WO2008146911A9 - Anticorps dirigé contre il13ra2 et agent de diagnostic/thérapie comprenant l'anticorps - Google Patents

Anticorps dirigé contre il13ra2 et agent de diagnostic/thérapie comprenant l'anticorps Download PDF

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WO2008146911A9
WO2008146911A9 PCT/JP2008/060009 JP2008060009W WO2008146911A9 WO 2008146911 A9 WO2008146911 A9 WO 2008146911A9 JP 2008060009 W JP2008060009 W JP 2008060009W WO 2008146911 A9 WO2008146911 A9 WO 2008146911A9
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antibody
il13ra2
cells
cancer
cell
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WO2008146911A1 (fr
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洋文 濱田
和則 加藤
公則 中村
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北海道公立大学法人札幌医科大学
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    • 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
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

  • the present invention relates to a composition for diagnosis and treatment of cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, which contains a substance that binds to IL13Ra2.
  • the present invention also relates to novel monoclonal antibodies that bind with high affinity to IL13Ra2 and compositions for the diagnosis and treatment of cancer comprising the same.
  • gene therapy that can demonstrate high specificity for cancer includes (1) introduction of suicide genes using cancer tissue-specific promoters, and (2) apoptosis specific to cancer cells. Activation of mechanism, (3) Immunotherapy targeting antigens specific to cancer cells, (4) Introduction of viral vectors that grow specifically in cancer cells, (5) Specific presence on the surface of cancer cells Strategies such as cancer cell-specific gene transfer using targets have been mainly proposed.
  • Non-Patent Document 1 adenoviral vector containing the Protein 33A Z33 motif that binds to the Fc domain of the antibody (IgG) in the HI loop of the knob, reporter genes such as lacZ and EGFP, or Adenovirus expressing UPRT (uracil phosphoribosyltransferase) that converts 5-fluorouracil into 5-fluoridine monophosphate, etc.
  • CEA human carcinoembryonic antigen
  • PAP2a phosphatidic acid phosphatase 2a
  • UPRT an adeno expressing FZ33 fiber mutant UPRT.
  • 5-fluorouracil was administered to a model animal of disseminated gastric cancer.
  • cancer targets such as CEA for gastric cancer and PAP2a for pancreatic cancer have been discovered.
  • the current status of many cancer targets has not yet been elucidated.
  • IL13Ra2 (interleukin 13 receptor ⁇ 2) is a single transmembrane type I membrane protein consisting of 380 amino acids cloned from human kidney cancer cell line Caki-1 in 1996, and has high binding ability to IL13. It is known to have (see Non-Patent Document 3). Furthermore, IL13Ra2 has been reported to have the internalization ability of IL13 (see Non-Patent Document 4), but is presumed to act as a decoy receptor (see Non-Patent Document 5).
  • IL13Ra2 is expressed in glioma (see non-patent document 6), kidney cancer, head and neck cancer (see non-patent document 7), ovarian cancer (see non-patent document 8), and Kaposi sarcoma (see non-patent document 9). It has been confirmed. However, there are no reports on the expression of IL13Ra2 for cancers other than those described above. In particular, regarding melanoma, it has been widely recognized in the art that it is a cell type that does not express IL13Ra2, such as using a melanoma cell line as a host cell for an IL13Ra2 gene introduction experiment system (see Non-Patent Document 10).
  • An object of the present invention is to find a suitable target for realizing highly specific diagnosis and targeted treatment of various cancers, and to provide an antibody against the target and a pharmaceutical composition for treatment using the target. There is.
  • the present inventors are diligently studying to solve the above-mentioned problems, and an antibody obtained from a hybridoma prepared from a mouse immunized with A375 cells, a human melanoma cell line, is used in combination with a Z33 fiber-modified adenovirus.
  • an antibody obtained from a hybridoma prepared from a mouse immunized with A375 cells, a human melanoma cell line is used in combination with a Z33 fiber-modified adenovirus.
  • the gene transfer efficiency to cancer cells is extremely high, and when used together with immunotoxin, the survival rate of cancer cells is significantly reduced, and the antigen of this antibody is the extracellular domain of IL13Ra2. It was identified.
  • IL13Ra2 is also expressed in other types of cancer than previously known, that IL13Ra2 can be used as a target molecule for these cancer cells, The inventors have found that cancer growth can be suppressed by administering an anti-IL13Ra2 antibody to a cancer-bearing animal, and have completed the present invention.
  • the present invention relates to: (1) A composition for diagnosing cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, comprising a substance that binds to IL13Ra2. (2) A composition for the treatment of cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, comprising a substance that binds to IL13Ra2. (3) The composition according to (1) or (2) above, wherein the substance that binds to IL13Ra2 is selected from the group consisting of an anti-IL13Ra2 antibody and IL13.
  • Hybridoma KH6g11 accession number: NITE P-368 deposited on May 18, 2007 as an accession number NITE AP-368 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, Anti-IL13Ra2 antibody, Hybridoma KH7B9 (Accession Number: NITE P-374) deposited on May 31, 2007 as receipt number NITE AP-374, and Hybridoma NS- deposited on February 14, 2008 as receipt number NITE AP-488 66.
  • the composition according to (3) above which is a monoclonal antibody produced by 66 (Accession Number: NITE P-488) or an antibody having binding properties equivalent to those of these antibodies.
  • Hybridoma deposited under the Accession Number NITE P-368, Hybridoma deposited under the Accession Number NITE P-374, and Deposited under the Accession Number NITE P-488 A monoclonal antibody produced by a hybridoma selected from the group consisting of hybridomas, or an antibody having binding properties equivalent to these antibodies. (6) A hybridoma that produces the antibody of (5) above. (7) The hybridoma deposited under the Accession Number NITE P-368, the hybridoma deposited under the Accession Number NITE P-374, and the deposit under the Accession Number NITE P-488. The hybridoma according to (6) above, which is selected from the group consisting of hybridomas.
  • a method for detecting and / or quantifying IL13Ra2 in a biological sample (A) contacting the biological sample with the composition of (1), (3), (4) or (8) or the anti-IL13Ra2 antibody of (5), and (b) in the biological sample. The method comprising the step of detecting and / or quantifying the binding of IL13Ra2 to the composition or the anti-IL13Ra2 antibody.
  • a method for treating cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, comprising the step of administering the composition of any one of (2) to (4) to a subject .
  • a method for treating cancer expressing IL13Ra2 comprising a step of administering the composition of (9) to a subject.
  • the present invention it becomes possible to perform diagnosis and treatment targeting the same protein for cancers (especially melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer) for which expression of IL13Ra2 has not been observed. Therefore, improvement in efficiency and expansion of options in diagnosis and treatment for these cancers can be expected.
  • a substance that binds to IL13Ra2 and an arbitrary anticancer agent can be combined to enhance the latter therapeutic effect, so that its application range is wide and convenience is extremely high.
  • the anti-IL13Ra2 monoclonal antibody of the present invention has excellent binding properties, and is an IL13Ra2-positive cancer (in particular, melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer, glioma, head and neck cancer, renal cancer) , Kaposi's sarcoma and ovarian cancer).
  • an IL13Ra2-positive cancer in particular, melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer, glioma, head and neck cancer, renal cancer
  • Kaposi's sarcoma and ovarian cancer Kaposi's sarcoma and ovarian cancer
  • the monoclonal antibody of the present invention specifically recognizes and binds to IL13Ra2 present on the cell membrane surface of living cancer cells, and is taken into the cells with high efficiency. Therefore, if the antibody of the present invention is used, gene introduction using a Z33 fiber mutant adenovirus capable of binding to the antibody, drugs capable of binding to the antibody (for example, various cytotoxins, immunotoxins, etc.) Therefore, the therapeutic agent for cancer comprising the antibody of the present invention has a therapeutic result that greatly exceeds conventional gene therapy and chemotherapy. It is possible to increase side effects and reduce side effects. In addition, such gene introduction and drug introduction using the antibody of the present invention are not only effective for primary cancers but also for metastatic cancers that have been difficult to cure by conventional treatment methods.
  • the diagnostic agent for cancer of the present invention can be diagnosed with extremely high accuracy due to the high specificity of the antibody of the present invention for IL13Ra2.
  • the antibody of the present invention is widely used in histopathological examinations, and can easily recognize formalin-fixed and denatured IL13Ra2 in paraffin-embedded samples. In addition, it has a special convenience that it can be used for a histopathological examination using a past sample whose time has passed since collection.
  • control represents an untreated 293T cell
  • pcDNA3.1 represents a 293T cell introduced with a control plasmid
  • pTARGET-IL13Ra2 represents a 293T cell in which IL13Ra2 is overexpressed by pTARGET-IL13Ra2.
  • 66 indicates NS-66 antibody. It is the figure which analyzed the coupling
  • control represents an untreated CHO cell
  • pcDNA3.1 represents a CHO cell into which a control plasmid was introduced
  • pTARGET-IL13Ra2 represents a CHO cell in which IL13Ra2 is overexpressed by pTARGET-IL13Ra2.
  • 66 indicates NS-66 antibody. It is the figure which analyzed the expression of IL13Ra2 in various cells with NS-66 antibody by FACS.
  • a) is 293T-IL13Ra2 and b) is A375. It is a photograph figure which shows the optical microscope image which immunostained the mouse
  • the present invention relates to a composition for diagnosing or treating a cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, comprising a substance that binds to IL13Ra2.
  • IL13Ra2 interleukin 13 receptor ⁇ 2
  • IL13Ra2 protein or “IL13Ra2 antigen” is an amino acid sequence registered under the accession number NM_002188 in the NCBI protein sequence database (SEQ ID NO: 8).
  • SEQ ID NO: 8 NCBI protein sequence database
  • derivative refers to mutation or substitution of one or more (for example, several (eg, 2, 3, 4, 5 or 6)) amino acid residues in the amino acid sequence of IL13Ra2 protein or a fragment thereof. Is intended to mean a peptide or polypeptide comprising deletions and / or additions and having substantially the same antigenicity as the IL13Ra2 protein.
  • substantially means the degree of specificity that is specifically recognized by an anti-IL13Ra2 antibody to the extent that it can be used for diagnosis and / or treatment of a disease characterized by IL13Ra2 expression. To do.
  • Typical examples of derivatives include IL13Ra2 polymorphism, sequence changes due to splicing and the like.
  • the length of the “fragment” is not limited as long as it can be recognized as an antigen specific to the anti-IL13Ra2 antibody, but is preferably 6 amino acids or more, more preferably 8 amino acids or more, and still more preferably. 10 amino acids or more.
  • These fragments may be any part of the IL13Ra2 protein, but more preferably correspond to an epitope of IL13Ra2 protein or include a part corresponding to the epitope.
  • the “substance that binds to IL13Ra2” includes any substance that binds to IL13Ra2, in particular, a substance that specifically binds to IL13Ra2.
  • examples of such substances include, but are not limited to, IL13, which is a natural ligand of IL13Ra2, and anti-IL13Ra2 antibody.
  • IL13 can be purified from a biological sample, for example, peripheral blood, but typically can be produced by genetic engineering using a nucleic acid encoding IL13.
  • a nucleic acid encoding IL13 is cloned from the gene of an IL13-expressing cell (eg, CD4 + T cell, CD8 + T cell, NK-T cell, basophil, mast cell, etc.) Can be expressed in an appropriate host cell (for example, E. coli, etc.), recovered and purified.
  • an IL13-expressing cell eg, CD4 + T cell, CD8 + T cell, NK-T cell, basophil, mast cell, etc.
  • An example of such a fabrication method is described in Example 5.
  • recombinant IL13 marketed by R & D Systems, PeproTech, etc. can also be used.
  • Anti-IL13Ra2 antibody or “antibody against IL13Ra2” refers to an antibody that specifically binds to IL13Ra2, and is a fragment of the antibody that exhibits substantially the same antigen specificity as the original antibody (referred to herein as “functional Also referred to as “fragment”) or derivatives.
