WO2016171242A1 - Détection d'epha2 - Google Patents

Détection d'epha2 Download PDF

Info

Publication number
WO2016171242A1
WO2016171242A1 PCT/JP2016/062719 JP2016062719W WO2016171242A1 WO 2016171242 A1 WO2016171242 A1 WO 2016171242A1 JP 2016062719 W JP2016062719 W JP 2016062719W WO 2016171242 A1 WO2016171242 A1 WO 2016171242A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
epha2
seq
amino acid
antigen
Prior art date
Application number
PCT/JP2016/062719
Other languages
English (en)
Japanese (ja)
Inventor
真由美 須江
朋子 渋谷
悌司 和田
臣哉 徳廣
Original Assignee
第一三共株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 第一三共株式会社 filed Critical 第一三共株式会社
Publication of WO2016171242A1 publication Critical patent/WO2016171242A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention introduces a novel antibody, a functional fragment of the antibody, a modified form of the antibody, a nucleotide containing a base sequence encoding the amino acid sequence of the antibody, a vector having the nucleotide inserted therein, the nucleotide or a vector.
  • the present invention relates to a method for producing the antibody, a pharmaceutical composition, a diagnostic or test composition, and the like, which comprise a step of culturing the cell.
  • EPHA2 is a receptor tyrosine kinase having a single transmembrane structure with a molecular weight of 130 kDa (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
  • Non-patent Document 2 Ephrin-A1 to A5 of GPI-anchored cell surface proteins are known (Non-patent Document 2). Activation of the tyrosine kinase domain of EPHA2 by ligand binding results in autophosphorylation of tyrosine residues present in the intracellular region of EPHA2, and induces intracellular signal transduction. In addition, it has been reported that EPHA2 is taken into cells by endocytosis after binding to a ligand and finally degraded by proteasome (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 (Non-Patent Documents 4 and 5). , 6, 7, 8, 9, 10, 11). Furthermore, in esophageal cancer, a significant correlation has been observed between EPHA2 expression and local lymph node metastasis, the number of lymph node metastases, and the low degree of differentiation of cancer, and the survival rate of EPHA2 positive patients is EPHA2 negative. It has been reported that it is lower than that of patients (Non-patent Document 5).
  • the expression level of EPHA2 is higher in patients less than 5 years compared to patients whose disease-free survival is longer than 5 years, and those who relapse have higher expression levels of EPHA2 than those who do not relapse, In patients who have progressed to brain metastasis, it has been reported that the expression level of EPHA2 is higher than those in patients who have no recurrence or in patients whose metastasis is confined to the contralateral lung (Non-patent Document 9). It has been reported that the expression level of EPHA2 also significantly correlates with liver metastasis, lymphatic vessel invasion and clinical stage in colorectal cancer (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.
  • cancer traits such as anchorage-independent growth ability, tubular morphogenesis on the extracellular matrix, and in vivo tumor growth ability.
  • Non-Patent Document 4 and cancer cells have been reported to be more invasive to the extracellular matrix (Non-Patent Documents 12 and 13).
  • Non-Patent Documents 13 and 14 suppression of EPHA2 expression with siRNA suppresses cancer cell invasion, anchorage-independent growth, in vivo tumor growth
  • Non-Patent Documents 13 and 14 ligand Ephrin-A1 EPHA2 is activated using a fusion protein of human and human IgG Fc region, and the degradation of EPHA2 by endocytosis suppresses the invasion, anchorage-independent growth, and tubular morphogenesis of cancer cells.
  • EPHA2 has been reported to be expressed not only in cancer cells but also in blood vessels in or around tumors (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 luminal 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 (Non-patent document 17).
  • EPHA2 may be an excellent therapeutic target for cancer, and some monoclonal antibodies against EPHA2 have actually been acquired and clinical trials are being conducted (Patent Documents 1 to 3).
  • the provision of a method capable of detecting the expression of EPHA2 is useful for testing or diagnosis of diseases related to EPHA2, such as cancer, and EPHA2 expression.
  • Non-patent Document 18 Many monoclonal antibodies that recognize human EPHA2 are known, but there are few that can be applied to immunohistochemical staining, and monoclonal antibodies that can be immunostained by recognizing modified EPHA2 fixed with formalin. As such, only clones D7 and B2D6 (Non-patent Document 18) are known.
  • One object of the present invention is to provide an antibody against EPHA2.
  • Another object of the present invention is to provide a diagnostic or test composition containing an anti-EPHA2 antibody.
  • the subject of the present invention also includes a nucleotide encoding the amino acid sequence of the antibody, a vector into which the nucleotide has been inserted, a cell into which the nucleotide or vector has been introduced, and a step of culturing the cell. Methods etc. are included.
  • Another object of the present invention is to provide a pharmaceutical composition and a treatment method.
  • the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, the CDRH1 is composed of an amino acid sequence represented by SEQ ID NO: 60, the CDRH2 is composed of an amino acid sequence represented by SEQ ID NO: 61, and the CDRH3 Consists of the amino acid sequence shown in SEQ ID NO: 62;
  • the light chain sequence includes a variable region having CDRL1, CDRL2, and CDRL3, wherein the CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 63, the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 64, and the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 65; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
  • the CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 69
  • the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 70
  • the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 71; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
  • An antibody or an antigen-binding fragment of the antibody (5) A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59
  • (6) A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59
  • the test specimen is determined to be positive, and EPHA2 is not detected or measured in the test specimen, or the expression level or expression level of EPHA2 in the test specimen is equal to or lower than a predetermined criterion
  • the subject from which the test specimen determined to be positive in the detection or measurement of EPHA2 by the test or diagnostic method for EPHA2-positive disease is an antibody that specifically binds to EPHA2, or an antigen-binding fragment of the antibody
  • Gray dotted line and gray solid line indicate the binding of isotype control antibody and anti-FLAG antibody to 293F cells transfected with pFLAG-GW, respectively.
  • the black dotted line and the black solid line indicate the binding of the isotype control antibody and the anti-FLAG antibody to 293F cells transfected with the gene shown in FIG.
  • Evaluation of binding specificity of mouse anti-human EPHA2 antibody A201 by flow cytometry Gray dotted line and gray solid line indicate binding of isotype control antibody and A201 antibody to 293F cells transfected with pFLAG-GW, respectively.
  • the black dotted line and the black solid line indicate the binding of the isotype control antibody and the A201 antibody to 293F cells transfected with the gene shown in FIG. Evaluation of Binding Specificity of Mouse Anti-Human EPHA2 Antibody A205 by Flow Cytometry
  • Gray dotted line and gray solid line indicate the binding of isotype control antibody and A205 antibody to 293F cells transfected with pFLAG-GW, respectively.
  • the black dotted line and the black solid line indicate the binding of the isotype control antibody and the A205 antibody to 293F cells transfected with the gene shown in FIG. 3, respectively. It is a figure which shows the EPHA2 specific binding property of the mouse
  • FIG. 6 is an immunostained image of mouse throat and laryngeal cancer and gastric cancer according to Example 5 with mouse anti-human EPHA2 antibody A205. It is the figure which showed the amino acid sequence (sequence number 51) of the heavy chain of mouse
  • FIG. 5 is an immunostained image of mouse anti-human EPHA2 antibodies A201 and A205 of human esophageal cancer according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). .
  • FIG. 5 is an immunostained image of mouse anti-human EPHA2 antibodies A201 and A205 of human esophageal cancer according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). .
  • FIG. 4 is an immunostaining image of mouse anti-EPHA2 antibodies A201 and A205 of human cervical cancer according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).
  • FIG. 9 shows immunostained images of mouse colonic cancer mouse anti-EPHA2 antibodies A201 and A205 according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).
  • FIG. 9 shows immunostained images of mouse colonic cancer mouse anti-EPHA2 antibodies A201 and A205 according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).
  • FIG. 9 shows immunostaining images of mouse gastric cancer mouse anti-EPHA2 antibodies A201 and A205 according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).
  • gene means a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof, for example, a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof.
  • Certain polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA and the like are included in the meaning of “gene”.
