing the vector into an appropriate host cell and expressing it (Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984). .
(ヒト化抗体作製)
 本発明の抗体がヒト化抗体の場合は、RAMP2を特異的に認識する非ヒト動物モノクローナル抗体のVH及びVLのCDRをコードするアミノ酸配列をヒト抗体のVH及びVLのFramework Region(FR)に移植したV領域をコードするDNAを構築し、構築したDNAをヒト由来免疫グロブリンの定常領域cDNAと結合して発現ベクターに組み込み、適当な宿主細胞に当該ベクターを導入して発現させることにより得ることができる(L.Rieohmannら,Nature,332,323,1988:Kettleborough,C.A.ら,Protein Eng.,4,773-783,1991;Clark M.,Immunol.Today.,21,397-402,2000参照)。非ヒト動物モノクローナル抗体のCDRは、上述の方法によって得られた非ヒト動物モノクローナル抗体のVH及びVLをコードするDNA配列から予測されるアミノ酸配列と、既知の抗体のVH及びVLの全アミノ酸配列とを比較して得ることができる。既知の抗体のアミノ酸配列は、例えば、プロテイン・データ・バンク等のデータベースに登録されている抗体のアミノ酸配列より得ることができる。また、ヒト化抗体のFRとしては、移植後の抗体が本発明の効果を奏するものであれば特に限定は無いが、好ましくは、ヒト化抗体の可変領域(以下、「V領域」という)がCDRが由来する非ヒト動物モノクローナル抗体のV領域と類似の立体構造となるヒト抗体のFR、又は、使用する非ヒト動物モノクローナル抗体のFRのアミノ酸配列と同一性が高いヒト抗体FRである。ヒト化抗体において、ヒト抗体に由来するFRを構成するアミノ酸の一部(特には、立体的にCDRと近接した位置に存在するアミノ酸)は、必要に応じてCDRが由来する非ヒト動物モノクローナル抗体のFR配列に置換されていてもよい(Queenら、米国特許第5585089号参照)。使用するヒト化抗体のV領域をコードするDNA配列は、非ヒト動物モノクローナル抗体のCDRのアミノ酸配列とヒト抗体のFRのアミノ酸配列を結合したアミノ酸配列に対応するDNA配列として設計する。ヒト化抗体のV領域をコードするDNAは、設計したDNA配列を基に、当業者周知の方法によって作製することができる。
(Humanized antibody production)
When the antibody of the present invention is a humanized antibody, the amino acid sequences encoding the CDRs of VH and VL of a non-human animal monoclonal antibody that specifically recognizes RAMP2 are transplanted into the framework regions (FR) of VH and VL of human antibody Obtained by constructing a DNA encoding the isolated V region, combining the constructed DNA with a constant region cDNA of a human-derived immunoglobulin, incorporating it into an expression vector, and introducing the vector into an appropriate host cell for expression (L. Rieohmann et al., Nature, 332, 323, 1988: Kettleborough, CA. et al., Protein Eng., 4, 773-783, 1991; Clark M., Immunol. Today., 21, 397-402, See 2000). The CDRs of the non-human animal monoclonal antibody are the amino acid sequences predicted from the DNA sequences encoding the VH and VL of the non-human animal monoclonal antibody obtained by the above-mentioned method, and the entire amino acid sequences of VH and VL of the known antibody Can be obtained by comparing. The amino acid sequences of known antibodies can be obtained, for example, from the amino acid sequences of antibodies registered in databases such as Protein Data Bank. Further, the FR of the humanized antibody is not particularly limited as long as the antibody after transplantation exerts the effect of the present invention, but preferably, the variable region of the humanized antibody (hereinafter referred to as "V region") is It is a human antibody FR having a steric structure similar to the V region of the non-human animal monoclonal antibody from which the CDRs are derived, or a human antibody FR having high identity to the amino acid sequence of the FR of the non-human animal monoclonal antibody used. In the humanized antibody, a part of the amino acids constituting the FR derived from the human antibody (in particular, the amino acids present at positions sterically close to the CDRs) are optionally non-human animal monoclonal antibodies from which the CDRs are derived (SEQ ID NO: 5) may be substituted by the FR sequence of (See, eg, Queen et al., US Pat. No. 5,855,089). The DNA sequence encoding the V region of the humanized antibody to be used is designed as a DNA sequence corresponding to the amino acid sequence obtained by combining the amino acid sequence of CDR of non-human animal monoclonal antibody and the amino acid sequence of FR of human antibody. The DNA encoding the V region of a humanized antibody can be prepared based on the designed DNA sequence by methods known to those skilled in the art.
(ヒト抗体)
 ヒト抗体は、例えば、ヒト抗体ファージライブラリー又はヒト抗体産生トランスジェニックマウスを利用することにより得ることができる(富塚ら,Nature Genet.,15,146-156(1997))。ヒト抗体ファージライブラリーを利用する場合、例えば、ヒトCRLR及びヒトRAMP2を発現している細胞を固相に固定化し、ファージ抗体ライブラリーを反応させて、非結合のファージを洗浄除去した後、結合したファージを回収することにより、所望のクローンを得ることができる(パンニング)。ヒト抗体産生トランスジェニックマウスは、内因性免疫グロブリン(Ig)遺伝子をノックアウトしたマウスにヒト抗体のIg遺伝子を導入したマウスである。ヒト抗体産生トランスジェニックマウスを免疫動物として、上述の本発明の抗体作製方法に準じて抗原(好ましくは、ヒトCRLR及びヒトRAMP2を発現している細胞)を免疫することにより、RAMP2を特異的に認識するヒト抗体を得ることができる。
(Human antibody)
Human antibodies can be obtained, for example, by using a human antibody phage library or a human antibody-producing transgenic mouse (Tomizuka et al., Nature Genet., 15, 146-156 (1997)). When using a human antibody phage library, for example, cells expressing human CRLR and human RAMP2 are immobilized on a solid phase, reacted with a phage antibody library, and washed to remove non-bound phage, and then bound. The desired clones can be obtained (panning) by recovering the isolated phages. A human antibody-producing transgenic mouse is a mouse in which an endogenous immunoglobulin (Ig) gene has been knocked out, into which an Ig gene of a human antibody has been introduced. RAMP2 is specifically expressed by immunizing an antigen (preferably, a cell expressing human CRLR and human RAMP2) according to the above-described antibody production method of the present invention, using a human antibody-producing transgenic mouse as an immunized animal. Human antibodies that recognize can be obtained.
(核酸、ベクター、宿主細胞)
 本発明の核酸は、上述において得られた抗体を産生するハイブリドーマからクローニングするか、あるいは、上述において得られた抗体またはその免疫反応性断片のアミノ酸配列を基に、適宜核酸配列を設計することにより得ることができる。本発明のベクターは、得られた核酸を適宜発現に適したベクターに組み込むことにより得ることができる。本発明のベクターは、本発明の核酸の他、発現に必要な領域(プロモーター、エンハンサー、ターミネーター等)を含んでいてもよい。また、本発明の宿主細胞は、本発明のベクターを適切な細胞株(例えば、動物細胞、昆虫細胞、植物細胞、酵母、大腸菌等の微生物)に導入することにより得ることができる。
(Nucleic acid, vector, host cell)
The nucleic acid of the present invention may be cloned from a hybridoma that produces the antibody obtained in the above, or, by appropriately designing a nucleic acid sequence based on the amino acid sequence of the antibody or its immunoreactive fragment obtained in the above. You can get it. The vector of the present invention can be obtained by incorporating the obtained nucleic acid into a vector suitable for expression as appropriate. The vector of the present invention may contain, in addition to the nucleic acid of the present invention, regions necessary for expression (promoter, enhancer, terminator, etc.). In addition, the host cell of the present invention can be obtained by introducing the vector of the present invention into an appropriate cell line (for example, animal cells, insect cells, plant cells, microorganisms such as yeast and E. coli).
(医薬組成物(治療薬、予防薬)及び治療方法)
 本発明の抗体又はその免疫反応性断片は、必要により精製した後、常法に従って製剤化することにより、医薬組成物として用いることができる。また、本発明は、医薬組成物を製造するための、本発明の抗体又はその免疫反応性断片の使用を含む。あるいは、本発明は、本発明の抗体又はその免疫反応性断片の、血管新生が発症又は増悪化に寄与する疾患又は障害の治療若しくは予防のための使用を含む。更に、本発明は、本発明の抗体又はその免疫反応性断片の有効量をそれを必要とする患者に投与することを含む、血管新生が発症又は増悪化に寄与する疾患又は障害の治療方法若しくは予防方法に関する。
(Pharmaceutical composition (therapeutic agent, prophylactic agent) and treatment method)
The antibody of the present invention or the immunoreactive fragment thereof can be used as a pharmaceutical composition by purifying it as necessary and then formulating it according to a conventional method. The present invention also includes the use of the antibody of the present invention or an immunoreactive fragment thereof for producing a pharmaceutical composition. Alternatively, the present invention includes the use of the antibody of the present invention or an immunoreactive fragment thereof for the treatment or prevention of a disease or disorder in which angiogenesis contributes to the onset or exacerbation. Furthermore, the present invention provides a method for treating a disease or disorder in which angiogenesis contributes to the onset or exacerbation, comprising administering to a patient in need thereof an effective amount of the antibody of the present invention or an immunoreactive fragment thereof It relates to the prevention method.
 例えば、本発明の医薬組成物(治療薬若しくは予防薬)は、注射剤として利用することができ、静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、硝子体内注射剤、点滴注射剤などの剤形を包含する。このような注射剤は、公知の方法に従って、例えば、上記抗体等を通常注射剤に用いられる無菌の水性もしくは油性液に溶解、懸濁または乳化することによって調製することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖、ショ糖、マンニトール、その他の補助薬を含む等張液等が用いることができ、適当な溶解補助剤、例えば、アルコール(例、エタノール)、ポリアルコール(例、プロピレングリコール、ポリエチレングリコール)、非イオン界面活性剤〔例、ポリソルベート80、ポリソルベート20、HCO-50(polyoxyethylene(50mol)adduct of hydrogenated castor oil)〕等を添加することができる。油性液としては、例えば、ゴマ油、大豆油などを用いることができ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を添加することができる。調製された注射液は、通常、適当なアンプル、バイアル、シリンジに充填される。また、本発明の抗体又はその免疫反応性断片に適当な賦形剤を添加することにより、凍結乾燥製剤を調製し、用時、注射用水、生理食塩水などで溶解して注射液とすることもできる。なお、一般的に抗体などのタンパク質の経口投与は消化器により分解されるため困難とされるが、抗体断片や修飾した抗体断片と剤形の創意工夫により、経口投与の可能性もある。経口投与の製剤としては、例えば、カプセル剤、錠剤、シロップ剤、顆粒剤等を挙げることができる。 For example, the pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention can be used as an injection, and intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravitreal injection, drip injection Including dosage forms such as agents. Such injections can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody etc. in a sterile aqueous or oily liquid usually used for injections, according to a known method. As the aqueous solution for injection, for example, physiological saline, glucose, sucrose, mannitol, isotonic solution containing other adjuvants, etc. can be used, and suitable solubilizers, such as alcohol (eg, ethanol, etc.) ), Polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. can be added. . As an oily liquid, for example, sesame oil, soybean oil and the like can be used, and as a solubilizer, benzyl benzoate, benzyl alcohol and the like can be added. The prepared injections are usually filled into appropriate ampoules, vials and syringes. In addition, a freeze-dried preparation is prepared by adding an appropriate excipient to the antibody of the present invention or the immunoreactive fragment thereof, and when used, it is dissolved in water for injection, physiological saline or the like to prepare an injection solution. You can also. Generally, oral administration of proteins such as antibodies is considered to be difficult because it is degraded by the digestive tract, but there is also the possibility of oral administration due to the inventive idea of antibody fragments and modified antibody fragments and dosage forms. Examples of preparations for oral administration include capsules, tablets, syrups, granules and the like.
