WO2012008397A1 - Anticorps anti-epha2 et utilisation de celui-ci - Google Patents

Anticorps anti-epha2 et utilisation de celui-ci Download PDF

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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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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

La présente invention concerne un anticorps ou équivalent, présentant une activité inhibitrice de la cancérisation des cellules et/ou une activité inhibitrice de la prolifération des cellules tumorales. La présente invention concerne : un anticorps anti-EPHA2 comprenant une chaîne lourde humanisée dérivée d'un anticorps SH 348-1 anti-EPHA2 qui est produit à partir d'un hybridome SH 348-1 (FERM BP-10836) et une chaîne légère humanisée dérivée d'un anticorps SH 357-1 anti-EPHA2 qui est produit à partir d'un hybridome SH 357-1 (FERM BP-10837); un fragment fonctionnel de l'anticorps; et un produit pharmaceutique ou équivalent, contenant l'anticorps comme principe actif.
PCT/JP2011/065766 2010-07-12 2011-07-11 Anticorps anti-epha2 et utilisation de celui-ci WO2012008397A1 (fr)

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WO2016171242A1 (fr) * 2015-04-24 2016-10-27 第一三共株式会社 Détection d'epha2

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