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WO2011003369A1 - 一种新的肿瘤标志物 - Google Patents

一种新的肿瘤标志物 Download PDF

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Publication number
WO2011003369A1
WO2011003369A1 PCT/CN2010/075896 CN2010075896W WO2011003369A1 WO 2011003369 A1 WO2011003369 A1 WO 2011003369A1 CN 2010075896 W CN2010075896 W CN 2010075896W WO 2011003369 A1 WO2011003369 A1 WO 2011003369A1
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Prior art keywords
hsp90a
tumor
polypeptide
cancer
antibody
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PCT/CN2010/075896
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English (en)
French (fr)
Inventor
罗永章
宋晓敏
王晓峰
卓巍
常国栋
付彦
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Tsinghua University
Protgen Ltd
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Tsinghua University
Protgen Ltd
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Priority to SG2012000642A priority Critical patent/SG177514A1/en
Priority to EP10796746.5A priority patent/EP2452948B1/en
Priority to AU2010268979A priority patent/AU2010268979B2/en
Priority to RU2012104024/10A priority patent/RU2567005C2/ru
Publication of WO2011003369A1 publication Critical patent/WO2011003369A1/zh
Priority to IL217276A priority patent/IL217276A/en
Anticipated expiration legal-status Critical
Priority to ZA2012/00888A priority patent/ZA201200888B/en
Priority to IL25361517A priority patent/IL253615B/en
Ceased legal-status Critical Current

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    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G01N33/5752—
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to the field of diagnosis and treatment of tumors, and in particular to a novel tumor marker and a method and kit for diagnosing the occurrence and metastasis of a tumor, and to a method and a medicament for treating tumors and tumor metastasis.
  • Tumor markers are antigens and other biologically active substances that are produced or reduced by tumor cells during the process of carcinogenesis due to changes in the expression levels of genes. They can be used for early diagnosis, staging, monitoring of tumor progression, and evaluation of therapeutic effects of drugs (ASCO). , 1996). It can have a dramatic impact on the clinical treatment of tumors, especially when it can be detected before a clinical condition occurs, or when it can be used for real-time detection of therapeutic effects. At present, in order to meet the needs of clinical diagnosis and treatment of tumors, the development of tumor markers needs to be accelerated.
  • Tumor markers currently used for early diagnosis of tumors are mostly not widely used in physical examination due to lack of sensitivity and specificity.
  • alpha-fetoprotein and ultrasonography are commonly used methods for diagnosing high-risk patients, and do significantly improve the survival rate of liver cancer patients, but the sensitivity is relatively low; tumor antigen CA-125 has higher sensitivity, but Lack of specificity.
  • the blood tumor marker CA15-3 used for breast cancer detection is almost useless in early diagnosis due to its low sensitivity. Therefore, early diagnosis of tumors, as well as differentiation of benign and malignant tumors, remains a clinical challenge, requiring new techniques and methods to discover new tumor markers and improve the sensitivity and confidence of tumor marker detection.
  • tumor proteomics The emergence of tumor proteomics has brought new hopes for the discovery of new tumor markers, and the screening, early diagnosis and prognosis of tumors. Malignant transformation of tumors is accompanied by certain protein expression Level changes, these changes can be detected qualitatively and quantitatively at the protein level, which is the main research content of tumor proteomics. The protein information of the tumor thus obtained can provide valuable assistance for more effective diagnosis, prognosis of the tumor and evaluation of the therapeutic effect.
  • Heat shock protein 90a is a molecular chaperone protein whose presence is essential for the stability and function of many tumor-associated proteins. Hsp90a is one of the most abundant chaperone proteins in the cytoplasm of eukaryotic cells, accounting for 1-2% of total protein in cells. The main functions of Hsp90a in cells include stabilizing proteins (such as estrogen receptors) and helping protein maturation (such as certain kinases and signaling proteins), but it has also been found in some other physiological processes in cells, including mutant proteins. Evolutionary stability, cytoskeletal rearrangement, nuclear protein transport, cell proliferation and apoptosis, protein degradation, antigen presentation, and lipopolysaccharide recognition.
  • Hsp90a is also linked to a variety of diseases, including cancer, autoimmune diseases and cardiovascular diseases.
  • a monoclonal antibody that uses the LKVIRK sequence of Hsp90a as an antigenic determinant is therapeutically active in antifungal infection, and is currently undergoing clinical trials by Neutec under the trade name Mycogrip®.
  • Hsp90a is secreted under extreme conditions (Liao et al. (2000) J. Biol. Chem. 275, 189-96), as a classical intracellular protein, in which Hsp90a occurs both extracellularly and in the cell. There are few reports on how to function outside. In previous reports, Hsp90a was found to be involved in antigen-presenting cells to aid antigen presentation, and was also found to be one of four proteins associated with lipid rafts on the cell surface, binding to lipopolysaccharides and causing intracellular protein responses (Triantafilou) Et al. (2002) Trends in Immunology 23, 301-4).
  • Hsp90a has also been found to be highly expressed on the surface of many tumor cells, including small cell carcinoma, melanoma, and liver cancer cell lines (Ferrarini et al. (1992) Int. J. Cancer 51, 613-19). The high expression of Hsp90a on the surface of these cell lines is presumed to be associated with antigen presentation, but there is no definitive evidence.
  • Hsp90a can help transmembrane protein transport (Schlatter et al. (2002) Biochem. J. 362, 675-84) and is associated with drug efflux in leukemia, lung cancer and ovarian cancer (Rappa) Et al (2002) Oncol. Res. 12, 113-9 and Rappa et al (2000) Anticancer Drug Des 15, 127-34).
  • Intracellular Hsp90a is also an important target for the development of anti-tumor drugs. Intracellular Hsp90a is involved in the regulation of many signaling pathways leading to cell carcinogenesis. Inhibition of Hsp90a can cause some selective degradation of signaling proteins involved in cell proliferation, cell cycle regulation, and apoptosis. Recently, many known antibiotics (such as Geldanamycin ⁇ Radicicol and Coumermycin A1) have been found to be inhibitors of Hsp90a, and WO 00/53169 describes this finding and proposes to inhibit chaperone by inhibiting the binding of chaperone to its substrate. A method of activity in which Coumarin and its derivatives are considered to have such a function. However, the inhibitors mentioned in WO 00/53169 are directed against intracellular Hsp90a.
  • antibiotics such as Geldanamycin ⁇ Radicicol and Coumermycin A1
  • WO 00/53169 describes this finding and proposes to inhibit chaperone by inhibiting the binding of chaperone to its substrate. A method
  • EP 1457499 A1 describes the function of extracellular Hsp90a in tumor cell invasion, and Hsp90a promotes tumor invasion by promoting the secretion or activation of matrix metalloproteinase MMP-2. Based on this finding, EP 1457499A1 proposes to inhibit tumor invasion and metastasis by inhibiting the activity of extracellular Hsp90a, and to determine the invasive ability of tumor cells by detecting the response of tumor cells to inhibitors of Hsp90a, as well as its invasion and Hsp90a. Relevance.
  • WO/2008/070472 proposes the detection and prognosis of anti-tumor therapeutic effects against Hsp90a by detecting Hsp90a in blood plasma and its related factors, in which the level of blood plasma Hsp90a and its inhibitor BA are provided in the examples (including 17- Correlation between the therapeutic effects of AAG and 17-DMAG), and the correlation of tumor volume size with the level of blood plasma Hsp90a in a mouse tumor model.
  • the presence of Hsp90a in the blood of tumor patients has not been identified, and the correlation between Hsp90a and tumor malignancy, especially metastasis, has not been found.
  • Hsp90a has not been proposed as an independent tumor marker in tumor diagnosis and prognosis. the use of.
  • Hsp90a in serum is associated with clinical stage of non-small cell lung cancer (Xu et al. (2007) J. Cancer Mol. 3, 107-112). Hsp90a in the serum of patients with lung cancer is significantly improved compared with patients with normal and benign tumors.
  • this article also did not identify the presence of Hsp90a in the blood of tumor patients, nor did it mention its correlation with tumor metastasis; in addition, this article only studied non-small cell lung cancer, for breast cancer, liver cancer, pancreas The correlation and specificity of cancer and blood plasma Hsp90a are not involved; and this article only qualitatively studies changes in serum Hsp90a levels in cancer patients, changes in absolute content, and normalization required for tumor diagnosis and prognosis. And the range of abnormal content is not defined. Summary of the invention
  • the present invention is based on the discovery that the level of Hsp90a in the blood is associated with the occurrence of various tumors and their degree of malignancy and metastasis. Therefore, Hsp90a in the blood can be used as a new tumor marker.
  • the present inventors have found that Hsp90a in the blood is different from intracellular Hsp90a, and the former has four amino acids deleted at the C-terminus.
  • the invention provides an isolated polypeptide, the polypeptide of the invention comprising or consisting of the amino acid sequence of SEQ ID No. 1.
  • the polypeptide of the present invention may be phosphorylated, wherein one or more amino acid residues selected from the group consisting of the amino acid sequence of SEQ ID No. 1 are phosphorylated: threonine at position 90, 231 Serine at position, serine at position 263, tyrosine at position 309, and combinations thereof.
  • threonine at position 90 of the polypeptide is phosphorylated.
  • the polypeptide of the invention can be used as a tumor marker. Detection of the level of the polypeptide of the present invention in the blood by using the specific composition of the polypeptide of the present invention can help to judge the occurrence of various tumors and the degree of malignancy and metastasis.
  • the invention relates to the use of a specific conjugate of a polypeptide of the invention in a kit for preparing, for example, for assisting in the determination of a tumor by detecting the level of a polypeptide of the invention in a blood paddle Presence, staging, and/or transfer for tumor screening of high-risk populations by detecting levels of the polypeptides of the invention in a blood paddle for use in detecting tumor patients by detecting levels of the polypeptides of the invention in a blood paddle
  • the prognosis is judged or used to determine whether the surgery, radiation or medication for the tumor patient is effective and/or when to stop treatment by detecting the level of the polypeptide of the invention in the blood paddle.
  • the specific conjugate of a polypeptide of the invention is a specific antibody to a polypeptide of the invention.
  • the antibody is a monoclonal antibody or antigen-binding fragment thereof, such as scFv, Fab, Fab' and F(ab':>2.
  • the antibody is deposited by CGMCC No.
  • the antibody specifically binds to a polypeptide of the invention, and preferably specifically binds to a polypeptide of the invention in a blood paddle.
  • the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein one or more amino acid residues of the polypeptide corresponding to SEQ ID No. 1 selected from the group consisting of the following are phosphorylated: Threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.
  • the antibody specifically binds to the threonine at position 90 is a phosphorylated polypeptide of the invention.
  • the invention relates to the use of an inhibitor of a polypeptide of the invention in the manufacture of a pharmaceutical composition for the prevention or treatment of tumor metastasis.
  • the inhibitor is a specific antibody to a polypeptide of the invention.
  • the antibody is a humanized antibody or antigen-binding fragment thereof.
  • the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein the polypeptide corresponds to
  • One or more amino acid residues selected from the group consisting of SEQ ID No. 1 are phosphorylated: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309 Amino acid and combinations thereof.
  • the antibody specifically binds to the threonine at position 90 which is a phosphorylated polypeptide of the invention.
  • the antibody of the invention is monoclonal antibody E9 or D10 produced by a cell line with accession number CGMCC No. 2903 or 2904.
  • the tumor may be, for example, lung cancer, liver cancer, gastric cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymphoma, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, Leukemia, malignant melanoma, sarcoma, kidney cancer, biliary cancer.
  • the invention also relates to an antibody that specifically binds to a polypeptide of the invention that specifically binds to a polypeptide of the invention in a blood paddle.
  • the antibody is monoclonal antibody E9 or D10 produced by a cell line with accession number CGMCC No. 2903 or 2904.
  • the antibody is a humanized antibody or antigen-binding fragment thereof.
  • the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein one or more amino acid residues of the polypeptide corresponding to SEQ ID No.
  • the antibody specifically binds to a threonine at position 90 which is a phosphorylated polypeptide of the invention.
  • the invention in another aspect, relates to a method of inhibiting invasion and metastasis of a tumor, the step comprising inhibiting phosphorylation of Hsp90a in tumor cells.
  • the method of the invention comprises inhibiting phosphorylation of threonine at position 90 of Hsp90a in tumor cells.
  • the method of the invention comprises overexpressing a nucleic acid encoding protein phosphorylase 5 (PP5) in a tumor cell, and preferably overexpressing PP5 by gene introduction.
  • PP5 nucleic acid encoding protein phosphorylase 5
  • Figure 1 The level of Hsp90a in the blood plasma of tumor-bearing mice is increased compared with normal mice.
  • Figure 2 The level of Hsp90a in the blood plasma of patients with malignant tumors is higher than that of normal people.
  • Figure 4 Standard curve for determination of Hsp90a concentration in blood plasma using murine mAb E9 and rabbit polyclonal antibody S2 (sandwich ELISA).
  • Figure 5 Quantitative comparison of Hsp90a in the blood plasma of patients with liver cancer, lung cancer, breast cancer, pancreatic adenocarcinoma, and benign breast cysts and uterine fibroids using a sandwich ELISA method.
  • Hsp90a in liver cancer patients was determined by sandwich ELISA.
  • the content of Hsp90a in patients with benign tumors was in the range of 2-10 ng/ml, most of them concentrated in 2-5 ng/ml; 69% (20/29)
  • the content of Hsp90a in the blood plasma of patients with liver cancer is above 50 ng/ml, and its mean value is >10 times higher than that of benign tumor patients. It is basically consistent with the results of immunoblotting, and also shows the level of Hsp90a in blood paddles. Correlation of the degree of malignancy of the tumor.
  • Hsp90a in lung cancer patients was determined by sandwich ELISA. Compared with patients with benign tumors, 64% (9/14) lung cancer patients had a blood plasma Hsp90a content above 50 ng/ml, and the mean value was compared with benign tumor patients. An increase of > 10 times indicates a correlation between the level of hemorrhagic paddle Hsp90a and the degree of malignancy of the tumor.
  • Hsp90a in breast cancer patients was determined by sandwich ELISA. Compared with patients with benign tumors, the highest rate was >5 times higher, and the overall level was significantly different from that of benign tumor patients.
  • Hsp90a in patients with pancreatic cancer was determined by sandwich ELISA. Compared with patients with benign tumors, the content of Hsp90a in patients with 100% (10/10) pancreatic cancer was above 50 ng/ml, and the mean and benign tumors. The patient had a >10-fold increase, indicating a correlation between the level of hemorrhagic paddle Hsp90a and the malignancy of the tumor.
  • Figure 6 Quantitative comparison of Hsp90a levels in the plasma paddles of patients with and without metastasis using a sandwich ELISA.
  • Liver cancer patients were grouped according to whether the tumors metastasized. Comparing the two groups, the patients with tumor metastasis had a blood plasma Hsp90a content above 200 ng/ml, and no metastasis between 50-200 ng/ml.
  • Figure 7 Quantitative comparison of Hsp90a in the blood plasma of patients with inflammation (including pneumonia, hepatic patients), normal subjects, and tumor patients using the sandwich ELISA method.
  • Hsp90a in the blood paddle of hepatitis patients (hepatitis A and hepatitis B) is between 2-10 ng/ml, and there is no significant difference compared with normal people.
  • FIG. 8 Hsp90a in the blood pad is secreted by tumor cells.
  • Figure 9 Determination of the absence of Hsp90a C-terminal secretion by tumor cells.
  • Hsp90a in the blood paddle is a 4-amino acid deletion at the C-terminus.
  • Figure 11 Detection of the phosphorylated form of Hsp90a in the blood paddle.
  • Figure 12 Increase in the level of threonine phosphorylated Hsp90a in the blood plasma of tumor patients.
  • Figure 13 The increase in the 90th threonine phosphorylated Hsp90a in the blood plasma of tumor patients is consistent with the increase in the total Hsp90a content in the blood plasma of tumor patients.
