WO2012008397A1 - Anti-epha2 antibody and use thereof - Google Patents

Anti-epha2 antibody and use thereof Download PDF

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Publication number
WO2012008397A1
WO2012008397A1 PCT/JP2011/065766 JP2011065766W WO2012008397A1 WO 2012008397 A1 WO2012008397 A1 WO 2012008397A1 JP 2011065766 W JP2011065766 W JP 2011065766W WO 2012008397 A1 WO2012008397 A1 WO 2012008397A1
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antibody
amino acid
seq
acid sequence
epha2
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PCT/JP2011/065766
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French (fr)
Japanese (ja)
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剛 滝沢
達司 松岡
長谷川　純
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第一三共株式会社
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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-EPHA2 antibody and a pharmaceutical composition containing the antibody.
  • EPHA2 is a receptor tyrosine kinase having a single transmembrane structure with a molecular weight of 130 kDa (see, for example, Non-Patent Document 1).
  • EPHA2 has an N-terminal extracellular region with a ligand binding domain and two fibronectin type 3 domains, and a C-terminal intracellular region with a tyrosine kinase domain and a sterile- ⁇ -motif (SAM) domain.
  • SAM sterile- ⁇ -motif
  • EPHA2 As a ligand for EPHA2, GPI-anchored cell membrane proteins Ephrin-A1 to A5 are known (for example, see Non-patent Document 2). By binding a ligand to EPHA2, the tyrosine kinase domain is activated, tyrosine residues existing in the intracellular region of EPHA2 are phosphorylated, and a signal is transmitted into the cell.
  • EPHA2 bound to a ligand has been reported to be taken up into cells by endocytosis and finally degraded by the proteasome (see, for example, Non-Patent Document 3).
  • EPHA2 is clinically highly expressed in many cancers, particularly breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme (for example, Non-Patent Document 4). 5, 6, 7, 8, 9, 10, 11).
  • esophageal cancer patients with positive expression of EPHA2 have a significantly high frequency of local lymph node metastasis and the number of lymph node metastases, and the degree of differentiation of cancer is significantly low. Patients with positive expression of EPHA2 survive 5 years.
  • Non-Patent Document 5 non-small cell lung cancer patients with a disease-free survival of less than 5 years have significantly higher expression of EPHA2 than patients with longer than 5 years
  • EPHA2 expression is significantly higher in patients who have metastasized to the brain (see, for example, Non-Patent Document 9)
  • liver metastasis, lymphatic vessel invasion, and lymph node metastasis are likely to occur in patients with colorectal cancer who are positive for EPHA2 expression.
  • it has been reported that there are many EPHA2 expression positive patients in patients with high clinical stage see, for example, Non-Patent Document 10).
  • non-cancerous cells can acquire cancer traits such as anchorage-independent growth ability, tubular morphogenesis on the extracellular matrix, and in vivo tumor growth ability. It has been reported (for example, see Non-patent Documents 4 and 13) and that the invasiveness of cancer cells to the extracellular matrix is enhanced (see Non-Patent Documents 12 and 13 for example).
  • EPHA2 is expressed not only in cancer cells but also in blood vessels in or around the tumor (for example, see Non-Patent Document 15).
  • EPHA2 signal is involved in angiogenesis induced by Ephrin-A1, especially that EPHA2 expressed in vascular endothelial cells is required for the formation and survival of vascular endothelial cells.
  • a fusion protein of the extracellular region of EPHA2 and the Fc region of human IgG suppresses angiogenesis in vivo and exhibits an antitumor effect (see, for example, Non-Patent Document 16) (for example, Non-patent document 17).
  • an agonist anti-EPHA2 monoclonal antibody having phosphorylation-inducing activity of EPHA2 tyrosine residue and degradation-inducing activity of EPHA2 is used for anchorage-independent growth of breast cancer cell lines and tubular form on the extracellular matrix. It has been reported to inhibit formation (see, for example, Non-Patent Document 14). Furthermore, an agonistic anti-EPHA2 monoclonal antibody that binds to an epitope on EPHA2 exposed in cancer cells as compared to non-cancer cells and has phosphorylation-inducing activity of EPHA2 tyrosine residue and EPHA2 degradation-inducing activity is anti-tumor in vivo.
  • Patent Document 2 describes LUCA19, SG5, LUCA40, and SPL1, which are anti-EPHA2 monoclonal antibodies obtained by immunizing cancer cells in mice.
  • LUCA19 and SG5 contain EPHA2 tyrosine residues. It does not affect the phosphorylation of the group, LUCA40 inhibits cancer cell growth in vitro, and LUCA19, SG5 and LUCA40 are internalized in cancer cells in the presence of anti-mouse antibody labeled with sapolin. It is described that it is done.
  • LUCA40 and SPL1 have been reported to show an antitumor effect in vivo, but it is not clear whether or not they have agonist activity.
  • Patent Document 4 a monoclonal antibody that binds to the extracellular region of EPHA2 and has antitumor activity and a humanized antibody thereof have been reported.
  • One object of the present invention is to provide an antibody against EPHA2. Another object of the present invention is to provide a pharmaceutical composition containing the anti-EPHA2 antibody having a therapeutic effect on cancer. Another object of the present invention is to provide a method for producing the antibody. Another object of the present invention is to provide a method for suppressing tumor growth using the antibody.
  • the present inventors diligently studied to solve the above-mentioned problems.
  • the anti-EPHA2 monoclonal antibody described in International Publication Pamphlet WO2009 / 028639 which is a heavy chain derived from SH348-1, and a light chain derived from SH357-1.
  • a humanized antibody (hereinafter referred to as a “swapped antibody”) in which a chain is combined was prepared. While maintaining the growth-inhibitory activity of cancer cells, the humanized antibody derived from SH348-1 and the derived from SH357-1 It was found that the binding activity to the antigen was improved and the ADCC activity was also improved as compared with the humanized antibody, and the present invention was completed.
  • the present invention (1) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. And a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and an amino acid sequence shown in SEQ ID NO: 52.
  • An antibody comprising a light chain comprising CDRL3 and specifically recognizing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, or a functional fragment of the antibody, (2) the antibody or the functional fragment of the antibody according to (1), which specifically recognizes a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing; (3) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing.
  • a Kd value for a polypeptide comprising a light chain comprising CDRL3 and comprising an amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.5 nM or less (1) or ( 2) an antibody or a functional fragment thereof, (4) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.0 nM or less Sex fragment, (5) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 0.5 nM or less Sex fragment
  • an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody (10) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a functional fragment of the antibody, (11) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody, (12) an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a
  • a pharmaceutical composition comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody
  • a pharmaceutical composition for treating cancer comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody, (19) Breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell, comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody
  • a pharmaceutical composition for treating lung cancer, colon cancer and / or glioblastoma multiforme comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody
  • (20) A method for suppressing tumor growth of a mammal by administering any one selected from (1) to (18), (21) The method for inhibiting tumor growth according to (19), wherein the tumor is a tumor expressing EPHA2.
  • (22) A polynucleotide encoding the antibody according to any one of (1) to (16) or a functional fragment of the antibody,
  • (23) A host cell transformed with the polynucleotide according to (22),
  • the present invention succeeded in obtaining an anti-EPHA2 swapped humanized antibody and a functional fragment of the antibody. And it was found that the antibody has excellent antitumor activity.
  • a pharmaceutical composition containing the antibody as an active ingredient and a pharmaceutical composition for cancer treatment containing the antibody were provided.
  • 357-L4 A diagram showing the amino acid sequence of the variable region of 357-L4.
  • 357-L5 A diagram showing the amino acid sequence of the variable region of 357-L5.
  • 357-H5 A diagram showing the amino acid sequence of the variable region of 357-H5.
  • SH348-1_CDRL1 A figure showing the amino acid sequence of CDRL1 of SH348-1.
  • SH348-1_CDRL2 A figure showing the amino acid sequence of CDRL2 of SH348-1.
  • SH348-1_CDRL3 A drawing showing the amino acid sequence of CDRL3 of SH348-1.
  • SH348-1_CDRH1 A figure showing the amino acid sequence of CDR4 of SH348-1.
  • SH348-1_CDRH2 A figure showing the amino acid sequence of CDR4 of SH348-1.
  • SH348-1_CDRH3 A figure showing the amino acid sequence of CDR4 of SH348-1.
  • 348-L4 A drawing showing the amino acid sequence of the variable region of 348-L4.
  • 348-L5 A drawing showing the amino acid sequence of the variable region of 348-L5.
  • 348-H4 A diagram showing the amino acid sequence of the variable region of 348-H4.
  • 348-H5 A diagram showing the amino acid sequence of the variable region of 348-H5.
  • SH357-1_CDR_L1 A diagram showing the amino acid sequence of CDRL1 of SH357-1.
  • SH357-1_CDR_L2 A diagram showing the amino acid sequence of CDRL2 of SH357-1.
  • SH357-1_CDR_L3 A diagram showing the amino acid sequence of CDRL3 of SH357-1.
  • SH357-1_CDR_H1 A drawing showing the amino acid sequence of CDR35 of SH357-1.
  • SH357-1_CDR_H2 A drawing showing the amino acid sequence of CDR35 of SH357-1.
  • SH357-1_CDR_H3 A drawing showing the amino acid sequence of CDR3 of SH357-1.
  • SEQ ID NO: 71 nucleotide sequence of DNA fragment encoding human ⁇ chain secretion signal, human ⁇ chain constant region and human polyA addition signal.
  • SEQ ID NO: 72 A diagram showing a nucleotide sequence of a DNA fragment containing a human IgG1 signal sequence and a DNA sequence encoding an amino acid in the constant region.
  • SEQ ID NO: 73 A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1L type light chain variable region fused with a secretion signal.
  • SEQ ID NO: 74 A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with a secretion signal.
  • SEQ ID NO: 75 A drawing showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1H type heavy chain variable region.
  • SEQ ID NO: 76 is a view showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1H type heavy chain variable region.
  • the black dotted line ( ⁇ ) is the control antibody hIgG1
  • the solid black line ( ⁇ ) is hSH357-1T1
  • the black square ( ⁇ ) is 357-H4-357-L4
  • the white circle ( ⁇ ) is 348-H4-357- L4
  • white triangle ( ⁇ ) indicates 348-H4-357-L5
  • white square ( ⁇ ) indicates 348-H5-357-L5.
  • A) shows the result of confirming the expression level of each protein on the cell membrane using Anti-FLAG mAB. Both proteins indicate that the same level of expression on the cell membrane was observed.
  • each antibody binds strongly to cells expressing human EPHA2, whereas pFLAG-GW, which is a negative control, is transfected into cells expressing human EPHA3a and human PTPRS-4. It shows that it showed only the same level of binding as the transfected cells. The figure which shows the result of the in vivo antitumor activity of each antibody.
  • A) shows results for hSH357-1-T1, B) for 348-H4-357-L4, C) for 348-H4-357-L5, and D) for 348-H5-357-L5.
  • the horizontal axis indicates the number of days after transplantation of human breast cancer cell line MDA-MB-231 cells, and the vertical axis indicates the tumor volume.
  • cancer and “tumor” are used interchangeably.
  • EPHA2 gene in the present invention includes EPHA2 DNA, mRNA, cDNA, and cRNA.
  • polynucleotide is used in the same meaning as a nucleic acid, and includes DNA, RNA, probes, oligonucleotides, and primers.
  • polypeptide and “protein” are used without distinction.
  • cell includes cells in an individual animal and cultured cells.
  • cell canceration means that the cell exhibits abnormal growth, such as loss of sensitivity to the contact-inhibiting phenomenon, or anchorage-independent growth, A cell exhibiting such abnormal growth is called a “cancer cell”.
  • EPHA2 a protein having a function equivalent to the cell carcinogenic activity and / or cell proliferation activity of EPHA2 is also referred to as EPHA2.
  • “swapped antibody” refers to an antibody in which heavy chains and / or light chains are exchanged among a plurality of types of antibodies, and the full lengths of heavy chains and / or light chains are exchanged. In addition to those, only variable regions, those in which only constant regions are exchanged, and those in which only complementarity determining regions are exchanged are included. That is, for example, in the full-length antibody sequence, when the heavy chain of antibody A is AH, the light chain is AL, the heavy chain of antibody B is BH, and the light chain is BL, the combination of heavy chain / light chain is AH / Antibodies that become BL and BH / AL are swapped antibodies.
  • CDR of the variable region of the heavy chain of antibody A is CDRH1A, CDRH2A, CDRH3A
  • CDR of the variable region of the light chain is CDRL1A, CDRL2A, CDRL3A
  • CDR of the variable region of the heavy chain of antibody B Is CDRH1B, CDRH2B, CDRH3B
  • the CDR of the light chain variable region is CDRL1B, CDRL2B, CDRL3B
  • the combination of CDRs is heavy chain CDRH1A, CDRH2A and CDRH3A
  • the light chain is CDRRL1B, CDRRL2B and CDRL3B
  • an antibody in which the heavy chain is CDRH1B, CDRH2B and CDRH3B, and the light chain is CDRL1A, CDRL2A and CDRL3A
  • an antibody in which the heavy chain is CDRH1B, CDRH2B and CDRH3B, and the light chain is CDRL1A, CD
  • the “functional fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ′) 2, scFv, and the like. Further, Fab ', which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody.
  • the molecule is not limited to these molecules as long as it has the ability to bind to an antigen.
  • These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.
  • phosphorylation of tyrosine residue means that a tyrosine residue contained in the amino acid sequence of a peptide is phosphorylated, and whether or not a tyrosine residue is phosphorylated is, for example, The binding of the peptide to an anti-phosphotyrosine antibody (for example, Anti-Phosphotyrosine, recombinant 4G10, HRP-conjugate (Millipore (Upsate) # 16-184)) was examined.
  • an anti-phosphotyrosine antibody for example, Anti-Phosphotyrosine, recombinant 4G10, HRP-conjugate (Millipore (Upsate) # 16-184
  • EPHA2 tyrosine residue phosphorylation ability refers to the ability to phosphorylate tyrosine residues in the amino acid sequence of EPHA2, and the antibody is EPHA2 Whether or not it has the ability to phosphorylate tyrosine residues of Eg to after incubation of the antibody and EPHA2, can be determined by whether or not there bind with anti-phosphotyrosine antibody and EPHA2.
  • the expression level of EPHA2 protein decreases means that the amount of EPHA2 protein decreases, and whether or not an antibody has an action of decreasing the amount of EPHA2 protein is, for example, an antibody Can be examined by quantifying the amount of EPHA2 after incubation with EPHA2.
  • EPHA2 ligand refers to a substance that can be a ligand of EPHA2, and specific examples thereof include GPI-anchored cell membrane proteins, Ephrin-A1 to A5 (Annual Review of Neuroscience, 1998, Vol. 21, pp. 309-345).
  • cytotoxicity refers to pathological changes in cells in some form, not just direct trauma, but also DNA cleavage, base dimer formation, chromosomes. This refers to any structural or functional damage to cells, such as cleavage of cells, damage to cell division apparatus, or reduction of various enzyme activities. As used herein, “cytotoxic activity” refers to causing the above-mentioned cytotoxicity.
  • ADCC is synonymous with antibody-dependent cellular cytotoxicity, and Fc ⁇ receptor-bearing cells are mediated through Fc ⁇ receptor in the Fc part of the antibody bound to the surface antigen of the target cell.
  • the ADCC activity is also referred to as antibody-dependent cytotoxic activity and refers to the activity of the above reaction.
  • ADCC activity can be measured by a method commonly used by those skilled in the art. For example, it can be measured by the method described in Example 8 herein.
  • CDC is synonymous with complement-dependent cytotoxicity
  • CDC activity refers to an activity that causes complement-dependent cytotoxicity. CDC activity can be measured by methods commonly used by those skilled in the art.
  • “having anti-tumor activity in vivo” means having an activity of suppressing or reducing the growth of a tumor of an animal individual having a tumor.
  • an anti-EPHA2 antibody has “in vivo antitumor activity” can be examined by a method commonly used by those skilled in the art, but can also be examined, for example, by the following method. That is, an anti-EPA2 antibody as a test substance is intraperitoneally injected into a nude mouse (for example, BALB / cAJc1-nu / nu; obtained from Clea Japan Co., Ltd.) into which tumor cells (for example, MDA-MB-231 cells) are transplanted subcutaneously.
  • a nude mouse for example, BALB / cAJc1-nu / nu; obtained from Clea Japan Co., Ltd.
  • tumor cells for example, MDA-MB-231 cells
  • the anti-EPA2 antibody can be determined to have “in vivo antitumor activity”.
  • CDRs complementarity determining regions
  • epitope means a partial peptide of EPHA2 having antigenicity and / or immunogenicity in an animal, preferably a mammal, more preferably a mouse or human body.
  • the epitope that is a partial peptide of EPHA2 having antigenicity can be determined by methods well known to those skilled in the art, such as immunoassay, and can be carried out, for example, by the following method.
  • Various partial structures of EPHA2 are produced. In producing the partial structure, a known oligopeptide synthesis technique can be used.
  • the epitope can be determined by synthesizing shorter peptides and examining their reactivity with those peptides.
  • a mutant antibody refers to an antibody in which one or several amino acids in the amino acid sequence of the antibody are substituted, deleted, or added.
  • Examples of the properties of antibodies in the present specification include biological, chemical, and physical properties, and more specific examples include biological activity, binding to antigens and epitopes, production, distribution, The stability at the time of storage, thermal stability, etc. can be mentioned.
  • hybridize under stringent conditions means to hybridize at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech) or use a filter on which DNA is fixed. After hybridization at 68 ° C in the presence of 0.7-1.0 M NaCl, 0.1-2 fold SSC solution (1 fold SSC consists of 150 mM NaCl, 15 mM sodium citrate) ) And hybridization under conditions that can be identified by washing at 68 ° C. or equivalent conditions.
  • EPHA2 gene The nucleotide sequence and amino acid sequence of the EPHA2 gene are registered in GenBank as EPH receptor A2 (accession numbers NM_004431 and NP_004422, respectively).
  • the nucleotide sequence of the open reading frame (ORF) of the EPHA2 gene is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence thereof is described in SEQ ID NO: 2 in the sequence listing.
  • EPHA2 includes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of EPHA2, and having a biological activity equivalent to those of these enzymes.
  • Cancer-specific expression of EPHA2 gene is reported to be highly expressed in many cancers, especially breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme Has been.
  • a substance that suppresses the expression level and / or activity of EPHA2 has an activity of suppressing canceration of cells caused by EPHA2 and / or suppressing proliferation of cancer cells.
  • EPHA2 EPHA2 which is an antigen of an anti-EPHA2 antibody
  • An outer region polypeptide (consisting of the amino acid sequence shown in amino acid Nos. 1 to 540 of SEQ ID No. 4 in the sequence listing), more preferably a polypeptide comprising the amino acid sequence shown in amino acid No. 426 to 540 of SEQ ID No.
  • polypeptide containing the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing, or a derivative in which any amino acid sequence or carrier is added to these preferably, a polypeptide containing the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing, or a derivative in which any amino acid sequence or carrier is added to these.
  • guide_body to which arbitrary amino acid sequences and carriers were added to these can be mentioned.
  • EPHA2 full-length polypeptide or a partial polypeptide thereof serving as an antigen can be obtained by causing a host cell to produce an EPHA2 gene or a gene of the partial polypeptide thereof by genetic manipulation.
  • EPHA2 can be directly purified from human tumor tissue or tumor cells, and EPHA2 full-length polypeptide or a partial polypeptide thereof can be synthesized in vitro or produced in a host cell by genetic manipulation. Can be obtained by:
  • EPHA2 or a partial polypeptide thereof is incorporated into a vector capable of expression, and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic organisms.
  • the protein can be obtained by expressing EPHA2 or a partial polypeptide thereof by transforming a eukaryotic host cell.
  • the cDNA of the partial polypeptide of EPHA2 for example, using a cDNA library expressing EPHA2 as a template, and using a primer that specifically amplifies the DNA encoding EPHA2 cDNA or the partial polypeptide (hereinafter referred to as polymerase chain reaction) (Referred to as “PCR”) (see Saiki, R. K., et al. Science (1988) 239, p. 487-489).
  • PCR polymerase chain reaction
  • Examples of in vitro synthesis of a polypeptide include, but are not limited to, a rapid translation system (RTS) manufactured by Roche Diagnostics.
  • RTS rapid translation system
  • Anti-EPHA2 Antibody (1) Acquisition of Anti-EPHA2 Monoclonal Antibody SH348-1 which is an anti-EPHA2 antibody can be obtained from hybridoma SH348-1. Moreover, SH357-1 which is an anti-EPHA2 antibody can be obtained from the hybridoma SH357-1.
  • Hybridoma SH348-1 and Hybridoma SH357-1 are dated June 8, 2007 to Japan National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan)
  • the hybridoma SH348-1 is assigned the deposit number FERM BP-10836 under the name SH348-1
  • the hybridoma SH357-1 is assigned the deposit number FERM BP-10837 under the name SH357-1.
  • the nucleotide sequence of the heavy chain of SH348-1 is described in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 6.
  • the nucleotide sequence of the light chain of SH348-1 is set forth in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 8.
  • the nucleotide sequence of the heavy chain variable region of SH348-1 is set forth in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 10.
  • the nucleotide sequence of the heavy chain constant region of SH348-1 is set forth in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 12.
  • the nucleotide sequence of the light chain variable region of SH348-1 is shown in SEQ ID NO: 13 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 14.
  • the nucleotide sequence of the light chain constant region of SH348-1 is shown in SEQ ID NO: 15 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 16.
  • the nucleotide sequence of CDRH1 of SH348-1 is SEQ ID NO: 17, the amino acid sequence is SEQ ID NO: 18, the nucleotide sequence of CDRH2 is SEQ ID NO: 19, the amino acid sequence is SEQ ID NO: 20, and the nucleotide sequence of CDRH3 is SEQ ID NO: In 21, the amino acid sequence is set forth in SEQ ID NO: 22.
  • the nucleotide sequence of CDRL1 of SH348-1 is SEQ ID NO: 23, the amino acid sequence is SEQ ID NO: 24, the nucleotide sequence of CDRL2 is SEQ ID NO: 25, the amino acid sequence is SEQ ID NO: 26, and the nucleotide sequence of CDRL3 is SEQ ID NO: 27, the amino acid sequence is set forth in SEQ ID NO: 28.
  • the nucleotide sequence of the heavy chain of SH357-1 is described in SEQ ID NO: 29 of the sequencing method, and the amino acid sequence is described in SEQ ID NO: 30.
  • the nucleotide sequence of the light chain of SH357-1 is set forth in SEQ ID NO: 31 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 32.
  • the nucleotide sequence of the heavy chain variable region of SH357-1 is set forth in SEQ ID NO: 33 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 34.
  • the nucleotide sequence of the heavy chain constant region of SH357-1 is set forth in SEQ ID NO: 35 in the Sequence Listing, and the amino acid sequence is set forth in SEQ ID NO: 36 in the Sequence Listing.
  • the nucleotide sequence of the light chain variable region of SH357-1 is set forth in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 38.
  • the nucleotide sequence of the light chain constant region of SH357-1 is set forth in SEQ ID NO: 39 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 40.
  • the nucleotide sequence of CDR357 of SH357-1 is SEQ ID NO: 41
  • the amino acid sequence is SEQ ID NO: 42
  • the nucleotide sequence of CDRH2 is SEQ ID NO: 43
  • the amino acid sequence is SEQ ID NO: 44
  • the nucleotide sequence of CDRH3 is SEQ ID NO: At 45, the amino acid sequence is set forth in SEQ ID NO: 46.
  • the nucleotide sequence of CDRL1 of SH357-1 is SEQ ID NO: 47
  • the amino acid sequence is SEQ ID NO: 48
  • the nucleotide sequence of CDRL2 is SEQ ID NO: 49
  • the amino acid sequence is SEQ ID NO: 50
  • the nucleotide sequence of CDRL3 is SEQ ID NO: In 51, the amino acid sequence is set forth in SEQ ID NO: 52.
  • anti-EPHA2 humanized swapped antibodies include humanized antibodies having a humanized heavy chain derived from SH348-1 and a humanized light chain derived from SH357-1. it can.
  • Examples of the anti-EPHA2 humanized swapped antibody include antibodies in which the heavy chain and the light chain are humanized in the above swapped antibody.
  • a humanized antibody only a complementarity determining region (CDR) is human.
  • CDR complementarity determining region
  • An antibody incorporated in an antibody derived from the antibody see Nature (1986) 321, p.522-525), an antibody in which some framework amino acid residues in addition to the CDR sequence are grafted to a human antibody by the CDR grafting method (WO90 No. 077861, US Pat. No. 6,972,323).
  • anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody include CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a sequence.
  • anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody include a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 54 of the Sequence Listing and the amino acid shown in SEQ ID NO: 66
  • the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention specifically recognizes EPHA2, preferably specifically recognizes the extracellular region of EPHA2, and more preferably, SEQ ID NO: in the sequence listing. 4 specifically, a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540, even more preferably a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing is specifically recognized.
  • the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention has a Kd value of 1.5 nM or less, preferably 1 with respect to the polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4. 0.0 nM or less, more preferably 0.5 nM or less.
  • eukaryotic cells are transformed with a cDNA encoding each of the heavy chain and light chain of the above humanized swapped antibody, preferably a vector containing the cDNA, and a gene recombinant human
  • This antibody can be obtained from the culture supernatant by culturing transformed cells that produce a monoclonal antibody.
  • eukaryotic cells preferably CHO cells
  • mammalian cells such as lymphocytes and myeloma can be used.
  • an antibody gene When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
  • eukaryotic cells When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.
  • animal cells examples include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblasts NIH3T3 (ATCC) No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220).
  • mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblasts NIH3T3 (ATCC) No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Ur
  • a host modified to express an antibody in which the sugar chain structure is modified and the ADCC activity (antibody-dependent cytotoxic activity) or CDC activity of the antibody is increased can be used.
  • ADCC activity antibody-dependent cytotoxic activity
  • CDC activity of the antibody is increased
  • a host among N-glycoside-bonded complex sugar chains that bind to the Fc region of an antibody, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more.
  • a CHO cell into which a gene encoding an antibody molecule that produces an antibody composition has been introduced can be mentioned (see WO02 / 31140).
  • prokaryotic cells for example, Escherichia coli and Bacillus subtilis can be mentioned.
  • An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.
  • the isotype of the antibody is not limited and includes, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE, and preferably IgG or IgM.
  • a functional fragment of the antibody may be used.
  • functional fragments of antibodies include Fab, F (ab ') 2, scFv and the like.
  • Fab ' which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody.
  • the molecule is not limited to these molecules as long as it has the ability to bind to an antigen.
  • These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.
  • a single chain antibody (also referred to as scFv), which is an example of an antibody, is obtained by linking a heavy chain V region and a light chain V region of an antibody with a linker of a polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenburg and Moore, Ed. Springer Verlag, New York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136).
  • peptide linker that links the V regions for example, any single chain peptide consisting of 12 to 19 residues is used.
  • the DNA encoding scFv is the DNA encoding the heavy chain or heavy chain V region of the antibody, and the DNA encoding the light chain or light chain V region.
  • Amplification is performed by PCR using a primer pair that defines both ends of the encoding DNA portion as a template, and then the DNA that encodes the peptide linker portion, and both ends thereof are connected to the heavy chain and light chain, respectively. Obtained by combining and amplifying the primer pairs defined in 1.
  • an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.
  • antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above.
  • an antibody it may be a multispecific antibody having specificity for at least two different antigens.
  • such a molecule binds two antigens (ie, bispecific antibody), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.
  • the antibody of the present invention may be a full-length multispecific antibody or a fragment of such an antibody (for example, F (ab ') 2 bispecific antibody).
  • Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by hybridizing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).
  • an antibody may be a polyclonal antibody that is a mixture of a plurality of types of anti-EPHA2 antibodies having different amino acid sequences.
  • a polyclonal antibody a mixture of plural kinds of antibodies having different CDRs can be mentioned.
  • a polyclonal antibody a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO 2004/061104).
  • an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • antibodies may further include those that form conjugates with these antibodies and other drugs (Immunoconjugate).
  • immunoconjugate examples include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).
  • the obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins.
  • antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (198 )) It is not intended to be limited thereto.
  • Chromatography includes affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like.
  • chromatography can be performed using liquid phase chromatography such as HPLC and FPLC.
  • Columns used for affinity chromatography include protein A columns and protein G columns.
  • Hyper D Hyper D
  • POROS Sepharose F. F. (Pharmacia) and the like.
  • the anti-EPHA2 humanized swapped antibody of the present invention or a functional fragment of the antibody has at least one of the following properties (1) to (5).
  • (1) It specifically binds to a polypeptide consisting of the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing, and has at least one property selected from the following a) to e) .
  • a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
  • b) Does not show an effect of decreasing the amount of EPHA2 protein.
  • d) Has CDC activity against EPHA2-expressing cells.
  • e) Has anti-tumor activity in vivo. (2) at least any two or more properties selected from the following a) to e), which specifically bind to a polypeptide having the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing Have a) It does not have the ability to phosphorylate EPHA2 tyrosine residues. b) Does not show an effect of decreasing the amount of EPHA2 protein. c) It has ADCC activity against EPHA2-expressing cells. d) Has CDC activity against EPHA2-expressing cells. e) Has anti-tumor activity in vivo.
  • the phosphorylation ability of EPHA2 tyrosine residues by the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody is, for example, (Example 3) 3) -1-1 to 3) -1- of WO2009 / 028639 It can be measured by the method described in 2. That is, MDA-MB-231 cells are cultured under appropriate conditions, and an anti-EPHA2 humanized swapped antibody or the antibody, as a soluble ligand of EPHA2, Recombinant Mouse Ephrin-A1 / Fc Chimera (hereinafter referred to as “Ephrin-A1 / Fc”).
  • R & D Systems (# 602-A1-200) diluted to an appropriate concentration with RPMI 1640 was added to MDA-MB-231 cells discarded with the medium, at 37 ° C. under 5% CO 2 conditions. Incubate for the specified time.
  • anti-EPHA2 antibody 8 ⁇ g of anti-Eck / EphA2, Clone D7 (hereinafter referred to as “anti-EPHA2 antibody (D7)”) was added to 25 ⁇ l of Protein G magnetic beads suspension (NEW ENGLAND Bio-Labs) per sample. Abbreviation: Millipore (Upstate) # 05-480) is added and mixed by inversion at 4 ° C. for 2 hours.
  • PVDF membrane polyvinylidene difluoride membrane
  • pore size 0.45 ⁇ m manufactured by Millipore
  • the PVDF membrane after transfer was dissolved in a blocking solution (Block Ace Powder (Dainippon Sumitomo Pharma Co., Ltd., Snow Brand Milk Products)) in 100 ml of ultrapure water, and then Tween 20 and sodium azide were each added at a final concentration of 0.1. % (V / v), 0.02% (w / v) added) to block.
  • a blocking solution Block Ace Powder (Dainippon Sumitomo Pharma Co., Ltd., Snow Brand Milk Products)
  • Tween 20 and sodium azide were each added at a final concentration of 0.1. % (V / v), 0.02% (w / v) added
  • the PVDF membrane after blocking is immersed in an anti-EPHA2 antibody solution diluted to 0.25 ⁇ g / ml with a blocking solution and shaken at room temperature for 1 hour.
  • the PVDF membrane was washed 3 times with TBST (50 mM Tris-HCl pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v / v) Tween 20) for 10 minutes, and then diluted 3000 times with TBST.
  • TBST 50 mM Tris-HCl pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v / v) Tween 20
  • the PVDF membrane is washed 3 times with TBST for 10 minutes, and then the signal is detected with a film for chemiluminescence using ECL Plus
  • the PVDF membrane is immersed in a Strip solution (50 mM Tris-HCl pH 6.8, 2% (w / v) SDS, 100 mM 2-mercaptoethanol) at 55 ° C. for 30 minutes. After shaking for 30 minutes, it was immersed in Quench solution (TBST containing 1% (v / v) H 2 O 2 , 0.1% (w / v) NaN 3 ) for 20 minutes at room temperature. Wash 3 times for 10 minutes.
  • Strip solution 50 mM Tris-HCl pH 6.8, 2% (w / v) SDS, 100 mM 2-mercaptoethanol
  • this PVDF membrane was dissolved in a sodium azide-free blocking solution (1 bag of Block Ace powder was dissolved in 100 ml of ultrapure water, and Tween 20 was added at a final concentration of 0.1%.
  • the PVDF membrane is washed 3 times with TBST for 10 minutes, then further washed 3 times with H 2 O for 5 minutes, and the signal is detected with a film for chemiluminescence using ECL Plus.
  • the effect of reducing the amount of EPHA2 protein can be measured, for example, by the method described in Example 3) 3) -1-3 of WO2009 / 028639. That is, after the cell lysate supernatant was separated by SDS-PAGE, the protein in the gel was transferred to a PVDF membrane, and Western blotting was performed with an anti-EPHA2 antibody to quantify the amount of protein decrease. It can be measured.
  • ADCC activity can be measured by the method currently performed normally, it can be measured by the method as described in Example 8 of this specification, for example.
  • CDC activity can be measured by a method commonly used by those skilled in the art. For example, it can be performed by the method described in Example 3) 3) -3 of WO2009 / 028639. That is, MDA-MB-231, A549, and PC-3 cells suspended in RPMI 1640 containing 10% FBS (with antibiotics) were seeded in a 96-well microplate at 5000 cells / well each, at 37 ° C., 5% CO 2.
  • SH348-1, SH357-1, and isotype control antibody diluted with 10% FBS-containing RPMI 1640 (with antibiotics) were added to a final concentration of 25 ⁇ g / ml after complement addition, and 4 ° C. Leave for 1 hour.
  • Rabbit complement (CEDARLANE # CL3051) diluted to 30% with RPMI 1640 was added thereto to a final concentration of 5%, incubated at 37 ° C. under 5% CO 2 for 1 hour, and further incubated at room temperature for 30 hours. Let stand for a minute.
  • CellTiter-Glo Luminescent Cell Viability Assay manufactured by Promega
  • the culture solution is stirred at room temperature for 10 minutes, and then the amount of luminescence is measured with a plate reader.
  • Cell viability is calculated by the following formula.
  • An equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added instead of the cell suspension at the time of cell seeding, and an equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added to the antibody dilution at the time of antibody addition. . Otherwise, perform the same operation as the sample well.
  • the antitumor activity in vivo can be confirmed, for example, by administering an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody to an experimental animal having a tumor and measuring a change in the volume of the tumor. For example, it can be measured by the method described in Example 10 of the present specification.
  • Anti-EPHA2 humanized swapped antibody or a functional fragment of said antibody is useful as a pharmaceutical composition, especially for the treatment of cancer, or for immunodiagnosis of such diseases. It is useful as an antibody.
  • the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody can be treated as a pharmaceutical as long as it is a cancer expressing EPHA2.
  • Examples of the types of cancer include breast cancer, esophageal cancer, Prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer and glioblastoma multiforme can be preferably mentioned, but are not limited thereto.