  • Such a gene is a single-stranded, double-stranded, or triple-stranded nucleotide, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) are mixed on a single nucleotide strand.
  • RNA ribonucleotide
  • DNA deoxyribonucleotide
  • EPHA2 gene include DNA, mRNA, cDNA, cRNA and the like containing a base sequence encoding the amino acid sequence of EPHA2 protein.
  • nucleotide and “nucleic acid” are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of “nucleotide”.
  • a nucleotide is a nucleotide composed of a single strand, a double strand, or three or more strands, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) on a single nucleotide strand.
  • RNA ribonucleotide
  • DNA deoxyribonucleotide
  • nucleotide also included within the meaning of “nucleotide” are those that are intermingled and aggregates of two or more strands containing such nucleotide strands.
  • polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
  • antigen is sometimes used to mean “immunogen”.
  • cell includes various cells derived from individual animals, subculture cells, primary culture cells, cell lines, recombinant cells, microorganisms, and the like.
  • an antibody that recognizes EPHA2 may be referred to as an “anti-EPHA2 antibody”.
  • anti-EPHA2 antibody Such antibodies include chimerized antibodies, humanized antibodies, human antibodies and the like.
  • the “functional fragment of an antibody” means an antibody fragment that exhibits at least a part of the function exhibited by the original antibody.
  • the “functional fragment of an antibody” include, but are not limited to, Fab, F (ab ′) 2, scFv, Fab ′, single chain immunoglobulin and the like.
  • Such functional fragments of antibodies are recombinant proteins produced in appropriate host cells using recombinant genes in addition to those obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin. May be.
  • the “site” to which the antibody binds that is, the “site” recognized by the antibody means a partial peptide or a partial higher order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site.
  • bonds or recognizes the partial peptide or partial higher order structure etc. on EPHA2 protein can be illustrated.
  • CDRs complementarity determining regions
  • the complementarity-determining region is also called a hypervariable domain, and is located in the variable region of the heavy and light chains of an antibody and has a particularly high primary structure variability. In the primary structure of the polypeptide chain, it is usually separated at three points.
  • the complementarity determining region of an antibody the complementarity determining region of an antibody
  • the complementarity determining region of the heavy chain is denoted as CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence
  • the complementarity determining region of the light chain is defined as the light chain amino acid.
  • CDRL1, CDRL2, and CDRL3 are represented from the amino terminal side of the sequence. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to be bound.
  • antibody variant has an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted (hereinafter collectively referred to as “mutation”) in the amino acid sequence of the original antibody. And a polypeptide that binds to the EPHA2 protein of the present invention.
  • the number of variant amino acids in such antibody variants is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50.
  • Such antibody variants are also included in the “antibody” of the present invention. *
  • “several” in “1 to several” refers to 3 to 10.
  • Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical properties, and the like. Specifically, various biological activities, binding activity to antigens and epitopes, stability during production and storage Property, heat stability, etc. can be raised.
  • hybridize under stringent conditions means hybridization at 65 ° C. in a solution containing 5 ⁇ SSC, and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS. 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS for 20 minutes at 65 ° C. and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS Means to hybridize under conditions of washing for 20 minutes or under equivalent conditions.
  • SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and nx SSC means n-fold concentration of SSC.
  • cytotoxicity refers to causing a pathological change in a cell in some form, and is not limited to direct trauma, but also includes DNA breakage, base dimer formation, chromosome breakage, It means any structural or functional damage of cells, such as damage to cell division apparatus or reduction of various enzyme activities.
  • cytotoxic activity means causing the above cytotoxicity.
  • antibody-dependent cytotoxic activity refers to “antibody dependent cellular cytotoxicity (ADCC) activity”, and means an activity in which NK cells damage target cells such as tumor cells via antibodies.
  • antibody-dependent cell-mediated phagocytic activity refers to “antibody dependent cell phagocytosis (ADCP) activity”, which is an activity that monocytes and macrophage cells phagocytose target cells such as tumor cells via antibodies. means. Also referred to as “antibody-dependent phagocytic activity”.
  • ADCP antibody dependent cell phagocytosis
  • complement-dependent cytotoxic activity refers to “complement dependent cytotoxicity (CDC) activity” and means an activity that complements a target cell such as a tumor cell via an antibody.
  • cancer and “tumor” are used interchangeably.
  • immunohistochemistry means a histological (histochemical) method for detecting an antigen in a tissue specimen, and is synonymous with “immunoantibody method”. “immunostaining”) is also used interchangeably.
  • denatured EPHA2 means an EPHA2 molecule in a specimen fixed with formalin. EPHA2 molecules in specimens fixed with formalin and then paraffin-treated and deparaffinized are also referred to as “denatured” EPHA2.
  • non-denatured EPHA2 means EPHA2 in a sample not fixed with formalin.
  • EPHA2 molecules in specimens not fixed with formalin are also referred to as “non-denaturing” EPHA2.
  • the cell membrane fraction of the above-mentioned cells can be prepared and used, and it can be obtained by synthesizing EPHA2 in vitro or producing it in a host cell by genetic manipulation.
  • EPHA2 cDNA 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 prokaryote or eukaryote.
  • the protein can be obtained by expressing EPHA2 by transforming a host cell of an organism.
  • the nucleotide sequence of human EPHA2 cDNA is registered in GenBank with accession number: NM_004431. Further, the amino acid sequence of human EPHA2 is registered in GenBank with an accession number: NP_004422.
  • EPHA2 cDNA is, for example, a polymerase chain reaction (hereinafter referred to as “PCR”) using a cDNA library of an organ expressing EPHA2 mRNA as a template and a primer that specifically amplifies EPHA2 cDNA (Saiki, R K., et al., Science, (1988) 239, 487-49).
  • PCR polymerase chain reaction
  • a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding human EPHA2 under stringent conditions and encodes a protein having biological activity equivalent to that of EPHA2, is also included in the EPHA2 cDNA. included.
  • a polynucleotide encoding a splicing variant transcribed from the human EPHA2 locus or a protein that hybridizes to this under stringent conditions and also has a biological activity equivalent to that of EPHA2 is also included in the EPHA2 cDNA. included.
  • EPHA2 amino acid sequence of human EPHA2
  • amino acid sequence obtained by substituting, deleting, or adding 1, 2, or 3, or 4 or 5 amino acids in the amino acid sequence obtained by removing the signal sequence from these sequences a protein having a biological activity equivalent to that of EPHA2 is also included in EPHA2.
  • an amino acid sequence encoded by a splicing variant transcribed from the human EPHA2 locus, or an amino acid sequence in which 1, 2 or 3 or 4 or 5 amino acids are substituted, deleted or added in the amino acid sequence EPHA2 is also included in the protein having a biological activity equivalent to that of EPHA2.
  • the nucleotide sequence encoding mature human EPHA2 that does not contain a signal peptide is set forth in SEQ ID NO: 1 in the Sequence Listing, and the amino acid sequence of the human EPHA2 is set forth in SEQ ID NO: 2 in the Sequence Listing. Has been. 2.
  • Production of anti-EPHA2 antibody The antibody against EPHA2 of the present invention is obtained by immunizing an animal with any polypeptide selected from the amino acid sequence of EPHA2 or EPHA2 using a conventional method, and collecting and purifying the antibody produced in vivo Can be obtained.
  • the species of EPHA2 serving as an antigen is not limited to humans, and animals can be immunized with EPHA2 derived from animals other than humans such as mice and rats.
  • an antibody applicable to a human disease can be selected by testing the cross-reactivity between the obtained antibody that binds to heterologous EPHA2 and human EPHA2.
  • EPHA2 used as an antigen can be obtained by causing a host cell to produce the EPHA2 gene by genetic manipulation. Specifically, a vector capable of expressing the EPHA2 gene is prepared, introduced into a host cell to express the gene, and the expressed EPHA2 may be purified.
  • the antibody against EPHA2 of the present invention can also be obtained using a DNA immunization method.
  • the DNA immunization method is a technique for inducing immunity to an antigen by introducing an antigen expression plasmid into an individual animal such as a mouse or a rat and expressing the antigen in the individual.
