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CELL ADHESION INHIBITOR, CELL PROLIFERATION INHIBITOR, AND METHOD AND KIT FOR TESTING CANCER

Foreign code F130007418
File No. S2011-0746-C0
Posted date Jun 20, 2013
Country WIPO
International application number 2012JP062226
International publication number WO 2012157589
Date of international filing May 11, 2012
Date of international publication Nov 22, 2012
Priority data
  • P2011-108685 (May 13, 2011) JP
Title CELL ADHESION INHIBITOR, CELL PROLIFERATION INHIBITOR, AND METHOD AND KIT FOR TESTING CANCER
Abstract [Problem] To provide: a means for preventing/treating cancer by controlling the expression/function of a target cell that is different from those employed in conventional means, as a novel means to be employed in a molecular targeting therapy for cancer; a means for screening for a substance having a prophylactic/therapeutic activity on cancer, which utilizes the target molecule; and a simple and rapid means for testing cancer utilizing the detection of the target molecule.
[Solution] According to one embodiment of the present invention, a cell proliferation inhibitor and a cell adhesion inhibitor for cancer cells are provided, each of which comprises an antibody against a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide of the protein. According to another embodiment of the present invention, a cell adhesion inhibitor for cancer cells is provided, which comprises an antisense polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to a nucleotide sequence for a polynucleotide that encodes a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a part of the nucleotide sequence. According to a further embodiment of the present invention, a cell adhesion inhibitor for cancer cells is provided, which comprises a substance capable of inhibiting the expression and/or activity of a protein that comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. According to a still another embodiment of the present invention, a method for testing cancer is provided, which comprises a step of detecting a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide of the protein, or detecting a polynucleotide that encodes a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. Further provided is a test kit for use in the method, which comprises an antibody against a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide of the protein, or comprises a primer or probe for detecting a polynucleotide that encodes a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2.
Outline of related art and contending technology BACKGROUND ART
In recent years, with rapid development of molecular biology, 'molecules target medicine' referred to as a new type of drug has been developed and, in some product already has been launched.
For example, cancer as an example, a conventional anticancer agent is, or mainly synthetic DNA target at a particular stage of the cell cycle was. This is because, compared to a normal cell growth has been more active that the use of the property of cancer cells, specific for cancer cells exhibit the toxicity of the present invention. Therefore, the same cancer cells but not the growth rate of cells with cancer cells (for example, in the bone marrow of the juvenile hematopoietic cells, oral cavity mucous membrane cell, gastrointestinal epithelial cells, hair follicle cells) and the like, there arises a fault by conventional anticancer agent, as a result myelosuppression (leukopenia, thrombocytopenia), digestive disorders, side effects such as hair loss problem.
On the other hand, the above-described 'molecules target medicine' is, specifically in cancer cells (or excessively) expressing the target molecule, the molecule to perform the selective anti-cancer action although it exhibits. Therefore, molecular target drugs according to the treatment of cancer, compared with the case where the conventional anti-cancer agent, such as those described above have the advantage that the occurrence of side effects can be suppressed.
A newly molecules target medicine as a means for the development of a potent anti-cancer, cancer cell, gene in a cancer tissue, protein expression analysis, is effective in functional analysis. Or by such an analysis, the particular therapeutic agent, and the effectiveness of the therapies for, a new drug-discovery target of the search can be considered.
Is acute myeloid leukemia (AML; Acute Myelogenous Leukemia), a type 1 leukemia of a hematologic cancer, tumor and hematopoietic cells of the myeloid series, differentiation, disease lose mature ability. Differentiation, mature hematopoietic cell is its ability, take the form of young leaves from the, 'blast' also referred to. In AML, the blasts in the bone marrow is accumulated, thereby lowering the production of normal blood cells. As a result, anemia due to lack of the red blood cells, shortness of breath, palpitations, and symptoms such as facial pallor, bleeding from the gums due to lack platelets, epistaxis, blood from the digestive tract under or bloody, expression of symptoms such as cerebral hemorrhage. Further, due to lack of white blood cells, reduced immunity to infection, such as high heat or heaviness become symptom occurs.
1976 Year according to the classification proposed FAB(French-American-British), AML is, the ratio of bone marrow cells blasts occupied by defined as greater than or equal to 30%. On the other hand, chromosome or gene mutations as a new leukemia including World Health Organization (WHO) classification methods published by classification according to WHO, the ratio of 20% or more states blastoma and defined AML. In recent years due to the development of chemotherapy or hematopoietic stem cell transplantation, AML is a significant improvement in the treatment result of the use of a chemical, is about a half of the anti-cancer agent resistance refractory AML leukemia.
Illustrating a mechanism for the onset of the leukemia, the so-called ' leukemia stem cells' concept has been proposed in recent years. According to this concept, significantly enhanced ability to self-replicate is one of the very small number of hematopoietic stem cells cells, leukemia cells for supplying 'stem cell' functions as, repeating self-replication and limiting differentiation, by maintaining the supply leukemia cells, which express leukemia pathology which will be described.
Then, in AML leukemia stem cells are, within the bone of the bone marrow cell adhesion molecule through the region where the film is fixed on the escape from apoptosis induced by a chemotherapeutic agent, that is, the acquired resistance to chemotherapeutic agents have been reported (for example, see non-patent document 1).
Conventional, having been subjected to chemotherapy in patients with AML, minimal residual disease (MRD; Minimal Residual Disease) due to a recurrence of disease state is known. Then, results in recurrence or the cause of the acquisition of drug resistance as one of the 1, leukemia VLA (Very Late Antigen) present on the cell surface of -4, bone marrow stromal cells (osteoblasts) to be adhered to the surface of the fibronectin involved is clarified. Such a based on the finding that, targeted VLA-4 AML therapy, relapse prevention method has been proposed. For example, an antibody specific for VLA-4 (VLA-4 neutralizing antibodies) administration, and inhibits adhesion of fibronectin VLA-4 and, thereby the treatment of AML, perform prevention has been proposed (for example, see non-patent document 2). In addition, the VLA-4, β 1 α 4 subunits of the integrin family and a subunit of a heterodimeric protein, subunit of these β 1, adhesion of bone marrow osteoblasts are involved.
Further, the inventors have found that, in the bone marrow of the mouse retrovirus integration sites as tumor (leukemia), Evi1 (ecotropic viral integration-1) genes are identified (see non-patent document 3).
Evi1 Gene, as described above a murine leukemia virus isolated from the insertion site, and from the 3099bp gene (Refseq Accession No.NM _007963), 1032 (Evi1 protein) proteins made of amino acids (RefSeq Accession No.NP _031908) encoding. Is in a human homologous gene (EVI1 gene), and from the 4873bp gene (Refseq Accession No.NM _001105077), a protein consisting of the amino acid 1115 (Refseq Accession No.NP _001098547) encoding (EVI1 protein). Evi1 Protein, EVI1 protein, each mouse, and human hematopoietic stem cells mainly expressed in the nucleus of leukemia cells can be confirmed.
Here, the relationship between the cancer gene EVI1, the inventors of the present invention, in human AML, 3 gene EVI1 (3q26) located in arm length No. staining by dislocations of the activation of the EVI1 gene has been discovered that by performing (see non-patent document 4). Then, EVI1 gene in cells of various types, play an important role in cell proliferation/differentiation are responsible for it has been found that, in addition, EVI1 gene is also expressed in bone marrow hematopoietic stem cells, hematopoietic stem cell regulation is responsible for clear that (for example, see non-patent document 5). The present inventors, this EVI1 gene GATA-2 by controlling the expression of the gene, contribute to the proliferation of hematopoietic stem cells to reveal (see non-patent document 6). In addition, in recent years, hematopoietic stem cells and transformed EVI1 gene in the proliferation of leukemia cells is an important control element have been reported (see non-patent document 7).
EVI1 The total AML AML cases of high expression is accounted for by the 8%, 5 - year survival rate as low as 20-30%, the existing chemotherapy or hematopoietic stem cell transplantation is difficult in the healing thereof. In addition, refractory or recurring AML such as higher than that of the known frequency. Conventional, EVI1 expression of the anti-cancer agent resistance in the high expression AML was unknown mechanism is, the inventors of the present invention, the microenvironment of the bone marrow of leukemia cells EVI1 gene (niche) in the non-differentiated to be essential for the maintenance or for cell adhesion, and, the adhesion ability of leukemia cells in the gene upregulation AML via resistance involved in the expression of the anti-cancer agent are revealed. Then, based on this knowledge, the expression of the protein expression of the gene EVI1 and EVI1, it is possible to inhibit the function, of leukemia cells through suppression of adhesion to the bone marrow niche, to treat dry AML, other anti-leukemia cells to improve the sensitivity can be proposed (see Patent Document 1) are.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドに対する抗体を含有する、癌細胞の細胞接着阻害剤。

[請求項2]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質をコードするポリヌクレオチドの塩基配列に、相補的もしくは実質的に相補的な塩基配列またはその一部を含むアンチセンスポリヌクレオチドを含有する、癌細胞の細胞接着阻害剤。

[請求項3]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質の発現および/または活性を阻害する物質を含有する、癌細胞の細胞接着阻害剤。

[請求項4]
 癌の予防または治療に用いられる、請求項1~3のいずれか1項に記載の阻害剤。

[請求項5]
 他の抗癌剤と併用された際に、癌細胞の前記抗癌剤に対する感受性を向上させるのに用いられる、請求項1~4のいずれか1項に記載の阻害剤。

[請求項6]
 癌が白血病である、請求項1~5のいずれか1項に記載の阻害剤。

[請求項7]
 前記白血病が急性骨髄性白血病である、請求項6に記載の阻害剤。

[請求項8]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質の発現および/または活性を阻害することを含む、癌細胞の細胞接着阻害方法。

[請求項9]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質をコードするポリヌクレオチドの発現および/または活性を阻害することを含む、癌細胞の細胞接着阻害方法。

[請求項10]
 癌の予防または治療に用いられる、請求項8または9に記載の阻害方法。

[請求項11]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドを用いる、癌細胞の細胞接着を阻害する物質のスクリーニング方法。

[請求項12]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドが、それを産生する細胞の形態で提供される、請求項11に記載のスクリーニング方法。

[請求項13]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドに対する抗体、並びに、前記タンパク質をコードするポリヌクレオチドまたはその塩基配列の一部を含むポリヌクレオチドからなる群より選択されるいずれかをさらに用いる、請求項12に記載のスクリーニング方法。

[請求項14]
 癌の予防または治療のための物質のスクリーニングに用いられる、請求項11~13のいずれか1項に記載のスクリーニング方法。

[請求項15]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドに対する抗体を含有する、細胞増殖抑制剤。

[請求項16]
 前記抗体が、細胞傷害活性を有する抗体である、請求項15に記載の抑制剤。

[請求項17]
 前記細胞傷害活性が、抗体依存性細胞性細胞傷害活性(ADCC活性)である、請求項16に記載の抑制剤。

[請求項18]
 癌細胞の増殖を抑制する、請求項15~17のいずれか1項に記載の抑制剤。

[請求項19]
 癌が白血病である、請求項18に記載の抑制剤。

[請求項20]
 前記白血病が急性骨髄性白血病である、請求項19に記載の抑制剤。

[請求項21]
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質あるいはその部分ペプチドに対する抗体を投与することを含む、細胞に障害を引き起こす方法。

[請求項22]
 以下の工程:
 (a)被験者から試料を採取する工程;および、
 (b)採取された試料に含まれる、配列番号:2で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むタンパク質またはその部分ペプチド、あるいは、配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質をコードするポリヌクレオチドを検出する工程、
を含む、癌の検査方法。

[請求項23]
 細胞表面に存在する、前記タンパク質またはその部分ペプチドを検出する、請求項22に記載の検査方法。

[請求項24]
 フローサイトメトリー法により検出を行う、請求項23に記載の検査方法。

[請求項25]
 癌が白血病である、請求項24に記載の検査方法。

[請求項26]
 前記白血病が急性骨髄性白血病である、請求項25に記載の検査方法。

[請求項27]
 請求項22~26のいずれか1項に記載の検査方法に用いるための検査用キットであって、
 配列番号:2で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むタンパク質またはその部分ペプチドに対する抗体、あるいは、
 配列番号:2で表されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を含むタンパク質をコードするポリヌクレオチドを検出するためのプライマーまたはプローブ、
を含む、検査用キット。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • UNIVERSITY OF MIYAZAKI
  • Inventor
  • MORISHITA, Kazuhiro
  • SAITO, Yusuke
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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