WO2012015008A1 - Anti-tumor agent - Google Patents

Anti-tumor agent Download PDF

Info

Publication number
WO2012015008A1
WO2012015008A1 PCT/JP2011/067360 JP2011067360W WO2012015008A1 WO 2012015008 A1 WO2012015008 A1 WO 2012015008A1 JP 2011067360 W JP2011067360 W JP 2011067360W WO 2012015008 A1 WO2012015008 A1 WO 2012015008A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
tniiia2
cells
amino acid
seq
Prior art date
Application number
PCT/JP2011/067360
Other languages
French (fr)
Japanese (ja)
Inventor
文雄 深井
吉田　利通
Original Assignee
学校法人東京理科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人東京理科大学 filed Critical 学校法人東京理科大学
Publication of WO2012015008A1 publication Critical patent/WO2012015008A1/en

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to an antitumor agent.
  • Patent Document 1 discloses a cancer metastasis-suppressing composition containing a nucleic acid that identifies a novel cancer metastasis-related gene and inhibits or suppresses the function in vivo.
  • Patent Document 2 relates to an antibody that selectively recognizes phosphorylated ⁇ -catenin, which is involved in cancer cell migration and is supposed to suppress cancer invasion and cancer metastasis, and a therapeutic agent for cancer containing the antibody, Cancer diagnostic agents and the like are disclosed.
  • Tenascin-C is a compound that is hardly expressed in normal cells excluding immune tissues, but is induced during pathologies such as inflammation and tumor.
  • As a method for detecting tenascin-C several anti-tenascin-C antibodies are known (see, for example, Patent Documents 3 and 4).
  • a method for diagnosing arthritis using tenascin C as a marker is known (see, for example, Patent Document 5).
  • the polypeptide constituting tenascin-C includes an epidermal growth factor-like domain, a fibronectin (FN) III-like domain, and a fibrinogen-like domain.
  • FN fibronectin
  • fibrinogen-like domain in the fibronectin III-like domain, eight basic repeat sequences (1 to 8) and nine spliced repeat sequences inserted between the fifth and sixth positions (A1, A2, A3, A4, B, AD2, AD1, C, D) are known to be continuous.
  • spliced repetitive sequence sites are sites that are easily cleaved by matrix metalloprotease (MMP), and it is considered that peptides produced by cleaving tenascin-C by MMP have various functions.
  • MMP matrix metalloprotease
  • a peptide derived from the A2 domain in the FNIII-like domain of tenascin-C (hereinafter sometimes referred to as “TNIIIA2”) is known as a peptide that causes integrin activation (see, for example, Non-Patent Document 1). .
  • Non-Patent Document 2 JP 2006-304716 A JP 2005-89354 A JP 2004-217546 A JP 2002-234900 A Japanese Patent Application Laid-Open No. 2004-138489 J. et al. Biol. Chem. , Vol. 282, pp. 34929-34937, (2007) J. et al. Pathol. , Vol. 162, pp. 1857-1867, (2003)
  • the present invention provides an antitumor agent that can effectively suppress malignant transformation of a tumor.
  • the present invention is as follows.
  • An antitumor agent comprising at least one selected from the group consisting of an antibody recognizing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing an antigen recognition site of the antibody.
  • At least one amino acid sequence of SEQ ID NO: 1 in a biological sample selected from the group consisting of an antibody recognizing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing an antigen recognition site of the antibody
  • a detection method means known in this technical field that can be used for quantitative analysis, qualitative analysis, visual observation and the like can be used as appropriate.
  • the antibody can be recognized by the immunohistochemical method. Presence of the polypeptide in the biological sample can be visually observed.
  • an antitumor agent that effectively suppresses malignancy of a tumor.
  • the antitumor agent of the present invention comprises an anti-TNIIIA2 antibody that recognizes TNIIIA2, which is a peptide derived from the A2 sequence site of the fibronectin III-like repeat of human tenascin-C. According to the present invention, since the anti-TNIIIA2 antibody has an inhibitory ability against tumor cell proliferation and invasion, the antitumor agent of the present invention containing the anti-TNIIIA2 antibody can be used as a therapeutic or prophylactic agent for tumors. .
  • TNIIIA2-like fragment is released from tenascin C, and integrins on tumor cells are activated, Not only is tumor cell growth excessive, but tumor cells migrate (infiltrate) across the basement membrane.
  • the anti-TNIIIA2 antibody inhibits the action of TNIIIA2 having such a function, thereby effectively suppressing tumor formation and malignant transformation thereof, resulting in a therapeutic effect on tumors. Presumed to be.
  • the amount of each component in the composition when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. Means the total amount.
  • the anti-TNIIIA2 antibody in the present invention is not particularly limited as long as it can recognize a peptide (TNIIIA2) derived from the A2 sequence portion of the FNIII-like domain of human tenascin C, and may be a monoclonal antibody or a polyclonal antibody. Good. Moreover, as long as the antigen recognition site is preserved, it may be an immunoreactive fragment.
  • TNIIIA2 is a peptide consisting of 22 amino acids of RSTDLPGLKAATHYTITIRGVC (SEQ ID NO: 1) (see J. Biol. Chem., Vol. 282, pp. 34929-34937 (2007)).
  • the anti-TNIIIA2 antibody of the present invention is an antibody that recognizes TNIIIA2, and has at least a part of the amino acid sequence of TNIIIA2 represented by SEQ ID NO: 1 as an epitope.
  • the epitope recognized by anti-TNIIIA2 may be a partial peptide containing the amino acid sequence of YTITIRGV (SEQ ID NO: 2).
  • the peptide used for preparing the antibody recognizing TNIIIA2, that is, the peptide (target peptide) recognized by the anti-TNIIIA2 antibody according to the present invention may be a peptide including SEQ ID NO: 2, and the full length (SEQ ID NO: 1) It may be.
  • one or more compounds having a linker function or a linker function, such as an amino acid may be added to the peptide having the amino acid sequence of SEQ ID NO: 2.
  • an additional amino acid include an amino acid capable of imparting an amino acid for binding a carrier protein to the peptide having the amino acid sequence of SEQ ID NO: 2, such as cysteine or threonine, and others such as an acidic amino acid.
  • the target peptide is preferably an amino acid sequence of CATHITITIRGVT (SEQ ID NO: 3) from the viewpoint of antibody production efficiency.
  • the antibody recognizing the TNIIIA2 can be prepared by a conventional method using the target peptide described above.
  • a polyclonal antibody may be obtained as follows. A peptide containing any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3 or a mixture thereof is used as a target peptide, and serum is obtained by immunizing a small animal such as a rabbit with the target peptide. The antibody is purified from the obtained serum by using known antibody purification means such as ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the specific peptide, etc. Can be prepared.
  • the target peptide is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells, using mouse myeloma cells and a reagent such as polyethylene glycol.
  • a clone that produces an antibody that binds to the target peptide is selected from the fused cells (hybridomas) formed by the fusion.
  • the selected hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is used, for example, by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and the specific peptide in the cup.
  • the antibody can be prepared by purifying the antibody by using a ringed affinity column or the like.
  • the target peptide may be used as an immunity as a fusion protein with a known carrier protein in order to enhance antigenicity.
  • a carrier protein can be used without particular limitation as long as it is a known molecule used for this purpose, and examples thereof include KLH, GST, and BSA.
  • the type of tumor whose growth or invasion is inhibited by the anti-TNIIIA2 antibody is not particularly limited and may be sarcoma or cancer, such as leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer , Hepatocellular carcinoma, colon cancer, pancreatic cancer, head and neck cancer and prostate cancer, and sarcomas such as fibrosarcoma and osteosarcoma, or glioma. It is preferably applied to breast cancer, lung cancer, glioma and the like.
  • sarcoma or cancer such as leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer , Hepatocellular carcinoma, colon cancer, pancreatic cancer, head and neck cancer and prostate cancer, and sarcomas such as fibrosarcoma and osteosarcoma, or glioma. It is preferably applied to breast cancer, lung cancer, glio
  • the antitumor agent of the present invention can be administered orally or parenterally systemically or locally.
  • intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc.
  • the administration method should be selected appropriately depending on the age and symptoms of the patient Can do.
  • the dose can also be appropriately selected depending on the patient's age, symptoms and the like.
  • the antitumor agent of the present invention may contain both pharmaceutically acceptable carriers and additives depending on the administration route.
  • carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, Examples include sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.
  • the additive to be used is appropriately selected from the above depending on the dosage form, but is not limited thereto.
  • the antitumor agent of the present invention only needs to contain an anti-TNIIIA2 antibody, and may be a fused anti-TNIIIA2 antibody in which another substance is bound to the anti-TNIIIA2 antibody.
  • substances that can be fused to the anti-TNIIIA2 antibody include other antitumor agents such as doxorubicin and cisplatin. Thereby, not only the direct antitumor activity by the anti-TNIIIA2 antibody but also the antitumor effect by the fused antitumor agent can be expected.
  • any fusion or conjugation method may be applied as long as the activity of the anti-TNIIIA2 antibody is not impaired, for example, covalent bonding via a linker Or by applying fusion or binding methods known in the art, such as coordinate binding to metal ions, to sites that do not impair the activity of the anti-TNIIIA2 antibody, such as the antibody constant region (C region).
  • Other anti-tumor agents can be fused or conjugated.
  • the present invention also includes a method for treating a tumor, comprising administering an antitumor agent according to the present invention to a patient suspected of having a tumor or a patient who has already developed a tumor.
  • treatment may be any improvement in symptoms, and the term includes the suppression or reduction of lesion hypertrophy, the reduction of metastasis rate, or the cessation of metastasis to the extent that it can be confirmed.
  • the method of administration to the patient varies depending on the dosage form of the drug applied, the sex, age, symptoms, etc. of the patient or a combination thereof, but oral administration, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, Suppository, enema, oral enteric solvent can be mentioned, and these administration methods may be appropriately selected according to the patient's condition. In certain embodiments, it can be preferably intravenous injection.
  • the therapeutically effective dose of the antibody of the present invention varies depending on the degree of symptoms and the condition of the patient, and can be, for example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, but is not limited thereto.
  • the administration frequency can be, for example, in the range of twice a day to once a week, but is not limited thereto.
  • Example 1 ⁇ Preparation of anti-human TNIIIA2 antibody> A peptide having the amino acid sequence CATHITITIRGVT (SEQ ID NO: 3) was synthesized by a conventional method. The synthesized peptide was fused to KLH as a hapten, and the obtained fusion peptide was used as an antigen peptide. Immunization was performed according to the usual method using rabbits. The obtained mixture containing the target antibody was purified by a conventional method. As described above, three types (No. 1 to No. 3) of polyclonal antibodies against human TNIIIA2 (anti-human TNIIIA2 antibody) were obtained.
  • the antibody activity of the anti-human TNIIIA2 polyclonal antibody obtained above was evaluated as follows using the inhibitory action of cell adhesion induced by human TNIIIA2 as an index. The results are shown in FIG.
  • RPMI1640 (trade name, Nissui Pharmaceutical Co., Ltd.) (20% FBS (JRH Biosciences) added) was used as the cell culture medium, and KOP2.16 cells (mouse bone marrow-derived vascular endothelial cell line) were placed 5 ⁇ in each well of a 96-well plate. The cells were seeded at a density of 10 4 cells / 200 ⁇ L / well and cultured at 37 ° C. and 5% CO 2 for 4 hours. 100 ⁇ L of the culture supernatant was removed, and 100 ⁇ L of 20% TCA aqueous solution was added, and then allowed to stand at 4 ° C. overnight or at room temperature for 1 hour to be solid-phased. This was washed sequentially with PBS ⁇ 5, RPMI 1640 (20% FBS added) ⁇ 1, PBS ⁇ 1 to prepare a 96-well plate in which each well was coated with KOP2.16 cells.
  • RPMI 1640 (20% FBS added) containing 12.5 ⁇ g / mL human TNIIIA2 was added to each well of the 96-well plate coated with KOP2.16 cells obtained above.
  • the anti-human TNIIIA2 antibody (No. 1 to 3) obtained above was added so that the concentrations were 1.5 ⁇ g / mL, 3 ⁇ g / mL, and 6 ⁇ g / mL, respectively, and K562 cells (leukemia cell line) were added as follows. Each of the cells was seeded at a density of 5 ⁇ 10 4 cells / well and cultured at 37 ° C. and 5% CO 2 for 1 hour.
  • Formalin was added and allowed to stand for 1 hour at room temperature and fixed, then washed 3 times with PBS, and stained with Lilymeyer's hematoxylin. Observed with an optical microscope, the number of adherent cells in a predetermined field of view was counted.
  • FIG. 1 shows that normal rabbit IgG does not inhibit cell adhesion induced by human TNIIIA2, whereas the anti-human TNIIIA2 antibody of the present invention inhibits cell adhesion in a dose-dependent manner.
  • any of the anti-human TNIIIA2 antibodies 1 to 3 show equivalent antibody activity.
  • the anti-human TNIIIA2 antibody of the present invention did not exhibit an inhibitory effect on cell adhesion induced by magnesium ions. From the above, it can be seen that the anti-human TNIIIA2 antibody of the present invention can specifically bind to human TNIIIA2 and suppress its function.
  • Example 2 ⁇ Evaluation of infiltration inhibitory activity> Using breast cancer cells, the inhibitory activity of anti-human TNIIIA2 antibody on the subbasement infiltration of tumor cells was evaluated as follows. As a basement membrane model, a membrane filter coated with EHS Matrigel was used. For comparison, 4F10TT, an anti-human tenascin-C antibody available from the Institute for Immunobiology, was used. 4F10TT is known as an antibody that can detect all human TN-C variants by using an EGF-like domain in the tenascin-C (TN-C) molecule as an epitope.
  • TN-C tenascin-C
  • Human tenascin-C (TN-C, or TNC) is a human melanoma cell line SK-MEL-28 cell (purchased from Riken Cell Bank), and after reaching confluence, the medium is replaced with serum-free DMEM. The culture supernatant collected after one week was purified according to a previously reported method (J. Tissue Culture Methods., Vol. 16, pp. 201-204 (1994)). It has been confirmed that the mouse breast cancer cell line MMT cells produce the proteolytic enzyme MMP (no data).
  • EHS sarcoma crude extract (6.0 mg / mL; Iwaki Glass Co., Ltd.) was diluted 5-fold with 0.15 M NaCl and 0.02 M HEPES to fibronectin (FN: J. Biol. Chem.,). Vol. 226, pp. 8807-8813 (1991)) was added at 10 ⁇ g / mL, the solution was dropped on a glass plate, and a porous (5.0 ⁇ m) membrane filter was removed from the solution. The filter was coated with a mixture of crude EHS sarcoma extract and fibronectin (hereinafter “Matrigel”) by leaving it overnight at 4 ° C. under humid conditions.
  • Mesatrigel a mixture of crude EHS sarcoma extract and fibronectin
  • This filter was set in the transwell chamber with the matrigel-coated surface down.
  • MMT cells mouse breast tumor cell line
  • RPMI 1640 0.5% dialyzed fetal bovine serum (dFBS) added
  • dFBS dialyzed fetal bovine serum
  • Example 3 ⁇ Evaluation of growth inhibitory activity> (1) Breast cancer cell proliferation test Using breast cancer cells, the anti-human TNIIIA2 antibody inhibitory activity against tumor cell proliferation was evaluated as follows. MMT cells adjusted to 1.0 ⁇ 10 4 cells / well with RPMI 1640 (0.5% dFBS) in a 96-well plate were mixed with TN-C (20 ⁇ g / ml), anti-TNIIIA2 antibody (20 ⁇ g / ml), Seed with 4F10TT antibody (20 ⁇ g / ml) or MMP-2 / -9 inhibitor (25 ⁇ M). After culturing at 37 ° C.
  • malignant normal cells by TNIIIA2 and TN-C are added with substances that block the activity of TNIIIA2 and TN-C, for example, the anti-TNIIIA2 antibody that induced infiltration inhibition of TNIIIA2 and TN-C in Example 2 Can be effectively prevented.
  • Example 4 ⁇ Expression of TNIIIA2 in breast cancer tissue>
  • the localization of TN-C and TNIIIA2 in the tumor tissue was confirmed as follows.
  • human breast cancer tissue specimens paraffin sections
  • tissues excised by diagnosis of breast cancer at Mie University Hospital were used with the consent of the patient with the approval of the Ethics Committee.
  • This section was deparaffinized according to a conventional method and then immunostained by the PAP (peroxidase-antiperoxidase complex) method using an anti-TN-C monoclonal antibody (4F10TT antibody) or an anti-TNIIIA2 polyclonal antibody, and TN-C and TNIIIA2 Localization was assessed.
  • the results are shown in FIGS. 5A and 5B (200 times).
  • the black arrowhead indicates the stained part.
  • TNIIIA2 is usually three-dimensionally concealed in the TN-C molecule and has been confirmed to be exposed by the action of proteolytic enzymes such as MMP-2. From these, it was shown that the exposure of TNIIIA2 was increased in breast cancer tissue by staining with anti-TNIIIA2 antibody in breast cancer tissue. This indicates that TNIIIA2 is exposed not only in vitro but also in tumor tissues, and has an effect on surrounding tumor cells.
  • TNIIIA2 has an effect of promoting cell proliferation accompanied by tumor cell invasion and stratification, and it was shown that such an activity of TNIIIA2 can be blocked by using an anti-TNIIIA2 antibody.
  • the anti-human TNIIA2 antibody of the present invention can inhibit tumor cell invasion and proliferation and effectively suppress tumor malignancy.
  • an antitumor agent capable of effectively suppressing tumor malignancy can be provided.