  • the present invention relates to a medicament comprising a therapeutically effective amount of an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.
  • a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant are also provided.
  • the substance used in the preparation acceptable in the pharmaceutical composition of the present invention is preferably a substance that is non-toxic to a person who is administered the pharmaceutical composition at a dosage or concentration.
  • the pharmaceutical composition of the present invention changes, maintains, or maintains pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, and penetration rate. Or a pharmaceutical substance for the preparation.
  • Substances for formulation may include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulfite
  • Antioxidants such as phosphate, citric acid, borate buffer, hydrogen carbonate, buffer such as tris-hydrochloric acid (Tris-Hcl) solution, filler such as mannitol and glycine, chelate such as ethylenediaminetetraacetic acid (EDTA)
  • EDTA ethylenediaminetetraacetic acid
  • Agents caffeine, polyvinylpyrrolidine, complexing agents such as ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides and glucose, mannose and dextrin, etc.
  • Carbohydrates, colorants, flavors Agents, diluents, emulsifiers and hydrophilic polymers such as polyvinylpyrrolidine, low molecular weight polypeptides, salt-forming counterions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid Preservatives such as hydrogen oxide, solvents such as glycerin, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspension agents, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Surfactants such as triton, tromethamine, lecithin or cholesterol, stabilization enhancers such as sucrose and sorbitol, sodium chloride, potassium chloride and mannini Elasticity enhancers such Lumpur sorbitol, transport agent,
  • the addition amount of the substance for these preparations is preferably 0.01 to 100 times, particularly 0.1 to 10 times the weight of the anti-EPHA2 humanized swapped antibody or functional fragment thereof.
  • a pharmaceutical composition containing an immunoliposome containing an anti-EPHA2 humanized swapped antibody in the liposome or an antibody in which the anti-EPHA2 antibody and the liposome are bound is also included in the present invention.
  • composition of a suitable pharmaceutical composition in the preparation can be appropriately determined by those skilled in the art according to the applied disease, the applied administration route, and the like.
  • Excipients and carriers in the pharmaceutical composition may be liquid or solid.
  • Appropriate excipients and carriers may be water for injection, physiological saline, artificial cerebrospinal fluid and other substances commonly used for parenteral administration.
  • Neutral physiological saline or physiological saline containing serum albumin can also be used as a carrier.
  • Pharmaceutical compositions include, for example, pH 7.0-8.5 Tris buffer, pH 4.0-5.5 acetate buffer, pH 5.0-8.0 citrate buffer, pH 5.0-8.0 histidine.
  • a buffer or a buffer containing sorbitol or other compounds can also be used.
  • the pharmaceutical composition of the present invention is prepared as a lyophilized product or liquid as an appropriate drug with the purity required for the selected composition.
  • a pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody can be molded as a lyophilized product using an appropriate excipient such as sucrose.
  • the pharmaceutical composition can be prepared for parenteral administration or can be prepared for oral digestive tract absorption.
  • composition and concentration of the preparation can be determined by the administration method, and the affinity of the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody contained in the pharmaceutical composition of the present invention for EPHA2, that is, dissociation for EPHA2
  • affinity Kd value
  • Kd value the affinity for the constant (Kd value)
  • the pharmaceutical composition of the present invention for humans The dosage can also be determined.
  • the pharmaceutical composition When the pharmaceutical composition is administered to humans, it is usually sufficient to administer about 0.1 to 100 mg / kg once every 1 to 180 days.
  • Examples of the form of the pharmaceutical composition include injections containing infusions, suppositories, nasal agents, sublingual agents, and transdermal absorption agents.
  • the pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be used simultaneously or sequentially with the chemotherapeutic agent. That is, after administering the chemotherapeutic agent, a pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be administered, or after administering the pharmaceutical composition, the chemotherapeutic agent In addition, the pharmaceutical composition and the chemotherapeutic agent may be administered simultaneously.
  • agents that can be used include antimetabolites including thymidylate synthase inhibitors, nucleic acid analogs, platinum cytotoxic agents, topoisomerase inhibitors, or anti-microtubule agents.
  • thymidylate synthase inhibitors that can be used in the present invention include 5-FU, MTA, and TDX.
  • An example of an antimetabolite that can be used is Tomdex (TDX).
  • platinum cytotoxic agents that can be used include cisplatin and oxaliplatin.
  • chemotherapeutic agents that can be used in the present invention include: alkylating agents; alkyl sulfonates; aziridines; ethyleneimines; methylamelamines; nitrogen mustards; Antimetabolites; Folic acid analogs; Purine analogs; Pyrimidine analogs; Androgens; Anti-adrenal substances; Folic acid supplements; Acegraton; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Vestlabsyl; Bisanthrene; May contain: diazicon; efomithine; ellipticin acetate; ethogluside; gallium nitrate; hydroxyurea; lentinan; ionidamine; Topoisomerase inhibitors may be used as chemotherapeutic agents, and the topoisomerase inhibitors used in certain embodiments are topoisomerase I inhibitors such as camptothecin.
  • a suitable topoisomerase I inhibitor that can be used in the present invention is ilenotecan (CPT-11) or its active metabolite SN-38.
  • CPT-11 acts specifically in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, called a cleavage or cleavage complex, and also arrests the cell cycle in the G2-M phase. Can be induced.
  • a chemotherapeutic agent that can be used in certain embodiments of the invention is a fluoropyrimidine, such as 5-FU. It should be understood that when a particular chemotherapeutic agent is used, analogs including biologically active derivatives and substantial equivalents thereof that retain the anti-tumor activity of a particular agent may be used.
  • Example 1 Design of humanized antibody a) Design of humanized antibody of SH348-1 a) -i) Molecular modeling of the variable region of SH348-1
  • the heavy chain variable region of SH348-1 is shown in SEQ ID NO: 10 of the Sequence Listing.
  • the light chain variable region of SH348-1 is shown in SEQ ID NO: 14 in the sequence listing.
  • variable region of SH348-1 was performed by a method generally known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)).
  • Primary sequence of variable region of human immunoglobulin registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) (three-dimensional structure derived from X-ray crystal structure is available) was compared to the variable region of SH348-1 determined above.
  • 2JEL and 1A4J were selected as the sequences with the highest sequence homology to the SH348-1 light and heavy chain variable regions, respectively.
  • the three-dimensional structure of the framework region was created by combining the 2JEL and 1A4J coordinates corresponding to the SH348-1 light and heavy chains to obtain a “framework model”.
  • the CDRs of SH348-1 are described in Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)), CDRL 1 , CDRL 2 , CDRL 3 , CDRH 1 and CDRH 2 are in clusters 16A, 7A, 9A, 10A, 10A, respectively.
  • CDRH 3 was classified as e (9) D using the H3 rule (FEBS letter 399, 1-8 (1996)).
  • a representative conformation for each CDR was then incorporated into the framework model.
  • the amino acid residues of the framework region for L chain 10060061A and H chain AAC09074 were aligned with the amino acid residues for SH348-1, and the position where the different amino acids were used was identified. The positions of these residues are analyzed using the SH348-1 three-dimensional model constructed above, and the donor residues to be grafted on the acceptor are described in Queen et al. (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)).
  • the humanized SH348-1 sequence was constructed as described in the examples below by transferring several selected donor residues into the acceptor antibody.
  • hSH348-1-T5H type heavy chain Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH348-1 heavy chain shown in SEQ ID NO: 6 in the sequence listing ), 76 (alanine), 80 (phenylalanine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 88 (asparagine), 93 (threonine), 114 (threonine), 115 (leucine), respectively, valine,
  • a humanized SH348-1 heavy chain designed with replacement of alanine, valine, alanine, serine, valine, glutamine, valine, tyrosine, leucine, serine, serine, alanine, valine, leucine, valine is replaced with “hSH348-1- It was named “T5
  • the amino acid sequence of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 55 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 56.
  • hSH348-1-T5L type light chain Amino acid numbers 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 79 (arginine), 88 (leucine), 105 (of the SH348-1 light chain shown in SEQ ID NO: 8 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with threonine, proline, glutamic acid, proline, lysine, valine, glutamine, valine and threonine, respectively.
  • 1-T5L type light chain ".
  • the amino acid sequence of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 59 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 60.
  • 2JEL and 1A4J were selected as the sequences having the highest sequence homology to the light and heavy chain variable regions of SH357-1, respectively, and CDRL 1 , CDRL 2 , CDRL 3 , CDRH 1 and CDRH 2 were assigned to clusters 16A, 7A, 9A, 10A, 10A, respectively.
  • CDRH 3 was classified as e (9) D.
  • amino acid sequence of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 61 in the sequence listing, and the amino acid sequence of the variable region of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 62.
  • hSH357-1-T5H type heavy chain Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH357-1 heavy chain shown in SEQ ID NO: 30 of the Sequence Listing ), 76 (alanine), 83 (isoleucine), 85 (asparagine), 88 (asparagine), 93 (serine), 114 (threonine), 115 (leucine), respectively, valine, alanine, valine, alanine, serine, valine,
  • the humanized SH357-1 heavy chain designed with replacement with glutamine, valine, leucine, serine, alanine, valine, leucine, valine was named “hSH357-1-T5H type heavy chain”.
  • amino acid sequence of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 63 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 64.
  • hSH357-1-T5L type light chain Amino acid numbers 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 88 (leucine), 105 (SH) light chain shown in SEQ ID NO: 32 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with serine, threonine, proline, glutamic acid, proline, valine, glutamine, valine, and threonine, respectively, and the humanized SH357-1 light chain designed with “hSH357- 1-T5L type light chain ".
  • amino acid sequence of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 67 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 68.
  • Example 2 Construction of universal expression vectors pEF6KCL and pEF1FCCU 1
  • Construction of human light chain expression vector pEF6KCL By PCR using plasmid pEF6 / V5-HisB (Invitrogen) as a template with the following primers (Sequence Position 2174) To DNA fragments up to Sma I (Sequence Position 2958) (DNA fragment containing f1 origin of replication and SV40 promoter and origin, hereinafter referred to as “fragment A”).
  • fragment B 5′-ccacgcgccctgttagcggcgcattaagc-3 ′ (primer EFF1) (SEQ ID NO: 69) 5′-aaacccggggagtttttgcaaaaggcctag-3 ′ (primer EFsmaR) (SEQ ID NO: 70)
  • the obtained fragment A overlaps with a DNA fragment (SEQ ID NO: 71, hereinafter referred to as “fragment B”) containing a DNA sequence encoding a human ⁇ chain secretion signal, a human ⁇ chain constant region, and a human polyA addition signal. Binding by PCR.
  • the obtained DNA fragment in which fragment A and fragment B were bound was digested with restriction enzymes KpnI and SmaI, and ligated with plasmid pEF6 / V5-HisB (Invitrogen) digested with restriction enzymes KpnI and SmaI, and downstream of the EF1 promoter.
  • a human light chain expression vector pEF6KCL having a signal sequence, a cloning site, a human ⁇ chain constant region, and a human polyA addition signal sequence was constructed.
  • human heavy chain expression vector pEF1FCCU A plasmid obtained by digesting a DNA fragment (SEQ ID NO: 72) containing a human IgG1 signal sequence and a DNA sequence encoding the amino acid of the constant region with restriction enzymes NheI and PmeI and digesting with NheI and PmeI
  • a human heavy chain expression vector pEF1FCCU was constructed by binding to pEF1KCL and having a signal sequence, a cloning site, a human heavy chain constant region, and a human polyA addition signal sequence downstream of the EF1 promoter.
  • Example 3 Construction of antibody expression vector 1 Construction of hSH348-1-T1L and hSH357-1-T1L type light chain expression vector hSH348-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 73 of the Sequence Listing And a DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 74 (Invitrogen Artificial Gene Synthesis Service), and was cleaved with restriction enzymes NdeI and BsiWI HSH348-1-T1L and hSH357-1-T1L type light chain expression vectors were constructed by inserting the DNA fragment into a site where a humanized antibody light chain expression general-purpose vector (pEF6KCL) was cleaved with restriction enzymes NdeI and BsiWI. .
  • the obtained expression vectors were named “pEF6KCL / hSH348-1-T1L
  • the hSH348-1-T1H and hSH357-1-T1H type heavy chain expression vectors were constructed by inserting them into the sites cleaved with.
  • the obtained expression vectors were named “pEF1FCCU / hSH348-1-T1H” and “pEF1FCCU / hSH357-1-T1H”, respectively.
  • a DNA comprising a type light chain variable region and a gene encoding a hSH357-1-T1L type light chain variable region (GENEART, artificial gene synthesis service), and a DNA fragment excised with restriction enzymes NdeI and BsiWI
  • a vector expressing hSH348-1-T1L and hSH357-1-T1L type light chains was constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI.
  • pEF6KCL humanized antibody light chain expression general-purpose vector
  • the hSH348-1-T1L type light chain was named “348-L4”
  • the hSH357-1-T1L type light chain was named “357-L4”.
  • the obtained vector expressing 348-L4 was named “pEF6KCL / 348-L4”, and the vector expressing 357-L4 was named “pEF6KCL / 357-L4”.
  • the nucleotide sequence of 348-L4 is shown in SEQ ID NO: 81 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 82.
  • the nucleotide sequence of 357-L4 is shown in SEQ ID NO: 83, and the amino acid sequence is shown in SEQ ID NO: 84.
  • Example 4 Preparation of humanized antibody 1
  • Production of humanized antibody 1.2 ⁇ 10 9 log-type freestyle 293F cells (Invitrogen) were seeded in fresh 1.2 L FreeStyle293 expression medium (Invitrogen) at 37 ° C. The cells were cultured with shaking at 90 rpm for 1 hour in an 8% CO 2 incubator.
  • 3.6 mg of Polyethyleneimine (Polyscience # 24765) dissolved in 20 ml of Opti-Pro SFM medium (Invitrogen), and then an H chain expression vector (0.4 mg) and L chain prepared using PureLink HiPure Plasmid kit (Invitrogen)
  • the expression vector (0.8 mg) was suspended in 20 ml of Opti-Pro SFM medium.
  • the humanized antibodies of SH348-1 obtained by the combination of pEF6KCL / hSH348-1-T1L and pEF1FCCU / hSH348-1-T1H are “hSH348-1-T1”, pEF6KCL / hSH357-1-T1L and pEF1FCCU / hSH357-
  • the humanized antibody of SH357-1 obtained by combination with 1-T1H was named “hSH357-1-T1”.
  • the substituted antibody solution was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT2-1 Hydroxyapatite Column: volume 2 ml) equilibrated with 5 mM NaPi / 50 mM MES / 20 mM NaCl / pH 6.5 buffer. Applied. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with CBS (10 mM citrate buffer / 140 mM sodium chloride, pH 6.0) with a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml ⁇ 2 linked).
  • CBS 10 mM citrate buffer / 140 mM sodium chloride, pH 6.0
  • desalting column manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml ⁇ 2 linked.
  • FIG. Construction of humanized antibody expression vector 1 Construction of 348-L5 and 357-L5 type light chain expression vector 348-L5 type light chain variable region represented by amino acid numbers 21 to 137 of SEQ ID NO: 86, and SEQ ID NO: 88 DNA comprising a gene encoding the 357-L5 type light chain variable region represented by amino acid numbers 21 to 137 (GENEART, artificial gene synthesis service), and a DNA fragment cleaved with restriction enzymes NdeI and BsiWI, 348-L5 and 357-L5 type light chain expression vectors were constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI. The obtained expression vectors were named “pEF6KCL / 348-L5” and “pEF6KCL / 357-L5”, respectively.
  • pEF6KCL humanized antibody light chain expression general-purpose vector
  • the obtained expression vectors were named “pEF1FCCU / 348-H4”, “pEF1FCCU / 348-H5”, “pEF1FCCU / 357-H4”, and “pEF1FCCU / 357-H5”, respectively.
  • Example 6 Preparation of humanized antibody 1 Production of humanized antibody Antibody was obtained by the same method as described in Example 4.
  • the humanized antibody of SH348-1 obtained by the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L4 is “348-H4-348-L4”, the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L5
  • the humanized antibody of SH348-1 obtained by the combination of “348-H4-348-L5”, the combination of pEF1FCCU / 348-H5 and pEF6KCL / 348-L4 was obtained by “348-H4-348-L5”.
  • the humanized antibody of SH357-1 obtained by the combination of pEF1FCCU / 357-H5 and pEF6KCL / 357-L4 is“ 357-H5-357-L4 ”, pEF1FCCU / 357-H5 SH357-1 obtained by combining “357-H5-357-L5”, a combination of pEF1FCCU / 348-H4 and pEF6KCL / 357-L4 with a humanized antibody of SH357-1 obtained by the combination of pEF6KCL / 357-L5 with pEF6KCL / 357-L5 From the humanized heavy chain of -1 and the humanized light chain of SH357-1
  • the antibody consists of a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H4-357-L4”, pEF1FCCU / 348-H4 and
  • the substituted antibody solution was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT2-1 Hydroxyapatite Column: volume 2 ml) equilibrated with 5 mM NaPi / 50 mM MES / 20 mM NaCl / pH 6.5 buffer. Applied. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with CBS (10 mM citrate buffer / 140 mM sodium chloride, pH 6.0) with a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml ⁇ 2 linked).
  • CBS 10 mM citrate buffer / 140 mM sodium chloride, pH 6.0
  • desalting column manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml ⁇ 2 linked.
  • Example 7 Measurement of Kd value of anti-EPHA2 antibody The dissociation constant of the anti-EPHA2 antibody obtained in Example 6 and Fibronctin type III domain was measured as follows.
  • His-Fibrinctin type III domain (426-540) (polypeptide having a histidine tag added to the amino terminus of a polypeptide consisting of the amino acid sequence of amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing) was prepared. Using Biacore T100 (GE Healthcare Bioscience Co., Ltd.), the antibody was captured (captured) on an immobilized anti-human IgG (Fc) antibody, and the capture method was performed in which the antigen was measured as an analyte.
  • Biacore T100 GE Healthcare Bioscience Co., Ltd.
  • An anti-human IgG (Fc) antibody (Human antibody capture kit, GE Healthcare Biosciences) was covalently bound to a sensor chip CM5 (BIAcore, Inc.) up to 10,000 RU by the amine coupling method.
  • the reference cell was similarly fixed.
  • HBS-EP (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer.
  • An anti-human IgG (Fc) antibody-immobilized chip was added with an antibody solution of about 20 nM at a flow rate of 10 ⁇ L / min for 60 seconds, and then a His-Fibroctin type III domain (426-540) dilution series solution (3.
  • the Kd value is higher than that of the humanized antibody of SH348-1 or the humanized antibody of SH357-1. It was found that the binding activity against the antigen was significantly decreased (Table 1).
  • Humanized antibody h348-1 Td Kd value 8.29 (nM), 348-H4-357-L4 0.37 (nM), 348H4-357-L5 0.37 (nM), 348- H5-357-L4 was 0.32 (nM) and 348-H5-357-L5 was 0.30 (nM), indicating that the binding activity was significantly increased.
  • Example 8 FIG. Measurement of ADCC activity
  • the prostate cancer cell line PC-3 introduced with the EGFP gene is used as a target cell, and human peripheral blood lymphocytes (BIOPREDIC # PBL003, hereinafter referred to as “human PBL”) are used as effector cells and released into the culture supernatant.
  • the antibody-dependent cytotoxic activity (ADCC activity) was calculated by measuring the amount of fluorescence of EGFP.
  • the cells were seeded and cultured overnight at 37 ° C. and 5% CO 2 .
  • humanized anti-EPHA2 antibody or isotype control antibody hIgG1 Alexis Biochemicals # ALX-804-133 diluted in fresh medium (final concentrations 0.1, 1.0, 10 ⁇ g, respectively) / ML), 2 ⁇ 10 5 cells / well of human PBL were added, and the cells were cultured overnight under conditions of 37 ° C. and 5% CO 2 .
  • the culture supernatant was collected and the amount of fluorescence (485/535 nm) in the supernatant was measured.
  • the cell lysis rate due to ADCC activity was calculated by the following formula.
  • Cell lysis rate (%) (AB) / (CB) ⁇ 100
  • FIG. 3 shows an average value of three samples, and error bars indicate standard deviation.
  • 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 were obtained from hSH357-1-T1 and 357-H4-357-L4 at an antibody concentration of 0.1 ⁇ g / mL.
  • the ADCC activity of 348-H4-357-L4, 348-H4-357-L5, and 348-H5-357-L5 at an antibody concentration of 0.1 ⁇ g / mL is 10 times higher than that. It was shown to be comparable to the ADCC activity of high concentrations of 1.0 ⁇ g / mL hSH357-1-T1 and 357-H4-357-L4.
  • Example 9 Antigen-specific binding
  • the humanized anti-EPHA2 antibody it is homologous to the amino acid sequence of amino acids 439 to 534 of human EPHA2 (the amino acid sequence shown in amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing)
  • the binding property to highly protein was examined by Cell-ELISA method.
  • the homology search was performed using NCBI BLAST, and the amino acid sequence from human 439 to 534 of human EPHA2 was searched for human protein.
  • EPHA3 isoforma
  • PPRS-4 human protein tyrosin phosphate, receptor type, sigma isoform 4
  • pFLAG-myc-CMV19 The reading frame B of Gateway Vector Conversion System (Invitrogen # 11828-029) was incorporated into a site obtained by cutting Sigma-Aldrich # E8658) with HindIII and BglII and smoothing to create “pFLAG-GW”.
  • human EPHA2 excluding the signal sequence (amino acid sequence from the start codon to the 23rd amino acid), human EPHA3a excluding the signal sequence (amino acid sequence from the start codon to the 20th), or the signal sequence (from the start codon to the 29th)
  • LR Clonase Invitrogen # 11791-019 between the entry vector in which the gene encoding the amino acid sequence of human PTPRS-4 (excluding the amino acid sequence of the above) was cloned and pFLAG-GW
  • Expression vectors “pFLAG-EPHA2”, “pFLAG-EPHA3a”, and “pFLAG-PTPRS-4” were prepared.
  • 293 ⁇ v ⁇ 3 cells transfected with pFLAG-GW, pFLAG-EPHA2, pFLAG-EPHA3a, or pFLAG-PTPRS-4 using Lipofectamin 2000 (Invitrogen # 11668-019) were expressed by INTGrin ⁇ V and integrin ⁇ 3 stably in HEK293 cells. Cells) were seeded in a 96-well plate and cultured overnight in DMEM containing 10% FBS under conditions of 37 ° C. and 5% CO 2 .
  • FIG. 4 shows an average value of three samples, and error bars indicate standard deviation.
  • Example 10 Confirmation of in vivo antitumor activity Human breast cancer cell line MDA-MB-231 cells were trypsinized and detached from the culture flask, suspended in Leibovitz's L-15 containing 10% FBS, and centrifuged to remove the supernatant. did.
  • the cells were washed once with the same medium, then suspended in a BD Matrigel basement membrane matrix (BD Biosciences # 354234), and 6-week-old nude mice (CAnN.Cg-Foxn1 [nu] / CrlCrlj [Foxn1nu / Foxn1nu]: Japan (Charles River) was transplanted subcutaneously between the second and third nipples of the right axilla at 5 ⁇ 10 6 cells / animal.
  • BD Matrigel basement membrane matrix BD Biosciences # 354234
  • 6-week-old nude mice CAnN.Cg-Foxn1 [nu] / CrlCrlj [Foxn1nu / Foxn1nu]: Japan (Charles River) was transplanted subcutaneously between the second and third nipples of the right axilla at 5 ⁇ 10 6 cells / animal.
  • Grouping was performed on day 17 with the day of transplantation as day 0, and on days 17, 24, 31 hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, or 348-H5 -357-L5 was administered at 0.01 mg / animal or 0.03 mg / animal.
  • the vehicle group was administered with the same amount of vehicle as the antibody.
  • Tumor volume was measured on days 17, 24, 31, and 38.
  • FIG. 5 shows the mean value of 9 tumor volumes per group, and error bars indicate standard errors. Further, statistical analysis was performed by Dunnett's multiple comparison test using values at 38 days (**: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001). .
  • the anti-EPHA2 antibody of the present invention has antitumor activity, and a pharmaceutical composition containing the anti-EPHA2 antibody can be an anticancer agent.
  • SEQ ID NO: 3 EPHA2 variant SEQ ID NO: 53: 348-H4 SEQ ID NO: 54: 348-H4 variable region SEQ ID NO: 55: 348-H5 SEQ ID NO: 56: 348-H5 variable region SEQ ID NO: 57: 348-L4 SEQ ID NO: 58: 348-L4 variable region SEQ ID NO: 59: 348-L5 SEQ ID NO: 60: 348-L5 variable region SEQ ID NO: 61: 357-H4 SEQ ID NO: 62: 357-H4 variable region SEQ ID NO: 63: 357-H5 SEQ ID NO: 64: 357-H5 variable region SEQ ID NO: 65: 357-L4 SEQ ID NO: 66: 357-L4 variable region SEQ ID NO: 67: 357-L5 SEQ ID NO: 68: 357-L5 variable region SEQ ID NO: 69: Primer EFF1 Sequence number 70: Primer EfsmaR S

Abstract

The present invention provides an antibody or the like, which has cell canceration inhibitory activity and/or tumor cell proliferation inhibitory activity. Provided are: an anti-EPHA2 antibody which comprises a humanized heavy chain derived from an anti-EPHA2 antibody SH 348-1 that is produced by a hybridoma SH 348-1 (FERM BP-10836) and a humanized light chain derived from an anti-EPHA2 antibody SH 357-1 that is produced by a hybridoma SH 357-1 (FERM BP-10837); a functional fragment of the antibody; and a pharmaceutical product or the like, which contains the antibody as an active ingredient.

Description

抗ＥＰＨＡ２抗体及びその用途Anti-EPHA2 antibody and use thereof
　本発明は、抗ＥＰＨＡ２抗体及び該抗体を含有する医薬組成物等に関する。 The present invention relates to an anti-EPHA2 antibody and a pharmaceutical composition containing the antibody.
　ＥＰＨＡ２は、分子量１３０ｋＤａの１回膜貫通構造を有するレセプター型チロシンキナーゼである（例えば、非特許文献１参照）。ＥＰＨＡ２のＮ末端側の細胞外領域には、リガンド結合ドメインと２ヶ所のフィブロネクチンタイプ３ドメインが存在し、Ｃ末端側の細胞内領域にはチロシンキナーゼドメインとｓｔｅｒｉｌｅ－α－ｍｏｔｉｆ（ＳＡＭ）ドメインが存在している。 EPHA2 is a receptor tyrosine kinase having a single transmembrane structure with a molecular weight of 130 kDa (see, for example, Non-Patent Document 1). EPHA2 has an N-terminal extracellular region with a ligand binding domain and two fibronectin type 3 domains, and a C-terminal intracellular region with a tyrosine kinase domain and a sterile-α-motif (SAM) domain. Existing.
　ＥＰＨＡ２のリガンドとしては、ＧＰＩアンカー型細胞膜蛋白質のＥｐｈｒｉｎ－Ａ１～Ａ５が知られている（例えば、非特許文献２参照）。ＥＰＨＡ２にリガンドが結合することによりチロシンキナーゼドメインが活性化され、ＥＰＨＡ２の細胞内領域に存在するチロシン残基がリン酸化され、細胞内にシグナルが伝達される。また、リガンドと結合したＥＰＨＡ２はエンドサイトーシスにより細胞内に取り込まれ、最終的にプロテアソームで分解されることが報告されている（例えば、非特許文献３参照）。 As a ligand for EPHA2, GPI-anchored cell membrane proteins Ephrin-A1 to A5 are known (for example, see Non-patent Document 2). By binding a ligand to EPHA2, the tyrosine kinase domain is activated, tyrosine residues existing in the intracellular region of EPHA2 are phosphorylated, and a signal is transmitted into the cell. In addition, EPHA2 bound to a ligand has been reported to be taken up into cells by endocytosis and finally degraded by the proteasome (see, for example, Non-Patent Document 3).
　ＥＰＨＡ２は臨床的に多くの癌、特に乳癌、食道癌、前立腺癌、胃癌、非小細胞肺癌、大腸癌、多形神経膠芽腫での高発現が報告されている（例えば、非特許文献４、５、６、７、８、９、１０、１１参照）。また、食道癌では、ＥＰＨＡ２発現陽性の患者で局所リンパ節転移の頻度やリンパ節転移の数が有意に高く、癌の分化度が有意に低いこと、また、ＥＰＨＡ２発現陽性の患者では５年生存率が有意に低いこと（例えば、非特許文献５参照）、非小細胞肺癌では無病生存期間が５年未満の患者では５年より長い患者よりもＥＰＨＡ２の発現が有意に高いこと、再発、特に脳への転移を起こした患者ではＥＰＨＡ２の発現が有意に高いこと（例えば、非特許文献９参照）、大腸癌においてはＥＰＨＡ２発現陽性の患者で肝転移、リンパ管浸潤、リンパ節転移が起こりやすいこと、臨床ステージの高い患者にＥＰＨＡ２発現陽性の患者が多いことが報告されている（例えば、非特許文献１０参照）。 EPHA2 is clinically highly expressed in many cancers, particularly breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme (for example, Non-Patent Document 4). 5, 6, 7, 8, 9, 10, 11). In esophageal cancer, patients with positive expression of EPHA2 have a significantly high frequency of local lymph node metastasis and the number of lymph node metastases, and the degree of differentiation of cancer is significantly low. Patients with positive expression of EPHA2 survive 5 years. The rate is significantly lower (see, for example, Non-Patent Document 5), non-small cell lung cancer patients with a disease-free survival of less than 5 years have significantly higher expression of EPHA2 than patients with longer than 5 years, EPHA2 expression is significantly higher in patients who have metastasized to the brain (see, for example, Non-Patent Document 9), and liver metastasis, lymphatic vessel invasion, and lymph node metastasis are likely to occur in patients with colorectal cancer who are positive for EPHA2 expression. In addition, it has been reported that there are many EPHA2 expression positive patients in patients with high clinical stage (see, for example, Non-Patent Document 10).
　また、ＥＰＨＡ２遺伝子を細胞に導入することによって、非癌細胞が足場非依存的増殖能や細胞外基質上での管状形態形成能、ｉｎ　ｖｉｖｏでの腫瘍増殖能といった癌形質を獲得するようになること（例えば、非特許文献４参照）、癌細胞の細胞外基質に対する浸潤性が亢進すること（例えば、非特許文献１２、１３参照）が報告されている。逆にＥＰＨＡ２の発現をｓｉＲＮＡで抑制すると癌細胞の浸潤性や足場非依存的増殖、ｉｎ　ｖｉｖｏでの腫瘍増殖が抑制されること（例えば、非特許文献１３、１４参照）及び、リガンドであるＥｐｈｒｉｎ－Ａ１とヒトＩｇＧのＦｃ領域との融合蛋白質を使用してＥＰＨＡ２を活性化し、エンドサイトーシスによるＥＰＨＡ２の分解を誘導することで癌細胞の浸潤性や足場非依存的増殖、管状形態形成能が抑制されること（例えば、非特許文献４、１１、１２参照）が報告されている。 Also, by introducing the EPHA2 gene into cells, non-cancerous cells can acquire cancer traits such as anchorage-independent growth ability, tubular morphogenesis on the extracellular matrix, and in vivo tumor growth ability. It has been reported (for example, see Non-patent Documents 4 and 13) and that the invasiveness of cancer cells to the extracellular matrix is enhanced (see Non-Patent Documents 12 and 13 for example). Conversely, suppression of EPHA2 expression with siRNA suppresses cancer cell invasion, anchorage-independent growth, and in vivo tumor growth (see, for example, Non-Patent Documents 13 and 14) and the ligand Ephrin -EPHA2 is activated using a fusion protein of A1 and the Fc region of human IgG, and the degradation of EPHA2 by endocytosis induces cancer cell invasion, anchorage-independent growth, and tubular morphogenesis It has been reported that it is suppressed (for example, see Non-Patent Documents 4, 11, and 12).
　一方でＥＰＨＡ２は、癌細胞のみならず腫瘍内または周囲の血管にも発現していることが報告されている（例えば、非特許文献１５参照）。マウスにおいてはＥｐｈｒｉｎ－Ａ１によって誘導される血管新生にＥＰＨＡ２シグナルが関与していること、特に血管内皮細胞に発現するＥＰＨＡ２が血管内皮細胞の管空形成や生存に必要とされることが報告されており（例えば、非特許文献１６参照）、ＥＰＨＡ２の細胞外領域とヒトＩｇＧのＦｃ領域との融合蛋白質がｉｎ　ｖｉｖｏで血管新生を抑制し、抗腫瘍効果を示すことも報告されている（例えば、非特許文献１７参照）。 On the other hand, it has been reported that EPHA2 is expressed not only in cancer cells but also in blood vessels in or around the tumor (for example, see Non-Patent Document 15). In mice, it has been reported that EPHA2 signal is involved in angiogenesis induced by Ephrin-A1, especially that EPHA2 expressed in vascular endothelial cells is required for the formation and survival of vascular endothelial cells. It has also been reported that a fusion protein of the extracellular region of EPHA2 and the Fc region of human IgG suppresses angiogenesis in vivo and exhibits an antitumor effect (see, for example, Non-Patent Document 16) (for example, Non-patent document 17).
　ＥＰＨＡ２に関しては、リガンドと同じようにＥＰＨＡ２チロシン残基のリン酸化誘導活性とＥＰＨＡ２の分解誘導活性を持つアゴニスト抗ＥＰＨＡ２モノクローナル抗体が乳癌細胞株の足場非依存的増殖や細胞外基質上での管状形態形成を阻害することが報告されている（例えば、非特許文献１４参照）。また、非癌細胞に比べ癌細胞で露出しているＥＰＨＡ２上のエピトープに結合し、ＥＰＨＡ２チロシン残基のリン酸化誘導活性とＥＰＨＡ２の分解誘導活性を有するアゴニスト抗ＥＰＨＡ２モノクローナル抗体がｉｎ　ｖｉｖｏで抗腫瘍効果を示すことが報告されている（例えば、非特許文献２３、特許文献１参照）。一方で、Ｋｉｅｗｌｉｃｈらの抗ＥＰＨＡ２モノクローナル抗体は、ＥＰＨＡ２チロシン残基のリン酸化誘導活性とＥＰＨＡ２の分解誘導活性を有するもののｉｎ　ｖｉｖｏで抗腫瘍効果を示さなかったことが報告されている（例えば、非特許文献２４参照）。 Regarding EPHA2, as in the case of the ligand, an agonist anti-EPHA2 monoclonal antibody having phosphorylation-inducing activity of EPHA2 tyrosine residue and degradation-inducing activity of EPHA2 is used for anchorage-independent growth of breast cancer cell lines and tubular form on the extracellular matrix. It has been reported to inhibit formation (see, for example, Non-Patent Document 14). Furthermore, an agonistic anti-EPHA2 monoclonal antibody that binds to an epitope on EPHA2 exposed in cancer cells as compared to non-cancer cells and has phosphorylation-inducing activity of EPHA2 tyrosine residue and EPHA2 degradation-inducing activity is anti-tumor in vivo. It has been reported that an effect is shown (for example, refer nonpatent literature 23 and patent documents 1). On the other hand, although the anti-EPHA2 monoclonal antibody of Kiewrich et al. Has phosphorylation-inducing activity of EPHA2 tyrosine residue and EPHA2 degradation-inducing activity, it has been reported that it did not exhibit an antitumor effect in vivo (eg, non- (See Patent Document 24).
