WO2018034332A1 - ANTICORPS FRAGMENT DE L'EXTRÉMITÉ N-TERMINALE EphA2 - Google Patents

ANTICORPS FRAGMENT DE L'EXTRÉMITÉ N-TERMINALE EphA2 Download PDF

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WO2018034332A1
WO2018034332A1 PCT/JP2017/029596 JP2017029596W WO2018034332A1 WO 2018034332 A1 WO2018034332 A1 WO 2018034332A1 JP 2017029596 W JP2017029596 W JP 2017029596W WO 2018034332 A1 WO2018034332 A1 WO 2018034332A1
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amino acid
acid sequence
epha2
seq
protein
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Japanese (ja)
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直彦 越川
元治 清木
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国立大学法人東京大学
地方独立行政法人神奈川県立病院機構
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Priority to JP2018534429A priority Critical patent/JP6729917B2/ja
Publication of WO2018034332A1 publication Critical patent/WO2018034332A1/fr

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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
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to an antibody that specifically binds to an N-terminal fragment of EphA2 cleaved by MT1-MMP and a method for producing the antibody.
  • EphA2 (Erythropoietin-producing hepatocellular receptor 2) is a member of the mammalian Eph receptor kinase family, including breast cancer, liver cancer, pancreatic cancer, prostate cancer, stomach cancer, glioma, melanoma and ovarian adenocarcinoma It is overexpressed in various tumor tissues and is related to cancer progression and metastasis (Non-Patent Document 1). The tyrosine kinase activity of EphA2 is activated when a ligand such as ephrin A1 binds.
  • EphA2 In normal epithelial cells, ligand-induced autophosphorylation of the EphA2 tyrosine residue (Tyr 594 ) plays a role in suppressing the growth signal mediated by the Erb-B receptor and maintaining the cell in a normal state. Yes. That is, EphA2 has a tumor suppressor-like activity in normal cells, but promotes cancer progression when overexpressed in cancer cells. Recent studies have revealed that EphA2 has two opposing activities.
  • EphA2 Unlike EphA2, which functions in a ligand-dependent manner, EphA2 in the absence of a ligand phosphorylates a serine residue (Ser 897 ) by Ras / MAPK after stimulation of the ErbB receptor (Non-patent Document 2), and ephexin-4 It acts as a docking site with / RhoG and induces activation of Rac1 (Non-patent Document 3).
  • EphA2 has attracted attention as a molecular target for anticancer agents because of the elucidation of the mechanism of action of EphA2 in the progression of cancer.
  • MT1-MMP membrane-type 1-matrix metalloproteinase 1
  • MT1-MMP membrane metalloprotease known to be overexpressed in tumor cells.
  • MT1-MMP regulates tumor progression through the processing of physiologically active proteins and extracellular matrix around the cell, and activation of HIF transcription factors via the cytoplasmic terminal.
  • EphA2 is a substrate of MT1-MMP, which is cleaved with MT1-MMP to remove the EphA2 ligand binding domain (Non-patent document 4, Non-patent document 5) and converted to a structure in which a ligand cannot bind.
  • EphA2 that is MT1-MMP and co-expressed in cancer tissues and lacking the ligand binding domain of N-terminal side was found that Ser 897 is phosphorylated (non-patent Reference 5).
  • the mutant EphA2 that was not cleaved by MT1-MMP was forcibly expressed in the epithelial carcinoma A431 cell line, the cell morphology changed to an epithelial cell-like morphology, and tumor growth and lung metastasis were suppressed (Non-patent Document 5). ). Therefore, EphA2 processing by MT1-MMP in cancer cells is an event closely related to cancer progression.
  • the N-terminal fragment of EphA2 cleaved by MT1-MMP circulates in the bloodstream away from the cell, the N-terminal fragment can be detected at an early stage using a conventional blood test. It can be used as a marker (Patent Document 1).
  • an object of the present invention is to provide an antibody that specifically binds to an N-terminal fragment of EphA2 cleaved with MT1-MMP and a method for producing the antibody.
  • Another object of the present invention is to provide a method for examining cancer using the antibody. Furthermore, this invention aims at provision of the kit for a test
  • EphA2 has also been reported to exist in exosomes, and the N-terminal fragment of EphA2 cleaved with MT1-MMP in blood (hereinafter referred to as “MT1-MMP-cleaved EphA2-N terminal fragment”) It was expected that full-length (intact) EphA2 was present.