  • Functional fragments or derivatives of antibodies include functional antibodies such as Fab, Fab ′, F (ab ′) 2 , single chain antibodies (scFv), disulfide stabilized V region fragments (dsFv), or peptides containing CDRs. Fragments, or derivatives such as humanized antibodies (eg, CDR-grafted fully human antibodies) are included.
  • the antibodies in the present invention comprise a conventional method comprising immunizing an animal and collecting serum (polyclonal) or spleen cells (for production of hybridomas by fusion with appropriate cells), as detailed below. Can be produced.
  • a substance eg, an antibody “specifically binds” to a protein or fragment thereof refers to a specific substance of these proteins or fragments thereof rather than its affinity for other amino acid sequences. It means to bind with substantially high affinity to the amino acid sequence.
  • substantially high affinity means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device.
  • Binding constant (K a ) of at least 10 7 M ⁇ 1 , preferably at least 10 8 M ⁇ 1 , more preferably 10 9 M ⁇ 1 , even more preferably 10 10 M ⁇ 1 , 10 11
  • binding affinity is meant such that it is M ⁇ 1 , 10 12 M ⁇ 1 or higher, for example up to 10 13 M ⁇ 1 or higher.
  • the anti-IL13Ra2 antibody in the present invention can be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the viewpoint of specificity, titer, staining, and the like.
  • a suitable example of a monoclonal antibody against IL13Ra2 is the receipt number at the National Institute of Technology and Evaluation (NPMD) (Postal Code 292-0818, 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan).
  • Antibodies produced by the hybridoma NS-66 Bay Biosciences Co., Ltd.-Part number: Bb-1211, Product name: anti human IL-13Ra2, Clone: NS-66 eBioscience-Catalog Number: 14-7136
  • Antibodies produced by the hybridoma KH7B9 Bay Bioscience Co., Ltd.-Product No .: Bb-1213, Product name: anti human IL-13Ra2, Clone: KH7B9 eBioscience-Catalog Number: 14-9937
  • Other antibodies having binding properties equivalent to those of these antibodies are also preferable as the anti-IL13Ra2 antibody of the present invention.
  • Commercially available anti-IL13Ra2 antibodies such as B-D13 (
  • One preferred embodiment of the antibody of the present invention has a higher cellular uptake rate than the conventional antibody.
  • the conventional antibody for example, B-D13, 83807,
  • the anti-IL13Ra2 monoclonal antibody exhibits cell killing activity 5 times or more, preferably 10 times or more, more preferably 15 times or more, and still more preferably 20 times or more than 83834.
  • Another preferred embodiment of the antibody of the present invention is an anti-IL13Ra2 monoclonal antibody having the property of recognizing IL13Ra2 in a formalin-fixed, paraffin-embedded sample.
  • formalin fixation and paraffin embedding mean what is usually performed in this technical field. Therefore, formalin fixation typically means fixing with formalin-based fixatives such as 10-20% formalin, neutral formalin, neutral buffered formalin, etc. for 24-72 hours. This means that the formalin-fixed sample is dehydrated with alcohol, de-alcoholized with xylene or chloroform, and then infiltrated with paraffin, but is not limited thereto.
  • Antibodies having the above properties can be screened by any known method such as the cell-ELISA method.
  • the cell-ELISA method can be performed, for example, by immobilizing IL13Ra2-expressing cells in formalin, allowing a test antibody to act on the cells, and detecting the bound antibody by color development with an enzyme-labeled secondary antibody.
  • the invention also relates to hybridomas producing monoclonal antibodies against IL13Ra2, in particular hybridoma KH6g11 (accession number: NITE P-368) deposited on May 18, 2007 as accession number NITE AP-368 with NPMD, accession number NITE.
  • a monoclonal antibody against IL13Ra2 can be produced, for example, as follows.
  • An antigen is administered to a mammal alone or together with a carrier or diluent at a site where antibody production is possible.
  • IL13Ra2 or a fragment thereof purified under denaturing or non-denaturing (native) conditions, for example, IL13Ra2-expressing cells can be used.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 2 to 6 weeks, for a total of 2 to 10 times.
  • Examples of the mammal to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • a monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained in the above with myeloma cells.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein (Kohler and Milstein (1975) Nature 256: 495).
  • Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%.
  • the cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.
  • the hybridoma culture supernatant is added to a solid phase (for example, a microplate) on which an antigen is adsorbed directly or together with a carrier, and then a radioactive substance is added.
  • a solid phase for example, a microplate
  • anti-immunoglobulin antibodies labeled with enzymes or enzymes if the cells used for cell fusion are mice, use anti-mouse immunoglobulin antibodies
  • protein A to detect monoclonal antibodies bound to the solid phase
  • anti-immunity Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, a protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
  • Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto, but can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium may be used as long as the hybridoma can grow.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal bovine serum, or serum-free for hybridoma culture
  • a culture medium SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually 20 to 40 ° C., preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
  • Separation and purification of monoclonal antibodies can be performed by separating and purifying immunoglobulins (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchange) in the same way as normal polyclonal antibodies.
  • immunoglobulins eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchange
  • Absorption / desorption method by body eg, DEAE
  • ultracentrifugation method eg, ultracentrifugation method
  • gel filtration method e.g, antigen-binding solid phase or specific adsorbents such as protein A or protein G by collecting only antibodies and dissociating the binding Purification method.
  • Polyclonal antibodies against IL13Ra2 can be produced according to known methods or modifications thereof. For example, a complex of an immunizing antigen (protein antigen) and a carrier protein is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and an antibody-containing product against the protein of the present invention is collected from the immunized animal. And it can manufacture by performing isolation
  • the kind of carrier protein and the mixing ratio of carrier and hapten should be such that an antibody can be efficiently produced against the hapten immunized by crosslinking to the carrier.
  • any of them may be cross-linked at an arbitrary ratio.
  • bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and the like are about 0.1 to 20, preferably about A method of coupling at a ratio of 1 to 5 is used.
  • various condensing agents can be used for coupling the hapten and the carrier, but an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, or dithiobilidyl group is used.
  • the condensation product is administered to a warm-blooded animal alone at a site where antibody production is possible, or together with a carrier and a diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.
  • Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably from blood.
  • Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (in the case of human IgG1, it is cleaved at the 224th amino acid residue of the H chain). It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000, in which the entire chain is bound by a disulfide bond.
  • the Fab of the present invention can be obtained by treating an antibody that specifically binds to IL13Ra2 with the proteolytic enzyme papain.
  • F (ab ') 2 is a fragment obtained by treating IgG with protein pepsin (in the case of human IgG1, it is cleaved at the 234th amino acid residue of the H chain).
  • Fab is a disulfide bond in the hinge region. This is an antibody fragment having an antigen binding activity with a molecular weight of about 100,000, which is slightly larger than that bound via the.
  • Fab ′ of the present invention can be obtained by treating an antibody that specifically binds to IL13Ra2 with the proteolytic enzyme pepsin.
  • Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 .
  • Fab ′ of the present invention can be obtained by treating an antibody that specifically binds to IL13Ra2 with a reducing agent dithiothreitol.
  • the above Fab, F (ab ′) 2 , or Fab ′ may also be a prokaryotic expression vector or eukaryotic expression vector that encodes the Fab, F (ab ′) 2 , or Fab ′ fragment of the full-length anti-IL13Ra2 antibody. It can also be produced by inserting into a vector and expressing the vector by introducing it into a prokaryotic or eukaryotic organism (eg, Co MSet al., J. Immunol. (1994) 152,2968-2976; Better M. & Horwitz AH Methods in Enzymology (1989) 178,476-496; Plueckthun, A. & Skerra A.
  • a prokaryotic or eukaryotic organism eg, Co MSet al., J. Immunol. (1994) 152,2968-2976; Better M. & Horwitz AH Methods in Enzymology (1989) 178,476-496; Plueckthun,
  • a single chain antibody (hereinafter sometimes abbreviated as scFv) is composed of a single heavy chain variable region (hereinafter abbreviated as VH) and a single light chain variable region (hereinafter referred to as VH).
  • the single-chain antibody of the present invention obtains cDNA encoding the VH and VL of an antibody that specifically binds to IL13Ra2, constructs a DNA encoding the single-chain antibody, and uses this to construct a prokaryotic expression vector or true DNA. It can be produced by inserting into an expression vector for nuclei and introducing the expression vector into a prokaryote or eukaryote for expression.
  • a disulfide-stabilized V region fragment (hereinafter sometimes abbreviated as dsFv) is obtained by binding a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7,697 (1994)).
  • an antibody that specifically binds to IL13Ra2 such as a humanized antibody or a human antibody, can be used.
  • the disulfide-stabilized V region fragment of the present invention obtains cDNA encoding the antibody VH and VL that specifically binds to IL13Ra2, constructs a DNA encoding the disulfide-stabilized V region fragment, and uses this for prokaryotes. It can be produced by inserting into an expression vector or an expression vector for eukaryotes, introducing this expression vector into a prokaryote or eukaryote and expressing it.
  • a peptide including a complementary determining region includes at least one region of an H chain or L chain CDR. Multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide containing CDR of the present invention is obtained by obtaining cDNA encoding VH and VL of an antibody that specifically binds to IL13Ra2, and then constructing DNA encoding CDR, and using this DNA as a prokaryotic expression vector or eukaryotic It can be produced by inserting into an expression vector for organisms and introducing the expression vector into a prokaryotic or eukaryotic organism for expression.
  • a peptide containing CDR can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method).
  • a humanized antibody obtained by using a non-human animal antibody as a human chimeric antibody or human CDR-grafted antibody using gene recombination technology can also be advantageously used in the present invention.
  • a human chimeric antibody is an antibody in which the variable region of an antibody (hereinafter referred to as V region) is an antibody of a non-human animal and the constant region (hereinafter referred to as C region) is a human antibody (Morrison SL et al., Proc Natl Acad Sci USA. 81 (21), 6851-6855, 1984), and human CDR-grafted antibody refers to the amino acid sequence of CDR in the V region of an antibody from a non-human animal.
  • humanized antibodies When administered to humans, humanized antibodies have fewer side effects than antibodies from non-human animals, and their therapeutic effects last for a long time.
  • humanized antibodies can be prepared as molecules of various forms using gene recombination techniques. For example, if a ⁇ 1 subclass is used as the heavy chain (hereinafter referred to as H chain) C region of a human antibody, a humanized antibody that is stable in blood and has high effector activity such as antibody-dependent cytotoxic activity. (Co MS et al., Cancer Research, 56 (5), 1118-1125, 1996). Humanized antibodies with high effector activity are useful when destruction of a target such as cancer is desired.
  • the ⁇ 4 subclass can be used as the H chain C region of a human antibody.
  • Subclasses generally have low effector activity (Bruggemann M et al., Journal of Experimental Medicine, 166 (5), 1351-1361, 1987; Bindon CI et al., Journal of Experimental Medicine, 168 (1), 127 -142, 1988), side effects can be avoided, and blood half-life is expected to be longer than that of mouse antibodies (Stephens S. et al., Immunology, 85 (4), 668-674, 1995).
  • Fab, Fab ′, F (ab ′) 2 , scFv prepared from antibodies including humanized antibodies using protein engineering and genetic engineering techniques. -426, 1988), dsFv (Webber KO et al., Molecular Immunology, 32 (4), 249-258, 1995), peptides containing CDRs (Monfardini C et al., Journal of Biological Chemistry, 271 (6), 2966-2971, 1996) and other antibody fragments having a lower molecular weight can also be used. Since these antibody fragments have a smaller molecular weight than a complete antibody molecule, they have excellent transferability to a target tissue and are advantageous (Cancer Research, 52, 3402-3408, 1992).
  • a radioisotope, a therapeutic protein, a small molecule drug, or the like is chemically or genetically bound to an antibody that specifically binds to IL13Ra2 of the present invention (for example, a humanized antibody, a human antibody, or an antibody fragment thereof).