Abstract

Provided is an anti-tumor agent which can inhibit the malignant transformation of a tumor effectively. Specifically provided is an anti-tumor agent comprising at least one component selected from the group consisting of an antibody capable of recognizing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 and antibody fragments of the antibody each of which contains an antigen recognition site.

Description

抗腫瘍剤Antitumor agent
　本発明は、抗腫瘍剤に関する。 The present invention relates to an antitumor agent.
　腫瘍又は癌の治療法としては、化学療法、放射線治療法及び外科療法などが知られており、例えば癌の発症メカニズム等に基づいて種々の治療方法が開発されている。癌の転移や再発などの問題を解決し得る、より効果的な治療方法を目指して、未だに開発が続けられている。
　特許文献１は、新規な癌転移関連遺伝子を同定し、このインビボにおける機能を阻害若しくは抑制する核酸を含む癌の転移抑制用組成物を開示している。
　特許文献２は、癌細胞の遊走に関与し、癌の浸潤及び癌の転移を抑制することが推測されているリン酸化βカテニンを選択的に認識する抗体と、これを含む癌の治療剤や癌の診断剤等とを開示している。 Known treatment methods for tumors or cancers include chemotherapy, radiotherapy, and surgical treatment. For example, various treatment methods have been developed based on the onset mechanism of cancer. Development is still ongoing with the aim of more effective treatment methods that can solve problems such as cancer metastasis and recurrence.
Patent Document 1 discloses a cancer metastasis-suppressing composition containing a nucleic acid that identifies a novel cancer metastasis-related gene and inhibits or suppresses the function in vivo.
Patent Document 2 relates to an antibody that selectively recognizes phosphorylated β-catenin, which is involved in cancer cell migration and is supposed to suppress cancer invasion and cancer metastasis, and a therapeutic agent for cancer containing the antibody, Cancer diagnostic agents and the like are disclosed.
　テネイシンＣは、免疫組織を除いた正常細胞においてはほとんど発現が認められないが、炎症、腫瘍等の病態時に発現が誘導される化合物である。
　テネイシンＣを検出する方法として、いくつかの抗テネイシンＣ抗体が知られている（例えば、特許文献３、４参照）。またテネイシンＣをマーカーとして、関節炎の診断を行なう方法が知られている（例えば、特許文献５参照）。 Tenascin-C is a compound that is hardly expressed in normal cells excluding immune tissues, but is induced during pathologies such as inflammation and tumor.
As a method for detecting tenascin-C, several anti-tenascin-C antibodies are known (see, for example, Patent Documents 3 and 4). A method for diagnosing arthritis using tenascin C as a marker is known (see, for example, Patent Document 5).
　テネイシンＣを構成するポリペプチドには、上皮増殖因子様ドメイン、フィブロネクチン（ＦＮ）ＩＩＩ様ドメイン、およびフィブリノーゲン様ドメインが含まれている。このうちフィブロネクチンＩＩＩ様ドメインでは、基本的な８種の反復配列（１～８）と、その５番目と６番目の間に挿入される９種のスプライシングされる反復配列（Ａ１、Ａ２、Ａ３、Ａ４、Ｂ、ＡＤ２、ＡＤ１、Ｃ、Ｄ）とが連続していることが知られている。 The polypeptide constituting tenascin-C includes an epidermal growth factor-like domain, a fibronectin (FN) III-like domain, and a fibrinogen-like domain. Among them, in the fibronectin III-like domain, eight basic repeat sequences (1 to 8) and nine spliced repeat sequences inserted between the fifth and sixth positions (A1, A2, A3, A4, B, AD2, AD1, C, D) are known to be continuous.
　これらのスプライシングされる反復配列部位は、マトリックスメタロプロテアーゼ（ＭＭＰ）で切断されやすい部位であり、テネイシンＣがＭＭＰにより切断されて生成するペプチドは種々の機能を有していると考えられている。
　例えば、このテネイシンＣのＦＮＩＩＩ様ドメインにおけるＡ２ドメインに由来するペプチド（以下、「ＴＮＩＩＩＡ２」ということがある）は、インテグリンの活性化を引き起こすペプチドとして知られている（例えば、非特許文献１参照）。
　また、テネイシンＣのＦＮＩＩＩの反復配列のうちＢドメインに由来するペプチドには、乳癌細胞株に対して遊走促進作用、及びＤＮＡ合成活性向上作用があることが知られている（非特許文献２参照）。
特開２００６－３０４７１６号公報 特開２００５－８９３５４号公報 特開２００４－２１７５４６号公報 特開２００２－２３４９００号公報 特開２００４－１３８４８９号公報 Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，　Ｖｏｌ．２８２，　ｐｐ．３４９２９－３４９３７，　（２００７） Ｊ．Ｐａｔｈｏｌ．，Ｖｏｌ．１６２，　ｐｐ．１８５７－１８６７，（２００３） These spliced repetitive sequence sites are sites that are easily cleaved by matrix metalloprotease (MMP), and it is considered that peptides produced by cleaving tenascin-C by MMP have various functions.
For example, a peptide derived from the A2 domain in the FNIII-like domain of tenascin-C (hereinafter sometimes referred to as “TNIIIA2”) is known as a peptide that causes integrin activation (see, for example, Non-Patent Document 1). .
In addition, it is known that a peptide derived from the B domain among the FNIII repetitive sequences of tenascin-C has a migration promoting effect and a DNA synthesis activity improving effect on breast cancer cell lines (see Non-Patent Document 2). ).
JP 2006-304716 A JP 2005-89354 A JP 2004-217546 A JP 2002-234900 A Japanese Patent Application Laid-Open No. 2004-138489 J. et al. Biol. Chem. , Vol. 282, pp. 34929-34937, (2007) J. et al. Pathol. , Vol. 162, pp. 1857-1867, (2003)
　腫瘍又は癌の治療には、周囲の正常細胞に対する影響を抑えつつ腫瘍細胞の活性のみを選択的に阻害することが求められる。このため、腫瘍細胞の悪性化に特徴的とも言える増殖態様や転移に対して効果的な阻害活性を示す抗腫瘍剤が必要となる。
　本発明は、腫瘍の悪性化を効果的に抑制し得る抗腫瘍剤を提供する。 In the treatment of a tumor or cancer, it is required to selectively inhibit only the activity of tumor cells while suppressing the influence on surrounding normal cells. For this reason, the antitumor agent which shows the effective inhibitory activity with respect to the growth mode and metastasis | transition which can be said to be malignant of a tumor cell is needed.
The present invention provides an antitumor agent that can effectively suppress malignant transformation of a tumor.
　本発明は以下のとおりである。
　［１］　配列番号１のアミノ酸配列からなるポリペプチドを認識する抗体および前記抗体の抗原認識部位を含む抗体断片からなる群より選択される少なくとも一つを含有する抗腫瘍剤。
　［２］　前記抗体が、配列番号２のアミノ酸配列を含むポリペプチドを認識する抗体である［１］に記載の抗腫瘍剤。
　［３］　前記抗体が、配列番号３のアミノ酸配列からなるポリペプチドを認識する抗体である［１］又は［２］に記載の抗腫瘍剤。
　［４］乳癌、肺癌、または神経膠腫に対して適用される［１］～［３］のいずれか１項に記載の抗腫瘍剤。
　［５］配列番号１のアミノ酸配列からなるポリペプチドを認識する抗体および前記抗体の抗原認識部位を含む抗体断片からなる群より選択される少なくとも一つの、生体試料中における配列番号１のアミノ酸配列の少なくとも一部を含むポリペプチドの検出における使用。ここで前記検出方法としては、定量的分析、定性的分析、視覚的観察等に用い得る本技術分野で公知の手段を適宜利用可能であり、例えば免疫組織化学的手法によって、前記抗体で認識されるポリペプチドの生体試料中における存在を視覚的に観察することが可能である。
　［６］前記抗体が、配列番号２のアミノ酸配列を含むポリペプチドを認識する抗体である［５］に記載の使用。
　［７］前記抗体が、配列番号３のアミノ酸配列からなるポリペプチドを認識する抗体である［５］に記載の使用。
　［８］前記生体試料が、腫瘍細胞を含む生体組織から得られた、［５］～［７］のいずれか１項に記載の使用。
　［９］前記生体試料が、腫瘍細胞を含む可能性を有する生体組織から得られた、［５］～［７］のいずれか１項に記載の使用。ここで前記ポリペプチドが検出されることは、前記試料中における腫瘍細胞の存在を意味し得る。
　［１０］前記腫瘍細胞が、乳癌細胞、肺癌細胞、または神経膠腫細胞である［８］または［９］に記載の使用。
　［１１］前記検出が、前記生体組織中に前記抗体を導入すること、及び、前記生体組織中における前記抗体の存在を視覚的に観察することを含む、［５］～［１０］のいずれか１項に記載の使用。
　［１２］前記観察が、免疫組織化学的手法による、［１１］に記載の使用。 The present invention is as follows.
[1] An antitumor agent comprising at least one selected from the group consisting of an antibody recognizing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing an antigen recognition site of the antibody.
[2] The antitumor agent according to [1], wherein the antibody recognizes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
[3] The antitumor agent according to [1] or [2], wherein the antibody recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
[4] The antitumor agent according to any one of [1] to [3], which is applied to breast cancer, lung cancer, or glioma.
[5] At least one amino acid sequence of SEQ ID NO: 1 in a biological sample selected from the group consisting of an antibody recognizing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing an antigen recognition site of the antibody Use in the detection of a polypeptide comprising at least a part. Here, as the detection method, means known in this technical field that can be used for quantitative analysis, qualitative analysis, visual observation and the like can be used as appropriate. For example, the antibody can be recognized by the immunohistochemical method. Presence of the polypeptide in the biological sample can be visually observed.
[6] The use according to [5], wherein the antibody recognizes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
[7] The use according to [5], wherein the antibody recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
[8] The use according to any one of [5] to [7], wherein the biological sample is obtained from a biological tissue containing tumor cells.