　また、特許文献２には、癌細胞をマウスに免疫して取得した抗ＥＰＨＡ２モノクローナル抗体であるＬＵＣＡ１９、ＳＧ５、ＬＵＣＡ４０、ＳＰＬ１が記載されており、これらの抗体については、ＬＵＣＡ１９とＳＧ５がＥＰＨＡ２チロシン残基のリン酸化に影響を及ぼさないこと、ＬＵＣＡ４０がｉｎ　ｖｉｔｒｏで癌細胞の増殖を阻害すること、ＬＵＣＡ１９、ＳＧ５及びＬＵＣＡ４０が毒素（ｓａｐｏｒｉｎ）標識された抗マウス抗体存在下で癌細胞内にインターナライズされることが記載されている。また、ＬＵＣＡ４０とＳＰＬ１については、ｉｎ　ｖｉｖｏで抗腫瘍効果を示すことが報告されているが、アゴニスト活性を有するか否かについては明らかとなっていない。 Patent Document 2 describes LUCA19, SG5, LUCA40, and SPL1, which are anti-EPHA2 monoclonal antibodies obtained by immunizing cancer cells in mice. For these antibodies, LUCA19 and SG5 contain EPHA2 tyrosine residues. It does not affect the phosphorylation of the group, LUCA40 inhibits cancer cell growth in vitro, and LUCA19, SG5 and LUCA40 are internalized in cancer cells in the presence of anti-mouse antibody labeled with sapolin. It is described that it is done. In addition, LUCA40 and SPL1 have been reported to show an antitumor effect in vivo, but it is not clear whether or not they have agonist activity.
　一方、ＥＰＨＡ２の細胞外領域に結合し、抗腫瘍活性を有するモノクローナル抗体及びそのヒト化抗体が報告されている（特許文献４）。 Meanwhile, a monoclonal antibody that binds to the extracellular region of EPHA2 and has antitumor activity and a humanized antibody thereof have been reported (Patent Document 4).
国際公開第ＷＯ０３／０９４８５９号パンフレットInternational Publication No. WO03 / 094859 Pamphlet 国際公開第ＷＯ２００６／０８４２２６号パンフレットInternational Publication No. WO2006 / 084226 Pamphlet 国際公開パンフレットＷＯ２００３／０５７８８１International publication pamphlet WO2003 / 057881 国際公開ＷＯ２００９／０２８６３９International Publication WO2009 / 028639
　本発明の１つの課題は、ＥＰＨＡ２に対する抗体を提供することである。本発明の他の１つの課題は癌に対して治療効果を有する該抗ＥＰＨＡ２抗体を含有する医薬組成物等を提供することである。本発明の他の一つの課題は、該抗体の製造方法を提供することである。本発明の他の一つの課題は、該抗体を用いた腫瘍の増殖の抑制方法等を提供することである。 One object of the present invention is to provide an antibody against EPHA2. Another object of the present invention is to provide a pharmaceutical composition containing the anti-EPHA2 antibody having a therapeutic effect on cancer. Another object of the present invention is to provide a method for producing the antibody. Another object of the present invention is to provide a method for suppressing tumor growth using the antibody.
　本発明者らは上記課題を解決するために鋭意、検討を行ったところ、国際公開パンフレットＷＯ２００９／０２８６３９に記載の抗ＥＰＨＡ２モノクローナル抗体である、ＳＨ３４８－１由来の重鎖及びＳＨ３５７－１由来の軽鎖を組み合わせたヒト化抗体（本明細書中において「スワップド抗体」という。）を作製したところ、ガン細胞の増殖抑制活性を維持しつつ、ＳＨ３４８－１由来のヒト化抗体及びＳＨ３５７－１由来のヒト化抗体に比べて抗原への結合活性が向上し、ＡＤＣＣ活性も向上していることを見出し、本発明を完成した。 The present inventors diligently studied to solve the above-mentioned problems. As a result, the anti-EPHA2 monoclonal antibody described in International Publication Pamphlet WO2009 / 028639, which is a heavy chain derived from SH348-1, and a light chain derived from SH357-1. A humanized antibody (hereinafter referred to as a “swapped antibody”) in which a chain is combined was prepared. While maintaining the growth-inhibitory activity of cancer cells, the humanized antibody derived from SH348-1 and the derived from SH357-1 It was found that the binding activity to the antigen was improved and the ADCC activity was also improved as compared with the humanized antibody, and the present invention was completed.
　すなわち、本発明は、
（１）配列表の配列番号１８に示されるアミノ酸配列からなるＣＤＲＨ１、配列表の配列番号２０に示されるアミノ酸配列からなるＣＤＲＨ２及び配列表の配列番号２２に示されるアミノ酸配列からなるＣＤＲＨ３を含むことからなる重鎖、並びに、配列表の配列番号４８に示されるアミノ酸配列からなるＣＤＲＬ１、配列表の配列番号５０に示されるアミノ酸配列からなるＣＤＲＬ２及び配列表の配列番号５２に示されるアミノ酸配列からなるＣＤＲＬ３を含むことからなる軽鎖からなり、配列表の配列番号４に示されるアミノ酸配列からなるポリペプチドを特異的に認識する抗体または該抗体の機能性断片、
（２）配列表の配列番号４のアミノ酸番号４３９乃至５３４に示されるアミノ酸配列からなるポリペプチドを特異的に認識する（１）に記載の抗体または該抗体の機能性断片、
（３）配列表の配列番号１８に示されるアミノ酸配列からなるＣＤＲＨ１、配列表の配列番号２０に示されるアミノ酸配列からなるＣＤＲＨ２及び配列表の配列番号２２に示されるアミノ酸配列からなるＣＤＲＨ３を含むことからなる重鎖、並びに、配列表の配列番号４８に示されるアミノ酸配列からなるＣＤＲＬ１、配列表の配列番号５０に示されるアミノ酸配列からなるＣＤＲＬ２及び配列表の配列番号５２に示されるアミノ酸配列からなるＣＤＲＬ３を含むことからなる軽鎖からなり、配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が１．５ｎＭ以下である（１）又は（２）に記載の抗体または該抗体の機能性断片、
（４）配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が１．０ｎＭ以下である、（３）に記載の抗体または該抗体の機能性断片、
（５）配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が０．５ｎＭ以下である、（３）に記載の抗体または該抗体の機能性断片、
（６）ＥＰＨＡ２リガンドにより誘導されるＥＰＨＡ２チロシン残基のリン酸化を抑制する、（１）乃至（５）のいずれか１項に記載の抗体または該抗体の機能性断片、
（７）配列表の配列番号５４、５６に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる重鎖可変領域を含む重鎖、及び、配列表の配列番号６６、６８に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる軽鎖可変領域を含む軽鎖からなる、（１）乃至（６）のいずれか１項に記載の抗体又は該抗体の機能性断片、
（８）配列表の配列番号５３、５５に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列を含む重鎖、及び配列表の配列番号６５、６７に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる軽鎖を含むことからなる、（１）乃至（７）のいずれか１項に記載の抗体又は該抗体の機能性断片。
（９）配列表の配列番号５３に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６５に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片、
（１０）配列表の配列番号５３に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６７に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片、
（１１）配列表の配列番号５５に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６５に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片、
（１２）配列表の配列番号５５に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６７に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片、
（１３）以下のａ）乃至ｄ）に示される性質を有する、（１）乃至（１２）のいずれか１項に記載の抗体又は該抗体の機能性断片：
ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない；
ｂ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する；
ｃ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する；
ｄ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。
（１４）以下のａ）乃至ｅ）に示される性質を有する、請求項１乃至１２のいずれか１項に記載の抗体又は該抗体の機能性断片：
ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない；
ｂ）ＥＰＨＡ２蛋白質量の減少作用を示さない；
ｃ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する；
ｄ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する；
ｅ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する、
（１５）ＩｇＧ抗体であることを特徴とする、請求項１乃至１４のいずれか１項に記載の抗体、
（１６）Ｆａｂ、Ｆ（ａｂ’）２、Ｆｖ、ｓｃＦＶ、ディアボディー、線状抗体及び多特異的抗体から選択されるいずれかであることを特徴とする、請求項１乃至１４のいずれか１項に記載の抗体又は該抗体の機能性断片、
（１７）（１）乃至（１６）から選択される抗体又は該抗体の機能性断片の少なくともいずれか１つを含有することを特徴とする、医薬組成物、
（１８）（１）乃至（１６）から選択される抗体又は該抗体の機能性断片の少なくともいずれか一つを含有することを特徴とする、癌の治療用医薬組成物、
（１９）（１）乃至（１６）から選択される抗体又は該抗体の機能性断片の少なくともいずれか一つを含有することを特徴とする、乳癌、食道癌、前立腺癌、胃癌、非小細胞肺癌、大腸癌、及び／又は多形神経膠芽腫の治療用医薬組成物。
（２０）（１）乃至（１８）から選択されるいずれかを投与することによる、哺乳動物の有する腫瘍の増殖の抑制方法、
（２１）腫瘍が、ＥＰＨＡ２を発現している腫瘍であることを特徴とする、（１９）に記載の腫瘍の増殖の抑制方法、
（２２）（１）乃至（１６）から選択されるいずれか１項に記載の抗体又は該抗体の機能性断片をコードするポリヌクレオチド、
（２３）（２２）に記載のポリヌクレオチドによって形質転換された宿主細胞、
（２４）（２３）に記載の宿主細胞を培養し、培養液より該抗体又は該抗体の機能性断片を回収する工程を含むことを特徴とする、該抗体又は該抗体の機能性断片の製造方法、
からなる。 That is, the present invention
(1) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. And a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and an amino acid sequence shown in SEQ ID NO: 52. An antibody comprising a light chain comprising CDRL3 and specifically recognizing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, or a functional fragment of the antibody,
(2) the antibody or the functional fragment of the antibody according to (1), which specifically recognizes a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing;
(3) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. And a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and an amino acid sequence shown in SEQ ID NO: 52. A Kd value for a polypeptide comprising a light chain comprising CDRL3 and comprising an amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.5 nM or less (1) or ( 2) an antibody or a functional fragment thereof,
(4) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.0 nM or less Sex fragment,
(5) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 0.5 nM or less Sex fragment,
(6) The antibody or the functional fragment of the antibody according to any one of (1) to (5), which suppresses phosphorylation of an EPHA2 tyrosine residue induced by an EPHA2 ligand,
(7) Heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 54 and 56 in the sequence listing or the amino acid sequence of any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added And the amino acid sequence shown in SEQ ID NOs: 66 and 68 in the sequence listing or any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence The antibody or the functional fragment of the antibody according to any one of (1) to (6), comprising a light chain comprising a light chain variable region,
(8) a heavy chain comprising any one of the amino acid sequences shown in SEQ ID NOs: 53 and 55 of the sequence listing or any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence; The amino acid sequence shown in SEQ ID NOs: 65 and 67 in the sequence listing, or a light chain comprising any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequence The antibody or the functional fragment of the antibody according to any one of (1) to (7).
(9) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody,
(10) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a functional fragment of the antibody,
(11) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody,
(12) an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a functional fragment of the antibody,
(13) The antibody or the functional fragment of the antibody according to any one of (1) to (12), which has the properties shown in the following a) to d):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) has ADCC activity against EPHA2 expressing cells;
c) has CDC activity against EPHA2 expressing cells;
d) Has anti-tumor activity in vivo.
(14) The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to e):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) Does not show an effect of decreasing the amount of EPHA2 protein;
c) has ADCC activity against EPHA2 expressing cells;
d) has CDC activity against EPHA2 expressing cells;
e) has anti-tumor activity in vivo,
(15) The antibody according to any one of claims 1 to 14, which is an IgG antibody,
(16) The method according to any one of claims 1 to 14, wherein the antibody is any one selected from Fab, F (ab ') 2, Fv, scFV, diabody, linear antibody, and multispecific antibody. Or a functional fragment of the antibody,
(17) A pharmaceutical composition comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody,
(18) A pharmaceutical composition for treating cancer, comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody,
(19) Breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell, comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody A pharmaceutical composition for treating lung cancer, colon cancer and / or glioblastoma multiforme.
(20) A method for suppressing tumor growth of a mammal by administering any one selected from (1) to (18),
(21) The method for inhibiting tumor growth according to (19), wherein the tumor is a tumor expressing EPHA2.
(22) A polynucleotide encoding the antibody according to any one of (1) to (16) or a functional fragment of the antibody,
(23) A host cell transformed with the polynucleotide according to (22),
(24) Production of the antibody or the functional fragment of the antibody, comprising culturing the host cell according to (23) and recovering the antibody or the functional fragment of the antibody from the culture solution. Method,
Consists of.
　本発明により、抗ＥＰＨＡ２スワップドヒト化抗体及び該抗体の機能性断片を取得することに成功した。そして、該抗体が優れた抗腫瘍活性を持つことが見出された。 The present invention succeeded in obtaining an anti-EPHA2 swapped humanized antibody and a functional fragment of the antibody. And it was found that the antibody has excellent antitumor activity.
　更に、該抗体を有効成分として含有する医薬組成物、更には、該抗体を含む癌治療用医薬組成物が提供された。 Furthermore, a pharmaceutical composition containing the antibody as an active ingredient and a pharmaceutical composition for cancer treatment containing the antibody were provided.
１）３５７－Ｌ４：３５７－Ｌ４の可変領域のアミノ酸配列を示す図。２）３５７－Ｌ５：３５７－Ｌ５の可変領域のアミノ酸配列を示す図。３）３５７－Ｈ４：３５７－Ｈ４の可変領域のアミノ酸配列を示す図。４）３５７－Ｈ５：３５７－Ｈ５の可変領域のアミノ酸配列を示す図。５）ＳＨ３４８－１＿ＣＤＲＬ１：ＳＨ３４８－１のＣＤＲＬ１のアミノ酸配列を示す図。６）ＳＨ３４８－１＿ＣＤＲＬ２：ＳＨ３４８－１のＣＤＲＬ２のアミノ酸配列を示す図。７）ＳＨ３４８－１＿ＣＤＲＬ３：ＳＨ３４８－１のＣＤＲＬ３のアミノ酸配列を示す図。８）ＳＨ３４８－１＿ＣＤＲＨ１：ＳＨ３４８－１のＣＤＲＨ１のアミノ酸配列を示す図。９）ＳＨ３４８－１＿ＣＤＲＨ２：ＳＨ３４８－１のＣＤＲＨ２のアミノ酸配列を示す図。1) 357-L4: A diagram showing the amino acid sequence of the variable region of 357-L4. 2) 357-L5: A diagram showing the amino acid sequence of the variable region of 357-L5. 3) The figure which shows the amino acid sequence of the variable region of 357-H4: 357-H4. 4) 357-H5: A diagram showing the amino acid sequence of the variable region of 357-H5. 5) SH348-1_CDRL1: A figure showing the amino acid sequence of CDRL1 of SH348-1. 6) SH348-1_CDRL2: A figure showing the amino acid sequence of CDRL2 of SH348-1. 7) SH348-1_CDRL3: A drawing showing the amino acid sequence of CDRL3 of SH348-1. 8) SH348-1_CDRH1: A figure showing the amino acid sequence of CDR4 of SH348-1. 9) SH348-1_CDRH2: A figure showing the amino acid sequence of CDR4 of SH348-1. １０）ＳＨ３４８－１＿ＣＤＲＨ３：ＳＨ３４８－１のＣＤＲＨ３のアミノ酸配列を示す図。１１）３４８－Ｌ４：３４８－Ｌ４の可変領域のアミノ酸配列を示す図。１２）３４８－Ｌ５：３４８－Ｌ５の可変領域のアミノ酸配列を示す図。１３）３４８－Ｈ４：３４８－Ｈ４の可変領域のアミノ酸配列を示す図。１４）３４８－Ｈ５：３４８－Ｈ５の可変領域のアミノ酸配列を示す図。１５）ＳＨ３５７－１＿ＣＤＲ＿Ｌ１：ＳＨ３５７－１のＣＤＲＬ１のアミノ酸配列を示す図。１６）ＳＨ３５７－１＿ＣＤＲ＿Ｌ２：ＳＨ３５７－１のＣＤＲＬ２のアミノ酸配列を示す図。１７）ＳＨ３５７－１＿ＣＤＲ＿Ｌ３：ＳＨ３５７－１のＣＤＲＬ３のアミノ酸配列を示す図。１８）ＳＨ３５７－１＿ＣＤＲ＿Ｈ１：ＳＨ３５７－１のＣＤＲＨ１のアミノ酸配列を示す図。10) SH348-1_CDRH3: A figure showing the amino acid sequence of CDR4 of SH348-1. 11) 348-L4: A drawing showing the amino acid sequence of the variable region of 348-L4. 12) 348-L5: A drawing showing the amino acid sequence of the variable region of 348-L5. 13) 348-H4: A diagram showing the amino acid sequence of the variable region of 348-H4. 14) 348-H5: A diagram showing the amino acid sequence of the variable region of 348-H5. 15) SH357-1_CDR_L1: A diagram showing the amino acid sequence of CDRL1 of SH357-1. 16) SH357-1_CDR_L2: A diagram showing the amino acid sequence of CDRL2 of SH357-1. 17) SH357-1_CDR_L3: A diagram showing the amino acid sequence of CDRL3 of SH357-1. 18) SH357-1_CDR_H1: A drawing showing the amino acid sequence of CDR35 of SH357-1. １９）ＳＨ３５７－１＿ＣＤＲ＿Ｈ２：ＳＨ３５７－１のＣＤＲＨ２のアミノ酸配列を示す図。２０）ＳＨ３５７－１＿ＣＤＲ＿Ｈ３：ＳＨ３５７－１のＣＤＲＨ３のアミノ酸配列を示す図。19) SH357-1_CDR_H2: A drawing showing the amino acid sequence of CDR35 of SH357-1. 20) SH357-1_CDR_H3: A drawing showing the amino acid sequence of CDR3 of SH357-1. １）配列番号７１：ヒトκ鎖分泌シグナル、ヒトκ鎖定常領域及びヒトｐｏｌｙＡ付加シグナルをコードするＤＮＡフラグメントのヌクレオチド配列を示す図。1) SEQ ID NO: 71: nucleotide sequence of DNA fragment encoding human κ chain secretion signal, human κ chain constant region and human polyA addition signal. ２）配列番号７２：ヒトＩｇＧ１シグナル配列及び定常領域のアミノ酸をコードするＤＮＡ配列を含むＤＮＡ断片のヌクレオオチド配列を示す図。３）配列番号７３：分泌シグナルと融合したｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖可変領域をコードする遺伝子を含むＤＮＡのヌクレオチド配列を示す図。2) SEQ ID NO: 72: A diagram showing a nucleotide sequence of a DNA fragment containing a human IgG1 signal sequence and a DNA sequence encoding an amino acid in the constant region. 3) SEQ ID NO: 73: A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1L type light chain variable region fused with a secretion signal. ４）配列番号７４：分泌シグナルと融合したｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖可変領域をコードする遺伝子を含むＤＮＡのヌクレオチド配列を示す図。５）配列番号７５：ｈＳＨ３４８－１－Ｔ１Ｈタイプ重鎖可変領域をコードする遺伝子を含むＤＮＡのヌクレオチド配列を示す図。６）配列番号７６：ｈＳＨ３５７－１－Ｔ１Ｈタイプ重鎖可変領域をコードする遺伝子を含むＤＮＡのヌクレオチド配列を示す図。4) SEQ ID NO: 74: A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with a secretion signal. 5) SEQ ID NO: 75: A drawing showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1H type heavy chain variable region. 6) SEQ ID NO: 76 is a view showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1H type heavy chain variable region. ７）配列番号７７：ｈＳＨ３４８－１－Ｔ１Ｌ：ｈＳＨ３４８－１－Ｔ１Ｌのヌクレオチド配列を示す図。８）配列番号７８：ｈＳＨ３４８－１－Ｔ１Ｌ：ｈＳＨ３４８－１－Ｔ１Ｌのアミノ酸配列を示す図。7) SEQ ID NO: 77: hSH348-1-T1L: a drawing showing the nucleotide sequence of hSH348-1-T1L. 8) SEQ ID NO: 78: hSH348-1-T1L: A drawing showing the amino acid sequence of hSH348-1-T1L. ９）配列番号７９：ｈＳＨ３５７－１－Ｔ１Ｌ：ｈＳＨ３５７－１－Ｔ１Ｌのヌクレオチド配列を示す図。１０）配列番号８０：ｈＳＨ３５７－１－Ｔ１Ｌ：ｈＳＨ３５７－１－Ｔ１Ｌのアミノ酸配列を示す図。9) A sequence showing the nucleotide sequence of SEQ ID NO: 79: hSH357-1-T1L: hSH357-1-T1L. 10) A sequence showing the amino acid sequence of SEQ ID NO: 80: hSH357-1-T1L: hSH357-1-T1L. １１）配列番号８１：３４８－Ｌ４：３４８－Ｌ４のヌクレオチド配列を示す図。１２）配列番号８２：３４８－Ｌ４：３４８－Ｌ４のアミノ酸配列を示す図。11) The figure which shows the nucleotide sequence of sequence number 81: 348-L4: 348-L4. 12) The figure which shows the amino acid sequence of sequence number 82: 348-L4: 348-L4. １３）配列番号８３：３５７－Ｌ４：３５７－Ｌ４のヌクレオチド配列を示す図。１４）配列番号８４：３５７－Ｌ４：３５７－Ｌ４のアミノ酸配列を示す図。13) The figure which shows the nucleotide sequence of sequence number 83: 357-L4: 357-L4. 14) The amino acid sequence of SEQ ID NO: 84: 357-L4: 357-L4. １５）配列番号８５：３４８－Ｌ５：３４８－Ｌ５のヌクレオチド酸配列を示す図。１６）配列番号８６：３４８－Ｌ５：３４８－Ｌ５のアミノ酸配列を示す図。15) A drawing showing the nucleotide acid sequence of SEQ ID NO: 85: 348-L5: 348-L5. 16) The figure which shows the amino acid sequence of sequence number 86: 348-L5: 348-L5. １７）配列番号８７：３５７－Ｌ５：３５７－Ｌ５のヌクレオチド配列を示す図。１８）配列番号８８：３５７－Ｌ５：３５７－Ｌ５のアミノ酸配列を示す図。17) The figure which shows the nucleotide sequence of sequence number 87: 357-L5: 357-L5. 18) The figure which shows the amino acid sequence of sequence number 88: 357-L5: 357-L5. １９）配列番号８９：３４８－Ｈ４：３４８－Ｈ４のヌクレオチド配列を示す図。19) The nucleotide sequence of SEQ ID NO: 89: 348-H4: 348-H4. ２０）配列番号９０：３４８－Ｈ４：３４８－Ｈ４のアミノ酸配列を示す図。20) The figure which shows the amino acid sequence of sequence number 90: 348-H4: 348-H4. ２１）配列番号９１：３４８－Ｈ５：３４８－Ｈ５のヌクレオチド配列を示す図。21) The nucleotide sequence of SEQ ID NO: 91: 348-H5: 348-H5. ２２）配列番号９２：３４８－Ｈ５：３４８－Ｈ５のアミノ酸配列示す図。22) The amino acid sequence which shows sequence number 92: 348-H5: 348-H5. ２３）配列番号９３：３５７－Ｈ４：３５７－Ｈ４のヌクレオチド配列を示す図。23) The nucleotide sequence of SEQ ID NO: 93: 357-H4: 357-H4. ２４）配列番号９４：３５７－Ｈ４のアミノ酸配列を示す図。24) The amino acid sequence of SEQ ID NO: 94: 357-H4. ２５）配列番号９５：３５７－Ｈ５：３５７－Ｈ５のヌクレオチド配列を示す図。25) The nucleotide sequence of SEQ ID NO: 95: 357-H5: 357-H5. ２６）配列番号９６：３５７－Ｈ５：３５７－Ｈ５のアミノ酸配列を示す図。26) The amino acid sequence of SEQ ID NO: 96: 357-H5: 357-H5. 各抗体のＡＤＣＣ活性の測定結果を示す図。横軸は抗体濃度を示し、縦軸は細胞様戒律を示す。黒丸破線（●）はコントロール抗体であるｈＩｇＧ１、黒丸実線（●）は、ｈＳＨ３５７－１Ｔ１を、黒四角（■）は３５７－Ｈ４－３５７－Ｌ４を、白丸（○）は３４８－Ｈ４－３５７－Ｌ４を、白三角（△）は３４８－Ｈ４－３５７－Ｌ５を、白四角（□）は３４８－Ｈ５－３５７－Ｌ５を示す。The figure which shows the measurement result of ADCC activity of each antibody. The horizontal axis indicates antibody concentration, and the vertical axis indicates cell-like discipline. The black dotted line (●) is the control antibody hIgG1, the solid black line (●) is hSH357-1T1, the black square (■) is 357-H4-357-L4, and the white circle (◯) is 348-H4-357- L4, white triangle (Δ) indicates 348-H4-357-L5, and white square (□) indicates 348-H5-357-L5. 各抗体の抗原特異的結合を示す図。Ａ）は各蛋白質の細胞膜上への発現量をＡｎｔｉ－ＦＬＡＧ　ｍＡＢを用いて確認した結果を示す。いずれの蛋白質も細胞膜上への同程度の発現が認められたことを示している。Ｂ）はヒト化抗ＥＰＨＡ２抗体であるｈＳＨ３５７－１－Ｔ１についての各蛋白質への結合結果を示し、Ｃ）は３４８－Ｈ４－３５７－Ｌ４についての、Ｄ）は３４８－Ｈ４－３５７－Ｌ５、Ｅ）は３４８－Ｈ５－３５７－Ｌ５についての結果を示す。Ｂ）～Ｅ）では、各抗体がヒトＥＰＨＡ２を発現する細胞に対して強く結合するのに対し、ヒトＥＰＨＡ３ａ及びヒトＰＴＰＲＳ－４を発現する細胞に対しては陰性コントロールであるｐＦＬＡＧ－ＧＷをトランスフェクションした細胞と同程度の結合性しか示さなかったことを示している。The figure which shows the antigen-specific binding of each antibody. A) shows the result of confirming the expression level of each protein on the cell membrane using Anti-FLAG mAB. Both proteins indicate that the same level of expression on the cell membrane was observed. B) shows the binding results to each protein for humanized anti-EPHA2 antibody hSH357-1-T1, C) for 348-H4-357-L4, D) 348-H4-357-L5, E) shows the results for 348-H5-357-L5. In B) to E), each antibody binds strongly to cells expressing human EPHA2, whereas pFLAG-GW, which is a negative control, is transfected into cells expressing human EPHA3a and human PTPRS-4. It shows that it showed only the same level of binding as the transfected cells. 各抗体のＩｎ　ｖｉｖｏ抗腫瘍活性の結果を示す図。Ａ）はｈＳＨ３５７－１－Ｔ１について、Ｂ）は３４８－Ｈ４－３５７－Ｌ４について、Ｃ）は３４８－Ｈ４－３５７－Ｌ５について、Ｄ）は３４８－Ｈ５－３５７－Ｌ５についての結果を示す。横軸はヒト乳癌細胞株ＭＤＡ－ＭＢ－２３１細胞移植後の経過日数を示し、縦軸は腫瘍の体積を示す。The figure which shows the result of the in vivo antitumor activity of each antibody. A) shows results for hSH357-1-T1, B) for 348-H4-357-L4, C) for 348-H4-357-L5, and D) for 348-H5-357-L5. The horizontal axis indicates the number of days after transplantation of human breast cancer cell line MDA-MB-231 cells, and the vertical axis indicates the tumor volume.
　１．定義
　本明細書中においては、「癌」と「腫瘍」は同じ意味に用いている。 1. Definitions In the present specification, “cancer” and “tumor” are used interchangeably.
　本明細書中において、「遺伝子」という語には、ＤＮＡのみならずそのｍＲＮＡ、ｃＤＮＡ及びそのｃＲＮＡも含まれるものとする。したがって、本発明における「ＥＰＨＡ２遺伝子」には、ＥＰＨＡ２のＤＮＡ、ｍＲＮＡ、ｃＤＮＡ及びｃＲＮＡが含まれる。 In this specification, the term “gene” includes not only DNA but also mRNA, cDNA and cRNA thereof. Therefore, “EPHA2 gene” in the present invention includes EPHA2 DNA, mRNA, cDNA, and cRNA.
　本明細書中において、「ポリヌクレオチド」という語は核酸と同じ意味で用いており、ＤＮＡ、ＲＮＡ、プローブ、オリゴヌクレオチド、及びプライマーも含まれている。 In the present specification, the term “polynucleotide” is used in the same meaning as a nucleic acid, and includes DNA, RNA, probes, oligonucleotides, and primers.
　本明細中においては、「ポリペプチド」と「蛋白質」は区別せずに用いている。 In the present specification, “polypeptide” and “protein” are used without distinction.
　本明細書中において、「細胞」には、動物個体内の細胞、培養細胞も含んでいる。 In the present specification, “cell” includes cells in an individual animal and cultured cells.
　本明細書中において、「細胞の癌化」とは、細胞が接触阻止現象への感受性を喪失することや、足場非依存性増殖を示すこと等、細胞が異常な増殖を示すことをいい、このような異常な増殖を示す細胞を「癌細胞」という。 In the present specification, “cell canceration” means that the cell exhibits abnormal growth, such as loss of sensitivity to the contact-inhibiting phenomenon, or anchorage-independent growth, A cell exhibiting such abnormal growth is called a “cancer cell”.
　本明細書中においては、ＥＰＨＡ２が有する細胞の癌化活性及び／又は細胞増殖活性等と同等の機能を有する蛋白質もＥＰＨＡ２という。 In the present specification, a protein having a function equivalent to the cell carcinogenic activity and / or cell proliferation activity of EPHA2 is also referred to as EPHA2.
　本明細書中における「スワップド抗体（ｓｗａｐｐｅｄ　ａｎｔｉｂｏｄｙ）」とは、複数の種類の抗体の間で重鎖及び／又は軽鎖を交換した抗体をいい、重鎖及び／又は軽鎖の全長を交換したものの他、可変領域のみ、定常領域のみを交換したもの、相補性決定領域のみを交換したものも含まれる。すなわち、例えば、抗体の全長配列においては、抗体Ａの重鎖をＡＨ、軽鎖をＡＬ、抗体Ｂの重鎖をＢＨ、軽鎖をＢＬとしたときには、重鎖／軽鎖の組み合わせがＡＨ／ＢＬ、ＢＨ／ＡＬとなる抗体はスワップド抗体である。また、他の例としては、抗体Ａの重鎖の可変領域のＣＤＲをＣＤＲＨ１Ａ、ＣＤＲＨ２Ａ、ＣＤＲＨ３Ａ、軽鎖の可変領域のＣＤＲをＣＤＲＬ１Ａ、ＣＤＲＬ２Ａ、ＣＤＲＬ３Ａとし、抗体Ｂの重鎖の可変領域のＣＤＲをＣＤＲＨ１Ｂ、ＣＤＲＨ２Ｂ、ＣＤＲＨ３Ｂ、軽鎖の可変領域のＣＤＲをＣＤＲＬ１Ｂ、ＣＤＲＬ２Ｂ、ＣＤＲＬ３Ｂとしたときには、ＣＤＲの組み合わせが、重鎖がＣＤＲＨ１Ａ、ＣＤＲＨ２Ａ及びＣＤＲＨ３Ａであり軽鎖がＣＤＲＬ１Ｂ、ＣＤＲＬ２Ｂ及びＣＤＲＬ３Ｂである抗体、並びに、ＣＤＲの組み合わせが、重鎖がＣＤＲＨ１Ｂ、ＣＤＲＨ２Ｂ及びＣＤＲＨ３Ｂであり、軽鎖がＣＤＲＬ１Ａ、ＣＤＲＬ２Ａ及びＣＤＲＬ３Ａである抗体はスワップド抗体である。本明細書中においては、特に記載がない限り、「抗ＥＰＨＡ２抗体」には抗ＥＰＨＡ２スワップド抗体も含まれ、「抗ＥＰＨＡ２ヒト化抗体」には抗ＥＰＨＡ２ヒト化スワップド抗体」も含まれる。 As used herein, “swapped antibody” refers to an antibody in which heavy chains and / or light chains are exchanged among a plurality of types of antibodies, and the full lengths of heavy chains and / or light chains are exchanged. In addition to those, only variable regions, those in which only constant regions are exchanged, and those in which only complementarity determining regions are exchanged are included. That is, for example, in the full-length antibody sequence, when the heavy chain of antibody A is AH, the light chain is AL, the heavy chain of antibody B is BH, and the light chain is BL, the combination of heavy chain / light chain is AH / Antibodies that become BL and BH / AL are swapped antibodies. As another example, CDR of the variable region of the heavy chain of antibody A is CDRH1A, CDRH2A, CDRH3A, CDR of the variable region of the light chain is CDRL1A, CDRL2A, CDRL3A, and CDR of the variable region of the heavy chain of antibody B Is CDRH1B, CDRH2B, CDRH3B, and the CDR of the light chain variable region is CDRL1B, CDRL2B, CDRL3B, the combination of CDRs is heavy chain CDRH1A, CDRH2A and CDRH3A, and the light chain is CDRRL1B, CDRRL2B and CDRL3B In addition, an antibody in which the heavy chain is CDRH1B, CDRH2B and CDRH3B, and the light chain is CDRL1A, CDRL2A and CDRL3A is a swapped antibody. In the present specification, unless otherwise specified, “anti-EPHA2 antibody” includes anti-EPHA2 swapped antibodies, and “anti-EPHA2 humanized antibody” also includes anti-EPHA2 humanized swapped antibodies.
　本明細書中における「抗体の機能性断片」とは、抗原との結合活性を有する抗体の部分断片を意味しており、Ｆａｂ、Ｆ（ａｂ’）２、ｓｃＦｖ等を含む。また、Ｆ（ａｂ’）２を還元条件下で処理した抗体の可変領域の一価の断片であるＦａｂ’も抗体の機能性断片に含まれる。但し、抗原との結合能を有している限りこれらの分子に限定されない。また、これらの機能性断片には、抗体蛋白質の全長分子を適当な酵素で処理したもののみならず、遺伝子工学的に改変された抗体遺伝子を用いて適当な宿主細胞において産生された蛋白質も含まれる。 In the present specification, the “functional fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ′) 2, scFv, and the like. Further, Fab ', which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody. However, the molecule is not limited to these molecules as long as it has the ability to bind to an antigen. These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.