  • the antibody specifically reacts with IL13Ra2, the N-terminal side or C-terminal side of the H chain or L chain of the antibody fragment, an appropriate substituent or side chain in the antibody and antibody fragment, and the antibody and It can be produced by chemically or genetically linking a radioisotope, therapeutic protein, or low molecular weight drug to a sugar chain in an antibody fragment (for example, Introduction to antibody engineering, Osamu Kanmitsu, Jinjinshokan) 1994).
  • the present invention provides a cancer diagnostic agent containing an anti-IL13Ra2 antibody.
  • the present invention also provides a therapeutic agent for cancer containing an anti-IL13Ra2 antibody.
  • the present invention relates to a diagnostic or therapeutic agent for cancer selected from the group consisting of melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer, comprising a substance that binds to IL13Ra2.
  • cancer and “tumor” are used as terms having the same meaning.
  • the therapeutic or diagnostic agent containing the anti-IL13Ra2 antibody of the present invention may be any disease characterized by high expression of IL13Ra2 (especially melanoma, bone Suitable for diagnosis or treatment of sarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma), glioma, head and neck cancer, renal cancer, Kaposi sarcoma, ovarian cancer, etc.
  • IL13Ra2 especially melanoma, bone Suitable for diagnosis or treatment of sarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma), glioma, head and neck cancer, renal cancer, Kaposi sarcoma, ovarian cancer, etc.
  • IL13Ra2 expression is newly confirmed, that is, melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer and lung cancer
  • anti-IL13Ra2 antibody not only the anti-IL13Ra2 antibody of the present invention but also known ones
  • Various substances that bind to IL13Ra2 antibodies such as various anti-IL13Ra2 antibodies, for example, 83834 antibody, 83807 antibody (R & D), B-D13 antibody (Diaclone), and IL13 protein can be included.
  • the anti-IL13Ra2 antibody of the present invention for use in the diagnosis or treatment of cancer is a labeling substance (for example, a radioisotope, a fluorescent substance, etc.) for monitoring or the like in the diagnostic or therapeutic drug of the present invention, if necessary. ).
  • the anti-IL13Ra2 antibody of the present invention may itself be an agent having a neutralizing activity that reduces the activity of the antigen in the diagnostic or therapeutic agent of the present invention.
  • Chemically or genetically engineered with other agents that exert therapeutic effects eg, radioisotopes, therapeutic proteins, small molecule agents, or viral or non-viral vectors for gene transfer to a target) Can be combined.
  • “chemical bond” includes ionic bond, hydrogen bond, covalent bond, bond by intermolecular force, bond by hydrophobic interaction, etc.
  • “genetic engineering bond” For example, a binding mode between an antibody and a therapeutic protein when a fusion protein composed of an antibody and a therapeutic protein is produced using a technique such as gene recombination is included.
  • antibody-chelating agents conjugates of antibodies labeled with indium-111 and chelating agents (antibody-chelating agents) have been described for use in radioimmunoscintigraphic imaging of tumors expressing carcinoembryonic antigen ( Sumerdon et al. Nucl. Med. Biol. 1990 17: 247-254).
  • these antibody-chelators have been used to detect tumors in patients suspected of having recurrent colorectal cancer (Griffin et al. J. Clin. Onc. 1991 9: 631-640) .
  • Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging have also been described (Lauffer, R.B. Magnetic Resonance in Medicine 1991 22: 339-342).
  • An antibody against IL13Ra2 can be used similarly. That is, a labeled antibody that specifically binds to IL13Ra2 can be injected, for example, into a patient suspected of having cancer for the purpose of diagnosing the disease state or staging of the patient.
  • the label used can be selected depending on the type of imaging used. For example, radioactive labels such as indium-111 ( 111 In), technetium- 99m ( 99m Tc) or iodine-131 ( 131 I) can be used for planar scanning or single photon tomography.
  • Positron emission labels such as Fluorine -18 (18 F) can be used in positron tomography.
  • the anti-IL13Ra2 antibody in the diagnostic or therapeutic agent of the present invention is chemically or genetically engineered with a radioisotope, a therapeutic protein, a small molecule drug, or a viral vector carrying a therapeutic gene. Are combined.
  • radioactive halogen elements such as fluorine-18, iodine-125 ( 125 I), and iodine-131. These radioactive halogen elements can be widely used as radiodiagnostic drugs or radiotherapeutic drugs by labeling antibodies and peptides in the same manner as the above-mentioned radiometal elements. For example, iodination with 125 I or 131 I can be bound to an antibody or antibody fragment by a known method such as the chloramine T method.
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  • therapeutic proteins include cytokines that activate cells responsible for immunity, such as human interleukin 2 (hIL2), human granulocyte-macrophage-colony stimulating factor (hGM-CSF), human macrophages Examples include colony stimulating factor (hM-CSF) and human interleukin 12 (hIL12).
  • cytokines that activate cells responsible for immunity
  • hIL2 human interleukin 2
  • hGM-CSF human granulocyte-macrophage-colony stimulating factor
  • hM-CSF colony stimulating factor
  • hIL12 human interleukin 12
  • toxins such as ricin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin (PE) can be used to directly kill cancer cells.
  • PE Pseudomonas aeruginosa exotoxin
  • a cDNA encoding the therapeutic protein is linked to a cDNA encoding the antibody or antibody fragment, and a DNA encoding the fusion antibody is constructed, and this DNA is used for prokaryotes or eukaryotes.
  • the fusion antibody can be produced by inserting the expression vector into a prokaryotic organism or a eukaryotic organism and expressing the expression vector.
  • Low molecular drug is used herein to mean a diagnostic or therapeutic compound other than “radioisotope”, “therapeutic protein” and the like.
  • small molecule drugs include alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, and antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin and doxorubicin.
  • plant alkaloids such as vincristine, vinblastine, vindesine
  • anticancer agents such as hormonal agents such as tamoxifen and dexamethasone, or steroids such as hydrocortisone and prednisone
  • non-steroidal agents such as aspirin and indomethacin
  • immunity such as gold thiomalate and penicillamine
  • regulators include regulators, immunosuppressants such as azathioprine, and anti-inflammatory agents such as chlorpheniramine maleate and antihistamines such as clemastine.
  • a method of binding daunomycin and antibody a method of binding between daunomycin and the amino group of the antibody via glutaraldehyde, a method of binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carbodiimide, etc. Is mentioned.
  • virus vectors include virus vectors modified to be able to bind to the anti-IL13Ra2 antibody of the present invention (for example, FZ33 fiber mutant adenovirus).
  • viral vectors include genes (therapeutic effects) such as cell proliferation-related genes, apoptosis-related genes, immunoregulatory genes and the like that induce therapeutic effects such as inducing apoptosis of cancer cells at target sites (for example, cancer).
  • a viral vector that binds to an anti-IL13Ra2 antibody can be delivered to a site where an antigen recognized by the anti-IL13Ra2 antibody (ie, IL13Ra2) is present when administered to a patient in need of gene therapy along with the anti-IL13Ra2 antibody.
  • the recombinant adenoviral vector typically used in the present invention can be prepared by molecular biological techniques well known in the art. For example, for general molecular biology techniques, see Sambrook, J. et al .: Molecular Cloning. A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Ausbel.
  • a cosmid in which a target gene is incorporated into a cosmid cassette such as pAxCw or pAxCAwt is produced.
  • DNA-TPC is prepared from the virus and cleaved with an appropriate restriction enzyme.
  • the above-mentioned cosmid and restriction enzyme-treated DNA-TPC are cotransfected into an appropriate host cell, for example, 293 cells. Thereafter, the cells are cultured for a certain period of time under appropriate conditions, and the recombinant adenovirus particles released as virus particles in the culture medium are collected.
  • the recombinant adenovirus particles typically used in the present invention are transfected with a nucleic acid molecule for expressing a recombinant adenovirus into an appropriate cultured cell, the cell is further cultured, and the culture supernatant is recovered.
  • E1A and E1B regions when an adenovirus deficient in the commonly used E1A and E1B regions is propagated, 293 cells that constantly express E1A and E1B can be used. Furthermore, if necessary, it can be concentrated and purified using a concentration gradient centrifugation method of cesium chloride. By concentrating, a high titer virus solution of about 10 9 to 10 11 particles / ml can be obtained.
  • the recombinant adenovirus expression nucleic acid molecule typically used in the present invention can incorporate a foreign gene, and the incorporated foreign gene can be efficiently introduced into a target cell.
  • the foreign gene incorporated into the nucleic acid molecule for expression of the recombinant adenovirus used in the present invention include a gene encoding a molecule that exhibits cytotoxicity directly or indirectly to the target cell, cell growth factor, and cell growth suppression. Factors, apoptosis regulatory genes, tumor suppressor genes, cell cycle regulatory genes, immune regulatory genes and the like.
  • Such combinations include herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (HSVtk / GCV), cytosine deaminase and 5-fluorocytosine (CD / 5FC), uracil phosphoribosyltransferase (UP) ) And 5-fluorouracil (5FU) (UP / 5FU) and HSVtk and UP (UPTK / 5FU + GCV).
  • HSVtk herpes simplex virus thymidine kinase
  • ganciclovir HSVtk / GCV
  • cytosine deaminase and 5-fluorocytosine CD / 5FC
  • UP uracil phosphoribosyltransferase
  • UPTK / 5FU + GCV 5-fluorouracil
  • Such foreign genes are generally replaced with or inserted into the E1 and / or E3 region of the adenovirus genome
  • Non-limiting examples of vectors preferably used in the present invention include FZ33 fiber mutant adenovirus modified so as to bind to the Fc domain of the antibody, adhering the antibody to any method (covalent binding, biotin- And modified adenoviruses that are bound by avidin crosslinking, polyethylene glycan with chemically bound antibodies, etc.).
  • the FZ33 fiber mutant adenovirus is based on the FZ33 fiber mutant Ad5 virus that contains the Z33 motif of protein A that binds to the Fc domain of the antibody in the Knob HI loop, and incorporates reporter genes such as lacZ and EGFP.
  • Adenovirus see Non-Patent Document 1.
  • viruses for example, retrovirus, lentivirus, herpes simplex virus, Sindbis virus, measles virus, Sendai virus, reovirus, poxvirus, poliovirus, coxsackie virus, adeno
  • viruses for example, retrovirus, lentivirus, herpes simplex virus, Sindbis virus, measles virus, Sendai virus, reovirus, poxvirus, poliovirus, coxsackie virus, adeno
  • modified viruses that have been chemically encapsulated with polyethylene glycol, etc. can also be used with the anti-IL13Ra2 antibodies of the present invention, or not limited to viral vectors, Sendai virus envelope
  • Non-viral vectors such as rope vectors and plasmid DNA naked vectors can also be used to bind antibodies in any way (methods for binding antibody binding molecules such as FZ33, chemical covalent bonding, biotin-avidin crosslinking, and antibody binding.
  • the present invention can be used together with the anti-IL13Ra2 antibody of the present invention by making it a modified non-viral vector conjugated by a method such as a method of wrapping a vector with a conjugated polyethylene glycol).
  • Advantages of using a viral vector modified so that it can bind to the anti-IL13Ra2 antibody of the present invention include the following points.
  • the cell surface receptor that the virus originally recognizes for example, CAR for adenovirus or high-affinity ⁇ laminin receptor (LAMR) for Sindbis virus
  • CD155 for poliovirus
  • ICAM / DAF for coxsackievirus A21
  • SLAM / CD46 for measles virus, and the like. Therefore, for cells that do not express cell surface receptors specific for the virus to be used or have a low level of expression, it is usually difficult to effectively introduce a gene using a viral vector.
  • a virus vector that can bind to or is modified to bind to the anti-IL13Ra2 antibody of the present invention is used, even if the cell does not express the original cell surface receptor of the virus, the cell is IL13Ra2.
  • the targeted introduction and expression of a therapeutic gene using a viral vector can be easily performed on the cell.