  • MT1-MMP-cleaved EphA2-N terminal fragment the N-terminal fragment of EphA2 cleaved with MT1-MMP in blood
  • the inventors have determined the structure of the MT1-MMP-cleaved EphA2-N terminal fragment, the structure of the N-terminal region of full-length EphA2, and the structure of the cleaved fragment if EphA2 is cleaved by membrane proteases (EphA2 extracellular).
  • EphA2 extracellular The preparation of an antibody that recognizes only the “MT1-MMP-cleaved EphA2-N terminal fragment” is made based on the hypothesis that the domain fragments are different or that there is a specific epitope for each. Tried.
  • EphA2 and EphA2 extracellular domain fragments As a result of conducting extensive screening and antibody screening, the inventors did not recognize full-length EphA2 and EphA2 extracellular domain fragments, but specifically recognized only the “MT1-MMP-cleaved EphA2-N terminal fragment”. The production of the antibody was successful and the present invention was completed.
  • the present invention relates to the following (1) to (12).
  • a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by the 28th to Xth positions As a specific example of the protein, a protein comprising the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Or a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, (B) a protein comprising the 28th to 9
  • a test kit for cancer comprising the antibody according to any one of (1) to (3) above.
  • (10) A method of measuring MT1-MMP protease activity in a sample based on the amount of MT1-MMP-cleaved EphA2-N terminal fragment measured by the measurement method of (9) above.
  • a method for producing an antibody comprising the following steps (i) and (ii): (I) a protein comprising an amino acid sequence represented by the 28th to Xth amino acid sequence represented by SEQ ID NO: 1 (where X is an integer of 328 to 435, the same shall apply hereinafter), represented by SEQ ID NO: 1
  • a protein comprising an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by positions 28 to X in the amino acid sequence, or in the amino acid sequence represented by SEQ ID NO: 1
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 2 an amino acid sequence represented by SEQ ID NO: 2
  • the antibody of the present invention can specifically bind to the MT1-MMP-cleaved EphA2-N terminal fragment. That is, the antibody of the present invention recognizes only the MT1-MMP-cleaved EphA2-N terminal fragment, not the full-length EphA2 or EphA2 extracellular domain fragment.
  • the antibody of the present invention it becomes possible to detect only the MT1-MMP-cleaved EphA2-N terminal fragment present in the sample, whether the subject from which the sample is derived is affected by cancer, cancer It is possible to examine and diagnose the degree of progression and the effect of cancer treatment.
  • EphA2 The structure of EphA2 and the reaction results of the antibody of the present invention and the antigen are shown.
  • A Schematic showing EphA2 domain and MT1-MMP cleavage site. “Recombinant antigen” is a figure showing to which region of EphA2 the prepared antigen protein corresponds. The arrow indicates the position reported to be cut by MT1-MMP. SP: Signal peptide, CRD; Cysteine-rich domain, TM; Transmembrane, Cytoplasmic; Recombinant antigen (Antigen # 1- # 5) produced by cytoplasmic region
  • B The results of detection by Western blotting using an anti-FLAG monoclonal antibody are shown.
  • the precipitate was analyzed by Western blotting using an anti-FLAG tag monoclonal antibody.
  • IP Immunoprecipitation, WB; Western blotting It is the result of having investigated the affinity with respect to the recombinant antigen (Antigens # 1-4) of the 46A1, 62A1, and 76A1 monoclonal antibodies by Western blotting (upper figure: reducing condition, lower figure; non-reducing condition). All antigens were detected under reducing conditions by anti-FLAG tag monoclonal antibody Results of sandwich ELISA assay.
  • (A) shows a standard curve of 76A1 binding reaction to Antigen # 1 (15-250 pg / mL).
  • the first embodiment of the present invention specifically binds to an MT1-MMP-cleaved EphA2-N terminal fragment, does not bind to full-length (intact) EphA2, and the EphA2 extracellular domain An antibody that does not bind to fragments.
  • the inventors have previously found that the MT1-MMP-cleaved EphA2-N terminal fragment circulates in the bloodstream away from cancer cells, and this MT1-MMP-cleaved EphA2-N terminal fragment It is clarified that it can be used as a detectable cancer marker (Patent Document 1).
  • an antibody prepared using the N-terminal fragment of EphA2 as an immunogen specifically recognizes the N-terminal fragment of EphA2, but at the same time, it is highly likely to recognize full-length EphA2.
  • Eph family molecules have been reported to be cleaved by membrane proteases (Bai G., Pfaff SL (2011). Protease regulation: the Yin and Yang of neural development and disease. Neuron 72, 9-21 ), EphA2 is also likely to be processed by membrane proteases, and antibodies produced simply using the N-terminal fragment as an immunogen recognizes fragments other than the MT1-MMP-cleaved EphA2-N terminal fragment. There was a possibility.