　本明細書中における、「チロシン残基のリン酸化」とは、ペプチドのアミノ酸配列に含まれるチロシン残基がリン酸化されることをいい、チロシン残基がリン酸化されているか否かは、例えば、ペプチドと抗リン酸化チロシン抗体（例えば、Ａｎｔｉ－Ｐｈｏｓｐｈｏｔｙｒｏｓｉｎｅ，ｒｅｃｏｍｂｉｎａｎｔ　４Ｇ１０　ＨＲＰ－ｃｏｎｊｕｇａｔｅ（Ｍｉｌｌｉｐｏｒｅ（Ｕｐｓａｔｅ）社製　＃１６－１８４）との結合性を調べ、該抗体と結合するときには、チロシン残基がリン酸化されていると判定することができる。本明細書中における「ＥＰＨＡ２チロシン残基のリン酸化能」とは、ＥＰＨＡ２のアミノ酸配列中のチロシン残基をリン酸化する能力をいい、抗体がＥＰＨＡ２のチロシン残基のリン酸化能を有するか否かは、例えば、抗体とＥＰＨＡ２をインキュベーションした後に、ＥＰＨＡ２と抗リン酸化チロシン抗体との結合性があるかないかで判定することができる。 In the present specification, “phosphorylation of tyrosine residue” means that a tyrosine residue contained in the amino acid sequence of a peptide is phosphorylated, and whether or not a tyrosine residue is phosphorylated is, for example, The binding of the peptide to an anti-phosphotyrosine antibody (for example, Anti-Phosphotyrosine, recombinant 4G10, HRP-conjugate (Millipore (Upsate) # 16-184)) was examined. In the present specification, the term “EPHA2 tyrosine residue phosphorylation ability” refers to the ability to phosphorylate tyrosine residues in the amino acid sequence of EPHA2, and the antibody is EPHA2 Whether or not it has the ability to phosphorylate tyrosine residues of Eg to after incubation of the antibody and EPHA2, can be determined by whether or not there bind with anti-phosphotyrosine antibody and EPHA2.
　本明細書中において、「ＥＰＨＡ２蛋白質の発現量が減少する」とは、ＥＰＨＡ２蛋白質の量が減少することをいい、抗体がＥＰＨＡ２蛋白質の量を減少させる作用を有するか否かは、例えば、抗体とＥＰＨＡ２をインキュベーションした後に、ＥＰＨＡ２の量を定量することによって調べることができる。 In the present specification, “the expression level of EPHA2 protein decreases” means that the amount of EPHA2 protein decreases, and whether or not an antibody has an action of decreasing the amount of EPHA2 protein is, for example, an antibody Can be examined by quantifying the amount of EPHA2 after incubation with EPHA2.
　本明細書中において、「ＥＰＨＡ２リガンド」とは、ＥＰＨＡ２のリガンドとなり得る物質を示し、その具体例としては、ＧＰＩアンカー型細胞膜蛋白質のＥｐｈｒｉｎ－Ａ１～Ａ５を挙げることができる（Ａｎｎｕａｌ　Ｒｅｖｉｅｗ　ｏｆ　Ｎｅｕｒｏｓｃｉｅｎｃｅ、１９９８年、第２１巻、ｐ．３０９－３４５）。 In the present specification, “EPHA2 ligand” refers to a substance that can be a ligand of EPHA2, and specific examples thereof include GPI-anchored cell membrane proteins, Ephrin-A1 to A5 (Annual Review of Neuroscience, 1998, Vol. 21, pp. 309-345).
　本明細書中における、「細胞傷害」とは、何らかの形で、細胞に病理的な変化をもたらすことをいい、直接的な外傷にとどまらず、ＤＮＡの切断や塩基の二量体の形成、染色体の切断、細胞***装置の損傷、各種酵素活性の低下などあらゆる細胞の構造や機能上の損傷をいう。本明細書中における「細胞障害活性」とは上記細胞傷害を引き起こすことをいう。 In the present specification, “cytotoxicity” refers to pathological changes in cells in some form, not just direct trauma, but also DNA cleavage, base dimer formation, chromosomes. This refers to any structural or functional damage to cells, such as cleavage of cells, damage to cell division apparatus, or reduction of various enzyme activities. As used herein, “cytotoxic activity” refers to causing the above-mentioned cytotoxicity.
　本明細書中における、ＡＤＣＣとは、抗体依存性細胞障害（Ａｎｔｉｂｏｄｙ－ｄｅｐｅｎｄｅｎｔ　ｃｅｌｌｕｌａｒ　ｃｙｔｏｔｏｘｉｃｉｔｙ）と同義であり、Ｆｃγレセプター保有細胞が、標的細胞の表面抗原に結合した抗体のＦｃ部分にＦｃγレセプターを介して付着し、標的細胞を殺す反応のことをいい、ＡＤＣＣ活性とは、抗体依存性細胞障害活性ともいい、上記反応の活性のことをいう。ＡＤＣＣ活性は当業者が通常行う方法で測定することができるが、例えば、本明細書中の実施例８に記載の方法によって測定することができる。 In the present specification, ADCC is synonymous with antibody-dependent cellular cytotoxicity, and Fcγ receptor-bearing cells are mediated through Fcγ receptor in the Fc part of the antibody bound to the surface antigen of the target cell. The ADCC activity is also referred to as antibody-dependent cytotoxic activity and refers to the activity of the above reaction. ADCC activity can be measured by a method commonly used by those skilled in the art. For example, it can be measured by the method described in Example 8 herein.
　本明細書中における、「ＣＤＣ」とは、補体依存性細胞障害（ｃｏｍｐｌｅｍｅｎｔ－ｄｅｐｅｎｄｅｎｔ　ｃｙｔｏｔｏｘｉｃｉｔｙ）と同義であり、ＣＤＣ活性とは補体依存性細胞障害を起こす活性のことをいう。ＣＤＣ活性は当業者が通常行う方法によって測定することができる。 As used herein, “CDC” is synonymous with complement-dependent cytotoxicity, and CDC activity refers to an activity that causes complement-dependent cytotoxicity. CDC activity can be measured by methods commonly used by those skilled in the art.
　本明細書中において、「Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。」とは、腫瘍を有する動物個体の腫瘍の増殖を抑制又は減少させる活性を有することを意味する。抗ＥＰＨＡ２抗体が「Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。」か否かは当業者が通常行う方法によって調べることができるが、例えば以下の方法によっても調べることができる。すなわち、腫瘍細胞（例えば、ＭＤＡ－ＭＢ－２３１細胞）が皮下移植されているヌードマウス（例えば、ＢＡＬＢ／ｃＡＪｃ１－ｎｕ／ｎｕ；日本クレア社より入手）に被験物質として抗ＥＰＡ２抗体を腹腔内に適当量投与し、腫瘍体積の経時変化を抗ＥＰＡ２抗体を投与しなかったコントロールと比較し、抗ＥＰＡ２抗体投与群でコントロールに比して腫瘍の体積が有意に小さい場合には、被験物質である該抗ＥＰＡ２抗体は「Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。」と判断することができる。 In the present specification, “having anti-tumor activity in vivo” means having an activity of suppressing or reducing the growth of a tumor of an animal individual having a tumor. Whether or not an anti-EPHA2 antibody has “in vivo antitumor activity” can be examined by a method commonly used by those skilled in the art, but can also be examined, for example, by the following method. That is, an anti-EPA2 antibody as a test substance is intraperitoneally injected into a nude mouse (for example, BALB / cAJc1-nu / nu; obtained from Clea Japan Co., Ltd.) into which tumor cells (for example, MDA-MB-231 cells) are transplanted subcutaneously. It is a test substance when the appropriate volume is administered and the change in the tumor volume over time is compared with the control in which the anti-EPA2 antibody was not administered, and the tumor volume is significantly smaller in the anti-EPA2 antibody administration group than in the control. The anti-EPA2 antibody can be determined to have “in vivo antitumor activity”.
　抗体分子の重鎖及び軽鎖にはそれぞれ３箇所の相補性決定領域（ＣＤＲ：Ｃｏｍｐｌｅｍｅｔａｒｙ　ｄｅｔｅｒｍｉｎｇ　ｒｅｇｉｏｎ）があることが知られている。本明細書中においては、抗体の相補性決定領域について、重鎖の相補性決定領域をＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３と表記し、軽鎖の相補性決定領域をＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３と表記する。 It is known that there are three complementarity determining regions (CDRs) in each of the heavy and light chains of an antibody molecule. In the present specification, with regard to the complementarity determining regions of antibodies, the complementarity determining regions of heavy chains are denoted as CDRH1, CDRH2, and CDRH3, and the complementarity determining regions of light chains are denoted as CDRL1, CDRL2, and CDRL3.
　本明細書における、「エピトープ」とは、動物、好ましくは、哺乳動物、より好ましくは、マウス又はヒトの体内において、抗原性及び／又は免疫原性を有するＥＰＨＡ２の部分ペプチドを意味する。抗原性を有するＥＰＨＡ２の部分ペプチドであるエピトープは、免疫アッセイ法等当業者によく知られている方法によって決定することができるが、例えば以下の方法によって行うことができる。ＥＰＨＡ２の様々な部分構造を作製する。部分構造の作製にあたっては、公知のオリゴペプチド合成技術を用いることができる。例えば、ＥＰＨＡ２のＣ末端あるいはＮ末端から適当な長さで順次短くした一連のポリペプチドを当業者に周知の遺伝子組み換え技術を用いて作製した後、それらに対する抗体の反応性を検討し、大まかな認識部位を決定した後に、さらに短いペプチドを合成してそれらのペプチドとの反応性を検討することによって、エピトープを決定することができる。 As used herein, “epitope” means a partial peptide of EPHA2 having antigenicity and / or immunogenicity in an animal, preferably a mammal, more preferably a mouse or human body. The epitope that is a partial peptide of EPHA2 having antigenicity can be determined by methods well known to those skilled in the art, such as immunoassay, and can be carried out, for example, by the following method. Various partial structures of EPHA2 are produced. In producing the partial structure, a known oligopeptide synthesis technique can be used. For example, after preparing a series of polypeptides sequentially shortened by an appropriate length from the C-terminal or N-terminal of EPHA2, using gene recombination techniques well known to those skilled in the art, the reactivity of antibodies against them is examined, After determining the recognition site, the epitope can be determined by synthesizing shorter peptides and examining their reactivity with those peptides.
　本明細書中における変異抗体とは、抗体のアミノ酸配列中の１又は数個のアミノ酸が置換・欠失・付加している抗体をいう。 As used herein, a mutant antibody refers to an antibody in which one or several amino acids in the amino acid sequence of the antibody are substituted, deleted, or added.
　本明細書中における抗体の性質の一例としては、生物的、化学的、物理的性質を挙げることができ、より具体的な例としては、生物活性、抗原・エピトープに対する結合性、製造、流通、保存時の安定性、熱安定性等を挙げることができる。 Examples of the properties of antibodies in the present specification include biological, chemical, and physical properties, and more specific examples include biological activity, binding to antigens and epitopes, production, distribution, The stability at the time of storage, thermal stability, etc. can be mentioned.
　本発明において、「ストリンジェントな条件下でハイブリダイズする」とは、市販のハイブリダイゼーション溶液ＥｘｐｒｅｓｓＨｙｂ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ　Ｓｏｌｕｔｉｏｎ（クロンテック社製）中、６８℃でハイブリダイズすること、又は、ＤＮＡを固定したフィルターを用いて０．７－１．０ＭのＮａＣｌ存在下６８℃でハイブリダイゼーションを行った後、０．１－２倍濃度のＳＳＣ溶液（１倍濃度ＳＳＣとは１５０　ｍＭ　ＮａＣｌ、１５　ｍＭ　クエン酸ナトリウムからなる）を用い、６８℃で洗浄することにより同定することができる条件又はそれと同等の条件でハイブリダイズすることをいう。 In the present invention, “hybridize under stringent conditions” means to hybridize at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech) or use a filter on which DNA is fixed. After hybridization at 68 ° C in the presence of 0.7-1.0 M NaCl, 0.1-2 fold SSC solution (1 fold SSC consists of 150 mM NaCl, 15 mM sodium citrate) ) And hybridization under conditions that can be identified by washing at 68 ° C. or equivalent conditions.
　２．ＥＰＨＡ２について
（１）ＥＰＨＡ２遺伝子
　ＥＰＨＡ２遺伝子のヌクレオチド配列及びアミノ酸配列はＧｅｎＢａｎｋにＥＰＨ　ｒｅｃｅｐｔｏｒ　Ａ２（アクセッション番号はそれぞれＮＭ＿００４４３１、ＮＰ＿００４４２２）として登録されている。また、ＥＰＨＡ２遺伝子のオープンリーディングフレーム（ＯＲＦ）のヌクレオチド配列は配列表の配列番号１に記載されており、そのアミノ酸配列は配列表の配列番号２に記載されている。 2. About EPHA2 (1) EPHA2 gene The nucleotide sequence and amino acid sequence of the EPHA2 gene are registered in GenBank as EPH receptor A2 (accession numbers NM_004431 and NP_004422, respectively). The nucleotide sequence of the open reading frame (ORF) of the EPHA2 gene is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence thereof is described in SEQ ID NO: 2 in the sequence listing.
　なお、ＥＰＨＡ２のアミノ酸配列において、１若しくは数個のアミノ酸が置換、欠失、付加されたアミノ酸配列からなり、これらの酵素と同等の生物活性を有する蛋白質もＥＰＨＡ２に含まれる。
（２）ＥＰＨＡ２遺伝子の癌部特異的発現
　ＥＰＨＡ２遺伝子は、多くの癌、特に乳癌、食道癌、前立腺癌、胃癌、非小細胞肺癌、大腸癌、多形神経膠芽腫での高発現が報告されている。 EPHA2 includes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of EPHA2, and having a biological activity equivalent to those of these enzymes.
(2) Cancer-specific expression of EPHA2 gene EPHA2 gene is reported to be highly expressed in many cancers, especially breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme Has been.
　また、ＥＰＨＡ２の発現量及び／又は活性を抑制する物質はＥＰＨＡ２に起因する細胞の癌化の抑制及び／又は癌細胞の増殖を抑制する活性を有する。 Further, a substance that suppresses the expression level and / or activity of EPHA2 has an activity of suppressing canceration of cells caused by EPHA2 and / or suppressing proliferation of cancer cells.
　（３）ＥＰＨＡ２の調製
　抗ＥＰＨＡ２抗体の抗原であるＥＰＨＡ２としてはＥＰＨＡ２の全長ポリペプチド又はその部分ポリペプチドを挙げることができ、より具体的には、ＥＰＨＡ２の全長ポリペプチド又は、好ましくはＥＰＨＡ２の細胞外領域ポリペプチド（配列表の配列番号４のアミノ酸番号１乃至５４０に示されるアミノ酸配列からなる。）、更に好ましくは配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むポリペプチド、更により好ましくは、配列表の配列番号４のアミノ酸番号４３９乃至５３４に示されるアミノ酸配列を含むポリペプチド、あるいはこれらに任意のアミノ酸配列や担体が付加された誘導体を挙げることができる。また、上記各ポリペプチドの少なくとも６個の連続した部分アミノ酸配列からなるポリペプチド、あるいはこれらに任意のアミノ酸配列や担体が付加された誘導体を挙げることができる。 (3) Preparation of EPHA2 EPHA2, which is an antigen of an anti-EPHA2 antibody, can include a full-length EPHA2 polypeptide or a partial polypeptide thereof, more specifically, a full-length EPHA2 polypeptide, or preferably an EPHA2 cell. An outer region polypeptide (consisting of the amino acid sequence shown in amino acid Nos. 1 to 540 of SEQ ID No. 4 in the sequence listing), more preferably a polypeptide comprising the amino acid sequence shown in amino acid No. 426 to 540 of SEQ ID No. 4 in the sequence listing Peptides, and more preferably, a polypeptide containing the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing, or a derivative in which any amino acid sequence or carrier is added to these. Moreover, the polypeptide which consists of at least 6 continuous partial amino acid sequences of each said polypeptide, or the derivative | guide_body to which arbitrary amino acid sequences and carriers were added to these can be mentioned.
　なお、抗原となる、ＥＰＨＡ２全長ポリペプチド又はその部分ポリペプチドはＥＰＨＡ２遺伝子又はその部分ポリペプチドの遺伝子を遺伝子操作により宿主細胞に産生させることによって得ることができる。 Note that the EPHA2 full-length polypeptide or a partial polypeptide thereof serving as an antigen can be obtained by causing a host cell to produce an EPHA2 gene or a gene of the partial polypeptide thereof by genetic manipulation.
　ＥＰＨＡ２は、ヒトの腫瘍組織あるいは腫瘍細胞から直接精製して使用することができ、また、ＥＰＨＡ２全長ポリペプチド又はその部分ポリペプチドはｉｎ　ｖｉｔｒｏにて合成する、あるいは遺伝子操作により宿主細胞に産生させることによって得ることができる。 EPHA2 can be directly purified from human tumor tissue or tumor cells, and EPHA2 full-length polypeptide or a partial polypeptide thereof can be synthesized in vitro or produced in a host cell by genetic manipulation. Can be obtained by:
　遺伝子操作では、具体的には、ＥＰＨＡ２又はその部分ポリペプチドを発現可能なベクターに組み込んだ後、転写と翻訳に必要な酵素、基質及びエネルギー物質を含む溶液中で合成する、あるいは他の原核生物、又は真核生物の宿主細胞を形質転換させることによってＥＰＨＡ２又はその部分ポリペプチドを発現させることにより、該蛋白質を得ることができる。 Specifically, in genetic manipulation, EPHA2 or a partial polypeptide thereof is incorporated into a vector capable of expression, and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic organisms. Alternatively, the protein can be obtained by expressing EPHA2 or a partial polypeptide thereof by transforming a eukaryotic host cell.
　ＥＰＨＡ２のその部分ポリペプチドのｃＤＮＡは例えば、ＥＰＨＡ２を発現しているｃＤＮＡライブラリーを鋳型として、ＥＰＨＡ２　ｃＤＮＡ又はその部分ポリペプチドをコードするＤＮＡを特異的に増幅するプライマーを用いてポリメラーゼ連鎖反応（以下「ＰＣＲ」という）（Ｓａｉｋｉ，　Ｒ．　Ｋ．，　ｅｔ　ａｌ．　Ｓｃｉｅｎｃｅ（１９８８）２３９，ｐ．４８７－４８９参照）を行なう、いわゆるＰＣＲ法により取得することができる。 The cDNA of the partial polypeptide of EPHA2, for example, using a cDNA library expressing EPHA2 as a template, and using a primer that specifically amplifies the DNA encoding EPHA2 cDNA or the partial polypeptide (hereinafter referred to as polymerase chain reaction) (Referred to as “PCR”) (see Saiki, R. K., et al. Science (1988) 239, p. 487-489).
　ポリペプチドのイン・ビトロ（ｉｎ　ｖｉｔｒｏ）合成としては、例えばロシュ・ダイアグノスティックス社製のラピッドトランスレーションシステム（ＲＴＳ）が挙げられるが、これに限定されない。 Examples of in vitro synthesis of a polypeptide include, but are not limited to, a rapid translation system (RTS) manufactured by Roche Diagnostics.
　３．抗ＥＰＨＡ２抗体
（１）抗ＥＰＨＡ２モノクローナル抗体の取得
　抗ＥＰＨＡ２抗体であるＳＨ３４８－１はハイブリドーマＳＨ３４８－１より取得することができる。また、抗ＥＰＨＡ２抗体であるＳＨ３５７－１はハイブリドーマＳＨ３５７－１より取得することができる。ハイブリドーマＳＨ３４８－１及びハイブリドーマＳＨ３５７－１は日本国独立行政法人産業技術総合研究所特許生物寄託センター（住所：日本国茨城県つくば市東１－１－１中央第６）に２００７年６月８日付けで寄託され、ハイブリドーマＳＨ３４８－１はＳＨ３４８－１の名称で受託番号ＦＥＲＭ　ＢＰ－１０８３６が付与され、ハイブリドーマＳＨ３５７－１はＳＨ３５７－１の名称で受託番号ＦＥＲＭ　ＢＰ－１０８３７が付与されている。 3. Anti-EPHA2 Antibody (1) Acquisition of Anti-EPHA2 Monoclonal Antibody SH348-1 which is an anti-EPHA2 antibody can be obtained from hybridoma SH348-1. Moreover, SH357-1 which is an anti-EPHA2 antibody can be obtained from the hybridoma SH357-1. Hybridoma SH348-1 and Hybridoma SH357-1 are dated June 8, 2007 to Japan National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan) The hybridoma SH348-1 is assigned the deposit number FERM BP-10836 under the name SH348-1, and the hybridoma SH357-1 is assigned the deposit number FERM BP-10837 under the name SH357-1.
　また、ＳＨ３４８－１の重鎖のヌクレオチド配列は配列表の配列番号５に、アミノ酸配列は配列番号６に記載されている。ＳＨ３４８－１の軽鎖のヌクレオチド配列は配列表の配列番号７に、アミノ酸配列は配列番号８に記載されている。ＳＨ３４８－１の重鎖可変領域のヌクレオチド配列は配列表の配列番号９に、アミノ酸配列は配列番号１０に記載されている。ＳＨ３４８－１の重鎖定常領域のヌクレオチド配列は配列表の配列番号１１に、アミノ酸配列は配列番号１２に記載されている。ＳＨ３４８－１の軽鎖可変領域のヌクレオチド配列は配列表の配列番号１３に、アミノ酸配列は配列番号１４に記載されている。ＳＨ３４８－１の軽鎖定常領域のヌクレオチド配列は配列表の配列番号１５に、アミノ酸配列は配列番号１６に記載されている。ＳＨ３４８－１のＣＤＲＨ１のヌクレオチド配列は配列表の配列番号１７に、アミノ酸配列は配列番号１８に、ＣＤＲＨ２のヌクレオチド配列は配列番号１９に、アミノ酸配列は配列番号２０に、ＣＤＲＨ３のヌクレオチド配列は配列番号２１に、アミノ酸配列は配列番号２２に記載されている。ＳＨ３４８－１のＣＤＲＬ１のヌクレオチド配列は配列表の配列番号２３に、アミノ酸配列は配列番号２４に、ＣＤＲＬ２のヌクレオチド配列は配列番号２５に、アミノ酸配列は配列番号２６に、ＣＤＲＬ３のヌクレオチド配列は配列番号２７に、アミノ酸配列は配列番号２８に記載されている。 The nucleotide sequence of the heavy chain of SH348-1 is described in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 6. The nucleotide sequence of the light chain of SH348-1 is set forth in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 8. The nucleotide sequence of the heavy chain variable region of SH348-1 is set forth in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 10. The nucleotide sequence of the heavy chain constant region of SH348-1 is set forth in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 12. The nucleotide sequence of the light chain variable region of SH348-1 is shown in SEQ ID NO: 13 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 14. The nucleotide sequence of the light chain constant region of SH348-1 is shown in SEQ ID NO: 15 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 16. The nucleotide sequence of CDRH1 of SH348-1 is SEQ ID NO: 17, the amino acid sequence is SEQ ID NO: 18, the nucleotide sequence of CDRH2 is SEQ ID NO: 19, the amino acid sequence is SEQ ID NO: 20, and the nucleotide sequence of CDRH3 is SEQ ID NO: In 21, the amino acid sequence is set forth in SEQ ID NO: 22. The nucleotide sequence of CDRL1 of SH348-1 is SEQ ID NO: 23, the amino acid sequence is SEQ ID NO: 24, the nucleotide sequence of CDRL2 is SEQ ID NO: 25, the amino acid sequence is SEQ ID NO: 26, and the nucleotide sequence of CDRL3 is SEQ ID NO: 27, the amino acid sequence is set forth in SEQ ID NO: 28.
　また、ＳＨ３５７－１の重鎖のヌクレオチド配列は配列法の配列番号２９に、アミノ酸配列は配列番号３０に記載されている。ＳＨ３５７－１の軽鎖のヌクレオチド配列は配列表の配列番号３１に、アミノ酸配列は配列番号３２に記載されている。ＳＨ３５７－１の重鎖可変領域のヌクレオチド配列は配列表の配列番号３３に、アミノ酸配列は配列番号３４に記載されている。ＳＨ３５７－１の重鎖定常領域のヌクレオチド配列は配列表の配列番号３５に、アミノ酸配列は配列表の配列番号３６に記載されている。ＳＨ３５７－１の軽鎖可変領域のヌクレオチド配列は配列表の配列番号３７に、アミノ酸配列は配列番号３８に記載されている。ＳＨ３５７－１の軽鎖定常領域のヌクレオチド配列は配列表の配列番号３９に、アミノ酸配列は配列番号４０に記載されている。ＳＨ３５７－１のＣＤＲＨ１のヌクレオチド配列は配列表の配列番号４１に、アミノ酸配列は配列番号４２に、ＣＤＲＨ２のヌクレオチド配列は配列番号４３に、アミノ酸配列は配列番号４４に、ＣＤＲＨ３のヌクレオチド配列は配列番号４５に、アミノ酸配列は配列番号４６に記載されている。ＳＨ３５７－１のＣＤＲＬ１のヌクレオチド配列は配列表の配列番号４７に、アミノ酸配列は配列番号４８に、ＣＤＲＬ２のヌクレオチド配列は配列番号４９に、アミノ酸配列は配列番号５０に、ＣＤＲＬ３のヌクレオチド配列は配列番号５１に、アミノ酸配列は配列番号５２に記載されている。 The nucleotide sequence of the heavy chain of SH357-1 is described in SEQ ID NO: 29 of the sequencing method, and the amino acid sequence is described in SEQ ID NO: 30. The nucleotide sequence of the light chain of SH357-1 is set forth in SEQ ID NO: 31 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 32. The nucleotide sequence of the heavy chain variable region of SH357-1 is set forth in SEQ ID NO: 33 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 34. The nucleotide sequence of the heavy chain constant region of SH357-1 is set forth in SEQ ID NO: 35 in the Sequence Listing, and the amino acid sequence is set forth in SEQ ID NO: 36 in the Sequence Listing. The nucleotide sequence of the light chain variable region of SH357-1 is set forth in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 38. The nucleotide sequence of the light chain constant region of SH357-1 is set forth in SEQ ID NO: 39 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 40. The nucleotide sequence of CDR357 of SH357-1 is SEQ ID NO: 41, the amino acid sequence is SEQ ID NO: 42, the nucleotide sequence of CDRH2 is SEQ ID NO: 43, the amino acid sequence is SEQ ID NO: 44, and the nucleotide sequence of CDRH3 is SEQ ID NO: At 45, the amino acid sequence is set forth in SEQ ID NO: 46. The nucleotide sequence of CDRL1 of SH357-1 is SEQ ID NO: 47, the amino acid sequence is SEQ ID NO: 48, the nucleotide sequence of CDRL2 is SEQ ID NO: 49, the amino acid sequence is SEQ ID NO: 50, and the nucleotide sequence of CDRL3 is SEQ ID NO: In 51, the amino acid sequence is set forth in SEQ ID NO: 52.
　（２）抗ＥＰＨＡ２ヒト化スワップド抗体の取得
　抗ＥＰＨＡ２ヒト化スワップド抗体の例としては、ＳＨ３４８－１由来のヒト化重鎖及びＳＨ３５７－１由来のヒト化軽鎖を有するヒト化抗体を挙げることができる。 (2) Acquisition of anti-EPHA2 humanized swapped antibody Examples of anti-EPHA2 humanized swapped antibodies include humanized antibodies having a humanized heavy chain derived from SH348-1 and a humanized light chain derived from SH357-1. it can.
　抗ＥＰＨＡ２ヒト化スワップド抗体としては、上記スワップド抗体において、重鎖及び軽鎖をヒト化した抗体を挙げることができ、ヒト化抗体としては、相補性決定領域（ＣＤＲ；ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）のみをヒト由来の抗体に組み込んだ抗体（Ｎａｔｕｒｅ（１９８６）３２１，ｐ．５２２－５２５参照）、ＣＤＲ移植法によって、ＣＤＲの配列に加え一部のフレームワークのアミノ酸残基もヒト抗体に移植した抗体（ＷＯ９０／０７８６１号、ＵＳ６９７２３２３号公報参照）を挙げることができる。 Examples of the anti-EPHA2 humanized swapped antibody include antibodies in which the heavy chain and the light chain are humanized in the above swapped antibody. As a humanized antibody, only a complementarity determining region (CDR) is human. An antibody incorporated in an antibody derived from the antibody (see Nature (1986) 321, p.522-525), an antibody in which some framework amino acid residues in addition to the CDR sequence are grafted to a human antibody by the CDR grafting method (WO90 No. 077861, US Pat. No. 6,972,323).
　抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片の具体例としては、配列表の配列番号１８に示されるアミノ酸配列からなるＣＤＲＨ１、配列表の配列番号２０に示されるアミノ酸配列からなるＣＤＲＨ２及び配列表の配列番号２２に示されるアミノ酸配列からなるＣＤＲＨ３を含むことからなる重鎖、並びに、配列表の配列番号４８に示されるアミノ酸配列からなるＣＤＲＬ１、配列表の配列番号５０に示されるアミノ酸配列からなるＣＤＲＬ２及び配列表の配列番号５２に示されるアミノ酸配列からなるＣＤＲＬ３を含むことからなる軽鎖からなり、配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むペプチドを特異的に認識する抗体又は該抗体の機能性断片を挙げることができる。 Specific examples of the anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody include CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a sequence. From the heavy chain comprising CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing, the CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, and the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing A peptide comprising an amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing, and comprising a light chain comprising CDRL3 comprising the amino acid sequence represented by SEQ ID NO: 52 in the sequence listing Or a functional fragment of the antibody.
　抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片の他の具体例としては、配列表の配列番号５４に示されるアミノ酸配列からなる重鎖可変領域を含む重鎖及び配列番号６６に示されるアミノ酸配列からなる軽鎖可変領域を含む軽鎖からなる抗体、配列表の配列番号５４に示されるアミノ酸配列からなる重鎖可変領域を含む重鎖及び配列表の配列番号６８に示されるアミノ酸配列からなる軽鎖可変領域を含む軽鎖、配列表の配列番号５６に示されるアミノ酸配列からなる重鎖可変領域を含む重鎖及び配列番号６６に示されるアミノ酸配列からなる軽鎖可変領域を含む軽鎖からなる抗体、配列表の配列番号５６に示されるアミノ酸配列からなる重鎖可変領域を含む重鎖及び配列番号６８に示されるアミノ酸配列からなる軽鎖可変領域を含む軽鎖からなる抗体、配列表の配列番号５３に示されるアミノ酸配列からなる重鎖及び配列番号６５に示される軽鎖からなる抗体、配列表の配列番号５３に示される重鎖及び配列番号６７に示される軽鎖からなる抗体、配列表の配列番号５５に示される重鎖及び配列番号６５に示される軽鎖からなる抗体、配列表の配列番号５５に示される重鎖及び配列番号６７に示される軽鎖からなる抗体、並びに、該抗体の機能性断片から選択される、少なくともいずれか一つを挙げることができる。また、上記抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片のアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列からなるものも本発明の抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片に含まれる。 Other specific examples of the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody include a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 54 of the Sequence Listing and the amino acid shown in SEQ ID NO: 66 An antibody comprising a light chain comprising a light chain variable region comprising a sequence, a heavy chain comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 54 in the sequence listing, and an amino acid sequence represented by SEQ ID NO: 68 in the sequence listing A light chain comprising a light chain variable region, a heavy chain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 56 of the Sequence Listing, and a light chain comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66 A heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 56 of the sequence listing and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68 An antibody comprising a light chain, a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 53 in the sequence listing and an antibody comprising a light chain represented by SEQ ID NO: 65, a heavy chain represented by SEQ ID NO: 53 in the sequence listing and SEQ ID NO: 67 An antibody consisting of the light chain shown in SEQ ID NO: 55, an antibody consisting of the heavy chain shown in SEQ ID NO: 55 and the light chain shown in SEQ ID NO: 65, the heavy chain shown in SEQ ID NO: 55 and the SEQ ID NO: 67 At least one selected from an antibody comprising a light chain and a functional fragment of the antibody. In addition, the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody may comprise an amino acid sequence in which one or several amino acids are deleted, substituted, or added. It is contained in an antibody or a functional fragment of the antibody.
　本発明の抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片は、ＥＰＨＡ２を特定的に認識し、好ましくは、ＥＰＨＡ２の細胞外領域を特異的に認識し、更に好ましくは、配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むポリペプチド、更により好ましくは配列表の配列番号４のアミノ酸番号４３９乃至５３４に示されるアミノ酸配列を含むポリペプチドを特異的に認識する。 The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention specifically recognizes EPHA2, preferably specifically recognizes the extracellular region of EPHA2, and more preferably, SEQ ID NO: in the sequence listing. 4 specifically, a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540, even more preferably a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing is specifically recognized.
　本発明の抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片は、配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むポリペプチドに対してＫｄ値として１．５ｎＭ以下、好ましくは１．０ｎＭ以下、更に好ましくは０．５ｎＭ以下である。 The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention has a Kd value of 1.5 nM or less, preferably 1 with respect to the polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4. 0.0 nM or less, more preferably 0.5 nM or less.
 
　（３）抗体の製造
　遺伝子組換え技術により、上記ヒト化スワップド抗体の重鎖及び軽鎖の各々をコードするｃＤＮＡ、好ましくは該ｃＤＮＡを含むベクターにより真核細胞を形質転換し、遺伝子組換えヒトモノクローナル抗体を産生する形質転換細胞を培養することにより、この抗体を培養上清中から得ることができる。
(3) Production of antibody By means of gene recombination technology, eukaryotic cells are transformed with a cDNA encoding each of the heavy chain and light chain of the above humanized swapped antibody, preferably a vector containing the cDNA, and a gene recombinant human This antibody can be obtained from the culture supernatant by culturing transformed cells that produce a monoclonal antibody.
　ここで、宿主としては例えば真核細胞、好ましくはＣＨＯ細胞、リンパ球やミエローマ等の哺乳動物細胞を用いることができる。 Here, as the host, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myeloma can be used.
　抗体遺伝子を一旦単離した後、適当な宿主に導入して抗体を作製する場合には、適当な宿主と発現ベクターの組み合わせを使用することができる。 When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
　真核細胞を宿主として使用する場合、動物細胞、植物細胞、真核微生物を用いることができる。 When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.
　動物細胞としては、（１）哺乳類細胞、例えば、サルの細胞であるＣＯＳ細胞（Ｇｌｕｚｍａｎ，　Ｙ．　Ｃｅｌｌ（１９８１）２３，ｐ．１７５－１８２、ＡＴＣＣ　ＣＲＬ－１６５０）、マウス繊維芽細胞ＮＩＨ３Ｔ３（ＡＴＣＣ　Ｎｏ．ＣＲＬ－１６５８）やチャイニーズ・ハムスター卵巣細胞（ＣＨＯ細胞、ＡＴＣＣ　ＣＣＬ－６１）のジヒドロ葉酸還元酵素欠損株（Ｕｒｌａｕｂ，Ｇ．ａｎｄ　Ｃｈａｓｉｎ，Ｌ．Ａ．　Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．（１９８０）７７，ｐ．４１２６－４２２０）を挙げることができる。 Examples of animal cells include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblasts NIH3T3 (ATCC) No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220).