  • the anti-IL13Ra2 antibody of the present invention can be used to deliver a drug useful for diagnosis and / or treatment of a disease to the target disease site. Accordingly, the present invention also provides a method of delivering an agent useful for diagnosis and / or treatment of a disease to a target disease site using an anti-IL13Ra2 antibody.
  • agents include the radioisotopes, therapeutic proteins, small molecule drugs, therapeutic genes, etc. already described.
  • the anti-IL13Ra2 antibody of the present invention is bound to a fiber mutant adenoviral vector incorporating a therapeutic gene to which the anti-IL13Ra2 antibody can bind.
  • the “target site” is a cell, tissue, organ or the like that highly expresses IL13Ra2 compared to normal cells, and in particular, a tumor cell that highly expresses IL13Ra2 compared to normal cells.
  • Non-limiting examples of such cells include melanoma cells, osteosarcoma cells, leukemia cells, lymphoma cells, prostate cancer cells, lung cancer cells (including malignant mesothelioma cells), glioma cells, head and neck cancer cells, kidneys Cancer cells, Kaposi's sarcoma cells, ovarian cancer cells and the like can be mentioned as suitable for the present invention.
  • the present invention also provides that, when an anti-IL13Ra2 antibody contained as an active ingredient in the therapeutic agent of the present invention is administered to a subject, lyses or grows an IL13Ra2-expressing cell via an immune effector cell.
  • therapeutic agents for diseases eg, cancer
  • IL13Ra2 characterized by high expression of IL13Ra2 that act to inhibit, and methods of treating such diseases by administering such therapeutic agents to a subject.
  • immune effector cell is used herein in the sense that is commonly used in the art, but particularly refers to an immune cell involved in the effector stage of the immune response, rather than the recognition and activation stage of the immune response
  • immune effector cells include T cells (eg, cytotoxic T cells (CTL), helper T cells (Th)), NK cells, NK-like T cells, B cells, monocytes, macrophages, Dendritic cells, Kupffer cells, Langerhans cells, polynuclear leukocytes (for example, neutrophils, eosinophils, basophils, mast cells) and the like are included.
  • the anti-IL13Ra2 antibody of the present invention can bind to an Fc receptor on the surface of immune effector cells.
  • Effector cells express specific Fc receptors and can exert specific immune functions through antibody binding, for example, neutrophils induce antibody-dependent cell-mediated cytotoxicity (ADCC) can do.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody of the present invention can phagocytose or lyse IL13Ra2-expressing cells via immune effector cells.
  • an anti-IL13Ra2 antibody contained as an active ingredient in the therapeutic agent of the present invention that acts to opsonize IL13Ra2-expressing cells when administered to a subject.
  • therapeutic agents for diseases characterized by expression eg, cancer
  • an anti-IL13Ra2 antibody of the invention can be a bispecific or multispecific molecule comprising at least one first binding specificity for IL13Ra2 and a second binding specificity for a second target epitope.
  • the second target epitope can be, for example, an Fc receptor such as a human Fc ⁇ RI or human Fc ⁇ receptor.
  • the present invention provides dual specificity that can bind to both effector cells expressing Fc ⁇ R, Fc ⁇ R or Fc ⁇ R (such as monocytes, macrophages or polynuclear leukocytes) and target cells expressing IL13Ra2.
  • Fc ⁇ R, Fc ⁇ R or Fc ⁇ R such as monocytes, macrophages or polynuclear leukocytes
  • target cells expressing IL13Ra2 may also be a complement binding part of an anti-IL13Ra2 antibody.
  • Anti-IL13Ra2 antibodies are capable of binding to both effector cells expressing complement receptors via complement (eg monocytes, macrophages or polynuclear leukocytes) and target cells expressing IL13Ra2. Specific or multispecific molecules are included.
  • bispecific or multispecific molecules target IL13Ra2-expressing cells to effector cells, and Fc receptor-mediated effector cell activities such as ADCC, cytokine release, phagocytosis of IL13Ra2-expressing cells, or superoxide anion production Can be evoked.
  • the present invention provides a method for diagnosing cancer, comprising a step of detecting and / or quantifying IL13Ra2 protein or a fragment thereof or a nucleic acid encoding the same in a biological sample derived from a subject as a diagnostic marker.
  • a “subject” refers to a disease characterized by high expression of IL13Ra2, typically suffering from, suspected of suffering from such a disease, or It shall mean a human subject at risk of suffering from such a disease.
  • Exemplary “cancers” in accordance with the purpose of the present invention include melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma), glioma, head and neck cancer, renal cancer, Kaposi sarcoma, Although ovarian cancer etc. are mentioned, it is not limited to these.
  • the “biological sample” means cells, tissues, organs, body fluids and the like derived from a subject collected for examination.
  • the body fluid includes blood, lymph, semen, saliva, sweat, and the like, and blood includes blood products such as serum and plasma in addition to whole blood.
  • the biological sample can be a cancer cell (or cancer tissue), preferably a melanoma cell, osteosarcoma cell, leukemia cell, lymphoma cell, prostate cancer cell, lung cancer cell (malignant mesothelioma cell).
  • a cancer cell or cancer tissue
  • a melanoma cell preferably a melanoma cell, osteosarcoma cell, leukemia cell, lymphoma cell, prostate cancer cell, lung cancer cell (malignant mesothelioma cell).
  • Glioma cells, head and neck cancer cells, kidney cancer cells, Kaposi sarcoma cells and ovarian cancer cells most preferably melanoma cells, osteosarcoma cells, leukemia cells, lymphoma cells, prostate cancer cells and lung cancer cells (malignant) Including dermatoma cells).
  • the present invention also provides a method for immunologically detecting and / or quantifying IL13Ra2 in a biological sample derived from a subject using an anti-IL13Ra2 antibody.
  • the method of the present invention according to a preferred embodiment comprises (1) a step of contacting a biological sample with an anti-IL13Ra2 antibody, and (2) detecting and / or quantifying the binding between IL13Ra2 and the anti-IL13Ra2 antibody in the biological sample. Process.
  • Such methods are characterized by high expression of IL13Ra2 compared to normal cells, typically melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma) It can be used for diagnosis of cancer including glioma, head and neck cancer, renal cancer, Kaposi's sarcoma, ovarian cancer and the like.
  • the level (or amount) of IL13Ra2 in a biological sample is evaluated based on the level (or amount) of a conjugate of IL13Ra2 and an anti-IL13Ra2 antibody.
  • the level of IL13Ra2 in the biological sample is compared with the level measured by a normal individual control, and the presence of cancer is diagnosed based on the change (or difference).
  • high IL13Ra2 levels compared to normal controls indicate the presence of cancer.
  • an IL13Ra2 level that is usually at least 2-fold, preferably about 5-fold higher, is a positive result indicating that the subject has cancer.
  • the presence of IL13Ra2 in the biological sample itself can indicate the presence of cancer in the subject.
  • Assay techniques that can be used to determine expression levels of a protein, such as IL13Ra2, in a biological sample are well-known to those of skill in the art.
  • Such assay methods include enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay, binding protein competition assay, reverse transcriptase PCR (RT-PCR) assay, immunohistochemical assay, in situ hybridization assay, And a proteomics approach.
  • ELISA is particularly preferably used for detection of a gene expression protein in a biological fluid. If not readily available commercially, ELISA analysis initially involves the preparation of an antibody, preferably a monoclonal antibody, that specifically binds to IL13Ra2.
  • a reporter antibody is prepared that specifically binds to IL13Ra2.
  • a detectable reagent such as a radioactive reagent, a fluorescent reagent or an enzyme reagent such as horseradish peroxidase or alkaline phosphatase.
  • an antibody that specifically binds to IL13Ra2 is incubated on a solid support to which the antibody is bound, such as a polystyrene dish.
  • a solid support to which the antibody is bound such as a polystyrene dish.
  • any free protein binding sites on the dish are coated (blocked) by incubating with a non-specific protein such as bovine serum albumin.
  • the sample to be analyzed is then incubated in the dish for the time that IL13Ra2 binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed away with buffer.
  • a reporter antibody that is specific for IL13Ra2 and linked to a detectable reagent such as horseradish peroxidase is placed in the dish, and the reporter antibody binds to any monoclonal antibody bound to IL13Ra2. Unbound reporter antibody is then washed away. Reagents for peroxidase activity including a colorimetric substrate are then added to the dish. Immobilized peroxidase linked to anti-IL13Ra2 antibody produces a colored reaction product. The amount of dye generated within a given time is proportional to the amount of IL13Ra2 protein in the sample. Quantitative results are usually obtained with reference to a standard curve.
  • a competitive assay can also be used, in which an antibody that specifically binds to IL13Ra2 is attached to a solid support, and the labeled IL13Ra2 and the sample from the subject or control pass over the solid support.
  • the amount of detection label attached to the solid support can be correlated to the amount of IL13Ra2 in the sample.
  • sandwich immunoassay fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA), luminescence immunoassay ( LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay, complement binding assay, immunization Any immunological assay such as a radiometric assay, a fluorescent immunoassay, or a protein A immunoassay can be used in the present invention.
  • the nucleic acid method uses all or part of the nucleic acid sequence of IL13Ra2 as a hybridization probe is used to detect IL13Ra2 mRNA as a marker for cancer such as melanoma.
  • Other nucleic acid methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) and nucleic acid sequence-based amplification (NASBA) to diagnose and monitor various malignancies Can be used to detect malignant cells.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • RT-PCR reverse transcriptase PCR
  • RT-PCR reverse transcriptase PCR
  • RT-PCR In RT-PCR, one type of mRNA is first reverse transcribed into complementary DNA (cDNA) using the enzyme reverse transcriptase, and then the cDNA is amplified as in a standard PCR reaction. Thus, RT-PCR can reveal the presence of a single species of mRNA by amplification. Thus, if this mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a particular type of cell.
  • the real-time PCR method can also be used for quantifying IL13Ra2 encoding nucleic acid (eg, mRNA) and diagnosing the subject's cancer using IL13Ra2 as a marker in comparison between the subject and a healthy individual.
  • IL13Ra2 it is also possible to monitor the progress or progress of treatment of the target cancer using IL13Ra2 as a marker.
  • Such cancers are not particularly limited as long as they are IL13Ra2-positive cancers.
  • IL13Ra2-positive melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma), glioma, head and neck cancer It can be suitably used for diagnosis of renal cancer, Kaposi's sarcoma, ovarian cancer and the like.
  • mRNA For example, by combining cell sorting and enrichment (enrichment) using antibodies and magnetic beads, and highly sensitive detection of mRNA by RT-PCR, it is present in, for example, 5 ml of the blood of a subject. It is possible to detect several tumor cells. By detecting tumor cells present in blood and detecting or measuring their IL13Ra2 expression level or mRNA level, tumors with high sensitivity and specificity can be diagnosed. For such methods, further ZieglschmidglV et al., Crit Rev Clin Lab Sci. 2005; 42 (2): 155-96, Waguri N et al., Clin Cancer Res. 2003 Aug 1; 9 (8): 3004-11, Zhang YL et al., World J Gastroenterol. 2005 Feb 21; 11 (7): 1023-7, Demel U et al., J Exp Clin Cancer Res. 2004 Sep; 23 (3): 465-8 Etc. can be referred to.
  • Hybridization to clones or oligonucleotides arrayed on a solid support can also be used for both detection of expression of the gene and quantification of expression levels.
  • cDNA encoding the IL13Ra2 gene is immobilized on a substrate.
  • the substrate may be of any suitable type, including but not limited to glass, nitrocellulose, nylon or plastic.
  • At least a portion of the DNA encoding the IL13Ra2 gene is attached to a substrate and then incubated with a sample that may be RNA or a copy of RNA that is complementary DNA (cDNA) isolated from the tissue.
  • Hybridization between the DNA bound to the substrate and the analyte can be detected and quantified by several means including, but not limited to, radiolabeling or fluorescent labeling of the analyte or hybrid detection secondary molecules.