 本発明の医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好適である。このような投薬単位の剤形としては、注射剤(アンプル、バイアル、プレフィルドシリンジ)が例示され、投薬単位剤形当たり通常5~500mg、5~100mg、10~250mgの本発明の抗体又はその免疫反応性断片を含有していても良い。 The pharmaceutical compositions of the present invention are preferably formulated in dosage unit forms that are compatible with the dose of the active ingredient. Examples of dosage forms of such dosage units include injections (ampoules, vials, prefilled syringes), and generally 5 to 500 mg, 5 to 100 mg, 10 to 250 mg of the antibody of the present invention or its immunity per dosage unit dosage form. It may contain a reactive fragment.
 本発明の抗体や医薬組成物(治療薬若しくは予防薬)の投与は、局所的であってもよく、全身的であってもよい。投与方法には特に制限はなく、上述のとおり非経口的又は経口的に投与される。非経口的投与経路としては、眼内、皮下、腹腔内、血中(静脈内、若しくは動脈内)又は脊髄液への注射又は点滴等が挙げられ、好ましくは、眼内又は血中への投与である。本発明の医薬組成物(治療薬若しくは予防薬)は、一時的に投与してもよいし、持続的又は断続的に投与してもよい。例えば、投与は、1分間~2週間の持続投与することもできる。また、本発明の抗体は、単独で投与されてもよいし、他の薬剤と共に併用により投与されてもよい。あるいは、本発明の抗体は当該他の薬剤が結合した抗体-薬剤コンジュゲート(ADC)であってもよい。他の薬剤としては、血管新生が発症又は増悪化に寄与する疾患又は障害への治療効果が知られている薬剤などを用いることができる。 Administration of the antibody or pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention may be local or systemic. There is no particular limitation on the method of administration, and as described above, it is administered parenterally or orally. Parenteral administration routes include injection or infusion into the eye, subcutaneously, intraperitoneally, in the blood (intravenous or intraarterial) or spinal fluid, and preferably in the intraocular or blood. It is. The pharmaceutical composition (therapeutic or prophylactic agent) of the present invention may be administered temporarily, or may be administered continuously or intermittently. For example, administration can be continuously performed for 1 minute to 2 weeks. Furthermore, the antibodies of the present invention may be administered alone or in combination with other agents. Alternatively, the antibody of the present invention may be an antibody-drug conjugate (ADC) bound to the other agent. As the other drug, a drug or the like which is known to have a therapeutic effect on a disease or disorder in which angiogenesis contributes to onset or exacerbation can be used.
 本発明の医薬組成物の投与量は、所望の治療効果又は予防効果が得られる投与量であれば特に限定は無く、症状、性別、年齢等により適宜決定することができる。本発明の医薬組成物の投与量は、例えば、血管新生が発症又は増悪化に寄与する疾患又は障害の治療効果又は予防効果を指標として決定することができる。例えば、血管新生が発症又は増悪化に寄与する疾患又は障害の患者の予防および/または治療のために使用する場合には、本発明の医薬組成物の有効成分の1回量として、通常0.01~20mg/kg体重程度、好ましくは0.1~10mg/kg体重程度、さらに好ましくは0.1~5mg/kg体重程度を、1月1~10回程度、好ましくは1月1~5回程度、静脈注射により投与するのが好都合である。他の非経口投与および経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量または投与回数を増加させてもよい。 The dose of the pharmaceutical composition of the present invention is not particularly limited as long as a desired therapeutic effect or prophylactic effect can be obtained, and can be appropriately determined depending on symptoms, sex, age, and the like. The dose of the pharmaceutical composition of the present invention can be determined, for example, using the therapeutic or prophylactic effect of a disease or disorder in which angiogenesis contributes to the onset or deterioration. For example, when used for the prevention and / or treatment of a patient with a disease or disorder where angiogenesis contributes to onset or exacerbation, the amount of the active ingredient of the pharmaceutical composition of the present invention is usually 0. About 01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 10 times a month, preferably 1 to 5 times a month To the extent it is convenient to administer by intravenous injection. In the case of other parenteral administration and oral administration, similar amounts can be administered. If the condition is particularly severe, the dose may be increased or the number of administrations may be increased depending on the condition.
 以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明はこれらの実施例に限定されるものではない。なお、本願全体を通して引用される全文献は参照によりそのまま本願に組み込まれる。また、本願は日本国特許出願第2017-175311号からの優先権を主張する。本願が優先権を主張する日本国特許出願第2017-175311号記載の内容は全て参照によりそのまま本願に組み込まれる。 Hereinafter, the present invention will be more specifically described based on examples. However, the present invention is not limited to these examples. All references cited throughout the present application are incorporated herein by reference in their entirety. The present application also claims priority from Japanese Patent Application No. 2017-175311. The contents of Japanese Patent Application No. 2017-175311 to which the present application claims priority are all incorporated by reference in their entirety.
(実施例1)抗RAMP2抗体の産生
 ハイブリドーマの作製にあたり、抗原として用いる細胞を以下の方法により調製した。ヒトCRLR(NM_005795)及びヒトRAMP2(NM_005854)をThermo社のpCHO1.0ベクターにタンデムに組み込み、Lipofectamine2000(Thermo社)を使用してNIH-3T3細胞(ATCC:CRL-1658)に遺伝子導入した(hCR2-NIH3T3)。安定発現株を作製するため、10μg/mL Puromycinと100nM Methotrexateを含むD-MEM培地(和光純薬、日本)で選択培養を行った。
Example 1 Production of Anti-RAMP2 Antibody In preparation of a hybridoma, cells used as an antigen were prepared by the following method. Human CRLR (NM — 005795) and human RAMP 2 (NM — 005854) were tandemly incorporated into pCHO 1.0 vector of Thermo, and transfected into NIH-3T3 cells (ATCC: CRL-1658) using Lipofectamine 2000 (Thermo) (hCR2) -NIH 3T3). In order to prepare a stable expression strain, selective culture was carried out in D-MEM medium (Wako Pure Chemical Industries, Japan) containing 10 μg / mL Puromycin and 100 nM Methotrexate.
 得られたhCR2-NIH3T3細胞を、MRL/lprマウス1匹あたり1×10cellsとなるように調製し、週一回で5週間腹腔内投与して抗原による免疫を行った。免疫前後の血清を採取し、Cell ELISAで抗原特異的な抗体価の上昇を確認した。抗体価の上昇確認後、最終免疫として1×10cellsのhCR2-NIH3T3細胞を抗原として再度免疫した。 The resulting hCR2-NIH3T3 cells were prepared to 1 × 10 6 cells / MRL / lpr mouse, and intraperitoneally administered once a week for 5 weeks to immunize with antigen. The sera before and after the immunization were collected, and an increase in antibody-specific antibody titer was confirmed by Cell ELISA. After confirming the increase in antibody titer, 1 × 10 6 cells of hCR2-NIH3T3 cells were again immunized as an antigen as a final immunization.
 細胞融合はマウスミエローマ細胞としてSP2ab細胞株を用い、脾臓及びリンパ節由来の細胞とPEG法により融合し、96well培養プレートに播種した。融合翌日からHAT含有10%FBS-TIL培地を添加し、3日毎に培地交換を行いハイブリドーマを作製した。 For cell fusion, SP2ab cell line was used as mouse myeloma cells, fused with cells derived from spleen and lymph node by PEG method, and seeded in 96 well culture plate. The day after fusion, HAT-containing 10% FBS-TIL medium was added, and the medium was changed every three days to prepare a hybridoma.
 抗ヒトRAMP2抗体の産生株をスクリーニングするために、NIH-3T3細胞と同様の方法で作製したhCRLR/hRAMP2発現CHO-K1細胞(hCR2-CHOK1)を96well培養プレートに播種した。4%PFAにより固定化し、1%BSA-PBSでブロッキング後、培養上清を添加して、二次抗体としてGoat Anti-mouse-IgG-HRP(SouthernBiotech社)を反応させた。TMB solution(eBioscience社)による発色で陽性wellをピックアップし、候補ハイブリドーマとした。 In order to screen anti-human RAMP2 antibody-producing strains, hCRLR / hRAMP2-expressing CHO-K1 cells (hCR2-CHOK1) prepared by the same method as NIH-3T3 cells were seeded on a 96-well culture plate. After immobilization with 4% PFA and blocking with 1% BSA-PBS, the culture supernatant was added to react with Goat Anti-mouse-IgG-HRP (SouthernBiotech) as a secondary antibody. The positive wells were picked up by coloring with TMB solution (eBioscience) and used as candidate hybridomas.
 候補ハイブリドーマのうち、3株をASF104培地(味の素株式会社、日本)に馴化した後、培養上清を回収し、ProteinA誘導体Ab-Capcher ExTra(プロテノバ株式会社、日本)でIgG抗体を精製し抗RAMP2抗体、25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体を得た。 Among the candidate hybridomas, after 3 strains are acclimated to ASF104 medium (Ajinomoto Co., Japan), the culture supernatant is recovered, and the IgG antibody is purified with Protein A derivative Ab-Capcher ExTra (Protenova Co., Japan) to obtain anti-RAMP2 An antibody, 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were obtained.
(実施例2)cAMP抑制試験
 hCR2-CHOK1細胞を用いて、得られた抗RAMP2抗体のcAMPの抑制試験を実施した。コントロール抗体として、公知の市販抗体(Novus bio社、4E5、CatNo.NBP2-01853;Abnova社、2F5、CatNo.H00010266-M05;及びSantacruz社、B5、CatNo.sc-365240)を用いた。測定にはHalf area 96well プレートを使用した。2500cellsのhCR2-CHOK1細胞を10%FBS含有D-MEM/Ham‘s F-12(和光純薬、日本)で一晩培養し、終濃度が0.001nM、0.01nM、0.1nM、1nM、10nM、100nM、及び1000nMとなるように、各抗体(25H4-4F9、85H7-2B11、33H4-1G3、4E5、2F5、及びB5)を添加し、終濃度150pMのヒトAM(株式会社ペプチド研究所、日本)を添加した。cAMPは、LANCE Ultra cAMP kit(Perkinelmer社)を用いて、製造者のマニュアルに従って測定した。コントロールとして、抗体非添加/AM添加、及び抗体非添加/AM非添加のhCR2-CHOK1細胞によるcAMP産生を同様に測定した。
Example 2 cAMP Suppression Test The hCR2-CHOK1 cells were used to conduct a cAMP suppression test of the obtained anti-RAMP2 antibody. As a control antibody, a known commercially available antibody (Novus bio, 4E5, Cat No. NBP2-01853; Abnova, 2F5, Cat No. H00010266-M05; and Santacruz, B5, Cat No. sc-365240) was used. The half area 96 well plate was used for the measurement. Incubate 2500 cells of hCR2-CHOK1 cells overnight in 10% FBS-containing D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Japan) to a final concentration of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM , 10 nM, 100 nM, and 1000 nM, each antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5, and B5) is added to a final concentration of 150 pM human AM (Peptide Research Laboratories, Inc.) , Japan) was added. cAMP was measured using the LANCE Ultra cAMP kit (Perkinelmer) according to the manufacturer's manual. As a control, cAMP production by hCR2-CHOK1 cells without antibody addition / AM addition and without antibody addition / AM addition was similarly measured.