[9] The use according to any one of [5] to [7], wherein the biological sample is obtained from a biological tissue having a possibility of containing tumor cells. Here, the detection of the polypeptide may mean the presence of tumor cells in the sample.
[10] The use according to [8] or [9], wherein the tumor cells are breast cancer cells, lung cancer cells, or glioma cells.
[11] Any of [5] to [10], wherein the detection includes introducing the antibody into the living tissue and visually observing the presence of the antibody in the living tissue. Use according to 1.
[12] The use according to [11], wherein the observation is performed by an immunohistochemical method.
　本発明によれば、腫瘍の悪性化を効果的に抑制する抗腫瘍剤を提供し得る。 According to the present invention, it is possible to provide an antitumor agent that effectively suppresses malignancy of a tumor.
本発明の実施例１にかかるポリペプチドＴＮＩＩＩＡ２誘導性の細胞接着に対する抗ヒトＴＮＩＩＩＡ２抗体の抑制効果を示す図である。It is a figure which shows the inhibitory effect of the anti-human TNIIIA2 antibody with respect to polypeptide TNIIIA2 induced cell adhesion concerning Example 1 of this invention. 本発明の実施例２にかかる基底膜下浸潤実験の結果を示すグラフである。It is a graph which shows the result of the subbasement infiltration experiment concerning Example 2 of this invention. 本発明の実施例３にかかる細胞増殖実験（ＷＳＴアッセイ）の結果を示すグラフである。It is a graph which shows the result of the cell proliferation experiment (WST assay) concerning Example 3 of this invention. 本発明の実施例３にかかるフォーカス形成実験（対照群）の結果を示す染色像である。It is a dyeing | staining image which shows the result of the focus formation experiment (control group) concerning Example 3 of this invention. 本発明の実施例３にかかるフォーカス形成実験（ＴＮＩＩＩＡ２添加群）の結果を示す染色像である。It is a dyeing | staining image which shows the result of the focus formation experiment (TNIIIA2 addition group) concerning Example 3 of this invention. 本発明の実施例３にかかるフォーカス形成実験（ＴＮ－Ｃ添加群）の結果を示す染色像である。It is a dyeing | staining image which shows the result of the focus formation experiment (TN-C addition group) concerning Example 3 of this invention. 本発明の実施例４にかかる乳癌組織の免疫染色像（抗ＴＮ－Ｃ抗体）の結果を示す染色像である。It is a dyed image which shows the result of the immuno-staining image (anti-TN-C antibody) of the breast cancer tissue concerning Example 4 of this invention. 本発明の実施例４にかかる乳癌組織の免疫染色像（抗ＴＮＩＩＩＡ２抗体）の結果を示す染色像である。It is a dyeing | staining image which shows the result of the immuno-staining image (anti-TNIIIA2 antibody) of the breast cancer tissue concerning Example 4 of this invention.
　本発明の抗腫瘍剤は、ヒトテネイシンＣのフィブロネクチンＩＩＩ様反復配列のＡ２配列部位に由来するペプチドであるＴＮＩＩＩＡ２を認識する抗ＴＮＩＩＩＡ２抗体を含むものである。
　本発明によれば、上記抗ＴＮＩＩＩＡ２抗体が、腫瘍細胞の増殖及び浸潤に対する阻害能を有するので、抗ＴＮＩＩＩＡ２抗体を含む本発明の抗腫瘍剤は、腫瘍の治療又は予防剤として使用することができる。
　即ち、腫瘍の転移などに大きく関与することが示唆されているＭＭＰ（マトリックス・メタロ・プロテアーゼ）が存在すると、テネイシンＣからＴＮＩＩＩＡ２様断片が遊離し、腫瘍細胞上のインテグリンが活性化することにより、腫瘍細胞の増殖が過剰となるばかりか、腫瘍細胞が基底膜を超えて移動（浸潤）する。特定の理論には拘束されないが、このような機能を有するＴＮＩＩＩＡ２の作用を抗ＴＮＩＩＩＡ２抗体が阻害することによって、腫瘍形成やその悪性化が効果的に抑制され、その結果、腫瘍の治療効果を奏するものと推測される。 The antitumor agent of the present invention comprises an anti-TNIIIA2 antibody that recognizes TNIIIA2, which is a peptide derived from the A2 sequence site of the fibronectin III-like repeat of human tenascin-C.
According to the present invention, since the anti-TNIIIA2 antibody has an inhibitory ability against tumor cell proliferation and invasion, the antitumor agent of the present invention containing the anti-TNIIIA2 antibody can be used as a therapeutic or prophylactic agent for tumors. .
That is, in the presence of MMP (matrix metalloprotease), which is suggested to be greatly involved in tumor metastasis and the like, TNIIIA2-like fragment is released from tenascin C, and integrins on tumor cells are activated, Not only is tumor cell growth excessive, but tumor cells migrate (infiltrate) across the basement membrane. Although not bound by a specific theory, the anti-TNIIIA2 antibody inhibits the action of TNIIIA2 having such a function, thereby effectively suppressing tumor formation and malignant transformation thereof, resulting in a therapeutic effect on tumors. Presumed to be.
　本発明において、組成物中の各成分の量について言及する場合、組成物中に各成分に該当する物質が複数存在する場合には、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。 In the present invention, when referring to the amount of each component in the composition, when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. Means the total amount.
　本発明における抗ＴＮＩＩＩＡ２抗体は、ヒトテネイシンＣのＦＮＩＩＩ様ドメインのＡ２配列部分に由来するペプチド（ＴＮＩＩＩＡ２）を認識できるものであれば特に制限はなく、モノクローナル抗体であってもポリクローナル抗体であってもよい。
　また、抗原認識部位が保存されていれば免疫反応性の断片であってもよい。 The anti-TNIIIA2 antibody in the present invention is not particularly limited as long as it can recognize a peptide (TNIIIA2) derived from the A2 sequence portion of the FNIII-like domain of human tenascin C, and may be a monoclonal antibody or a polyclonal antibody. Good.
Moreover, as long as the antigen recognition site is preserved, it may be an immunoreactive fragment.
　ＴＮＩＩＩＡ２は、ＲＳＴＤＬＰＧＬＫＡＡＴＨＹＴＩＴＩＲＧＶＣ（配列番号１）の２２個のアミノ酸からなるペプチドである（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，　Ｖｏｌ．２８２，　ｐｐ．３４９２９－３４９３７（２００７）参照）。本発明の抗ＴＮＩＩＩＡ２抗体は、ＴＮＩＩＩＡ２を認識する抗体であって、配列番号１で示されるＴＮＩＩＩＡ２のアミノ酸配列のうち少なくとも一部をエピトープとするものである。抗ＴＮＩＩＩＡ２に認識されるエピトープは、ＹＴＩＴＩＲＧＶ（配列番号２）のアミノ酸配列を含む部分ペプチドであればよい。 TNIIIA2 is a peptide consisting of 22 amino acids of RSTDLPGLKAATHYTITIRGVC (SEQ ID NO: 1) (see J. Biol. Chem., Vol. 282, pp. 34929-34937 (2007)). The anti-TNIIIA2 antibody of the present invention is an antibody that recognizes TNIIIA2, and has at least a part of the amino acid sequence of TNIIIA2 represented by SEQ ID NO: 1 as an epitope. The epitope recognized by anti-TNIIIA2 may be a partial peptide containing the amino acid sequence of YTITIRGV (SEQ ID NO: 2).
　前記ＴＮＩＩＩＡ２を認識する抗体を作製するために用いられるペプチド、即ち本発明にかかる抗ＴＮＩＩＩＡ２抗体が認識するペプチド（標的ペプチド）は、配列番号２を含むペプチドであればよく、全長（配列番号１）であってもよい。抗原性や安定性を高める観点から、配列番号２のアミノ酸配列を有するペプチドに、リンカー機能又はリンカー機能を有する１つ以上の化合物、たとえばアミノ酸を追加してもよい。このような付加的なアミノ酸としては、キャリアタンパク質を結合するためのアミノ酸を配列番号２のアミノ酸配列を有するペプチドに付与可能とするアミノ酸を挙げることができ、例えばシステイン又はスレオニン、その他、例えば酸性アミノ酸または塩基性アミノ酸等を挙げることができ、これらを１種又は２種以上を組み合わせて使用することができる。標的ペプチドは、中でも、ＣＡＴＨＹＴＩＴＩＲＧＶＴ（配列番号３）のアミノ酸配列であることが、抗体作製効率の観点から好ましい。 The peptide used for preparing the antibody recognizing TNIIIA2, that is, the peptide (target peptide) recognized by the anti-TNIIIA2 antibody according to the present invention may be a peptide including SEQ ID NO: 2, and the full length (SEQ ID NO: 1) It may be. From the viewpoint of enhancing antigenicity and stability, one or more compounds having a linker function or a linker function, such as an amino acid, may be added to the peptide having the amino acid sequence of SEQ ID NO: 2. Examples of such an additional amino acid include an amino acid capable of imparting an amino acid for binding a carrier protein to the peptide having the amino acid sequence of SEQ ID NO: 2, such as cysteine or threonine, and others such as an acidic amino acid. Or a basic amino acid etc. can be mentioned, These can be used 1 type or in combination of 2 or more types. In particular, the target peptide is preferably an amino acid sequence of CATHITITIRGVT (SEQ ID NO: 3) from the viewpoint of antibody production efficiency.
　前記ＴＮＩＩＩＡ２を認識する抗体は、上述した標的ペプチドを用いて、通常行われる方法によって調製することができる。
　例えば、ポリクローナル抗体であれば、次のようにして得てもよい。前記配列番号１～配列番号３のアミノ酸配列のいずれかを含むペプチド又はこれらの混合物を標的ペプチドとして使用し、この標的ペプチドをウサギ等の小動物に免疫して血清を得る。得られた血清から、公知の抗体精製手段、例えば、硫安沈殿、プロテインＡ、プロテインＧカラム、ＤＥＡＥイオン交換クロマトグラフィー、前記特定ペプチドをカップリングしたアフィニティカラム等を用いることによって本抗体を精製することで調製できる。 The antibody recognizing the TNIIIA2 can be prepared by a conventional method using the target peptide described above.