  • the inventors have identified the MT1-MMP-cleaved EphA2-N terminal fragment, and the EphA2 extracellular domain conformation that may occur when processed by full-length EphA2 and membrane proteases.
  • Antibodies that do not react to either full-length EphA2 or the extracellular domain of EphA2 among antibodies obtained using MT1-MMP-cleaved EphA2-N terminal fragments as immunogens considering the possibility of different structures Only tried to get.
  • a part of the MT1-MMP-cleaved EphA2-N terminal fragment is expressed in animal cells, and this is used as an immunogen to obtain several antibodies that can immunoprecipitate the fragment.
  • EphA2 did not immunoprecipitate full-length EphA2 from the extract of cells expressing full-length EphA2, and did not immunoprecipitate the extracellular domain of EphA2 expressed in animal cells.
  • the antibody obtained by the method as described above is confirmed to react specifically with a blood sample derived from a cancer patient, as shown in the examples below, and is expected to be used as a cancer marker. It specifically binds to the MT1-MMP-cleaved EphA2-N terminal fragment.
  • the first embodiment of the present invention is specifically an antibody that binds to the following protein (a) and does not bind to the proteins (b) and (c).
  • A a protein comprising an amino acid sequence represented by the 28th to Xth amino acid sequence represented by SEQ ID NO: 1 (where X is an integer of 328 to 435, the same shall apply hereinafter), represented by SEQ ID NO: 1
  • a protein comprising an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by positions 28 to X in the amino acid sequence, or in the amino acid sequence represented by SEQ ID NO: 1
  • a protein comprising an amino acid sequence having at least 80% sequence identity with the 28th to Xth amino acid sequences As a specific example of the protein, a protein comprising the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Or a protein comprising an
  • the MT1-MMP-cleaved EphA2-N terminal fragment of EphA2 refers to, for example, the 28th to 385th amino acids in the amino acid sequence shown in SEQ ID NO: 1. , Fragments containing the amino acid sequence from 28th to 395th, 28th to 432th, or 28th to 435th.
  • an antigen for producing an antibody that binds to the MT1-MMP-cleaved EphA2-N terminal fragment for example, the 28th to Xth amino acids in the amino acid sequence represented by SEQ ID NO: 1 (where X is 328 or more)
  • a protein consisting of an amino acid sequence represented by an integer of 435 or less (the same shall apply hereinafter), or adding or replacing one or several amino acids in the amino acid sequence represented by positions 28 to X in the amino acid sequence represented by SEQ ID NO: 1.
  • a protein comprising an inserted or deleted amino acid sequence, or a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by positions 28 to X in the amino acid sequence represented by SEQ ID NO: 1 Can be used.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (the 28th to 328th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1) (hereinafter referred to as “Antigen # 1”) is described.
  • the antigen for producing an antibody that binds to the MT1-MMP-cleaved EphA2-N terminal fragment may be prepared in any way, but is preferably expressed in animal cells.
  • full-length (intact) intact EphA2 is a protein consisting of the 28th to 976th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1, and in the amino acid sequence represented by SEQ ID NO: 1.
  • a protein comprising an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the 28th to 976th amino acid sequence, or the 28th to 976th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 And an amino acid sequence having 80% or more sequence identity.
  • “Full-length EphA2” refers to the full-length EphA2 functioning in the cell relative to the EphA2 fragment. EphA2 is expressed in the cell, or expressed in the cell. A protein that retains the three-dimensional structure of EphA2.
  • the “full-length EphA2” is, for example, preferably expressed in animal cells, and examples thereof include those present in cell extracts expressing EphA2.
  • EphA2 extracellular domain is a region of EphA2 protruding outside the cell.
  • it is represented by SEQ ID NO: 3 (the 28th to 537th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1).
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 3 a protein comprising the amino acid sequence represented by SEQ ID NO: 3 with one or several amino acids added, substituted, inserted or deleted, or the amino acid sequence represented by SEQ ID NO: 3.
  • an amino acid sequence having 80% or more sequence identity may be prepared in any way, but is preferably expressed in animal cells.