 抗体を10nM添加した時のcAMP抑制を表す結果を図1に示す。図1は、抗体非添加の場合のcAMP濃度を100とした場合の各抗体添加群のcAMP濃度の割合を示す。コントロール抗体はほとんどcAMP産生を抑制しなかったのに対し、25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体は、AM非添加群と同等までcAMP産生を抑制した。また、各抗体の濃度とcAMP抑制率との関係を図2に示す。25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体のcAMP抑制に対するIC50値は、それぞれ、0.15nM、0.03nM、及び0.045nMであった。 The result showing cAMP suppression when 10 nM of antibody is added is shown in FIG. FIG. 1 shows the ratio of the cAMP concentration in each antibody-added group when the cAMP concentration in the case of no antibody addition is 100. The control antibody hardly suppressed cAMP production, whereas the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody suppressed cAMP production to the same level as that in the group without AM. Further, the relationship between the concentration of each antibody and the cAMP suppression rate is shown in FIG. The IC50 values for cAMP inhibition of the 25H4-4F9, 85H7-2B11 and 33H4-1G3 antibodies were 0.15 nM, 0.03 nM and 0.045 nM, respectively.
(実施例3)HUVECの増殖抑制試験
 増殖因子を除いた2%FBS含有Humedia-EG2培地中のHUVEC(KURABO社)を96well培養プレートに1well当たり2500cellsで播種し、3時間の前培養により接着させた。終濃度が0.0001nM、0.001nM、0.01nM、0.1nM、1nM、10nM、100nM、及び1000nMとなるように、各抗体(25H4-4F9、85H7-2B11、33H4-1G3、4E5、2F5、及びB5)を添加し、終濃度100nMのAMを添加して、96時間後におけるHUVECの増殖を観察することで、HUVECの増殖抑制試験を行った。細胞数の測定にはCell Counting Kit-8(DOJINDO社)を用い、増殖因子を除いた2%FBS含有Humedia-EG2培地で10倍希釈した溶液を100μL添加し、COインキュベーターで4時間発色させ、OD450として計測した。
(Example 3) Growth inhibition test of HUVEC HUVEC (KURABO) in Humedia-EG2 medium containing 2% FBS excluding growth factor was seeded at 2500 cells per well on 96 well culture plate and adhered by pre-incubation for 3 hours The Each antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5 so that the final concentration is 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM. And B5) were added, and a final concentration of 100 nM of AM was added to observe HUVEC proliferation after 96 hours, thereby conducting a HUVEC proliferation inhibition test. To measure the number of cells, use Cell Counting Kit-8 (DOJINDO), add 100 μL of a 10-fold diluted solution in Humedia-EG2 medium containing 2% FBS excluding growth factors, and color in a CO 2 incubator for 4 hours , OD 450 was measured.
 抗体を10nM添加した時の増殖抑制を表す結果を図3に示す。図3は、抗体非添加の場合のOD450(HUVEC細胞数)を100とした場合の各抗体添加群のOD450(HUVEC細胞数)の割合を示す。コントロール抗体はほとんどHUVECの増殖を抑制しなかったのに対し、25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体は、30%程度にまで増殖を抑制した。また、各抗体の濃度とHUVEC増殖抑制率との関係を図4に示す。25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体のHUVEC増殖抑制に対するIC50値は、それぞれ、1.2nM、0.8nM、及び1.6nMであった。 The results showing growth inhibition when 10 nM of antibody is added are shown in FIG. FIG. 3 shows the percentage of OD450 (number of HUVEC cells) in each antibody-added group, where OD450 (number of HUVEC cells) in the case of no antibody addition is 100. The control antibody hardly inhibited the proliferation of HUVEC, whereas the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody inhibited the proliferation to about 30%. Further, the relationship between the concentration of each antibody and the HUVEC growth inhibition rate is shown in FIG. The IC50 values for inhibition of HUVEC proliferation of the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were 1.2 nM, 0.8 nM, and 1.6 nM, respectively.
(実施例4)配列解析
 各抗体産生ハイブリドーマのセルペレットからSV-Total RNA Isolation kit(Promega社)を用いてRNAを抽出し、Transcriptor First Strand cDNA Synthesis Kit(Roche社)によりcDNAを合成して、ハイブリドーマの抗体配列を解析した。既存マウスのCDR配列よりwobble primer(縮重プライマー)を設計し、重鎖・軽鎖それぞれの配列をタカラバイオ社のEx Taq polymeraseを用いて増幅した。得られた塩基配列からアミノ酸配列を決定し、KABAT法によりCDR領域を特定した。
Example 4 Sequence Analysis RNA is extracted from cell pellets of each antibody-producing hybridoma using SV-Total RNA Isolation kit (Promega), and cDNA is synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche), The antibody sequences of the hybridomas were analyzed. Wobble primers (degenerate primers) were designed from the CDR sequences of existing mice, and the sequences of heavy chain and light chain were amplified using Ex Taq polymerase from Takara Bio Inc., respectively. The amino acid sequence was determined from the obtained base sequence, and the CDR region was specified by the KABAT method.
 決定された25H4-4F9抗体のVH(配列番号2)及びVL(配列番号4)、85H7-2B11抗体のVH(配列番号12)及びVL(配列番号14)、及び33H4-1G3抗体のVH(配列番号19)及びVL(配列番号21)の配列を図5に示す。また、決定された各CDR配列を以下に示す。
25H4-4F9抗体 重鎖CDR配列:
CDRH1:GYFMN(配列番号5)
CDRH2:RNNPYNGDSIYNQKFKG(配列番号6)
CDRH3:LMIFDAYYAMDY(配列番号7)
25H4-4F9抗体 軽鎖CDR配列:
CDRL1:RASQDIRNYLN(配列番号8)
CDRL2:YTSRLHS(配列番号9)
CDRL3:QQDSKHPWT(配列番号10)

85H7-2B11抗体 重鎖CDR配列:
CDRH1:GYFMN(配列番号5)
CDRH2:RINPYNGDTFYNQKFKG(配列番号15)
CDRH3:LGIYDVYHYVMEN(配列番号16)
85H7-2B11抗体 軽鎖CDR配列:
CDRL1:RASQDIRNYLN(配列番号8)
CDRL2:YTSRLHS(配列番号9)
CDRL3:QQDSKNPWT(配列番号17)

33H4-1G3抗体 重鎖CDR配列:
CDRH1:GYFMN(配列番号5)
CDRH2:RINPYNGDTLYNQKFKG(配列番号22)
CDRH3:LGIYDVYHYVMEN(配列番号16)
33H4-1G3抗体 軽鎖CDR配列:
CDRL1:RASQDIRNYLN(配列番号8)
CDRL2:YTSRLHS(配列番号9)
CDRL3:QQDSKNPWT(配列番号17)
The determined VH (SEQ ID NO: 2) and VL (SEQ ID NO: 4) of 25H4-4F9 antibody, VH (SEQ ID NO: 12) and VL (SEQ ID NO: 14) of 85H7-2B11 antibody, and VH (sequence of 33H4-1G3 antibody) The sequences of No. 19) and VL (SEQ ID No. 21) are shown in FIG. Moreover, each determined CDR sequence is shown below.
25H4-4F9 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RNNPYNGDSIYNQKFKG (SEQ ID NO: 6)
CDRH3: LMIFDAYYAMDY (SEQ ID NO: 7)
25H4-4F9 Antibody Light Chain CDR Sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKHPWT (SEQ ID NO: 10)

85H7-2B11 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINPYNGDTFYNQKFKG (SEQ ID NO: 15)
CDRH3: LGIYDV YHY VMEN (SEQ ID NO: 16)
85H7-2B11 Antibody Light Chain CDR Sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)

33H4-1G3 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINPYNGDTLYNQKFKG (SEQ ID NO: 22)
CDRH3: LGIYDV YHY VMEN (SEQ ID NO: 16)
33H4-1G3 Antibody Light Chain CDR Sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)
(実施例5)抗RAMP2特異的な抗体の確認
 取得した抗体が抗RAMP2特異的である事をCell ELISAによって確認した。具体的には、実施例1に準じて、ハムスター卵巣細胞(CHO-K1)に、ヒトCRLR及びヒトRAMP2を遺伝子導入した。ヒトCRLR及びヒトRAMP2を遺伝子導入した細胞と、ヒトCRLRのみを遺伝子導入した細胞との間で、抗体の結合性を比較した。
(Example 5) Confirmation of anti-RAMP2 specific antibody It was confirmed by Cell ELISA that the obtained antibody was anti-RAMP2 specific. Specifically, according to Example 1, human CRLR and human RAMP2 were transfected into hamster ovary cells (CHO-K1). The binding properties of the antibodies were compared between cells transfected with human CRLR and human RAMP2 and cells transfected with human CRLR alone.
 結果を図6に示す。25H4-4F9抗体、85H7-2B11抗体、及び33H4-1G3抗体は、ヒトCRLRのみを発現させたCHO-K1細胞にはほとんど結合せず、ヒトCRLRとヒトRAMP2を共発現させたCHO-K1細胞には特異的に結合する事を示している。 The results are shown in FIG. The 25H4-4F9 antibody, the 85H7-2B11 antibody, and the 33H4-1G3 antibody hardly bind to CHO-K1 cells in which only human CRLR is expressed, but in CHO-K1 cells in which human CRLR and human RAMP2 are coexpressed Indicates that it specifically binds.
(実施例6)Cell ELISAによる細胞膜表面タンパクに対する結合親和性解析
 10%FCS含有DMEM/F-12培地中のhCR2-CHOK1細胞を1×10cells/wellで96well培養プレート(BD-FALCON社)に添加し、一晩培養した。等量の4%パラホルムアルデヒド溶液(MUTO化学)を添加し4℃で20分間静置して細胞を固定し、PBSTで洗浄した後、1%BSA-PBSで1時間以上のブロッキング処理を行った。抗体としては、実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3、並びに、市販の抗RAMP2抗体である、4E5、2F5、及びB5を用いた。精製した各抗体を67.5nMから1/2段階希釈して添加し、二次抗体としてGoat Anti-mouse-IgG-HRP(SouthernBiotech社)を反応させた。TMB solution(eBioscience社)を加えて発色させた後、2NのHSO溶液を添加して反応を停止した。OD450は、iMark(BioRAD社)を用いて測定した。得られた結合データから、GraphPad Prism7を用いて結合親和性(EC50)を算出した。
(Example 6) Binding affinity analysis for cell membrane surface protein by Cell ELISA 96-well culture plate (BD-FALCON) with 1 × 10 4 cells / well of hCR2-CHOK1 cells in DMEM / F-12 medium containing 10% FCS And cultured overnight. Equal volume of 4% paraformaldehyde solution (MUTO Chemical) was added, cells were fixed by standing at 4 ° C for 20 minutes, and after washing with PBST, blocking treatment was performed for 1 hour or more with 1% BSA-PBS . As antibodies, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1, and 4E5, 2F5, and B5, which are commercially available anti-RAMP2 antibodies, were used. Each purified antibody was added in a 1/2 serial dilution from 67.5 nM, and reacted with Goat Anti-mouse-IgG-HRP (SouthernBiotech) as a secondary antibody. After coloring by adding TMB solution (eBioscience), the reaction was stopped by adding 2N H 2 SO 4 solution. OD450 was measured using iMark (BioRAD). From the binding data obtained, binding affinity (EC50) was calculated using GraphPad Prism 7.