  • Gene transfer methods include direct injection of plasmids into muscle, intravenous injection of liposomes and polyethyleneimine and other introduction reagents, viral vector methods, and gold particles with plasmids attached to them by Gene Gun.
  • the amount of expression is further improved by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug; 8 (16) : 1264-70).
  • Hybridomas can be established by fusing antibody-producing cells that produce antibodies against EPHA2 and myeloma cells to obtain monoclonal antibodies. Specific examples of such a method are described in International Publication No. WO 09/48072 (published on April 16, 2009) and WO 10/11711 (published on October 14, 2010).
  • Examples of the mouse anti-human EPHA2 antibody thus established include A201 antibody and A205 antibody.
  • the amino acid sequence of the A201 antibody heavy chain is shown in SEQ ID NO: 51 of the Sequence Listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 50 of the Sequence Listing.
  • the amino acid sequence of the A201 antibody light chain is shown in SEQ ID NO: 54 of the sequence listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 53 of the sequence listing.
  • the amino acid sequence of the A205 antibody heavy chain is shown in SEQ ID NO: 57 in the sequence listing, and the nucleotide sequence encoding this is shown in SEQ ID NO: 56 in the sequence listing.
  • the amino acid sequence of the A205 antibody light chain is shown in SEQ ID NO: 59 in the sequence listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 58 in the sequence listing.
  • the antibody of the present invention may be an antibody that retains all six CDR sequences derived from A201 and has an activity of binding to EPHA2. That is, the heavy chain variable region of the antibody of the present invention is represented by CDRH1 (DTYVH) consisting of the amino acid sequence shown in SEQ ID NO: 60, CDRH2 (RIDPANANTKYDPKFQG) consisting of the amino acid sequence shown in SEQ ID NO: 61, and SEQ ID NO: 62. It possesses CDRH3 (YGKSAWFY) consisting of an amino acid sequence.
  • the light chain variable region of the antibody comprises CDRL1 (RASQDIGNYLN) consisting of the amino acid sequence shown in SEQ ID NO: 63, CDRL2 (YTSRLHS) consisting of the amino acid sequence shown in SEQ ID NO: 64, and amino acids shown in SEQ ID NO: 65. It has CDRL3 (QQGHSLPPT) consisting of the sequence.
  • CDRL1 RASQDIGNYLN
  • YTSRLHS amino acid sequence shown in SEQ ID NO: 64
  • QQGHSLPPT amino acid sequences of these CDRs are also shown in FIG.
  • the antibody of the present invention may be an antibody that retains all six CDR sequences derived from A205 and has an activity of binding to EPHA2. That is, the heavy chain variable region of the antibody of the present invention is shown in CDRH1 (DYSMN) consisting of the amino acid sequence shown in SEQ ID NO: 66, CDRH2 (WINTYTGEPTYADDFKG) consisting of the amino acid sequence shown in SEQ ID NO: 67, and SEQ ID NO: 68. It possesses CDRH3 (PISLLLRLDY) consisting of an amino acid sequence.
  • the light chain variable region of the above-mentioned antibody has CDRL1 (RSSQSLVHSNGNTYLH) consisting of the amino acid sequence shown in SEQ ID NO: 69, CDRL2 (KVSNRFS) consisting of the amino acid sequence shown in SEQ ID NO: 70, and amino acids shown in SEQ ID NO: 71. It possesses CDRL3 (SQSTHVPWT) consisting of the sequence.
  • CDRL1 RSSQSLVHSNGNTYLH
  • KVSNRFS the amino acid sequence shown in SEQ ID NO: 70
  • SEQ ID NO: 71 amino acids shown in SEQ ID NO: 71.
  • CDRL3 SQSTHVPWT
  • suitable antibodies specifically bind to both undenatured human EPHA2 and denatured human EPHA2 in formalin-fixed specimens. More preferable antibodies include antibodies that specifically bind to both non-denatured human EPHA2 and denatured human EPHA2 in formalin-fixed specimens and that do not specifically bind to other Eph families. However, it is not limited to them.
  • the antibodies of the present invention include genetically modified antibodies that have been artificially modified for the purpose of reducing heteroantigenicity against humans, such as chimeric antibodies, or humans. Also included are humanized antibodies. These antibodies can be produced using known methods.
  • chimeric antibody examples include antibodies in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad). Sci.U.S.A., 81, 6851-6855, (1984)).
  • the above-described chimeric antibody sequence against EPHA2 can be artificially modified for the purpose of, for example, reducing the heterologous antigenicity to humans, thereby producing a humanized antibody that is a recombinant antibody.
  • the antibody of the present invention includes an antibody obtained by modifying the CDR of the humanized antibody. These antibodies can be produced using known methods.
  • humanized antibodies antibodies (see Nature (1986) 321, p.522-525) in which only complementarity determining regions (CDRs) are incorporated into human-derived antibodies (see Nature (1986) 321, p.522-525), some sequences in addition to CDR sequences
  • CDRs complementarity determining regions
  • Examples of the amino acid residues of the framework include an antibody grafted on a human antibody (International Publication No. WO90 / 07861 pamphlet).
  • the present invention also includes antibodies having such modifications, including deletions in which one or two amino acids have been deleted at the heavy chain carboxyl terminus, and such a deletions that have been amidated (eg, at the carboxyl terminus site).
  • Heavy chain in which a proline residue is amidated e.g., at the carboxyl terminus site.
  • the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention is not limited to the above type.
  • the two heavy chains constituting the antibody according to the present invention may be either one of the full length and the heavy chain selected from the group consisting of the above-mentioned deletion forms, or a combination of any two of them. It may be a thing.
  • the amount ratio of each deletion can be influenced by the type and culture conditions of the cultured mammalian cells that produce the antibody according to the present invention, but the main component of the antibody according to the present invention is a carboxyl in both two heavy chains. A case where one terminal amino acid residue is deleted can be mentioned.
  • the antibody obtained by the above method can be evaluated for binding to an antigen and a suitable antibody can be selected.
  • An example of another index for comparing antibody properties is antibody stability.
  • Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is an indicator of good relative structural stability of proteins. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It is known that the storage stability of an antibody shows a certain degree of correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p.265-273), and heat.
  • a suitable antibody can be selected using stability as an index.
  • Other indicators for selecting antibodies include high yields in appropriate host cells and low aggregation in aqueous solutions. For example, since the antibody with the highest yield does not always exhibit the highest thermal stability, it is necessary to select the most suitable antibody based on a comprehensive judgment based on the above-described indicators.
  • the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence.
  • Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and are actually observed in camels or llamas and reported to retain antigen-binding ability.
  • sdAbs single domain antibodies
  • nanobodies are actually observed in camels or llamas and reported to retain antigen-binding ability.
  • the above-described antibody can also be interpreted as a kind of antigen-binding fragment of the antibody in the present invention.
  • an antibody gene When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
  • Specific examples of the antibody gene include a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence.
  • the heavy chain sequence gene and the light chain sequence gene When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
  • eukaryotic cells When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.
  • ATCC CRL-1650 which are monkey cells, mouse fibroblasts NIH3T3 (ATCC). No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strains (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220).
  • Escherichia coli and Bacillus subtilis can be mentioned, for example.
  • An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. In the above culture method, the yield may vary depending on the sequence of the antibody. From the antibodies having equivalent binding activity, those that can be easily produced as pharmaceuticals can be selected using the yield as an index.
  • IgG IgG1, IgG2, IgG3, IgG4
  • IgM IgA (IgA1, IgA2)
  • IgD or IgE preferably IgG or IgM
  • IgG1 or IgG2 More preferably, IgG1 or IgG2 can be mentioned.
  • the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof.
  • a fragment of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or modifying the antibody gene by a genetic engineering technique and expressing it in an appropriate cultured cell.
  • a fragment that retains all or part of the functions of the full-length antibody molecule can be called an antigen-binding fragment of an antibody.
  • Antibody functions generally include antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and complement-dependence Mention may be made of cellular cytotoxic activity.
  • the function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to EPHA2.
  • antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv), diabodies (diabodies), linear antibody in which heavy and light chain Fvs are linked by an appropriate linker.
  • Fab ' which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antibody fragment.