  • amino acid sequence having 80% or more sequence identity may be any percentage as long as it is an amino acid sequence having 80% or more sequence identity. For example, 90% or more 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the antibody according to the first embodiment of the present invention (which may be referred to as “the antibody of the present invention”) is, for example, “28th to Xth in the amino acid sequence represented by SEQ ID NO: 1 (where X Is a protein consisting of an amino acid sequence represented by an integer of 328 or more and 435 or less (the same shall apply hereinafter), and one or several amino acids in the amino acid sequence represented by positions 28 to X in the amino acid sequence represented by SEQ ID NO: 1
  • a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 one or several amino acids added in the amino acid sequence represented by SEQ ID NO: 2
  • Several antibodies are produced using “a protein comprising a substituted, inserted or deleted amino acid sequence, or a protein comprising an amino acid sequence having
  • a protein comprising an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3
  • the method for confirming the presence or absence of the binding between the antibody and the antigen protein is not particularly limited, and a method known to those skilled in the art may be adopted.
  • the binding to the antigen protein by immunoprecipitation or Western blotting may be employed.
  • the method of confirming the presence or absence can be mentioned.
  • the antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, or fragments of these antibodies. In addition, so-called chimeric antibodies modified by genetic engineering are also included.
  • the antibody fragment of the present invention means a partial region of the antibody of the present invention. For example, Fab, Fab ′, F (ab ′) 2 , Fv (variable fragment of antibody), single chain antibody (heavy chain, light chain) Chain, heavy chain variable region, light chain variable region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide stabilization variable region), and a peptide containing CDR at least in part.
  • monoclonal is indicative of the properties of an antibody obtained from a substantially homogeneous population of antibodies, and is not intended to limit the production of antibodies by a particular method.
  • the monoclonal antibody used in the present invention may be produced, for example, by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) or the recombinant method (US Pat. No. 4,816,567).
  • Monoclonal antibodies of the present invention may also be isolated from phage antibody libraries (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581- 597 (1991)).
  • the antibody of the present invention is a polyclonal antibody
  • it can be prepared, for example, by injecting a mixture of an immunogen and an adjuvant into a mammalian host animal.
  • an immunogen and an adjuvants as immunogens are injected multiple times subcutaneously or intraperitoneally into the host animal.
  • adjuvants include complete Freud and monophosphoryl lipid A synthesis-trehalose dicorynomycolate (MPL-TDM).
  • the antibody of the present invention is a monoclonal antibody
  • it can be prepared, for example, using a hybridoma method.
  • This method includes the following four steps: (1) immunize the host animal with the immunogen, (2) collect monoclonal antibody secreting (or potentially secreting) lymphocytes, (3 (1) fuse lymphocytes to immortalized cells; (4) select cells that secrete the desired monoclonal antibody; A mouse, rat, guinea pig, hamster or other suitable host animal is selected as the immunized animal and the immunogen is injected. After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusing agent such as polyethylene glycol in order to establish hybridoma cells.
  • a fusing agent such as polyethylene glycol
  • fusion cells for example, rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines.
  • a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines.
  • Conventional techniques use parental cells that lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hybridomas.
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase
  • hybridoma that produces a desired antibody is selected, and the target monoclonal antibody can be obtained from the medium in which the hybridoma grows according to a conventional method.
  • the hybridoma thus prepared can be cultured in vitro or in vivo in ascites such as mouse, rat, guinea pig, hamster, etc., and the desired antibody can be prepared from the culture supernatant or ascites.
  • the second embodiment of the present invention comprises a step of measuring the amount of the MT1-MMP-cleaved EphA2-N terminal fragment present in a sample derived from a subject using the antibody according to the first embodiment of the present invention. Including cancer screening methods.
  • the measurement of the amount of “MT1-MMP-cleaved EphA2-N terminal fragment” by an antibody is carried out in a sample derived from a subject. It can be carried out by any method known to those skilled in the art, which can detect the fragment present in, for example, a method using an immunological technique.
  • an immunological method for example, immunochromatography, Western blotting, surface plasmon method, ELISA method (for example, directly) using the antibody of the present invention and, optionally, the detectably labeled antibody of the present invention.
  • ELISA immunochromatography
  • Western blotting Western blotting
  • surface plasmon method ELISA method (for example, directly) using the antibody of the present invention and, optionally, the detectably labeled antibody of the present invention.
  • Competitive ELISA for example, indirect competitive ELISA, sandwich ELISA, etc., radioimmunoassay (RIA), fluorescent immunoassay (FIA), Quartz crystal microbalance (QCM) and methods using magnetic beads can be mentioned.
  • RIA radioimmunoassay
  • FFA fluorescent immunoassay
  • QCM Quartz crystal microbalance
  • the sandwich ELISA method is particularly preferable because it can be measured easily and quickly.