 図7に示すように実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3は、市販抗体である4E5、2F5、及びB5と比較して明らかに優れた結合活性を示した。縦軸は同一試験において得られた抗体の最大結合量(OD値)を100%として、最大結合量に対する割合として算出した。各抗体の結合親和性を表すEC50は、25H4-4F9が0.40nM、85H7-2B11が0.14nM、33H4-1G3が0.30nMだった。市販抗体は、いずれも結合能が低いことから正常な結合曲線が描けず、EC50値としては、は4E5が37.7nM以上、2F5とB5は66.7nM以上であった。 As shown in FIG. 7, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 clearly showed superior binding activity as compared with the commercially available antibodies 4E5, 2F5, and B5. . The vertical axis is calculated as a ratio to the maximum binding amount, where the maximum binding amount (OD value) of the antibody obtained in the same test is 100%. The EC 50 representing binding affinity of each antibody was 0.40 nM for 25H4-4F9, 0.14 nM for 85H7-2B11, and 0.30 nM for 33H4-1G3. None of the commercially available antibodies can draw a normal binding curve due to their low binding ability, and EC50 values are 37.7 nM or more for 4E5, and 66.7 nM or more for 2F5 and B5.
(実施例7)FACSによる細胞膜表面タンパクに対する結合解析
 各抗体(実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3、並びに、市販の抗RAMP2抗体である、4E5、2F5、及びB5)のhCR2-CHOK1細胞の膜表面RAMP2タンパクに対する結合能をフローサイトメーター(ACEA社)にて測定した。3×10cellsの剥離したhCR2-CHOK1細胞と3μg/mLの抗体溶液50μLを96ウェルV底プレート(Thermo Fishcer Scientific社)に加え、氷上で1時間静置し抗体を結合させた。FACSバッファー(1%BSA/0.05%NaN/PBS)にて3回洗浄し、2.5μg/mLに調製したR-Phycoerythrin-conjugated Affinipure F(ab’) Fragment Goat Anti-Mouse IgG(H+L)(Jackson Immunoresearch Laboratories社)を加え、氷上遮光下で30分間静置し二次抗体を結合させた。一回洗浄した後、500μLのFACSバッファーに懸濁して、実施例7と同様の方法でFACSにより蛍光を測定した。
(Example 7) Binding analysis to cell membrane surface proteins by FACS Each antibody (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1, and 4E5, 2F5, which are commercially available anti-RAMP2 antibodies, And B5) hCR2-CHOK1 cells' binding ability to membrane surface RAMP2 protein was measured with a flow cytometer (ACEA). 3 × 10 5 cells of exfoliated hCR2-CHOK1 cells and 50 μL of 3 μg / mL antibody solution were added to a 96-well V-bottom plate (Thermo Fishcer Scientific) and allowed to stand on ice for 1 hour to allow antibody to bind. R-Phycoerythrin-conjugated Affinipure F (ab ') 2 Fragment Goat Anti-Mouse IgG (washed with FACS buffer (1% BSA / 0.05% NaN 3 / PBS) three times and adjusted to 2.5 μg / mL H + L) (Jackson Immunoresearch Laboratories) was added, and the mixture was allowed to stand for 30 minutes under light shielding on ice to bind a secondary antibody. After washing once, the cells were suspended in 500 μL of FACS buffer, and fluorescence was measured by FACS in the same manner as in Example 7.
 図8に示すように、実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3は、全て細胞膜表面タンパクに結合した。また、市販抗体のうち4E5は細胞膜表面タンパクへの結合が認められたが、市販抗体2F5とB5は結合が観察されなかった。 As shown in FIG. 8, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 were all bound to cell membrane surface proteins. Among the commercially available antibodies, 4E5 was found to bind to cell membrane surface proteins, but the commercially available antibodies 2F5 and B5 were not observed to bind.
(実施例8)FACSによるエピトープの競合解析
 ビオチン化した85H7-2B11とビオチン化していない各抗体(実施例1で得られた25H4-4F9、85H7-2B11、及び33H4-1G3、並びに市販抗体4E5)をhCR2-CHOK1に同時に反応させることにより、抗原に対する競合試験を行った。製造者のマニュアルに従って、Biotin Labeling kit-NH(Dojindo社)を用いて85H7-2B11抗体をビオチン化した。ビオチン化85H7-2B11抗体3μg/mLとビオチン化していない各抗体の0、0.1、0.3、1、3、10、及び30μg/mLをhCR2-CHOK1細胞に加え、氷上で1時間静置し、競合条件下で抗体を結合させた。3回洗浄の後、2.5μg/mLに調製したStreptavidin-Fluorescein Isothiocyanate(Biolegend社)を加え、氷上遮光下で30分間静置し二次抗体を結合させた。一回洗浄の後、500μLのFACSバッファーに懸濁して、実施例7と同様の方法でFACSにより蛍光を測定した。
(Example 8) Competitive analysis of epitope by FACS Biotinylated 85H7-2B11 and non-biotinylated antibodies (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and commercial antibody 4E5) The competition test for the antigen was performed by simultaneously reacting hCR2-CHOK1 with hCR2-CHOK1. The 85H7-2B11 antibody was biotinylated using Biotin Labeling kit-NH 2 (Dojindo) according to the manufacturer's manual. Add 3 μg / mL of biotinylated 85H7-2B11 antibody and 0, 0.1, 0.3, 1, 3, 10, and 30 μg / mL of each non-biotinylated antibody to hCR2-CHOK1 cells, and stand on ice for 1 hour The antibodies were allowed to bind under competitive conditions. After washing three times, Streptavidin-Fluorescein Isothiocyanate (Biolegend) prepared to 2.5 μg / mL was added, and the mixture was allowed to stand for 30 minutes on a light-shielded light to bind a secondary antibody. After washing once, the cells were suspended in 500 μL of FACS buffer, and fluorescence was measured by FACS in the same manner as in Example 7.
 図9に示すように25H4-4F9、85H7-2B11、及び33H4-1G3は抗原に対する結合において85H7-2B11と競合したが、市販抗体の4E5は85H7-2B11と競合しなかった。 As shown in FIG. 9, 25H4-4F9, 85H7-2B11, and 33H4-1G3 competed with 85H7-2B11 for binding to the antigen, but the commercial antibody 4E5 did not compete with 85H7-2B11.
(実施例9)ヒト化抗体のデザイン
 25H4-4F9及び85H7-2B11抗体について、ヒト化抗体(ヒト化25H4-4F9、ヒト化85H7-2B11抗体)をデザインした。ヒト化配列のデザインは、Xoma corporationで考案された方法(US Patent5,766,886;MODIFIED ANTIBODY VARIABLE DOMAINS、特許期間満了)に従い、アミノ酸配列の設計を行った。25H4-4F9マウス抗体重鎖及びVLまたは85H7-2B11マウス抗体重鎖及びVLのアミノ酸配列を用いてヒト免疫グロブリンGERMLINEデータベース(NCBI:IGBLAST)で相同性検索を行い、最も高い相同性を示したアミノ酸配列(25H4-4F9重鎖:IGHV1-69-2*01、IGHV1-18*04及びIGHJ6*01、25H4-4F9軽鎖:IGKV1-33*01及びIGKJ2*01、85H7-2B11重鎖:IGHV1-46*01、IGHV1-18*04及びIGHJ6*01、85H7-2B11軽鎖:IGKV1-33*01及びIGKJ2*01、なお、25H4-4F9と85H7-2B11のVLのアミノ酸配列は一か所を除いて相同であるため、同一配列をフレームワークとして選択した)をヒト化抗体のフレームワークとした。これらフレームワークの各アミノ酸残基につき、マウスの配列をヒトの配列に変更した場合に抗体の3次元構造や抗原への結合性が失われてしまうリスクを3段階(HIGH,MODERATE,LOW)で評価し、LOWリスクのアミノ酸残基をはじめにヒト型のアミノ酸配列に変換し、その後、MODERATE及びHIGHリスクのアミノ酸残基の変換を行い、各抗体の重鎖については5種類(vh1~vh5)、軽鎖は4種類(vk1~vk4)の配列をデザインした。さらに、重鎖と軽鎖を組み合わせて、各抗体につき計8種類のヒト化抗体を作製した。
Example 9 Design of Humanized Antibody A humanized antibody (humanized 25H4-4F9, humanized 85H7-2B11 antibody) was designed for the 25H4-4F9 and 85H7-2B11 antibodies. For the design of the humanized sequence, the design of the amino acid sequence was performed according to the method (US Patent 5,766, 886; MODIFIED ANTIBODY VARIABLE DOMAINS, expiration of patent term) devised by Xoma corporation. Amino acid sequences of 25H4-4F9 mouse antibody heavy chain and VL or amino acids of 85H7-2B11 mouse antibody heavy chain and VL were used for homology search with human immunoglobulin GERMLINE database (NCBI: IGBLAST) and amino acids showing the highest homology Sequence (25H4-4F9 heavy chain: IGHV1-69-2 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 25H4-4F9 light chain: IGKV1-33 * 01 and IGKJ2 * 01, 85H7-2B11 heavy chain: IGHV1- 46 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 85H7-2B11 light chains: IGKV1-33 * 01 and IGKJ2 * 01, except that the amino acid sequences of the 25H4-4F9 and 85H7-2B11 VL are excluded from one place Of the same sequence, It was selected as a work) and the humanized antibody framework. For each amino acid residue of these frameworks, there is a three-step risk (HIGH, MODERATE, LOW) of losing the three-dimensional structure of the antibody and the binding to the antigen when the mouse sequence is changed to the human sequence. Evaluate and convert LOW risk amino acid residues to human type amino acid sequence first, then convert MODERATE and HIGH risk amino acid residues, and for each antibody heavy chain, 5 types (vh1 to vh5), The light chain was designed with four types (vk1 to vk4) of sequences. In addition, the heavy and light chains were combined to produce a total of eight humanized antibodies for each antibody.