  • labels that can be used in the present invention include radioisotopes, fluorescent groups, luminescent groups, free radical groups, particles, bacteriophages, cells, metals, enzymes, or coenzymes. Quantification of gene expression levels is done by comparing the intensity of the signal from the specimen to a level determined from a known standard. Standards can be obtained by in vitro transcription of the target gene, quantification of yield, and using this material to generate a standard curve.
  • two-dimensional electrophoresis is a technique well known to those skilled in the art. That is, individual proteins from a sample such as serum can be isolated by continuously performing separations according to different properties of the protein, usually on a polyacrylamide gel. First, proteins are separated by size using an electric current. The current acts equally on all proteins, so that small proteins move faster on the gel than larger proteins. In the second dimension, an electric current is applied at right angles to the first dimension and the proteins are separated based on the specific charge each protein has rather than based on size. Because no two proteins of different sequence are identical in both size and charge, the result of the two-dimensional separation is a square gel where each protein occupies a unique spot. Analysis of the spot with a chemical or antibody probe, or subsequent protein microsequencing can reveal the relative abundance of a given protein and identify the protein in the sample.
  • the detection and / or quantification methods detailed above are samples derived from various cells, body fluids and / or tissue extracts (homogenate or solubilized tissue) obtained from subjects including tissue biopsy material and autopsy material. Can be done about.
  • Body fluids useful in the present invention include blood, urine, saliva or any other body secretion or derivative thereof.
  • Blood includes whole blood, plasma, serum, or any blood derivative.
  • the present invention also provides a method for detecting and / or quantifying autoantibodies against IL13Ra2 (anti-IL13Ra2 autoantibodies) in a biological sample derived from a subject.
  • Detection of anti-IL13Ra2 autoantibodies in a biological sample from a subject can be performed in any of a number of ways, but representative methods include immunoassays, such as Western blot, radioimmunoassay, ELISA, sandwich Immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA), luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination Methods, immunoprecipitation assays, sedimentation reaction methods, gel diffusion sedimentation reaction methods, immunodiffusion assays, agglutinin assays, complement binding assays, immunoradiometric assays, protein A immunoassays, and the like.
  • immunoassays such as Western blot, radioimmunoassay, ELISA, sandwich Immunoassay, fluor
  • Such an immunoassay can be performed in various ways.
  • one method for performing such an assay involves tethering IL13Ra2 protein on a solid support and detecting anti-IL13Ra2 antibodies specific thereto.
  • the IL13Ra2 protein used in the assay of the present invention can be prepared by recombinant DNA techniques well known in the art.
  • IL13Ra2 protein can be expressed on a large scale by introducing DNA encoding IL13Ra2 protein into an appropriate expression vector by a gene recombination technique.
  • a fusion protein that can facilitate the labeling, immobilization or detection of IL13Ra2 is genetically engineered (eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Laboratory Manual, Cold Spring Spring Press, Cold Spring Harbor, NY See the technology described in).
  • IL13Ra2 protein can be purified from natural sources. For example, it is purified from melanoma cells using a protein separation technique well known in the art. Such purification techniques include, but are not limited to, molecular sieve chromatography and / or ion exchange chromatography.
  • a microtiter plate is advantageously used as the solid support for the IL13Ra2 protein.
  • methods specifically exemplified for the generation of hybridomas producing NS-66 as well as the subsequent identification of antigens generally include candidate molecules targeted for drug targeted therapy. This is useful as a systematic search method.
  • the antibody screening method of the present invention enables direct and rapid screening of antibodies and corresponding antigen molecules that are highly optimized for highly sensitive and highly specific target therapies as diagnostic markers. Accordingly, the present invention provides a method for systematically searching for candidate molecular candidates for targeted drug therapy. More specifically, the present invention provides methods for identifying monoclonal antibodies against tumor-specific antigens and methods for identifying tumor-specific antigens.
  • the present invention provides a method for producing a hybridoma that produces a monoclonal antibody against a tumor-specific antigen, the method comprising: (1) a step of immunizing a mammal with a tumor-specific antigen or a tumor cell expressing the antigen and fusing lymphocytes from the immunized mammal with myeloma cells to produce a library of hybridomas; (2) In the presence of the hybridoma-derived product (eg, antibody) derived from the library, the fiber mutation is brought into contact with the tumor cell by contacting the fiber mutant adenovirus modified to bind to the antibody with the tumor cell.
  • the hybridoma-derived product eg, antibody
  • the present invention also provides a method of screening for a monoclonal antibody against a tumor-specific antigen.
  • This screening method is (1) immunizing a mammal with a tumor-specific antigen or a tumor cell expressing the antigen, lysing lymphocytes from the immunized mammal with myeloma cells, and preparing a hybridoma library; and (2 ) In the presence of the hybridoma-derived product (eg, antibody) derived from the library, the fiber mutant adenovirus is modified by contacting the fiber mutant adenovirus modified to bind to the antibody with the tumor cell. Infecting the tumor cells with a virus, and evaluating the infection efficiency of the fiber mutant adenovirus for the tumor cells.
  • the hybridoma-derived product eg, antibody
  • the infection of the tumor cells with the fiber mutant adenovirus in the presence of the hybridoma-derived product derived from the library (A) The hybridoma-derived product from the library is contacted with the tumor cell, and then the fiber mutant adenovirus modified to bind to the antibody is contacted with the tumor cell. (B) The hybridoma-derived product derived from the library is reacted in advance with a fiber mutant adenovirus modified so as to bind to an antibody, and then contacted with the tumor cell, or (C) the library Contacting the tumor cells by simultaneously administering the hybridoma-derived product derived from and a fiber mutant adenovirus modified to bind to an antibody, Is done by.
  • the target monoclonal antibody is usually selected by cloning and cloning a hybridoma whose infection efficiency is increased compared to the case where a hybridoma-derived product (such as an antibody) is not contacted with tumor cells.
  • Antibodies can be obtained.
  • the step of selecting a hybridoma producing the target monoclonal antibody comprises evaluating the infection efficiency of the fiber mutant adenovirus with respect to the tumor cells by a reporter gene expression measurement assay.
  • the fiber mutant adenovirus is prepared so that a reporter gene for evaluating the infection efficiency can be expressed during the infection.
  • the reporter gene expression measurement assay can measure the expression of a reporter gene (for example, EGFP) using, for example, a spectrophotometer or flow cytometry.
  • a reporter gene for example, EGFP
  • the expression of the lacZ gene product is expressed by a commercially available chemiluminescent ⁇ -Gal reporter measurement kit (for example, Galacto Examples include the use of Light Plus Reporter Gene Assay System (Roche, code number T1011).
  • a luciferase gene when a luciferase gene is used as a reporter gene, it can be measured using Luciferase assay system (Promega, Cat No. E1500) as a commercially available luciferase assay system.
  • the fiber mutant adenovirus modified to bind to the antibody includes, for example, FZ33 fiber mutant adenovirus containing the Z33 motif of protein A that binds to the Fc domain of the antibody at the HI loop site of the knob.
  • FZ33 fiber mutant adenovirus containing the Z33 motif of protein A that binds to the Fc domain of the antibody at the HI loop site of the knob.
  • an antibody having a certain affinity for example, a binding constant (K a ) of at least 10 7 M ⁇ 1 , preferably at least 10 8 M ⁇ 1 , more preferably Any kind of vector can be used as long as it has a property capable of binding with a binding affinity of 10 9 M ⁇ 1 or more.
  • an antibody-binding molecule or peptide such as Z33 is adsorbed to adenovirus by any method (method of covalently binding Z33, etc., method of cross-linking Z33, etc. with biotin-avidin, or wrapping the virus with polyethylene glycol chemically bound to Z33, etc. Modified adenoviruses such as FZ33 bound by the method etc.).
  • a fiber mutant adenovirus lacking the binding site of adenovirus fiber to the CAR receptor ie Kirby I et al. J. Virol. 73: 9508-9514, 1999 .; Mizuguchi et al. Gene Therapy 9 : 769-776, 2002 .; can bind to antibodies with a certain affinity based on fiber mutant adenoviruses based on references such as RoelvinkPW et al. Science, 286: 1568-1571, 1999.
  • Vectors imparted with properties can be used for the purposes of the present invention as well.
  • a vector having a property capable of binding to an antibody with a certain degree of affinity based on a penton base protein mutant adenovirus lacking reactivity with the integrin molecule group of adenovirus is also used for the purpose of the present invention.
  • fiber mutant adenoviruses lacking adenovirus uptake into the liver namely Smith TA et al (Smith TA, Idamakanti N, Rollence ML, Marshall-Neff J, Kim J, Mulgrew K, Nemerow GR, Kaleko M , Stevenson SC.
  • Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum Gene Ther. 2003 May 20; 14 (8): 777-87.
  • vectors imparted with the property of being able to bind to antibodies with a certain degree of affinity can also be used for the purposes of the present invention.
  • viruses for example, retrovirus, lentivirus, herpes simplex virus, Sindbis virus, measles virus, Sendai virus, reovirus, poxvirus, poliovirus, coxsackie virus, adeno Mutant virus with modified envelope (envelope), capsid, etc., or any other method (such as Z33, Z33, etc.)
  • viruses for example, retrovirus, lentivirus, herpes simplex virus, Sindbis virus, measles virus, Sendai virus, reovirus, poxvirus, poliovirus, coxsackie virus, adeno Mutant virus with modified envelope (envelope), capsid, etc., or any other method (such as Z33, Z33, etc.)
  • Modified viruses bound by a method of crosslinking with biotin-avidin, a method of wrapping a virus with polyethylene glycol chemically bound with Z33 or the like can also be used in the method of the present invention.
  • an antibody-binding molecule or peptide is chemically covalently bound to any method (antibody-binding molecule is chemically bound to non-viral vectors such as liposome vectors, Sendai virus envelope vectors, plasmid DNA naked vectors, etc.
  • non-viral vectors such as liposome vectors, Sendai virus envelope vectors, plasmid DNA naked vectors, etc.
  • Modified non-viral vectors bound by a method, a method of cross-linking with biotin-avidin, a method of wrapping a vector with polyethylene glycol chemically bound to an antibody binding molecule, etc. can also be advantageously used in the present invention.
  • reporter genes include fluorescent proteins such as lacZ and EGFP, and luciferase genes. These reporter genes can be detected using reporter gene assay systems well known to those skilled in the art.
  • the infection efficiency of FZ33 fiber mutant adenovirus prepared to express lacZ can be determined by using a commercially available chemiluminescent ⁇ -Gal reporter kit (for example, Galacto Light Plus Reporter Gene Assay System (Roche, code number T1011)) ) Can be measured using.
  • a luciferase quantification system it can measure using Luciferase assay system (Promega, Cat No E1500) etc.
  • the expression level of fluorescent proteins such as EGFP can be measured by a spectrophotometer or flow cytometry.
  • the present invention provides a method for identifying a tumor specific antigen.
  • the method for identifying a tumor-specific antigen of the present invention comprises the steps of identifying an antigen protein that specifically binds to a monoclonal antibody purified from the hybridoma-derived product, determining the amino acid sequence thereof, and the determined amino acid sequence.
  • the method includes a step of identifying the antigen protein by performing a homology search on a sequence database.
  • the step of identifying the antigen protein includes immunoprecipitation between the antigen and the monoclonal antibody, Western blot analysis, and the like.
  • the step of determining the amino acid sequence is performed by any known amino acid sequencing method (for example, a method for determining an amino acid sequence using a gas phase sequencer (for example, Procise 490cLC ABI, HP241 HP, etc.), cleavage by enzyme or chemical degradation. Separation of the peptides obtained by HPLC, peptide mapping by gel electrophoresis, amino acid sequence analysis of peptides separated by HPLC using a mass spectrometer, etc., but preferably by mass spectrometry The amino acid sequence is determined.
  • a gas phase sequencer for example, Procise 490cLC ABI, HP241 HP, etc.