  • the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens.
  • a molecule binds to two types of antigens (ie, bispecific antibodies), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.
  • the multispecific antibody of the present invention may be a full-length antibody or a fragment of such an antibody (for example, F (ab ') 2 bispecific antibody).
  • Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by hybridizing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).
  • the antibody of the present invention may be a single chain antibody (also referred to as scFv).
  • a single chain antibody is obtained by linking an antibody heavy chain variable region and a light chain variable region with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, edited by Springer Verlag, New). York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136)
  • a BiscFv fragment produced by linking two scFvs with a polypeptide linker is used as a bispecific antibody. It can also be used.
  • the heavy chain variable region and the light chain variable region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci.U.S.A. (1988), 85, p.5879-5883).
  • the heavy chain variable region and the light chain variable region in scFv may be derived from the same antibody or different antibodies.
  • the polypeptide linker that links the variable regions for example, any single chain peptide consisting of 12 to 19 residues is used.
  • the DNA encoding the scFv is the DNA encoding the heavy chain or heavy chain variable region of the antibody, and the DNA encoding the light chain or light chain variable region.
  • Amplification is performed by PCR using a coding DNA portion as a template and a primer pair defining both ends thereof, and then the DNA encoding the polypeptide linker portion and both ends thereof are connected to the heavy chain and light chain, respectively. Obtained by combining and amplifying the primer pairs defined in 1.
  • an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.
  • These antibody fragments can be produced by a host after obtaining and expressing the gene in the same manner as described above.
  • the antibody of the present invention may be one that has been increased in quantity and has increased affinity for the antigen.
  • the antibody that multiplies may be one type of antibody or a plurality of antibodies that recognize multiple epitopes of the same antigen. Examples of the method for increasing the amount of antibody include binding of IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of helix-turn-helix motif.
  • the antibody of the present invention may be a polyclonal antibody that is a mixture of a plurality of types of anti-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
  • the antibody of the present invention may be one in which these antibody and another drug form a conjugate (Immunoconjugate).
  • conjugate examples include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).
  • the obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies) for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid chromatography, such as HPLC and FPLC.
  • column used for affinity chromatography include a protein A column and a protein G column.
  • a column using a protein A column Hyper D, POROS, Sepharose F.R. F. (Pharmacia) and the like. It is also possible to purify an antibody using a carrier on which an antigen is immobilized, utilizing the binding property to the antigen.
  • composition comprising an anti-EPHA2 antibody or a functional fragment thereof or a modified form thereof.
  • the pharmaceutical composition of the present invention can be used in various diseases (hereinafter referred to as “induced or exacerbated” by EPHA2 signal abnormality or enhancement by EPHA2 or its ligand overexpression or EPHA2 mutation or gene amplification, or by switching of EPHA2 isoforms). , Referred to as “diseases associated with EPHA2”), especially for the treatment or prevention of various cancers.
  • SNP single nucleotide substitution
  • cancer types include lung cancer such as breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colon cancer, esophageal cancer, bladder cancer, uterus.
  • lung cancer such as breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colon cancer, esophageal cancer, bladder cancer, uterus.
  • Cervical cancer, hematological cancer, lymphoma, malignant melanoma and the like can be mentioned, and those cancers expressing the EPHA2 protein can be mentioned preferably.
  • the onset of such a disease preferably the onset of such a disease in an individual expressing the EPHA2 protein, the suppression or inhibition of progression or progression, and the individual suffering from such a disease
  • examples include, but are not limited to, alleviation of one or more symptoms present, suppression or remission of progression or progression, treatment or prevention of secondary diseases, and the like.
  • the pharmaceutical composition of the present invention comprises a therapeutically or prophylactically effective amount of an anti-EPHA2 antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Can be contained.
  • “Therapeutically or prophylactically effective amount” means an amount that exhibits a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is synonymous with “pharmacologically effective amount”.
  • the pharmaceutical composition of the present invention has pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or antibody contained therein, solubility, sustained release, absorbability, penetration.
  • Substances for changing, maintaining, and maintaining properties, dosage forms, strength, properties, shapes, etc. can be included.
  • the substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance.
  • non-toxicity or low toxicity is a property that a substance for preparation preferably comprises.
  • Substances for formulation include, for example, amino acids, antibacterial agents, antioxidants, buffers, fillers, chelating agents, complexing agents, bulking agents, monosaccharides, disaccharides, carbohydrates, coloring agents, flavoring agents, diluents , Emulsifier, hydrophilic polymer, preservative, solvent, sugar alcohol, suspending agent, surfactant, stabilization enhancer, elasticity enhancer, transport agent, diluent, excipient, and / or pharmaceutical adjuvant
  • the amount of these substances to be added is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably the weight of the anti-EPHA2 antibody or functional fragment or modified product thereof. Is 0.1 to 10 times.
  • a pharmaceutical composition containing an anti-EPHA2 antibody or a functional fragment thereof or a modified product thereof in an liposome, or an antibody modified product (US Pat. No. 6,214,388, etc.) in which an antibody and a liposome are bound is also present. Included in the pharmaceutical composition of the invention.
  • the excipient or carrier is usually liquid or solid, and is not particularly limited as long as it is a substance used for water for injection, physiological saline, artificial cerebrospinal fluid, and other preparations for oral administration or parenteral administration.
  • physiological saline include neutral ones and those containing serum albumin.
  • the buffer examples include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetate buffer prepared so as to be 4.0 to 5.5, and 5. Examples thereof include a citrate buffer prepared to be 0 to 8.0, a histidine buffer prepared to be 5.0 to 8.0, and the like.
  • the pharmaceutical composition of the present invention is a solid, liquid, suspension or the like. Freeze-dried preparations can be mentioned. An excipient such as sucrose can be used to mold the lyophilized preparation.
  • the administration route of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration and parenteral administration.
  • enteral administration intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration Administration, transdermal administration, intraosseous administration, intraarticular administration and the like can be mentioned.
  • composition of such a pharmaceutical composition can be determined according to the administration method, the EPHA2 protein binding affinity of the antibody, and the like.
  • the dose of the anti-EPHA2 antibody of the present invention is not limited as long as it is a pharmacologically effective amount, and the species of the individual, the type of disease, the symptom, sex, age, prevalence, EPHA2 protein binding affinity of the antibody or Although it can be appropriately determined depending on the biological activity and other factors, it is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg once every 1 to 180 days, or 1 It can be administered twice or more times a day.
  • the form of the pharmaceutical composition includes injections (including lyophilized preparations and infusions), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols. Examples thereof include pills, pills, powders, suspensions, emulsions, eye drops, and implantable preparations.
  • a pharmaceutical composition comprising an anti-EPHA2 antibody or a functional fragment thereof or a modified form thereof as an active ingredient can be administered simultaneously with or separately from other drugs.
  • a pharmaceutical composition containing an anti-EPHA2 antibody or a functional fragment of the antibody as an active ingredient is administered, or after administering such a pharmaceutical composition, another pharmaceutical is administered, or You may administer the said pharmaceutical composition and another pharmaceutical simultaneously.