 デザインされた25H4-4F9のVH及びVLの配列を図10に、デザインされた85H7-2B11のVH及びVLの配列を図11に示す。また、作製したヒト化抗体における重鎖と軽鎖及びVH,VL,CDRの配列の組み合わせを以下の表2に示す。 Sequences of the designed 25H4-4F9 VH and VL are shown in FIG. 10, and sequences of the designed 85H7-2B11 VH and VL are shown in FIG. In addition, combinations of heavy chain and light chain and VH, VL and CDR sequences in the produced humanized antibody are shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
(実施例10)リコンビナントヒト化抗体の作製
 リコンビナント抗体を作製するために重鎖をコードする遺伝子配列(配列番号102,104,106,108,110,120,122,124,126,及び128)及び軽鎖をコードする遺伝子配列(配列番号112,114,116,118,130,132,134,及び136)を哺乳類発現ベクターpcDNA3.4(Thermo)にクローニングした。重鎖及び軽鎖をクローニングしたベクターを重量比1:1になるように混合し、10μgから30μgのベクターDNAを用いて、哺乳類細胞CHO-S(Thermo)にトランスフェクションした。トランスフェクションは、ExpiFectamineTMCHO Transfection Kit(Thermo)を用い、10mLから30mLの細胞懸濁液を14日間フラスコによる旋回培養を行った。14日間培養した培養液から遠心操作により細胞を除き、0.45μmのフィルターを通した培養上清をProteinAレジン(Amsphere A3;JSR Life Sciences)により精製した。ProteinAレジンと培養上清を4℃で一晩混合し、リコンビナント抗体を結合させた後、20mMリン酸緩衝液pH7.0で洗浄した。レジンに結合したリコンビナント抗体を0.1M Glycine-HCl pH3.2のElution bufferで溶出し、1/50容量の1M Tris-HCl pH9.0で中和した。中和後、30kDaの限外濾過膜(Amicon Ultra:ミリポア)を用いてリン酸緩衝液にBufferを置換し、280nmの吸光度を測定してリコンビナント抗体の濃度を決定した。
Example 10 Preparation of Recombinant Humanized Antibody Gene sequence encoding heavy chain for preparation of recombinant antibody (SEQ ID NOs: 102, 104, 106, 108, 110, 120, 122, 124, 126, and 128) and The gene sequence encoding the light chain (SEQ ID NOS: 112, 114, 116, 118, 130, 132, 134 and 136) was cloned into the mammalian expression vector pcDNA3.4 (Thermo). The heavy and light chain cloned vectors were mixed at a weight ratio of 1: 1, and 10 μg to 30 μg of vector DNA was used to transfect mammalian cell CHO-S (Thermo). For transfection, 10 ml to 30 ml of the cell suspension was subjected to swirl culture in a flask for 14 days using ExpiFectamineTM CHO Transfection Kit (Thermo). The cells were removed from the culture solution cultured for 14 days by centrifugation, and the culture supernatant filtered through a 0.45 μm filter was purified by Protein A resin (Amsphere A3; JSR Life Sciences). Protein A resin and the culture supernatant were mixed overnight at 4 ° C. to allow binding of the recombinant antibody, and then washed with 20 mM phosphate buffer pH 7.0. The resin-bound recombinant antibody was eluted with 0.1 M Glycine-HCl pH 3.2 Elution buffer and neutralized with 1/50 volume of 1 M Tris-HCl pH 9.0. After neutralization, Buffer was replaced with phosphate buffer using a 30 kDa ultrafiltration membrane (Amicon Ultra: Millipore), and the concentration of recombinant antibody was determined by measuring the absorbance at 280 nm.
(実施例11)ヒト化抗体のcAMP抑制試験
 hCR2-CHOK1細胞を用いて、ヒト化抗体のcAMPの抑制試験を実施した。測定にはマウス抗体の活性測定と同様に、Perkinelmer社製のHalf area 96wellプレートを使用し、2500cellsのhCR2-CHOK1細胞を10%FBS含有D-MEM/Ham’s F-12(和光純薬、日本)中で一晩培養し、各抗体(ヒト化25H4-4F9、ヒト化85H7-2B11、4E5、2F5、及びB5)を添加し、終濃度150pMのヒトAM(株式会社ペプチド研究所、日本)を添加した。cAMPは、LANCE Ultra cAMP kit(Perkinelmer社)を用いて、製造者のマニュアルに従って測定した。コントロールとして、抗体非添加/AM添加、及び抗体非添加/AM非添加のhCR2-CHOK1細胞によるcAMP産生を同様に測定した。
Example 11 cAMP Suppression Test of Humanized Antibody The hCR2-CHOK1 cells were used to conduct a suppression test of cAMP of humanized antibody. For measurement, as in the measurement of mouse antibody activity, use of Perkinelmer's Half area 96 well plate, and using 2500 cells of hCR2-CHOK1 cells in D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd., 10% FBS) After overnight culture in Japan, each antibody (humanized 25H4-4F9, humanized 85H7-2B11, 4E5, 2F5, and B5) was added, and human AM with a final concentration of 150 pM (Peptide Research Laboratories, Japan) Was added. cAMP was measured using the LANCE Ultra cAMP kit (Perkinelmer) according to the manufacturer's manual. As a control, cAMP production by hCR2-CHOK1 cells without antibody addition / AM addition and without antibody addition / AM addition was similarly measured.
 図12及び図13は、それぞれヒト化25H4-4F9抗体及びヒト化85H7-2B11抗体を16.7nM添加した時のcAMP抑制率を示す。cAMP抑制率は、抗体非添加の場合のcAMP濃度を100とした場合の各抗体添加群のcAMP濃度の割合で示した。コントロールとして用いた市販抗体(4E5、2F5、及びB5)はほとんどcAMP産生を抑制しなかったのに対し、ヒト化25H4-4F9抗体、及びヒト化85H7-2B11抗体は、AM非添加群と同等までcAMP産生を抑制した。 FIG. 12 and FIG. 13 show the cAMP inhibition rate when 16.7 nM of humanized 25H4-4F9 antibody and humanized 85H7-2B11 antibody were added, respectively. The cAMP inhibition rate was shown as a ratio of the cAMP concentration of each antibody-added group when the cAMP concentration in the case of no antibody addition was 100. The commercially available antibodies (4E5, 2F5, and B5) used as controls hardly suppressed cAMP production, whereas the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were equivalent to the non-AM added group. It suppressed cAMP production.
 また、各抗体の濃度とcAMP抑制率との関係を図14に示す。ヒト化25H4-4F9抗体、及びヒト化85H7-2B11抗体のcAMP抑制に対するIC50値は、それぞれ、h25H4-4F9v1が0.17nM、h25H4-4F9v2が0.14nM、h25H4-4F9v3が0.26nM、h25H4-4F9v4が0.17nM、h25H4-4F9v5が0.50nM、h25H4-4F9v6が1.35nM、h25H4-4F9v8が1.07nM、そして、h85H7-2B11v1が0.07nM、h85H7-2B11v2が0.11nM、h85H7-2B11v3が0.08nM、h85H7-2B11v4が0.13nM、h85H7-2B11v5が0.14nM、h85H7-2B11v6が0.18nM、h85H7-2B11v7が0.12nM、h85H7-2B11v8が0.18nMであった。 In addition, the relationship between the concentration of each antibody and the cAMP suppression rate is shown in FIG. The IC50 values for cAMP inhibition of the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were 0.17 nM for h25H4-4F9v1, 0.14 nM for h25H4-4F9v2, and 0.26 nM for h25H4-4F9v3, respectively. 4F9v4 is 0.17 nM, h25H4-4F9v5 is 0.50 nM, h25H4-4F9v6 is 1.35 nM, h25H4-4F9v8 is 1.07 nM, and h85H7-2B11v1 is 0.07 nM, h85H7-2B11v2 is 0.11 nM, h85H7- 2B11v3 is 0.08 nM, h85H7-2B11v4 is 0.13 nM, h85H7-2B11v5 is 0.14 nM, h85H7-2B11v6 is 0.18 nM, h85H7-2B11v7 is 0.12 nM, h85 7-2B11v8 was 0.18nM.
(実施例12)ヒト化抗体のCell ELISAによる結合親和性解析
 96wellプレート(BD-FALCON社)に10%FCS含有DMEM/F-12培地(和光純薬)中のhCR2-CHOK1細胞を1×10cells/wellで添加し、一晩培養した。等量の4%パラホルムアルデヒド溶液(MUTO化学)を添加し、4℃で20分間静置して細胞を固定し、PBSTで洗浄した後、1%BSA-PBSで1時間以上のブロッキング処理を行った。精製抗体を67nMから1/10段階希釈して添加し、二次抗体として40.0ng/mLのGoat-Anti-Human-IgG-Fc-Fragment-HRP-conjugated(Bethyl社)を100μL添加して反応させた。TMB solution(eBioscience社)により発色させ、2NのHSO溶液で反応を停止してiMark(BioRAD社)を用いてOD450を測定した。得られた結合データについて、GraphPad Prism7を用いて結合親和性(EC50)を算出した。
Example 12 Binding Affinity Analysis of Humanized Antibody by Cell ELISA Binding to 96-well plate (BD-FALCON) hCR2-CHOK1 cells in DMEM / F-12 medium (Wako Pure Chemical Industries) containing 10% FCS 1 × 10 The cells were added at 4 cells / well and cultured overnight. Add an equal volume of 4% paraformaldehyde solution (MUTO Chemical), fix at 20 ° C for 20 minutes to fix cells, wash with PBST, and then block with 1% BSA-PBS for 1 hour or more The The purified antibody was added in 1/10 serial dilution from 67 nM, and 100 μL of 40.0 ng / mL Goat-Anti-Human-IgG-Fc-Fragment-HRP-conjugated (Bethyl) was added as a secondary antibody for reaction. I did. The color was developed with TMB solution (eBioscience), the reaction was stopped with 2N H 2 SO 4 solution, and OD450 was measured using iMark (BioRAD). The binding affinity (EC50) was calculated using GraphPad Prism 7 for the obtained binding data.
 図15に示すように、抗体を6.7nM添加した際の結合は、ヒト化25H4-4F9及びヒト化85H7-2B11において、市販抗体の4E5、2F5、及びB5と比較して明らかに優れていた。 As shown in FIG. 15, the binding when adding 6.7 nM of antibody was clearly superior to humanized 25H4-4F9 and humanized 85H7-2B11 in comparison with the commercially available antibodies 4E5, 2F5, and B5. .
 また、各濃度での結合を図16に示す。各抗体の結合活性(EC50)は、それぞれh25H4-4F9v1が0.73nM、h25H4-4F9v2が1.05nM、h25H4-4F9v3が0.83nM、h25H4-4F9v4が1.83nM、h25H4-4F9v5が2.32nM、h25H4-4F9v6が3.19nM、h25H4-4F9v8が3.03nM、そして、h85H7-2B11v1が0.18nM、h85H7-2B11v2が0.24nM、h85H7-2B11v3が0.23nM、h85H7-2B11v4が0.22nM、h85H7-2B11v5が0.25nM、h85H7-2B11v6が0.24nM、h85H7-2B11v7が0.23nM、h85H7-2B11v8が0.20nMであった。 Also, binding at each concentration is shown in FIG. The binding activity (EC50) of each antibody is 0.73 nM for h25H4-4F9v1, 1.05 nM for h25H4-4F9v2, 0.83 nM for h25H4-4F9v3, 1.83 nM for h25H4-4F9v4, and 2.32 nM for h25H4-4F9v5. H25H4-4F9v6 3.19 nM h25H4-4F9v8 3.03 nM h85H7-2B11 v1 0.18 nM h85 H7-2 B11 v2 0.24 nM h85 H7-2 B11 v3 0.23 nM h85 H7-2 B11 v4 0.22 nM H85H7-2B11v5 was 0.25 nM, h85H7-2B11v6 was 0.24 nM, h85H7-2B11v7 was 0.23 nM, and h85H7-2B11v8 was 0.20 nM.
(実施例13)ヒト化抗体のHUVECの増殖抑制試験
 ヒト化抗体のHUVEC増殖抑制能について、実施例3と同様の方法で調べた。96well培養プレートに2,500cells/wellでHUVECを播種し、3時間の前培養により接着させた。その後、各濃度のヒト化25H4-4F9、及びヒト化85H7-2B11を添加し、終濃度100nMのAMを添加し、96時間後におけるHUVECの増殖を観察して、HUVECの増殖抑制能を確認した。細胞数の測定にはCell Counting Kit-8(DOJINDO社)を用いた。増殖因子を除いた2%FBS含有Humedia-EG2培地で10倍希釈した発色溶液を100μL添加し、COインキュベーターで4時間発色させOD450として計測した。
Example 13 Growth Inhibition Test of HUVEC of Humanized Antibody The HUVEC growth inhibitory ability of the humanized antibody was examined in the same manner as in Example 3. HUVECs were seeded at 2,500 cells / well in 96-well culture plates and adhered by pre-incubation for 3 hours. Thereafter, each concentration of humanized 25H4-4F9 and humanized 85H7-2B11 was added, and a final concentration of 100 nM of AM was added, and the proliferation of HUVEC after 96 hours was observed to confirm the proliferation inhibitory ability of HUVEC. . Cell Counting Kit-8 (DOJINDO) was used to measure the number of cells. 100 μL of a 10-fold diluted color solution was added to Humedia-EG2 medium containing 2% FBS excluding growth factors, and coloring was performed for 4 hours in a CO 2 incubator to measure OD450.
 抗体非添加の場合のOD450(HUVEC細胞数)を100として、各抗体の添加濃度の割合からHUVEC増殖抑制率(IC50)を算出した。その結果、ヒト化25H4-4F9抗体、及びヒト化85H7-2B11抗体のIC50値は、それぞれh25H4-4F9v1が16.0nM、h25H4-4F9v2が10.9nM、そして、h85H7-2B11v1が5.1nM、h85H7-2B11v2が9.5nM、h85H7-2B11v3が1.5nM、h85H7-2B11v4が1.4nM、h85H7-2B11v5が21.4nM、h85H7-2B11v6が14.8nM、h85H7-2B11v7が32.7nM、h85H7-2B11v8が22.6nMであった。 Assuming that the OD450 (number of HUVEC cells) in the case of no antibody addition is 100, the HUVEC growth inhibition rate (IC50) was calculated from the ratio of the concentration of each antibody. As a result, the IC50 values of the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were 16.0 nM for h25H4-4F9v1, 10.9 nM for h25H4-4F9v2, and 5.1 nM for h85H7-2B11v1, respectively. -2B11v2 is 9.5 nM, h85H7-2B11v3 is 1.5 nM, h85H7-2B11v4 is 1.4 nM, h85H7-2B11v5 is 21.4 nM, h85H7-2B11v6 is 14.8 nM, h85H7-2B11v7 is 32.7 nM, h85H7-2B11v8 Was 22.6 nM.
(実施例14)抗体のエピトープ解析
 85H7-2B11抗体が認識する特定のエピトープ一次配列が存在するか否かを決定するために、ヒトRAMP2タンパクの細胞外領域について、12アミノ酸が重複するように3アミノ酸ずらした以下の31個の13~15アミノ酸からなるペプチド断片(配列番号71~101)を作製し、それらのペプチドに対する抗体の結合能をELISA法により調べた。ペプチドとタンパクの固相は1.0μg/mLの十分量を用い、結合には85H7-2B11抗体を20ng/mLで反応させた。二次抗体としてGoat Anti-mouse-IgG-HRPを50.0ng/mLで反応させ、結合した抗体をTMB溶液で発色させ、2NのHSO溶液で反応を停止した後にOD450を測定した。
QPLPTTGTPGSEGGT(配列番号71)
PTTGTPGSEGGTVKN(配列番号72)
GTPGSEGGTVKNYET(配列番号73)
GSEGGTVKNYETAVQ(配列番号74)
GGTVKNYETAVQFSW(配列番号75)
VKNYETAVQFSWNHY(配列番号76)
YETAVQFSWNHYKDQ(配列番号77)
AVQFSWNHYKDQMDP(配列番号78)
FSWNHYKDQMDPIEK(配列番号79)
NHYKDQMDPIEKDWS(配列番号80)
KDQMDPIEKDWSDWA(配列番号81)
MDPIEKDWSDWAMIS(配列番号82)
IEKDWSDWAMISRPY(配列番号83)
DWSDWAMISRPYSTL(配列番号84)
DWAMISRPYSTLRDS(配列番号85)
MISRPYSTLRDSLEH(配列番号86)
RPYSTLRDSLEHFAE(配列番号87)
STLRDSLEHFAELFD(配列番号88)
RDSLEHFAELFDLGF(配列番号89)
LEHFAELFDLGFPNP(配列番号90)
FAELFDLGFPNPLAE(配列番号91)
LFDLGFPNPLAERII(配列番号92)
LGFPNPLAERIIFET(配列番号93)
PNPLAERIIFETHQI(配列番号94)
LAERIIFETHQIHFA(配列番号95)
RIIFETHQIHFANSS(配列番号96)
FETHQIHFANSSLVQ(配列番号97)
HQIHFANSSLVQPTF(配列番号98)
HFANSSLVQPTFSDP(配列番号99)
NSSLVQPTFSDPPED(配列番号100)
LVQPTFSDPPEDV(配列番号101)
(Example 14) Epitope analysis of antibody In order to determine whether or not a specific epitope primary sequence recognized by the 85H7-2B11 antibody is present, 12 amino acids are overlapped for the extracellular region of human RAMP2 protein 3 The following 31 13-15 amino acid peptide fragments (SEQ ID NOS: 71-101) shifted by amino acid were prepared, and the binding ability of the antibodies to these peptides was examined by ELISA. A sufficient amount of 1.0 μg / mL was used for the solid phase of peptide and protein, and 85H7-2B11 antibody was reacted at 20 ng / mL for binding. As a secondary antibody, Goat Anti-mouse-IgG-HRP was reacted at 50.0 ng / mL, the bound antibody was developed with TMB solution, and after stopping the reaction with 2N H 2 SO 4 solution, OD450 was measured.
QPLPTTGTPGSEGGT (SEQ ID NO: 71)
PTTGTPGSEGGTVKN (SEQ ID NO: 72)
GTPGSEGGTVKNYET (SEQ ID NO: 73)
GSEGGTVKNYETAVQ (SEQ ID NO: 74)
GGTVKNYETAVQFSW (SEQ ID NO: 75)
VKNYETAVQFSWNHY (SEQ ID NO: 76)
YETAVQFSWNHYKDQ (SEQ ID NO: 77)
AVQFSWNHYKDQMDP (SEQ ID NO: 78)
FSWNHYKDQMDPIEK (SEQ ID NO: 79)
NHYKDQMDPIEKDWS (SEQ ID NO: 80)
KDQMDPIEKDWSDWA (SEQ ID NO: 81)
MDPIEKDWSDWAMIS (SEQ ID NO: 82)
IEKDWS DWAMISRPY (SEQ ID NO: 83)
DWSDWAMISRPYSTL (SEQ ID NO: 84)
DWAMISRPYSTLRDS (SEQ ID NO: 85)
MISRPYSTRDLSHEH (SEQ ID NO: 86)
RPYSTRDSLEHFAE (SEQ ID NO: 87)
STLRDSLEHFAELFD (SEQ ID NO: 88)
RDSLEHFAELFDLGF (SEQ ID NO: 89)
LEHFAELFDLGFPNP (SEQ ID NO: 90)
FAELFDLGFPNPLAE (SEQ ID NO: 91)
LFDLGFPNPLAERII (SEQ ID NO: 92)
LGFPNPLAERIIFET (SEQ ID NO: 93)
PNPLAERII FET HQI (SEQ ID NO: 94)
LAERII FET HQI HFA (SEQ ID NO: 95)
RIIFETHQIHFANSS (SEQ ID NO: 96)
FETHQIHFANSSLVQ (SEQ ID NO: 97)
HQIHFANSL VQPTF (SEQ ID NO: 98)
HFANSSLVQPTFSDP (SEQ ID NO: 99)
NSSLVQPTFSDPPED (SEQ ID NO: 100)
LVQPTFSDPPEDV (SEQ ID NO: 101)
 図17に示すように、等量のヒトRAMP2タンパクに対しては十分量の85H7-2B11抗体が結合した。一方で、85H7-2B11抗体はいずれのヒトRAMP2ペプチド断片にも結合しなかった。このことから、85H7-2B11抗体がRAMP2タンパク質の特定の一次構造ではなく、立体構造などの高次構造により構成されるエピトープを認識していることが示された。また、実施例8の結果から、本発明の抗体が抗原への結合において互いに競合することから、本発明の抗体はいずれもRAMP2タンパク質の特定の一次構造ではなく、立体構造などの高次構造により構成されるエピトープを認識すると考えられた。これらの結果から、HUVECの増殖を抑制するためには、RAMP2タンパク質の特定の一次構造ではなく、細胞外の立体構造により構成されるエピトープを認識することが重要であることが示された。 As shown in FIG. 17, a sufficient amount of 85H7-2B11 antibody bound to an equal amount of human RAMP2 protein. On the other hand, the 85H7-2B11 antibody did not bind to any human RAMP2 peptide fragment. From this, it was shown that the 85H7-2B11 antibody recognizes not the specific primary structure of the RAMP2 protein but an epitope constituted by a conformation such as a three-dimensional structure. Further, from the results of Example 8, since the antibodies of the present invention compete with each other for binding to the antigen, all of the antibodies of the present invention are not the specific primary structure of the RAMP2 protein but the higher order structure such as three-dimensional structure. It was thought to recognize the constructed epitope. From these results, it has been shown that it is important to recognize an epitope composed of an extracellular three-dimensional structure rather than the specific primary structure of the RAMP2 protein in order to suppress the proliferation of HUVEC.
 本抗体は、ヒトアドレノメジュリン受容体であるヒトRAMP2の機能を特異的に抑制できることから、RAMP2に関連する疾患の予防、診断又は治療方法の提供や、該疾患の予防薬、診断薬又は治療薬の開発において重要な役割を果たす事が期待される。 Since the present antibody can specifically suppress the function of human RAMP2 which is a human adrenomedullin receptor, provision of a method for preventing, diagnosing or treating a disease associated with RAMP2, a preventive agent, a diagnostic agent or a treatment for the disease It is expected to play an important role in drug development.

Claims (29)

  1.  in vitroのアドレノメジュリン添加による血管内皮細胞増殖試験において、血管内皮細胞の増殖抑制作用を有する、受容体活性調節タンパク質2(RAMP2)に特異的に結合する抗体又はその免疫反応性断片。 An antibody or an immunoreactive fragment thereof specifically binding to receptor activity regulatory protein 2 (RAMP2), which has a growth inhibitory effect on vascular endothelial cells in a vascular endothelial cell proliferation test by addition of adrenomedullin in vitro.
  2.  前記血管内皮細胞の増殖抑制作用が、35nM以下のIC50値である、請求項1に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to claim 1, wherein the growth inhibitory effect of the vascular endothelial cells is an IC 50 value of 35 nM or less.
  3.  アドレノメジュリン添加による動物細胞によるcAMP産生を抑制する作用を有する、請求項1又は請求項2に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to claim 1 or 2, which has an action of suppressing cAMP production by animal cells by addition of adrenomedullin.
  4.  in vitroのアドレノメジュリン添加による動物細胞によるcAMP産生試験において、cAMP産生抑制作用が、2nM以下のIC50値である、請求項3に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to claim 3, wherein the inhibitory effect on cAMP production is an IC50 value of 2 nM or less in a test of cAMP production by animal cells by addition of adrenomedullin in vitro.
  5.  ヒトCRLRとヒトRAMP2を共発現する細胞に結合する、請求項1~請求項4のいずれか1項に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to any one of claims 1 to 4, which binds to cells co-expressing human CRLR and human RAMP2.
  6.  ヒトCRLRとヒトRAMP2を共発現する細胞を用いたCell ELISA試験による結合親和性(EC50)が3.5nM以下である、請求項5に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to claim 5, which has a binding affinity (EC50) of 3.5 nM or less according to a Cell ELISA test using cells co-expressing human CRLR and human RAMP2.
  7.  配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載のアミノ酸配列を有する重鎖可変領域(VH)及び配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載のアミノ酸配列を有する軽鎖可変領域(VL)を含む抗体、とRAMP2への結合において競合する抗体又はその免疫反応性断片、あるいは、
     配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載のアミノ酸配列を有するVH及び配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載のアミノ酸配列を有するVLを含む抗体、と同じエピトープに結合する抗体又はその免疫反応性断片。
    A heavy chain variable region (VH) having the amino acid sequence set forth in any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And an antibody comprising a light chain variable region (VL) having the amino acid sequence set forth in any one selected from SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 , And an antibody or immunoreactive fragment thereof that competes for binding to RAMP2, or
    SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. VH having the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14 , 21, 37, 39, 41, 43, 55, 57, 59, and 61. An antibody comprising the VL having an amino acid sequence according to any one selected from the group consisting of Sex fragments.
  8.  RAMP2の細胞外領域を構成するアミノ酸配列の一次構造を認識しない、請求項1~請求項7のいずれか1項に記載の抗体又はその免疫反応性断片。 8. The antibody or immunoreactive fragment thereof according to any one of claims 1 to 7, which does not recognize the primary structure of the amino acid sequence constituting the extracellular domain of RAMP2.
  9.  配列番号71~101に記載の各アミノ酸配列からなるペプチドの全てと結合しない、請求項8に記載の抗体又はその免疫反応性断片。 The antibody or immunoreactive fragment thereof according to claim 8, which does not bind to all of the peptides consisting of the amino acid sequences set forth in SEQ ID NOs: 71 to 101.
  10.  LXIXDXYXYXMX(配列番号24)で表されるアミノ酸配列であって、ここで、XはG又はMであり、XはF又はYであり、XはA又はVであり、Xは存在しないかHであり、XはAかVであり、XはDかEであり、かつ、XはYかNである、アミノ酸配列からなる重鎖相補性決定領域(CDRH)3を有する重鎖を含む、請求項1~請求項9のいずれか1項に記載の抗体又はその免疫反応性断片。 An amino acid sequence represented by LX 1 IX 2 DX 3 YX 4 YX 5 MX 6 X 7 (SEQ ID NO: 24), wherein X 1 is G or M and X 2 is F or Y, Amino acid sequence wherein X 3 is A or V, X 4 is absent or H, X 5 is A or V, X 6 is D or E, and X 7 is Y or N The antibody or immunoreactive fragment thereof according to any one of claims 1 to 9, comprising a heavy chain having a heavy chain complementarity determining region (CDRH) 3 consisting of
  11.  LMIFDAYYAMDY(配列番号7)又はLGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有する重鎖を含む、請求項10に記載の抗体又はその免疫反応性断片。 11. The antibody or immunoreactive fragment thereof according to claim 10, which comprises a heavy chain having a CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDV YHYVMEN (SEQ ID NO: 16).
  12.  更に、重鎖がGYFMN(配列番号5)のアミノ酸配列からなるCDRH1、及びRXNPYNGDX10YX11QKFX12G(配列番号66)のアミノ酸配列からなるCDRH2を有する、請求項10又は請求項11に記載の抗体又はその免疫反応性断片、
     ここで、XはN又はIであり、XはS又はTであり、X10はI、F又はLであり、X11はA又はNであり、X12はQ又はKである。
    11. The method according to claim 10, wherein the heavy chain has CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5), and CDRH2 consisting of the amino acid sequence of RX 8 NPYNGDX 9 X 10 YX 11 QKFX 12 G (SEQ ID NO: 66). 11, or the immunoreactive fragment thereof
    Here, X 8 is N or I, X 9 is S or T, X 10 is I, F or L, X 11 is A or N, and X 12 is Q or K.
  13.  更に、X13ASQDIRNYLN(配列番号69)のアミノ酸配列からなる軽鎖相補性決定領域(CDRL)1、YTSRLX1415(配列番号70)のアミノ酸配列からなるCDRL2、及びQQDSKX16PWT(配列番号25)のアミノ酸配列からなるCDRL3を有する軽鎖を含む、請求項12に記載の抗体又はその免疫反応性断片、
     ここで、X13はQ又はRであり、X14はE又はHであり、X15はS又はTであり、X16はH又はNである。
    Furthermore, a light chain complementarity determining region (CDRL) 1 consisting of the amino acid sequence of X 13 ASQDIRNYLN (SEQ ID NO: 69), a CDRL 2 consisting of the amino acid sequence of YTSRLX 14 X 15 (SEQ ID NO: 70), and QQDSKX 16 PWT (SEQ ID NO: 25) The antibody according to claim 12, or an immunoreactive fragment thereof, comprising a light chain having a CDR L3 consisting of the amino acid sequence of
    Here, X 13 is Q or R, X 14 is E or H, X 15 is S or T, and X 16 is H or N.
  14.  GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RNNPYNGDSIYNQKFKG(配列番号6)、RINPYNGDTFYNQKFKG (配列番号15)、RINPYNGDTLYNQKFKG(配列番号22)、RNNPYNGDSIYNEKFQG(配列番号62)、RNNPYNGDSIYAEKFQG(配列番号63)、RINPYNGDTFYNQKFQG(配列番号64)、及びRINPYNGDTFYAQKFQG(配列番号65)からなる群から選択されるいずれか1つのアミノ酸配列からなるCDRH2、及び
     LMIFDAYYAMDY(配列番号7)又はLGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3を有する、請求項12に記載の抗体又はその免疫反応性断片。
    CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    RNNPYNGDSIYNQKFQ (SEQ ID NO: 6), RINPYNGDTFYNQKFKG (SEQ ID NO: 15), RINPYNGDTLYNQKFKG (SEQ ID NO: 22), RNNPYNGDSIYNEKFQG (SEQ ID NO: 63), RINPYNGDTFYNQQ QQQ 13. The antibody according to claim 12, which has a CDRH2 consisting of any one amino acid sequence selected from the group consisting of: and a CDRH3 consisting of an amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO: 16). Sex fragments.
  15.  更に、RASQDIRNYLN(配列番号8)又はQASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、
     YTSRLHS(配列番号9)又はYTSRLET(配列番号68)のアミノ酸配列からなるCDRL2、及び
     QQDSKHPWT(配列番号10)又はQQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3を有する、請求項14に記載の抗体又はその免疫反応性断片。
    Furthermore, a CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO: 67),
    The antibody according to claim 14, having a CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9) or YTSRLET (SEQ ID NO: 68), and a CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT (SEQ ID NO: 17). Or an immunoreactive fragment thereof.
  16.  以下の(i)~(vii)のいずれか1つの重鎖CDRの組み合わせを有する、請求項14に記載の抗体又はその免疫反応性断片:
    (i)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RNNPYNGDSIYNQKFKG(配列番号6)のアミノ酸配列からなるCDRH2、及び
     LMIFDAYYAMDY(配列番号7)のアミノ酸配列からなるCDRH3;
    (ii)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RNNPYNGDSIYNEKFQG(配列番号62)のアミノ酸配列からなるCDRH2、及び
     LMIFDAYYAMDY(配列番号7)のアミノ酸配列からなるCDRH3;
    (iii)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RNNPYNGDSIYAEKFQG(配列番号63)のアミノ酸配列からなるCDRH2、及び
     LMIFDAYYAMDY(配列番号7)のアミノ酸配列からなるCDRH3
    (iv)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RINPYNGDTFYNQKFKG (配列番号15)のアミノ酸配列からなるCDRH2、及び
     LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3;
    (v)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RINPYNGDTFYNQKFQG(配列番号64)、のアミノ酸配列からなるCDRH2、及び
     LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3;
    (vi)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RINPYNGDTFYAQKFQG(配列番号65)のアミノ酸配列からなるCDRH2、及び
     LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3;又は、
    (vii)GYFMN(配列番号5)のアミノ酸配列からなるCDRH1、
     RINPYNGDTLYNQKFKG(配列番号22)のアミノ酸配列からなるCDRH2、及び
     LGIYDVYHYVMEN(配列番号16)のアミノ酸配列からなるCDRH3。
    15. The antibody or immunoreactive fragment thereof according to claim 14, which has a combination of heavy chain CDRs of any one of the following (i) to (vii):
    (I) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RNNP YNGDSI YNQ KF KF (SEQ ID NO: 6), and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7);
    (Ii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RNNP YNGDSI YNEKF QG (SEQ ID NO: 62), and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7);
    (Iii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RNNP YNGDSIYAEKF QG (SEQ ID NO: 63), and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7)
    (Iv) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RINPYNGDTFYNQKFKG (SEQ ID NO: 15), and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16);
    (V) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RINPYNGDTFYNQKFQG (SEQ ID NO: 64), and CDRH3 consisting of the amino acid sequence of LGIYDV YHYVMEN (SEQ ID NO: 16);
    (Vi) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RINPYNGDTFYAQFKQG (SEQ ID NO: 65), and CDRH3 consisting of the amino acid sequence of LGIYDV YHYVMEN (SEQ ID NO: 16);
    (Vii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
    CDRH2 consisting of the amino acid sequence of RINPYNGDTLYNQKFKG (SEQ ID NO: 22), and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16).