　また、これらの宿主としては、糖鎖構造が改変され、抗体のＡＤＣＣ活性（抗体依存性細胞障害活性）やＣＤＣ活性が高められた抗体を発現するように改変された宿主を用いることもでき、そのような宿主としては、抗体のＦｃ領域に結合するＮ－グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ－アセチルグルコサミンにフコースが結合していない糖鎖の割合が２０％以上である抗体組成物を産生する抗体分子をコードする遺伝子を導入されたＣＨＯ細胞を挙げることができる（ＷＯ０２／３１１４０号参照。）
　原核細胞を使用する場合は、例えば、大腸菌、枯草菌を挙げることができる。 In addition, as these hosts, a host modified to express an antibody in which the sugar chain structure is modified and the ADCC activity (antibody-dependent cytotoxic activity) or CDC activity of the antibody is increased can be used. As such a host, among N-glycoside-bonded complex sugar chains that bind to the Fc region of an antibody, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more. A CHO cell into which a gene encoding an antibody molecule that produces an antibody composition has been introduced can be mentioned (see WO02 / 31140).
When prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be mentioned.
　これらの細胞に、目的とする抗体遺伝子を形質転換により導入し、形質転換された細胞をｉｎ　ｖｉｔｒｏで培養することにより抗体が得られる。 An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.
　抗体のアイソタイプとしては制限はなく、例えばＩｇＧ（ＩｇＧ１，ＩｇＧ２，ＩｇＧ３，ＩｇＧ４）、ＩｇＭ、ＩｇＡ（ＩｇＡ１，ＩｇＡ２）、ＩｇＤあるいはＩｇＥ等が挙げられるが、好ましくはＩｇＧまたはＩｇＭを挙げることができる。 The isotype of the antibody is not limited and includes, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE, and preferably IgG or IgM.
　また本発明には、上記抗体に加え、該抗体の機能性断片であってもよい。抗体の機能性断片の例としては、Ｆａｂ、Ｆ（ａｂ’）２、ｓｃＦｖ等を含む。また、Ｆ（ａｂ’）２を還元条件下で処理した抗体の可変領域の一価の断片であるＦａｂ’も抗体の機能性断片に含まれる。但し、抗原との結合能を有している限りこれらの分子に限定されない。また、これらの機能性断片には、抗体蛋白質の全長分子を適当な酵素で処理したもののみならず、遺伝子工学的に改変された抗体遺伝子を用いて適当な宿主細胞において産生された蛋白質も含まれる。 In the present invention, in addition to the above antibody, a functional fragment of the antibody may be used. Examples of functional fragments of antibodies include Fab, F (ab ') 2, scFv and the like. Further, Fab ', which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody. However, the molecule is not limited to these molecules as long as it has the ability to bind to an antigen. These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.
　抗体の一例である一本鎖抗体（ｓｃＦｖとも記載する）は、抗体の重鎖Ｖ領域と軽鎖Ｖ領域とをポリペプチドのリンカーで連結することにより得られる（Ｐｌｕｃｋｔｈｕｎ，Ｔｈｅ　Ｐｈａｒｍａｃｏｌｏｇｙ　ｏｆ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ，１１３（Ｒｏｓｅｎｂｕｒｇ及びＭｏｏｒｅ編、Ｓｐｒｉｎｇｅｒ　Ｖｅｒｌａｇ，Ｎｅｗ　Ｙｏｒｋ，ｐ．２６９－３１５（１９９４），Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５），２３，ｐ．１１２６－１１３６）。 A single chain antibody (also referred to as scFv), which is an example of an antibody, is obtained by linking a heavy chain V region and a light chain V region of an antibody with a linker of a polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenburg and Moore, Ed. Springer Verlag, New York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136).
　一本鎖抗体を作成する方法は当技術分野において周知である（例えば、米国特許第４，９４６，７７８号、米国特許第５，２６０，２０３号、米国特許第５，０９１，５１３号、米国特許第５，４５５，０３０号等を参照）。このｓｃＦｖにおいて、重鎖Ｖ領域と軽鎖Ｖ領域は、コンジュゲートを作らないようなリンカー、好ましくはポリペプチドリンカーを介して連結される（Ｈｕｓｔｏｎ，Ｊ．Ｓ．ｅｔ　ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．（１９８８），８５，ｐ．５８７９－５８８３）。ｓｃＦｖにおける重鎖Ｖ領域および軽鎖Ｖ領域は、同一の抗体に由来してもよく、別々の抗体に由来してもよい。 Methods for making single chain antibodies are well known in the art (eg, US Pat. No. 4,946,778, US Pat. No. 5,260,203, US Pat. No. 5,091,513, US (See Japanese Patent No. 5,455,030). In this scFv, the heavy chain V region and the light chain V region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci.U.S.A. (1988), 85, p.5879-5883). The heavy chain V region and the light chain V region in scFv may be derived from the same antibody or different antibodies.
　Ｖ領域を連結するペプチドリンカーとしては、例えば１２～１９残基からなる任意の一本鎖ペプチドが用いられる。 As the peptide linker that links the V regions, for example, any single chain peptide consisting of 12 to 19 residues is used.
　ｓｃＦｖをコードするＤＮＡは、前記抗体の重鎖または重鎖Ｖ領域をコードするＤＮＡ、および軽鎖または軽鎖Ｖ領域をコードするＤＮＡのうち、それらの配列のうちの全部又は所望のアミノ酸配列をコードするＤＮＡ部分を鋳型とし、その両端を規定するプライマー対を用いてＰＣＲ法により増幅し、次いで、さらにペプチドリンカー部分をコードするＤＮＡ、およびその両端が各々重鎖、軽鎖と連結されるように規定するプライマー対を組み合わせて増幅することにより得られる。 The DNA encoding scFv is the DNA encoding the heavy chain or heavy chain V region of the antibody, and the DNA encoding the light chain or light chain V region. Amplification is performed by PCR using a primer pair that defines both ends of the encoding DNA portion as a template, and then the DNA that encodes the peptide linker portion, and both ends thereof are connected to the heavy chain and light chain, respectively. Obtained by combining and amplifying the primer pairs defined in 1.
　また、一旦ｓｃＦｖをコードするＤＮＡが作製されると、それらを含有する発現ベクター、および該発現ベクターにより形質転換された宿主を常法に従って得ることができ、また、その宿主を用いることにより、常法に従ってｓｃＦｖを得ることができる。 In addition, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.
　これらの抗体断片は、前記と同様にして遺伝子を取得し発現させ、宿主により産生させることができる。 These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above.
　さらに、抗体の例としては少なくとも２種類の異なる抗原に対して特異性を有する多特異性抗体であってもよい。 Furthermore, as an example of an antibody, it may be a multispecific antibody having specificity for at least two different antigens.
　通常このような分子は２個の抗原を結合するものであるが（即ち、二重特異性抗体（ｂｉｓｐｅｃｉｆｉｃ　ａｎｔｉｂｏｄｙ））、本発明における「多特異性抗体」は、それ以上（例えば、３種類）の抗原に対して特異性を有する抗体を包含するものである。 Usually, such a molecule binds two antigens (ie, bispecific antibody), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.
　本発明の抗体は、多特異性抗体は全長からなる抗体、またはそのような抗体の断片（例えば、Ｆ（ａｂ’）２二特異性抗体）でもよい。二重特異性抗体は２種類の抗体の重鎖と軽鎖（ＨＬ対）を結合させて作製することもできるし、異なるモノクローナル抗体を産生するハイブリドーマを融合させて、二重特異性抗体産生融合細胞を作製することによっても、作製することができる（Ｍｉｌｌｓｔｅｉｎ　ｅｔ　ａｌ．，Ｎａｔｕｒｅ（１９８３）３０５，ｐ．５３７－５３９）。 The antibody of the present invention may be a full-length multispecific antibody or a fragment of such an antibody (for example, F (ab ') 2 bispecific antibody). Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by hybridizing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).
　抗体の例としては、アミノ酸配列が異なる複数種類の抗ＥＰＨＡ２抗体の混合物である、ポリクローナル抗体であってもよい。ポリクローナル抗体の一例としては、ＣＤＲが異なる複数種類の抗体の混合物を挙げることができる。そのようなポリクローナル抗体としては、異なる抗体を産生する細胞の混合物を培養し、該培養物から精製された抗体を用いることができる（ＷＯ２００４／０６１１０４号参照）。 An example of an antibody may be a polyclonal antibody that is a mixture of a plurality of types of anti-EPHA2 antibodies having different amino acid sequences. As an example of the polyclonal antibody, a mixture of plural kinds of antibodies having different CDRs can be mentioned. As such a polyclonal antibody, a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO 2004/061104).
　抗体の修飾物として、ポリエチレングリコール（ＰＥＧ）等の各種分子と結合した抗体を使用することもできる。 As the modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
　抗体の例としては、更にこれらの抗体と他の薬剤とコンジュゲートを形成しているもの（Ｉｍｍｕｎｏｃｏｎｊｕｇａｔｅ）でもよい。このような抗体の例としては、該抗体が放射性物質や薬理作用を有する化合物と結合している物を挙げることができる（Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５）２３，ｐ．１１３７－１１４６）。 Examples of antibodies may further include those that form conjugates with these antibodies and other drugs (Immunoconjugate). Examples of such an antibody include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).
　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、精製は通常の蛋白質で使用されている分離、精製方法を使用すればよい。 The obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins.
　例えばクロマトグラフィーカラム、フィルター、限外濾過、塩析、透析、調製用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、組み合わせれば、抗体を分離、精製することができる（Ｓｔｒａｔｅｇｉｅｓ　ｆｏｒ　Ｐｒｏｔｅｉｎ　Ｐｕｒｉｆｉｃａｔｉｏｎ　ａｎｄ　Ｃｈａｒｃｔｅｒｉｚａｔｉｏｎ：Ａ　Ｌａｂｏｒａｔｏｙ　Ｃｏｕｒｓｅ　Ｍａｎｕａｌ，Ｄａｎｉｅｌ　Ｒ．Ｍａｒｓｈａｋ　ｅｔ　ａｌ．ｅｄｓ．，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９９６）；Ａｎｔｉｂｏｄｉｅｓ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ．Ｅｄ　Ｈａｒｌｏｗ　ａｎｄ　Ｄａｖｉｄ　Ｌａｎｅ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８））が、これらに限定されるものではない。 For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (198 )) It is not intended to be limited thereto.
　クロマトグラフィーとしては、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過、逆相クロマトグラフィー、吸着クロマトグラフィー等が挙げられる。 Chromatography includes affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like.
　これらのクロマトグラフィーは、ＨＰＬＣやＦＰＬＣ等の液相クロマトグラフィーを用いて行うことができる。 These chromatography can be performed using liquid phase chromatography such as HPLC and FPLC.
　アフィニティークロマトグラフィーに用いるカラムとしては、プロテインＡカラム、プロテインＧカラムが挙げられる。 Columns used for affinity chromatography include protein A columns and protein G columns.
　例えばプロテインＡカラムを用いたカラムとして、Ｈｙｐｅｒ　Ｄ，ＰＯＲＯＳ，Ｓｅｐｈａｒｏｓｅ　Ｆ．Ｆ．（Ｐｈａｒｍａｃｉａ）等が挙げられる。 For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.
　また抗原を固定化した担体を用いて、抗原への結合性を利用して抗体を精製することも可能である。 It is also possible to purify the antibody using the antigen-immobilized carrier using the binding property to the antigen.
　４．抗ＥＰＨＡ２ヒト化スワップド抗体の性質
　本発明の抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片は以下（１）乃至（５）の少なくともいずれか１つの性質を有する。
（１）配列表の配列番号８のアミノ酸番号４２６乃至５３４に示されるアミノ酸配列からなるポリペプチドに特異的に結合し、下記のａ）乃至ｅ）から選択される少なくともいずれか一つの性質を有する。
ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない。
ｂ）ＥＰＨＡ２蛋白質量の減少作用を示さない。
ｃ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する。
ｄ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する。
ｅ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。
（２）配列表の配列番号８のアミノ酸番号４２６乃至５３４に示されるアミノ酸配列からなるポリペプチドに特異的に結合し、下記のａ）乃至ｅ）から選択される少なくともいずれか２つ以上の性質を有する。
ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない。
ｂ）ＥＰＨＡ２蛋白質量の減少作用を示さない。
ｃ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する。
ｄ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する。
ｅ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。
（３）配列表の配列番号８のアミノ酸番号４２６乃至５３４に示されるアミノ酸配列からなるポリペプチドに特異的に結合し、下記のａ）乃至ｅ）の性質を有する。
ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない。
ｂ）ＥＰＨＡ２蛋白質量の減少作用を示さない。
ｃ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する。
ｄ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する。
ｅ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。 4). Properties of anti-EPHA2 humanized swapped antibody The anti-EPHA2 humanized swapped antibody of the present invention or a functional fragment of the antibody has at least one of the following properties (1) to (5).
(1) It specifically binds to a polypeptide consisting of the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing, and has at least one property selected from the following a) to e) .
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.
(2) at least any two or more properties selected from the following a) to e), which specifically bind to a polypeptide having the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing Have
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.
(3) It specifically binds to a polypeptide consisting of the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing, and has the following properties a) to e).
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.
　ここで、抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片によるＥＰＨＡ２チロシン残基のリン酸化能は、例えば、ＷＯ２００９／０２８６３９の（実施例３）３）－１－１～３）－１－２に記載の方法によって測定することができる。すなわち、ＭＤＡ－ＭＢ－２３１細胞を適当な条件下で培養し、抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体、ＥＰＨＡ２の可溶性リガンドとしてＲｅｃｏｍｂｉｎａｎｔ　Ｍｏｕｓｅ　Ｅｐｈｒｉｎ－Ａ１／Ｆｃ　Ｃｈｉｍｅｒａ（以下「Ｅｐｈｒｉｎ－Ａ１／Ｆｃ」と略す；Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社製：＃６０２－Ａ１－２００）を適当な濃度にＲＰＭＩ１６４０で希釈した溶液を、培地を捨てたＭＤＡ－ＭＢ－２３１細胞に添加し、３７℃、５％　ＣＯ２の条件下で指定の時間インキュベーションする。 Here, the phosphorylation ability of EPHA2 tyrosine residues by the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody is, for example, (Example 3) 3) -1-1 to 3) -1- of WO2009 / 028639 It can be measured by the method described in 2. That is, MDA-MB-231 cells are cultured under appropriate conditions, and an anti-EPHA2 humanized swapped antibody or the antibody, as a soluble ligand of EPHA2, Recombinant Mouse Ephrin-A1 / Fc Chimera (hereinafter referred to as “Ephrin-A1 / Fc”). Abbreviated; R & D Systems (# 602-A1-200) diluted to an appropriate concentration with RPMI 1640 was added to MDA-MB-231 cells discarded with the medium, at 37 ° C. under 5% CO 2 conditions. Incubate for the specified time.
　ＥＰＨＡ２を免疫沈降するため、まず１サンプル当たり２５μｌのＰｒｏｔｅｉｎ　Ｇ　ｍａｇｎｅｔｉｃ　ｂｅａｄｓ懸濁液（ＮＥＷ　ＥＮＧＬＡＮＤ　Ｂｉｏ－Ｌａｂｓ社製）に８μｇのａｎｔｉ－Ｅｃｋ／ＥｐｈＡ２，ｃｌｏｎｅ　Ｄ７（以下「抗ＥＰＨＡ２抗体（Ｄ７）」と略す：Ｍｉｌｌｉｐｏｒｅ（Ｕｐｓｔａｔｅ）社製　＃０５－４８０）を加え、４℃で２時間転倒混和する。 In order to immunoprecipitate EPHA2, first, 8 μg of anti-Eck / EphA2, Clone D7 (hereinafter referred to as “anti-EPHA2 antibody (D7)”) was added to 25 μl of Protein G magnetic beads suspension (NEW ENGLAND Bio-Labs) per sample. Abbreviation: Millipore (Upstate) # 05-480) is added and mixed by inversion at 4 ° C. for 2 hours.
　その後、終濃度で１０％になるようＦＢＳを加え、さらに４℃で３０分間転倒混和する。ビーズを１ｍＭ　ＰＭＳＦ含有１×Ｃｅｌｌ　Ｌｙｓｉｓ　Ｂｕｆｆｅｒで３回洗浄したあと、上記で調製した細胞溶解液上清を２００μｇ加え、４℃で一晩転倒混和する。翌日、ビーズを１ｍＭ　ＰＭＳＦ含有１×Ｃｅｌｌ　Ｌｙｓｉｓ　Ｂｕｆｆｅｒで３回洗浄した後、ビーズにＳＤＳ－ｓａｍｐｌｅ　ｂｕｆｆｅｒ（５６．３ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ６．８、１．８％（ｗ／ｖ）　ＳＤＳ、９％　ｇｌｙｃｅｒｏｌ、０．７２Ｍ　２‐メルカプトエタノール、０．０４５ｍｇ／ｍｌ　ブロモフェノールブルー）を加えて９８℃で５分間過熱し、ビーズから解離した蛋白質をＳＤＳ－ＰＡＧＥで分離する。 Then, add FBS so that the final concentration is 10%, and mix by inverting at 4 ° C. for 30 minutes. After washing the beads 3 times with 1 × Cell Lysis Buffer containing 1 mM PMSF, 200 μg of the cell lysate supernatant prepared above is added and mixed by inversion at 4 ° C. overnight. The next day, the beads were washed with 1 mM PMSF-containing 1 × Cell Lysis Buffer three times, and then the beads were washed with SDS-sample buffer (56.3 mM Tris-HCl pH 6.8, 1.8% (w / v) SDS, 9% glycerol. , 0.72M 2-mercaptoethanol, 0.045 mg / ml bromophenol blue) and heated at 98 ° C. for 5 minutes, and the protein dissociated from the beads is separated by SDS-PAGE.
　分離後のゲルから蛋白質をポリビニリデンジフルオリド膜（以下「ＰＶＤＦ膜」と略す；ポアサイズ　０．４５μｍ：Ｍｉｌｌｉｐｏｒｅ社製）に転写する。転写後のＰＶＤＦ膜をブロッキング液（ブロックエース粉末（大日本住友製薬（雪印乳業）社製）１袋を１００ｍｌの超純水に溶解後、Ｔｗｅｅｎ　２０、アジ化ナトリウムをそれぞれ終濃度で０．１％（ｖ／ｖ）、０．０２％（ｗ／ｖ）になるよう添加）中で振とうし、ブロッキングする。初めに、免疫沈降されたＥＰＨＡ２を検出するため、ブロッキング後のＰＶＤＦ膜をブロッキング液で０．２５μｇ／ｍｌに希釈した抗ＥＰＨＡ２抗体溶液に浸し、室温で１時間振とうする。ＰＶＤＦ膜をＴＢＳＴ（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０、１３８ｍＭ　ＮａＣｌ、２．７ｍＭ　ＫＣｌ、０．１％（ｖ／ｖ）　Ｔｗｅｅｎ　２０）で１０分間、３回洗浄した後、ＴＢＳＴで３０００倍に希釈したＡｎｔｉ－Ｍｏｕｓｅ　Ｉｇ，ＨＲＰ－Ｌｉｎｋｅｄ　Ｗｈｏｌｅ　Ａｂ　Ｓｈｅｅｐ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ－Ｓｃｉｅｎｃｅｓ社製）溶液に浸して室温で３０分間振とうする。さらにＰＶＤＦ膜をＴＢＳＴで１０分間、３回洗浄した後、ＥＣＬ　Ｐｌｕｓ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ－Ｓｃｉｅｎｃｅｓ社製）を使用し化学発光用フィルムでシグナルを検出する。 The protein is transferred from the gel after separation to a polyvinylidene difluoride membrane (hereinafter abbreviated as “PVDF membrane”; pore size 0.45 μm: manufactured by Millipore). The PVDF membrane after transfer was dissolved in a blocking solution (Block Ace Powder (Dainippon Sumitomo Pharma Co., Ltd., Snow Brand Milk Products)) in 100 ml of ultrapure water, and then Tween 20 and sodium azide were each added at a final concentration of 0.1. % (V / v), 0.02% (w / v) added) to block. First, in order to detect immunoprecipitated EPHA2, the PVDF membrane after blocking is immersed in an anti-EPHA2 antibody solution diluted to 0.25 μg / ml with a blocking solution and shaken at room temperature for 1 hour. The PVDF membrane was washed 3 times with TBST (50 mM Tris-HCl pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v / v) Tween 20) for 10 minutes, and then diluted 3000 times with TBST. Immerse in Anti-Mouse Ig, HRP-Linked Whole Ab Sheep (GE Healthcare Bio-Sciences) solution and shake at room temperature for 30 minutes. Further, the PVDF membrane is washed 3 times with TBST for 10 minutes, and then the signal is detected with a film for chemiluminescence using ECL Plus (GE Healthcare Bio-Sciences).
　次に、このＰＶＤＦ膜上から抗体を除去するため、ＰＶＤＦ膜をＳｔｒｉｐ液（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ６．８、２％（ｗ／ｖ）　ＳＤＳ、１００ｍＭ　２‐メルカプトエタノール）に浸して５５℃で３０分間振とうした後、Ｑｕｅｎｃｈ液（１％（ｖ／ｖ）　Ｈ２Ｏ２、０．１％（ｗ／ｖ）　ＮａＮ３含有ＴＢＳＴ）に浸して室温で２０分間振とう処理を行い、さらにＴＢＳＴで１０分間、３回洗浄する。ＥＰＨＡ２チロシン残基のリン酸化状態を検出するため、このＰＶＤＦ膜をアジ化ナトリウム不含ブロッキング液（ブロックエース粉末１袋を１００ｍｌの超純水に溶解後、Ｔｗｅｅｎ　２０を終濃度で０．１％（ｖ／ｖ）になるよう添加）中で振とうしてブロッキングした後、アジ化ナトリウム不含ブロッキング液で１００００倍に希釈したＡｎｔｉ－Ｐｈｏｓｐｈｏｔｙｒｏｓｉｎｅ，ｒｅｃｏｍｂｉｎａｎｔ　４Ｇ１０　ＨＲＰ－ｃｏｎｊｕｇａｔｅ（図中で「４Ｇ１０抗体」と略す；Ｍｉｌｌｉｐｏｒｅ　（Ｕｐｓａｔｅ）社製　＃１６－１８４）溶液に浸し室温で１時間振とうする。ＰＶＤＦ膜をＴＢＳＴで１０分間、３回洗浄後、さらにＨ２Ｏで５分間、３回洗浄し、ＥＣＬ　Ｐｌｕｓを使用し化学発光用フィルムでシグナルを検出する。 Next, in order to remove the antibody from the PVDF membrane, the PVDF membrane is immersed in a Strip solution (50 mM Tris-HCl pH 6.8, 2% (w / v) SDS, 100 mM 2-mercaptoethanol) at 55 ° C. for 30 minutes. After shaking for 30 minutes, it was immersed in Quench solution (TBST containing 1% (v / v) H 2 O 2 , 0.1% (w / v) NaN 3 ) for 20 minutes at room temperature. Wash 3 times for 10 minutes. In order to detect the phosphorylation state of the EPHA2 tyrosine residue, this PVDF membrane was dissolved in a sodium azide-free blocking solution (1 bag of Block Ace powder was dissolved in 100 ml of ultrapure water, and Tween 20 was added at a final concentration of 0.1%. Anti-Phosphotyrosine, recombinant 4G10 HRP-conjugate (“4G10 antibody” in the figure) diluted with a sodium azide-free blocking solution 10,000 times after blocking. Dipped in Millipore (Upsate) # 16-184) solution and shaken at room temperature for 1 hour. The PVDF membrane is washed 3 times with TBST for 10 minutes, then further washed 3 times with H 2 O for 5 minutes, and the signal is detected with a film for chemiluminescence using ECL Plus.
　また、ＥＰＨＡ２タンパク質量の減少作用は、例えば、ＷＯ２００９／０２８６３９の実施例３の３）－１－３に記載の方法によって測定することが出来る。すなわち、細胞溶解液上清をＳＤＳ－ＰＡＧＥで分離後、ゲル中の蛋白質をＰＶＤＦ膜に転写し、抗ＥＰＨＡ２抗体でウエスタンブロッティングを行うことによってＥＰＨＡ２タンパク質の量を定量することによってタンパク質の減少量を測定することが出来る。 The effect of reducing the amount of EPHA2 protein can be measured, for example, by the method described in Example 3) 3) -1-3 of WO2009 / 028639. That is, after the cell lysate supernatant was separated by SDS-PAGE, the protein in the gel was transferred to a PVDF membrane, and Western blotting was performed with an anti-EPHA2 antibody to quantify the amount of protein decrease. It can be measured.
　また、ＡＤＣＣ活性は、通常行われている方法で測定することが出来るが、例えば、本明細書の実施例８に記載の方法によって測定することが出来る。 Moreover, although ADCC activity can be measured by the method currently performed normally, it can be measured by the method as described in Example 8 of this specification, for example.
　また、ＣＤＣ活性は、当業者が通常行う方法で測定することが出来る。また、例えば、ＷＯ２００９／０２８６３９の実施例３の３）－３の記載の方法によって行うことができる。すなわち、１０％　ＦＢＳ含有ＲＰＭＩ１６４０（抗生物質入り）に懸濁したＭＤＡ－ＭＢ－２３１、Ａ５４９、ＰＣ－３細胞を９６穴マイクロプレートに各５０００細胞／ウェルで播種し、３７℃、５％　ＣＯ２の条件下で一晩培養する。翌日、補体添加後の終濃度で２５μｇ／ｍｌになるように１０％　ＦＢＳ含有ＲＰＭＩ１６４０（抗生物質入り）で希釈したＳＨ３４８－１、ＳＨ３５７－１、アイソタイプコントロール抗体（ｍＩｇＧ２ａ）を添加し、４℃で１時間静置する。そこにＲＰＭＩ１６４０で３０％に希釈したウサギ補体（ＣＥＤＡＲＬＡＮＥ社製　＃ＣＬ３０５１）を終濃度で５％になるよう添加し３７℃、５％　ＣＯ２の条件下で１時間インキュベートし、さらに室温で３０分間静置する。細胞生存率を測定するため、培養液と等量のＣｅｌｌＴｉｔｅｒ－Ｇｌｏ　Ｌｕｍｉｎｅｓｃｅｎｔ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ　Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製）を添加し、室温で１０分間攪拌した後、プレートリーダーで発光量を計測する。細胞生存率は次式で算出する。 Further, CDC activity can be measured by a method commonly used by those skilled in the art. For example, it can be performed by the method described in Example 3) 3) -3 of WO2009 / 028639. That is, MDA-MB-231, A549, and PC-3 cells suspended in RPMI 1640 containing 10% FBS (with antibiotics) were seeded in a 96-well microplate at 5000 cells / well each, at 37 ° C., 5% CO 2. Incubate overnight under the conditions of The following day, SH348-1, SH357-1, and isotype control antibody (mIgG2a) diluted with 10% FBS-containing RPMI 1640 (with antibiotics) were added to a final concentration of 25 μg / ml after complement addition, and 4 ° C. Leave for 1 hour. Rabbit complement (CEDARLANE # CL3051) diluted to 30% with RPMI 1640 was added thereto to a final concentration of 5%, incubated at 37 ° C. under 5% CO 2 for 1 hour, and further incubated at room temperature for 30 hours. Let stand for a minute. In order to measure the cell viability, CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) equivalent to the culture solution is added, and the mixture is stirred at room temperature for 10 minutes, and then the amount of luminescence is measured with a plate reader. Cell viability is calculated by the following formula.
　細胞生存率（％）＝（ａ－ｂ）／（ｃ－ｂ）×１００
　ａ：サンプルウェルの発光量
　ｂ：バックグラウンド（細胞・抗体非添加ウェル）発光量の平均値（ｎ＝８）。細胞播種時に細胞懸濁液の代わりに等量の１０％　ＦＢＳ含有ＲＰＭＩ１６４０（抗生物質入り）を添加し、抗体添加時に抗体希釈液と等量の１０％　ＦＢＳ含有ＲＰＭＩ１６４０（抗生物質入り）を添加する。それ以外はサンプルウェルと同様の操作を行う。 Cell viability (%) = (ab) / (cb) × 100
a: Amount of light emitted from the sample well b: Average value of the amount of light emitted from the background (well without cells / antibodies) (n = 8). An equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added instead of the cell suspension at the time of cell seeding, and an equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added to the antibody dilution at the time of antibody addition. . Otherwise, perform the same operation as the sample well.
　ｃ：抗体非添加ウェルの発光量の平均値（ｎ＝３）。抗体添加時に抗体希釈液と等量の１０％　ＦＢＳ含有ＲＰＭＩ１６４０（抗生物質入り）を添加する。それ以外はサンプルウェルと同様の操作を行う。 C: Average value of the amount of luminescence in wells without antibody added (n = 3). At the time of antibody addition, 10% FBS-containing RPMI 1640 (with antibiotics) is added in an amount equal to that of the antibody diluent. Otherwise, perform the same operation as the sample well.
　また、ｉｎ　ｖｉｖｏにおける抗腫瘍活性は例えば、腫瘍を有する実験動物に抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片を投与し、腫瘍の体積の変化を測定することによって確認することが出来る。例えば、本明細書の実施例１０に記載の方法によって測定することが出来る。 Moreover, the antitumor activity in vivo can be confirmed, for example, by administering an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody to an experimental animal having a tumor and measuring a change in the volume of the tumor. For example, it can be measured by the method described in Example 10 of the present specification.
　５．ＥＰＨＡ２ヒト化スワップド抗体を含有する医薬
　抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片は、医薬として特に癌の治療を目的とした医薬組成物として、あるいはこのような疾患の免疫学的診断のための抗体として有用である。 5. Pharmaceutical containing EPHA2 humanized swapped antibody Anti-EPHA2 humanized swapped antibody or a functional fragment of said antibody is useful as a pharmaceutical composition, especially for the treatment of cancer, or for immunodiagnosis of such diseases. It is useful as an antibody.
　抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片は医薬としては、ＥＰＨＡ２を発現している癌であれば全てが治療の対象になるが、癌の種類としては、例えば、乳癌、食道癌、前立腺癌、胃癌、非小細胞肺癌、大腸癌及び多形神経膠芽腫を好適に挙げることができるがこれらに限定されない。 The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody can be treated as a pharmaceutical as long as it is a cancer expressing EPHA2. Examples of the types of cancer include breast cancer, esophageal cancer, Prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer and glioblastoma multiforme can be preferably mentioned, but are not limited thereto.
　本発明は、治療に有効な量の抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片と薬学上許容される希釈剤、担体、可溶化剤、乳化剤、保存剤及び／又は補助剤を含む医薬組成物も提供する。 The present invention relates to a medicament comprising a therapeutically effective amount of an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Compositions are also provided.
　本発明の医薬組成物において許容される製剤に用いる物質としては好ましくは投与量や投与濃度において、医薬組成物を投与される者に対して非毒性のものが好ましい。 The substance used in the preparation acceptable in the pharmaceutical composition of the present invention is preferably a substance that is non-toxic to a person who is administered the pharmaceutical composition at a dosage or concentration.
　本発明の医薬組成物は、ｐＨ、浸透圧、粘度、透明度、色、等張性、無菌性、安定性、溶解率、徐放率、吸収率、浸透率を変えたり、維持したり、保持したりするための製剤用の物質を含むことができる。 The pharmaceutical composition of the present invention changes, maintains, or maintains pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, and penetration rate. Or a pharmaceutical substance for the preparation.
　製剤用の物質として以下のものを挙げることができるが、これらに制限されない；グリシン、アラニン、グルタミン、アスパラギン、ヒスチジン、アルギニン又はリジン等のアミノ酸類、抗菌剤、アスコルビン酸、硫酸ナトリウム又は亜硫酸水素ナトリウム等の抗酸化剤、リン酸、クエン酸、ホウ酸バッファー、炭酸水素、トリス－塩酸（Ｔｒｉｓ－Ｈｃｌ）溶液等の緩衝剤、マンニトールやグリシン等の充填剤、エチレンジアミン四酢酸（ＥＤＴＡ）等のキレート剤、カフェイン、ポリビニルピロリジン、β－シクロデキストリンやヒドロキシプロピル－β－シクロデキストリン等の錯化剤、グルコース、マンノース又はデキストリン等の増量剤、単糖類、二糖類やグルコース、マンノースやデキストリン等の他の炭水化物、着色剤、香味剤、希釈剤、乳化剤やポリビニルピロリジン等の親水ポリマー、低分子量ポリペプチド、塩形成対イオン、塩化ベンズアルコニウム、安息香酸、サリチル酸、チメロサール、フェネチルアルコール、メチルパラベン、プロピルパラベン、クロレキシジン、ソルビン酸又は過酸化水素等の防腐剤、グリセリン、プロピレン・グリコール又はポリエチレングリコール等の溶媒、マンニトール又はソルビトール等の糖アルコール、懸濁剤、ＰＥＧ、ソルビタンエステル、ポリソルビテート２０やポリソルビテート８０等ポリソルビテート、トリトン（ｔｒｉｔｏｎ）、トロメタミン（ｔｒｏｍｅｔｈａｍｉｎｅ）、レシチン又はコレステロール等の界面活性剤、スクロースやソルビトール等の安定化増強剤、塩化ナトリウム、塩化カリウムやマンニトール・ソルビトール等の弾性増強剤、輸送剤、希釈剤、賦形剤、及び／又は薬学上の補助剤。 Substances for formulation may include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulfite Antioxidants such as phosphate, citric acid, borate buffer, hydrogen carbonate, buffer such as tris-hydrochloric acid (Tris-Hcl) solution, filler such as mannitol and glycine, chelate such as ethylenediaminetetraacetic acid (EDTA) Agents, caffeine, polyvinylpyrrolidine, complexing agents such as β-cyclodextrin and hydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides and glucose, mannose and dextrin, etc. Carbohydrates, colorants, flavors Agents, diluents, emulsifiers and hydrophilic polymers such as polyvinylpyrrolidine, low molecular weight polypeptides, salt-forming counterions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid Preservatives such as hydrogen oxide, solvents such as glycerin, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspension agents, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Surfactants such as triton, tromethamine, lecithin or cholesterol, stabilization enhancers such as sucrose and sorbitol, sodium chloride, potassium chloride and mannini Elasticity enhancers such Lumpur sorbitol, transport agent, diluent, excipient, and / or pharmaceutically adjuvants.
　これらの製剤用の物質の添加量は、抗ＥＰＨＡ２ヒト化スワップド抗体又はその機能性断片の重量に対して０．０１～１００倍、特に０．１～１０倍添加するのが好ましい。 The addition amount of the substance for these preparations is preferably 0.01 to 100 times, particularly 0.1 to 10 times the weight of the anti-EPHA2 humanized swapped antibody or functional fragment thereof.