  • other medicaments include various anticancer agents such as chemotherapeutic agents and radiotherapy. These are collectively referred to as “the combined use of the antibody of the present invention with another drug”, and a pharmaceutical composition containing an additional drug in addition to the antibody of the present invention, a functional fragment thereof or a modified form thereof is also included in the present invention.
  • the present invention relates to a method for treating or preventing a disease associated with EPHA2, such as cancer, the use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, and the present invention for treating or preventing the disease.
  • a disease associated with EPHA2 such as cancer
  • the use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease and the present invention for treating or preventing the disease.
  • a therapeutic or prophylactic kit containing the antibody of the present invention is also included in the present invention.
  • Diagnostic Composition A diagnostic or diagnostic composition (hereinafter collectively referred to as “diagnostic composition”) comprising the anti-EPHA2 antibody of the present invention or a functional fragment thereof or a modified form thereof is provided.
  • the diagnostic composition of the present invention is useful for testing or diagnosis of diseases related to EPHA2, such as cancer, and EPHA2 expression.
  • the examination or diagnosis in the present invention includes, for example, determination or measurement of morbidity risk, determination of the presence or absence of morbidity, measurement of the degree of progression or deterioration, measurement or determination of the effect of drug treatment with a pharmaceutical composition such as an anti-EPHA2 antibody.
  • measurement or determination of the effect of treatment other than drug treatment measurement of recurrence risk, determination of the presence or absence of recurrence, and the like are included, but the test or diagnosis is not limited thereto.
  • the diagnostic composition of the present invention is useful for identification of an individual to which the antibody of the present invention or a functional fragment thereof or a modified product thereof, a composition containing them, or a pharmaceutical composition containing them is administered.
  • Such a diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a developer, a sensitizer, an aggregation inhibitor, and the like.
  • the present invention relates to a method for testing or diagnosing a disease associated with EPHA2, such as cancer, use of the antibody of the present invention for preparing a diagnostic composition for the disease, use of the antibody of the present invention for testing or diagnosing the disease , Also provide.
  • a test or diagnostic kit containing the antibody of the present invention is also included in the present invention.
  • a sandwich ELISA is preferable as a test or diagnostic method including the antibody of the present invention, but a normal ELISA method, RIA method, ELISPOT (Enzyme-Linked ImmunoSpot) method, dot blot method, octalony method, CIE (Counterimmunoelectrophoresis) method, CLIA Detection methods using antibodies such as (Chemiluminescent immunoassay) and FCM (Flow Cytometry) can be used.
  • labeling method for antibodies in addition to biotin, labeling methods that can be used for biochemical analysis such as labels for fluorophores such as HRP, alkaline phosphatase, FITC and ALEXA, and radioisotopes can be used.
  • TMB (3,3 ′, 5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indoyl phosphate), ⁇ -NPP ( ⁇ -nitrophenyl phosphate), OPD Fluorescent substrates such as (O-Phenylenediamine), ABTS (3-Ethylbenzothiazine-6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate TM (Sermo Fisher Scientific) Substrate In addition, it is possible to use chemiluminescent substrate.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate
  • ⁇ -NPP ⁇ -nitrophenyl phosphate
  • OPD Fluorescent substrates such as (O-Phenylenediamine), ABTS (3-Ethylbenzothiazine-6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Subst
  • test samples derived from living organisms include, but are not limited to, blood, joint fluid, ascites, lymph, cerebrospinal fluid, tissue homogenate supernatant, tissue section, and the like.
  • the test or diagnostic sandwich ELISA kit containing the antibody of the present invention may contain an EPHA2 protein standard solution, a coloring reagent, a dilution buffer, a solid phase antibody, a detection antibody, a washing solution, and the like.
  • an absorption method, a fluorescence method, a luminescence method, an RI (Radioisotope) method or the like is preferably applied.
  • An RI liquid scintillation counter or the like is preferably used.
  • a soluble protein is prepared from cells, tissues or organs or a part thereof in a sample according to a conventional method, and the soluble protein is reacted with a labeled antibody in the soluble protein. It can also be used in Western blotting or dot blotting to confirm the presence or absence of EPHA2.
  • the present invention provides antibodies useful for immunohistochemistry (IHC) analysis, functional fragments thereof and modifications thereof, and compositions containing them. Such a composition is also included in the “diagnostic composition” of the present invention.
  • IHC immunohistochemistry
  • Immunohistochemistry is not particularly limited as long as it is a technique in which a tissue section is reacted with an antibody (primary antibody) that binds to an antigen to detect the primary antibody bound to the antigen.
  • Tissue sections are preferably paraffin-embedded after formalin fixation. After embedding in paraffin, the sliced tissue section is deparaffinized and then subjected to antigen activation treatment and nonspecific reaction suppression treatment.
  • the antigen activation treatment method include heat treatment, enzyme treatment with protease, etc., and heat treatment is preferred.
  • As conditions for the heat treatment a temperature range of 90 to 110 ° C., a pH of 8 to 10 and a treatment time of 20 to 60 minutes are usually preferable.
  • Tris-EDTA buffer for example, 10 mM Tris buffer containing 1 mM EDTA
  • a method of inactivating an endogenous enzyme having a catalytic activity similar to or similar to the enzyme used for color development is usually used.
  • H 2 O 2 As the H 2 O 2 solvent, water, methanol or the like can be used, and the concentration of H 2 O 2 is 0.1 to 3%, preferably 0.3 to 3%.
  • Sodium azide can be added to the H 2 O 2 solution.
  • a method of blocking with serum or casein can also be used as a nonspecific reaction suppression treatment.
  • Serum and casein can treat tissues before the primary antibody reaction, but can also be included in a solvent that dilutes the primary antibody.
  • the reaction conditions for the primary antibody are not particularly limited, but the temperature is 4 to 50 ° C, preferably 20 to 37 ° C, more preferably 24 ° C.
  • the reaction time is 5 minutes to 1 day, preferably 10 minutes to 4 hours, more preferably 30 minutes to 1 hour.
  • an antibody that can be visualized and binds to the primary antibody can be preferably used.
  • the reaction is performed three or more times using an antibody (tertiary antibody) that binds to the secondary antibody itself.
  • the secondary or tertiary antibody can be visualized by binding an enzyme such as peroxidase or alkaline phosphatase to these antibodies, or by adding biotin or the like to these antibodies and binding to the aforementioned enzymes such as streptavidin.
  • a method of reacting a chromogenic substrate corresponding to those enzymes can be preferably used.
  • Examples of a method for binding an enzyme to a secondary antibody or a tertiary antibody include a method using a reagent in which a large number of the enzyme and secondary antibody are bound to a dextrin polymer or an amino acid polymer (polymer method).
  • polymer method a method of reacting a biotinylated secondary antibody and peroxidase-labeled streptavidin (LSAB method), DAB or the like can be used as a chromogenic substrate.
  • a secondary antibody labeled with a fluorescent dye or the like can also be used. When treated with a fluorescently labeled secondary antibody, positive cells are detected using a fluorescence microscope after the treatment.
  • the isolated cells are applied to glass or separated by a centrifugal separator, divided into cell components and liquid components, and immunostaining is performed on the cell components. That is, cell components can be applied on a slide glass and fixed with an ethanol solution or a 10% formalin solution, and then immunostaining similar to a tissue section can be performed.
  • the excised tissue is rapidly frozen with liquid nitrogen after embedding with an OCT compound and sliced with a cryostat to prepare a slide specimen. After fixing this specimen with 10% formalin or ethanol solution, immunostaining similar to the tissue section can be performed.
  • the operation related to immunohistochemistry can be performed automatically by programming the reaction solution, reaction conditions, number of washings, etc., and incorporating it into the immune device.
  • diagnostic imaging label the antibody with a pharmaceutically acceptable radionuclide or illuminant, administer the antibody to a subject, take an image using diagnostic imaging techniques such as PET / CT, and then the presence of EPHA2 Can be determined or inspected.
  • the antibody, functional fragment thereof or modified product thereof contained in the diagnostic composition of the present invention is preferably an antibody that binds to or recognizes EPHA2, that is, an antibody having EPHA2 selectivity, a functional fragment thereof, or a modified product thereof.