  • the antibody of the present invention (used as a capture antibody) is a solid phase carrier (for example, a microtiter plate made of glass, metal or resin, a substrate, a resin or magnetic bead, nylon or a membrane made of PVDF. Etc.), and the carrier part to which the antibody is not adsorbed is blocked. Then, a sample containing the antigen is added to the carrier for reaction, the carrier is then washed, and another antibody that recognizes the antigen (detection antibody) An antibody that binds to another epitope) is added to the carrier and allowed to react.
  • a solid phase carrier for example, a microtiter plate made of glass, metal or resin, a substrate, a resin or magnetic bead, nylon or a membrane made of PVDF. Etc.
  • an unlabeled primary antibody (detection antibody) is used to detect a signal derived from the enzyme-labeled secondary antibody, for example, as absorbance, and based on a standard curve, It is also possible to quantify the amount of antigen in it.
  • the label include HRP.
  • HRP 3,3′-diaminobenzidine
  • TMB 3,3 ′, 5,5′-tetramethylbenzidine
  • OPD o -Phenylenediamine
  • p-nitrophenyl phosphate When labeling with alkaline phosphatase, p-nitrophenyl phosphate (NPP) or the like can be used as a substrate.
  • the antibody may be labeled via a biotin-avidin bond (ie, avidin-HRP or avidin-alkaline phosphatase is added to the biotinylated antibody).
  • the antibody of the present invention is an antibody that can be used for testing for cancer depends on a sample derived from a known cancer patient and a known healthy person (a person who is clearly not affected by the cancer to be tested). This can be determined by performing ROC analysis (Receiver operating characteristic analysis) based on the reactivity of the antibody to these samples using the samples derived from the samples.
  • ROC analysis Receiveiver operating characteristic analysis
  • the cancer testing method of the present invention compares the amount of MT1-MMP-cleaved EphA2-N terminal fragment present in a subject-derived sample with the amount of the N-terminal fragment present in a healthy sample.
  • the amount of fragments present in the sample derived from the subject is significantly large, it can be determined that the subject may have cancer.
  • the inventors have found that when the expression of MT1-MMP is increased, the frequency of EphA2 cleavage is increased, the proliferation, survival and movement of cancer cells are promoted, and the progression of cancer cells to malignant traits progresses. (Patent Document 1 etc.). Therefore, by applying the cancer testing method of the present invention over time to a patient-derived sample, it is also possible to obtain data for determining the degree of cancer progression or the therapeutic effect of cancer treatment.
  • Examples of materials used in the cancer testing method of the present invention include blood, lymph, urine, saliva, sputum and other body fluids, feces, and cell and tissue extracts.
  • the cancer to be subjected to the cancer testing method of the present invention may be any cancer as long as cells expressing EphA2 are cancerated, such as pancreatic cancer, stomach cancer, and the like. Esophageal cancer, colon cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, thyroid cancer, uterine cancer, prostate cancer, bladder cancer, kidney cancer, melanoma or glioma it can.
  • the third embodiment of the present invention is a cancer test kit (also referred to as “cancer test kit of the present invention”).
  • the third embodiment of the present invention includes the antibody according to the first embodiment of the present invention.
  • reagents, instruments and the like used in the cancer testing method of the present invention may be included.
  • the cancer testing kit of the present invention includes, for example, the MT1-MMP-cleaved EphA2-N terminal side in addition to the antibody of the present invention.
  • an antibody that binds to a fragment may be included.
  • a solid phase carrier for example, glass, metal, or resin
  • Microtiter plates, substrates, resins or magnetic beads, nylon, PVDF membranes, solid phase carrier blocking reagents, labeled secondary antibodies or labels used for antibody labeling eg HRP, alkaline phosphatase
  • Reagents, and substrates for color development eg, 3,3'-diaminobenzidine (DAB), 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-phenyle Diamine (OPD), p-nitrophenyl phosphate (NPP), etc.
  • DAB 3,3'-diaminobenzidine
  • TMB 3,3 ', 5,5'-tetramethylbenzidine
  • OPD o-phenyle Diamine
  • NPP p-nitrophenyl phosphate
  • the fourth embodiment of the present invention is a method for measuring the amount of the MT1-MMP-cleaved EphA2-N terminal fragment present in a sample using the antibody according to the first embodiment of the present invention.
  • the amount of the N-terminal fragment of the EphA2 protein present in the sample can be measured by an immunological method (for example, ELISA method) using the antibody of the present invention.
  • the activity of MT1-MMP is closely related to the onset of cancer and the progression of cancer, and MT1-MMP is considered to be important as a target molecule for anticancer agents.