For example, a polyclonal antibody may be obtained as follows. A peptide containing any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3 or a mixture thereof is used as a target peptide, and serum is obtained by immunizing a small animal such as a rabbit with the target peptide. The antibody is purified from the obtained serum by using known antibody purification means such as ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the specific peptide, etc. Can be prepared.
　また、例えばモノクローナル抗体であれば、前記標的ペプチドを、マウスなどの小動物に免疫を行い、同マウスより脾臓を摘出し、これをすりつぶして細胞を分離し、マウスミエローマ細胞とポリエチレングリコールなどの試薬により融合させ、これにより形成された融合細胞（ハイブリドーマ）の中から、前記標的ペプチドと結合する抗体を産生するクローンを選択する。次いで選択したハイブリドーマをマウス腹腔内に移植し、同マウスより腹水を回収し、得られたモノクローナル抗体を、例えば、硫安沈殿、プロテインＡ、プロテインＧカラム、ＤＥＡＥイオン交換クロマトグラフィー、前記特定ペプチドをカップリングしたアフィニティカラム等を用いることによって本抗体を精製することで調製できる。 In addition, for example, in the case of a monoclonal antibody, the target peptide is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells, using mouse myeloma cells and a reagent such as polyethylene glycol. A clone that produces an antibody that binds to the target peptide is selected from the fused cells (hybridomas) formed by the fusion. Next, the selected hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is used, for example, by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and the specific peptide in the cup. The antibody can be prepared by purifying the antibody by using a ringed affinity column or the like.
　なお標的ペプチドは、抗原性を高めるために公知のキャリアタンパク質との融合タンパク質として免役に用いてもよい。このようなキャリアタンパク質としては、この目的で使用される公知の分子であれば特に制限なく使用することができ、例えば、ＫＬＨ、ＧＳＴ、ＢＳＡなどを挙げることができる。 The target peptide may be used as an immunity as a fusion protein with a known carrier protein in order to enhance antigenicity. Such a carrier protein can be used without particular limitation as long as it is a known molecule used for this purpose, and examples thereof include KLH, GST, and BSA.
　本抗ＴＮＩＩＩＡ２抗体によって増殖又は浸潤が阻害される腫瘍の種類は、特に制限はなく、肉腫又は癌のいずれであってもよく、例えば、白血病、リンパ腫、乳癌、肺癌、胃癌、食道癌、卵巣癌、肝細胞癌、大腸癌、膵臓癌、頭頚部癌及び前立腺癌などの癌や、線維肉腫、骨肉腫などの肉腫類、或いは神経膠腫などを挙げることができるが、特に、浸潤を起こしやすい乳癌、肺癌、神経膠腫などに対して適用されることが好ましい。 The type of tumor whose growth or invasion is inhibited by the anti-TNIIIA2 antibody is not particularly limited and may be sarcoma or cancer, such as leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer , Hepatocellular carcinoma, colon cancer, pancreatic cancer, head and neck cancer and prostate cancer, and sarcomas such as fibrosarcoma and osteosarcoma, or glioma. It is preferably applied to breast cancer, lung cancer, glioma and the like.
　本発明の抗腫瘍剤は、経口的にまたは非経口的に全身あるいは局所的に投与することができる。例えば、点滴などの静脈内注射、筋肉内注射、腹腔内注射、皮下注射、坐薬、注腸、経口性腸溶剤などを選択することができ、患者の年齢、症状により適宜投与方法を選択することができる。また投与量についても患者の年齢、症状等により適宜選択することができる。 The antitumor agent of the present invention can be administered orally or parenterally systemically or locally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient Can do. The dose can also be appropriately selected depending on the patient's age, symptoms and the like.
　本発明の抗腫瘍剤は、投与経路に応じて医薬的に許容される担体や添加物を共に含むものであってもよい。このような担体および添加物の例として、水、医薬的に許容される有機溶媒、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、ポリアクリル酸ナトリウム、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ペクチン、メチルセルロース、エチルセルロース、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、ジグリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン（ＨＳＡ）、マンニトール、ソルビトール、ラクトース、医薬添加物として許容される界面活性剤などが挙げられる。使用される添加物は、剤形に応じて上記の中から適宜選択されるが、これらに限定されるものではない。 The antitumor agent of the present invention may contain both pharmaceutically acceptable carriers and additives depending on the administration route. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, Examples include sorbitol, lactose, and surfactants acceptable as pharmaceutical additives. The additive to be used is appropriately selected from the above depending on the dosage form, but is not limited thereto.
　本発明の抗腫瘍剤は、抗ＴＮＩＩＩＡ２抗体を含むものであればよく、抗ＴＮＩＩＩＡ２抗体に他の物質を結合した融合型抗ＴＮＩＩＩＡ２抗体としてもよい。抗ＴＮＩＩＩＡ２抗体に融合可能な物質としては、例えば、ドキソルビシン、シスプラチン等の他の抗腫瘍剤を挙げることができる。これにより、抗ＴＮＩＩＩＡ２抗体による直接的な抗腫瘍活性のみならず、融合された抗腫瘍剤による抗腫瘍効果も期待できる。抗ＴＮＩＩＩＡ２抗体にこれらの他の抗腫瘍剤を融合する場合には、抗ＴＮＩＩＩＡ２抗体の活性を損なわない限りいずれの融合又は結合方法を適用してもよく、例えば、リンカーを介して共有結合することによって、或いは金属イオンとの配位結合などの当業界で既知の融合又は結合方法を適用して、抗ＴＮＩＩＩＡ２抗体の活性を損なわない部位、例えば抗体の定常域（Ｃ領域）などに、対象となる他の抗腫瘍剤を融合又は結合することができる。 The antitumor agent of the present invention only needs to contain an anti-TNIIIA2 antibody, and may be a fused anti-TNIIIA2 antibody in which another substance is bound to the anti-TNIIIA2 antibody. Examples of substances that can be fused to the anti-TNIIIA2 antibody include other antitumor agents such as doxorubicin and cisplatin. Thereby, not only the direct antitumor activity by the anti-TNIIIA2 antibody but also the antitumor effect by the fused antitumor agent can be expected. When these other anti-tumor agents are fused to the anti-TNIIIA2 antibody, any fusion or conjugation method may be applied as long as the activity of the anti-TNIIIA2 antibody is not impaired, for example, covalent bonding via a linker Or by applying fusion or binding methods known in the art, such as coordinate binding to metal ions, to sites that do not impair the activity of the anti-TNIIIA2 antibody, such as the antibody constant region (C region). Other anti-tumor agents can be fused or conjugated.
　また、本発明は、腫瘍の疑いのある患者又は腫瘍が既に発生している患者に対して、本発明にかかる抗腫瘍剤を投与することを含む腫瘍の治療方法を含む。ここで、「治療」には、症状の改善であればよく、病巣の肥大抑制又は縮小、転移速度の緩和又は転移の停止も、確認できる範囲でこの用語に包含される。 The present invention also includes a method for treating a tumor, comprising administering an antitumor agent according to the present invention to a patient suspected of having a tumor or a patient who has already developed a tumor. Here, the term “treatment” may be any improvement in symptoms, and the term includes the suppression or reduction of lesion hypertrophy, the reduction of metastasis rate, or the cessation of metastasis to the extent that it can be confirmed.
　患者への投与方法は、適用される薬剤の剤形や、患者の性別、年齢、症状等又はこれらの組み合わせによって異なるが、経口投与、静脈内注射、筋肉内注射、腹腔内注射、皮下注射、坐薬、注腸、経口性腸溶剤を挙げることができ、これらの投与方法から患者の状態によって適宜選択すればよい。ある実施形態では、好ましくは静脈内注射とすることができる。
　本発明の抗体の治療上有効用量は、症状の程度や患者の状態によって異なり、例えば、約０．１ｍｇ／ｋｇ体重～約５０ｍｇ／ｋｇ体重とすることができるが、これに限定されない。また、投与頻度は、例えば、毎日２回ないし１週間に１回の範囲とすることができるが、これに限定されない。 The method of administration to the patient varies depending on the dosage form of the drug applied, the sex, age, symptoms, etc. of the patient or a combination thereof, but oral administration, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, Suppository, enema, oral enteric solvent can be mentioned, and these administration methods may be appropriately selected according to the patient's condition. In certain embodiments, it can be preferably intravenous injection.
The therapeutically effective dose of the antibody of the present invention varies depending on the degree of symptoms and the condition of the patient, and can be, for example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, but is not limited thereto. In addition, the administration frequency can be, for example, in the range of twice a day to once a week, but is not limited thereto.
　以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例に限定されるものではない。尚、特に断りのない限り、「％」は質量基準である。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples. Unless otherwise specified, “%” is based on mass.
［実施例１］
＜抗ヒトＴＮＩＩＩＡ２抗体の作製＞
　アミノ酸配列ＣＡＴＨＹＴＩＴＩＲＧＶＴ（配列番号３）を有するペプチドを、常法により合成した。合成したペプチドを、ハプテンとしてＫＬＨに融合し、得られた融合ペプチドを抗原ペプチドとして用いた。免疫は家兎を用いた通常の方法に従って行った。得られた目的とする抗体を含む混合物を通常の方法により精製した。
　以上のようにして、ヒトＴＮＩＩＩＡ２に対するポリクローナル抗体（抗ヒトＴＮＩＩＩＡ２抗体）を３種類（Ｎｏ．１～Ｎｏ．３）得た。 [Example 1]
<Preparation of anti-human TNIIIA2 antibody>