  • the homology search on the sequence database is performed using a program well known to those skilled in the art (for example, FASTA, BLAST, MASCOT, etc.) and a sequence database (for example, PIR, SWISS-PROT, NCBI, etc.). be able to.
  • a program well known to those skilled in the art for example, FASTA, BLAST, MASCOT, etc.
  • a sequence database for example, PIR, SWISS-PROT, NCBI, etc.
  • amino acid sequences of the identified antibodies and target molecules and the nucleic acid sequences encoding them can be determined by sequencing methods well known to those skilled in the art.
  • Amino acid sequencing methods include determination of amino acid sequences using gas phase sequencers (eg Procise 490cLC ABI, HP241 HP, etc.), separation of peptides obtained by enzymatic or chemical cleavage by HPLC, gel electrophoresis Peptide map methods by electrophoresis, amino acid sequence analysis of peptides separated by HPLC using a mass spectrometer, and the like can be used.
  • a method for determining a nucleic acid sequence a method for determining a nucleic acid sequence well known to those skilled in the art, such as a cycle sequencing method using PCR, can be used.
  • the antibody of the present invention or a fusion with another molecule is prepared by genetic engineering techniques based on the determined base sequence. be able to.
  • the target DNA can be obtained in large quantities using molecular biological techniques well known to those skilled in the art, such as a cloning method using a plasmid vector (eg, Sambrook, J. et al .: Molecular Cloning. A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, etc.).
  • a therapeutic agent that is linked to a target or genetically engineered can be formulated based on a known method.
  • a pharmaceutically acceptable carrier can be added as necessary according to a conventional method.
  • surfactants for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, taste masking
  • the present invention is not limited thereto, and other commonly used carriers can be appropriately used.
  • Examples of the dosage form of the therapeutic agent of the present invention include tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coating agents, pellets, and sublingual agents as oral preparations.
  • pastes such as injections, suppositories, transdermal agents, ointments, plasters, liquids for external use and the like, and those skilled in the art will determine the optimal dosage form according to the route of administration, administration target, etc. You can choose.
  • the function (or expression) inhibitor of IL13Ra2 protein as an active ingredient can be contained in the preparation in an amount of 0.1 to 99.9% by weight.
  • the dose of the active ingredient of the drug of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, per day for a patient (as 60 kg) About 0.1 mg to 1,000 mg, preferably about 1.0 to 100 mg, more preferably about 1.0 to 50 mg.
  • the single dose varies depending on the administration subject, target organ, symptom, administration method, etc.
  • a patient for example, a patient (relative to 60 kg)
  • the final decision can be made as appropriate based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
  • the preparation thus obtained can be administered to, for example, humans and other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). In the case of animals other than humans, the amount converted per 60 kg can be administered.
  • the therapeutic agent of the present invention is not limited to a specific type of cell, tissue, organ, or cancer, but melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, lung cancer (including malignant mesothelioma) It is preferably used for the treatment of glioma, head and neck cancer, renal cancer, Kaposi's sarcoma, and ovarian cancer.
  • the viral vector particles typically used in the present invention can be used as a component of a pharmaceutical composition in combination with an anti-IL13Ra2 antibody for treatment, particularly for tumor treatment.
  • an anti-IL13Ra2 antibody for treatment
  • these may be used alone, but are generally used with a pharmaceutically acceptable carrier.
  • a carrier a carrier as described above and an aqueous isotonic solution such as water, physiological saline, glucose, human albumin and the like are preferable.
  • additives, preservatives, preservatives, fragrances and the like that are commonly used in pharmaceutics can also be added.
  • the pharmaceutical composition thus prepared can be administered by an appropriate administration form and administration route depending on the disease to be treated.
  • the dosage form include emulsion, syrup, capsule, tablet, granule, injection, ointment and the like.
  • the number of administrations may be once to several times a day, the administration period may be from one day to several months or more, and one to several injections may be set as one set, and multiple sets may be administered intermittently over a long period of time.
  • the viral vector particles or viral vector nucleic acid molecules used in the present invention can be used for detection of specific cells and / or tissues, or diagnosis of disease states.
  • viral vector particles obtained by incorporating a detectable marker gene into a nucleic acid molecule of a viral vector and transfecting it into an appropriate host cell can be used to detect and diagnose tumor cells in combination with an anti-IL13Ra2 antibody. can do.
  • it can be used to detect and diagnose tumor cells by binding a detectable label to the anti-IL13Ra2 antibody.
  • Example 1 Preparation of targeted antibody against IL13Ra2 ⁇ 1> Preparation of hybridoma (i) Immunization of mice was carried out by the following procedure. 1) 5 ⁇ 10 6 A375 cells (human melanoma (malignant melanoma) cell line, ATCC CRL-1619) were cultured in 10 ml of DMEM (Dulbecco's Modified Eagle Medium) medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • CD40L-expressing adenovirus Ax3CAhCD40L-F / RGD
  • BAFF-expressing adenovirus Ax3CAhBAFF-F / RGD
  • adenovirus expressing BAFF (the B cell-activating factor of tumor necrosis factor family) is based on nucleotide sequence information from GenBank (LOCUS NM_006573 tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B), etc.)
  • GenBank LCUS NM_006573 tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B), etc.
  • a cDNA region corresponding to the cDNA coding region of human BAFF was prepared by PCR, and using this, a method similar to the CD40L-expressing adenovirus described in Tomihara K et al. Was prepared. 3) Virus was infected to A375 cells at 37 ° C. for 48 hours.
  • Virus-infected cells were collected, washed twice with PBS (phosphate buffered saline), and resuspended in 500 ⁇ l of PBS.
  • PBS phosphate buffered saline
  • BALB / c mice (6 weeks old) were immunized intraperitoneally with virus-infected cells 7 times every 2 weeks.
  • the 8th round was the final immunization, and virus-uninfected A375 cells were immunized intravenously with 1 ⁇ 10 6 cells / 200 ⁇ l PBS.
  • (Ii) -2 Preparation of myeloma cells 6) P3U1 cells (mouse myeloma cell line) were collected and centrifuged at 1500 rpm for 5 minutes at room temperature. 7) 10 ml of EDTA (0.02%) solution was added to P3U1 cells and treated at 37 ° C. for 5 minutes. 8) The P3U1 cell suspension was centrifuged at 1500 rpm for 5 minutes at room temperature. 9) The supernatant was discarded and resuspended in 10 ml of RPMI 1640 FBS ( ⁇ ) medium. (Ii) -3. Cell fusion 10) Spleen cells and myeloma cells were mixed at a ratio of 5: 1.
  • Hybridoma screening was performed as follows. (Iii) -1. Virus infection 1) A375 cells were seeded at 3 ⁇ 10 3 cells / well in a 96-well plate and cultured at 37 ° C. for 48 hours. 2) Cells were washed twice with 150 ⁇ l / well PBS. 3) 50 ⁇ l of each hybridoma culture supernatant was added to each well and reacted at 4 ° C. for 1 hour. 4) Washed twice with 150 ⁇ l / well of PBS.
  • Adenovirus Ax3CAZ3-FZ33 ( ⁇ -galactosidase-expressing adenovirus modified with Z33 fiber so as to bind to the antibody, see Non-Patent Documents 1 and 2) 3 ⁇ 10 6 vp / 100 ⁇ l (1 ⁇ 10 3 vp / Cells) was diluted with DMEM FBS ( ⁇ ) medium to a concentration of 100), and this diluted solution was added at 100 ⁇ l / well. 6) Adenovirus was reacted at 4 ° C for 1 hour. 7) Washed twice with 150 ⁇ l / well of PBS. 8) DMEM FBS (+) medium was added at 100 ⁇ l / well and cultured at 37 ° C. for 24 hours.
  • clones having a measured value (RLU) of 5000 RLU or more were selected from the entire group (minimum value 179 RLU, median value 904 LU, maximum value 45381 RLU).
  • RLU measured value
  • 134 positive wells were selected from 960 well hybridoma wells obtained by one cell fusion.
  • a duplicate assay is performed in the same manner as in the primary screening, and both wells having a measured value of 5000 RLU or more are made positive, and from the 134 wells obtained in the primary screening, 64 Positive wells were selected.
  • the selected clones were subcloned twice to establish 9 monoclonal hybridoma strains.
  • NS-66 antibody monoclonal NS-66 antibody obtained from one of the nine hybridoma strains obtained above.
  • the subclass of NS-66 antibody was mouse IgG2b. Therefore, in the following experiments, unless otherwise specified, mouse IgG2b control antibody (Mouse IgG2b Isotype control, clone eBMG2b, Cat No 16-4732) commercially available from eBioscience is used as the same subclass control of NS-66 antibody. It was.
  • ⁇ 2> Determination of antigen molecule of NS-66 antibody (i) Immunoprecipitation of biotin-labeled cell surface protein using NS-66 antibody was performed by the following procedure. 1) 5 ⁇ 10 6 A375 cells were collected and washed 3 times with PBS. 2) EZ-Link Sulfo-NHS-Biotin (PIERCE) was suspended in PBS at a concentration of 0.1 mg / ml. 3) A375 cells were rotated in Biotin / PBS solution for 30 minutes at room temperature. 4) A375 cells were washed twice with 25 ml of 100 mM glycine / PBS solution. 5) A375 cells were washed 3 times with 25 ml of PBS.
  • A375 cells were resuspended in 2 ml of lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH7.6, 1% NP-40 + Protease inhibitor, Complete EDTA free (Roche) 1 tablet / 50 ml) at 4 ° C. Treated for 30 minutes.
  • the buffer of Protein G Sepharose (Protein G Sepharose 4 Fast Flow (GE Healthcare)) was replaced with a lysis buffer.
  • 300 ⁇ l of Protein G Sepharose / lysis buffer (50% slurry) was added to the cell lysate obtained in 6) and rotated at 4 ° C. for 1 hour.
  • Centrifugation was performed at 4 ° C.
  • Silver staining The silver staining MS kit (wako) was used as follows. 31) SDS-PAGE was performed on the sample obtained in (iii) -3 above, and the gel after electrophoresis was shaken with 100 ml of fixative 20 for 20 minutes. 32) The gel was shaken with 100 ml of fixative 2 for 10 minutes and then with 100 ml of DDW for 10 minutes. 33) The gel was shaken with 100 ml of the sensitizing solution for 1 minute, and then shaken twice with 100 ml of DDW for 1 minute. 34) The gel was shaken with 100 ml of staining solution for 20 minutes and then shaken with 100 ml of DDW for 1 minute.
  • Mass spectrometry was requested and analyzed by Hitachi Science Co., Ltd. The conditions are as follows. Model used MALDI-Qq-TOF MS / MS QSTAR Pulsar I (Applied Biosystems) Database search software MASCOT (Matrix Science) Database NCBIInr (Taxonomy: human) The SDS-PAGE separation gel was digested with trypsin and desalted, followed by TOF measurement and database search. The results of mass spectrometry are shown in FIG. As shown in the figure, two specific peaks different from keratin and trypsin were obtained (peak 1: 921.5016 and peak 2: 1206.7056).
  • the amino acid sequence corresponding to the mass of peak 1 was QLCFVVR (SEQ ID NO: 24), and the amino acid sequence corresponding to the mass of peak 2 was WSIPLGPIPAR (SEQ ID NO: 25).
  • Peak 1 matched the amino acid sequence from the 303rd to the 309th amino acid sequence of human IL13Ra2, and peak 2 matched the amino acid sequence from the 258th to 268th amino acid sequence of human IL13Ra2.
  • the 58 kDa band excised from the gel in panel C of FIG. 1 was shown to contain human IL13Ra2 protein.
  • DEPC DW diethyl pyrocarbon
  • sequence of the cDNA incorporated into pTARGET-IL13Ra2 was determined.
  • the nucleotide sequence (1179 bp) of cDNA encoding human IL13Ra2 incorporated into pTARGET-IL13Ra2 was as shown in SEQ ID NO: 3.
  • the amino acid sequence (380 amino acids) of human IL13Ra2 encoded by this cDNA sequence is as shown in SEQ ID NO: 4.