  • FIG. 14 Phosphorylation of the threonine at position 90 is essential for the secretion of Hsp90a.
  • Figure 15 PP5 dephosphorylation of threonine at position 90 of Hsp90a.
  • human PP5 was overexpressed, or the expression of human PP5 was inhibited by RNA interference technology, and the phosphorylation of threonine at position 90 of Hsp90a was observed.
  • the 90th threonine phosphorylated Hsp90a (pT90-Hsp90a) was significantly reduced (0.55 in the control group); when the expression of endogenous human PP5 was inhibited, the 90th threonine phosphate Hsp90a (pT90-Hsp90o was significantly increased (1.58 for control).
  • Figure 16 Intracellular PP5 regulates the secretion of Hsp90a.
  • Figure 17 Relationship between the expression level of PP5 and the secretion of Hsp90a by cells.
  • Figure 18 Relationship between the expression level of PP5 and tumor cell migration ability.
  • Figure 19 Specific antibodies to Hsp90a are able to inhibit tumor cell migration.
  • Figure 20 Activity of Hsp90a-specific antibodies to inhibit tumor metastasis. Microbial material preservation information
  • the SP2/0-Agl4 mouse hybridoma cell line of monoclonal antibody E9 was deposited on February 24, 2009 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Datun Road, Chaoyang District, Beijing, China) Microbiology Institute:), the deposit number is CGMCC No. 2903.
  • the SP2/0-Agl4 mouse hybridoma cell line of monoclonal antibody D10 was deposited on February 24, 2009 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Datun Road, Chaoyang District, Beijing, China) Microbiology Institute:), the deposit number is CGMCC No. 2904
  • Cellular carcinogenesis is caused by changes in certain signal transduction pathways in cells, accompanied by changes in protein expression levels, modification types, and spatial distribution. These protein changes can be used to monitor tumor development and progression. Called a tumor marker.
  • proteomics methods and techniques qualitative and quantitative monitoring of changes in tumor proteomes has become possible, and many new tumor markers have been discovered, providing more accurate and reliable clinical diagnosis and prognosis of tumors. Basis.
  • the present invention is based on the discovery of a novel blood tumor tumor marker, namely Hsp90a o present in the blood plasma and Hsp90a in the cell (the amino acid sequence is SEQ ID No. 3, and the encoding nucleic acid sequence is SEQ ID No. 4).
  • the C-terminus of Hsp90a in the blood paddle is missing 4 amino acids.
  • the invention provides an isolated polypeptide which is Hsp90a in a blood plasma or serum, the polypeptide comprising or consisting of the amino acid sequence of SEQ ID No. 1.
  • polypeptide of the invention refers to Hsp90a in blood plasma or serum comprising the amino acid sequence of SEQ ID No. 1 or ammonia by SEQ ID No. 1.
  • the composition of the base acid sequence Preferably, the term "polypeptide of the invention” as used in this application refers to a polypeptide consisting of the amino acid sequence of SEQ ID No. 1.
  • Hsp90a in blood plasma or “Hsp90a in serum” are equivalently referred to as non-intracellular and cell surface Hsp90a proteins present in the blood, which may be freely present alone or in combination with other blood.
  • the extracellular protein in the form of a combined form exists.
  • polypeptide is used interchangeably with "protein”.
  • the present invention also relates to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 or consisting of the amino acid sequence of SEQ ID No. 1.
  • the polynucleotide comprises the sequence of SEQ ID No. 2 or consists of the sequence of SEQ ID No. 2.
  • the polypeptide of the present invention is phosphorylated in the form of a blood plasma, wherein one or more amino acid residues selected from the group consisting of the amino acid sequence of SEQ ID No. 1 are phosphoric acid.
  • the threonine at position 90 of the polypeptide is phosphorylated.
  • EP1457499A1 describes extracellular Hsp90a and suggests that its inhibitors can be used to treat tumor metastasis, as well as to diagnose the invasive ability of tumor cells, and whether this ability is dependent on Hsp90a.
  • the method described in EP 1457499A1 detects Hsp90a on the cell membrane surface, does not involve Hsp90a in the blood plasma, and does not involve determining the malignant level of the tumor based on the level of Hsp90a, and is used for early diagnosis, staging, efficacy detection and prognosis of the tumor.
  • WO/2008/070472 proposes to determine the effect of anti-tumor therapy against Hsp90a by detecting Hsp90a in blood plasma and its related factors, but does not mention the use of Hsp90a as an independent tumor marker in tumor diagnosis and prognosis.
  • Hsp90a in the blood paddle is a new tumor marker that can be used for the diagnosis and prognosis of tumors and their metastasis.
  • kits of the invention are resistant to specific conjugates comprising, for example, the polypeptides of the invention.
  • the kit of the present invention can be used to detect the amount of Hsp90a in a blood plasma.
  • the invention also relates to the use of a specific conjugate of a polypeptide of the invention in the preparation of a kit
  • the kit can be used, for example, to detect the presence, degree of malignancy or metastasis of a tumor by detecting the level of Hsp90a in the blood paddle; for screening a high-risk population by detecting the level of Hsp90a in the blood pad; for detecting The prognosis of the tumor patient is judged by the level of Hsp90a in the blood paddle; or it is used to determine whether the surgery, radiotherapy or drug treatment for the tumor patient is effective and/or decide when to stop the treatment by detecting the level of Hsp90a in the blood paddle.
  • the term "specific conjugate” refers to a molecule which binds to a polypeptide of the present invention with high affinity, and also includes a molecule which binds Hsp90a intracellularly or on the cell surface with high affinity.
  • the specific conjugate is, for example, a protein, and preferably, the specific conjugate is an Hsp90a-specific antibody.
  • the antibody is a monoclonal antibody or antigen-binding fragment thereof, such as scFv, Fab, Fab' and F (ab': > 2.
  • the antibody is deposited by The monoclonal antibody E9 or D10 produced by the cell line of CGMCC No. 2903 or 2904.
  • Monoclonal antibodies are obtained by selecting cells secreting the desired antibodies and culturing them in vitro. Methods for preparing monoclonal antibodies are well known in the art (; K6hler G & Milstein C. (1975) Nature. 256, 495-7).
  • the specific procedure for the preparation of a monoclonal antibody that specifically recognizes Hsp90a is as follows: BALB/C mice were immunized with recombinant human Hsp90a, and primary immunization with the antigen 100 g plus Freund's complete adjuvant was injected subcutaneously in the back; the second immunization after 3 weeks, dose Same as above, Jiafu's incomplete adjuvant adjuvant intraperitoneal injection; the third immunization after 3 weeks, the dose is the same as above, no adjuvant adjuvant intraperitoneal injection (5 ⁇ 7 days after blood sampling to measure its titer); another 3 weeks after booster immunization, The dose was 200 ⁇ 8 and injected intraperitoneally.
  • SP2/0 hybridoma cells source: ATCC, number: CRL-1581
  • HAT cloning of hybridoma cells by limiting dilution
  • immunoblotting It was identified by the method of ELISA, and finally a hybridoma cell strain secreting an antibody which specifically recognizes Hsp90a was obtained.
  • an antibody which can be used to prepare the above kit specifically binds to Hsp90a, and preferably specifically binds to Hsp90a in a blood plasma.
  • the antibody specifically binds to phosphorylated Hsp90a, wherein one or more amino acid residues of the Hsp90a selected from the group consisting of phosphorylated: threonine at position 90, Serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.
  • the antibody specifically binds to the threonine at position 90 which is phosphorylated Hsp90a.
  • the invention also relates to a method of detecting the level of a polypeptide of the invention in a blood paddle.
  • the amount of Hsp90a in the blood plasma can be detected by any suitable method.
  • the method comprises directly or indirectly determining the level of a polypeptide of the invention in a blood paddle to help determine the occurrence of the tumor and its degree of malignancy and Transfer.
  • a method for direct assay comprises the use of a specific conjugate of the polypeptide to detect a polypeptide of the invention, e.g., using an antibody specific for the recognition of the polypeptide for immunoblotting or ELISA.
  • Indirect assays include, for example, by measuring the activity of Hsp90a to reflect the concentration of Hsp90a, such as luciferase thermal denaturation assay based on detection of Hsp90a chaperone activity.
  • the content of Hsp90a in the blood plasma is detected by ELISA or immunoblotting, which mainly comprises the following steps:
  • step b) ELISA or immunoblotting method to detect the content of Hsp90a in the blood paddle or serum obtained in step a), and use the blood paddle of a healthy normal person as a negative control, and the blood paddle of the patient who has been diagnosed as a malignant tumor is used as a positive control, optionally Generating a standard curve for the concentration of Hsp90a;
  • the steps may also use other detection means based on the principle of antigen-antibody reaction, as well as other detection means which can directly or indirectly reflect the concentration of Hsp90a, for example, by detecting the activity of Hsp90a to reflect the concentration of Hsp90a.
  • the Hsp90a standard used to determine the Hsp90a concentration standard is purified from the blood plasma of tumor patients and can also be obtained by recombinant expression, including the full length, fragment of Hsp90a, and recombinant proteins containing Hsp90a sequences and other groups. Coupled complex.
  • the "standard curve of the concentration of Hsp90a” refers to a corresponding curve of the concentration and the absorbance measurement value determined by an ELISA method using a known concentration of the Hsp90a standard sample.
  • “Hsp90a Standard” refers to a sample of blood plasma Hsp90a protein, recombinant Hsp90a protein, fragment and derivative with a purity greater than 95%.
  • Determining the degree of malignancy of a tumor means determining the level of the patient's blood plasma Hsp90a according to the concentration of the sample Hsp90a, the value of the negative control and the positive control, and thus the tumor properties of the patient (ie, benign or malignant:) Make a judgment.
  • the ELISA methods that can be used include both sandwich and competition methods. Among them, a competitive method with high sensitivity is preferred.
  • the general steps of the sandwich method include: a) attaching a specific antibody to a solid phase carrier to form a solid phase antibody, and washing to remove unbound antibody and impurities; b) adding a test specimen: making it with a solid phase antibody Contacting the reaction for a period of time, allowing the antigen in the specimen to bind to the antibody on the solid phase carrier to form a solid phase antigen-antibody immune complex, and washing to remove other unbound substances; C) adding the enzyme-labeled antibody: making the solid phase immune complex The antigen is bound to the enzyme-labeled antibody, and the unbound enzyme-labeled antibody is thoroughly washed.
  • the amount of the enzyme carried on the solid phase carrier is positively correlated with the amount of the test substance in the specimen; d) the substrate is added, and the sandwich compound is added.
  • the enzyme catalyzed substrate in the material becomes a colored product.
  • Qualitative or quantitative determination of the antigen is based on the extent of the color reaction.
  • the general steps of the competition method include: a) attaching a specific antibody to a solid phase carrier to form a solid phase antibody, and washing; b) adding a mixed solution of the sample to be tested and a certain amount of the enzyme-labeled antigen to the solid phase Antibody response. If there is no antigen in the specimen, the enzyme antigen can be successfully combined with the solid phase antibody. If the sample contains an antigen, it binds to the solid phase antibody at the same opportunity as the enzyme-labeled antigen, competitively occupies the opportunity to bind the enzyme-labeled antigen to the solid-phase carrier, and combines the enzyme-labeled antigen with the solid-phase carrier. The amount is reduced.
  • the reference tube Only the enzyme-labeled antigen is added to the reference tube, and after incubation, the binding of the enzyme-labeled antigen to the solid-phase antibody can reach the most sufficient amount and wash. c) Adding a substrate to develop color, the reference tube has the largest number of antigens due to binding, so the color is the deepest. The difference between the reference tube color depth and the color depth of the tube to be tested represents the amount of the sample antigen to be tested. The lighter the color of the tube to be tested, the more antigen content in the specimen.
  • the method is detected by ELISA sandwich method, and the antibody used is derived from a specific antibody of two different species of blood plasma Hsp90a, wherein the plating antibody is preferably a rabbit polyclonal antibody with strong binding ability, and the other antibody preferably has good recognition specificity.
  • the plating antibody is preferably a rabbit polyclonal antibody with strong binding ability, and the other antibody preferably has good recognition specificity.
  • Mouse monoclonal antibody Both antibodies must not cross-react.
  • the antibody used is a specific antibody of the blood plasma Hsp90a, and has strong recognition ability and specificity for recognition of the competitor and Hsp90a.
  • the competitor used is a blood paddle.
  • a marker for the Hsp90a standard This marker does not interfere with the binding of the Hsp90a standard to its antibody.
  • the antibody used is a specific antibody of blood plasma Hsp90a; the secondary antibody may be coupled with horseradish peroxidase or alkaline phosphatase;
  • the substrate includes DAB and a fluorescent substrate, and preferably the sensitivity of the above-mentioned detection method is as high as 10 ng/ml or less.
  • the specific antibody of the blood plasma Hsp90a used in the present invention includes the full length of the antibody, a fragment and a derivative thereof.
  • the antibodies of the invention may also be replaced by other Hsp90a specific conjugates, including small molecule compounds, polypeptides and derivatives thereof.
  • the marker of the blood plasma Hsp90a standard used in the present invention wherein the labeling group includes biotin and various fluorescent labeling reagents. Among them, biotin-labeled competitors are preferred.
  • Cancers detectable using the kit of the present invention include, but are not limited to, lung cancer, liver cancer, gastric cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymphoma, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal carcinoma , cervical cancer, leukemia, malignant melanoma, sarcoma, kidney cancer, biliary cancer, etc.
  • the degree of tumor and its malignancy refers to whether the tumor is benign, malignant, or whether it metastasizes.
  • the preliminary study by the present inventors confirmed that the normal human blood paddle Hsp90a ranged from 2-50 ng/ml, more concentrated to 2-10 ng/ml, and the bloodstain Hsp90a level of patients diagnosed with tumor was higher than the normal level.
  • the level of blood plasma Hsp90a is higher than 50 ng/ml in patients with metastasis, and most of them are above 200 ng/ml. This makes the Hsp90a level of the blood paddle a new tumor marker that can be used to help determine the presence of a tumor, especially if there is a metastasis of the tumor.
  • the kit or method of the invention can be used to determine if an individual has a tumor, particularly a malignant tumor, and whether the tumor has metastasized.
  • the kit or method of the present invention can be used to determine the level of Hsp90a in a blood sample from a suspected tumor patient or a suspected tumor metastasis, and optionally compared to a normal control, and then judged based on the level of Hsp90a in the sample.
  • the patient has a possibility of tumor or tumor metastasis. Elevated levels of Hsp90a suggest that patients are more likely to have malignant tumors, whereas in patients with known tumors, a significant increase in Hsp90a levels suggests a greater likelihood of tumor metastasis.
  • the kit or method of the invention can be used to screen a high risk population for tumors by detecting levels of Hsp90a in the blood plasma.
  • the kit or method of the present invention can be used to determine the level of Hsp90a in a blood sample from a high-risk population, and optionally compared to a normal control, and then based on the level of Hsp90a in the sample, which individuals in the population may have been A tumor appears. Elevated levels of Hsp90a suggest a greater likelihood of malignancy in individuals.
  • the kit or method of the invention can be used to judge the prognosis of a tumor patient by detecting the level of Hsp90a in the blood paddle.
  • the kit or method of the invention can be used to determine the level of Hsp90a in a blood sample from a tumor patient, and optionally with normal The control or the patient's previous blood plasma Hsp90a level was compared, and then the prognosis of the tumor patient was judged based on the Hsp90a level in the sample. Maintaining high levels of Hsp90a or further elevation of Hsp90a levels may be associated with an unfavorable prognosis. Therefore, the clinician can be reminded to take a closer look at the patient and, if necessary, change the current treatment plan.
  • the kit or method of the invention can be used to determine whether surgery, radiation or medication for a tumor patient is effective and/or to decide when to stop treatment by detecting the level of Hsp90a in the blood paddle.
  • the Hsp90a level in a blood plasma sample from a tumor patient can be determined using the kit or method of the invention, and optionally compared to a normal control or the patient's previous blood plasma Hsp90a level, and then based on the Hsp90a within the sample.