A peptide having the amino acid sequence CATHITITIRGVT (SEQ ID NO: 3) was synthesized by a conventional method. The synthesized peptide was fused to KLH as a hapten, and the obtained fusion peptide was used as an antigen peptide. Immunization was performed according to the usual method using rabbits. The obtained mixture containing the target antibody was purified by a conventional method.
As described above, three types (No. 1 to No. 3) of polyclonal antibodies against human TNIIIA2 (anti-human TNIIIA2 antibody) were obtained.
＜抗体活性の評価＞
　上記で得られた抗ヒトＴＮＩＩＩＡ２ポリクローナル抗体の抗体活性を、ヒトＴＮＩＩＩＡ２によって誘導される細胞接着の抑制作用を指標とし、以下のようにして評価した。結果を図１に示した。 <Evaluation of antibody activity>
The antibody activity of the anti-human TNIIIA2 polyclonal antibody obtained above was evaluated as follows using the inhibitory action of cell adhesion induced by human TNIIIA2 as an index. The results are shown in FIG.
　細胞培地としてＲＰＭＩ１６４０（商品名、日水製薬社）（２０％ＦＢＳ（ＪＲＨ　Ｂｉｏｓｃｉｅｎｃｅｓ社）添加）を用い、ＫＯＰ２．１６細胞（マウス骨髄由来血管内皮細胞株）を９６穴プレートの各ウェルに５×１０４ｃｅｌｌｓ／２００μＬ／ｗｅｌｌの密度で播種し、３７℃、５％ＣＯ２で４時間培養した。培養上清の１００μＬを取り除き、２０％ＴＣＡ水溶液を１００μＬ添加した後、４℃で一晩もしくは常温で１時間放置して固相化した。これをＰＢＳ×５、ＲＰＭＩ１６４０（２０％ＦＢＳ添加）×１、ＰＢＳ×１で順次洗浄し、各ウェルをＫＯＰ２．１６細胞でコートした９６穴プレートを作製した。 RPMI1640 (trade name, Nissui Pharmaceutical Co., Ltd.) (20% FBS (JRH Biosciences) added) was used as the cell culture medium, and KOP2.16 cells (mouse bone marrow-derived vascular endothelial cell line) were placed 5 × in each well of a 96-well plate. The cells were seeded at a density of 10 4 cells / 200 μL / well and cultured at 37 ° C. and 5% CO 2 for 4 hours. 100 μL of the culture supernatant was removed, and 100 μL of 20% TCA aqueous solution was added, and then allowed to stand at 4 ° C. overnight or at room temperature for 1 hour to be solid-phased. This was washed sequentially with PBS × 5, RPMI 1640 (20% FBS added) × 1, PBS × 1 to prepare a 96-well plate in which each well was coated with KOP2.16 cells.
　上記で得られたＫＯＰ２．１６細胞でコートした９６穴プレートの各ウェルに１２．５μｇ／ｍＬのヒトＴＮＩＩＩＡ２を含むＲＰＭＩ１６４０（２０％ＦＢＳ添加）を加えた。上記で得られた抗ヒトＴＮＩＩＩＡ２抗体（Ｎｏ．１～３）を濃度が１．５μｇ／ｍＬ、３μｇ／ｍＬ、６μｇ／ｍＬとなるようにそれぞれ添加し、Ｋ５６２細胞（白血病細胞株）を１．５×１０４ｃｅｌｌｓ／ｗｅｌｌの密度でそれぞれ播種して、３７℃、５％ＣＯ２で１時間培養した。
　ホルマリンを添加してさらに１時間常温で放置して固定した後、ＰＢＳで３回洗浄し、リリーマイヤー・ヘマトキシリン染色を行なった。光学顕微鏡で観察し、所定の視野内の接着細胞数を計数した。 RPMI 1640 (20% FBS added) containing 12.5 μg / mL human TNIIIA2 was added to each well of the 96-well plate coated with KOP2.16 cells obtained above. The anti-human TNIIIA2 antibody (No. 1 to 3) obtained above was added so that the concentrations were 1.5 μg / mL, 3 μg / mL, and 6 μg / mL, respectively, and K562 cells (leukemia cell line) were added as follows. Each of the cells was seeded at a density of 5 × 10 4 cells / well and cultured at 37 ° C. and 5% CO 2 for 1 hour.
Formalin was added and allowed to stand for 1 hour at room temperature and fixed, then washed 3 times with PBS, and stained with Lilymeyer's hematoxylin. Observed with an optical microscope, the number of adherent cells in a predetermined field of view was counted.
　図１から、正常ウサギＩｇＧではヒトＴＮＩＩＩＡ２によって誘導される細胞接着は抑制されないのに対して、本発明の抗ヒトＴＮＩＩＩＡ２抗体は細胞接着を用量依存的に抑制することが分かる。またＮｏ．１～３のいずれの抗ヒトＴＮＩＩＩＡ２抗体も同等の抗体活性を示すことが分かる。さらに本発明の抗ヒトＴＮＩＩＩＡ２抗体は、マグネシウムイオンで誘導される細胞接着の抑制作用を示さなかった。
　以上から、本発明の抗ヒトＴＮＩＩＩＡ２抗体は、ヒトＴＮＩＩＩＡ２に特異的に結合し、その機能を抑制できることが分かる。 FIG. 1 shows that normal rabbit IgG does not inhibit cell adhesion induced by human TNIIIA2, whereas the anti-human TNIIIA2 antibody of the present invention inhibits cell adhesion in a dose-dependent manner. No. It can be seen that any of the anti-human TNIIIA2 antibodies 1 to 3 show equivalent antibody activity. Furthermore, the anti-human TNIIIA2 antibody of the present invention did not exhibit an inhibitory effect on cell adhesion induced by magnesium ions.
From the above, it can be seen that the anti-human TNIIIA2 antibody of the present invention can specifically bind to human TNIIIA2 and suppress its function.
［実施例２］
＜浸潤阻害活性評価＞
　乳癌細胞を用いて、腫瘍細胞の基底膜下浸潤に対する抗ヒトＴＮＩＩＩＡ２抗体の阻害活性を以下のようにして評価した。
　なお、基底膜モデルとしては、ＥＨＳマトリゲルを被覆したメンブレンフィルターを使用した。比較対象として免疫生物研究所から入手可能な抗ヒトテネイシンＣ抗体である４Ｆ１０ＴＴを用いた。４Ｆ１０ＴＴは、テネイシンＣ（ＴＮ－Ｃ）分子内のＥＧＦ様ドメインをエピトープとし、全てのヒトＴＮ－Ｃバリアントを検出できる抗体として知られている。ヒトテネイシンＣ（ＴＮ－Ｃ、又はＴＮＣ）は、ヒト由来黒色皮腫細胞ＳＫ－ＭＥＬ－２８細胞（理研セルバンクから購入）を大量に培養し、コンフレント到達後に培地を無血清のＤＭＥＭと入れ替え、その１週間後に回収された培養上清を、以前に報告された方法（Ｊ．　Ｔｉｓｓｕｅ　Ｃｕｌｔｕｒｅ　Ｍｅｔｈｏｄｓ．，　Ｖｏｌ．１６，　ｐｐ．２０１－２０４　（１９９４））に従い精製したものを使用した。マウス乳癌細胞株ＭＭＴ細胞がタンパク質分解酵素ＭＭＰを産生することは、確認済みである（データなし）。 [Example 2]
<Evaluation of infiltration inhibitory activity>
Using breast cancer cells, the inhibitory activity of anti-human TNIIIA2 antibody on the subbasement infiltration of tumor cells was evaluated as follows.
As a basement membrane model, a membrane filter coated with EHS Matrigel was used. For comparison, 4F10TT, an anti-human tenascin-C antibody available from the Institute for Immunobiology, was used. 4F10TT is known as an antibody that can detect all human TN-C variants by using an EGF-like domain in the tenascin-C (TN-C) molecule as an epitope. Human tenascin-C (TN-C, or TNC) is a human melanoma cell line SK-MEL-28 cell (purchased from Riken Cell Bank), and after reaching confluence, the medium is replaced with serum-free DMEM. The culture supernatant collected after one week was purified according to a previously reported method (J. Tissue Culture Methods., Vol. 16, pp. 201-204 (1994)). It has been confirmed that the mouse breast cancer cell line MMT cells produce the proteolytic enzyme MMP (no data).
　基底膜マトリックスとしてＥＨＳ肉腫粗抽出物（６．０ｍｇ／ｍＬ；岩城硝子社）を０．１５Ｍ　ＮａＣｌ、０．０２Ｍ　ＨＥＰＥＳで５倍に希釈したものに、フィブロネクチン（ＦＮ：Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　Ｖｏｌ．２２６，　ｐｐ．８８０７－８８１３　（１９９１）に記載された方法に従って精製）を１０μｇ／ｍＬで加え、その溶液をガラス板上に滴下し、その上から多孔性（５．０μｍ）メンブレンフィルターを被せ、４℃かつ湿潤条件下で一晩放置することによって、フィルターにＥＨＳ肉腫粗抽出物とフィブロネクチンとの混合物（以下、「マトリゲル」）を被覆した。
　このフィルターを、マトリゲル被覆面を下にしてトランスウェルチャンバーにセットした。ＲＰＭＩ１６４０（０．５％透析ウシ胎児血清（ｄＦＢＳ）添加）を用いて３．０×１０４ｃｅｌｌｓ／ｗｅｌｌとなるように調整したＭＭＴ細胞（マウス乳腫瘍細胞株）を、チャンバーの上室に播種した。次いで、ＴＮ－Ｃ（２０μｇ／ｍｌ）、抗ＴＮＩＩＩＡ２抗体（２０μｇ／ｍｌ）及び４Ｆ１０ＴＴ抗体（２０μｇ／ｍｌ）を添加した。 As a basement membrane matrix, EHS sarcoma crude extract (6.0 mg / mL; Iwaki Glass Co., Ltd.) was diluted 5-fold with 0.15 M NaCl and 0.02 M HEPES to fibronectin (FN: J. Biol. Chem.,). Vol. 226, pp. 8807-8813 (1991)) was added at 10 μg / mL, the solution was dropped on a glass plate, and a porous (5.0 μm) membrane filter was removed from the solution. The filter was coated with a mixture of crude EHS sarcoma extract and fibronectin (hereinafter “Matrigel”) by leaving it overnight at 4 ° C. under humid conditions.
This filter was set in the transwell chamber with the matrigel-coated surface down. MMT cells (mouse breast tumor cell line) adjusted to 3.0 × 10 4 cells / well using RPMI 1640 (0.5% dialyzed fetal bovine serum (dFBS) added) are seeded in the upper chamber of the chamber did. Then TN-C (20 μg / ml), anti-TNIIIA2 antibody (20 μg / ml) and 4F10TT antibody (20 μg / ml) were added.
　３７℃、５％ＣＯ２インキュベーターで６時間インキュベートした後、フィルターの上側を綿棒で擦り、非浸潤細胞を取り除いた。その後フィルターを回収し、４％ホルマリン／５％スクロースを含むＰＢＳ（－）溶液に浸し、室温で１時間放置することでフィルターの下側へ浸潤した細胞を固定した。クリスタルバイオレット染色液で細胞を染色した後、１００倍顕微鏡下であらかじめ決められた４領域の浸潤細胞数を計測した。
　結果を表１及び図２に示す。 After incubating at 37 ° C. in a 5% CO 2 incubator for 6 hours, the upper side of the filter was rubbed with a cotton swab to remove non-invasive cells. Thereafter, the filter was recovered, immersed in a PBS (−) solution containing 4% formalin / 5% sucrose, and allowed to stand at room temperature for 1 hour to fix cells infiltrating the lower side of the filter. After staining the cells with a crystal violet staining solution, the number of infiltrating cells in four predetermined areas was counted under a 100-fold microscope.
The results are shown in Table 1 and FIG.
Figure JPOXMLDOC01-appb-T000001
 