  • MT1-MMP protease
  • MT1-MMP-cleaved EphA2- present in a sample (eg, body fluid) derived from a subject. It is possible to measure the amount of the N-terminal fragment and evaluate the MT1-MMP activity in the subject's living body based on the amount.
  • the MT1-MMP activity in the presence of anti-cancer drug candidate molecules is monitored by the above-described method, so that the anti-cancer drug candidate molecules become MT1-MMP activity. The impact can be evaluated.
  • the method according to the fourth embodiment of the present invention can also be used for screening anticancer drug candidate molecules.
  • the fifth embodiment of the present invention is a method for producing an antibody that specifically binds to an MT1-MMP-cleaved EphA2-N terminal fragment.
  • the inventors have identified the three-dimensional structure of the extracellular domain of EphA2 that may occur when processed by MT1-MMP-cleaved EphA2-N terminal fragment and full-length EphA2 and membrane proteases. Each was expected to be different.
  • an antibody preparation method including the following step (ii) is performed to obtain a full length (intact (intact)
  • a protein comprising an amino acid sequence represented by the 28th to Xth amino acid sequence represented by SEQ ID NO: 1 (where X is an integer of 328 to 435, the same shall apply hereinafter), represented by SEQ ID NO: 1
  • a protein comprising an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by positions 28 to X in the amino acid sequence, or in the amino acid sequence represented by SEQ ID NO: 1
  • Producing an antibody that binds to any one of the proteins consisting of the amino acid sequence having 80% or more of the amino acid sequence represented by the 28th to Xth amino acids As a specific example of the protein, a protein comprising the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2
  • the proteins (b) and (c) described in the antibody (ii) of the fifth embodiment are: The protein of (b) and (c) of each of the embodiments is the same.
  • the antibody obtained by the fifth embodiment specifically binds only to the MT1-MMP-cleaved EphA2-N terminal fragment, and was found to be very useful in cancer diagnosis (Examples). checking).
  • Steps (i) and (ii) included in the fifth embodiment of the present invention are not steps that require undue trial and error by those skilled in the art, and can be easily performed within the scope of ordinary practice in the art. Is possible.
  • EphA2 antibody anti- EphA2 monoclonal antibody (mAb) 96-1 and polyclonal antibody (pAb) C309 were provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). It has been confirmed that the epitopes of mAb 96-1 and pAb C309 are present in the ligand binding domain and the cysteine-rich domain (CRD), respectively.
  • Other antibodies against EphA2, pAbs 371805 and pAbs C20 were purchased from R & D Systems (Minneapolis, Minn., USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively.
  • the epitopes of the pAbs 371805 and pAbs C20 antibodies are present in the EphA2 ligand binding domain and the cytoplasmic domain, respectively. All antibodies except pAb 309 can be used for immunoprecipitation.
  • Recombinant antigen human EphA2 cDNA was purchased from Open Biosystems (Huntsville, AL, USA).
  • the coding region of the FLAG tag is ligated to the 3 'end of the fragment corresponding to nucleotides 1-981 and nucleotides 1-1578 of the coding region (antigen # 1 and antigen # 5, respectively), and amplified by PCR Then, it was subcloned into pcDNA3.2 DEST mammalian-expression vector using Gateway System (Invitrogen, Carlsbad, CA, USA) (FIG. 1A).
  • Ephrin binding domain + CRD Antigen # 1
  • Ephrin binding domain + CRD + stem region Antigen # 5; EphA2 extracellular domain
  • the expressed recombinant antigen was collected in a serum-free conditioned medium and collected and purified using anti-FLAG agarose affinity chromatography. The purity of the recombinant antigen was confirmed by CBB staining after SDS-PAGE.
  • Immunization, cell fusion and hybridoma screening Ligand binding domain and Antigen # 1 are emulsified with an equal volume of complete Freund's adjuvant (Difco; Diagnostic Systems, Sparks, MD, USA) and 50 ⁇ g injected into the peritoneal cavity of BALB / c mice did. Then boosted twice every 2 weeks with an equal volume of Antigen # 1 emulsified with incomplete Freund's adjuvant. Six weeks after the third immunization, the same amount of laminin- ⁇ 2 protein was injected intravascularly in the mice without adjuvant. Three days after the intravascular injection, the collected blood was centrifuged at 3,000 rpm at room temperature to obtain serum.
  • complete Freund's adjuvant Difco; Diagnostic Systems, Sparks, MD, USA
  • the resulting serum showing specificity for the ligand binding domain was used as a positive control during hybridoma screening.
  • Cell fusion between the mouse myeloma cell line (P3U1) and spleen cells was performed according to the method reported by IBL Co. Ltd. (Gunma, Japan).