  • antibodies having human EPHA2 selectivity include heavy chain CDRH1 to CDRH3 of the mouse A201 antibody (amino acid sequence shown in SEQ ID NOs: 60 to 62 (FIG. 11)), and light chain CDRL1 to CDRRL3 ( An antibody comprising a light chain comprising the amino acid sequence shown in SEQ ID NO: 63 to 65 (FIG. 11), a heavy chain variable region of the mouse A201 antibody (amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 51) and light Examples include an antibody containing a chain variable region (amino acid sequence shown at amino acid numbers 20 to 128 of SEQ ID NO: 54), an antibody containing the heavy and light chains of mouse A201 antibody, and the like. Examples of such antibodies include mouse A201 antibody and chimeric A201 antibody, but are not limited thereto.
  • Antibodies having human EPHA2 selectivity include heavy chain CDRH1 to CDRH3 (amino acid sequence shown in SEQ ID NOs: 66 to 68 (FIG. 12)) of mouse A205 antibody, and light chain CDRL1 to CDRRL3 ( An antibody comprising a light chain comprising the amino acid sequence shown in SEQ ID NOs: 69 to 71 (FIG. 12), a heavy chain variable region of the mouse A205 antibody (amino acid sequence shown in amino acids 20 to 138 of SEQ ID NO: 57), and Examples include an antibody containing a light chain variable region (amino acid sequence shown in amino acid numbers 20 to 133 of SEQ ID NO: 59), an antibody containing the heavy chain and light chain of mouse A205 antibody, and the like. Examples of such antibodies include, but are not limited to, mouse A205 antibody and chimeric A205 antibody.
  • the diagnostic composition is for EPHA2 detection or measurement.
  • the present invention provides a method for detecting or measuring human EPHA2 in a test sample.
  • the diagnostic composition of the present invention can be used for these detection or measurement methods. Such measurement methods and diagnostic compositions are also included in the present invention for diagnosis or testing of human EPHA2-positive cancer.
  • a method for identifying an individual to which the pharmaceutical composition of the present invention is administered is also encompassed by the present invention.
  • human EPHA2 in the sample derived from the individual is measured, and human EPHA2 is detected in the sample, or compared with the amount of human EPHA2 detected in the sample derived from a healthy individual. If many human EPHA2s are detected, the individual can be determined as positive.
  • the diagnostic composition of the present invention can be used.
  • the individual is suffering from or at risk of having cancer.
  • the pharmaceutical composition of the present invention can be administered to an individual who has been determined to be positive by such an identification method.
  • Reagent The antibody of the present invention or a functional fragment thereof or a modified form thereof is also useful as a reagent. Such reagents are used in the above-described examination or diagnostic, research and other applications.
  • Example 1 Production of Mouse Anti-Human EPHA2 Antibody 1 -1 Immunization Fcgr2b KO (BALB / c) Homo mouse female (Immuno-Biological Laboratories) was used for immunization. A mixture of Recombinant Human EPHA2 (R & D SYSTEM; 3035-A2) and Freund's Complete Adjuvant (Wako Pure Chemical Industries) was collected subcutaneously and used for hybridoma production.
  • Example 2 Evaluation of antigen-binding ability of mouse anti-human EPHA2 antibody 2) -1 Construction of vector expressing human EPHA2 and protein having sequence similarity to human EPHA2 2) -1-1 Construction of expression vector pFLAG- Reading frame of Gateway Vector Conversion System (Life Technologies, Thermo Fisher Scientific) on blind-ended HindIII and BglII sites of myc-CMV19 (Sigma). This vector was named “pFLAG-GW”.
  • the gene encoding EPHA2 was transferred from this entry vector to pFLAG-GW by LR recombination reaction.
  • the resulting vector was named pFLAG-EPHA2.
  • the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • PTPRS-2 A DNA fragment (SEQ ID NO: 39) encoding human PTPRS isoform 2 (hereinafter referred to as PTPRS-2) without a signal sequence was used as a primer set 5'-GGGACAAGTTTGTACAAAAAAAGCAGCCTTCGAGT 3 ′ (PTPRS-2 / 4-Fw: SEQ ID NO: 41) 5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTTAGGTTGCATAGTGTCAAAG-3 ′ (PTPRS-2 / 4-Re: SEQ ID NO: 42) And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction.
  • PTPRF-2-Fw SEQ ID NO: 47
  • PTPRF-2-Re SEQ ID NO: 48
  • This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction.
  • the gene encoding PTPRF-2 was transferred from this entry vector to pFLAG-GW by LR recombination reaction.
  • the resulting vector was named pFLAG-PTPRF-2.
  • the amino acid sequence of SEQ ID NO: 45 is shown in SEQ ID NO: 46.
  • Sections are prepared from these blocks and using an automated immunostaining device Ventana Discovery ULTRA and DAB map kit (above, Roche Diagnostics) or Autostainer Link 48 and EnVisionFLEX Mini Kit (DAKO) using the instrument's instructions.
  • IHC screening of the antibody contained in the culture supernatant derived from was performed. First, among the 67 hybridoma-derived culture supernatants obtained in Example 1) -2, 12 antibodies that react strongly with EPHA2-expressing 293 ⁇ cells and do not react with non-transfected 293 ⁇ cells were obtained. Selected. Further, 6 cells that react with MDA-MB-231 cells were selected, and 3 cells that strongly reacted with endogenous EPHA2 were selected.
  • Example 2 Three antibodies selected in 2-1 were purified from the culture supernatant of hybridomas producing these antibodies, Example 2) -2-1 In the same manner as human esophageal cancer (CR2), stomach cancer (CQ2), colon cancer (CDA3), cervical cancer (CZA2), lung cancer (CCA4), pharyngeal laryngeal cancer (CH3), malignant melanoma (LM481) and commercially available paraffin sections of normal tissues (MAN, MBN, MCN) (above, SuperBioChip Laboratories) were stained. Among the three, A201 and A205 that responded strongly to tumor cells and had low background staining were selected.
  • A201 and A205 which are mouse anti-human EPHA2 antibodies, were determined by the Mouse monoclonal isotyping test kit (AbD Serotec). As a result, it was shown that the isotype of A201 is IgG1, and the isotype of A205 is IgG2b.
  • pFLAG-PTPRS-4, pFLAG-PTPRF-2 and pFLAG-GW as controls were transfected into 293F cells using 293fectin (Life Technologies, Thermo Fisher Scientific), 37 ° C., 125 rpm, 8% CO 2, respectively. Cultured overnight under 2 conditions. The next day, these 293F cells were collected, washed, and then used for flow cytometry analysis.
  • each cell prepared by the method shown in Example 2) -4 was treated with A201, A205, a mouse IgG control antibody (Becton Dickinson), and Anti-FLAG M2 antibody (Sigma-Aldrich) were added and incubated at 4 ° C. for 30 minutes. These cells were washed twice with PBS containing 5% FBS, Anti-Mouse IgG FITC conjugate (MP Bio) diluted 500 times with PBS containing 5% FBS was added, and incubated at 4 ° C. for 20 minutes.
  • A205 binds to EPHA2, while for molecules with sequence similarity to EPHA2 (EPHA3, EPHA5, EPHA6, EPHA7, EPHAB1, EPHAB3, EPHB4, PTPRS-2, PTPRS-4, and PTPRF-2) It was shown not to bind ( Figure 3). A205 also bound to cells transfected with the negative control pFLAG-GW, which was presumed to be due to A205 binding to EPHA2 that is endogenously expressed in 293F cells. .
  • mG1VR1 SEQ ID NO: 49
  • mG1VR1 was designed from the sequence of the constant region of mouse heavy chain (IgG1) in the database.
  • cDNA encoding the heavy chain fragment of A201 was amplified by 5'-RACE PCR. This PCR was performed using a touch-down PCR program according to the manual of SMARTER RACE cDNA Amplification Kit using KOD-Plus- (TOYOBO) as Polymerase.
  • a cDNA encoding a heavy chain fragment amplified by 5'-RACE PCR is purified using MinElute PCR Purification Kit (QIAGEN), then cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and cloned cDNA was sequenced.
  • 5'-CATCCCCAGGGTCACCCATGGAGTTTAGTTTG-3 '(mG1VR1: SEQ ID NO: 49) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.
  • the sequencing reaction was performed with GeneAmp 9700 (Applied Biosystems), and the DNA sequencing was performed with ABI PRISM 3700 DNA Analyzer (Applied Biosystems) or Applied Biosystems 3730xxl Analyzer (Applied Biosystems).
  • the nucleotide sequence of the cDNA encoding the determined heavy chain of A201 is shown in SEQ ID NO: 50, and the amino acid sequence is shown in SEQ ID NO: 51.
  • the amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 51 corresponds to the signal sequence of the A201 heavy chain, and the amino acid sequence consisting of the 20th to 137th amino acid residues corresponds to the heavy chain variable region of A201.
  • SEQ ID NO: 50 and SEQ ID NO: 51 are also shown in FIG.
  • cDNA encoding the light chain fragment of A201 was amplified by 5'-RACE PCR. This PCR was performed using a touch-down PCR program according to the manual of SMARTER RACE cDNA Amplification Kit using KOD-Plus- (TOYOBO) as Polymerase.
  • a cDNA encoding a light chain fragment amplified by 5'-RACE PCR is purified using MinElute PCR Purification Kit (QIAGEN), then cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and cloned cDNA was sequenced.
  • 5'-AGTCCAACTGTTCAGGACGCCCATTTGTCG-3 '(mKVR2: SEQ ID NO: 52) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.
  • the sequencing reaction was performed with GeneAmp 9700 (Applied Biosystems), and the DNA sequencing was performed with ABI PRISM 3700 DNA Analyzer (Applied Biosystems) or Applied Biosystems 3730xxl Analyzer (Applied Biosystems).
  • the determined nucleotide sequence of cDNA encoding the light chain of A201 is shown in SEQ ID NO: 53 of the Sequence Listing, and the amino acid sequence is shown in SEQ ID NO: 54.