  • the cells were resuspended in 100 ⁇ l of the secondary antibody dilution and allowed to react at 4 ° C. for 30 minutes in the dark. 7) The cells were washed twice with 1 ml of 2% FBS / PBS buffer and resuspended with 400 ⁇ l of 2% FBS / PBS buffer. 8) 1 ⁇ l of propidium iodide (0.1 mg / ml) was added to the cell suspension. 9) Using a FACSCalibur (Becton, Dickinson) as a flow cytometer, the fluorescence of propidium iodide was excluded and the fluorescence of RPE was measured.
  • FACSCalibur Becton, Dickinson
  • FIGS. 3A and B The results are shown in FIGS. 3A and B.
  • the upper panel shows the reactivity between IgG and each transfectant
  • the lower panel shows the reactivity between NS-66 antibody and each transfectant.
  • control is 293T cell to which no gene is introduced
  • pcDNA3.1 is 293T cell to which control plasmid pcDNA3.1 is introduced
  • pTARGET-IL13Ra2 is FACS of 293T cell to which plasmid pTARGET-IL13Ra2 expressing human IL13Ra2 is introduced
  • Upper left panel (IgG / control): The control antibody does not react in non-gene-transfected cells.
  • Upper middle of the panel (IgG / pcDNA3.1): The control antibody does not react with cells into which control DNA has been introduced.
  • Lower middle panel (66 / pcDNA3.1): NS-66 antibody does not react with cells into which control DNA has been introduced.
  • Upper right of the panel (IgG / pTARGET-IL13Ra2): The control antibody does not react with 293T cells expressing human IL13Ra2.
  • control is a CHO cell in which no gene is introduced
  • pcDNA3.1 is a CHO cell in which a control plasmid pcDNA3.1 is introduced
  • pTARGET-IL13Ra2 is a FACS of a CHO cell in which a plasmid pTARGET-IL13Ra2 expressing human IL13Ra2 is introduced The analysis results are shown respectively. As understood from the results of FIG. 3B, when CHO cells were used as cells for gene transfer, as in FIG.
  • the experimental procedure was as follows. 1) The collected cells were washed twice with 1 ml of 2% FBS / PBS buffer. 2) NS-66 antibody and control antibody were diluted 100 times with 2% FBS / PBS buffer. 3) Cells were resuspended with 100 ⁇ l of diluted antibody solution and reacted at 4 ° C. for 30 minutes. 4) The cells were washed twice with 1 ml of 2% FBS / PBS buffer. 5) A secondary antibody (Polyclonal Goat Anti-Mouse Immunoglobulins / RPE Goat F (ab ′) 2 (Dako)) was diluted 100 times with 2% FBS / PBS buffer.
  • the cells were resuspended in 100 ⁇ l of the secondary antibody dilution and allowed to react at 4 ° C. for 30 minutes in the dark. 7) The cells were washed twice with 1 ml of 2% FBS / PBS buffer, and the cells were resuspended with 400 ⁇ l of 2% FBS / PBS buffer. 8) 1 ⁇ l of propidium iodide (0.1 mg / ml) was added to the cell suspension. 9) Using FACS Calibur (Becton, Dickinson) as a flow cytometer, the fluorescence of propidium iodide was excluded, and the fluorescence of RPE was measured.
  • FACS Calibur Becton, Dickinson
  • Example 3 Gene transfer to melanoma cells using NS-66 antibody Genes using IL-Ra13 expressing adenovirus (Ax3CAZ3-FZ33) modified with Z33 fiber and NS-66 antibody for IL13Ra2 expressing melanoma cells (A375) The introduction efficiency was examined by ⁇ -Gal reporter gene assay. Using eBMG2b as a control IgG antibody, 83824 (R & D), 83807 (R & D) and B-D13 (Diaclone), which are commercially available anti-IL13Ra2 monoclonal antibodies, were also examined for their gene transfer efficiency. The experimental procedure was performed as follows.
  • Virus infection 1 A375 cells were seeded in a 96-well plate at a density of 3 ⁇ 10 3 cells / well and cultured at 37 ° C. for 48 hours. 2) Cells were washed twice with 150 ⁇ l / well PBS. 3) To each well, 100 ⁇ l of each antibody dilution (diluted so that the final concentrations were 3, 10, 30, 100, 300, and 1000 ng / well, respectively) was added and reacted at 4 ° C. for 1 hour. . 4) Washed twice with 150 ⁇ l / well of PBS.
  • Adenovirus Ax3CAZ3-FZ33 was diluted with DMEM FBS ( ⁇ ) medium to a concentration of 3 ⁇ 10 6 vp / 100 ⁇ l (300 vp / cell), and this diluted solution was added at 100 ⁇ l / well. 6) Adenovirus was reacted at 4 ° C for 1 hour. 7) Washed twice with 150 ⁇ l / well of PBS. 8) DMEM FBS (+) medium was added at 100 ⁇ l / well and cultured at 37 ° C. for 24 hours.
  • NS-66 antibody showed a gene transfer efficiency that was 4.30 times higher than that of B-D13 and 18.8 times higher than that of 83807. From the above results, it is understood that the NS-66 antibody according to the present invention is a highly specific antibody that is markedly more suitable for targeted treatment / diagnosis of melanoma and other cancers than other commercially available anti-IL13Ra2 antibodies. Is done.
  • Example 4 Targeted treatment of melanoma cells by IL13Ra2 selective immunotoxin introduction using NS-66 antibody Immunotoxin (saporin-binding IgG) when NS-66 antibody is used against melanoma cells expressing IL13Ra2 (A375)
  • the introduction efficiency was examined by measuring the cell viability.
  • eBMG2b as a control IgG antibody, the introduction efficiency of commercially available anti-IL13Ra2 monoclonal antibodies 83824 (R & D), 83807 (R & D) and B-D13 (Diaclone) was also examined. The experiment was performed as follows.
  • A375 cells in the logarithmic growth phase were collected from a culture dish using a trypsin-EDTA solution. 2) The supernatant was discarded by centrifugation, and the cells were suspended in DMEM medium (containing 10% FBS). 3) The cell concentration in DMEM medium was measured and adjusted to 3 ⁇ 10 4 cells / ml using DMEM medium (containing 10% FBS). 4) Cells were seeded in a 96-well plate (Nunc) at 100 ⁇ l / well (3 ⁇ 10 3 cells / well).
  • the NS-66 antibody according to the present invention is a highly specific antibody that is incorporated into cells with extremely high efficiency in cancer cells such as melanoma, compared with other commercially available anti-IL13Ra2 antibodies. It is understood that it is very effective for targeted therapy using such drugs.
  • Embodiment 5 In order to evaluate whether IL13Ra2 can be used as a target molecule in melanoma etc., recombinant human IL13 (rhIL13) was added as a targeting molecule to Pseudomonas aeruginosa exotoxin (PE) A fusion protein rhIL13-PE40 was prepared, and its cell killing effect on cells expressing or not expressing IL13Ra2 was examined.
  • rhIL13 recombinant human IL13
  • PE Pseudomonas aeruginosa exotoxin
  • PE Pseudomonas aeruginosa exotoxin
  • PA103 type E genomic DNA (provided by Professor Akihiro Fujii, Department of Microbiology, Sapporo Medical University School of Medicine) as a template for PE40 (Pseudomonas exotoxin A First, PCR (1st) with a DNA fragment encoding domains 2 and 3) with primers # 2291 / # 2292 and then PCR (2nd) with primers # 2295 / # 2296 that anneal inside Obtained (nested PCR). The reaction conditions are shown below. Reaction solution composition PCR conditions
  • the 1400 bp DNA amplified by PCR was purified and then ligated to the cloning vector pGEM (registered trademark) -T Easy, which was transformed into E.
  • the base sequence (1440 bp) from the partial BamHI site to the KpnI site encoding the fusion protein is shown as SEQ ID NO: 15, and the amino acid sequence (477aa) of the fusion protein encoded thereby is shown as SEQ ID NO: 16.
  • the first to second amino acids (GS) and the 115th to 116th amino acids (KL) are artificial amino acid sequences corresponding to BamHI and HindIII, respectively. Since it is linked to pQE30, a 6 ⁇ His tag is added to the N-terminus.
  • the obtained pQE30hIL13PE40 was transformed into E. coli M15 (pREP4) to prepare pQE-hIL13-PE (see FIG. 8).
  • the cells were collected using a centrifuge.
  • the collected E. coli is suspended in a solubilization buffer (50 mM NaH 2 PO 4 ⁇ 2H 2 O, 300 mM NaCl, 10 mM imidazole), pulverized by sonication, and the supernatant is purified with Ni-NTA. And recovered at respective imidazole concentrations of 250 mM and 500 mM. Thereafter, each fraction was replaced with PBS (-) with a dialysis membrane. In order to measure the protein concentration of each fraction, CBB staining was performed after SDS-PAGE using BSA as a standard (see FIG. 10). As a result, when recovered with 250 mM imidazole, 300 ⁇ g / ml (1 ml) of rIL13-PE was obtained.
  • the mixture was allowed to stand for 30 minutes, and 10 ⁇ l of Lipofectamin 2000 (Gibco) suspended in 250 ⁇ l of OPTI-MEM (Gibco) were mixed and allowed to stand at room temperature for 30 minutes. After the cell culture medium was replaced with 2 ml of DMEM FBS (+) penicillin / streptomycin (-) medium, the entire amount of the plasmid DNA / Lipofectamin 2000 / OPTI-MEM mixture was added dropwise to each cell and cultured at 37 ° C. for 48 hours. 293T-cDNA3.1 and 293T-TARGET-IL13Ra2 were obtained.
  • rhIL13-PE40 Cell killing effect of rhIL13-PE40 on other cell lines
  • the cell killing effect of rhIL13-PE40 on A375 (melanoma cell line) cells and HUVEC (human umbilical vein endothelial cells) is similar to the above ⁇ 6>
  • rIL13-PE After inducing expression of rIL13-PE, the cells were collected using a centrifuge. The collected Escherichia coli was solubilized with 8 M urea, purified with Ni-NTA, and recovered at imidazole concentrations of 250 mM and 500 mM. Thereafter, each fraction was replaced with PBS (-) with a dialysis membrane. The cell killing effect of the obtained highly effective rhIL13-PE40 on A375 (melanoma cell line) cells was evaluated by the same method as in the above ⁇ 6>. That is, A375 cells were seeded in a 96-well plate at a density of 5 ⁇ 10 3 cells / well, cultured at 37 ° C.
  • Example 6 Preparation of anti-hIL13Ra2 antibody Antibodies with diagnostic and therapeutic properties that are effective for more specific purposes, such as monoclonal anti-hIL13Ra2 antibodies that can be diagnosed by immunostaining in formalin-fixed, paraffin-embedded samples was made.
  • PCR Polymerase chain reaction
  • the nucleotide sequence was confirmed with ABI PRISM (registered trademark) 3100 Genetic Analyzer.
  • the extracellular domain of human IL13Ra2 consists of 316aa represented by SEQ ID NO: 20, and the base sequence encoding this consists of 948 bp represented by SEQ ID NO: 19.
  • the obtained expression vector expresses a fusion protein having the amino acid sequence (337aa) represented by SEQ ID NO: 21.
  • 1 to 12 amino acids (MRGSHHHHHHGS) in the above amino acid sequence is a His ⁇ 6 tag sequence derived from pQE30
  • the VDLQPSLIS sequence at the C terminal is a sequence derived from a multi-restriction enzyme site derived from pQE30.
  • IPTG was added to Escherichia coli (M15 (pREP4) -QE30-hIL13Ra2) in which pQE30-hIL13Ra2 was incorporated to induce expression.
  • the culture solution was centrifuged at 4000 g for 20 minutes to recover the cells as pellets, and half of the cells were suspended in 20 ml of solubilization buffer (8M urea).