Figure JPOXMLDOC01-appb-T000001
 
　表１及び図２に示されるように、ＴＮ－Ｃを加えると浸潤する細胞数が増加していることから、ＴＮ－Ｃの存在によって癌が転移することが示唆された。このようなＴＮ－Ｃの存在下に抗ＴＮＩＩＩＡ２抗体を添加すると、浸潤する細胞数が低下することから、抗ＴＮＩＩＩＡ２抗体によってＴＮ－Ｃの効果が抑制されることがわかった。それに対して、同様に抗ＴＮ－Ｃ抗体であるがＴＮＩＩＩＡ２以外の領域を認識する公知の抗体４Ｆ１０ＴＴを加えても、浸潤する細胞数は低下しなかった。
　このことから、抗ＴＮＩＩＩＡ２抗体は、基底膜を越えて細胞が浸潤することを抑制できるとわかる。抗ＴＮ－Ｃ抗体である公知の抗体４Ｆ１０ＴＴを使用することにより得られた結果からは、ＴＮ－Ｃのうち特にＴＮＩＩＩＡ２を認識する本願発明の抗体を使用することにより当該抑制効果を得うることは、全く予測不能であった。 As shown in Table 1 and FIG. 2, when TN-C was added, the number of infiltrating cells increased, suggesting that cancer was metastasized by the presence of TN-C. When the anti-TNIIIA2 antibody was added in the presence of such TN-C, the number of cells invading was reduced, and it was found that the effect of TN-C was suppressed by the anti-TNIIIA2 antibody. On the other hand, the number of infiltrating cells did not decrease even when the well-known antibody 4F10TT, which is an anti-TN-C antibody but recognizes a region other than TNIIIA2, was added.
From this, it can be seen that the anti-TNIIIA2 antibody can suppress the infiltration of cells across the basement membrane. From the results obtained by using the known antibody 4F10TT, which is an anti-TN-C antibody, it is possible to obtain the inhibitory effect by using the antibody of the present invention that specifically recognizes TNIIIA2 among TN-C. It was totally unpredictable.
［実施例３］
＜増殖阻害活性評価＞
（１）乳癌細胞の増殖試験
　乳癌細胞を用いて、腫瘍細胞の増殖に対する抗ヒトＴＮＩＩＩＡ２抗体の阻害活性を以下のようにして評価した。
　９６穴プレートにＲＰＭＩ１６４０（０．５％ｄＦＢＳ）で１．０×１０４　ｃｅｌｌｓ／ｗｅｌｌとなるように調整したＭＭＴ細胞を、ＴＮ－Ｃ（２０μｇ／ｍｌ）、抗ＴＮＩＩＩＡ２抗体（２０μｇ／ｍｌ）、４Ｆ１０ＴＴ抗体（２０μｇ／ｍｌ）又はＭＭＰ－２／－９阻害剤（２５μＭ）とともに播種した。３７℃、５％ＣＯ２インキュベーターで２４時間培養した後、ＷＳＴ法に準じてＣｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ（商品名、同人化学社）を用いて評価した。
　結果を表２及び図３に示す。なお吸光度はＯ．Ｄ．４５０ｎｍの値である。 [Example 3]
<Evaluation of growth inhibitory activity>
(1) Breast cancer cell proliferation test Using breast cancer cells, the anti-human TNIIIA2 antibody inhibitory activity against tumor cell proliferation was evaluated as follows.
MMT cells adjusted to 1.0 × 10 4 cells / well with RPMI 1640 (0.5% dFBS) in a 96-well plate were mixed with TN-C (20 μg / ml), anti-TNIIIA2 antibody (20 μg / ml), Seed with 4F10TT antibody (20 μg / ml) or MMP-2 / -9 inhibitor (25 μM). After culturing at 37 ° C. in a 5% CO 2 incubator for 24 hours, evaluation was performed using Cell Counting Kit (trade name, Doujin Chemical Co., Ltd.) according to the WST method.
The results are shown in Table 2 and FIG. The absorbance is O.D. D. The value is 450 nm.
Figure JPOXMLDOC01-appb-T000002
 