  • the amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 54 corresponds to the signal sequence of the A201 light chain, and the amino acid sequence consisting of the 20th to 128th amino acid residues corresponds to the light chain variable region of A201.
  • SEQ ID NO: 53 and SEQ ID NO: 54 are also shown in FIG.
  • mG2bVR2 was designed from the sequence of the constant region of the mouse heavy chain (IgG2b) in the database.
  • 5′-TGCACACTGCTGGACAGGGGATCCAGAGTTC-3 ′ (mG2bVR2: SEQ ID NO: 55) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.
  • the nucleotide sequence of the cDNA encoding the determined heavy chain of A205 is shown in SEQ ID NO: 56 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 57.
  • the amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 57 corresponds to the signal sequence of the A205 heavy chain, and the amino acid sequence consisting of the 20th to 138th amino acid residues corresponds to the heavy chain variable region of A205.
  • SEQ ID NO: 56 and SEQ ID NO: 57 are also shown in FIG.
  • Example 3 3) -2-4 Amplification of cDNA encoding light chain fragment of A205 by 5'-RACE PCR and determination of nucleotide sequence Example 3) cDNA synthesized in 2-2 (5'-RACE-Ready cDNA) Using the template, the cDNA encoding the light chain fragment of antibody A205 was amplified and sequenced in the same manner as in Example 3) -1-4.
  • the nucleotide sequence of the determined cDNA encoding the light chain of A205 is shown in SEQ ID NO: 58 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 59.
  • the amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 59 corresponds to the signal sequence of the A205 light chain, and the amino acid sequence consisting of the 20th to 133rd amino acid residues corresponds to the light chain variable region of A205.
  • SEQ ID NO: 58 and SEQ ID NO: 59 are also shown in FIG.
  • Example 4 Production of mouse anti-human EPHA2 antibody
  • Mouse anti-human EPHA2 antibodies A201 and A205 were purified from the hybridoma culture supernatant.
  • A201 and A205-producing hybridomas were grown to a sufficient amount with ClonCell-HY Selection Medium E, and then Ultra Low IgG FBS (Life Technologies, ThermoFisher Scifi) was added to 20% of the world's TiMeMeSriMiMeFriSriMeMriFiSriMiSriFiSriMiSriMiSriMiSriFiSriMiSriFlM. The medium was changed to Fisher Scientific) and cultured for 5 days. The main culture supernatant was collected and sterilized through a 0.45 ⁇ m filter.
  • the antibody was purified from the above hybridoma supernatant by Protein G affinity chromatography (under 4-6 ° C.) in a one-step process.
  • the buffer substitution step after protein G affinity chromatography purification was performed at 4 to 6 ° C.
  • the culture supernatant of the hybridoma was applied to a column packed with Protein G (GE Healthcare Bioscience) equilibrated with 0.2 M sodium phosphate buffer (pH 7.0). After all of the culture supernatant liquid entered the column, the column was washed with a sodium phosphate buffer having a column volume of 2 times or more.
  • FIG. 4 shows the specificity of the mouse anti-EPHA2 antibody in immunohistochemical staining.
  • the Eph family molecules shown in FIG. 4 were transiently expressed in 293F cells, and the pellets of these cells were embedded in paraffin after formalin fixation.
  • Sections prepared from these blocks were subjected to antigen activation treatment with Tris-EDTA solution (DAKO) at 97 ° C for 40 minutes, endogenous peroxidase (HRP) blocking reagent (DAKO) 5 minutes, nonspecific protein adsorption blocking reagent (DAKO) After reacting in order of 30 minutes, mouse anti-EPHA2 antibody A201 or A205 prepared to a concentration of 2.5 ⁇ g / mL with an antibody diluent (DAKO) was reacted for 1 hour. Immunohistochemical staining was performed by a conventional method using an HRP-labeled universal secondary antibody (DAKO) and DAB chromogenic substrate (DAKO).
  • FIG. 5 shows the results of immunohistochemical staining with mouse anti-EPHA2 antibody A201.
  • a commercially available human micropharyngeal cancer and gastric cancer tissue microarray (SuperBioChips Laboratories) was subjected to antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, and an endogenous peroxidase (HRP) blocking reagent (DAKO) After reacting in the order of 5 minutes and non-specific protein adsorption blocking reagent (DAKO) for 30 minutes, mouse anti-EPHA2 antibody A201 prepared to a concentration of 5 ⁇ g / mL with an antibody diluent (DAKO) was reacted for 1 hour.
  • DAKO endogenous peroxidase
  • FIG. 5 is a photomicrograph showing specific staining of part of the cell membrane and cytoplasm of tumor cells of laryngeal squamous cell carcinoma and gastric adenocarcinoma by mouse anti-EPHA2 antibody A201. There is no staining in the tissues and cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.
  • FIG. 6 shows the results of immunohistochemical staining with mouse anti-EPHA2 antibody A205.
  • a commercially available human micropharyngeal cancer and gastric cancer tissue microarray (SuperBioChips Laboratories) was subjected to an antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, and an endogenous HRP blocking reagent (DAKO) 5 minutes.
  • DAKO Tris-EDTA solution
  • DAKO endogenous HRP blocking reagent
  • mouse anti-EPHA2 antibody A205 prepared to a concentration of 15 ⁇ g / mL with an antibody diluent (DAKO) was reacted for 1 hour.
  • FIG. 6 is a photomicrograph showing specific staining of the cell membrane and part of the cytoplasm of tumor cells of laryngeal squamous cell carcinoma and gastric adenocarcinoma by the mouse anti-EPHA2 antibody A205. There is no staining in the tissues and cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.
  • mice anti-EPHA2 antibodies A201 and A205 The same site is stained with mouse anti-EPHA2 antibodies A201 and A205, but A201 is more sensitive than A205, and more tumor cells are stained.
  • FIGS. 13 and 14 show the staining conditions and staining properties of the anti-EPHA2 antibody in immunohistochemical staining.
  • EPHA2 non-expressing 293 ⁇ cells, EPHA2 overexpressing 293 ⁇ cells, and EPHA2 endogenously expressed MDA-MB-231 cell pellets were embedded in paraffin after formalin fixation. Sections prepared from these blocks were subjected to antigen activation treatment with Tris-EDTA solution (DAKO or Roche Diagnostics) at 97 ° C. for 36 to 40 minutes, and endogenous peroxidase (HRP) blocking, non-blocking was performed by a conventional method.
  • Tris-EDTA solution DAKO or Roche Diagnostics
  • concentration optimized by preliminary examination was made to react for 1 hour.
  • immunohistochemical staining was carried out by the LSAB method or the polymer method using a kit of Roche Diagnostics or DAKO. By microscopic observation, a positive positive reaction was observed in the cell membrane or cytoplasm, and a positive positive reaction was observed when a weak positive reaction was observed.
  • Both A201 and A205 antibodies show strong positive reaction to both EPHA2 overexpressing 293 ⁇ cells and EPHA2 endogenously expressed MDA-MB-231 cells, and recognize EPHA2 with higher sensitivity than various commercially available EPHA2 antibodies compared. Was confirmed (FIG. 13).
  • FIG. 14 is a photomicrograph of cells. In all antibodies, no positive reaction is observed in EPHA2 non-expressing 293 ⁇ cells, whereas in EPHA2 overexpressing 293 ⁇ cells, a strong positive reaction is observed in the cell membrane and cytoplasm.
  • the mouse anti-EPHA2 antibody (D4) manufactured by Millipore high non-specific staining was observed throughout the cells.
  • 15 to 18 show the results of immunohistochemical staining with mouse anti-EPHA2 antibodies A201 and A205, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).
  • a commercially available human tumor tissue microarray (SuperBioChips Laboratories) was subjected to antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, blocked in the same manner as described above, and then diluted with an antibody diluent (DAKO). Each anti-EPHA2 antibody diluted in 1) was reacted for 1 hour.
  • Immunohistochemical staining was performed by a conventional method using an HRP-labeled universal secondary antibody (DAKO) or an HRP-labeled anti-goat IgG antibody (Nichirei Bioscience) or a DAB chromogenic substrate (DAKO).
  • DAKO an anti-mouse linker antibody
  • 15 to 18 are micrographs of tumor tissues, which are stained images of human esophageal cancer (FIG. 15), cervical cancer (FIG. 16), colon cancer (FIG. 17), and stomach cancer (FIG. 18).
  • A201 antibody and A205 antibody showed specific staining in the cell membrane and part of cytoplasm of tumor cells, and A201 was more sensitive than A205, and more tumor cells were stained. Yes. There is little or no specific staining with the AF3035 and sc-924 antibodies.
  • the A201 antibody, A205 antibody, and AF3035 antibody show no staining in the tissues and cells of the tumor stroma, but the sc-924 antibody also shows a cross reaction in the non-tumor cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.
  • the use of the antibody provided by the present invention enables examination or diagnosis of various cancers.
  • SEQ ID NO: 1 Nucleotide sequence encoding human EPHA2 SEQ ID NO: 2: Amino acid sequence of human EPHA2 SEQ ID NO: 3: PCR primer EPHA2-Fw SEQ ID NO: 4: PCR primer EPHA2-Re SEQ ID NO: 5: nucleotide sequence encoding human EPHA3 SEQ ID NO: 6: amino acid sequence of human EPHA3 SEQ ID NO: 7: PCR primer EPHA3-Fw SEQ ID NO: 8: PCR primer EPHA3-Re SEQ ID NO: 9: nucleotide sequence encoding human EPHA4 SEQ ID NO: 10: amino acid sequence of human EPHA4 SEQ ID NO: 11: PCR primer EPHA4-Fw SEQ ID NO: 12: PCR primer EPHA4-Re SEQ ID NO: 13: nucleotide sequence encoding human EPHA5 SEQ ID NO: 14: amino acid sequence of human EPHA5 SEQ ID NO: 15: PCR primer EPHA5-Fw SEQ ID NO: 16