  17.  更に、以下の(a)~(f)のいずれか1つの軽鎖CDRの組み合わせを有する、請求項16に記載の抗体又はその免疫反応性断片:
    (a)RASQDIRNYLN(配列番号8)のアミノ酸配列からなるCDRL1、
     YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
     QQDSKHPWT(配列番号10)のアミノ酸配列からなるCDRL3;
    (b)QASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、
     YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
     QQDSKHPWT(配列番号10)のアミノ酸配列からなるCDRL3;
    (c)QASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、
     YTSRLET(配列番号68)のアミノ酸配列からなるCDRL2、及び
     QQDSKHPWT(配列番号10)のアミノ酸配列からなるCDRL3;
    (d)RASQDIRNYLN(配列番号8)のアミノ酸配列からなるCDRL1、
     YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
     QQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3;
    (e)QASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、
     YTSRLHS(配列番号9)のアミノ酸配列からなるCDRL2、及び
     QQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3;又は、
    (f)QASQDIRNYLN(配列番号67)のアミノ酸配列からなるCDRL1、
     YTSRLET(配列番号68)のアミノ酸配列からなるCDRL2、及び
     QQDSKNPWT(配列番号17)のアミノ酸配列からなるCDRL3。
    Furthermore, the antibody or the immunoreactive fragment thereof according to claim 16, which has a combination of light chain CDRs of any one of the following (a) to (f):
    (A) CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
    CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10);
    (B) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67),
    CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10);
    (C) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67),
    CDRL2 consisting of the amino acid sequence of YTSRLET (SEQ ID NO: 68), and CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10);
    (D) CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
    CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17);
    (E) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67),
    CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9), and CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17);
    (F) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67),
    CDRL2 consisting of the amino acid sequence of YTSRLET (SEQ ID NO: 68), and CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17).
  18.  以下のいずれかである、請求項1~請求項17のいずれか1項に記載の抗体:
     VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有し、かつ、VLが配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載されたアミノ酸配列をコードする核酸配列とストリンジェントな条件下でハイブリダイズする核酸配列によりコードされたアミノ酸配列を有する抗体又はその免疫反応性断片;あるいは、
     VHが、配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つのアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、かつ、VLが、配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つのアミノ酸配列と80%以上の同一性を有するアミノ酸配列を有する抗体又はその免疫反応性断片。
    The antibody according to any one of claims 1 to 17, which is any of the following:
    A nucleic acid sequence encoding the amino acid sequence set forth in any one of VH selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. And has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with the SEQ ID NO: 4, and wherein VL has the sequence of SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 An antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding an amino acid sequence described in any one selected from: or an immunoreactive fragment thereof;
    VH has at least 80% identity with any one amino acid sequence selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 And has an amino acid sequence and 80% or more of any one amino acid sequence selected from SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 An antibody having an amino acid sequence with identity or an immunoreactive fragment thereof.
  19.  配列番号2、12、19、27、29,31,33、35、45,47,49、51,及び53から選択されるいずれか1つに記載のアミノ酸配列を有するVHを有する、請求項1~請求項17のいずれか1項に記載の抗体又はその免疫反応性断片。 2. A VH having an amino acid sequence according to any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. The antibody or immunoreactive fragment thereof according to any one of the above.
  20.  更に、配列番号4、14,21、37,39,41、43、55,57,59,及び61から選択されるいずれか1つに記載のアミノ酸配列を有するVLを含む、請求項19に記載の抗体又はその免疫反応性断片。 20. The method according to claim 19, further comprising a VL having an amino acid sequence set forth in any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. Or an immunoreactive fragment thereof.
  21.  以下の群から選択されるいずれか1つのVH及びVLの組み合わせを含む、請求項20に記載の抗体又はその免疫反応性断片:
     配列番号2に記載のアミノ酸配列を有するVH及び配列番号4に記載のアミノ酸配列を有するVL;
     配列番号27に記載のアミノ酸配列を有するVH及び配列番号37に記載のアミノ酸配列を有するVL;
     配列番号29に記載のアミノ酸配列を有するVH及び配列番号37に記載のアミノ酸配列を有するVL;
     配列番号29に記載のアミノ酸配列を有するVH及び配列番号39に記載のアミノ酸配列を有するVL;
     配列番号31に記載のアミノ酸配列を有するVH及び配列番号39に記載のアミノ酸配列を有するVL;
     配列番号31に記載のアミノ酸配列を有するVH及び配列番号41に記載のアミノ酸配列を有するVL;
     配列番号33に記載のアミノ酸配列を有するVH及び配列番号41に記載のアミノ酸配列を有するVL;
     配列番号35に記載のアミノ酸配列を有するVH及び配列番号43に記載のアミノ酸配列を有するVL;
     配列番号12に記載のアミノ酸配列を有するVH及び配列番号14に記載のアミノ酸配列を有するVL;
     配列番号45に記載のアミノ酸配列を有するVH及び配列番号55に記載のアミノ酸配列を有するVL;
     配列番号47に記載のアミノ酸配列を有するVH及び配列番号55に記載のアミノ酸配列を有するVL;
     配列番号47に記載のアミノ酸配列を有するVH及び配列番号57に記載のアミノ酸配列を有するVL;
     配列番号49に記載のアミノ酸配列を有するVH及び配列番号57に記載のアミノ酸配列を有するVL;
     配列番号49に記載のアミノ酸配列を有するVH及び配列番号59に記載のアミノ酸配列を有するVL;
     配列番号51に記載のアミノ酸配列を有するVH及び配列番号59に記載のアミノ酸配列を有するVL;
     配列番号51に記載のアミノ酸配列を有するVH及び配列番号61に記載のアミノ酸配列を有するVL;
     配列番号53に記載のアミノ酸配列を有するVH及び配列番号61に記載のアミノ酸配列を有するVL;
     配列番号19に記載のアミノ酸配列を有するVH及び配列番号21に記載のアミノ酸配列を有するVL。
    21. The antibody or immunoreactive fragment thereof according to claim 20, which comprises a combination of any one of VH and VL selected from the following group:
    A VH having the amino acid sequence set forth in SEQ ID NO: 2 and a VL having the amino acid sequence set forth in SEQ ID NO: 4;
    A VH having the amino acid sequence set forth in SEQ ID NO: 27 and a VL having the amino acid sequence set forth in SEQ ID NO: 37;
    A VH having the amino acid sequence set forth in SEQ ID NO: 29 and a VL having the amino acid sequence set forth in SEQ ID NO: 37;
    A VH having the amino acid sequence set forth in SEQ ID NO: 29 and a VL having the amino acid sequence set forth in SEQ ID NO: 39;
    A VH having the amino acid sequence set forth in SEQ ID NO: 31 and a VL having the amino acid sequence set forth in SEQ ID NO: 39;
    A VH having the amino acid sequence set forth in SEQ ID NO: 31 and a VL having the amino acid sequence set forth in SEQ ID NO: 41;
    A VH having the amino acid sequence set forth in SEQ ID NO: 33 and a VL having the amino acid sequence set forth in SEQ ID NO: 41;
    A VH having the amino acid sequence set forth in SEQ ID NO: 35 and a VL having the amino acid sequence set forth in SEQ ID NO: 43;
    A VH having the amino acid sequence set forth in SEQ ID NO: 12 and a VL having the amino acid sequence set forth in SEQ ID NO: 14;
    A VH having the amino acid sequence set forth in SEQ ID NO: 45 and a VL having the amino acid sequence set forth in SEQ ID NO: 55;
    A VH having the amino acid sequence set forth in SEQ ID NO: 47 and a VL having the amino acid sequence set forth in SEQ ID NO: 55;
    A VH having the amino acid sequence set forth in SEQ ID NO: 47 and a VL having the amino acid sequence set forth in SEQ ID NO: 57;
    A VH having the amino acid sequence set forth in SEQ ID NO: 49 and a VL having the amino acid sequence set forth in SEQ ID NO: 57;
    A VH having the amino acid sequence set forth in SEQ ID NO: 49 and a VL having the amino acid sequence set forth in SEQ ID NO: 59;
    A VH having the amino acid sequence set forth in SEQ ID NO: 51 and a VL having the amino acid sequence set forth in SEQ ID NO: 59;
    A VH having the amino acid sequence set forth in SEQ ID NO: 51 and a VL having the amino acid sequence set forth in SEQ ID NO: 61;
    A VH having the amino acid sequence set forth in SEQ ID NO: 53 and a VL having the amino acid sequence set forth in SEQ ID NO: 61;
    A VH having the amino acid sequence set forth in SEQ ID NO: 19 and a VL having the amino acid sequence set forth in SEQ ID NO: 21.
  22.  請求項1~請求項21のいずれか1項に記載の抗体又はその免疫反応性断片のアミノ酸配列をコードする核酸分子。 A nucleic acid molecule encoding an amino acid sequence of the antibody according to any one of claims 1 to 21 or an immunoreactive fragment thereof.
  23.  以下の群から選択されるいずれか1つのポリヌクレオチドを含有する、請求項22に記載の核酸分子:
     配列番号1に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号3に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号26に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号36に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号28に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号36に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号28に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号38に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号30に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号38に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号30に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号40に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号32に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号40に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号34に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号42に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号11に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号13に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号44に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号54に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号46に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号54に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号46に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号56に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号48に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号56に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号48に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号58に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号50に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号58に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号50に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号60に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;
     配列番号52に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号60に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド;及び
     配列番号18に記載のヌクレオチド配列を有するVHをコードするポリヌクレオチド、及び配列番号20に記載のヌクレオチド配列を有するVLをコードするポリヌクレオチド。
    23. The nucleic acid molecule according to claim 22, containing any one polynucleotide selected from the following group:
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 1, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 3;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 26, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 36;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 28; and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 36;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 28; and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 38;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 30, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 38;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 30, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 40;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 32, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 40;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 34, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 42;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 11, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 13;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 44, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 54;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 46, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 54;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 46, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 56;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 48, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 56;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 48, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 58;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 50, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 58;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 50, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 60;
    A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 52, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 60; and a VH encoding the nucleotide sequence set forth in SEQ ID NO: 18 A polynucleotide, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 20.
  24.  請求項22又は請求項23に記載の核酸分子を有するベクター。 A vector comprising the nucleic acid molecule of claim 22 or claim 23.
  25.  請求項24に記載のベクターを有する宿主細胞。 A host cell comprising the vector of claim 24.
  26.  請求項25に記載の宿主細胞を培養することを含む、請求項1~請求項21のいずれか1項に記載の抗体又はその免疫反応性断片の製造方法。 A method of producing the antibody or the immunoreactive fragment thereof according to any one of claims 1 to 21, comprising culturing the host cell according to claim 25.
  27.  請求項1~請求項21のいずれか1項に記載の抗体又はその免疫反応性断片を有効成分として含有する、医薬組成物。 A pharmaceutical composition comprising the antibody according to any one of claims 1 to 21 or an immunoreactive fragment thereof as an active ingredient.
  28.  血管新生抑制剤である、請求項27に記載の医薬組成物。 The pharmaceutical composition according to claim 27, which is an angiogenesis inhibitor.
  29.  癌、癌転移、糖尿病性網膜症、未熟児網膜症、加齢黄斑変性症、糖尿病網膜症、網膜静脈閉塞症、血管新生緑内障、炎症性皮膚疾患、関節リウマチ、又は変形性関節症の治療薬又は予防薬である、請求項27に記載の医薬組成物。 Therapeutic agents for cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis The pharmaceutical composition according to claim 27, which is a prophylactic agent.
PCT/JP2018/034076 2017-09-13 2018-09-13 Anti-ramp2 antibody WO2019054460A1 (en)

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