Figure JPOXMLDOC01-appb-T000002
 
　表２及び図３に示されるように、ＭＭＰ－２／－９阻害剤および抗ＴＮＩＩＩＡ２抗体は、ＴＮ－Ｃが誘導するＭＭＴの増殖促進を抑制することが分かった。　一方、同様に抗ＴＮ－Ｃ抗体であるがＴＮＩＩＩＡ２以外の領域を認識する公知の抗体４Ｆ１０ＴＴを用いた場合は、ＴＮ－Ｃが誘導するＭＭＴの増殖促進は抑制されなかった。
　抗ＴＮ－Ｃ抗体である公知の抗体４Ｆ１０ＴＴを使用することにより得られた結果からは、ＴＮ－Ｃのうち特にＴＮＩＩＩＡ２を認識する本願発明の抗体を使用することにより当該抑制効果を得うることは、全く予測不能であった。
　さらに、ペプチドＴＮＩＩＩＡ２を単独で添加すると、ＭＭＴの増殖および／または生存は、ＴＮＩＩＩＡ２の濃度依存的に促進された（データ示さず）。従って、ＴＮ－Ｃ分子中のＴＮＩＩＩＡ２活性部位がＭＭＴの増殖および／または生存を促進することがわかった。 As shown in Table 2 and FIG. 3, it was found that the MMP-2 / -9 inhibitor and the anti-TNIIIA2 antibody suppressed the growth promotion of MMT induced by TN-C. On the other hand, when the known antibody 4F10TT, which is an anti-TN-C antibody but recognizes a region other than TNIIIA2, was used, the promotion of MMT proliferation induced by TN-C was not suppressed.
From the results obtained by using the known antibody 4F10TT, which is an anti-TN-C antibody, it is possible to obtain the inhibitory effect by using the antibody of the present invention that specifically recognizes TNIIIA2 among TN-C. It was totally unpredictable.
Furthermore, when the peptide TNIIIA2 was added alone, MMT proliferation and / or survival was promoted in a TNIIIA2 concentration-dependent manner (data not shown). Thus, it was found that the TNIIIA2 active site in the TN-C molecule promotes MMT proliferation and / or survival.
（２）正常細胞の増殖試験
　正常細胞株を用いて、正常細胞の悪性化に対する抗ヒトＴＮＩＩＩＡ２抗体の阻害活性を以下のようにして検討した。
　９６穴プレートにＤＭＥＭ（日水製薬社）（１０％ＣＳ（ＪＲＨ　Ｂｉｏｓｃｉｅｎｃｅｓ社）添加）で２．０×１０４　ｃｅｌｌｓ／ｗｅｌｌとなるように調整したＮＩＨ３Ｔ３細胞を、ＴＮＩＩＩＡ２（５０μｇ／ｍｌ）またはＴＮ－Ｃ（５μｇ／ｍｌ）とともに播種し、３７℃、５％ＣＯ２インキュベーターで培養した。１週間後に培地の半分を入れ替え、更に１週間後にクリスタルバイオレットで細胞を染色した後、１００倍顕微鏡下で細胞塊の形成を評価した。
　結果を図４に示す。 (2) Proliferation test of normal cells Using normal cell lines, the inhibitory activity of anti-human TNIIIA2 antibody against malignant normal cells was examined as follows.
NIH3T3 cells adjusted to 2.0 × 10 4 cells / well with DMEM (Nissui Pharmaceutical) (10% CS (JRH Biosciences) added) in a 96-well plate were mixed with TNIIIA2 (50 μg / ml) or TN Seeded with -C (5 μg / ml) and cultured in a 37 ° C., 5% CO 2 incubator. One week later, half of the medium was replaced, and after another week, the cells were stained with crystal violet, and the formation of cell clusters was evaluated under a 100-fold microscope.
The results are shown in FIG.
　図４Ａに示されるように、対照群では、細胞は増殖して単一層を形成後、単一層を維持していた。即ち、対照群では増殖しても細胞間の接触が生じると増殖を停止する接触阻害を示し、正常細胞の性状を示した。
　これに対してＴＮＩＩＩＡ２を添加した細胞群（図４Ｂ）及びＴＮ－Ｃを添加した細胞群（図４Ｃ）ではいずれも、フォーカス形成と思われる細胞の重層化の促進が認められた。即ち、ＴＮＩＩＩＡ２及びＴＮ－Ｃは、正常細胞の悪性化に密接に関与していることが示唆された。
　従って、ＴＮＩＩＩＡ２及びＴＮ－Ｃによる正常細胞の悪性化は、ＴＮＩＩＩＡ２及びＴＮ－Ｃの活性を阻止する物質、例えば、実施例２においてＴＮＩＩＩＡ２及びＴＮ－Ｃの浸潤阻害を誘導した抗ＴＮＩＩＩＡ２抗体を添加することにより、効果的に阻止できる。 As shown in FIG. 4A, in the control group, cells proliferated to form a monolayer and then maintained a monolayer. That is, the control group exhibited contact inhibition that stopped proliferation when contact between cells occurred even when it proliferated, indicating the properties of normal cells.
On the other hand, in both the cell group to which TNIIIA2 was added (FIG. 4B) and the cell group to which TN-C was added (FIG. 4C), promotion of cell stratification that was considered to be focus formation was observed. That is, it was suggested that TNIIIA2 and TN-C are closely involved in the malignant transformation of normal cells.
Therefore, malignant normal cells by TNIIIA2 and TN-C are added with substances that block the activity of TNIIIA2 and TN-C, for example, the anti-TNIIIA2 antibody that induced infiltration inhibition of TNIIIA2 and TN-C in Example 2 Can be effectively prevented.
［実施例４］
＜乳癌組織におけるＴＮＩＩＩＡ２の発現＞
　実際の腫瘍組織における抗ＴＮＩＩＩＡ２抗体の効果を検討するため、腫瘍組織におけるＴＮ－Ｃ及びＴＮＩＩＩＡ２の局在を以下のようにして確認した。
　ヒト乳がん組織標本（パラフィン切片）には、三重大学医学部付属病院で乳がんの診断で切除された組織を、倫理委員会の承認の下、患者の同意を得て用いた。本切片を常法にしたがい脱パラフィン後、抗ＴＮ－Ｃモノクローナル抗体（４Ｆ１０ＴＴ抗体）又は抗ＴＮＩＩＩＡ２ポリクローナル抗体を用いてＰＡＰ（ペルオキシダーゼ－抗ペルオキシダーゼ複合体）法により免疫染色し、ＴＮ－ＣおよびＴＮＩＩＩＡ２の局在を評価した。
　結果を図５Ａ及び図５Ｂに示す（２００倍）。黒矢頭は染色された部分を示す。 [Example 4]
<Expression of TNIIIA2 in breast cancer tissue>
In order to examine the effect of the anti-TNIIIA2 antibody in the actual tumor tissue, the localization of TN-C and TNIIIA2 in the tumor tissue was confirmed as follows.
For human breast cancer tissue specimens (paraffin sections), tissues excised by diagnosis of breast cancer at Mie University Hospital were used with the consent of the patient with the approval of the Ethics Committee. This section was deparaffinized according to a conventional method and then immunostained by the PAP (peroxidase-antiperoxidase complex) method using an anti-TN-C monoclonal antibody (4F10TT antibody) or an anti-TNIIIA2 polyclonal antibody, and TN-C and TNIIIA2 Localization was assessed.
The results are shown in FIGS. 5A and 5B (200 times). The black arrowhead indicates the stained part.
　図５Ｂに示されるように、乳癌組織に対する抗ＴＮＩＩＩＡ２抗体による染色は、基底膜及び胞巣間質に比較的広範に認められた。また図５Ａに示されるように、抗ＴＮ－Ｃ抗体による染色も同様であった。ＴＮＩＩＩＡ２は、通常ＴＮ－Ｃ分子内では立体構造的に隠蔽されており、ＭＭＰ－２を始めとするタンパク質分解酵素の作用等により露出することが確認されている。これらのことから、乳癌組織での抗ＴＮＩＩＩＡ２抗体による染色によって、乳癌組織においてＴＮＩＩＩＡ２の露出が増大していることが示された。このことは、ｉｎ　ｖｉｔｒｏ　だけでなく、腫瘍組織においてもＴＮＩＩＩＡ２が露出しており、周囲の腫瘍細胞に対して影響を与えていることを示しており、生体への抗ＴＮＩＩＩＡ２抗体を投与することによりその影響を阻害できることが示唆された。 As shown in FIG. 5B, staining with an anti-TNIIIA2 antibody against breast cancer tissue was relatively widely observed in the basement membrane and alveolar stroma. Also, as shown in FIG. 5A, the staining with anti-TN-C antibody was the same. TNIIIA2 is usually three-dimensionally concealed in the TN-C molecule and has been confirmed to be exposed by the action of proteolytic enzymes such as MMP-2. From these, it was shown that the exposure of TNIIIA2 was increased in breast cancer tissue by staining with anti-TNIIIA2 antibody in breast cancer tissue. This indicates that TNIIIA2 is exposed not only in vitro but also in tumor tissues, and has an effect on surrounding tumor cells. By administering anti-TNIIIA2 antibody to the living body, It was suggested that the influence could be inhibited.
　このように、ＴＮＩＩＩＡ２は、腫瘍細胞の浸潤及び重層化を伴う細胞増殖を促進する作用を有することが明らかとなり、抗ＴＮＩＩＩＡ２抗体を用いることによって、このようなＴＮＩＩＩＡ２の活性を阻止できることが示された。
　以上から、本発明の抗ヒトＴＮＩＩＡ２抗体は、腫瘍細胞の浸潤及び増殖を阻害し、腫瘍の悪性化を効果的に抑制できることが分かる。 Thus, it was revealed that TNIIIA2 has an effect of promoting cell proliferation accompanied by tumor cell invasion and stratification, and it was shown that such an activity of TNIIIA2 can be blocked by using an anti-TNIIIA2 antibody. .
From the above, it can be seen that the anti-human TNIIA2 antibody of the present invention can inhibit tumor cell invasion and proliferation and effectively suppress tumor malignancy.
　従って、本発明によれば、腫瘍の悪性化を効果的に抑制し得る抗腫瘍剤を提供できる。 Therefore, according to the present invention, an antitumor agent capable of effectively suppressing tumor malignancy can be provided.