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'objectif de la présente invention est de fournir un anticorps capable de se lier à l'EPHA2 humain, une composition médicinale comprenant l'anticorps, une composition de diagnostic comprenant l'anticorps, etc.
PCT/JP2016/062719 2015-04-24 2016-04-22 Détection d'epha2 WO2016171242A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015089156 2015-04-24
JP2015-089156 2015-04-24

Publications (1)

Publication Number Publication Date
WO2016171242A1 true WO2016171242A1 (fr) 2016-10-27

Family

ID=57143160

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/062719 WO2016171242A1 (fr) 2015-04-24 2016-04-22 Détection d'epha2

Country Status (1)

Country Link
WO (1) WO2016171242A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110198956A (zh) * 2017-01-23 2019-09-03 学校法人东洋大学 抗epha2抗体和使用该抗epha2抗体的epha2免疫检测
WO2021250418A1 (fr) * 2020-06-12 2021-12-16 Bicycletx Limited Traitement de maladies caractérisées par la surexpression d'un récepteur hépatocellulaire a2 produisant de l'érythropoïétine (epha2)
US11623012B2 (en) 2017-12-19 2023-04-11 Bicyclerd Limited Bicyclic peptide ligands specific for EphA2
US11814447B2 (en) 2018-06-22 2023-11-14 Bicyclerd Limited Peptide ligands for binding to EphA2
US11833211B2 (en) 2017-12-19 2023-12-05 Bicycletx Limited Methods of suppression and treatment of disease comprising administering bicycle peptide ligands specific for EphA2
US11912792B2 (en) 2018-06-22 2024-02-27 Bicycletx Limited Bicyclic peptide ligands specific for nectin-4
US11970553B2 (en) 2019-07-30 2024-04-30 Bicycletx Limited Heterotandem bicyclic peptide complex

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008500021A (ja) * 2003-11-20 2008-01-10 メディミューン，インコーポレーテッド ＥｐｈＡ２アゴニストモノクローナル抗体およびその使用法
WO2012008397A1 (fr) * 2010-07-12 2012-01-19 第一三共株式会社 Anticorps anti-epha2 et utilisation de celui-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008500021A (ja) * 2003-11-20 2008-01-10 メディミューン，インコーポレーテッド ＥｐｈＡ２アゴニストモノクローナル抗体およびその使用法
WO2012008397A1 (fr) * 2010-07-12 2012-01-19 第一三共株式会社 Anticorps anti-epha2 et utilisation de celui-ci

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110198956A (zh) * 2017-01-23 2019-09-03 学校法人东洋大学 抗epha2抗体和使用该抗epha2抗体的epha2免疫检测
EP3572426A4 (fr) * 2017-01-23 2020-12-23 Toyo University Anticorps anti-epha2 et détection immunologique d'epha2 faisant appel audit anticorps
US11623012B2 (en) 2017-12-19 2023-04-11 Bicyclerd Limited Bicyclic peptide ligands specific for EphA2
US11833211B2 (en) 2017-12-19 2023-12-05 Bicycletx Limited Methods of suppression and treatment of disease comprising administering bicycle peptide ligands specific for EphA2
US11814447B2 (en) 2018-06-22 2023-11-14 Bicyclerd Limited Peptide ligands for binding to EphA2
US11912792B2 (en) 2018-06-22 2024-02-27 Bicycletx Limited Bicyclic peptide ligands specific for nectin-4
US11970553B2 (en) 2019-07-30 2024-04-30 Bicycletx Limited Heterotandem bicyclic peptide complex
WO2021250418A1 (fr) * 2020-06-12 2021-12-16 Bicycletx Limited Traitement de maladies caractérisées par la surexpression d'un récepteur hépatocellulaire a2 produisant de l'érythropoïétine (epha2)

Similar Documents

Publication Publication Date Title
WO2016171242A1 (fr) Détection d'epha2
CA3027417C (fr) Anticorps anti-trop-2 humain presentant une activite antitumorale in vivo
CA2669731C (fr) Anticorps anti-humain specifiques de dlk1 presentant une activite anti-tumorale in vivo
JP2021524242A (ja) 抗−ｒｏｒ１抗体およびその用途
KR102029248B1 (ko) 암세포 특이적 항체, 항암제 및 암 검사 방법
EP2275537A1 (fr) ANTICORPS ANTI-HUMAIN SPÉCIFIQUES DE DLK1 PRÉSENTANT UNE ACTIVITÉ ANTI-TUMORALE IN VIVO& xA;
US20230331836A1 (en) Anti-cldn18.2 antibodies and diagnostic uses thereof
EP3282014B1 (fr) Anticorps contre la podocalyxine spécifique des cellules cancéreuses et procédé de production de ce dernier
EP3543259A2 (fr) Anticorps se liant à l'anhydrase carbonique, et son utilisation
JP2024020436A (ja) Ｈｅｒ２標的化剤
CA2948743C (fr) Anticorps se liant a une anhydrase carbonique et utilisation associee
WO2016171107A1 (fr) Détection de fgfr2
JP7082112B2 (ja) 抗ｇｐｒ２０抗体
EP4294839A1 (fr) Molécules de liaison à vegfa
JP2022504472A (ja) Ｈｅｒ２結合四量体ポリペプチド
WO2023219147A1 (fr) Nouveaux anticorps anti-ccr8 pour la détection de ccr8
WO2023190465A1 (fr) Anticorps anti-sema7a humain
NZ716839A (en) Anti-human trop-2 antibody having an antitumor activity in vivo

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16783263

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16783263

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP