WO2019054460A1 - Anticorps anti-ramp2 - Google Patents

Anticorps anti-ramp2 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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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 JP2019542293A priority Critical patent/JP7202011B2/ja
Priority to CA3075867A priority patent/CA3075867A1/fr
Publication of WO2019054460A1 publication Critical patent/WO2019054460A1/fr
Priority to JP2022136364A priority patent/JP2022169737A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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

La présente invention a trait à un anticorps anti-RAMP2 ayant un effet inhibiteur de cAMP et un effet antiprolifératif HUVEC. La présente invention concerne un inhibiteur d'angiogenèse contenant ledit anticorps en tant que principe actif. Particulièrement, la présente invention concerne, dans un test de prolifération in vitro : un anticorps contre une protéine 2 de modification d'activité de récepteur (RAMP2) ayant un effet anti-prolifératif de cellules endothéliales vasculaires, ou des fragments immunoréactifs de ceux-ci; ou un anticorps ayant un effet d'inhibition de la production de cAMP provoquée par des cellules animales par ajout d'adrénomédulline ou de fragments immunoréactifs de celle-ci.
PCT/JP2018/034076 2017-09-13 2018-09-13 Anticorps anti-ramp2 WO2019054460A1 (fr)

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FR2934597B1 (fr) * 2008-07-31 2013-04-19 Univ Aix Marseille Ii Anticorps se liant aux recepteurs de l'adrenomedulline et leurs utilisations comme medicament.
FR2964103B1 (fr) * 2010-08-30 2018-11-23 Universite D'aix-Marseille Anticorps se liant a l'adrenomedulline et aux recepteurs de l'adrenomedulline et leurs utilisations comme medicament

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WO2004050834A2 (fr) * 2002-11-27 2004-06-17 Genpath Pharmaceuticals, Incorporated Gpc99 et gpc99a: compositions et methodes de traitement du cancer
JP2009504681A (ja) * 2005-08-11 2009-02-05 アミリン・ファーマシューティカルズ，インコーポレイテッド 選択可能な特性を有するハイブリッドポリペプチド
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WO2009069546A1 (fr) * 2007-11-26 2009-06-04 Japan Science And Technology Agency Agent thérapeutique ou prophylactique, procédé de détection et agent de détection pour le syndrome métabolique, et procédé de criblage d'un composé candidat pour un agent thérapeutique pour le syndrome métabolique

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