  • the prepared hybridoma cells were cultured in a HAT selection medium for 10 days, and then re-plated into a 96-well plate. The supernatant of each well containing proliferating cells was used to confirm the produced antibody by ELISA using Antigen # 1 as an antigen.
  • Cell limiting dilution and cloning were repeated twice, resulting in three hybridomas (mAb 46A1, 62A1 and 76A1) producing antibodies against Antigen # 1.
  • Western blotting was performed using non-reducing conditions and reducing conditions using recombinant antigens (Antigens # 2, # 3 and # 4).
  • Immunoprecipitation radioimmunoprecipitation assay (RIPA; radioimmunoprecipitation assay) buffer (final volume; 200 [mu] L) in, Antigen # 1 included in the A431 cell extract: the (1 [mu] g) or full-length EphA2 protein: (1 ⁇ g), Antigen # 5 Incubate overnight at 4 ° C. with monoclonal or polyclonal antibodies.
  • the suspended Protein-G magnetic beads were then added to 40 ⁇ L of the incubation mixture and incubated for 1 hour at 4 ° C. with agitation. Protein-G magnetic beads were washed three times with RIPA buffer, and the beads containing the antigen / antibody complex were removed with a magnetic rack. The precipitated antigen / antibody complex was released from the magnetic beads by adding 2 ⁇ SDS sample buffer containing 5% ⁇ 2-mercaptoethanol.
  • Sandwich ELISA 96-well plates were treated overnight at 4 ° C. with mAb 76A1 or mAb 46A1 (20 ⁇ g / mL in PBS), preimmune serum as a negative control. Next, at room temperature, the wells were blocked with protein-free locking buffer (Pierce Biotechnology, Rockford, IL, USA) for 1 hour and then dried for 40 minutes. Each well was washed with PBS / 0.05% Tween-20 Tris, added to each well with increasing amounts of MT1-MMP cleavage fragment (Antigen # 1: 0-1,000 pg / mL), then biotinylated mAb The mixture was reacted with 62A1 at 37 ° C for 1 hour.
  • EphA2 extracellular domain present following the purified SP of the recombinant antigen (signal peptide, amino acids 1-27; SEQ ID NO: 1) consists of a ligand (Ephrin) binding domain (amino acids 28-201), CRD in order from the N-terminal side. (Cysteine rich domain, amino acids 202-328) and a stem region (amino acids 329-537) (FIG. 1A).
  • MT1-MMP cleavage sites mapped by the inventors and other researchers are indicated by arrows.
  • Antigens used for antibody preparation were expressed in HKE293 cells with amino acid 28-328 (Antigen # 1) and amino acid 28-537 (Antigen # 5) bound to the C-terminus of the FLAG tag. (FIG. 1A). Each antigen was purified using anti-FLAG mAb and Western blotted with anti-FLAG mAb (FIG. 1B). Furthermore, three types of antigens (Antigen # 2, # 3, and # 4) with a FLAG tag at the C terminus and a GST tag at the N terminus were expressed in E. coli. Each antigen was purified using anti-FLAG mAb, and the purity was confirmed by Western blotting using anti-FLAG mAb (FIG. 1B).
  • a monoclonal antibody mouse against the cleaved fragment of EphA2 was immunized with Antigen # 1 (5 ⁇ g / mouse) to obtain 400 hybridoma cells.
  • Antibodies secreted from each hybridoma were screened by ELISA using plates coated with Antigen # 1 (0.5 ⁇ g / plate).
  • three hybridoma clones (46A1, 62A1 and 76A1) producing the IgG1 class were obtained and further analyzed.
  • immunoprecipitation was performed with the antibody obtained with Antigen # 1, the precipitate was subjected to PAGE under reducing conditions, and then Western blotting was performed using anti-FLAG mAb (FIG. 2A).
  • the extracellular domain of A431 cells expressing endogenous EphA2 and the EphA2 FLAG-labeled EphA2 was prepared.
  • the prepared cell extract was subjected to an immunoprecipitation experiment using the monoclonal antibody prepared here, and the precipitate was subjected to PAGE under non-reducing conditions.
  • Positive control antibodies (96-1, 371805 and C20) were immunoprecipitated with full-length EphA2 and detected by Western blotting using C20 antibody (FIG. 2B).
  • 46A1 binds to both full-length EphA2 and extracellular domain (Antigen # 5) in addition to the MT1-MMP cleaved form (Antigen # 1), and 62A1 cleaves to MT1-MMP (Antigen # 1) It was found to bind to the extracellular domain (Antigen # 5).