　なお、抗ＥＰＨＡ２ヒト化スワップド抗体をリポソーム中に含有するようにしたイムノリポソームや抗ＥＰＨＡ２抗体とリポソームを結合した抗体（米国特許第６２１４３８８号）を含有する医薬組成物も本発明に含まれる。 A pharmaceutical composition containing an immunoliposome containing an anti-EPHA2 humanized swapped antibody in the liposome or an antibody in which the anti-EPHA2 antibody and the liposome are bound (US Pat. No. 6,214,388) is also included in the present invention.
　製剤中の好適な医薬組成物の組成は当業者によって、適用疾患、適用投与経路などに応じて適宜決定することができる。 The composition of a suitable pharmaceutical composition in the preparation can be appropriately determined by those skilled in the art according to the applied disease, the applied administration route, and the like.
　医薬組成物中の賦形剤や担体は液体でも固体でもよい。
適当な賦形剤や担体は注射用の水や生理食塩水、人工脳脊髄液や非経口投与に通常用いられている他の物質でもよい。 Excipients and carriers in the pharmaceutical composition may be liquid or solid.
Appropriate excipients and carriers may be water for injection, physiological saline, artificial cerebrospinal fluid and other substances commonly used for parenteral administration.
　中性の生理食塩水や血清アルブミンを含む生理食塩水を担体に用いることもできる。
医薬組成物には例えば、ｐＨ７．０－８．５のＴｒｉｓバッファー、ｐＨ４．０－５．５の酢酸バッファー、ｐＨ５．０－８．０のクエン酸バッファー、ｐＨ５．０－８．０のヒスチジンバッファー、又はそれらにソルビトールや他の化合物を含んだバッファーを用いることもできる。 Neutral physiological saline or physiological saline containing serum albumin can also be used as a carrier.
Pharmaceutical compositions include, for example, pH 7.0-8.5 Tris buffer, pH 4.0-5.5 acetate buffer, pH 5.0-8.0 citrate buffer, pH 5.0-8.0 histidine. A buffer or a buffer containing sorbitol or other compounds can also be used.
　本発明の医薬組成物は選択された組成で必要な純度で適当な薬剤として、凍結乾燥品あるいは液体として準備される。 The pharmaceutical composition of the present invention is prepared as a lyophilized product or liquid as an appropriate drug with the purity required for the selected composition.
　抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片を含む医薬組成物はスクロースのような適当な賦形剤を用いた凍結乾燥品として成型されることもできる。 A pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody can be molded as a lyophilized product using an appropriate excipient such as sucrose.
　医薬組成物は非経口投与用に調製することもできるし、経口による消化管吸収用に調製することもできる。 The pharmaceutical composition can be prepared for parenteral administration or can be prepared for oral digestive tract absorption.
　製剤の組成及び濃度は投与方法によって決定することができるし、本発明の医薬組成物に含まれる、抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片のＥＰＨＡ２に対する親和性、即ち、ＥＰＨＡ２に対する解離定数（Ｋｄ値）に対し、親和性が高い（Ｋｄ値が低い）ほど、ヒトへの投与量を少なく薬効を発揮することができるので、この結果に基づいて本発明の医薬組成物の人に対する投与量を決定することもできる。 The composition and concentration of the preparation can be determined by the administration method, and the affinity of the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody contained in the pharmaceutical composition of the present invention for EPHA2, that is, dissociation for EPHA2 The higher the affinity (Kd value) for the constant (Kd value), the smaller the dose to humans, the more effective the drug can be. Therefore, based on this result, the pharmaceutical composition of the present invention for humans The dosage can also be determined.
　投与量は、医薬組成物をヒトに対して投与する際には、通常約０．１～１００ｍｇ／ｋｇを１～１８０日間に１回投与すればよい。 When the pharmaceutical composition is administered to humans, it is usually sufficient to administer about 0.1 to 100 mg / kg once every 1 to 180 days.
　医薬組成物の形態としては、点滴を含む注射剤、坐剤、経鼻剤、舌下剤、経皮吸収剤などが挙げられる。
抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片を有効成分として含む医薬組成物は化学療法剤と同時にあるいは順番に用いてもよい。すなわち、化学療法剤を投与した後に抗ＥＰＨＡ２ヒト化スワップド抗体又は該抗体の機能性断片を有効成分として含む医薬組成物を投与してもよいし、当該医薬組成物を投与した後に、化学療法剤を投与してもよく、更に、当該医薬組成物と化学療法剤を同時に投与してもよい。例えば、用い得る作用薬は、チミジル酸シンターゼ阻害剤を含む代謝拮抗剤、核酸類似体、白金細胞毒性剤、トポイソメラーゼ阻害剤、又は抗微小管剤を含む。本発明で用い得るチミジル酸シンターゼ阻害剤の例は、５－ＦＵ、ＭＴＡ、及びＴＤＸを含む。用い得る代謝拮抗剤の例は、トムデックス（ＴＤＸ）である。用い得る白金細胞毒性剤の例は、シスプラチン及びオキサリプラチンを含む。上記に挙げた特定の作用薬に加えて、又はその代りに本発明で用い得る化学療法剤は、アルキル化剤；スルホン酸アルキル；アジリジン；エチレンイミン；メチルアメラミン；ナイトロジェンマスタード；ニトロスレアス；代謝拮抗剤；葉酸類似体；プリン類似体；ピリミジン類似体；アンドロゲン；抗副腎物質；葉酸補填剤；アセグラトン；アルドホスファミドグリコシド；アミノレブリン酸；アムサクリン；ベストラブシル；ビスアントレン；エダトラキセート；デホファミン；デメコルシン；ジアジコン；エフロミチン（ｅｌｆｏｍｉｔｈｉｎｅ）；酢酸エリプチニウム；エトグルシド；硝酸ガリウム；ヒドロキシ尿素；レンチナン；イオニダミン；ミトグアゾン；ミトキサントロンを含み得る。化学療法剤として、トポイソメラーゼ阻害剤を用いてもよく、特定の実施形態において用いるトポイソメラーゼ阻害剤は、トポイソメラーゼＩ阻害剤、例えば、カンプトテシンである。本発明で用い得る適切なトポイソメラーゼＩ阻害剤は、イレノテカン（ＣＰＴ－１１）又はその活性代謝物であるＳＮ－３８である。ＣＰＴ－１１は、開裂又は開裂複合体と称する、可逆的な共有結合反応中間体を安定化させることにより、細胞周期のＳ期において特異的に作用し、Ｇ２～Ｍ期における細胞周期の停止も誘導し得る。本発明の特定の実施形態において用い得る、化学療法剤は、フルオロピリミジン、例えば、５－ＦＵである。特定の化学療法剤を用いる場合、特定の作用薬の抗腫瘍活性を保持する、生物学的に活性な誘導体及びその実質的な同等物を含む類似体を用いてよいことを理解されたい。 Examples of the form of the pharmaceutical composition include injections containing infusions, suppositories, nasal agents, sublingual agents, and transdermal absorption agents.
The pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be used simultaneously or sequentially with the chemotherapeutic agent. That is, after administering the chemotherapeutic agent, a pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be administered, or after administering the pharmaceutical composition, the chemotherapeutic agent In addition, the pharmaceutical composition and the chemotherapeutic agent may be administered simultaneously. For example, agents that can be used include antimetabolites including thymidylate synthase inhibitors, nucleic acid analogs, platinum cytotoxic agents, topoisomerase inhibitors, or anti-microtubule agents. Examples of thymidylate synthase inhibitors that can be used in the present invention include 5-FU, MTA, and TDX. An example of an antimetabolite that can be used is Tomdex (TDX). Examples of platinum cytotoxic agents that can be used include cisplatin and oxaliplatin. In addition to or in place of the specific agents listed above, chemotherapeutic agents that can be used in the present invention include: alkylating agents; alkyl sulfonates; aziridines; ethyleneimines; methylamelamines; nitrogen mustards; Antimetabolites; Folic acid analogs; Purine analogs; Pyrimidine analogs; Androgens; Anti-adrenal substances; Folic acid supplements; Acegraton; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Vestlabsyl; Bisanthrene; May contain: diazicon; efomithine; ellipticin acetate; ethogluside; gallium nitrate; hydroxyurea; lentinan; ionidamine; Topoisomerase inhibitors may be used as chemotherapeutic agents, and the topoisomerase inhibitors used in certain embodiments are topoisomerase I inhibitors such as camptothecin. A suitable topoisomerase I inhibitor that can be used in the present invention is ilenotecan (CPT-11) or its active metabolite SN-38. CPT-11 acts specifically in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, called a cleavage or cleavage complex, and also arrests the cell cycle in the G2-M phase. Can be induced. A chemotherapeutic agent that can be used in certain embodiments of the invention is a fluoropyrimidine, such as 5-FU. It should be understood that when a particular chemotherapeutic agent is used, analogs including biologically active derivatives and substantial equivalents thereof that retain the anti-tumor activity of a particular agent may be used.
　本明細書で開示される医薬組成物を投与することによってＥＰＨＡ２を発現している腫瘍の増殖を抑制することが可能になる。 It is possible to suppress the growth of tumors expressing EPHA2 by administering the pharmaceutical composition disclosed herein.
　以下、実施例において本発明を更に詳細に説明するが、本発明はこれらに限定されない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
　なお、下記実施例において遺伝子操作に関する各操作は特に明示がない限り、「モレキュラークローニング（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ）」（Ｓａｍｂｒｏｏｋ，Ｊ．，Ｆｒｉｔｓｃｈ，Ｅ．Ｆ．およびＭａｎｉａｔｉｓ，Ｔ．著，Ｃｏｌｄ　ＳｐｒｉｎｇＨａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓより１９８９年発刊）に記載の方法及びその他の当業者が使用する実験書に記載の方法により行うか、または、市販の試薬やキットを用いる場合には市販品の指示書に従って行った。 In the following examples, unless otherwise specified, each operation relating to gene manipulation is from “Molecular Cloning” (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press). (Published in 1989) and the methods described in other experimental documents used by those skilled in the art, or when using commercially available reagents and kits, they were performed according to the instructions for commercial products.
　実施例１．ヒト化抗体の設計
　ａ）ＳＨ３４８－１のヒト化抗体の設計
　ａ）－ｉ）　ＳＨ３４８－１の可変領域の分子モデリング
　ＳＨ３４８－１の重鎖可変領域は配列表の配列番号１０に示されている。ＳＨ３４８－１の軽鎖可変領域は配列表の配列番号１４に示されている。 Example 1. Design of humanized antibody a) Design of humanized antibody of SH348-1 a) -i) Molecular modeling of the variable region of SH348-1 The heavy chain variable region of SH348-1 is shown in SEQ ID NO: 10 of the Sequence Listing. . The light chain variable region of SH348-1 is shown in SEQ ID NO: 14 in the sequence listing.
　ＳＨ３４８－１の可変領域の分子モデリングは、相同性モデリングとして一般的に公知の方法（Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，２０３，１２１－１５３，（１９９１））によって実行された。Ｐｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｎｋ（Ｎｕｃ．Ａｃｉｄ　Ｒｅｓ．３５，Ｄ３０１－Ｄ３０３（２００７））に登録されるヒト免疫グロブリンの可変領域の１次配列（Ｘ線結晶構造から誘導される三次元構造が入手可能である）を、上で決定されたＳＨ３４８－１の可変領域と比較した。結果として、２ＪＥＬおよび１Ａ４Ｊが、それぞれ、ＳＨ３４８－１の軽鎖および重鎖の可変領域に対して最も高い配列相同性を有する配列として選択された。フレームワーク領域の三次元構造は、ＳＨ３４８－１の軽鎖及び重鎖に対応する２ＪＥＬ及び１Ａ４Ｊの座標を組み合わせて、「フレームワークモデル」を得ることによって作製された。ＳＨ３４８－１のＣＤＲは、Ｔｈｏｒｎｔｏｎ　ｅｔ　ａｌ．（Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２６３，８００－８１５，（１９９６））の分類に従って、ＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３、ＣＤＲＨ１及びＣＤＲＨ２は、それぞれクラスター１６Ａ、７Ａ、９Ａ、１０Ａ、１０Ａに割り当てられた。ＣＤＲＨ３は、Ｈ３ルール（ＦＥＢＳ　ｌｅｔｔｅｒ　３９９，１－８（１９９６））を使用して、ｅ（９）Ｄに分類された。次いで、それぞれのＣＤＲについての代表的なコンホメーションがフレームワークモデルに組み込まれた。 Molecular modeling of the variable region of SH348-1 was performed by a method generally known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Primary sequence of variable region of human immunoglobulin registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) (three-dimensional structure derived from X-ray crystal structure is available) Was compared to the variable region of SH348-1 determined above. As a result, 2JEL and 1A4J were selected as the sequences with the highest sequence homology to the SH348-1 light and heavy chain variable regions, respectively. The three-dimensional structure of the framework region was created by combining the 2JEL and 1A4J coordinates corresponding to the SH348-1 light and heavy chains to obtain a “framework model”. The CDRs of SH348-1 are described in Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)), CDRL 1 , CDRL 2 , CDRL 3 , CDRH 1 and CDRH 2 are in clusters 16A, 7A, 9A, 10A, 10A, respectively. Assigned. CDRH 3 was classified as e (9) D using the H3 rule (FEBS letter 399, 1-8 (1996)). A representative conformation for each CDR was then incorporated into the framework model.
　最後に、エネルギーの点でＳＨ３４８－１の可変領域の可能性のある分子モデルを得るために、不利な原子間接触を除くためのエネルギー計算を行った。上記手順を、市販の蛋白質立体構造予測プログラムＰｒｉｍｅ及び配座探索プログラムＭａｃｒｏＭｏｄｅｌ（Ｓｃｈｒｏｄｉｎｇｅｒ，　ＬＬＣ）を用いて行った。 Finally, in order to obtain a molecular model that may be a variable region of SH348-1 in terms of energy, an energy calculation was performed to eliminate unfavorable interatomic contact. The above procedure was performed using a commercially available protein tertiary structure prediction program Prime and a conformational search program MacroModel (Schrodinger, LLC).
　ａ）－ｉｉ）　ヒト化ＳＨ３４８－１に対するアミノ酸配列の設計
　ヒト化ＳＨ３４８－１抗体の構築を、ＣＤＲグラフティング（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６，１００２９－１００３３（１９８９））として一般的に公知の方法によって行った。アクセプター抗体は、フレームワーク領域内のアミノ酸相同性に基づいて選択された。ＳＨ３４８－１のフレームワーク領域の配列を、ＩｇＢＬＡＳＴ（Ｎｕｃ．Ａｃｉｄ　Ｒｅｓ．３６，Ｄ２５－Ｄ３０（２００７））の全てのヒトフレームワークと比較し、結果として、Ｌ鎖は１００６２６１Ａがフレームワーク領域についての９６％配列相同性に起因して、アクセプターとして選択された。Ｈ鎖はＡＡＣ０９０７４がフレームワーク領域についての８６％の配列相同性に起因して、アクセプターとして選択された。Ｌ鎖は１００６２６１Ａについて、Ｈ鎖はＡＡＣ０９０７４についてのフレームワーク領域のアミノ酸残基を、ＳＨ３４８－１についてのアミノ酸残基と整列させ、異なるアミノ酸が使用される位置を同定した。これらの残基の位置は、上で構築されたＳＨ３４８－１の三次元モデルを使用して分析され、そしてアクセプター上にグラフティングされるべきドナー残基が、Ｑｕｅｅｎ　ｅｔ　ａｌ．（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６，１００２９－１００３３（１９８９））によって与えられる基準によって選択された。選択されたいくつかのドナー残基をアクセプター抗体に移入することによって、ヒト化ＳＨ３４８－１配列を以下の実施例に記載されるように構築した。 a) -ii) Design of amino acid sequence for humanized SH348-1 The construction of humanized SH348-1 antibody is commonly described as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). By a known method. Acceptor antibodies were selected based on amino acid homology within the framework regions. The sequence of the framework region of SH348-1 was compared with all human frameworks of IgBLAST (Nuc. Acid Res. 36, D25-D30 (2007)). As a result, the light chain was 1006261A for the framework region. Selected as acceptor due to 96% sequence homology. The heavy chain was selected as an acceptor due to 86% sequence homology with AAC09074 for the framework region. The amino acid residues of the framework region for L chain 10060061A and H chain AAC09074 were aligned with the amino acid residues for SH348-1, and the position where the different amino acids were used was identified. The positions of these residues are analyzed using the SH348-1 three-dimensional model constructed above, and the donor residues to be grafted on the acceptor are described in Queen et al. (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). The humanized SH348-1 sequence was constructed as described in the examples below by transferring several selected donor residues into the acceptor antibody.
　ｂ）　ＳＨ３４８－１重鎖のヒト化
　ｂ）－ｉ）　ｈＳＨ３４８－１－Ｔ４Ｈタイプ重鎖：
　配列表の配列番号６に示されるＳＨ３４８－１重鎖のアミノ酸番号２（イソロイシン）、９（プロリン）、１１（ロイシン）、１６（グルタミン酸）、１７（スレオニン）、２０（イソロイシン）、３８（リジン）、４３（リジン）、４６（リジン）、６９（アラニン）、７３（グルタミン酸）、７６（アラニン）、８０（フェニルアラニン）、８３（イソロイシン）、８４（アスパラギン）、８５（アスパラギン）、８８（アスパラギン）、９３（スレオニン）、９５（フェニルアラニン）、１１４（スレオニン）、１１５（ロイシン）をそれぞれバリン、アラニン、バリン、アラニン、セリン、バリン、アルギニン、グルタミン、グルタミン酸、バリン、アスパラギン酸、バリン、チロシン、ロイシン、セリン、セリン、アラニン、バリン、チロシン、ロイシン、バリンに置き換えることを伴い設計されたヒト化ＳＨ３４８－１重鎖を「ｈＳＨ３４８－１－Ｔ４Ｈタイプ重鎖」と命名した。ｈＳＨ３４８－１－Ｔ４Ｈタイプ重鎖のアミノ酸配列を配列表の配列番号５３に、ｈＳＨ３４８－１－Ｔ４Ｈタイプ重鎖の可変領域のアミノ酸配列を配列番号５４に示す。 b) Humanization of SH348-1 heavy chain b) -i) hSH348-1-T4H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine) of the SH348-1 heavy chain shown in SEQ ID NO: 6 in the sequence listing ), 43 (lysine), 46 (lysine), 69 (alanine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 88 (asparagine) ), 93 (threonine), 95 (phenylalanine), 114 (threonine), 115 (leucine) are valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, aspartic acid, valine, tyrosine, Leucine, serine, serine, alanine, burr , Tyrosine, leucine, and with designed humanized SH348-1 heavy chain to replace the valine designated as "hSH348-1-T4H-type heavy chain". The amino acid sequence of the hSH348-1-T4H type heavy chain is shown in SEQ ID NO: 53, and the amino acid sequence of the variable region of the hSH348-1-T4H type heavy chain is shown in SEQ ID NO: 54.
　ｂ）－ｉｉ）　ｈＳＨ３４８－１－Ｔ５Ｈタイプ重鎖：
　配列表の配列番号６に示されるＳＨ３４８－１重鎖のアミノ酸番号２（イソロイシン）、９（プロリン）、１１（ロイシン）、１６（グルタミン酸）、１７（スレオニン）、２０（イソロイシン）、４３（リジン）、７６（アラニン）、８０（フェニルアラニン）、８３（イソロイシン）、８４（アスパラギン）、８５（アスパラギン）、８８（アスパラギン）、９３（スレオニン）、１１４（スレオニン）、１１５（ロイシン）をそれぞれバリン、アラニン、バリン、アラニン、セリン、バリン、グルタミン、バリン、チロシン、ロイシン、セリン、セリン、アラニン、バリン、ロイシン、バリンに置き換えることを伴い設計されたヒト化ＳＨ３４８－１重鎖を「ｈＳＨ３４８－１－Ｔ５Ｈタイプ重鎖」と命名した。ｈＳＨ３４８－１－Ｔ５Ｈタイプ重鎖のアミノ酸配列を配列表の配列番号５５に、ｈＳＨ３４８－１－Ｔ５Ｈタイプ重鎖の可変領域のアミノ酸配列を配列番号５６に示す。 b) -ii) hSH348-1-T5H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH348-1 heavy chain shown in SEQ ID NO: 6 in the sequence listing ), 76 (alanine), 80 (phenylalanine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 88 (asparagine), 93 (threonine), 114 (threonine), 115 (leucine), respectively, valine, A humanized SH348-1 heavy chain designed with replacement of alanine, valine, alanine, serine, valine, glutamine, valine, tyrosine, leucine, serine, serine, alanine, valine, leucine, valine is replaced with “hSH348-1- It was named “T5H type heavy chain”. The amino acid sequence of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 55 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 56.
　ｃ）　ＳＨ３４８－１軽鎖のヒト化
　ｃ）－ｉ）　ｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖：
　ＷＯ２００９／０２８６３９（実施例１０）、１０－１－３－１）に記載のｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖のアミノ酸配列を配列表の配列番号５７に、ｈＳＨ３４８－１－Ｔ１Ｌタイプ重鎖の可変領域のアミノ酸配列を配列番号５８に示す。 c) Humanization of SH348-1 light chain c) -i) hSH348-1-T1L type light chain:
The amino acid sequence of the hSH348-1-T1L type light chain described in WO2009 / 028639 (Example 10), 10-1-3-1) is represented by SEQ ID NO: 57 in the Sequence Listing, and the variable of the hSH348-1-T1L type heavy chain. The amino acid sequence of the region is shown in SEQ ID NO: 58.
 
　ｃ）－ｉｉ）　ｈＳＨ３４８－１－Ｔ５Ｌタイプ軽鎖：
　配列表の配列番号８に示されるＳＨ３４８－１軽鎖のアミノ酸番号１４（セリン）、１５（ロイシン）、１７（アスパラギン酸）、１８（グルタミン）、７９（アルギニン）、８８（ロイシン）、１０５（グリシン）、１０９（ロイシン）、１１４（アラニン）をそれぞれスレオニン、プロリン、グルタミン酸、プロリン、リジン、バリン、グルタミン、バリン、スレオニンに置き換えることを伴い設計されたヒト化ＳＨ３４８－１軽鎖を「ｈＳＨ３４８－１－Ｔ５Ｌタイプ軽鎖」と命名した。ｈＳＨ３４８－１－Ｔ５Ｌタイプ軽鎖のアミノ酸配列を配列表の配列番号５９に、ｈＳＨ３４８－１－Ｔ５Ｌタイプ軽鎖の可変領域のアミノ酸配列を配列番号６０に示す。
c) -ii) hSH348-1-T5L type light chain:
Amino acid numbers 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 79 (arginine), 88 (leucine), 105 (of the SH348-1 light chain shown in SEQ ID NO: 8 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with threonine, proline, glutamic acid, proline, lysine, valine, glutamine, valine and threonine, respectively. 1-T5L type light chain ". The amino acid sequence of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 59 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 60.
　ｄ）ＳＨ３５７－１のヒト化抗体の設計
　ｄ）－ｉ）　ＳＨ３５７－１の可変領域の分子モデリング
　ＳＨ３５７－１の重鎖可変領域は配列表の配列番号３４に記載されている。ＳＨ３５７－１の軽鎖可変領域は配列表の配列番号３８に記載されている。 d) Design of humanized antibody of SH357-1 d) -i) Molecular modeling of the variable region of SH357-1 The heavy chain variable region of SH357-1 is set forth in SEQ ID NO: 34 of the Sequence Listing. The light chain variable region of SH357-1 is set forth in SEQ ID NO: 38 in the sequence listing.
　上記ａ）－ｉ）と同様の方法で、２ＪＥＬ及び１Ａ４Ｊが、それぞれＳＨ３５７－１の軽鎖および重鎖の可変領域に対して最も高い配列相同性を有する配列として選択され、ＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３、ＣＤＲＨ１及びＣＤＲＨ２は、それぞれクラスター１６Ａ、７Ａ、９Ａ、１０Ａ、１０Ａに割り当てられた。ＣＤＲＨ３は、ｅ（９）Ｄに分類された。 In the same manner as in a) -i) above, 2JEL and 1A4J were selected as the sequences having the highest sequence homology to the light and heavy chain variable regions of SH357-1, respectively, and CDRL 1 , CDRL 2 , CDRL 3 , CDRH 1 and CDRH 2 were assigned to clusters 16A, 7A, 9A, 10A, 10A, respectively. CDRH 3 was classified as e (9) D.
　ａ）－ｉｉと同様の方法によって、Ｌ鎖は１００６２６１Ａがアクセプターとして選択され、Ｈ鎖はＡＡＣ０９０７４がアクセプターとして選択され、ヒト化ＳＨ３５７－１の配列を決定した。その結果、以下に示すように、軽鎖について１タイプ、重鎖について２タイプのヒト化配列を取得した。 A) The sequence of humanized SH357-1 was determined by the same method as in -ii, in which 1006661A was selected as the acceptor for the L chain and AAC09074 was selected as the acceptor for the H chain. As a result, as shown below, one type of humanized sequence for the light chain and two types of humanized sequences for the heavy chain were obtained.
　ｅ）　ＳＨ３５７－１重鎖のヒト化
　ｅ）－ｉ）　ｈＳＨ３５７－１－Ｔ４Ｈタイプ重鎖：
　配列表の配列番号３０に示されるＳＨ３５７－１重鎖のアミノ酸番号２（イソロイシン）、９（プロリン）、１１（ロイシン）、１６（グルタミン酸）、１７（スレオニン）、２０（イソロイシン）、３８（リジン）、４３（リジン）、４６（リジン）、６９（アラニン）、７３（グルタミン酸）、７６（アラニン）、８３（イソロイシン）、８５（アスパラギン）、８８（アスパラギン）、９３（セリン）、９５（フェニルアラニン）、１１４（スレオニン）、１１５（ロイシン）をそれぞれバリン、アラニン、バリン、アラニン、セリン、バリン、アルギニン、グルタミン、グルタミン酸、バリン、アスパラギン酸、バリン、ロイシン、セリン、アラニン、バリン、チロシン、ロイシン、バリンに置き換えることを伴い設計されたヒト化ＳＨ３５７－１重鎖を「ｈＳＨ３５７－１－Ｔ４Ｈタイプ重鎖」と命名した。ｈＳＨ３５７－１－Ｔ４Ｈタイプ重鎖のアミノ酸配列を配列表の配列番号６１に、ｈＳＨ３５７－１－Ｔ４Ｈタイプ重鎖の可変領域のアミノ酸配列を配列番号６２に示す。 e) Humanization of SH3577-1 heavy chain e) -i) hSH357-1-T4H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine) of the SH357-1 heavy chain shown in SEQ ID NO: 30 of the Sequence Listing ), 43 (lysine), 46 (lysine), 69 (alanine), 73 (glutamic acid), 76 (alanine), 83 (isoleucine), 85 (asparagine), 88 (asparagine), 93 (serine), 95 (phenylalanine) ), 114 (threonine), 115 (leucine) are valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, aspartic acid, valine, leucine, serine, alanine, valine, tyrosine, leucine, Humanization designed with valine replacement The H357-1 heavy chain was named "hSH357-1-T4H-type heavy chain". The amino acid sequence of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 61 in the sequence listing, and the amino acid sequence of the variable region of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 62.
　ｅ）－ｉｉ）　ｈＳＨ３５７－１－Ｔ５Ｈタイプ重鎖：
　配列表の配列番号３０に示されるＳＨ３５７－１重鎖のアミノ酸番号２（イソロイシン）、９（プロリン）、１１（ロイシン）、１６（グルタミン酸）、１７（スレオニン）、２０（イソロイシン）、４３（リジン）、７６（アラニン）、８３（イソロイシン）、８５（アスパラギン）、８８（アスパラギン）、９３（セリン）、１１４（スレオニン）、１１５（ロイシン）をそれぞれバリン、アラニン、バリン、アラニン、セリン、バリン、グルタミン、バリン、ロイシン、セリン、アラニン、バリン、ロイシン、バリンに置き換えることを伴い設計されたヒト化ＳＨ３５７－１重鎖を「ｈＳＨ３５７－１－Ｔ５Ｈタイプ重鎖」と命名した。ｈＳＨ３５７－１－Ｔ５Ｈタイプ重鎖のアミノ酸配列を配列表の配列番号６３に、ｈＳＨ３５７－１－Ｔ５Ｈタイプ重鎖の可変領域のアミノ酸配列を配列番号６４に示す。 e) -ii) hSH357-1-T5H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH357-1 heavy chain shown in SEQ ID NO: 30 of the Sequence Listing ), 76 (alanine), 83 (isoleucine), 85 (asparagine), 88 (asparagine), 93 (serine), 114 (threonine), 115 (leucine), respectively, valine, alanine, valine, alanine, serine, valine, The humanized SH357-1 heavy chain designed with replacement with glutamine, valine, leucine, serine, alanine, valine, leucine, valine was named “hSH357-1-T5H type heavy chain”. The amino acid sequence of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 63 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 64.
　ｆ）　ＳＨ３５７－１軽鎖のヒト化
　ｆ）－ｉ）　ｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖：
　ＷＯ２００９／０２８６３９（実施例１０）、１０－２－３－１）に記載のｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖のアミノ酸配列を配列表の配列番号６５に、ｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖の可変領域のアミノ酸配列を配列番号６６に示す。 f) Humanization of SH3577-1 light chain f) -i) hSH357-1-T1L type light chain:
The amino acid sequence of the hSH357-1-T1L type light chain described in WO2009 / 028639 (Example 10), 10-2-3-1) is represented by SEQ ID NO: 65 in the sequence listing, and the variable of the hSH357-1-T1L type light chain. The amino acid sequence of the region is shown in SEQ ID NO: 66.
　ｆ）－ｉｉ）　ｈＳＨ３５７－１－Ｔ５Ｌタイプ軽鎖：
　配列表の配列番号３２に示されるＳＨ３５７－１軽鎖のアミノ酸番号７（スレオニン）、１４（セリン）、１５（ロイシン）、１７（アスパラギン酸）、１８（グルタミン）、８８（ロイシン）、１０５（グリシン）、１０９（ロイシン）、１１４（アラニン）をそれぞれセリン、スレオニン、プロリン、グルタミン酸、プロリン、バリン、グルタミン、バリン、スレオニンに置き換えることを伴い設計されたヒト化ＳＨ３５７－１軽鎖を「ｈＳＨ３５７－１－Ｔ５Ｌタイプ軽鎖」と命名した。ｈＳＨ３５７－１－Ｔ５Ｌタイプ軽鎖のアミノ酸配列を配列表の配列番号６７に、ｈＳＨ３５７－１－Ｔ５Ｌタイプ軽鎖の可変領域のアミノ酸配列を配列番号６８に示す。 f) -ii) hSH357-1-T5L type light chain:
Amino acid numbers 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 88 (leucine), 105 (SH) light chain shown in SEQ ID NO: 32 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with serine, threonine, proline, glutamic acid, proline, valine, glutamine, valine, and threonine, respectively, and the humanized SH357-1 light chain designed with “hSH357- 1-T5L type light chain ". The amino acid sequence of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 67 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 68.
　実施例２．汎用発現ベクターｐＥＦ６ＫＣＬ及びｐＥＦ１ＦＣＣＵの作製
　１）ヒト軽鎖発現ベクターｐＥＦ６ＫＣＬの構築
　プラスミドｐＥＦ６／Ｖ５－ＨｉｓＢ（Ｉｎｖｉｔｒｏｇｅｎ社）を鋳型として下記プライマーを用いてＰＣＲを行うことにより、ＢＧＨｐＡの直後（Ｓｅｑｕｅｎｃｅ　Ｐｏｓｉｔｉｏｎ　２１７４）から、Ｓｍａ　Ｉ（Ｓｅｑｕｅｎｃｅ　Ｐｏｓｉｔｉｏｎ　２９５８）までのＤＮＡ断片（ｆ１　ｏｒｉｇｉｎ　ｏｆ　ｒｅｐｌｉｃａｔｉｏｎ及びＳＶ４０　ｐｒｏｍｏｔｏｒ　ａｎｄ　ｏｒｉｇｉｎを含むＤＮＡフラグメント、以下、「フラグメントＡ」とする。）を取得した。
５’－ｃｃａｃｇｃｇｃｃｃｔｇｔａｇｃｇｇｃｇｃａｔｔａａｇｃ－３’（プライマーＥＦＦ１）（配列番号６９）
５’－ａａａｃｃｃｇｇｇａｇｃｔｔｔｔｔｇｃａａａａｇｃｃｔａｇｇ－３’（プライマーＥＦｓｍａＲ）（配列番号７０）
　得られたフラグメントＡと、ヒトκ鎖分泌シグナル、ヒトκ鎖定常領域及びヒトｐｏｌｙＡ付加シグナルをコードするＤＮＡ配列を含むＤＮＡフラグメント（配列番号７１、以下、「フラグメントＢ」とする。）をオーバーラップＰＣＲにより結合した。得られたフラグメントＡとフラグメントＢとが結合したＤＮＡ断片を制限酵素ＫｐｎＩ及びＳｍａＩで消化し、制限酵素ＫｐｎＩ及びＳｍａＩで消化したプラスミドｐＥＦ６／Ｖ５－ＨｉｓＢ　（Ｉｎｖｉｔｒｏｇｅｎ）とライゲーションし、ＥＦ１プロモーターの下流にシグナル配列、クローニングサイト、ヒトκ鎖定常領域、及びヒトｐｏｌｙＡ付加シグナル配列をもつ、ヒト軽鎖発現ベクターｐＥＦ６ＫＣＬを構築した。 Example 2 Construction of universal expression vectors pEF6KCL and pEF1FCCU 1) Construction of human light chain expression vector pEF6KCL By PCR using plasmid pEF6 / V5-HisB (Invitrogen) as a template with the following primers (Sequence Position 2174) To DNA fragments up to Sma I (Sequence Position 2958) (DNA fragment containing f1 origin of replication and SV40 promoter and origin, hereinafter referred to as “fragment A”).
5′-ccacgcgccctgttagcggcgcattaagc-3 ′ (primer EFF1) (SEQ ID NO: 69)
5′-aaacccggggagtttttgcaaaaggcctag-3 ′ (primer EFsmaR) (SEQ ID NO: 70)
The obtained fragment A overlaps with a DNA fragment (SEQ ID NO: 71, hereinafter referred to as “fragment B”) containing a DNA sequence encoding a human κ chain secretion signal, a human κ chain constant region, and a human polyA addition signal. Binding by PCR. The obtained DNA fragment in which fragment A and fragment B were bound was digested with restriction enzymes KpnI and SmaI, and ligated with plasmid pEF6 / V5-HisB (Invitrogen) digested with restriction enzymes KpnI and SmaI, and downstream of the EF1 promoter. A human light chain expression vector pEF6KCL having a signal sequence, a cloning site, a human κ chain constant region, and a human polyA addition signal sequence was constructed.