  • Levels determine whether surgery, radiation therapy, or medication for the tumor patient is effective and/or decide when to stop treatment.
  • the present invention determines whether a patient has a malignant tumor or tumor by detecting the level of Hsp90a in a blood sample.
  • the method of metastasis is an auxiliary detection method in the process of tumor diagnosis. According to the results of this method, it is not directly determined whether the patient has a malignant tumor, nor can it directly determine whether there is metastasis of the malignant tumor, nor can it directly determine the tissue origin of the malignant tumor and its pathological type or the location and number of metastatic lesions. Therefore, for clinicians in athereurology, the establishment of diagnosis still relies on imaging, pathology and other methods as well as the judgment of clinical doctors.
  • the methods of the present invention provide additional reference information only to the clinician or researcher.
  • the detection of Hsp90a levels in the blood plasma can provide valuable supplementary reference information for determining whether a patient has a malignant tumor, especially if there is a tumor metastasis.
  • the above-described methods for performing screening of a high-risk population, judging the prognosis of a tumor patient, and evaluating the therapeutic effect of the present invention are also intended to provide supplementary reference information for a clinician or a scientific researcher.
  • Hsp90a in the blood plasma is derived from tumor cells, but is different from Hsp90a in tumor cells, and thus it is possible to inhibit the development and metastasis of tumor by inhibiting Hsp90a in the blood plasma.
  • the present inventors have also demonstrated this by experiments in which the specific antibody of the blood plasma Hsp90a can inhibit tumor metastasis in mice, and further, the blood plasma Hsp90a can be used as a new target for screening new anticancer drugs.
  • the invention relates to the use of an inhibitor of a polypeptide of the invention in the manufacture of a pharmaceutical composition for preventing or treating tumor metastasis.
  • the inhibitor is an Hsp90a specific antibody.
  • the antibody is a humanized antibody or antigen-binding fragment thereof.
  • the antibody specifically binds to phosphorylated Hsp90a, wherein one or more amino acid residues of the Hsp90a selected from the group consisting of phosphorylated: threonine at position 90, Serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.
  • the antibody specifically binds to the threonine at position 90 which is phosphorylated Hsp90a.
  • the antibody is a monoclonal antibody E9 or D10 produced by a cell line with accession number CGMCC No. 2903 or 2904. As described in the examples below, this antibody completely inhibits lymph node metastasis in mouse tumors, and the efficiency of inhibiting lung metastasis is up to 56%.
  • the invention also relates to an antibody that specifically binds to a polypeptide of the invention that specifically binds to Hsp90a in a blood paddle.
  • the antibody is a monoclonal antibody E9 or D10 produced by a cell line with accession number CGMCC No. 2903 or 2904.
  • the antibody is a humanized antibody or antigen-binding fragment thereof.
  • the antibody specifically binds to phosphorylated Hsp90a, wherein one or more amino acid residues of the Hsp90a selected from the group consisting of phosphorylated: threonine at position 90, Serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.
  • the antibody specifically binds position 90 is threonine phosphorylated Hsp90a o
  • the antibody inhibits tumor growth, in particular metastasis.
  • the invention also relates to a conjugate comprising an antibody of the invention and a detection moiety or therapeutic moiety.
  • the detection moiety is, for example, a fluorophore
  • the therapeutic moiety is, for example, a tumor chemotherapeutic agent.
  • the present inventors also found that there is a certain correlation between the level of Hsp90a secreted by tumor cells and the expression level of intracellular protein phosphatase 5 (PP5).
  • PP5 protein phosphatase 5
  • the secretion of Hsp90a is lower, while the expression level of PP5 is higher.
  • the secretion of Hsp90a is high, while the expression level of PP5 is very low. Therefore, the level of Hsp90a secreted by tumor cells is negatively correlated with the level of intracellular PP5. Therefore, by detecting the expression level of PP5 in tumor tissues, the level of Hsp90a secreted by the tumor cells into the blood plasma can be predicted, so that the early diagnosis of the tumor can be performed.
  • the present invention also demonstrates by experiments that the secretion of Hsp90a is inhibited by intracellular PP5.
  • the nucleic acid encoding PP5 was overexpressed in the cells, the secretion of Hsp90a was inhibited, and the expression level of PP5 in the cells was decreased, and the amount of Hsp90a secreted by the cells was significantly increased. Therefore, by overexpressing the nucleic acid encoding PP5, thereby inhibiting the secretion of Hsp90a, it is possible to inhibit the development and metastasis of the tumor.
  • overexpression of PP5 can inhibit The migration of breast cancer cells MCF-7, therefore PP5 can be used as a new tumor treatment target.
  • the invention relates to a method of inhibiting invasion and metastasis of a tumor, the step comprising inhibiting phosphorylation of Hsp90a in tumor cells.
  • the methods of the invention comprise inhibiting phosphorylation of threonine at position 90 of Hsp90a in tumor cells.
  • the method of the invention comprises overexpressing a nucleic acid encoding PP5 in a tumor cell, and preferably overexpressing PP5 by gene introduction.
  • the method of the invention comprises overexpressing a nucleic acid encoding a PP5 comprising the amino acid sequence of SEQ ID No. 5 in a tumor cell, in a specific embodiment, the nucleic acid comprises the SEQ ID No.
  • Nucleotide sequence The present invention also relates to the use of a vector carrying the above-described nucleic acid encoding PP5 operably linked to a promoter for the preparation of a medicament for inhibiting phosphorylation of Hsp90a in tumor cells by overexpressing PP5 in tumor cells.
  • the drug can be used to inhibit tumor invasion and metastasis.
  • the present invention also provides methods and models for screening antitumor drugs using blood plasma Hsp90a and its derivatives, including but not limited to finding binding proteins, small peptides and small molecule compounds of blood plasma Hsp90a, and inhibiting inhibition of blood plasma Hsp90a activity. Screening of agents.
  • Example 1 Collection, preparation, and detection of blood paddles of mouse blood samples
  • Balb/c mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • an average body weight of about 20 grams were randomly divided into two groups, three in each group, one of which was connected to H22 mouse hepatoma cells.
  • CTCC, No.: GDC091 The number of cells per access was 10 6 and the control group was not inoculated with tumors.
  • the tumor diameter of the mouse grows to an average of 2 cm (about 20 days)
  • blood is taken from the fundus venous plexus, and an anticoagulant is added to the blood to avoid hemolysis. If the sample is hemolyzed, recollect it.
  • Whole blood was collected and centrifuged twice at 6000 °C at 4 ° C.
  • Example 2 Collection, preparation, and detection of blood paddle Hsp90a in normal and tumor patients Take whole blood from normal or cancer patients and send them to the laboratory within 24 hours and at low temperature (about 4 °C). Hemolysis should be avoided.
  • test results will be compared with clinical diagnosis to verify the correlation between the content of Hsp90a in the blood plasma and the degree of tumor malignancy.
  • the specific method of immunoblot detection is as follows: The blood paddle sample is mixed with the loading buffer 1:1, and 1-2 microliters is loaded for SDS-PAGE, the anti-specific antibody for identifying the blood plasma Hsp90a (rat monoclonal antibody) SPA-840, Stressgen), the second antibody is a horseradish peroxidase-conjugated goat anti-rat antibody (purchased from Nakasugi Jinqiao).
  • Hsp90a rat monoclonal antibody
  • SPA-840 serum-840
  • Stressgen the second antibody is a horseradish peroxidase-conjugated goat anti-rat antibody (purchased from Nakasugi Jinqiao).
  • the results are shown in Figure 2.
  • the content of Hsp90a in the blood plasma of liver cancer patients was detected by immunoblotting, which was about 10 times higher than that of normal people (A).
  • the content of Hsp90a in patients with benign breast cysts and uterine fibroids About 2 times higher than normal people (B
  • Hsp90a-Sall-Re ACGCGTCGACTTAGTCTACTTCTTCCATGC (SEQ ID ⁇ 8) and Hsp90a-Sphl-For: ACATGCATGCATGCCTGAGGAAAC CCAGACC (SEQ ID No. 9) (synthesized from Invitrogen) and Pfu DNA polymerase were used.
  • Source: NEB The full-length Hsp90a sequence was amplified from human liver cDNA library (source: Stratagene), and the fragment and pQE80L vector (source: Qiagen) were double-digested using Sphl and Sail (source: NEB).
  • the fragment was ligated using T4 ligase (source: NEB).
  • the ligation product was transformed into ToplO E. coli competent cells (source: Transgen) for amplification and validation, and the verified plasmid was transformed into BL21DE3 E. coli competent cells (source: Transgen) for expression to obtain recombinant human Hsp90a protein.
  • Method for purification of recombinant human Hsp90a protein ion exchange chromatography SP HP, pH 6.8, collection of conductivity 10 ms/ml elution peak; Q HP, pH 7.8, collection of conductivity 19 ms/ml elution peak.
  • the purified rabbit polyclonal antibody was named S2.
  • BALB/C mice were immunized with recombinant human Hsp90a, and the primary immunization was performed with subcutaneous multi-point injection of the antigen 100 g plus Freund's complete adjuvant on the back; the second immunization after 3 weeks, the dose was the same as above, and the incomplete injection of Jiafu's incomplete adjuvant; After 3 weeks, the third immunization was performed at the same dose as above.
  • the adjuvant was intraperitoneally injected without adjuvant (5 to 7 days after blood sampling). After another 3 weeks, the immunization was given at a dose of 200 ⁇ 8 and intraperitoneal injection.
  • SP2/0 hybridoma cells source: ATCC, number: CRL-1581
  • HAT cloning of hybridoma cells by limiting dilution
  • immunoblotting The ELISA method was used to identify hybridoma cell lines E9 and D10 secreting antibodies that specifically recognize Hsp90a, and the accession numbers were: CGMCC No. 2903 and 2904, respectively, which were deposited on CGMCC on February 24, 2009.
  • the indirect ELISA was used to detect the titers of E9 and D10. The results are shown in Figure 3.
  • the E9 and D10 titers can reach 500,000, which can be used to detect Hsp90a in the blood plasma.
  • the specific steps of the indirect method ELISA were as follows: using recombinant human Hsp90a at 4 ° C overnight, coating concentration of 10 g / ml; blocking at 37 ° C for one hour and then according to 1:400, 1: 1600, 1: 6400, 1 :25600, 1 : 102400, 1 :509600 Gradient dilution of E9 or D10, incubate for 2 hours at room temperature; then incubate with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (purchased from Nakasujinjin) for 1 hour at room temperature , using o-phenylenediamine to develop color, read at OD 490 nm.
  • Example 4 Determin
  • the antibody used is derived from a specific antibody of two different species of blood plasma Hsp90a, wherein the plating antibody uses a rabbit polyclonal antibody S2 with strong binding ability (preparation method is As described in Example 3, another antibody preferably recognizes a specific specific murine mAb E9 (preparation method is described in Example 3).
  • the two antibodies did not cross-react, and the repeatability was good and the detection sensitivity was high. From the standard curve in Figure 4, the detection level of this method can reach 5 ng/ml.
  • Example 5 Collection and preparation of human blood paddle samples, and determination of blood plasma Hsp90a and determination of tumor malignancy (sandwich ELISA)
  • Sandwich ELISA uses two different sources of anti-Hsp90a antibodies, of which self-made rabbit-derived polyclonal anti-S2 (preparation method is described in Example 3) for overnight plating at 4 ° C, and one hour after blocking at 37 ° C
  • the blood sample and the standard curve are sampled, the blood sample to be tested is diluted 10 times, and 100 ⁇ l is added to each well; the standard curve sample is filled with a known amount of standard Hsp90a sample and 10 ⁇ L of blank negative serum per well to exclude serum.
  • patients with benign tumors including 7 patients with benign breast cysts and uterine fibroids
  • the blood plasma Hsp90a content is above 50 ng/ml, and its mean value is >10 times higher than that of benign tumor patients, P O.
  • patients with inflammation including 10 cases of pneumonia (A:), hepatitis patients (5 cases of hepatitis A and 5 cases of hepatitis B) (B) Hsp90a content in the blood paddle is between 2-10 ng / ml, and normal people There were no significant differences in (3 cases), P values were 0.2988, 0.5177, 0.138, Student's t test.
  • mice with an average body weight of 20 grams (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into two groups, 6 in each group, and the underarms were connected to Hela cervical cancer cells (Source: ATCC, Code: CCL -2), the number of cells per access is 10 6 .
  • the control group was a normal mouse that was not inoculated with tumors.
  • Hsp90a in the paddle As a result, as shown in Fig. 8, Hsp90a in the blood plasma of a mouse inoculated with a human-derived tumor can be recognized by an antibody that specifically recognizes a human source but does not recognize a mouse-derived Hsp90a, indicating that Hsp90a in the blood plasma is composed of tumor cells. Secreted.
  • Example 7 Determination of Hsp90a secreted by tumor cells as a C-terminal deletion
  • Primers consisting of the nucleotide sequences of AAGAC (SEQ ID No. 10) and Hsp90a-pc3.1-Xhol-Re-nostop: CCCGCTCGA GTGTCTACTTCTTCCATGCGTGATG (SEQ ID No.
  • Example 12 (synthesized from Invitrogen) and Pfu DNA polymerase (source) : NEB), using the pQE80L-Hsp90a plasmid obtained in Example 3 as a template, the full-length Hsp90a sequence was amplified, and digested and ligated into pcDNA3.1/Myc-His (-) (vector source: Invitrogen) , to obtain an additional Hc90a with a Myc tag at the N-terminus, named Myc-H; or use Hsp90a-pc3.1 -Nhe 1 -For-His-Myc:GCTAGCTAGCGCCACCATGCATCATCA
  • the two vectors were transiently transfected for overexpression, and the secretion of exogenous Hsp90a (i.e., overexpressed Hsp90a, which is different from endogenous and background Hsp90a) was observed. Changes in Hsp90a secreted into the extracellular medium were detected by anti-Hsp90a, anti-Myc tag, anti-His tagged antibodies. The results showed that Hsp90a secreted extracellularly was a C-terminal deletion form (Fig. 9A).
  • Myc-His-H was used as a template to mutate the last four amino acids (EEVD) at the C-terminus, where EE->AA indicates that two EEs are mutated into two Ala (using SEQ ID No. 11 and Hsp90a-EE) -AA: Amplification of the nucleotide sequence consisting of GGCCGCTCGAGTGTCTACTGCTGCCATGCGTGATGTG (SEQ ID No. 13), VD->AA indicates two VDs Mutated into two Ala (amplified by primers consisting of the nucleotide sequences of SEQ ID No. 11 and Hsp90a-VD-AA: GGCCGCTCGA GTTGCTGCTTCTTCCATGCGTGATGTG (SEQ ID No.
  • All Ala indicates that EEVD is mutated into Ala (amplified using primers consisting of the nucleotide sequence of SEQ ID No. ll and Hsp90a-EEVD-AAAA: GGCCGCTCGAGTTGCTGCTGCTGCCATGCG TGATGTG (SEQ ID No. 15)), CA4 indicates the last EEVD deletion of four amino acids (using SEQ ID) No. 11 and Hsp90a-CA4-Xho: CCGCTCGAGTCATGCGTGATGTGTCGTCATCTC (SEQ ID ⁇ . 16) primers of nucleotide sequence composition were amplified).
  • Hsp90a in the blood pad was detected by immunoprecipitation and immunoblotting. That is, first, a rabbit polyclonal antibody (Source: Labvision) that specifically recognizes Hsp90a was used to immunoprecipitate Hsp90a in a blood plasma, and a rabbit polyclonal antibody (Anti-C4) that specifically recognizes the C-terminal 4 amino acid EEVD of Hsp90a (Anti-C4) was used ( Self-made, the antigen used for immunization was a three-repeat peptide of EEVD coupled to a carrier protein, synthesized from Cypress) to detect the loss of the C-terminal end of Hsp90a in the blood plasma.
  • a rabbit polyclonal antibody (Source: Labvision) that specifically recognizes Hsp90a was used to immunoprecipitate Hsp90a in a blood plasma
  • Anti-C4 rabbit polyclonal antibody that specifically recognizes the C-terminal 4 amino acid EEVD of Hsp90a
  • a rabbit polyclonal antibody that specifically recognizes the C-terminal 4 amino acid EEVD of Hsp90a recognizes Hsp90a in whole cells, but does not recognize Hsp90a in the blood plasma, indicating that Hsp90a in the blood plasma is C-terminal deletion 4
  • the form of the amino acid is different from the intracellular Hsp90a (Fig. 10).
  • Example 9 Detection of phosphorylated forms of Hsp90a in human blood paddles
  • Hsp90a in the blood pad was detected by immunoprecipitation and immunoblotting. That is, first, a rabbit polyclonal antibody (Source: Labvision) that specifically recognizes Hsp90a is used to immunoprecipitate Hsp90a in a blood plasma, and an antibody that specifically recognizes the phosphorylation of threonine at the 90th position of Hsp90a (Rabbit) Anti-phospho-(Ser/Thr) PKA substrate pAb, Cell signaling) The threonine phosphorylation at the 90th position of Hsp90a in the blood plasma was examined.
  • a rabbit polyclonal antibody Source: Labvision
  • an antibody that specifically recognizes the phosphorylation of threonine at the 90th position of Hsp90a Rabbit
  • Anti-phospho-(Ser/Thr) PKA substrate pAb Cell signaling
  • Hsp90a in the blood plasma can be recognized by an antibody that specifically recognizes threonine phosphorylation at the 90th position of Hsp90a, indicating that Hsp90a in the blood plasma is the threonine phosphorylation at the 90th position.
  • Example 10 Determination of the content of threonine phosphorylated Hsp90a at the 90th point in a human blood paddle sample. Whole blood collected within 24 hours of normal human and liver cancer patients was taken, and the blood was taken twice by centrifugation, using a sandwich ELISA method. Methods The relative content of Hsp90a in blood plasma was measured.
  • the operation steps are firstly using a self-made rabbit-derived polyclonal antibody S2 (preparation method is described in Example 3), plating at 4 ° C overnight, and sealing at 37 ° C for one hour, adding a blood sample to be tested, and the blood sample to be tested is diluted 10 Times, add 100 ⁇ l per well; after incubating for 2 hours at 37 ° C, add an antibody (Cell signaling) that specifically recognizes the 90th point of threonine phosphorylation of Hsp90a, incubate for 2 hours at 37 ° C; The root peroxidase-conjugated goat anti-rabbit secondary antibody was incubated for 1 hour, developed with o-phenylenediamine, and read at OD 490 nm.
  • the blood volume of the same (8) liver cancer patients was simultaneously tested for the Hsp90a content and the total Hsp90a content at the 90th point of threonine phosphorylation.
  • the method for detecting the total content of Hsp90a was the same as in Example 5; the method for detecting the content of threonine phosphorylation of Hsp90a at the 90th position was the same as in Example 10.
  • the results showed that the content of threonine phosphorylation of Hsp90a and the total content of Hsp90a at the 90th point in the blood plasma of patients with liver cancer were consistent, further confirming that Hsp90a in the blood plasma is phosphorylated by threonine at the 90th position.
  • an increase in the total Hsp90a content may represent an increase in the content of threonine phosphorylated Hsp90a at the 90th position (Fig. 13).
  • Example 12 Threonine phosphorylation at position 90 is required for secretion of Hsp90a
  • Hsp90a-T89A-Sense GATCGA Primer consisting of the nucleotide sequence of ACTCTTGCAATTGTGGATACTGGAATTGGAATG (SEQ ID No. 17) and Hsp90a-T89-AntiSense: CATTCCAATTCCAGTATCCACAATTGCAAGAGT TCGATC (SEQ ID No.
  • mutant Hsp90a (T90A), a threonine mutation at position 90 Alanine, which cannot be phosphorylated, was named (T90A Hsp90a) o Overexpressing wild-type Hsp90a (WT) and mutations in the human breast cancer cell line MCF-7 (purchasing white ATCC, number HTB-22) Type and Hsp90a (T90A), the culture medium was collected, and the secretion of exogenous Hsp90a (ie, overexpressed Hsp90a, which is different from endogenous and background Hsp90a) was observed. Changes in Hsp90a secreted into the extracellular medium were detected with an antibody against Hsp90a.
  • pT90-Hsp90o After incubation of recombinant human Hsp90a protein and recombinant protein kinase A (Promega, USA) in reaction buffer (NEB, UK) for 30 hours, The pT90-Hsp90a protein was purified and the free phosphate group was removed by three times of dialysis. The purified pT90-Hsp90a protein and recombinant human PP5 protein were mixed at 30 degrees, and the non-radioactive serine/threonine phosphatase activity detection kit was used. Promega) detects the released free phosphate group.
  • the peptide substrate is part of the kit and serves as a positive control.
  • PP5 is incubated with the peptide substrate, and the release of free phosphate groups is significantly increased.
  • the full-length sequence of PP5 (SEQ ID No. 6) was amplified from the human liver cDNA library, and the gene was sequenced correctly and constructed into pcDNA3.1/Myc-His (-) (vector source: Invitrogen).
  • the vector was transferred into the human breast cancer cell line MCF-7 and overexpressed.
  • 5'-ACTCGAACACCTCGCTAAAGAGCTC-3' (SEQ ID ⁇ 7) was used as the target sequence of the siRNA of ⁇ 5, and the specific small RNA against human PP5 (synthesized by Invitrogen) was expressed, and the expression of human PP5 was expressed.
  • PP5 can cause phosphorylation and dephosphorylation of threonine at position 90 of Hsp90a.
  • the full-length sequence was sequenced and correctly constructed into pcDNA3.1/Myc-His (-) (vector source: Invitrogen).
  • the vector was transfected into the human breast cancer cell line MCF-7, and overexpressed, and the level of Hsp90a secreted by the cells was observed. The results showed that the amount of Hsp90a secreted by the cells was significantly decreased after overexpression of human PP5 (Fig. 16A).
  • RNA interference technology ie, transduction into a specific small RNA against human PP5, Invitrogen
  • Hsp90a secreted by the cells was regulated.
  • the expression of the source human PP5 was inhibited, the secretion of Hsp90a by the cells was significantly increased (Fig. 16B).
  • Example 15 Detection of PP5 content and determination of tumor malignancy
  • MCF-7, SKBR3, MDA-MB-453, 435s and 23KATCC numbered HTB-22, -30, -131, -129 and HTB-26, respectively.
  • MCF-7, SKBR3 is a less malignant breast cancer cell line.
  • these two cells can only form in situ tumors, but can not metastasize; MDA-MB-453, 435s and 231 are In the malignant breast cancer cell line, in the nude mouse tumor formation model, the two cells can form in situ tumors and metastasis, and MDA-MB-435S and 231 are commonly used to establish a tumor metastasis model.
  • the five breast cancer cell lines are arranged in descending order of malignancy.
  • Example 17 Detection of tumor cell migration activity by a blood plasma Hsp90a-specific antibody.
  • the wound healing model was used to detect the activity of the blood-splitting Hsp90a-specific antibody to inhibit tumor cell migration.
  • MCF-7 and MDA-MB-231 cells were inoculated into 12-well plates. When the cells were close to the bottom of the culture dish, some cells were scraped off with a gun tip. Form a "wound”, aspirate the scraped cells, replace the fresh DMEM medium (GIBCO), and add the specific mouse monoclonal antibody E9 of the blood plasma Hsp90a (20 ⁇ or control IgG (20 ⁇ ) The culture was continued in a 37 ° C incubator. The "wounds" were photographed at 0h, 6h, 12h, 24h, 48h and 72h.
  • Hsp90a antibody The inhibition of Hsp90a antibody on cell migration was detected by the rate of "wound" healing. The results are shown in Figure 19.
  • the specific antibody to the plasma paddle Hsp90a had >40% inhibition of MDA-MB-231 ( Figure 19A) and MCF-7 ( Figure 19B) cell migration.
  • Example 18 Blood paddle Hsp90a Specific antibodies inhibit the detection of tumor metastatic activity.

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一种新的肿瘤标志物 技术领域
本发明涉及肿瘤的诊断和治疗领域， 并具体涉及一种新的肿瘤标志物 以及用于诊断肿瘤的发生和转移的方法和试剂盒， 并涉及治疗肿瘤以及肿 瘤转移的方法和药物。
说
背景技术
目前， 全世界每年约有 1100 万人被诊断为肿瘤。并且预计在 2020 年 以前每年将会有 1600万新增病例。 2005书年， 在全世界 5800 万的总死亡 人数中， 有 760 万是由癌症导致的 （约占总死亡人数的 13%) , 并且这一 数字还有逐年增加的趋势， 预计到 2015 年将会有 900 万人死于癌症， 而 到 2030 年将会达到 1140 万 （World Health Organization, 2006)。
肿瘤标志物是肿瘤细胞在癌变过程中由于基因的表达水平的变化而生 成或减少的抗原和其他生物活性物质， 可用于肿瘤的早期诊断、 分期、 监 测肿瘤进程， 和评价药物的治疗效果（ASCO, 1996)。 它会对肿瘤的临床治 疗带来巨大的影响， 尤其当它能够在临床病症出现之前被检测到， 或者可 以用于治疗效果的实时检测时。 目前， 为了满足肿瘤的临床诊断和治疗的 需求， 肿瘤标志物的研发亟待加速。
目前用于肿瘤早期诊断的肿瘤标志物， 大多由于缺乏灵敏度和特异性 而不能在体检中广泛应用。 例如， 对于肝癌， 甲胎蛋白和超声波检查是普 遍采用的诊断高危病人的方式， 并且确实显著提高了肝癌病人的生存率， 但灵敏度比较低； 肿瘤抗原 CA-125 有更高的灵敏度， 但却缺乏特异性。 同样地， 用于乳腺癌检测的血液肿瘤标志物 CA15-3 因灵敏度低在早期诊 断中几乎没用。 因此， 肿瘤的早期诊断、 以及良性和恶性肿瘤的区分仍然 是一个临床难题， 需要新的技术和方法来发现新的肿瘤标志物和提高肿瘤 标志物检测的灵敏度和可信度。
肿瘤蛋白质组学的出现为新的肿瘤标志物的发现， 和肿瘤的普查、 早 期诊断以及预后带来了新的希望。 肿瘤的恶性转化会伴随有某些蛋白表达 水平的变化， 这些变化能够在蛋白水平被定性和定量的检测到， 这就是肿 瘤蛋白质组学的主要研究内容。 由此获得的肿瘤的蛋白质信息能够为更加 有效的诊断、 预后肿瘤和评价治疗效果提供有价值的帮助。
热休克蛋白 90a (Heat shock protein 90α， Hsp90a)是一种分子伴侣蛋白， 其存在对于许多肿瘤相关蛋白的稳定性和行使功能都是必需的。 Hsp90a是 真核细胞胞质当中含量最为丰富的伴侣蛋白之一， 约占细胞内总蛋白的 1-2%。 细胞内 Hsp90a 的主要功能包括稳定蛋白 （如雌激素受体） 和帮助 蛋白成熟 （如某些激酶和信号蛋白） ， 但在细胞的一些其他生理过程中也 发现有它的参与， 包括突变蛋白的进化稳定， 细胞骨架的重排， 细胞核蛋 白的转运， 细胞增殖和凋亡， 蛋白降解， 抗原呈递， 和脂多糖的识别等。 Hsp90a还和多种疾病相联系，包括癌症、 自身免疫性疾病和心血管疾病等。 例如， 以 Hsp90a的 LKVIRK序列为抗原决定簇的单克隆抗体在抗真菌感 染方面有治疗活性， 目前 Neutec 公司在对其进行临床试验， 商品名为 Mycogrip®。
有文章发现 Hsp90a在极端条件刺激下会被分泌 （Liao et al. (2000) J. Biol. Chem. 275, 189-96), 作为一个经典的胞内蛋白， 有关 Hsp90a出现在 胞外以及在胞外如何行使功能的报道很少。 在以往报道中， Hsp90a被发现 在抗原呈递细胞中可以帮助抗原呈递， 还被发现是与细胞表面的脂筏相联 系的四个蛋白之一， 与脂多糖结合， 引起胞内蛋白的响应 （Triantafilou et al. (2002) Trends in Immunology 23, 301-4)。
Hsp90a还被发现在许多肿瘤细胞的表面高表达， 包括小细胞癌， 黑色 素瘤，和肝癌细胞株 (Ferrarini et al. (1992) Int. J. Cancer 51, 613-19)。 Hsp90a 在这些细胞系表面的高表达被推测是与抗原提呈相联系的， 但目前还没有 确切的证据。
还有报道说 Hsp90a 能够帮助穿膜蛋白的转运 （Schlatter et al. (2002) Biochem. J. 362, 675-84) , 并且与白血病、 肺癌和卵巢癌的药物外排 (drug efflux)有关 （Rappa et al (2002) Oncol. Res. 12, 113-9 and Rappa et al (2000) Anticancer Drug Des 15,127-34)。
胞内 Hsp90a目前也是抗肿瘤药物开发的一个重要靶点。 胞内 Hsp90a 参与了导致细胞癌变的许多信号通路的调节。对 Hsp90a的抑制能够引起一 些与细胞增殖、 细胞周期调节和细胞凋亡相关的信号蛋白的选择性降解。 最近发现许多已知的抗生素（如 Geldanamycin^ Radicicol禾卩 Coumermycin A1 等)是 Hsp90a的抑制剂， WO 00/53169 描述了这一发现， 并提出了通过抑 制伴侣蛋白与其底物的结合来抑制伴侣蛋白活性的方法， 其中 Coumarin及 其衍生物被认为具有这样的功能。 但 WO 00/53169所提到的抑制剂是针对 于胞内的 Hsp90a的。
Geldanamycin的类似物 17-AAG也是 Hsp90a的抑制剂， 目前已经进 入临床试验， 但有文章认为 17-AAG 因与多种细胞组分的蛋白相结合产生 的非特异性的抑制作用而具有较大的毒性 （Dunn (2002) J. Natl. Cancer Inst. 94, 1194-5)。 另外， 因对胞内 Hsp90a与其底物结合所参与的多种细胞信号 过程缺乏足够的认识， 直接抑制胞内 Hsp90a的活性也确实存有风险。
EP1457499A1描述了胞外 Hsp90a在肿瘤细胞侵袭中的功能， Hsp90a 通过促进基质金属蛋白酶 MMP-2 的分泌或激活来促进肿瘤侵袭。 依据这 一发现， EP1457499A1 提出可以通过抑制胞外 Hsp90a的活性来抑制肿瘤 的侵袭和转移， 并且可以通过检测肿瘤细胞对 Hsp90a的抑制剂的响应， 来 确定肿瘤细胞的侵袭能力， 以及其侵袭与 Hsp90a的相关性。
WO/2008/070472提出通过检测血桨中的 Hsp90a以及其相关因子来对 针对 Hsp90a 的抗肿瘤治疗效果进行检测和预后， 其实施例中提供了血桨 Hsp90a的水平与其抑制剂 BA (包括 17-AAG和 17-DMAG)的治疗效果的 相关性， 以及在小鼠肿瘤模型中肿瘤体积大小与血桨 Hsp90a的水平的相关 性。 但并未鉴定出 Hsp90a 在肿瘤病人的血液中的存在形式， 也未发现 Hsp90a与癌症病人肿瘤恶性程度尤其是转移的相关性；也并未提出 Hsp90a 作为独立的肿瘤标志物在肿瘤诊断和预后中的用途。
另有文章报道说血清中的 Hsp90a 与非小细胞肺癌的临床分期相关 (Xu et al. (2007) J. Cancer Mol. 3,107-112)。 肺癌病人血清中的 Hsp90a与正 常人和良性肿瘤患者相比有显著提高。但此文章同样未鉴定出 Hsp90a在肿 瘤病人的血液中的存在形式， 也未提及其与肿瘤转移的相关性； 另外此文 章只就非小细胞肺癌进行了研究， 对乳腺癌、 肝癌、 胰腺癌与血桨 Hsp90a 的相关性以及特异性并未涉及；并且此文章仅对血清 Hsp90a水平在肿瘤病 人中的变化进行了定性研究， 对其绝对含量的变化， 以及肿瘤诊断和预后 所必需的正常和非正常含量范围没有进行定义。 发明内容
本发明基于这样一个发现， 即血液中的 Hsp90a的水平与多种肿瘤的发 生及其恶性程度和转移相关。 因此， 血液中的 Hsp90a可以作为一种新的肿 瘤标志物。 本发明人发现， 血液中的 Hsp90a与细胞内的 Hsp90a不同， 前 者 C末端缺失 4个氨基酸。
因此， 在一个方面， 本发明提供一种分离的多肽， 本发明的多肽包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组成。
本发明的多肽可以是磷酸化的， 其中 SEQ ID No.l的氨基酸序列中的 选自以下一组中的一或多个氨基酸残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、第 263位的丝氨酸、第 309位的酪氨酸及其组合。优选地， 所述多肽的第 90位的苏氨酸是磷酸化的。
本发明的多肽可作为肿瘤标志物。 通过使用本发明的多肽的特异性结 合物来检测本发明的多肽在血液中的水平， 可有助于判断多种肿瘤的发生 及其恶性程度和转移。
因此， 在另一个方面， 本发明涉及本发明的多肽的特异性结合物在制 备试剂盒中的用途， 所述试剂盒例如可用于通过检测血桨中的本发明的多 肽的水平而辅助确定肿瘤的存在、 分期和 /或转移， 用于通过检测血桨中的 本发明的多肽的水平而对高危人群进行肿瘤筛查， 用于通过检测血桨中的 本发明的多肽的水平而对肿瘤患者的预后进行判断， 或用于通过检测血桨 中的本发明的多肽的水平而判断对肿瘤病人的手术、 放疗或药物治疗是否 有效和 /或决定何时停止治疗。
优选地， 本发明的多肽的特异性结合物是本发明的多肽的特异性抗体。 优选地， 所述抗体是单克隆抗体或其抗原结合片段， 例如 scFv、 Fab, Fab' 和 F(ab':>2。在一个具体的实施方式中， 所述抗体是由保藏号为 CGMCC No. 2903或 2904的细胞系产生的单克隆抗体 E9或 D10。
根据本发明， 所述抗体特异性结合本发明的多肽， 且优选地特异性结 合血桨中的本发明的多肽。 优选地， 所述抗体特异性结合磷酸化的本发明 的多肽， 其中所述多肽的相应于 SEQ ID No.l的选自以下一组中的一或多 个氨基酸残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、 第 263 位的丝氨酸、 第 309位的酪氨酸及其组合。 更优选地， 所述抗体特异性结 合第 90位的苏氨酸是磷酸化的本发明的多肽。 在另一个方面， 本发明涉及本发明的多肽的抑制剂在制备用于预防或 治疗肿瘤转移的药物组合物中的用途。
根据本发明的一个实施方式， 所述抑制剂是本发明的多肽的特异性抗 体。 优选地， 所述抗体是人源化抗体或其抗原结合片段。 在一个实施方式 中， 所述抗体特异性结合磷酸化的本发明的多肽， 其中所述多肽的相应于
SEQ ID No.l的选自以下一组中的一或多个氨基酸残基是磷酸化的： 第 90 位的苏氨酸、 第 231位的丝氨酸、 第 263位的丝氨酸、 第 309位的酪氨酸 及其组合。 在一个优选的实施方式中， 所述抗体特异性结合第 90位的苏氨 酸是磷酸化的本发明的多肽。 在一个具体的实施方式中， 本发明的抗体是 由保藏号为 CGMCC No. 2903或 2904的细胞系产生的单克隆抗体 E9或 D10。
在本发明的各种用途中， 肿瘤可以是例如肺癌、 肝癌、 胃癌、 食道癌、 骨肉瘤、 胰腺癌、 淋巴癌、 结肠癌、 乳腺癌、 前列腺癌、 口腔癌、 鼻咽癌、 宫颈癌、 白血病、 恶性黑素瘤、 肉瘤、 肾癌、 胆癌。
本发明也涉及特异性结合本发明的多肽的抗体， 所述抗体特异性结合 血桨中的本发明的多肽。 在一个具体的实施方式中， 所述抗体是由保藏号 为 CGMCC No. 2903或 2904的细胞系产生的单克隆抗体 E9或 D10。 优选 地， 所述抗体是人源化抗体或其抗原结合片段。 在一个实施方式中， 所述 抗体特异性结合磷酸化的本发明的多肽， 其中所述多肽的相应于 SEQ ID No.l的选自以下一组中的一或多个氨基酸残基是磷酸化的：第 90位的苏氨 酸、 第 231位的丝氨酸、 第 263位的丝氨酸、 第 309位的酪氨酸及其组合。 在一个优选的实施方式中， 所述抗体特异性结合第 90位的苏氨酸是磷酸化 的本发明的多肽。
在另一个方面， 本发明涉及一种抑制肿瘤的侵袭和转移的方法， 其步 骤包括抑制肿瘤细胞内 Hsp90a的磷酸化。在一个实施方式中， 本发明的方 法包括抑制肿瘤细胞内 Hsp90a第 90位的苏氨酸的磷酸化。 在一个具体的 实施方式中，本发明的方法包括在肿瘤细胞内过量表达编码蛋白磷酸化酶 5 (PP5)的核酸， 且优选地可通过基因导入的方式过量表达 PP5。 附图说明
图 1 : 与正常小鼠相比， 荷瘤小鼠血桨中 Hsp90a的含量升高。 图 2: 与正常人相比， 恶性肿瘤病人血桨中 Hsp90a的含量升高。
图 3 : 鼠单抗 E9和 D10的效价的检测
图 4: 使用鼠单抗 E9和兔多抗 S2 (夹心法 ELISA) 测定血桨 Hsp90a 浓度的标准曲线。
图 5: 使用夹心法 ELISA的方法， 定量比较肝癌、 肺癌、 乳腺癌、 胰 腺癌病人和良性乳腺囊肿和子宫肌瘤病人血桨中 Hsp90a的含量。
A:使用夹心法 ELISA测定肝癌病人血桨 Hsp90a的含量， 良性肿瘤患 者血桨 Hsp90a的含量在 2-10 ng/ml 区间内， 多数集中于 2-5 ng/ml; 69% (20/29) 肝癌病人血桨中 Hsp90a的含量在 50 ng/ml以上， 其均值与良性 肿瘤病人相比有> 10倍的升高， 与免疫印迹的结果基本一致， 也初步显示 了血桨 Hsp90a的含量水平与肿瘤的恶性程度的相关性。
B:使用夹心法 ELISA测定肺癌病人血桨 Hsp90a的含量， 与良性肿瘤 患者相比， 64% (9/14)肺癌病人血桨 Hsp90a的含量在 50 ng/ml以上， 其 均值与良性肿瘤病人相比有 > 10 倍的升高， 显示出血桨 Hsp90a的含量水 平与肿瘤的恶性程度的相关性。
C:使用夹心法 ELISA测定乳腺癌病人血桨 Hsp90a的含量， 与良性肿 瘤患者相比， 最高者可有 > 5倍的升高， 总体水平与良性肿瘤患者相比有 显著性差异。
D:使用夹心法 ELISA测定胰腺癌病人血桨 Hsp90a的含量，与良性肿 瘤患者相比， 100% ( 10/10) 胰腺癌病人血桨 Hsp90a的含量在 50 ng/ml以 上， 其均值与良性肿瘤病人相比有>10倍的升高， 显示出血桨 Hsp90a的含 量水平与肿瘤的恶性程度的相关性。
图 6: 使用夹心法 ELISA的方法， 定量比较肿瘤发生转移和未发生转 移的病人血桨中 Hsp90a的含量。
A: 将肝癌患者按照肿瘤是否发生转移进行分组， 比较两组发现， 肿瘤 发生转移的病人血桨 Hsp90a的含量在 200ng/ml以上， 而未发生转移的在 50-200ng/ml之间。
B: 将肺癌患者按照肿瘤是否发生转移进行分组， 比较两组发现， 肿瘤 发生转移的病人血桨 Hsp90a的含量在 200ng/ml以上， 而未发生转移的在 50-200ng/ml之间。
C: 将乳腺癌患者按照肿瘤是否发生转移进行分组， 比较两组发现， 肿 瘤发生转移的病人血桨 Hsp90a的含量有显著的升高。
图 7: 使用夹心法 ELISA的方法， 定量比较炎症病人 （包括肺炎、 肝 炎病人）、 正常人和肿瘤病人血桨中 Hsp90a的含量。
A: 为了确证血桨中 Hsp90a的含量在肿瘤病人中的升高是特异性的， 我们比较了肺炎病人、 正常人和肿瘤病人血桨中 Hsp90a的含量， 发现肺炎 病人血桨中的 Hsp90a的水平在 2-10 ng/ml之间， 与正常人相比， 没有显著
B: 肝炎病人（甲肝和乙肝）血桨中 Hsp90a的含量在 2-10 ng/ml之间， 与正常人相比， 亦没有显著差异。
图 8: 血桨中的 Hsp90a由肿瘤细胞分泌而来。
图 9: 肿瘤细胞分泌的 Hsp90aC末端缺失的情况的确定。
图 10: 血桨中的 Hsp90a是 C末端缺失 4个氨基酸的形式。
图 11 : 血桨中的 Hsp90a磷酸化形式的检测。
图 12: 肿瘤病人血桨中第 90位苏氨酸磷酸化的 Hsp90a的含量升高。 图 13 : 肿瘤病人血桨中第 90位苏氨酸磷酸化的 Hsp90a的含量升高与 肿瘤病人血桨中 Hsp90a的总含量升高是一致的。
图 14: 第 90位苏氨酸的磷酸化对于 Hsp90a的分泌是必需的。
图 15: PP5去磷酸化 Hsp90a的第 90位苏氨酸。
A: 将纯化的 PP5和第 90位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90a)混 合孵育， 检测释放出来的游离磷酸基团。 Peptide作为阳性对照。 结果显示， PP5可以直接去磷酸化 Hsp90a的第 90位苏氨酸。
B: 在人乳腺癌细胞系 MCF-7中， 过量表达人 PP5, 或者利用 RNA干 扰技术， 对人 PP5的表达进行抑制， 观察 Hsp90a的第 90位苏氨酸的磷酸 化情况， 结果显示， 过量表达人 PP5后， 第 90位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90a)明显减少（为对照组的 0.55 ); 当内源的人 PP5的表达被抑制 的时候， 第 90位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90o 明显增加 （为对照 的 1.58)。
图 16: 细胞内 PP5调节 Hsp90a的分泌。
A:在乳腺癌细胞系 MCF-7中，过量表达人 PP5 ,观察细胞分泌 Hsp90a 的变化， 结果显示， 过量表达人 PP5后， 细胞分泌的 Hsp90a明显减少。
B: 在乳腺癌细胞系 MCF-7中， 利用 RNA干扰技术， 对人 PP5的表达 进行抑制， 观察细胞分泌 Hsp90a的变化， 结果显示， 当内源的人 PP5的表 达被抑制的时候， 细胞分泌的 Hsp90a明显增加。
图 17: PP5的表达水平和细胞分泌 Hsp90a的关系。
图 18: PP5的表达水平和肿瘤细胞迁移能力的关系。
图 19: Hsp90a的特异性抗体能够抑制肿瘤细胞迁移。
图 20: Hsp90a特异性抗体抑制肿瘤转移的活性。 微生物材料保藏信息
产生的单克隆抗体 E9的 SP2/0-Agl4小鼠杂交瘤细胞系于 2009年 2月 24 日保藏于中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC, 北 京市朝阳区大屯路， 中国科学院微生物研究所：)， 保藏号为 CGMCC No. 2903。
产生的单克隆抗体 D10的 SP2/0-Agl4小鼠杂交瘤细胞系于 2009年 2 月 24日保藏于中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC, 北京市朝阳区大屯路， 中国科学院微生物研究所：)， 保藏号为 CGMCC No. 2904 发明详述
细胞的癌变是由细胞某些信号转导途径的变化所引起的， 伴随有蛋白 质表达水平、 修饰类型以及空间分布的变化， 这些蛋白质的变化信息可以 用来监测肿瘤的发生和进展， 这些蛋白质被称为肿瘤标志物。 随着蛋白质 组学方法和技术的进步和发展， 使得定性和定量的监测肿瘤蛋白质组的变 化成为可能， 许多新的肿瘤标志物从而被发现， 为肿瘤临床诊断和预后提 供了更为准确可信的依据。
本发明基于发现了一种新的血桨肿瘤标志物， 即存在于血桨中的 Hsp90ao 与细胞内的 Hsp90a (其氨基酸序列为 SEQ ID No.3， 编码核酸序 列为 SEQ ID No.4)比较， 血桨中的 Hsp90a的 C末端缺失 4个氨基酸。
因此， 在一个方面， 本发明提供一种分离的多肽， 该多肽是血桨或血 清中的 Hsp90a，所述多肽包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.1 的氨基酸序列组成。 在本申请中， 术语 "本发明的多肽"指的是血桨或血 清中的 Hsp90a， 其包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.1的氨 基酸序列组成。优选地，术语"本发明的多肽"在本申请中指的是由 SEQ ID No.l的氨基酸序列组成的多肽。在本申请中，术语"血桨中的 Hsp90a"或"血 清中的 Hsp90a"等价地指存在于血液中的非细胞内和细胞表面的 Hsp90a蛋 白， 它可单独游离存在， 也可以与其他血液中的细胞外蛋白相结合的形式 存在。 在本申请中， 术语 "多肽"可与 "蛋白质"互换使用。
本发明还涉及多核苷酸， 其编码包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组成的多肽。在一具体的实施方式中， 所述多核 苷酸包含 SEQ ID No.2的序列或由 SEQ ID No.2的序列组成。
本发明人还发现， 本发明的多肽在血桨中的形式是磷酸化的， 其中相 应于 SEQ ID No.l的氨基酸序列中的选自以下一组中的一或多个氨基酸残 基是磷酸化的：第 90位的苏氨酸、第 231位的丝氨酸、第 263位的丝氨酸、 第 309位的酪氨酸及其组合。 优选地， 所述多肽的第 90位的苏氨酸是磷酸 化的。
以往从未发现血桨 Hsp90a的特殊存在形式，也未有报道其水平与肿瘤 发生、 发展相关。 EP1457499A1 对细胞外 Hsp90a进行了描述， 并提出其 抑制剂可以用于治疗肿瘤转移， 以及诊断肿瘤细胞的侵袭能力， 及该能力 是否依赖于 Hsp90a。 但 EP1457499A1 所述的方法检测的是细胞膜表面 Hsp90a, 并未涉及血桨中的 Hsp90a， 也并未涉及依据 Hsp90a的水平判定 肿瘤的恶性水平， 及用于肿瘤早期诊断、 分期、 疗效检测和预后。 WO/2008/070472提出通过检测血桨中的 Hsp90a及其相关因子来判断针对 Hsp90a的抗肿瘤治疗的效果， 但未提及 Hsp90a作为独立的肿瘤标志物在 肿瘤诊断和预后中的用途。
本发明人通过对来自近百名肿瘤患者 （患有乳腺癌、 肝癌、 胰腺癌和 肺癌等 4种类型的肿瘤）中的血液进行检测发现，血桨中的 Hsp90a水平与 肿瘤的恶性程度， 尤其是转移具有相关性， 而炎症反应对血桨 Hsp90a的水 平没有影响。因此血桨中的 Hsp90a是一种新的肿瘤标志物，可以用于肿瘤 及其转移的诊断和预后。
因此， 在另一个方面， 本发明涉及检测血桨中的本发明多肽的水平的 试剂盒。 本发明的试剂盒抗包括例如本发明的多肽的特异性结合物。 本发 明的试剂盒可用于检测血桨中 Hsp90a的含量。
本发明还涉及本发明的多肽的特异性结合物在制备试剂盒中的用途， 所述试剂盒例如可用于通过检测血桨中的 Hsp90a水平而辅助确定肿瘤的存 在、 恶性程度或转移； 用于通过检测血桨中的 Hsp90a水平而对高危人群进 行肿瘤筛查；用于通过检测血桨中的 Hsp90a水平而对肿瘤患者的预后进行 判断； 或用于通过检测血桨中的 Hsp90a水平而判断对肿瘤病人的手术、放 疗或药物治疗是否有效和 /或决定何时停止治疗。
在本申请中， 术语 "特异性结合物"指的是以高亲和力结合本发明的 多肽的分子，其也包括以高亲和力结合细胞内或细胞表面的 Hsp90a的分子。 特异性结合物例如是蛋白质，优选地，特异性结合物是 Hsp90a特异性抗体。 在优选的实施方式中， 所述抗体是单克隆抗体或其抗原结合片段， 例如 scFv、 Fab、 Fab'和 F(ab':>2。 在一个具体的实施方式中， 所述抗体是由保藏 号为 CGMCC No. 2903或 2904的细胞系产生的单克隆抗体 E9或 D10。
单克隆抗体是通过将分泌所需要的抗体的细胞选出并在体外进行培养 获得的。 制备单克隆抗体的方法是本领域熟知的 (； K6hler G & Milstein C. (1975) Nature. 256, 495-7)。 制备特异性识别 Hsp90a的单克隆的具体过程如 下： 使用重组人 Hsp90a免疫 BALB/C小鼠，初次免疫使用抗原 lOO g加福 氏完全佐剂背部皮下多点注射； 3周后第二次免疫， 剂量同上， 加福氏不完 全佐剂腹腔内注射； 再过 3 周后第三次免疫， 剂量同上， 不加佐剂腹腔内 注射 （5〜7天后采血测其效价）； 另 3周后加强免疫， 剂量 200μ8， 腹腔内 注射。 3天后取脾细胞与 SP2/0-Agl4 (SP2/0)杂交瘤细胞（来源： ATCC, 编 号： CRL-1581 )融合， 使用 HAT进行筛选， 有限稀释法进行杂交瘤细胞的 克隆化， 免疫印迹和 ELISA 的方法进行鉴定， 最终得到分泌特异性识别 Hsp90a的抗体的杂交瘤细胞株。
根据本发明， 可用于制备上述试剂盒的抗体特异性结合 Hsp90a， 且优 选地特异性结合血桨中的 Hsp90a。 在一个实施方式中， 所述抗体特异性结 合磷酸化的 Hsp90a， 其中所述 Hsp90a的选自以下一组中的一或多个氨基 酸残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、 第 263位的丝 氨酸、 第 309位的酪氨酸及其组合。 在优选的实施方式中， 所述抗体特异 性结合第 90位的苏氨酸是磷酸化的 Hsp90a。
本发明同时也涉及检测血桨中的本发明多肽的水平的方法。 可通过任 何合适的方法检测血桨中 Hsp90a的含量。所述方法包括直接或间接测定本 发明的多肽在血桨中的水平， 以便有助于判断肿瘤的发生及其恶性程度和 转移。
直接测定的方法包括使用所述多肽的特异性结合物来检测本发明的多 肽， 例如使用识别所述多肽的特异性抗体进行免疫印迹或 ELISA检测。
间接测定的方法包括例如通过检测 Hsp90a的活性来反映 Hsp90a的浓 度， 例如以检测 Hsp90a 分子伴侣活性为基础的荧光素酶热变性检测
(Johnson et al. (2000) J. Biol. Chem., 275, 32499-32507)。
优选地， 通过 ELISA 或免疫印迹方法检测血桨中 Hsp90a的含量， 其 主要包括以下步骤：
a) 采集个体如肿瘤病人的全血， 按常规方法离心处理获得血桨或血 清；
b) 用 ELISA或免疫印迹方法检测步骤 a)所得血桨或血清中 Hsp90a 的含量， 并使用健康正常人的血桨作阴性对照， 已确诊恶性肿瘤患者的血 桨作阳性对照， 任选地可产生 Hsp90a浓度的标准曲线；
c) 根据所测定到的血桨 Hsp90a的含量， 判定肿瘤的发生及恶性程度 及分期。 从而对肿瘤的诊断、 预后和治疗效果做出判断。
步骤 也可使用以抗原抗体反应为原理的其他检测手段， 以及其他原 理的、 可以直接或间接的反映 Hsp90a 的浓度的检测手段， 比如通过检测 Hsp90a的活性来反映 Hsp90a的浓度。
测定 Hsp90a浓度的标准曲线所使用的 Hsp90a标准品纯化于肿瘤病 人的血桨， 也可以通过基因重组表达获得， 包括 Hsp90a的全长、 片段， 以 及包含有 Hsp90a序列的重组蛋白和与其他基团相偶联的复合物。 "Hsp90a 浓度的标准曲线"指的是使用已知浓度的 Hsp90a标准样品，用 ELISA方法 测定的浓度和吸光度测定值的对应曲线。 "Hsp90a标准品 "指的是纯度大于 95% 的血桨 Hsp90a蛋白、 重组 Hsp90a蛋白、 片段以及衍生物样品。
"判定肿瘤的恶性程度"， 是指依据测定样品 Hsp90a的浓度， 阴性对照 和阳性对照的值， 判定患者血桨 Hsp90a的含量属于何种水平， 从而对患者 的肿瘤性质 (即良性或恶性:)进行判断。
可使用的 ELISA方法包括夹心法和竞争法两种。其中优选灵敏度高的 竞争法。
夹心法的一般步骤包括： a) 将特异性抗体与固相载体连接， 形成固相 抗体， 并洗涤除去未结合的抗体及杂质； b) 加受检标本： 使之与固相抗体 接触反应一段时间， 让标本中的抗原与固相载体上的抗体结合， 形成固相 抗原抗体免疫复合物， 并洗涤除去其他未结合的物质； C ) 加酶标抗体： 使 固相免疫复合物上的抗原与酶标抗体结合， 并彻底洗涤未结合的酶标抗体， 此时固相载体上带有的酶量与标本中受检物质的量正相关； d ) 加底物， 夹 心式复合物中的酶催化底物成为有色产物。 根据颜色反应的程度进行该抗 原的定性或定量。
竞争法的一般步骤包括： a) 将特异抗体与固相载体连接， 形成固相抗 体， 洗涤； b ) 待测管中加受检标本和一定量酶标抗原的混合溶液， 使之与 固相抗体反应。 如受检标本中无抗原， 则酶标抗原能顺利地与固相抗体结 合。 如受检标本中含有抗原， 则与酶标抗原以同样的机会与固相抗体结合， 竞争性地占去了酶标抗原与固相载体结合的机会， 使酶标抗原与固相载体 的结合量减少。 参考管中只加酶标抗原， 保温后， 酶标抗原与固相抗体的 结合可达最充分的量， 洗涤。 c ) 加底物显色， 参考管中由于结合的酶标抗 原最多， 故颜色最深。 参考管颜色深度与待测管颜色深度之差， 代表受检 标本抗原的量。 待测管颜色越淡， 表示标本中抗原含量越多。
所述方法使用 ELISA夹心法进行检测，所使用的抗体来源于两种不同 种属的血桨 Hsp90a 的特异性抗体， 其中铺板抗体优选结合能力强的兔多 抗， 另外一个抗体优选识别特异性好的鼠单抗。 两种抗体须没有交叉反应。
所述方法若使用 ELISA 竞争法进行检测， 所使用的抗体为血桨 Hsp90a的特异性抗体， 对竞争物和 Hsp90a都要有较强的识别能力和识别 的特异性。
所述方法若使用 ELISA 竞争法进行检测， 所使用的竞争物为血桨
Hsp90a标准品的标记物。 且此标记不会干扰 Hsp90a标准品与其抗体的结 合。
所述方法若使用免疫印迹法进行检测， 所使用的抗体为血桨 Hsp90a 的特异性抗体； 二抗可以是辣根过氧化物酶偶联的， 也可以是碱性磷酸酶 偶联的； 反应底物包括 DAB 以及荧光底物， 其中优选灵敏度高的荧光底 上述检测方法的灵敏度需达到 10 纳克 /毫升或者更低。
本发明中所使用的血桨 Hsp90a的特异性抗体，包括抗体全长、片段及 其衍生物。 本发明中的抗体还可以由其他的 Hsp90a特异性结合物所代替，所述结 合物包括小分子化合物、 多肽及其衍生物。
本发明中所使用的血桨 Hsp90a标准品的标记物，其中的标记基团包括 生物素和各种荧光标记试剂。 其中优选生物素标记的竞争物。
可采用本发明的试剂盒进行检测的癌症包括， 但不仅限于， 肺癌、 肝 癌、 胃癌、 食道癌、 骨肉瘤、 胰腺癌、 淋巴癌、 结肠癌、 乳腺癌、 前列腺 癌、 口腔癌、 鼻咽癌、 宫颈癌、 白血病、 恶性黑素瘤、 肉瘤、 肾癌、 胆癌 等。 "肿瘤及其恶性程度"是指肿瘤是否良性、 是否恶性、 是否转移。
本发明人经初步的研究证实，正常人血桨 Hsp90a的范围是 2-50 ng/ml, 更集中于 2-10 ng/ml, 确诊为肿瘤的患者的血桨 Hsp90a水平高于正常人水 平， 而血桨 Hsp90a的水平在发生转移的患者体内高于 50 ng/ml， 多数高于 200ng/ml水平以上。 这使得血桨的 Hsp90a水平成为一种新的肿瘤标志物， 可以用于辅助判断肿瘤是否存在， 尤其是是否存在肿瘤的转移。
因此， 在一个实施方式中， 本发明的试剂盒或方法可用于确定个体是 否存在肿瘤、 尤其是恶性肿瘤以及肿瘤是否转移。 为此， 可采用本发明的 试剂盒或方法测定来自肿瘤疑似患者或肿瘤转移疑似患者的血桨样品内的 Hsp90a水平， 并任选地与正常对照进行比较， 然后根据样品内的 Hsp90a 水平判断该患者出现肿瘤或肿瘤转移的可能性。 Hsp90a水平升高提示患者 患有恶性肿瘤的可能性较大， 而对于已知患有肿瘤的患者， Hsp90a水平显 著升高提示肿瘤转移的可能性较大。
在另一个实施方式中， 本发明的试剂盒或方法可用于通过检测血桨中 的 Hsp90a水平而对高危人群进行肿瘤筛查。 为此， 可采用本发明的试剂盒 或方法测定来自高危人群的血桨样品内的 Hsp90a水平，并任选地与正常对 照进行比较，然后根据样品内的 Hsp90a水平判断该人群中哪些个体可能已 经出现肿瘤。 Hsp90a水平升高提示个体发生恶性肿瘤的可能性较大。 本领 域人员已知， 不同类型的肿瘤有其相应的高危人群， 这取决于肿瘤的类型， 个体因素如年龄、 家族史、 生活习惯、 工作环境、 有害物的接触史等等。 例如， 慢性乙型肝炎或丙型肝炎患者是肝细胞癌的高危人群。