  • the obtained suspension was centrifuged at 10,000 g for 20 minutes to recover the supernatant, and 2 ml of 50% Ni-NTA was added thereto, and the mixture was rotated at room temperature for 60 minutes.
  • the resulting mixture was loaded onto a column (Poly-Prep Chromatography Columns: BioRad, Cat. No.
  • rhIL13Ra2 protein was quantified by SDS-PAGE and protein assay. In SDS-PAGE, the amount of rhIL13Ra2 protein was calculated by comparing the density of the BSA band with a known protein quantity with the density of the rhIL13Ra2 band, and was about 150 ng / ⁇ l. The amount of rhIL13Ra2 protein was quantified using the BCA protein Assay Kit (PIERCE) and found to be 160 ng / ⁇ l.
  • PIERCE BCA protein Assay Kit
  • the membrane surface expressed protein encoded by this vector has the amino acid sequence (415aa) represented by SEQ ID NO: 22.
  • amino acids 1-21 (METDTLLLWVLLLWVPGSTGD) is the leader sequence of immunoglobulin ⁇ chain
  • amino acids 22-30 (YPYDVPDYA) is the HA (influenza virus hemagglutinin clot) tag sequence
  • amino acids 31-37 (GAQPARS)
  • amino acids 38 to 353 are the extracellular domain sequences of human IL13Ra2
  • amino acids 354 to 355 are sequences derived from the restriction enzyme SalI site, 356 to 365.
  • the amino acid (EQKLISEEDL) is a myc tag sequence, and the remaining amino acids 366 to 415 are a transmembrane peptide sequence derived from PDGFR (platelet-derived growth factor receptor).
  • the part encoding the expressed protein of pDisplay-hIL13Ra2 has the base sequence (1248 bp) represented by SEQ ID NO: 23. Of these, bases 106 to 1065 are BglII / SalI fragments prepared by PCR.
  • plasmids pCAcchCD40Lm3 and pCAcchBax-a were prepared as adjuvants.
  • CDNA was synthesized from the obtained total RNA using Superscript II Reverse Transcriptase (Invitrogen). Using this cDNA as a template, polymerase chain reaction (PCR) was performed using primers # 1795 and # 1796. Primer: # 1795: 5'-CGGAATTCAGCATGATCGAAACATACAACCAAAC-3 '(sense: SEQ ID NO: 26) # 1796: 5'-CGGGATCCTCAGAGTTTGAGTAAGCCAAAGGACG-3 '(antisense: SEQ ID NO: 27)
  • pDisplay-hIL13Ra2 obtained in the above ⁇ 4>, and pCAcchCD40Lm3 and pCAcchBax-a were introduced into mice by a gene gun twice 1 week and 3 weeks after the initial immunization with rhIL13Ra2 protein. did.
  • the introduction of DNA vaccine by gene gun was performed as follows. (I) Introduction by gene gun (i) -1. Preparation of cartridge 100 ⁇ l of spermidine (0.05 M) was added to 30 mg of gold particles (1 micron), and sufficiently suspended by vortex and ultrasonic treatment.
  • plasmid DNA mix (pTarget-IL13Ra2IL-13 (100 ⁇ g) as target substance, pCAcchBAX ⁇ (50 ⁇ g) and pCAcchCD40Lm3 (50 ⁇ g) as adjuvants, vortex for 5 seconds, and then gently vortex the gold particle-DNA suspension. While adding 100 ⁇ l of CaCl 2 (1M). After incubating at room temperature for 10 minutes, it was washed with ethanol three times, and the gold particle-DNA ethanol suspension was sealed in a gold coat tube (Bio-Rad). After drying with nitrogen gas, it was cut into about 0.5 inches and stored at room temperature. (I) -2. Immunization After shaving the abdomen of BALB / c mice, the above cartridge was mounted on HELIOS TM GENE GUN, and gene introduction into the abdominal dermis was performed with helium gas (300 psi).
  • Each mouse serum sample was serially diluted to x100, x300, x1000, x3000, x10000, and x30000, seeded in duplicate at 100 ⁇ l / well, and incubated at room temperature for 1 hour.
  • Washed 5 times with 200 ⁇ l / well wash buffer 6)
  • a 2000-fold diluted secondary antibody (sheep anti-mouse IgG-HRP, GE Healthcare) was added at 100 ⁇ l / well and incubated at room temperature for 1 hour.
  • 1 ⁇ TMB Substrate Solution (KPL) was added at 100 ⁇ l / well and incubated at room temperature for 10 minutes.
  • COS1-Target-IL13Ra2 cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 cells / well and cultured at 37 ° C. for 24 hours. 2) The cells were washed twice with 200 ⁇ l / well of PBS ( ⁇ ). 3) The cells were fixed with 100 ⁇ l / well of 10% neutral buffered formalin at room temperature for 20 minutes. 4) Washed twice with 200 ⁇ l / well PBS. 5) Blocking was performed with 200 ⁇ l / well of 1% BSA for 1 hour at room temperature. 6) 50 ⁇ l / well of hybridoma supernatant was reacted at room temperature for 1 hour.
  • the solution was collected, electrophoresed at 20 mA using Ready Gels J 5-20% (Bio-Rad), and then blotted from the gel to a PVDF membrane at 0.1 mA / cm 2 .
  • KH6g11 and KH7B9 (10 ⁇ g / ml) were reacted at room temperature for 1 hour, and then secondary antibody and sheep anti-mouse IgG-HRP (GE Healthcare) were diluted 2000 times. For 1 hour at room temperature.
  • the target band was detected using ECL western blotting detection reagents (GE Healthcare) and Hyperfilm ECL GE Healthcare). The results are shown in FIG.
  • lane 1 is a cell extract sample not expressing IL13Ra2 (negative control), and 2 lanes is a cell extract sample expressing IL13Ra2 by gene transfer.
  • a clear band was observed at the 50 kDa position corresponding to IL13Ra2 only in 2 lanes.
  • KH6g11 antibody and KH7B9 antibody showed clear bands at 50 kDa. Therefore, it was confirmed that the KH6g11 antibody and the KH7B9 antibody are antibodies that specifically recognize IL13Ra2.
  • a KH6g11 or KH7B9 antibody (both 100 ⁇ l of 10 ⁇ g / ml) was used as the primary antibody and reacted at 4 ° C. for 16 hours. After completion of immunostaining, counterstaining with hematoxylin was performed, followed by dehydration, penetration, and encapsulation. As is clear from the stained images shown in FIG. 15, the membrane surface of the A375 cell cluster that highly expresses IL13Ra2 present on the right side of each image is localized and strongly stained, but the mouse skin tissue, stromal cells Inflammatory cells are not stained. From this, it was confirmed that the KH6g11 and KH7B9 antibodies were antibodies capable of fixing formalin and immunostaining paraffin-embedded samples. Since there are no known anti-IL13Ra2 antibodies having such characteristics, the effect of the antibody of the present invention is surprising.
  • KH7B9 antibody (10 ⁇ g / ml) was used as the primary antibody and reacted at 4 ° C. for 16 hours. After completion of immunostaining, counterstaining with hematoxylin was performed, followed by dehydration, penetration, and encapsulation by a conventional method. From the stained image shown in FIG. 16, in a), the melanoma cells are specifically strongly stained in DAB brown, but the normal lymph node cells are not stained, and also in b), the melanoma cells are specifically stained. Although it is strongly stained, it can be seen that normal human cells including inflammatory cells, stromal cells, vascular endothelial cells, vascular smooth muscle cells, alveoli and bronchiole cells are not stained.
  • IL13Ra2 was expressed in more than 50% of both primary and metastatic tissues, and normal tissues such as lymphocytes and lung tissues were not stained in the metastatic lesions, and only melanoma was stained. Therefore, the above results indicate that IL13Ra2 can be applied to the diagnosis and treatment of melanoma as a target molecule of melanoma.
  • testis showed strong staining on the cell membrane as reported.
  • the cytoplasm was weakly stained.
  • IL13Ra2 Localization Site in Liver Tissue In order to confirm that the positive result of immunostaining in liver tissue is not a nonspecific reaction but is due to the presence of IL13Ra2, cell lysate of normal hepatocytes Were immunoblotted with KH7B9 antibody. In addition, to know whether IL13Ra2 expressed in the liver is localized in the cell or on the cell membrane, NS-66 is reacted with intact hepatocytes and permeabilized hepatocytes, Flow cytometry analysis was performed. The immunoblot was performed as follows.
  • the flow cytometry analysis was performed as follows. 1) Normal hepatocytes (normal human hepatocytes (hNHeps), Sanko Junyaku Co., Ltd.) were washed twice with 1 ml of 2% FBS / PBS buffer. 2) Cell fixation and cell membrane permeabilization were performed using BD Cytofix / Cytoperm TM Kit (Becton, Dickinson). 3) NS-66 antibody and control antibody (Mouse IgG1 Isotype control, eBioscience, catalog number 16-4714) were diluted 100-fold with 2% FBS / PBS buffer. 4) The cells were resuspended with 100 ⁇ l of diluted antibody solution and reacted at 4 ° C. for 30 minutes.
  • FIG. 25 The result of immunoblotting is shown in FIG. 25, and the result of flow cytometry analysis is shown in FIG. Although the immunoblotting was thin, a band was observed at the corresponding molecular weight of IL13Ra2 (FIG. 25). Intact hepatocytes showed no reaction, but permeabilized hepatocytes showed a reaction (FIG. 26). From these results, it was revealed that IL13Ra2 is expressed in hepatocytes but localized in the cells. Therefore, the above results indicate that in the normal tissues examined this time, IL13Ra2 is not strongly expressed in the membrane except for the testis, and even if slightly expressed, it is localized in the cytoplasm .
  • Example 7 Anti-tumor effect of NS-66 antibody in melanoma subcutaneous transplantation model
  • Melanoma cells expressing IL13Ra2 (A375) are transplanted subcutaneously into immunodeficient mice KSN / Slc, and NS-66 antibody and immunotoxin (saporin-binding IgG) against in vivo proliferation
  • the therapeutic effect of was examined using the increase in tumor volume as an index.
  • the experimental procedure is as follows. 1) A375 cells in the logarithmic growth phase were collected from a culture dish using a trypsin-EDTA solution. 2) The collected solution was centrifuged to discard the supernatant, and the cells were suspended in PBS to prepare 1 ⁇ 10 7 cells / ml.
  • mice were subcutaneously inoculated with a syringe equipped with a 26G needle so that the cell preparation was 100 ⁇ l / mouse (1 ⁇ 10 6 cells / mouse).
  • the mice used were immunodeficient mice KSN Slc purchased from Charles River Japan, which were raised for two weeks in a sterile animal room at the Sapporo Medical University Animal Experiment Facility, and were administered to a total of 18 mice.
  • mice were randomly classified into 3 groups (6 mice / group).
  • the tumor diameter was measured with an electronic caliper (manufactured by MITSUTOYO) at a frequency of twice a week from the day of tumor inoculation, and the tumor volume was calculated by the following formula.
  • Tumor volume (mm 3 ) 0.4 ⁇ tumor major axis ⁇ tumor minor axis ⁇ tumor minor axis.
  • NS-66 antibody according to the present invention is very effective for targeted treatment of cancer such as melanoma using a drug such as immunotoxin.

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Abstract

L'invention concerne une composition de diagnostic et de traitement d'un cancer du type mélanome, ostéosarcome, leucémie, lymphome, cancer de la prostate et cancer du poumon. La composition inclut une substance pouvant se lier à IL13Ra2. Un nouvel anticorps monoclonal pouvant se lier à IL13Ra2 avec une affinité élevée est également décrit. Une composition de diagnostic et de traitement du cancer qui comprend l'anticorps est aussi décrite, qui permet de réaliser le diagnostic très spécifique ou le traitement ciblé de divers types de cancer.
PCT/JP2008/060009 2007-06-01 2008-05-30 Anticorps dirigé contre il13ra2 et agent de diagnostic/thérapie comprenant l'anticorps WO2008146911A1 (fr)

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