　２）ｐＥＦ１／ＫＣＬの構築
　上記の方法で得られたｐＥＦ６ＫＣＬから制限酵素ＫｐｎＩ及びＳｍａＩで切り出されたＤＮＡ断片を、ＫｐｎＩ及びＳｍａＩで消化したｐＥＦ１／ｍｙｃ－ＨｉｓＢ（Ｉｎｖｉｔｒｏｇｅｎ社）とライゲーションし、プラスミドｐＥＦ１ＫＣＬを構築した。 2) Construction of pEF1 / KCL A DNA fragment cleaved from the restriction enzyme KpnI and SmaI from pEF6KCL obtained by the above method was ligated with pEF1 / myc-HisB (Invitrogen) digested with KpnI and SmaI, and the plasmid pEF1KCL Built.
　３）ヒト重鎖発現ベクターｐＥＦ１ＦＣＣＵの構築
　ヒトＩｇＧ１シグナル配列及び定常領域のアミノ酸をコードするＤＮＡ配列を含むＤＮＡ断片（配列番号７２）を制限酵素ＮｈｅＩ及びＰｍｅＩで消化し、ＮｈｅＩ及びＰｍｅＩで消化したプラスミドｐＥＦ１ＫＣＬと結合し、ＥＦ１プロモーターの下流にシグナル配列、クローニングサイト、ヒト重鎖定常領域、及びヒトｐｏｌｙＡ付加シグナル配列をもつヒト重鎖発現ベクターｐＥＦ１ＦＣＣＵを構築した。 3) Construction of human heavy chain expression vector pEF1FCCU A plasmid obtained by digesting a DNA fragment (SEQ ID NO: 72) containing a human IgG1 signal sequence and a DNA sequence encoding the amino acid of the constant region with restriction enzymes NheI and PmeI and digesting with NheI and PmeI A human heavy chain expression vector pEF1FCCU was constructed by binding to pEF1KCL and having a signal sequence, a cloning site, a human heavy chain constant region, and a human polyA addition signal sequence downstream of the EF1 promoter.
実施例３．抗体発現ベクターの構築
　１）　ｈＳＨ３４８－１－Ｔ１Ｌ、及びｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖発現ベクターの構築
　配列表の配列番号７３に示される分泌シグナルと融合したｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖可変領域及び配列番号７４に示される分泌シグナルと融合したｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖可変領域をコードする遺伝子を含むＤＮＡを合成し（Ｉｎｖｉｔｒｏｇｅｎ社　人工遺伝子合成サービス）、制限酵素ＮｄｅＩおよびＢｓｉＷＩで切り出されたＤＮＡ断片を、ヒト化抗体軽鎖発現汎用ベクター（ｐＥＦ６ＫＣＬ）を制限酵素ＮｄｅＩおよびＢｓｉＷＩで切断した箇所に挿入することにより、ｈＳＨ３４８－１－Ｔ１Ｌ、ｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖発現ベクターを構築した。得られた発現ベクターをそれぞれ「ｐＥＦ６ＫＣＬ／ｈＳＨ３４８－１－Ｔ１Ｌ」、「ｐＥＦ６ＫＣＬ／ｈＳＨ３５７－１－Ｔ１Ｌ」と命名した。 Example 3 Construction of antibody expression vector 1) Construction of hSH348-1-T1L and hSH357-1-T1L type light chain expression vector hSH348-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 73 of the Sequence Listing And a DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 74 (Invitrogen Artificial Gene Synthesis Service), and was cleaved with restriction enzymes NdeI and BsiWI HSH348-1-T1L and hSH357-1-T1L type light chain expression vectors were constructed by inserting the DNA fragment into a site where a humanized antibody light chain expression general-purpose vector (pEF6KCL) was cleaved with restriction enzymes NdeI and BsiWI. . The obtained expression vectors were named “pEF6KCL / hSH348-1-T1L” and “pEF6KCL / hSH357-1-T1L”, respectively.
　２）　ｈＳＨ３４８－１－Ｔ１Ｈ、ｈＳＨ３５７－１－Ｔ１Ｈ　タイプ重鎖発現ベクターの構築
　配列表の配列番号７５に示されるｈＳＨ３４８－１－Ｔ１Ｈタイプ重鎖可変領域及び配列番号７６に示されるｈＳＨ３５７－１－Ｔ１Ｈタイプ重鎖可変領域をコードする遺伝子を含むＤＮＡを合成し（Ｉｎｖｉｔｒｏｇｅｎ社　人工遺伝子合成サービス）、制限酵素ＢｌｐＩで切り出されるＤＮＡ断片を、ヒト化抗体Ｈ鎖発現汎用ベクター（ｐＥＦ１ＦＣＣＵ）を制限酵素ＢｌｐＩで切断した箇所に挿入することにより、ｈＳＨ３４８－１－Ｔ１Ｈ、ｈＳＨ３５７－１－Ｔ１Ｈタイプ重鎖発現ベクターを構築した。得られた発現ベクターをそれぞれ「ｐＥＦ１ＦＣＣＵ／ｈＳＨ３４８－１－Ｔ１Ｈ」、「ｐＥＦ１ＦＣＣＵ／ｈＳＨ３５７－１－Ｔ１Ｈ」と命名した。 2) Construction of hSH348-1-T1H, hSH357-1-T1H type heavy chain expression vector hSH348-1-T1H type heavy chain variable region shown in SEQ ID NO: 75 of the sequence listing and hSH357-1- shown in SEQ ID NO: 76 A DNA containing a gene encoding a T1H type heavy chain variable region was synthesized (Invitrogen Artificial Gene Synthesis Service), and a DNA fragment excised with the restriction enzyme BlpI was used as a humanized antibody H chain expression general-purpose vector (pEF1FCCU) as a restriction enzyme BlpI. The hSH348-1-T1H and hSH357-1-T1H type heavy chain expression vectors were constructed by inserting them into the sites cleaved with. The obtained expression vectors were named “pEF1FCCU / hSH348-1-T1H” and “pEF1FCCU / hSH357-1-T1H”, respectively.
　３）３４８―Ｌ４、及び３５７－Ｌ４タイプ軽鎖発現ベクターの構築
配列表の配列番号７８のアミノ酸番号２１乃至１３７、及び配列番号８０のアミノ酸番号２１乃至１３７にそれぞれ示される、ｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖可変領域、及びｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖可変領域をコードする遺伝子を含むＤＮＡを合成し（ＧＥＮＥＡＲＴ社、人工遺伝子合成サービス）、制限酵素ＮｄｅＩ及びＢｓｉＷＩで切り出されたＤＮＡ断片を、ヒト化抗体軽鎖発現汎用ベクター（ｐＥＦ６ＫＣＬ）を制限酵素ＮｄｅＩ及びＢｓｉＷＩで切断した箇所に挿入することにより、ｈＳＨ３４８－１－Ｔ１Ｌ、及びｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖を発現するベクターを構築した。ここで、ｈＳＨ３４８－１－Ｔ１Ｌタイプ軽鎖を「３４８－Ｌ４」、ｈＳＨ３５７－１－Ｔ１Ｌタイプ軽鎖を「３５７－Ｌ４」と命名した。また、得られた３４８－Ｌ４を発現するベクターを「ｐＥＦ６ＫＣＬ／３４８－Ｌ４」、３５７－Ｌ４を発現するベクターを「ｐＥＦ６ＫＣＬ／３５７－Ｌ４」と命名した。３４８－Ｌ４のヌクレオチド配列は配列表の配列番号８１に、アミノ酸配列は配列番号８２に記載されている。３５７－Ｌ４のヌクレオチド配列は配列番号８３にアミノ酸配列は配列番号８４に記載されている。 3) Construction of 348-L4 and 357-L4 type light chain expression vectors hSH348-1-T1L represented by amino acid numbers 21 to 137 of SEQ ID NO: 78 and amino acid numbers 21 to 137 of SEQ ID NO: 80, respectively. A DNA comprising a type light chain variable region and a gene encoding a hSH357-1-T1L type light chain variable region (GENEART, artificial gene synthesis service), and a DNA fragment excised with restriction enzymes NdeI and BsiWI, A vector expressing hSH348-1-T1L and hSH357-1-T1L type light chains was constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI. Here, the hSH348-1-T1L type light chain was named “348-L4” and the hSH357-1-T1L type light chain was named “357-L4”. The obtained vector expressing 348-L4 was named “pEF6KCL / 348-L4”, and the vector expressing 357-L4 was named “pEF6KCL / 357-L4”. The nucleotide sequence of 348-L4 is shown in SEQ ID NO: 81 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 82. The nucleotide sequence of 357-L4 is shown in SEQ ID NO: 83, and the amino acid sequence is shown in SEQ ID NO: 84.
　実施例４．ヒト化抗体の調製
　１）ヒト化抗体の生産
　１．２×１０９個の対数増殖期のＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞（Ｉｎｖｉｔｒｏｇｅｎ）を新鮮な１．２ＬのＦｒｅｅＳｔｙｌｅ２９３　ｅｘｐｒｅｓｓｉｏｎ　ｍｅｄｉｕｍ　（Ｉｎｖｉｔｒｏｇｅｎ）に播種し、３７℃、８％ＣＯ２インキュベーター内で９０ｒｐｍで一時間振とう培養した。Ｐｏｌｙｅｔｈｙｌｅｎｅｉｍｉｎｅ（Ｐｏｌｙｓｃｉｅｎｃｅ　＃２４７６５）３．６ｍｇをＯｐｔｉ－Ｐｒｏ　ＳＦＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）２０ｍｌに溶解し、次にＰｕｒｅＬｉｎｋ　ＨｉＰｕｒｅ　Ｐｌａｓｍｉｄキット（Ｉｎｖｉｔｒｏｇｅｎ社）を用いて調製したＨ鎖発現ベクター（０．４ｍｇ）及びＬ鎖発現ベクター（０．８ｍｇ）を２０ｍｌのＯｐｔｉ－Ｐｒｏ　ＳＦＭ培地に懸濁した。Ｐｏｌｙｅｔｈｙｌｅｎｅｉｍｉｎｅ／Ｏｐｔｉ－Ｐｒｏ　ＳＦＭ混合液２０ｍｌに、発現ベクター／Ｏｐｔｉ－Ｐｒｏ　ＳＦＭ混合液２０ｍｌを加え穏やかに攪拌し、さらに５分間放置した後にＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞に添加した。３７℃、８％ＣＯ２インキュベーターで７日間、９０ｒｐｍで振とう培養して得られた培養上清をＤｉｓｐｏｓａｂｌｅ　Ｃａｐｓｕｌｅ　Ｆｉｌｔｅｒ　（Ａｄｖａｎｔｅｃ　＃ＣＣＳ－０４５－Ｅ１Ｈ）でろ過した。 Example 4 Preparation of humanized antibody 1) Production of humanized antibody 1.2 × 10 9 log-type freestyle 293F cells (Invitrogen) were seeded in fresh 1.2 L FreeStyle293 expression medium (Invitrogen) at 37 ° C. The cells were cultured with shaking at 90 rpm for 1 hour in an 8% CO 2 incubator. 3.6 mg of Polyethyleneimine (Polyscience # 24765) dissolved in 20 ml of Opti-Pro SFM medium (Invitrogen), and then an H chain expression vector (0.4 mg) and L chain prepared using PureLink HiPure Plasmid kit (Invitrogen) The expression vector (0.8 mg) was suspended in 20 ml of Opti-Pro SFM medium. 20 ml of an expression vector / Opti-Pro SFM mixture was added to 20 ml of a polyethyleneimine / Opti-Pro SFM mixture, gently stirred, allowed to stand for another 5 minutes, and then added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 90 rpm in a 37 ° C., 8% CO 2 incubator for 7 days was filtered with a Disposable Capsule Filter (Advantec # CCS-045-E1H).
　ｐＥＦ６ＫＣＬ／ｈＳＨ３４８－１－Ｔ１ＬとｐＥＦ１ＦＣＣＵ／ｈＳＨ３４８－１－Ｔ１Ｈとの組合せによって取得されたＳＨ３４８－１のヒト化抗体を「ｈＳＨ３４８－１－Ｔ１」、ｐＥＦ６ＫＣＬ／ｈＳＨ３５７－１－Ｔ１ＬとｐＥＦ１ＦＣＣＵ／ｈＳＨ３５７－１－Ｔ１Ｈとの組合せによって取得されたＳＨ３５７－１のヒト化抗体を「ｈＳＨ３５７－１－Ｔ１」と命名した。 The humanized antibodies of SH348-1 obtained by the combination of pEF6KCL / hSH348-1-T1L and pEF1FCCU / hSH348-1-T1H are “hSH348-1-T1”, pEF6KCL / hSH357-1-T1L and pEF1FCCU / hSH357- The humanized antibody of SH357-1 obtained by combination with 1-T1H was named “hSH357-1-T1”.
　２）ヒト化抗体の精製
　上記１）で得られた培養上清を、ｒＰｒｏｔｅｉｎＡアフィニティークロマトグラフィー（４－６℃下）とセラミックヒドロキシルアパタイト（室温下）の２段階工程で精製した。ｒＰｒｏｔｅｉｎＡアフィニティークロマトグラフィー精製後とセラミックヒドロキシルアパタイト精製後のバッファー置換工程は室温下で実施した。最初に、培養上清１１００－１２００ｍｌを、ＰＢＳで平衡化したＭａｂＳｅｌｅｃｔＳｕＲｅ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐカラム：容積１ｍｌ×２連結）にアプライした。培養液がカラムに全て入ったのち、ＰＢＳ１５－３０ｍｌでカラムを洗浄した。次に２Ｍアルギニン塩酸塩溶液（ｐＨ４．０）で溶出し、抗体の含まれる画分を集めた。その画分を脱塩カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐ　Ｄｅｓａｌｔｉｎｇカラム：容積５ｍｌ×２連結）で５ｍＭ　りん酸ナトリウム／５０ｍＭ　ＭＥＳ／２０ｍＭ　ＮａＣｌ／ｐＨ６．５のバッファーへ置換を行った。さらにその置換した抗体溶液を、５ｍＭ　ＮａＰｉ／５０ｍＭ　ＭＥＳ／２０ｍＭ　ＮａＣｌ／ｐＨ６．５のバッファーで平衡化されたセラミックハイドロキシルアパタイトカラム（日本バイオラッド、Ｂｉｏ－Ｓｃａｌｅ　ＣＨＴ２－１　Ｈｙｄｒｏｘｙａｐａｔｉｔｅ　Ｃｏｌｕｍｎ：容積２ｍｌ）にアプライした。塩化ナトリウムによる直線的濃度勾配溶出を実施し、抗体の含まれる画分を集めた。その画分を脱塩カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐ　Ｄｅｓａｌｔｉｎｇカラム：容積５ｍｌｘ２連結）でＣＢＳ（１０ｍＭクエン酸緩衝液／１４０ｍＭ塩化ナトリウム、ｐＨ６．０）への液置換を行った。最後にＣｅｎｔｒｉｆｕｇａｌ　ＵＦ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅ　ＶＩＶＡＳＰＩＮ２０（分画分子量３０Ｋ，Ｓａｒｔｏｒｉｕｓ社，４℃下）にて濃縮し、ＩｇＧ濃度を１．０ｍｇ／ｍｌ以上に調製し精製サンプルとした。 2) Purification of humanized antibody The culture supernatant obtained in 1) above was purified in two steps, rProtein A affinity chromatography (4-6 ° C.) and ceramic hydroxylapatite (room temperature). The buffer replacement step after purification of rProtein A affinity chromatography and after purification of ceramic hydroxylapatite was carried out at room temperature. First, 1100-1200 ml of the culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column: volume 1 ml × 2 linked) equilibrated with PBS. After all of the culture solution entered the column, the column was washed with 15-30 ml of PBS. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected. The fraction was replaced with a 5 mM sodium phosphate / 50 mM MES / 20 mM NaCl / pH 6.5 buffer using a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Further, the substituted antibody solution was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT2-1 Hydroxyapatite Column: volume 2 ml) equilibrated with 5 mM NaPi / 50 mM MES / 20 mM NaCl / pH 6.5 buffer. Applied. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with CBS (10 mM citrate buffer / 140 mM sodium chloride, pH 6.0) with a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Finally, it was concentrated with Centrifugal UF Filter Device VASPASP20 (fractionated molecular weight 30K, Sartorius, 4 ° C.), and the IgG concentration was adjusted to 1.0 mg / ml or more to obtain a purified sample.
　実施例５．ヒト化抗体の発現ベクターの構築
　１）３４８－Ｌ５、及び３５７－Ｌ５タイプ軽鎖発現ベクターの構築
　配列番号８６のアミノ酸番号２１乃至１３７に示される３４８－Ｌ５タイプ軽鎖可変領域、及び配列番号８８のアミノ酸番号２１乃至１３７に示される３５７－Ｌ５タイプ軽鎖可変領域をコードする遺伝子を含むＤＮＡを合成し（ＧＥＮＥＡＲＴ社、人工遺伝子合成サービス）、制限酵素ＮｄｅＩ及びＢｓｉＷＩで切り出されたＤＮＡ断片を、ヒト化抗体軽鎖発現汎用ベクター（ｐＥＦ６ＫＣＬ）を制限酵素ＮｄｅＩ及びＢｓｉＷＩで切断した箇所に挿入することにより、３４８－Ｌ５、及び３５７－Ｌ５タイプ軽鎖発現ベクターを構築した。得られた発現ベクターをそれぞれ「ｐＥＦ６ＫＣＬ／３４８－Ｌ５」、及び「ｐＥＦ６ＫＣＬ／３５７－Ｌ５」と命名した。 Example 5 FIG. Construction of humanized antibody expression vector 1) Construction of 348-L5 and 357-L5 type light chain expression vector 348-L5 type light chain variable region represented by amino acid numbers 21 to 137 of SEQ ID NO: 86, and SEQ ID NO: 88 DNA comprising a gene encoding the 357-L5 type light chain variable region represented by amino acid numbers 21 to 137 (GENEART, artificial gene synthesis service), and a DNA fragment cleaved with restriction enzymes NdeI and BsiWI, 348-L5 and 357-L5 type light chain expression vectors were constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI. The obtained expression vectors were named “pEF6KCL / 348-L5” and “pEF6KCL / 357-L5”, respectively.
　２）３４８―Ｈ４、３４８－Ｈ５、３５７－Ｈ４、及び３５７－Ｈ５タイプ重鎖発現ベクターの構築
　配列表の配列番号９０のアミノ酸番号２０乃至１３８に示される３４８－Ｈ４タイプ重鎖可変領域、配列番号９２のアミノ酸番号２０乃至１３８に示される３４８－Ｈ５タイプ重鎖可変領域、配列番号９４のアミノ酸番号２０乃至１３８に示される３５７－Ｈ４タイプ重鎖可変領域、及び配列番号９６のアミノ酸番号２０乃至１３８にそれぞれ示される、３５７－Ｈ５タイプ重鎖可変領域をコードする遺伝子を含むＤＮＡを合成し（ＧＥＮＥＡＲＴ社　人工遺伝子合成サービス）、制限酵素ＢｌｐＩで切り出されるＤＮＡ断片を、ヒト化抗体重鎖発現汎用ベクター（ｐＥＦ１ＦＣＣＵ）を制限酵素ＢｌｐＩで切断した箇所に挿入することにより、３４８―Ｈ４、３４８－Ｈ５、３５７－Ｈ４、及び３５７－Ｈ５タイプ重鎖発現ベクターを構築した。得られた発現ベクターをそれぞれ「ｐＥＦ１ＦＣＣＵ／３４８―Ｈ４」、「ｐＥＦ１ＦＣＣＵ／３４８―Ｈ５」、「ｐＥＦ１ＦＣＣＵ／３５７―Ｈ４」、及び「ｐＥＦ１ＦＣＣＵ／３５７―Ｈ５」と命名した。 2) Construction of 348-H4, 348-H5, 357-H4, and 357-H5 type heavy chain expression vectors 348-H4 type heavy chain variable region shown in amino acid numbers 20 to 138 of SEQ ID NO: 90 in the sequence listing, sequence 348-H5 type heavy chain variable region represented by amino acid numbers 20 to 138 of number 92, 357-H4 type heavy chain variable region represented by amino acid numbers 20 to 138 of SEQ ID NO: 94, and amino acid numbers 20 to 20 of SEQ ID NO: 96 A DNA fragment containing a gene encoding the 357-H5 type heavy chain variable region shown in 138 (synthetic gene synthesis service of GENEART) and a DNA fragment cleaved by the restriction enzyme BlpI By inserting the vector (pEF1FCCU) into the site cut with the restriction enzyme BlpI It was constructed 348-H4,348-H5,357-H4, and 357-H5-type heavy chain expression vectors. The obtained expression vectors were named “pEF1FCCU / 348-H4”, “pEF1FCCU / 348-H5”, “pEF1FCCU / 357-H4”, and “pEF1FCCU / 357-H5”, respectively.
　各抗体の配列を図１、図２に記載した。 The sequence of each antibody is shown in FIG. 1 and FIG.
　実施例６．ヒト化抗体の調製
　１）ヒト化抗体の生産
　実施例４に記載の方法と同様の方法によって抗体を取得した。 Example 6 Preparation of humanized antibody 1) Production of humanized antibody Antibody was obtained by the same method as described in Example 4.
　ｐＥＦ１ＦＣＣＵ／３４８―Ｈ４とｐＥＦ６ＫＣＬ／３４８－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化抗体を「３４８－Ｈ４－３４８－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ４とｐＥＦ６ＫＣＬ／３４８－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化抗体を「３４８－Ｈ４－３４８－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ５とｐＥＦ６ＫＣＬ／３４８－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化抗体を「３４８－Ｈ５－３４８－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ５とｐＥＦ６ＫＣＬ／３４８－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化抗体を「３４８－Ｈ５－３４８－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ４とｐＥＦ６ＫＣＬ／３５７－Ｌ４との組合せによって取得されたＳＨ３５７－１のヒト化抗体を「３５７－Ｈ４－３５７－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ４とｐＥＦ６ＫＣＬ／３５７－Ｌ５との組合せによって取得されたＳＨ３５７－１のヒト化抗体を「３５７－Ｈ４－３５７－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ５とｐＥＦ６ＫＣＬ／３５７－Ｌ４との組合せによって取得されたＳＨ３５７－１のヒト化抗体を「３５７－Ｈ５－３５７－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ５とｐＥＦ６ＫＣＬ／３５７－Ｌ５との組合せによって取得されたＳＨ３５７－１のヒト化抗体を「３５７－Ｈ５－３５７－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ４とｐＥＦ６ＫＣＬ／３５７－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化重鎖とＳＨ３５７－１のヒト化軽鎖からなる抗体を「３４８－Ｈ４－３５７－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ４とｐＥＦ６ＫＣＬ／３５７－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化重鎖とＳＨ３５７－１のヒト化軽鎖からなる抗体を「３４８－Ｈ４－３５７－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ５とｐＥＦ６ＫＣＬ／３５７－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化重鎖とＳＨ３５７－１のヒト化軽鎖からなる抗体を「３４８－Ｈ５－３５７－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３４８―Ｈ５とｐＥＦ６ＫＣＬ／３５７－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化重鎖とＳＨ３５７－１のヒト化軽鎖からなる抗体を「３４８－Ｈ５－３５７－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ４とｐＥＦ６ＫＣＬ／３４８－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化軽鎖とＳＨ３５７－１のヒト化重鎖からなる抗体を「３５７－Ｈ４－３４８－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ４とｐＥＦ６ＫＣＬ／３４８－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化軽鎖とＳＨ３５７－１のヒト化重鎖からなる抗体を「３５７－Ｈ４－３４８－Ｌ５」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ５とｐＥＦ６ＫＣＬ／３４８－Ｌ４との組合せによって取得されたＳＨ３４８－１のヒト化軽鎖とＳＨ３５７－１のヒト化重鎖からなる抗体を「３５７－Ｈ５－３４８－Ｌ４」、ｐＥＦ１ＦＣＣＵ／３５７―Ｈ５とｐＥＦ６ＫＣＬ／３４８－Ｌ５との組合せによって取得されたＳＨ３４８－１のヒト化軽鎖とＳＨ３５７－１のヒト化重鎖からなる抗体を「３５７－Ｈ５－３４８－Ｌ５」と命名した。 The humanized antibody of SH348-1 obtained by the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L4 is “348-H4-348-L4”, the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L5 The humanized antibody of SH348-1 obtained by the combination of “348-H4-348-L5”, the combination of pEF1FCCU / 348-H5 and pEF6KCL / 348-L4 was obtained by “348-H4-348-L5”. -H5-348-L4 ", the humanized antibody of SH348-1 obtained by the combination of pEF1FCCU / 348-H5 and pEF6KCL / 348-L5," 348-H5-348-L5 ", pEF1FCCU / 357-H4 and In combination with pEF6KCL / 357-L4 The humanized antibody of SH357-1 obtained by the combination of “357-H4-357-L4”, pEF1FCCU / 357-H4 and pEF6KCL / 357-L5 was obtained as the humanized antibody of SH357-1. 357-H4-357-L5 ”, the humanized antibody of SH357-1 obtained by the combination of pEF1FCCU / 357-H5 and pEF6KCL / 357-L4 is“ 357-H5-357-L4 ”, pEF1FCCU / 357-H5 SH357-1 obtained by combining “357-H5-357-L5”, a combination of pEF1FCCU / 348-H4 and pEF6KCL / 357-L4 with a humanized antibody of SH357-1 obtained by the combination of pEF6KCL / 357-L5 with pEF6KCL / 357-L5 From the humanized heavy chain of -1 and the humanized light chain of SH357-1 The antibody consists of a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H4-357-L4”, pEF1FCCU / 348-H4 and pEF6KCL / 357-L5 An antibody comprising a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H4-357-L5”, pEF1FCCU / 348-H5 and pEF6KCL / 357-L4 An antibody comprising a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H5-357-L4”, pEF1FCCU / 348-H5 and pEF6KCL / 357-L5 “348-H5-357-L5”, pEF1FCCU / 357-H4 and pEF6KCL / 348-L4 An antibody comprising a humanized light chain of SH348-1 and a humanized heavy chain of SH357-1 obtained by the combination of “357-H4-348-L4”, pEF1FCCU / 357-H4 and pEF6KCL / 348-L5 An antibody comprising a humanized light chain of SH348-1 and a humanized heavy chain of SH357-1 obtained by the combination is “357-H4-348-L5”, a combination of pEF1FCCU / 357-H5 and pEF6KCL / 348-L4 The antibody consisting of the humanized light chain of SH348-1 and the humanized heavy chain of SH357-1 obtained by the method described above by “357-H5-348-L4”, a combination of pEF1FCCU / 357-H5 and pEF6KCL / 348-L5 The obtained antibody comprising the humanized light chain of SH348-1 and the humanized heavy chain of SH357-1 is designated as “357. It was named H5-348-L5 ".
　２）ヒト化抗体の精製
　上記１）で得られた培養上清を、ｒＰｒｏｔｅｉｎＡアフィニティークロマトグラフィー（４－６℃下）とセラミックヒドロキシルアパタイト（室温下）の２段階工程で精製した。ｒＰｒｏｔｅｉｎＡアフィニティークロマトグラフィー精製後とセラミックヒドロキシルアパタイト精製後のバッファー置換工程は室温下で実施した。最初に、培養上清１１００－１２００ｍｌを、ＰＢＳで平衡化したＭａｂＳｅｌｅｃｔＳｕＲｅ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐカラム：容積１ｍｌ×２連結）にアプライした。培養液がカラムに全て入ったのち、ＰＢＳ１５－３０ｍｌでカラムを洗浄した。次に２Ｍアルギニン塩酸塩溶液（ｐＨ４．０）で溶出し、抗体の含まれる画分を集めた。その画分を脱塩カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐ　Ｄｅｓａｌｔｉｎｇカラム：容積５ｍｌ×２連結）で５ｍＭ　リン酸ナトリウム／５０ｍＭ　ＭＥＳ／２０ｍＭ　ＮａＣｌ／ｐＨ６．５のバッファーへ置換を行った。さらにその置換した抗体溶液を、５ｍＭ　ＮａＰｉ／５０ｍＭ　ＭＥＳ／２０ｍＭ　ＮａＣｌ／ｐＨ６．５のバッファーで平衡化されたセラミックハイドロキシルアパタイトカラム（日本バイオラッド、Ｂｉｏ－Ｓｃａｌｅ　ＣＨＴ２－１　Ｈｙｄｒｏｘｙａｐａｔｉｔｅ　Ｃｏｌｕｍｎ：容積２ｍｌ）にアプライした。塩化ナトリウムによる直線的濃度勾配溶出を実施し、抗体の含まれる画分を集めた。その画分を脱塩カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製、ＨｉＴｒａｐ　Ｄｅｓａｌｔｉｎｇカラム：容積５ｍｌｘ２連結）でＣＢＳ（１０ｍＭクエン酸緩衝液／１４０ｍＭ塩化ナトリウム、ｐＨ６．０）への液置換を行った。最後にＣｅｎｔｒｉｆｕｇａｌ　ＵＦ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅ　ＶＩＶＡＳＰＩＮ２０（分画分子量３０Ｋ，Ｓａｒｔｏｒｉｕｓ社，４℃下）にて濃縮し、ＩｇＧ濃度を１．０ｍｇ／ｍｌ以上に調製し精製サンプルとした。 2) Purification of humanized antibody The culture supernatant obtained in 1) above was purified in two steps, rProtein A affinity chromatography (4-6 ° C.) and ceramic hydroxylapatite (room temperature). The buffer replacement step after purification of rProtein A affinity chromatography and after purification of ceramic hydroxylapatite was carried out at room temperature. First, 1100-1200 ml of the culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column: volume 1 ml × 2 linked) equilibrated with PBS. After all of the culture solution entered the column, the column was washed with 15-30 ml of PBS. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected. The fraction was replaced with a 5 mM sodium phosphate / 50 mM MES / 20 mM NaCl / pH 6.5 buffer using a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Further, the substituted antibody solution was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT2-1 Hydroxyapatite Column: volume 2 ml) equilibrated with 5 mM NaPi / 50 mM MES / 20 mM NaCl / pH 6.5 buffer. Applied. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with CBS (10 mM citrate buffer / 140 mM sodium chloride, pH 6.0) with a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Finally, it was concentrated with Centrifugal UF Filter Device VASPASP20 (fractionated molecular weight 30K, Sartorius, 4 ° C.), and the IgG concentration was adjusted to 1.0 mg / ml or more to obtain a purified sample.
　実施例７．抗ＥＰＨＡ２抗体のＫｄ値の測定
　実施例６で得られた抗ＥＰＨＡ２抗体とＦｉｂｒｏｎｅｃｔｉｎ　ｔｙｐｅ　ＩＩＩ　ｄｏｍａｉｎとの解離定数測定を以下のように行った。 Example 7 Measurement of Kd value of anti-EPHA2 antibody The dissociation constant of the anti-EPHA2 antibody obtained in Example 6 and Fibronctin type III domain was measured as follows.
　Ｈｉｓ－Ｆｉｂｒｏｎｅｃｔｉｎ　ｔｙｐｅ　ＩＩＩ　ｄｏｍａｉｎ　（４２６－５４０）（配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列からなるポリペプチドのアミノ末端にヒスチジンタグを付加したポリペプチド）を調製した。Ｂｉａｃｏｒｅ　Ｔ１００　（ＧＥヘルスケアバイオサイエンス（株））を使用し、固定化した抗ヒトＩｇＧ（Ｆｃ）抗体に抗体を捕捉（キャプチャー）し、抗原をアナライトとして測定するキャプチャー法にて行った。抗ヒトＩｇＧ（Ｆｃ）抗体（Ｈｕｍａｎ　ａｎｔｉｂｏｄｙ　ｃａｐｔｕｒｅ　ｋｉｔ、ＧＥヘルスケアバイオサイエンス（株））は、センサーチップＣＭ５（ＢＩＡｃｏｒｅ，Ｉｎｃ．）へアミンカップリング法にて～１０，０００ＲＵ共有結合させた。リファレンスセルにも同様に固定化した。ランニングバッファーとしてＨＢＳ－ＥＰ　（１０ｍＭ　ＨＥＰＥＳ　ｐＨ７．４、０．１５Ｍ　ＮａＣｌ，　３ｍＭ　ＥＤＴＡ、　０．０５％　Ｓｕｒｆａｃｔａｎｔ　Ｐ２０）を用いた。抗ヒトＩｇＧ（Ｆｃ）抗体を固定化したチップ上に、約２０ｎＭの抗体溶液を流速１０μＬ／分で６０秒間添加した後、Ｈｉｓ－Ｆｉｂｒｏｎｅｃｔｉｎ　ｔｙｐｅ　ＩＩＩ　ｄｏｍａｉｎ　（４２６－５４０）の希釈系列溶液（３．１３－　５０ｎＭ）を流速３０μｌ／分で１２０秒間添加し、引き続き１８０秒間の解離相をモニターした。再生溶液として、３Ｍ　塩化マグネシウムを流速１０μｌ／分で３０秒間添加した。データの解析には、分析ソフトウェア（Ｂｉａｃｏｒｅ　Ｔ１００　Ｅｖａｌｕａｔｉｏｎ　ｓｏｆｔｗａｒｅ，　ｖｅｒｓｉｏｎ　２．０．１）の１対１結合モデルを用いて、結合速度定数ｋｏｎ、解離速度定数ｋｏｆｆおよび解離定数（Ｋｄ；Ｋｄ＝ｋｏｆｆ／ｋｏｎ）を算出した。 His-Fibrinctin type III domain (426-540) (polypeptide having a histidine tag added to the amino terminus of a polypeptide consisting of the amino acid sequence of amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing) was prepared. Using Biacore T100 (GE Healthcare Bioscience Co., Ltd.), the antibody was captured (captured) on an immobilized anti-human IgG (Fc) antibody, and the capture method was performed in which the antigen was measured as an analyte. An anti-human IgG (Fc) antibody (Human antibody capture kit, GE Healthcare Biosciences) was covalently bound to a sensor chip CM5 (BIAcore, Inc.) up to 10,000 RU by the amine coupling method. The reference cell was similarly fixed. HBS-EP (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer. An anti-human IgG (Fc) antibody-immobilized chip was added with an antibody solution of about 20 nM at a flow rate of 10 μL / min for 60 seconds, and then a His-Fibroctin type III domain (426-540) dilution series solution (3. 13-50 nM) was added at a flow rate of 30 μl / min for 120 seconds, followed by monitoring the dissociation phase for 180 seconds. As a regeneration solution, 3M magnesium chloride was added at a flow rate of 10 μl / min for 30 seconds. For the analysis of the data, a one-to-one binding model of analysis software (Biacore T100 Evaluation software, version 2.0.1) was used, and the association rate constant kon, the dissociation rate constant koff and the dissociation constant (Kd; Kd = koff / kon) was calculated.