在另一个实施方式中， 本发明的试剂盒或方法可用于通过检测血桨中 的 Hsp90a水平而对肿瘤患者的预后进行判断。 为此， 可采用本发明的试剂 盒或方法测定来自肿瘤患者的血桨样品内的 Hsp90a水平，并任选地与正常 对照或该患者以往的血桨 Hsp90a水平进行比较，然后根据样品内的 Hsp90a 水平判断该肿瘤患者的预后。 Hsp90a维持高水平或 Hsp90a水平进一步升 高可能与不利的预后相关。 因此， 可提醒临床医生对该患者进行更加密切 的观察， 且必要时改变目前的治疗方案。
在另一个实施方式中， 本发明的试剂盒或方法可用于通过检测血桨中 的 Hsp90a水平而判断对肿瘤病人的手术、 放疗或药物治疗是否有效和 /或 决定何时停止治疗。 为此， 可采用本发明的试剂盒或方法测定来自肿瘤患 者的血桨样品内的 Hsp90a水平，并任选地与正常对照或该患者以往的血桨 Hsp90a水平进行比较， 然后根据样品内的 Hsp90a水平判断对该肿瘤病人 的手术、 放疗或药物治疗是否有效和 /或决定何时停止治疗。
本领域人员了解， 与所有已知的肿瘤标记物或利用肿瘤标记物对肿瘤 进行筛查和诊断的方法一样，本发明的通过检测血桨样品内的 Hsp90a水平 来判断患者是否存在恶性肿瘤或肿瘤转移的方法是肿瘤诊断过程中的一种 辅助检测方法。 依据该方法的结果， 并不能直接确定患者是否患有恶性肿 瘤， 也不能直接确定是否存在恶性肿瘤的转移， 同样也不能直接确定恶性 肿瘤的组织来源及其病理类型或者转移病灶的位置和数量。 因此， 对于肿 瘤学临床医生而言， 诊断的确立仍依靠影像学、 病理学等手段以及临床医 生的判断。 就此而言， 本发明的方法仅为临床医生或科研人员提供辅助的 参考信息。 不过， 正如说明书中所提到的并如实施例中所证明的那样， 检 测血桨中的 Hsp90a水平能够为判断患者是否存在恶性肿瘤、尤其是是否存 在肿瘤的转移提供有价值的辅助的参考信息。 同样， 本发明的上述对高危 人群进行肿瘤筛查、 对肿瘤患者的预后进行判断以及对治疗效果进行评价 等方法的目的也是为临床医生或科研人员提供辅助的参考信息。
本发明人进一步证实，血桨中的 Hsp90a来源于肿瘤细胞，但与肿瘤细 胞内的 Hsp90a不同，因此通过抑制血桨中的 Hsp90a有可能抑制肿瘤的发 展和转移。本发明人还通过血桨 Hsp90a的特异性抗体能够抑制小鼠肿瘤转 移的实验证明了这一点，从而更进一步的，血桨 Hsp90a可以作为一个新的 靶点， 用于筛选新的抗癌药物。
在另一个方面， 本发明涉及本发明的多肽的抑制剂在制备用于预防或 治疗肿瘤转移的药物组合物中的用途。
根据本发明的一个实施方式， 所述抑制剂是 Hsp90a特异性抗体。优选 地， 所述抗体是人源化抗体或其抗原结合片段。 在一个实施方式中， 所述 抗体特异性结合磷酸化的 Hsp90a， 其中所述 Hsp90a的选自以下一组中的 一或多个氨基酸残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、 第 263位的丝氨酸、 第 309位的酪氨酸及其组合。 在一个优选的实施方式 中，所述抗体特异性结合第 90位的苏氨酸是磷酸化的 Hsp90a。在一个具体 的实施方式中， 所述抗体是由保藏号为 CGMCC No. 2903或 2904的细胞系 产生的单克隆抗体 E9或 D10。 正如下文实施例所述， 此抗体可以完全抑制 小鼠肿瘤的淋巴节转移， 抑制肺转移的效率可达 56%。
因此， 在另一个方面本发明也涉及特异性结合本发明的多肽的抗体， 所述抗体特异性结合血桨中的 Hsp90a。 在一个具体的实施方式中， 所述抗 体是由保藏号为 CGMCC No. 2903或 2904的细胞系产生的单克隆抗体 E9 或 D10。 优选地， 所述抗体是人源化抗体或其抗原结合片段。 在一个实施 方式中， 所述抗体特异性结合磷酸化的 Hsp90a， 其中所述 Hsp90a的选自 以下一组中的一或多个氨基酸残基是磷酸化的： 第 90位的苏氨酸、 第 231 位的丝氨酸、 第 263位的丝氨酸、 第 309位的酪氨酸及其组合。 在一个优 选的实施方式中， 所述抗体特异性结合第 90 位的苏氨酸是磷酸化的 Hsp90ao 优选地， 所述抗体抑制肿瘤的生长， 特别是转移。 本发明还涉及 一种缀合物， 其包含本发明的抗体和一种检测部分或治疗部分。 所述检测 部分例如为荧光团， 所述治疗部分例如为肿瘤化疗剂。
本发明人还发现，肿瘤细胞分泌 Hsp90a的水平高低和细胞内蛋白磷酸 酶 5 (Protein phosphatase 5 , PP5 ) 的表达水平存在一定的相关性。 在良性 肿瘤中， Hsp90a的分泌量较低， 而 PP5的表达水平却较高； 在恶性肿瘤中， Hsp90a的分泌量很高，而 PP5的表达水平却很低。因此肿瘤细胞分泌 Hsp90a 的水平和胞内 PP5的水平呈负相关。 因此通过检测肿瘤组织中 PP5的表达 水平， 可以预测肿瘤细胞分泌到血桨中的 Hsp90a的水平， 从而可以进行肿 瘤的早期诊断。
本发明还通过实验证明了 Hsp90a的分泌受到细胞内 PP5 的抑制性调 节。 当在细胞内过量表达编码 PP5的核酸的时候， Hsp90a的分泌受到了抑 制， 而降低细胞内 PP5的表达水平， 细胞分泌的 Hsp90a有明显的增加。 因 此通过过量表达编码 PP5的核酸， 从而抑制 Hsp90a的分泌， 有可能抑制肿 瘤的发展和转移。 而通过我们的实验， 我们也发现过量表达 PP5可以抑制 乳腺癌细胞 MCF-7的迁移， 因此 PP5可以作为一个新的肿瘤治疗靶点。 因此， 在另一个方面， 本发明涉及一种抑制肿瘤的侵袭和转移的方法， 其步骤包括抑制肿瘤细胞内 Hsp90a的磷酸化。在一个实施方式中， 本发明 的方法包括抑制肿瘤细胞内 Hsp90a第 90位的苏氨酸的磷酸化。 在一个具 体的实施方式中， 本发明的方法包括在肿瘤细胞内过量表达编码 PP5 的核 酸， 且优选地可通过基因导入的方式过量表达 PP5。在一个实施方式中， 本 发明的方法包括在肿瘤细胞内过量表达编码包含 SEQ ID No.5的氨基酸序 列的 PP5的核酸， 在一个具体的实施方式中， 核酸包含 SEQ ID No.6所示 的核苷酸序列。 本发明也涉及携带可操纵地连接于启动子的上述编码 PP5 的核酸的载体在制备用于通过在肿瘤细胞内过量表达 PP5而抑制肿瘤细胞 内 Hsp90a的磷酸化的药物中的用途。所述药物可用于抑制肿瘤的侵袭和转 移。
本发明还提供使用血桨 Hsp90a及其衍生物进行抗肿瘤药物的筛选的 方法和模型，包括但不仅限于寻找血桨 Hsp90a的结合蛋白、小肽和小分子 化合物， 以及抑制血桨 Hsp90a活性的抑制剂的筛选。 实施例
实施例 1 : 小鼠血桨样品的采集、 制备， 以及血桨 Hsp90a的检测
选用平均体重 20克左右的 Balb/c小鼠（购自北京维通利华实验动物技 术有限公司）， 随机分为两组， 每组 3只， 其中一组腋下接入 H22小鼠肝癌 细胞 （CCTCC, 编号： GDC091 ) 每只接入细胞数 106个， 对照组不接种肿 瘤。 当小鼠肿瘤直径生长至平均 2厘米（约 20天）时， 自眼底静脉丛取血， 血中加入抗凝剂， 须避免溶血。 样品如果发生溶血， 则重新采集。 全血收 集后于 4°C， 6000g离心两次， 取上清， 用免疫印迹方法检测血桨中 Hsp90a 的含量， 抗体为 Rabbit anti-human Hsp90a pAb (Labvasion)。 使用 BCA法 (Pierce) 测定样品总蛋白的含量， 使得每个样品的上样量 （蛋白量） 保持 一致。 结果如图 1所示， 与正常小鼠相比， 荷瘤小鼠血桨中 Hsp90a的含量 升高。 实施例 2: 正常人和肿瘤患者血桨样品的采集、 制备， 以及血桨 Hsp90a的 检测 取正常人或癌症病人的全血， 在 24小时以内、 低温条件下 （4°C左右） 送至实验室， 须避免溶血， 样品如果发生溶血， 则重新采集。于 4°C、 6000g 离心两次， 取上清， 采用免疫印迹方法检测 Hsp90a在血桨中的含量， 若不 能立即检测则分装后保存于 -80 °C。 检测结果将与临床诊断进行对照， 以验 证血桨中 Hsp90a的含量与肿瘤恶性程度的相关性。
免疫印迹检测的具体操作方法为： 血桨样品与上样缓冲液 1 : 1混合， 上样 1-2微升进行 SDS-PAGE, —抗为识别血桨 Hsp90a的特异性抗体 （大 鼠单抗 SPA-840, Stressgen), 二抗为辣根过氧化物酶偶联的山羊抗大鼠的 抗体 （购自中杉金桥）。 结果如图 2所示， 使用免疫印迹检测肝癌病人血桨 中 Hsp90a的含量， 与正常人比较有 10倍左右的升高 (A); 良性乳腺囊肿和 子宫肌瘤病人的血桨 Hsp90a的含量， 与正常人相比约有 2倍左右的升高 (B)。 实施例 3 : Hsp90a特异性兔多抗和小鼠单抗的制备
使用由 Hsp90a-Sall-Re: ACGCGTCGACTTAGTCTACTTCTTCCATGC (SEQ ID Νο·8)和 Hsp90a-Sphl-For: ACATGCATGCATGCCTGAGGAAAC CCAGACC (SEQ ID No.9)的核苷酸序列组成的引物（合成自 Invitrogen)和 Pfu DNA聚合酶（来源： NEB)从人肝脏 cDNA文库中 （来源： Stratagene) 扩增得到 Hsp90a全长序列，使用 Sphl和 Sail (来源： NEB )对片段和 pQE80L 载体 （来源： Qiagen) 进行双酶切， 得到的片段使用 T4 连接酶 （来源： NEB ) 进行连接。 连接产物转化到 ToplO 大肠杆菌感受态细胞 （来源： Transgen) 中进行扩增和验证， 验证后的质粒再转化到 BL21DE3大肠杆菌 感受态细胞 （来源： Transgen) 中进行表达， 得到重组人 Hsp90a蛋白。 重 组人 Hsp90a蛋白的纯化方法： 离子交换层析 SP HP, pH6.8, 收集电导 10 ms/ml的洗脱峰； Q HP, pH7.8, 收集电导 19 ms/ml的洗脱峰。
使用纯度 > 95% 的重组人 Hsp90a蛋白免疫成年雄性新西兰白兔， 经 背部皮内多点注射新西兰白兔， 每次抗原剂量为 100μ8/只。 2 周后以相同 方法进行第 2次免疫 (抗原剂量减半：)， 之后每隔 1周加强免疫 1次， 共加强 2次， 并于加强免疫后 7〜10天耳缘静脉取血， 测定血清中抗体的效价。 于 最后 1次免疫后 8天， 颈动脉插管放血， 分离血清， 置 - 20°C保存。 使用偶 联有抗原的亲和柱料对血清进行纯化。 纯化后的兔多抗命名为 S2。 使用重组人 Hsp90a免疫 BALB/C小鼠，初次免疫使用抗原 lOO g加福 氏完全佐剂背部皮下多点注射； 3周后第二次免疫， 剂量同上， 加福氏不完 全佐剂腹腔内注射； 再过 3 周后第三次免疫， 剂量同上， 不加佐剂腹腔内 注射 （5〜7天后采血测其效价）； 另 3周后加强免疫， 剂量 200μ8， 腹腔内 注射。 3天后取脾细胞与 SP2/0-Agl4(SP2/0)杂交瘤细胞 （来源： ATCC, 编 号： CRL-1581 )融合， 使用 HAT进行筛选， 有限稀释法进行杂交瘤细胞的 克隆化， 免疫印迹和 ELISA 的方法进行鉴定， 最终得到分泌特异性识别 Hsp90a的抗体的杂交瘤细胞株 E9和 D10,保藏号分别为： CGMCC No. 2903 和 2904， 于 2009年 2月 24日保藏于 CGMCC。
使用间接法 ELISA检测 E9和 D10的效价， 结果如图 3所示， E9和 D10的效价均可以达到 500,000， 可用其对血桨中的 Hsp90a进行检测。 间 接法 ELISA的具体步骤如下： 使用重组人 Hsp90a于 4°C包板过夜， 包被浓 度为 10 g/ml; 37°C封闭一小时后加入按照 1 :400， 1 : 1600， 1 :6400， 1 :25600， 1 : 102400， 1 :509600梯度稀释的 E9或 D10, 室温孵育 2小时； 然后用辣根 过氧化物酶偶联的山羊抗小鼠二抗 （购自中杉金桥） 室温孵育 1 小时， 使 用邻苯二胺显色， 于 OD 490nm读数。 实施例 4: 使用鼠单抗 E9和兔多抗 S2 (夹心法 ELISA) 测血桨 Hsp90a浓 度的标准曲线的测定
在使用夹心法 ELISA测定血桨 Hsp90a浓度的方法中， 所使用的抗体 来源于两种不同种属的血桨 Hsp90a的特异性抗体，其中铺板抗体使用结合 能力强的兔多抗 S2 (制备方法在实施例 3中进行了描述） ， 另外一个抗体 优选识别特异性好的鼠单抗 E9 (制备方法在实施例 3中进行了描述） 。 两 种抗体没有交叉反应， 且重复性好， 检测灵敏度高。 由图 4 的标准曲线可 见此方法的检测水平可达 5 ng/ml。 实施例 5 : 人血桨样品的采集、 制备， 以及血桨 Hsp90a的测定和肿瘤恶性 程度的判定 （夹心法 ELISA)
取正常人、 癌症病人或炎症病人 24小时以内采集的全血， 在低温条件 下送至实验室，须避免溶血。样品如果发生溶血，则重新采集。于 4°C、6000g 离心两次， 取上清， 采用 ELISA的方法检测 Hsp90a在血桨中的含量， 若 不能立即检测则分装后保存于 -80°C。 检测结果将与临床诊断进行对照， 以 验证血桨中 Hsp90a的含量与肿瘤恶性程度的相关性。
夹心法 ELISA使用两种不同来源的抗 Hsp90a的抗体， 其中自制兔源 多抗 S2 (制备方法在实施例 3中进行了描述） 用于 4°C铺板过夜， 37°C封 闭一小时后加待测血桨样品和标准曲线的样品， 待测血桨稀释 10倍， 每孔 加入 100微升；标准曲线的样品每孔加入已知量的标准 Hsp90a样品以及 10 微升空白阴性血清， 以排除血清背景； 于 37°C孵育 2小时后， 加入另外一 种抗体， 自制鼠单抗 E9 (制备方法在实施例 3中进行了描述）， 37°C孵育 2 小时； 然后用辣根过氧化物酶偶联的山羊抗小鼠二抗孵育 1 小时， 使用邻 苯二胺显色， 于 OD 450nm读数。 结果如图 5、 6、 7所示。
如图 5所示， 良性肿瘤患者 （包括良性乳腺囊肿和子宫肌瘤病人， 共 7 例）血桨：¾ 9001的含量在2-101¾/1111 区间内， 多数集中于 2-5 ng/ml; 69% (20/29) 肝癌病人 （29例） 血桨中 Hsp90a的含量在 50 ng/ml以上， 其均 值与良性肿瘤病人相比有 > 10倍的升高， P =0.00263，学生 t检验 （A); 64% (9/14)肺癌病人 ( 14例)血桨 9001的含量在50 1¾/1111以上， 其均值与 良性肿瘤病人相比有 > 10倍的升高， P =0.0497，学生 t检验 （B); 78%(25/32) 乳腺癌病人 （32例） 血桨 Hsp90a的含量在 50 ng/ml以上， 其均值与良性 肿瘤病人相比有> 10倍的升高， P O.001 , 学生 t检验 （C); 100% ( 10/10) 胰腺癌病人 （10例） 血桨 Hsp90a的含量在 50 ng/ml以上， 其均值与良性 肿瘤病人相比有> 10倍的升高， P <0.05， 学生 t检验 （D：)。
如图 6所示， 在肝癌 （共 17例， 其中发生转移 7例） （A)、 肺癌 （共 10例， 其中发生转移 2例） （B)和乳腺癌 （共 21例， 其中发生转移 10例） (C)病人中， 发生转移的病人的血桨 Hsp90a 的含量较未发生转移的病人显 著提高， 其中肝癌 P值 =0.003， 乳腺癌 P值 =0.002， 学生 t检验。
如图 7所示， 炎症病人， 包括肺炎 10例 (A：)、肝炎病人（甲肝 5例和乙 肝 5例） （B)血桨中 Hsp90a的含量在 2-10ng/ml之间， 与正常人 （3例） 相 比， 亦没有显著差异， P值分别为 0.2988， 0.5177， 0.138， 学生 t检验。
以上血桨样品， 其中正常人样品来自健康志愿者， 肿瘤病人和炎症病 人样品来自北京肿瘤医院和厦门第一医院。 选用平均体重 20克左右的裸鼠（购自北京维通利华实验动物技术有限 公司）， 分为两组， 每组 6只， 腋下接入 Hela宫颈癌细胞 （来源： ATCC, 编号： CCL-2), 每只接入细胞数 106个。 对照组为不接种肿瘤正常鼠。 当 小鼠肿瘤直径生长至平均 2厘米 （约 20天） 时， 眼底静脉丛取血， 使用能 够特异性识别人源、但不能识别鼠源的 Hsp90a的抗体 (大鼠单抗， Stressgen) 检测血桨中的 Hsp90a。 结果如图 8所示， 接种人源的肿瘤的小鼠血桨中的 Hsp90a可以被特异性识别人源、但不能识别鼠源的 Hsp90a的抗体所识别， 说明血桨中的 Hsp90a是由肿瘤细胞分泌而来的。 实施例 7: 肿瘤细胞分泌的 Hsp90a是 C末端缺失情况的确定
使用由 Hsp90a-pc3.1-Nhel-For-Myc: GCTAGCTAGCGCCACCATGGA