Claims (12)

  1. 　配列番号１のアミノ酸配列からなるポリペプチドを認識する抗体および前記抗体の抗原認識部位を含む抗体断片からなる群より選択される少なくとも一つを含有する抗腫瘍剤。 An antitumor agent comprising at least one selected from the group consisting of an antibody recognizing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing an antigen recognition site of the antibody.
  2. 　前記抗体が、配列番号２のアミノ酸配列を含むポリペプチドを認識する抗体である請求項１に記載の抗腫瘍剤。 The antitumor agent according to claim 1, wherein the antibody recognizes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  3. 　前記抗体が、配列番号３のアミノ酸配列からなるポリペプチドを認識する抗体である請求項１に記載の抗腫瘍剤。 The antitumor agent according to claim 1, wherein the antibody recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
  4. 　乳癌、肺癌、または神経膠腫に対して適用される請求項１～３のいずれか１項に記載の抗腫瘍剤。 The antitumor agent according to any one of claims 1 to 3, which is applied to breast cancer, lung cancer, or glioma.
  5. 　配列番号１のアミノ酸配列からなるポリペプチドを認識する抗体および前記抗体の抗原認識部位を含む抗体断片からなる群より選択される少なくとも一つの、生体試料中における配列番号１のアミノ酸配列の少なくとも一部を含むポリペプチドの検出における使用。 At least a part of the amino acid sequence of SEQ ID NO: 1 in a biological sample selected from the group consisting of an antibody that recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an antibody fragment containing the antigen recognition site of the antibody In the detection of polypeptides comprising
  6. 　前記抗体が、配列番号２のアミノ酸配列を含むポリペプチドを認識する抗体である請求項５に記載の使用。 The use according to claim 5, wherein the antibody recognizes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  7. 　前記抗体が、配列番号３のアミノ酸配列からなるポリペプチドを認識する抗体である請求項５に記載の使用。 The use according to claim 5, wherein the antibody recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
  8. 　前記生体試料が、腫瘍細胞を含む生体組織から得られた、請求項５～７のいずれか１項に記載の使用。 The use according to any one of claims 5 to 7, wherein the biological sample is obtained from a biological tissue containing tumor cells.
  9. 　前記生体試料が、腫瘍細胞を含む可能性を有する生体組織から得られた、請求項５～７のいずれか１項に記載の使用。 The use according to any one of claims 5 to 7, wherein the biological sample is obtained from a biological tissue having a possibility of containing tumor cells.
  10. 　前記腫瘍細胞が、乳癌細胞、肺癌細胞、または神経膠腫細胞である請求項８または９に記載の使用。 The use according to claim 8 or 9, wherein the tumor cells are breast cancer cells, lung cancer cells, or glioma cells.
  11. 　前記検出が、前記生体組織中に前記抗体を導入すること、及び、前記生体組織中における前記抗体の存在を視覚的に観察することを含む、請求項５～１０のいずれか１項に記載の使用。 The detection according to any one of claims 5 to 10, wherein the detection includes introducing the antibody into the living tissue and visually observing the presence of the antibody in the living tissue. use.
  12. 　前記視覚的観察が、免疫組織化学的手法による、請求項１１に記載の使用。 The use according to claim 11, wherein the visual observation is by an immunohistochemical technique.
PCT/JP2011/067360 2010-07-29 2011-07-28 Anti-tumor agent WO2012015008A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010170975A JP2012031084A (en) 2010-07-29 2010-07-29 Anti-tumor agent
JP2010-170975 2010-07-29

Publications (1)

Publication Number Publication Date
WO2012015008A1 true WO2012015008A1 (en) 2012-02-02

Family

ID=45530208

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/067360 WO2012015008A1 (en) 2010-07-29 2011-07-28 Anti-tumor agent

Country Status (2)

Country Link
JP (1) JP2012031084A (en)
WO (1) WO2012015008A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007212438A (en) * 2006-01-10 2007-08-23 Meneki Seibutsu Kenkyusho:Kk Cancer diagnostic agent and cancer diagnostic kit
JP2008506355A (en) * 2004-02-27 2008-03-06 テクノジェン・ソシエタ・ペル・アチオニ Anti-human tenascin monoclonal antibody
WO2010090272A1 (en) * 2009-02-06 2010-08-12 学校法人東京理科大学 Remedy for chronic inflammation and antibody to be used therein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008506355A (en) * 2004-02-27 2008-03-06 テクノジェン・ソシエタ・ペル・アチオニ Anti-human tenascin monoclonal antibody
JP2007212438A (en) * 2006-01-10 2007-08-23 Meneki Seibutsu Kenkyusho:Kk Cancer diagnostic agent and cancer diagnostic kit
WO2010090272A1 (en) * 2009-02-06 2010-08-12 学校法人東京理科大学 Remedy for chronic inflammation and antibody to be used therein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MARIKO SAKAI ET AL.: "Nyugan Saibo no Seizon/ Zoshoku Oyobi Kiteimakuka Shinjun ni Okeru Tenascin-C no Kan'yo", JAPANESE SOCIETY OF CONNECTIVE TISSUE RESEARCH GAKUJUTSU TAIKAI SHOROKUSHU, vol. 42, 2 August 2010 (2010-08-02), pages 104 *
MARIKO SAKAI ET AL.: "T-lymphocyte no Naihika Shinjun ni Okeru Tenascin-C no Kan'yo", ABSTRACTS OF ANNUAL MEETING OF PHARMACEUTICAL SOCIETY OF JAPAN, vol. 130, 5 March 2010 (2010-03-05) *
SAITO Y. ET AL.: "A peptide derived from Tenascin-C induces beta1 Integrin activation through Syndecan-4", J.BIOL.CHEM., vol. 282, 2007, pages 34929 - 34937, XP008152259, DOI: doi:10.1074/jbc.M705608200 *

Also Published As

Publication number Publication date
JP2012031084A (en) 2012-02-16

Similar Documents

Publication Publication Date Title
EP3489261B1 (en) Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis
AU2012341450B2 (en) Anti-human TROP-2 antibody having antitumor activity in vivo
TWI485161B (en) Anti-epithelial cell adhesion molecule (epcam) antibodies and methods of use thereof
JP5615169B2 (en) Anti-human Dlk-1 antibody having antitumor activity in vivo
JP5367776B2 (en) Binding assay and isolation method for cells exhibiting CD44 surface expression
US7399469B2 (en) Anti-LFL2 antibodies for the diagnosis, prognosis and treatment of cancer
Sun et al. A novel mouse CD133 binding-peptide screened by phage display inhibits cancer cell motility in vitro
JP5816188B2 (en) Anti-clusterin antibodies and antigen-binding fragments and their use to reduce tumor volume
BR112012029281B1 (en) antibody to human trop-2, monoclonal antibody to human trop-2, antibody-derived antibody fragment, hybridoma, conjugate, pharmaceutical composition, use of pharmaceutical composition, method to detect a tumor and kit to treat, diagnose or detect a tumor
KR20140054238A (en) Antibodies that bind integrin alpha-v beta-8
JP2017505107A (en) Antibodies against CCR9 and uses thereof
KR20090010194A (en) Methods of treating, diagnosing or detecting cancer
WO2019086480A1 (en) Anti-cd5l antibody and uses thereof
WO2017062496A2 (en) Anti-sas1b antibodies, associated methods of use, and compositions and methods for detecting and treating cancer
US8679495B2 (en) Use of anti-90K monoclonal antibodies for the prevention and treatment of tumors and metastases thereof
JP4695982B2 (en) Method for detecting liver cancer, liver cancer diagnostic agent, and cancer therapeutic agent
JP2019512210A (en) EGFL6-specific monoclonal antibodies and methods of using them
JP2022530339A (en) Integrin α10 and invasive cancer type
DK2984108T3 (en) Anti-s100a7 antibodies for the treatment and diagnosis of cancer
JP7357347B2 (en) Liver cancer cell proliferation inhibitor that targets extracellular PKCδ and a novel liver cancer treatment drug containing the same
WO2012015008A1 (en) Anti-tumor agent
US20160090412A1 (en) Antibodies against the s100p protein for the treatment and diagnosis of cancer
WO2020171171A1 (en) Anti-hla-dr antibody, and use thereof for cancer therapy
WO2021251459A1 (en) Humanized anti-gpc-1 antibody
CA3142238A1 (en) Anti-adam8 antibodies and uses of the same

Legal Events

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

Ref document number: 11812597

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11812597

Country of ref document: EP

Kind code of ref document: A1