　その結果、ＳＨ３４８－１由来のヒト化重鎖とＳＨ３５７－１由来のヒト化軽鎖を組み合わせた抗体において、ＳＨ３４８－１のヒト化抗体やＳＨ３５７－１のヒト化抗体に比してＫｄ値が顕著に低下し、抗原に対する結合活性が上昇していることが明らかとなった（表１）。ヒト化抗体ｈ３４８－１　Ｔ１のＫｄ値８．２９（ｎＭ）に対して、３４８－Ｈ４－３５７－Ｌ４で０．３７（ｎＭ）、３４８Ｈ４－３５７－Ｌ５で０．３７（ｎＭ）、３４８－Ｈ５－３５７－Ｌ４で０．３２（ｎＭ）、３４８－Ｈ５－３５７－Ｌ５で、０．３０（ｎＭ）となっており、結合活性が顕著に上昇していた。 As a result, in the combination of the humanized heavy chain derived from SH348-1 and the humanized light chain derived from SH357-1, the Kd value is higher than that of the humanized antibody of SH348-1 or the humanized antibody of SH357-1. It was found that the binding activity against the antigen was significantly decreased (Table 1). Humanized antibody h348-1 Td Kd value 8.29 (nM), 348-H4-357-L4 0.37 (nM), 348H4-357-L5 0.37 (nM), 348- H5-357-L4 was 0.32 (nM) and 348-H5-357-L5 was 0.30 (nM), indicating that the binding activity was significantly increased.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　実施例８．ＡＤＣＣ活性の測定
　ＥＧＦＰ遺伝子を導入した前立腺癌細胞株ＰＣ－３を標的細胞、ヒト末梢血リンパ球（ＢＩＯＰＲＥＤＩＣ　＃ＰＢＬ００３　以下、「ヒトＰＢＬ」と記す）をエフェクター細胞として、培養上清中に放出されるＥＧＦＰの蛍光量を測定することで抗体依存性細胞傷害活性（ＡＤＣＣ活性）を算出した。１０％　Ｕｌｔｒａｌｏｗ　ＩｇＧ　ＦＢＳ（Ｉｎｖｉｔｒｏｇｅｎ　＃１６２５０－０７８）を含有するフェノールレッド不含ＲＰＭＩ１６４０（Ｉｎｖｉｔｒｏｇｅｎ　＃１１８３５－０３０）培地に懸濁したＥＧＦＰ発現ＰＣ－３細胞を１×１０４個／ウェルで９６穴マイクロプレートに播種し、３７℃、５％ＣＯ２の条件下で一晩培養した。培地を取り除いた後、新鮮な培地で希釈されたヒト化抗ＥＰＨＡ２抗体またはアイソタイプコントロール抗体であるｈＩｇＧ１（Ａｌｅｘｉｓ　Ｂｉｏｃｈｅｍｉｃａｌｓ　＃ＡＬＸ－８０４－１３３）（それぞれ、終濃度で０．１、１．０、１０μｇ／ｍＬ）を添加し、さらに２×１０５個／ウェルのヒトＰＢＬを添加して、３７℃、５％ＣＯ２の条件下で一晩培養した。培養上清を回収し、上清中の蛍光量（４８５／５３５ｎｍ）を測定した。ＡＤＣＣ活性による細胞溶解率は次式で算出した。
細胞溶解率（％）＝（Ａ－Ｂ）／（Ｃ－Ｂ）×１００
Ａ：サンプルウェルの培養上清の蛍光量
Ｂ：抗体・エフェクター細胞非添加ウェルの培養上清の蛍光量の平均値。
Ｃ：標的細胞を終濃度１％のＴｒｉｔｏｎ－Ｘ１００で溶解させたウェルの培養上清の蛍光量の平均値。
図３は、３サンプルの平均値で、エラーバーは、標準偏差を示す。その結果、０．１μｇ／ｍＬの抗体濃度において３４８－Ｈ４－３５７－Ｌ４、３４８－Ｈ４－３５７－Ｌ５、３４８－Ｈ５－３５７－Ｌ５はｈＳＨ３５７－１－Ｔ１および３５７－Ｈ４－３５７－Ｌ４よりも高いＡＤＣＣ活性を示し、０．１μｇ／ｍＬの抗体濃度での３４８－Ｈ４－３５７－Ｌ４、３４８－Ｈ４－３５７－Ｌ５、３４８－Ｈ５－３５７－Ｌ５のＡＤＣＣ活性は、それよりも１０倍高い濃度である１．０μｇ／ｍＬのｈＳＨ３５７－１－Ｔ１および３５７－Ｈ４－３５７－Ｌ４のＡＤＣＣ活性と同程度であることが示された。 Example 8 FIG. Measurement of ADCC activity The prostate cancer cell line PC-3 introduced with the EGFP gene is used as a target cell, and human peripheral blood lymphocytes (BIOPREDIC # PBL003, hereinafter referred to as “human PBL”) are used as effector cells and released into the culture supernatant. The antibody-dependent cytotoxic activity (ADCC activity) was calculated by measuring the amount of fluorescence of EGFP. 96-well microwells of EGFP-expressing PC-3 cells suspended in phenol red-free RPMI 1640 (Invitrogen # 11835-030) medium containing 10% Ultralow IgG FBS (Invitrogen # 16250-078) at 1 × 10 4 cells / well. The cells were seeded and cultured overnight at 37 ° C. and 5% CO 2 . After removing the medium, humanized anti-EPHA2 antibody or isotype control antibody hIgG1 (Alexis Biochemicals # ALX-804-133) diluted in fresh medium (final concentrations 0.1, 1.0, 10 μg, respectively) / ML), 2 × 10 5 cells / well of human PBL were added, and the cells were cultured overnight under conditions of 37 ° C. and 5% CO 2 . The culture supernatant was collected and the amount of fluorescence (485/535 nm) in the supernatant was measured. The cell lysis rate due to ADCC activity was calculated by the following formula.
Cell lysis rate (%) = (AB) / (CB) × 100
A: Fluorescence amount of culture supernatant of sample well B: Average value of fluorescence amount of culture supernatant of well without antibody / effector cells.
C: Average value of the fluorescence amount of the culture supernatant of a well in which target cells were lysed with Triton-X100 at a final concentration of 1%.
FIG. 3 shows an average value of three samples, and error bars indicate standard deviation. As a result, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 were obtained from hSH357-1-T1 and 357-H4-357-L4 at an antibody concentration of 0.1 μg / mL. The ADCC activity of 348-H4-357-L4, 348-H4-357-L5, and 348-H5-357-L5 at an antibody concentration of 0.1 μg / mL is 10 times higher than that. It was shown to be comparable to the ADCC activity of high concentrations of 1.0 μg / mL hSH357-1-T1 and 357-H4-357-L4.
　実施例９．抗原特異的結合
　ヒト化抗ＥＰＨＡ２抗体の結合特異性を検証するため、ヒトＥＰＨＡ２の４３９から５３４番目までのアミノ酸配列（配列表の配列番号４のアミノ酸番号４３９乃至５３４に示されるアミノ酸配列）と相同性の高い蛋白質との結合性をＣｅｌｌ－ＥＬＩＳＡ法により検討した。相同性検索はＮＣＢＩのＢＬＡＳＴを利用して行い、ヒトＥＰＨＡ２の４３９から５３４番目までのアミノ酸配列をヒト蛋白質に対して検索した。ヒットした蛋白質のうち、上位２蛋白質（ＥＰＨＡ３　ｉｓｏｆｏｒｍ　ａ（以下及び図中で「ＥＰＨＡ３ａ」と記す）及びヒトＰｒｏｔｅｉｎ　ｔｙｒｏｓｉｎｅ　ｐｈｏｓｐｈａｔａｓｅ，ｒｅｃｅｐｔｏｒ　ｔｙｐｅ，ｓｉｇｍａ　ｉｓｏｆｏｒｍ　４（以下及び図中で「ＰＴＰＲＳ－４」と記す））を用いて解析した。
これらの蛋白質はいずれもＮ末端が細胞外領域となる膜貫通蛋白質であることから、Ｎ末端にＦＬＡＧ－Ｔａｇが付加された蛋白質を発現するコンストラクトを作製するため、まずはじめにｐＦＬＡＧ－ｍｙｃ－ＣＭＶ１９（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ　＃Ｅ８６５８）をＨｉｎｄＩＩＩとＢｇｌＩＩで切断して平滑化したサイトにＧａｔｅｗａｙ　Ｖｅｃｔｏｒ　Ｃｏｎｖｅｒｓｉｏｎ　Ｓｙｓｔｅｍ（Ｉｎｖｉｔｒｏｇｅｎ　＃１１８２８－０２９）のＲｅａｄｉｎｇ　Ｆｒａｍｅ　Ｂを組み込み、「ｐＦＬＡＧ－ＧＷ」を作成した。次にシグナル配列（開始コドンから２３番目までのアミノ酸配列）を除いたヒトＥＰＨＡ２、シグナル配列（開始コドンから２０番目までのアミノ酸配列）を除いたヒトＥＰＨＡ３ａ、またはシグナル配列（開始コドンから２９番目までのアミノ酸配列）を除いたヒトＰＴＰＲＳ－４のアミノ酸配列をコードする遺伝子がクローニングされたエントリーベクターとｐＦＬＡＧ－ＧＷとの間でＬＲ　Ｃｌｏｎａｓｅ（Ｉｎｖｉｔｒｏｇｅｎ　＃１１７９１－０１９）を用いた組換え反応を行い、発現ベクター「ｐＦＬＡＧ－ＥＰＨＡ２」、「ｐＦＬＡＧ－ＥＰＨＡ３ａ」、「ｐＦＬＡＧ－ＰＴＰＲＳ－４」を作成した。 Example 9 Antigen-specific binding In order to verify the binding specificity of the humanized anti-EPHA2 antibody, it is homologous to the amino acid sequence of amino acids 439 to 534 of human EPHA2 (the amino acid sequence shown in amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing) The binding property to highly protein was examined by Cell-ELISA method. The homology search was performed using NCBI BLAST, and the amino acid sequence from human 439 to 534 of human EPHA2 was searched for human protein. Among the hit proteins, the top 2 proteins (EPHA3 isoforma (hereinafter referred to as “EPHA3a” in the figure and in the figure)) and human protein tyrosin phosphate, receptor type, sigma isoform 4 (hereinafter referred to as “PTPRS-4” in the figure). )).
Since these proteins are transmembrane proteins whose N-terminus is an extracellular region, in order to produce a construct expressing a protein with FLAG-Tag added to the N-terminus, pFLAG-myc-CMV19 ( The reading frame B of Gateway Vector Conversion System (Invitrogen # 11828-029) was incorporated into a site obtained by cutting Sigma-Aldrich # E8658) with HindIII and BglII and smoothing to create “pFLAG-GW”. Next, human EPHA2 excluding the signal sequence (amino acid sequence from the start codon to the 23rd amino acid), human EPHA3a excluding the signal sequence (amino acid sequence from the start codon to the 20th), or the signal sequence (from the start codon to the 29th) A recombination reaction using LR Clonase (Invitrogen # 11791-019) between the entry vector in which the gene encoding the amino acid sequence of human PTPRS-4 (excluding the amino acid sequence of the above) was cloned and pFLAG-GW, Expression vectors “pFLAG-EPHA2”, “pFLAG-EPHA3a”, and “pFLAG-PTPRS-4” were prepared.
　Ｌｉｐｏｆｅｃｔａｍｉｎ２０００（Ｉｎｖｉｔｒｏｇｅｎ　＃１１６６８－０１９）を用いてｐＦＬＡＧ－ＧＷ、ｐＦＬＡＧ－ＥＰＨＡ２、ｐＦＬＡＧ－ＥＰＨＡ３ａ、またはｐＦＬＡＧ－ＰＴＰＲＳ－４をトランスフェクションした２９３αｖβ３細胞（ＨＥＫ２９３細胞にｉｎｔｅｇｒｉｎ　αＶ及びｉｎｔｅｇｒｉｎ　β３を安定的に発現させた細胞）を９６穴プレートに播種し、１０％　ＦＢＳ含有ＤＭＥＭ中で３７℃、５％　ＣＯ２の条件下で一晩培養した。培地を捨て、５％　ＦＢＳ含有ＰＢＳで希釈したヒト化抗ＥＰＨＡ２抗体（５μｇ／ｍＬまたは５０μｇ／ｍＬ）、ヒト化抗体の陰性コントロールとしてｈＩｇＧ（５μｇ／ｍＬまたは５０μｇ／ｍＬ）（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ　＃１０９－０３５－０９８）、Ｍｏｎｏｃｌｏｎａｌ　ＡＮＴＩ－ＦＬＡＧ　Ｍ２　ａｎｔｉｂｏｄｙ（０．０２μｇ／ｍＬまたは０．１μｇ／ｍＬ）（Ｓｉｇｍａ　＃Ｆ３１６５：及び図中で「Ａｎｔｉ－ＦＬＡＧ　ｍＡｂ」と記す）、Ａｎｔｉ－ＦＬＡＧ　ｍＡｂの陰性コントロールとしてｍＩｇＧ１（０．０２μｇ／ｍＬまたは０．１μｇ／ｍＬ）（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅ　＃５５３４４７）を添加し、４℃で１時間インキュベーションした。５％　ＦＢＳ含有ＰＢＳで細胞を洗浄後、ヒト化抗ＥＰＨＡ２抗体及びその陰性コントロール抗体の検出には５％　ＦＢＳ含有ＰＢＳで５００倍希釈したＰｅｒｏｘｉｄａｓｅ　ＡｆｆｉｎｉＰｕｒｅ　Ｇｏａｔ　Ａｎｔｉ－Ｈｕｍａｎ　ＩｇＧ　Ｆｃγ　Ｆｒａｇｍｅｎｔ　Ｓｐｅｃｉｆｉｃ（Ｊａｃｋｓｏｎ　ＩｍｍｕｎｏＲｅｓｅａｒｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ　＃１０９－０３５－０９８）を、Ａｎｔｉ－ＦＬＡＧ　ｍＡｂ及びその陰性コントロール抗体の検出には５００倍希釈したＧｏａｔ　ａｎｔｉ－Ｍｏｕｓｅ　ＩｇＧ　Ｐｅｒｏｘｉｄａｓｅ　Ｃｏｎｊｕｇａｔｅ（Ｍｉｌｌｉｐｏｒｅ　＃ＡＰ１８１Ｐ）を添加し、４℃で１時間インキュベーションした。５％　ＦＢＳ含有ＰＢＳで細胞を洗浄後、ＯＰＤにより発色反応を行い、１Ｍ　ＨＣｌで反応を停止後、４９０ｎｍの吸光度を測定した。図４は、３サンプルの平均値で、エラーバーは、標準偏差を示す。 293αvβ3 cells transfected with pFLAG-GW, pFLAG-EPHA2, pFLAG-EPHA3a, or pFLAG-PTPRS-4 using Lipofectamin 2000 (Invitrogen # 11668-019) were expressed by INTGrin αV and integrin β3 stably in HEK293 cells. Cells) were seeded in a 96-well plate and cultured overnight in DMEM containing 10% FBS under conditions of 37 ° C. and 5% CO 2 . Discard the medium and humanized anti-EPHA2 antibody (5 μg / mL or 50 μg / mL) diluted in PBS containing 5% FBS, hIgG (5 μg / mL or 50 μg / mL) as a negative control for humanized antibody (Jackson ImmunoResearch Laboratories # 109 -035-098), Monoclonal ANTI-FLAG M2 antibody (0.02 μg / mL or 0.1 μg / mL) (Sigma # F3165: and indicated as “Anti-FLAG mAb” in the figure), Anti-FLAG mAb negative As a control, mIgG1 (0.02 μg / mL or 0.1 μg / mL) (BD Bioscience # 553447) was added and incubated at 4 ° C. for 1 hour. After washing the cells with PBS containing 5% FBS, for detection of humanized anti-EPHA2 antibody and its negative control antibody, Peroxidase AffiniPure Goat Anti-Human IgG Fcγ Fragment Specific (Jackson Immense Immense) was diluted 500 times with PBS containing 5% FBS. 109-035-098) was added 500-fold diluted Goat anti-Mouse IgG Peroxidase Conjugate (Millipore # AP181P) for the detection of Anti-FLAG mAb and its negative control antibody and incubated at 4 ° C. for 1 hour. After washing the cells with PBS containing 5% FBS, color reaction was performed by OPD, and the reaction was stopped with 1M HCl, and the absorbance at 490 nm was measured. FIG. 4 shows an average value of three samples, and error bars indicate standard deviation.
　各蛋白質の細胞膜上への発現量をＡｎｔｉ－ＦＬＡＧ　ｍＡｂを用いて確認したところ、いずれの蛋白質も細胞膜上への同程度の発現が認められた（図４　Ａ）。一方で、ヒト化抗ＥＰＨＡ２抗体であるｈＳＨ３５７－１－Ｔ１、３４８－Ｈ４－３５７－Ｌ４、３４８－Ｈ４－３５７－Ｌ５、３４８－Ｈ５－３５７－Ｌ５は、ヒトＥＰＨＡ２を発現する細胞に対して強く結合するのに対し、ヒトＥＰＨＡ３ａ及びヒトＰＴＰＲＳ－４を発現する細胞に対しては陰性コントロールであるｐＦＬＡＧ－ＧＷをトランスフェクションした細胞と同程度の結合性しか示さなかった（図４　Ｂ－Ｅ）。以上の結果より、これらのヒト化抗ＥＰＨＡ２抗体がヒトＥＰＨＡ２を特異的に認識し結合することが示された。 When the expression level of each protein on the cell membrane was confirmed using Anti-FLAG mAb, the same level of expression on the cell membrane was observed for each protein (FIG. 4A). On the other hand, the humanized anti-EPHA2 antibodies hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 are against human EPHA2 expressing cells. While binding strongly, cells expressing human EPHA3a and human PTPRS-4 showed only the same level of binding as cells transfected with pFLAG-GW, which is a negative control (FIG. 4, BE). ). From the above results, it was shown that these humanized anti-EPHA2 antibodies specifically recognize and bind to human EPHA2.
　実施例１０．Ｉｎ　ｖｉｖｏ抗腫瘍活性の確認
　ヒト乳癌細胞株ＭＤＡ－ＭＢ－２３１細胞をトリプシン処理して培養フラスコより剥がした後、１０％　ＦＢＳ含有Ｌｅｉｂｏｖｉｔｚ‘ｓ　Ｌ－１５に懸濁後遠心し、上清を除去した。細胞を同培地で１回洗浄したあと、ＢＤマトリゲル基底膜マトリックス（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ　＃３５４２３４）に懸濁し、６週齢のヌードマウス（ＣＡｎＮ．Ｃｇ－Ｆｏｘｎ１［ｎｕ］／ＣｒｌＣｒｌｊ〔Ｆｏｘｎ１ｎｕ／Ｆｏｘｎ１ｎｕ〕：日本チャールスリバー）の右腋窩部第２第３乳頭間皮下に５×１０６細胞／匹で移植した。移植日を０日目として１７日目に群分けを行い、１７、２４、３１日目にｈＳＨ３５７－１－Ｔ１、３４８－Ｈ４－３５７－Ｌ４、３４８－Ｈ４－３５７－Ｌ５、または３４８－Ｈ５－３５７－Ｌ５を０．０１ｍｇ／匹または０．０３ｍｇ／匹で投与した。Ｖｅｈｉｃｌｅ群は抗体と等量のｖｅｈｉｃｌｅを投与した。腫瘍体積は１７、２４、３１、３８日目に測定した。図５は、１群あたり９匹の腫瘍体積の平均値で、エラーバーは標準誤差を示す。また、統計解析は３８日時点の値を用い、Ｄｕｎｎｅｔｔ‘ｓ　ｍｕｌｔｉｐｌｅ　ｃｏｍｐａｒｉｓｏｎ　ｔｅｓｔにより行った（＊：Ｐ＜０．０５、＊＊：Ｐ＜０．０１、＊＊＊：Ｐ＜０．００１）。 Example 10 Confirmation of in vivo antitumor activity Human breast cancer cell line MDA-MB-231 cells were trypsinized and detached from the culture flask, suspended in Leibovitz's L-15 containing 10% FBS, and centrifuged to remove the supernatant. did. The cells were washed once with the same medium, then suspended in a BD Matrigel basement membrane matrix (BD Biosciences # 354234), and 6-week-old nude mice (CAnN.Cg-Foxn1 [nu] / CrlCrlj [Foxn1nu / Foxn1nu]: Japan (Charles River) was transplanted subcutaneously between the second and third nipples of the right axilla at 5 × 10 6 cells / animal. Grouping was performed on day 17 with the day of transplantation as day 0, and on days 17, 24, 31 hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, or 348-H5 -357-L5 was administered at 0.01 mg / animal or 0.03 mg / animal. The vehicle group was administered with the same amount of vehicle as the antibody. Tumor volume was measured on days 17, 24, 31, and 38. FIG. 5 shows the mean value of 9 tumor volumes per group, and error bars indicate standard errors. Further, statistical analysis was performed by Dunnett's multiple comparison test using values at 38 days (**: P <0.05, **: P <0.01, ***: P <0.001). .
　その結果、ｈＳＨ３５７－１－Ｔ１、３４８－Ｈ４－３５７－Ｌ４、３４８－Ｈ４－３５７－Ｌ５、３４８－Ｈ５－３５７－Ｌ５の全てのヒト化抗ＥＰＨＡ２抗体において０．０３ｍｇ／匹の投与量で有意な抗腫瘍効果が確認され、ｈＳＨ３５７－１－Ｔ１、３４８－Ｈ４－３５７－Ｌ５、３４８－Ｈ５－３５７－Ｌ５については０．０１ｍｇ／匹でも有意な抗腫瘍効果が認められた（図５　Ａ－Ｄ）。 As a result, all humanized anti-EPHA2 antibodies of hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 were administered at a dose of 0.03 mg / animal. A significant antitumor effect was confirmed, and for hSH357-1-T1, 348-H4-357-L5, 348-H5-357-L5, a significant antitumor effect was observed even at 0.01 mg / animal (FIG. 5). AD).
　本発明の抗ＥＰＨＡ２抗体は抗腫瘍活性を有し、該抗ＥＰＨＡ２抗体を含む医薬組成物は抗癌剤となり得る。 The anti-EPHA2 antibody of the present invention has antitumor activity, and a pharmaceutical composition containing the anti-EPHA2 antibody can be an anticancer agent.
ＦＥＲＭ　ＢＰ－１０８３６
ＦＥＲＭ　ＢＰ－１０８３７ FERM BP-1083
FERM BP-10837
配列番号３：ＥＰＨＡ２変異体配列
配列番号５３：３４８－Ｈ４
配列番号５４：３４８－Ｈ４可変領域
配列番号５５：３４８－Ｈ５
配列番号５６：３４８－Ｈ５可変領域
配列番号５７：３４８－Ｌ４
配列番号５８：３４８－Ｌ４可変領域
配列番号５９：３４８－Ｌ５
配列番号６０：３４８－Ｌ５可変領域
配列番号６１：３５７－Ｈ４
配列番号６２：３５７－Ｈ４可変領域
配列番号６３：３５７－Ｈ５
配列番号６４：３５７－Ｈ５可変領域
配列番号６５：３５７－Ｌ４
配列番号６６：３５７－Ｌ４可変領域
配列番号６７：３５７－Ｌ５
配列番号６８：３５７－Ｌ５可変領域
配列番号６９：プライマーＥＦＦ１
配列番号７０：プライマーＥｆｓｍａＲ
配列番号７１：ヒトκシグナル配列＋κ定常領域＋ポリＡ
配列番号７２：ヒトＩｇＧ　シグナル配列＋＋定常領域
配列番号７３：シグナル配列＋ｈＳＨ３４８－１－Ｔ１Ｌ可変領域
配列番号７４：シグナル配列＋３５７－Ｔ１Ｌ可変領域
配列番号７５：ｈＳＨ３４８－１－Ｔ１Ｈ可変領域
配列番号７６：ｈ３５７－１－Ｔ１Ｈ可変領域
配列番号７７：ｈＳＨ３４８－Ｔ１Ｌ
配列番号７９：ｈＳＨ３５７－Ｔ１Ｌ
配列番号８１：３４８－Ｌ４
配列番号８３：３５７－Ｌ５
配列番号８５：３４８－Ｌ５
配列番号８７：３５７－Ｌ５
配列番号８９：３４８－Ｈ４
配列番号９１：３４８－Ｈ５
配列番号９３：３５７－Ｈ４
配列番号９５：３５７－Ｈ５ SEQ ID NO: 3: EPHA2 variant SEQ ID NO: 53: 348-H4
SEQ ID NO: 54: 348-H4 variable region SEQ ID NO: 55: 348-H5
SEQ ID NO: 56: 348-H5 variable region SEQ ID NO: 57: 348-L4
SEQ ID NO: 58: 348-L4 variable region SEQ ID NO: 59: 348-L5
SEQ ID NO: 60: 348-L5 variable region SEQ ID NO: 61: 357-H4
SEQ ID NO: 62: 357-H4 variable region SEQ ID NO: 63: 357-H5
SEQ ID NO: 64: 357-H5 variable region SEQ ID NO: 65: 357-L4
SEQ ID NO: 66: 357-L4 variable region SEQ ID NO: 67: 357-L5
SEQ ID NO: 68: 357-L5 variable region SEQ ID NO: 69: Primer EFF1
Sequence number 70: Primer EfsmaR
SEQ ID NO: 71: human κ signal sequence + κ constant region + poly A
SEQ ID NO: 72: human IgG signal sequence + + constant region SEQ ID NO: 73: signal sequence + hSH348-1-T1L variable region SEQ ID NO: 74: signal sequence + 357-T1L variable region SEQ ID NO: 75: hSH348-1-T1H variable region SEQ ID NO: 76 h357-1-T1H variable region SEQ ID NO: 77: hSH348-T1L
SEQ ID NO: 79: hSH357-T1L
SEQ ID NO: 81: 348-L4
SEQ ID NO: 83: 357-L5
SEQ ID NO: 85: 348-L5
SEQ ID NO: 87: 357-L5
SEQ ID NO: 89: 348-H4
SEQ ID NO: 91: 348-H5
SEQ ID NO: 93: 357-H4
SEQ ID NO: 95: 357-H5
[規則26に基づく補充 11.10.2011]　
Figure WO-DOC-RO134
[Supplement under rule 26 11.10.2011]
Figure WO-DOC-RO134

Claims (24)

  1. 配列表の配列番号１８に示されるアミノ酸配列からなるＣＤＲＨ１、配列表の配列番号２０に示されるアミノ酸配列からなるＣＤＲＨ２及び配列表の配列番号２２に示されるアミノ酸配列からなるＣＤＲＨ３を含むことからなる重鎖、並びに、配列表の配列番号４８に示されるアミノ酸配列からなるＣＤＲＬ１、配列表の配列番号５０に示されるアミノ酸配列からなるＣＤＲＬ２及び配列表の配列番号５２に示されるアミノ酸配列からなるＣＤＲＬ３を含むことからなる軽鎖からなり、配列表の配列番号４に示されるアミノ酸配列からなるポリペプチドを特異的に認識する抗体または該抗体の機能性断片。 A CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, a CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. Chain, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 52 in the sequence listing. An antibody or a functional fragment of the antibody that specifically recognizes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
  2. 配列表の配列番号４のアミノ酸番号４３９乃至５３４に示されるアミノ酸配列からなるポリペプチドを特異的に認識する、請求項１に記載の抗体または該抗体の機能性断片。 The antibody or the functional fragment of the antibody according to claim 1, which specifically recognizes a polypeptide comprising an amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing.
  3. 配列表の配列番号１８に示されるアミノ酸配列からなるＣＤＲＨ１、配列表の配列番号２０に示されるアミノ酸配列からなるＣＤＲＨ２及び配列表の配列番号２２に示されるアミノ酸配列からなるＣＤＲＨ３を含むことからなる重鎖、並びに、配列表の配列番号４８に示されるアミノ酸配列からなるＣＤＲＬ１、配列表の配列番号５０に示されるアミノ酸配列からなるＣＤＲＬ２及び配列表の配列番号５２に示されるアミノ酸配列からなるＣＤＲＬ３を含むことからなる軽鎖からなり、配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が１．５ｎＭ以下である請求項１又は２に記載の抗体または該抗体の機能性断片。 A CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, a CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. Chain, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 52 in the sequence listing. The Kd value for a polypeptide consisting of the light chain and comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.5 nM or less. An antibody or a functional fragment of the antibody.
  4. 配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が１．０ｎＭ以下である、請求項３に記載の抗体または該抗体の機能性断片。 The antibody or the functional fragment of the antibody according to claim 3, wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.0 nM or less.
  5. 配列表の配列番号４のアミノ酸番号４２６乃至５４０に示されるアミノ酸配列を含むことからなるポリペプチドに対するＫｄ値が０．５ｎＭ以下である、請求項３に記載の抗体または該抗体の機能性断片。 The antibody or the functional fragment of the antibody according to claim 3, wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 0.5 nM or less.
  6. ＥＰＨＡ２リガンドにより誘導されるＥＰＨＡ２チロシン残基のリン酸化を抑制する、請求項１乃至５のいずれか１項に記載の抗体または該抗体の機能性断片。 The antibody or the functional fragment of the antibody according to any one of claims 1 to 5, which suppresses phosphorylation of an EPHA2 tyrosine residue induced by an EPHA2 ligand.
  7. 配列表の配列番号５４、５６に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる重鎖可変領域を含む重鎖、及び、配列表の配列番号６６、６８に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる軽鎖可変領域を含む軽鎖からなる、請求項１乃至６のいずれか１項に記載の抗体又は該抗体の機能性断片。 A heavy chain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 54 and 56 in the sequence listing or the amino acid sequence of any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added. Light chain variable consisting of the amino acid sequence shown in SEQ ID NOs: 66 and 68 in the sequence listing or the amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence. The antibody or the functional fragment of the antibody according to any one of claims 1 to 6, comprising a light chain comprising a region.
  8. 配列表の配列番号５３、５５に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列を含む重鎖、及び配列表の配列番号６５、６７に示されるアミノ酸配列又は該アミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、付加されたアミノ酸配列のいずれかのアミノ酸配列からなる軽鎖を含むことからなる、請求項１乃至７のいずれか１項に記載の抗体又は該抗体の機能性断片。 A heavy chain comprising any one of the amino acid sequences shown in SEQ ID NOs: 53 and 55 in the sequence listing or an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence; The amino acid sequence shown in SEQ ID NOs: 65 and 67 or a light chain comprising any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequence. 8. The antibody or the functional fragment of the antibody according to any one of 1 to 7.
  9. 配列表の配列番号５３に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６５に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片。 An antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the Sequence Listing, or a functional fragment of the antibody.
  10. 配列表の配列番号５３に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６７に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片。 An antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the Sequence Listing, or a functional fragment of the antibody.
  11. 配列表の配列番号５５に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６５に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片。 An antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the Sequence Listing, or a functional fragment of the antibody.
  12. 配列表の配列番号５５に示されるアミノ酸配列からなる重鎖、及び配列表の配列番号６７に示されるアミノ酸配列からなる軽鎖からなる抗体又は該抗体の機能性断片。 An antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the Sequence Listing, or a functional fragment of the antibody.
  13. 以下のａ）乃至ｄ）に示される性質を有する、請求項１乃至１２のいずれか１項に記載の抗体又は該抗体の機能性断片：
    ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない；
    ｂ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する；
    ｃ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する；
    ｄ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。     The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to d):
    a) not having the ability to phosphorylate EPHA2 tyrosine residues;
    b) has ADCC activity against EPHA2 expressing cells;
    c) has CDC activity against EPHA2 expressing cells;
    d) Has anti-tumor activity in vivo.
  14. 以下のａ）乃至ｅ）に示される性質を有する、請求項１乃至１２のいずれか１項に記載の抗体又は該抗体の機能性断片：
    ａ）ＥＰＨＡ２チロシン残基のリン酸化能を有さない；
    ｂ）ＥＰＨＡ２蛋白質量の減少作用を示さない；
    ｃ）ＥＰＨＡ２発現細胞に対してＡＤＣＣ活性を有する；
    ｄ）ＥＰＨＡ２発現細胞に対してＣＤＣ活性を有する；
    ｅ）Ｉｎ　ｖｉｖｏで抗腫瘍活性を有する。     The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to e):
    a) not having the ability to phosphorylate EPHA2 tyrosine residues;
    b) Does not show an effect of decreasing the amount of EPHA2 protein;
    c) has ADCC activity against EPHA2 expressing cells;
    d) has CDC activity against EPHA2 expressing cells;
    e) Has anti-tumor activity in vivo.
  15. ＩｇＧ抗体であることを特徴とする、請求項１乃至１４のいずれか１項に記載の抗体。 The antibody according to claim 1, which is an IgG antibody.
  16. Ｆａｂ、Ｆ（ａｂ’）２、Ｆｖ、ｓｃＦＶ、ディアボディー、線状抗体及び多特異的抗体から選択されるいずれかであることを特徴とする、請求項１乃至１４のいずれか１項に記載の抗体又は該抗体の機能性断片。 The method according to any one of claims 1 to 14, wherein the antibody is any one selected from Fab, F (ab ') 2, Fv, scFV, diabody, linear antibody, and multispecific antibody. Or a functional fragment thereof.
  17. 請求項１乃至１６から選択される抗体又は該抗体の機能性断片の少なくともいずれか１つを含有することを特徴とする、医薬組成物。 A pharmaceutical composition comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody.
  18. 請求項１乃至１６から選択される抗体又は該抗体の機能性断片の少なくともいずれか一つを含有することを特徴とする、癌の治療用医薬組成物。 A pharmaceutical composition for treating cancer comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody.
  19. 請求項１乃至１６から選択される抗体又は該抗体の機能性断片の少なくともいずれか一つを含有することを特徴とする、乳癌、食道癌、前立腺癌、胃癌、非小細胞肺癌、大腸癌、及び／又は多形神経膠芽腫の治療用医薬組成物。 A breast cancer, an esophageal cancer, a prostate cancer, a stomach cancer, a non-small cell lung cancer, a colon cancer, comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody, And / or a pharmaceutical composition for the treatment of glioblastoma multiforme.
  20. 請求項１乃至１８から選択されるいずれかを投与することによる、哺乳動物の有する腫瘍の増殖の抑制方法。 A method for suppressing the growth of a tumor possessed by a mammal, by administering any one selected from claims 1 to 18.
  21. 腫瘍が、ＥＰＨＡ２を発現している腫瘍であることを特徴とする、請求項１９に記載の腫瘍の増殖の抑制方法。 The method for inhibiting tumor growth according to claim 19, wherein the tumor is a tumor expressing EPHA2.
  22. 請求項１乃至１６から選択されるいずれか１項に記載の抗体又は該抗体の機能性断片をコードするポリヌクレオチド。 A polynucleotide encoding the antibody or the functional fragment of the antibody according to any one of claims 1 to 16.
  23. 請求項２２に記載のポリヌクレオチドによって形質転換された宿主細胞。 23. A host cell transformed with the polynucleotide of claim 22.
  24. 請求項２３に記載の宿主細胞を培養し、培養液より該抗体又は該抗体の機能性断片を回収する工程を含むことを特徴とする、該抗体又は該抗体の機能性断片の製造方法。
          A method for producing the antibody or a functional fragment of the antibody, comprising the steps of culturing the host cell according to claim 23 and recovering the antibody or the functional fragment of the antibody from the culture solution.
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