  • Antigen # 5 antibodies that do not bind to the full-length EphA2 or EphA2 extracellular domain (Antigen # 5) are screened among antibodies obtained using MT1-MMP-cleaved EphA2 fragment (Antigen # 1) as an immunogen.
  • MT1-MMP-cleaved EphA2 fragment Antigen # 1
  • the AUC area under the curve
  • the sensitivity and specificity of serum samples from pancreatic cancer patients relative to serum samples from healthy subjects were 89.0% and 90.0%, respectively (FIG. 5A).
  • the sensitivity and specificity of the serum samples from gastric cancer patients relative to the serum samples from healthy subjects were 88.2% and 84.0%, respectively (FIG. 5B).
  • the level of CA19-9 a known marker for digestive system tumors (including pancreatic cancer), was measured (FIG. 5C).
  • the detection sensitivity and specificity of CA19-9 for sera from patients with pancreatic cancer were 88.9% and 72.0%, respectively.
  • the AUC of CA19-9 was 0.83. From this result, it is clear that the diagnostic accuracy of pancreatic cancer based on the result of measuring the amount of soluble EphA2 fragment using the antibody of the present invention is superior to the diagnostic accuracy based on detecting CA19-9. (FIGS. 5A and C).
  • the antibody of the present invention that is, an antibody that specifically recognizes only EphA2 cleaved fragment by MT1-MMP
  • the antibody of the present invention specifically binds to an N-terminal fragment of EphA2 useful as a cancer marker.
  • diagnosis of cancer for example, pancreatic cancer

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Abstract

L'objectif de la présente invention est de fournir un anticorps qui se lie spécifiquement à un fragment du côté N-terminal de EphA2 coupé par MT1-MMP, l'anticorps se liant à la protéine de (a) mais pas aux protéines de (b) et (c), où : (a) est une protéine comprenant une séquence d'acides aminés représentée par les acides aminés 28 à X (dans ce cas, X est un entier de 328 à 435; également ci-dessous) dans une séquence d'acides aminés représentée par SEQ ID NO : 1; une protéine comprenant une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés ont été ajoutés, substitués, insérés ou supprimés dans la séquence d'acides aminés représentée par les acides aminés 28 à X dans la séquence d'acides aminés représentée par SEQ ID NO: 1; ou une protéine comprenant une séquence d'acides aminés qui présente au moins 80 % d'identité de séquence avec la séquence d'acides aminés représentée par acides aminés 28 à X dans la séquence d'acides aminés représentée par SEQ ID NO : 1; (b) est une protéine comprenant une séquence d'acides aminés représentée par les acides aminés 28 à 976 dans la séquence d'acides aminés représentée par SEQ ID NO: 1; une protéine comprenant une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés ont été ajoutés, substitués, insérés, ou supprimés dans la séquence d'acides aminés représentée par les acides aminés 28 à 976 dans la séquence d'acides aminés représentée par SEQ ID NO : 1; ou une protéine comprenant une séquence d'acides aminés qui présente au moins 80 % d'identité de séquence avec la séquence d'acides aminés représentée par les acides aminés 28 à 976 dans la séquence d'acides aminés représentée par SEQ ID NO : 1; et (c) est une protéine comprenant une séquence d'acides aminés représentée par SEQ ID NO : 3; une protéine comprenant une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés ont été ajoutés, substitués, insérés, ou supprimés dans la séquence d'acides aminés représentée par SEQ ID NO : 3; ou une protéine comprenant une séquence d'acides aminés qui présente au moins 80 % d'identité de séquence avec la séquence d'acides aminés représentée par SEQ ID NO : 3.
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Citations (4)

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US20120122112A1 (en) * 1999-08-17 2012-05-17 Purdue Research Foundation Antibodies as a cancer diagnostic
JP2012103264A (ja) * 1999-08-17 2012-05-31 Purdue Res Found 癌診断用抗体
JP2014178301A (ja) * 2013-02-13 2014-09-25 Univ Of Tokyo がんの検査方法及び検査用キット

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JP2003516930A (ja) * 1999-08-17 2003-05-20 パーデュー・リサーチ・ファウンデーション 転移性疾患の治療
US20120122112A1 (en) * 1999-08-17 2012-05-17 Purdue Research Foundation Antibodies as a cancer diagnostic
JP2012103264A (ja) * 1999-08-17 2012-05-31 Purdue Res Found 癌診断用抗体
JP2014178301A (ja) * 2013-02-13 2014-09-25 Univ Of Tokyo がんの検査方法及び検査用キット

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