AAGAC (SEQ ID No.lO)和 Hsp90a-pc3.1-Xhol-Re-nostop: CCCGCTCGA GTGTCTACTTCTTCCATGCGTGATG (SEQ ID No.12)的核苷酸序列组成的 引物 （合成自 Invitrogen) 和 Pfu DNA聚合酶 （来源： NEB), 以实施例 3 中得到的 pQE80L-Hsp90a质粒为模版， 扩增得到 Hsp90a全长序列， 经酶 切、 连接构建到 pcDNA3.1/Myc-His (-) (载体来源： Invitrogen) 中， 得到在 N末端额外增加一个 Myc 标签的 Hsp90a， 命名为 Myc-H; 或者使用由 Hsp90a-pc3.1 -Nhe 1 -For-His-Myc:GCTAGCTAGCGCCACCATGCATCATCA
AACCCAGACCCAAGAC (SEQ ID No.11)和 SEQ ID No.12的核苷酸序列组 成的引物（合成自 Invitrogen)在其 N末端额外增加一个连续的 His-Myc标 签， 命名为 His-Myc-H。 在人乳腺癌细胞系 MCF-7中， 瞬时转染这两种载 体使之过量表达， 观察外源 Hsp90a (；即过量表达的 Hsp90a， 区别于内源、 本底的 Hsp90a) 的分泌情况。用抗 Hsp90a， 抗 Myc标签， 抗 His标签的抗 体检测分泌到胞外培养基中的 Hsp90a 的变化。 结果显示分泌到胞外的 Hsp90a是 C末端缺失的形式 （图 9A)。
以 Myc-His-H为模板， 对其 C末端的最后四个氨基酸 （EEVD) 进行 突变， 其中 EE->AA表示两个 EE突变成两个 Ala (使用 SEQ ID No.11和 Hsp90a-EE-AA: GGCCGCTCGAGTGTCTACTGCTGCCATGCGTGATGTG (SEQ ID No.13)的核苷酸序列组成的引物扩增得到）， VD->AA表示两个 VD 突变成两个 Ala (使用 SEQ ID No.11和 Hsp90a-VD-AA: GGCCGCTCGA GTTGCTGCTTCTTCCATGCGTGATGTG (SEQ ID No.14)的核苷酸序列组 成的引物扩增得到）， All Ala表示 EEVD都突变成 Ala (使用 SEQ ID No.ll 和 Hsp90a-EEVD-AAAA: GGCCGCTCGAGTTGCTGCTGCTGCCATGCG TGATGTG (SEQ ID No.15)的核苷酸序列组成的引物扩增得到）， CA4表示 最后的 EEVD四个氨基酸缺失 （使用 SEQ ID No.11和 Hsp90a-CA4-Xho: CCGCTCGAGTCATGCGTGATGTGTCGTCATCTC (SEQ ID Νο·16)的核苷 酸序列组成的引物扩增得到）。 在人乳腺癌细胞系 MCF-7 中， 瞬时转染这 几种突变体， 使之过量表达， 观察外源 Hsp90a (即过量表达的 Hsp90a， 区 别于内源、本底的 Hsp90o 的分泌情况。用抗 Hsp90a的抗体检测分泌到胞 外培养基中的 Hsp90a 的变化。 结果显示， C 末端的四个氨基酸调节了 Hsp90a的分泌， 这四个氨基酸上任意的点突变或者缺失可以导致分泌到胞 外的 Hsp90a不再以 C末端缺失的形式存在， 证明分泌到胞外的 Hsp90aC 末端缺失的是 EEVD四个氨基酸 （图 9B)。 实施例 8: 人血桨中的 Hsp90a存在形式的检测
取肝癌病人 24小时以内采集的全血， 离心两次取血桨， 采用免疫共沉 淀和免疫印迹方法检测 Hsp90a在血桨中的形式。 即首先使用特异性识别 Hsp90a的兔多克隆抗体 （来源： Labvision) 将血桨中的 Hsp90a进行免疫 沉淀，进而使用特异性识别 Hsp90a C末端 4个氨基酸 EEVD的兔多克隆抗 体 （Anti-C4) (自制， 免疫所用抗原为与载体蛋白偶联的 EEVD三次重复 的肽段， 合成自赛百盛）检测血桨中的 Hsp90a C末端缺失的情况。 结果如 图 10所示， 特异性识别 Hsp90a C末端 4个氨基酸 EEVD的兔多克隆抗体 能够识别全细胞中的 Hsp90a， 而不能识别血桨中的 Hsp90a， 说明血桨中的 Hsp90a是 C末端缺失 4个氨基酸的形式，与细胞内的 Hsp90a不同（图 10)。 实施例 9: 人血桨中的 Hsp90a磷酸化形式的检测
取肝癌病人 24小时以内采集的全血， 离心两次取血桨， 采用免疫共沉 淀和免疫印迹方法检测 Hsp90a在血桨中的形式。 即首先使用特异性识别 Hsp90a的兔多克隆抗体 （来源： Labvision) 将血桨中的 Hsp90a进行免疫 沉淀，进而使用特异性识别 Hsp90a第 90位点的苏氨酸磷酸化的抗体 (Rabbit anti-phospho-(Ser/Thr) PKA substrate pAb, Cell signaling ) 检测血桨中的 Hsp90a第 90位点的苏氨酸磷酸化情况。
结果如图 11所示， 血桨中的 Hsp90a能够被特异性识别 Hsp90a第 90 位点的苏氨酸磷酸化的抗体所识别， 说明血桨中的 Hsp90a是第 90位点的 苏氨酸磷酸化的形式。 实施例 10: 人血桨样品中第 90位点的苏氨酸磷酸化的 Hsp90a的含量检测 取正常人和肝癌病人 24小时以内采集的全血， 离心两次取血桨， 采用 夹心法 ELISA的方法检测 Hsp90a在血桨中相对含量。 操作步骤为首先使 用自制兔源多抗 S2 (制备方法在实施例 3中进行了描述）于 4°C铺板过夜， 37°C封闭一小时后加待测血桨样品， 待测血桨稀释 10倍， 每孔加入 100微 升；于 37°C孵育 2小时后，加入特异性识别第 90位点苏氨酸磷酸化的 Hsp90a 的抗体 （Cell signaling) , 37°C孵育 2小时； 然后用辣根过氧化物酶偶联的 山羊抗兔二抗孵育 1小时， 使用邻苯二胺显色， 于 OD 490nm读数。 结果 显示， 肝癌病人血桨的检测值明显高于正常人的检测值， P值 =0.003， 学生 t检验，表明肝癌病人血桨中第 90位点的苏氨酸磷酸化的 Hsp90a的含量升 高 （图 12)。 实施例 11 : 人血桨样品中第 90 位点的苏氨酸磷酸化的 Hsp90a 含量和 Hsp90a总含量的一致性检测
将同样一份 （共 8份）肝癌病人的血桨同时进行第 90位点的苏氨酸磷 酸化的 Hsp90a含量和 Hsp90a总含量的检测。 Hsp90a总含量的检测方法同 实施例 5;第 90位点的苏氨酸磷酸化 Hsp90a含量的检测方法同实施例 10。 结果显示， 肝癌病人血桨中第 90 位点的苏氨酸磷酸化的 Hsp90a含量和 Hsp90a总含量是一致的， 进一步证明血桨中的 Hsp90a是在第 90位点的苏 氨酸发生磷酸化， 且 Hsp90a总含量升高可代表第 90位点的苏氨酸磷酸化 的 Hsp90a的含量升高 （图 13 )。 实施例 12: 第 90位点的苏氨酸磷酸化对于 Hsp90a的分泌是必需的
以实施例 3中得到的 pcDNA3.1-Myc-His -Hsp90a质粒为模版（又名野 生型 Hsp90a ( WT Hsp90a ) )， 使用 Hsp90a-T89A-Sense: GATCGA ACTCTTGCAATTGTGGATACTGGAATTGGAATG (SEQ ID No.17) 和 Hsp90a-T89-AntiSense: CATTCCAATTCCAGTATCCACAATTGCAAGAGT TCGATC (SEQ ID No.18)的核苷酸序列组成的引物构建突变型 Hsp90a (T90A), 即第 90位点的苏氨酸突变为丙氨酸， 使之不能被磷酸化， 命名为 (T90A Hsp90a) o 在人乳腺癌细胞系 MCF-7 (购白 ATCC, 编号 HTB-22) 中， 过量表达野生型 Hsp90a (WT) 和突变型和 Hsp90a (T90A), 收集培养 基，观察外源 Hsp90a (即过量表达的 Hsp90a， 区别于内源、本底的 Hsp90a) 的分泌情况。 用抗 Hsp90a的抗体检测分泌到胞外培养基中的 Hsp90a的变 化。
结果显示， 外源野生型 Hsp90a可以在胞外被检测到， 而 T90A突变体 不能被检测到， 证实第 90位苏氨酸的磷酸化对于 Hsp90a的分泌是必需的 (图 14)。 实施例 13 : PP5去磷酸化 Hsp90a的第 90位苏氨酸
第 90 位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90o 的制备： 将重组人 Hsp90a蛋白和重组蛋白激酶 A (美国 Promega公司） 在反应缓冲液 （英国 NEB公司 )中 30度混合孵育 1小时后， 纯化出 pT90-Hsp90a蛋白， 三次透 析去除游离的磷酸基团。 将纯化得到的 pT90-Hsp90a蛋白和重组人 PP5蛋 白混合 30度孵育， 利用非放射性丝氨酸 /苏氨酸磷酸酶活性检测试剂盒（美 国 Promega公司） 检测释放出来的游离磷酸基团。 多肽底物是该试剂盒的 组成部分， 作为阳性对照。 结果如图 15A所示， PP5与多肽底物共同孵育， 游离磷酸基团的释放显著增加， ？值<0.005， 学生 t检验， 表明 PP5可以将 多肽底物直接去磷酸化（阳性对照）；将 pT90-Hsp90a蛋白与 PP5共同孵育， 游离磷酸基团的释放也显著增加， ？值<0.005， 学生 t检验， 表明 PP5可以 直接去磷酸化 Hsp90a的第 90位苏氨酸。
从人肝脏 cDNA文库中扩增得到 PP5的全长序列 (SEQ ID No.6), 基因 测序正确后构建到 pcDNA3.1/Myc-His (-) (载体来源： Invitrogen)。 在人乳 腺癌细胞系 MCF-7中转入该载体， 进行过量表达。 或者利用 RNA干扰技 术， 以 5'-ACTCGAACACCTCGCTAAAGAGCTC-3' (SEQ ID Νο·7)作为 ΡΡ5 的 siRNA的靶序列，转入针对人 PP5的特异性小 RNA (由 Invitrogen合成)， 对人 PP5的表达进行抑制， 观察 Hsp90a的第 90位苏氨酸的磷酸化情况， 结果如图 15B所示， 过量表达人 PP5后， 第 90位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90a)明显减少（为对照组的 0.55 ); 当内源的人 PP5的表达被抑制 的时候， 第 90位苏氨酸磷酸化的 Hsp90a (pT90-Hsp90o 明显增加 （为对照 的 1.58)。 实施例 14: 通过促进或抑制 PP5的表达调节分泌 Hsp90a的水平
PP5 能够导致 Hsp90a 第 90 位苏氨酸的磷酸化去磷酸化。 使用 PP5-NheI-For: CTAGCTAGCATGTACCCATACGACGTCCCAGACTACGCT (SEQ ID No.19)和 PP5-XhoI-Re: CCGCTCGAGTTAATGATGATGATGATG ATGCACGTGTACC (SEQ ID No.20)的核苷酸序列组成的引物从人肝脏 cDNA 文库中扩增得到 PP5 的全长序列， 基因测序正确后构建到 pcDNA3.1/Myc-His (-) (载体来源： Invitrogen)。 在人乳腺癌细胞系 MCF-7 中转入该载体， 进行过量表达， 观察细胞分泌 Hsp90a的水平， 结果显示， 过量表达人 PP5后， 细胞分泌的 Hsp90a明显减少 （图 16A)。
在乳腺癌细胞系 MCF-7中， 利用 RNA干扰技术 （即转入针对人 PP5 的特异性小 RNA， Invitrogen), 对人 PP5的表达进行抑制， 调节细胞分泌 Hsp90a的水平， 结果显示， 当内源的人 PP5的表达被抑制的时候， 细胞分 泌的 Hsp90a明显增加 (图 16B)。 实施例 15: PP5 含量的检测和肿瘤恶性程度的判定
在人乳腺癌细胞系 MCF-7，SKBR3，MDA-MB-453，435s和 23KATCC, 编号分别为 HTB-22, -30， -131， -129和 HTB-26) 中， 利用免疫印迹技术， 检测细胞内 PP5 的表达水平和细胞分泌 Hsp90a的关系。 MCF-7, SKBR3 是恶性程度较低的乳腺癌细胞系， 在裸鼠成瘤模型中， 这两株细胞只能形 成原位肿瘤， 而不能发生转移； MDA-MB-453 , 435s和 231是恶性程度较 高的乳腺癌细胞系， 在裸鼠成瘤模型中， 这两株细胞即可以形成原位肿瘤， 又可以发生转移， 其中 MDA-MB-435S和 231常用来建立肿瘤转移模型。 在图 17中， 这五株乳腺癌细胞系按照恶性程度由低到高依次排列。
结果显示，当细胞内 PP5的表达量高的时候，细胞分泌较少的 Hsp90a， 而当细胞内 PP5的表达量低的时候， 细胞分泌较多的 Hsp90a (图 17); 同 时， 分泌型 Hsp90a的水平与肿瘤恶性程度正相关， 而 PP5与肿瘤恶性程度 负相关 （图 17)， 分泌型 Hsp90a及其调节因子 PP5的水平可用于肿瘤恶性 程度的判定。 实施例 16 : PP5的表达水平和肿瘤细胞迁移能力的关系
采用伤口愈合模型（wound healing model )检测 PP5的表达水平和肿瘤 细胞迁移能力的关系。
在人乳腺癌细胞系 MCF-7中， 过量表达人 PP5 , 或者利用小 RNA干 扰， 降低内源 PP5的表达， 然后分别接种 12孔板， 当细胞接近长满培养皿 底的时候， 使用枪尖刮掉部分细胞， 形成"伤口"， 将刮下的细胞吸走， 换新 鲜的 DMEM培养基（GIBCO ) , 置于 37°C培养箱中继续培养。 在 0h、 12h、 24h的时候拍照纪录"伤口" （图 18A)。 通过"伤口"愈合的速度来检测 PP5 的表达水平对细胞迁移的影响。 结果显示， 过表达 PP5可以抑制 MCF-7的 细胞迁移能力， 而 PP5干扰可以促进 MCF-7细胞的迁移能力 （图 18B)。 实施例 17 : 血桨 Hsp90a特异性抗体抑制肿瘤细胞迁移活性的检测。
采用伤口愈合模型 （wound healing model) 检测血桨 Hsp90a特异性抗 体抑制肿瘤细胞迁移的活性。
将 MCF-7 和 MDA-MB-231 细胞 （ATCC , 编号分别为 HTB-22 和 HTB-26 )分别接种 12孔板， 当细胞接近长满培养皿底的时候， 使用枪尖刮 掉部分细胞， 形成"伤口"， 将刮下的细胞吸走， 换新鲜的 DMEM培养基 ( GIBCO ) , 同时加入血桨 Hsp90a的特异性小鼠单克隆抗体 E9 (20 μ^πύ 或对照 IgG (20 μ^Ώύ ) , 置于 37°C培养箱中继续培养。 在 0h、 6h、 12h、 24h、 48h和 72h 的时候拍照纪录"伤口"。 通过"伤口"愈合的速度来检测 Hsp90a抗体对细胞迁移的抑制作用。 结果如图 19所示。 血桨 Hsp90a的特 异性抗体对于 MDA-MB-231 (图 19A) 和 MCF-7 (图 19B ) 细胞的迁移有 >40%的抑制。 实施例 18 : 血桨 Hsp90a的特异性抗体抑制肿瘤转移活性的检测。
选用平均体重 20克左右的裸鼠（购自北京维通利华实验动物技术有限 公司）， 尾静脉接种 B16/F10 小鼠恶性黑色素瘤细胞 （ATCC , 编号： CRL-6475 ) 每只接入细胞数 2χ 105个。 第二天随机分组， 每组 8只， 分别 设定阴性对照组 （IgG) 和给药组 （Hsp90aAb) (小鼠单克隆抗体 E9)， 每 隔一天给药一次， 给药剂量为 40 μ8/只 /次， 15天后检查转移情况。 结果如 图 20所示， 血桨 Hsp90a的特异性抗体能够完全抑制 B16/F10细胞的淋巴 转移 （A), 对肺转移的抑制率为 56% (B)。

Claims

权 利 要 求 书
1. 一种分离的多肽， 其包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组成。
2. 权利要求 1所述的多肽， 其中 SEQ ID No.l的氨基酸序列中的选自 以下一组中的一或多个氨基酸残基是磷酸化的： 第 90位的苏氨酸、 第 231 位的丝氨酸、 第 263位的丝氨酸、 第 309位的酪氨酸及其组合。
3. 权利要求 2所述的多肽， 其中第 90位的苏氨酸是磷酸化的。
4. 权利要求 1-3 中任一项的多肽的特异性结合物在制备用于通过检测 血桨中的包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组 成的多肽的水平而确定肿瘤的存在、 分期和 /或转移的试剂盒中的用途。
5. 权利要求 1-3 中任一项的多肽的特异性结合物在制备用于通过检测 血桨中的包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组 成的多肽的水平而对高危人群进行肿瘤筛查的试剂盒中的用途。
6. 权利要求 1-3 中任一项的多肽的特异性结合物在制备用于通过检测 血桨中的包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组 成的多肽的水平而对肿瘤患者的预后进行判断的试剂盒中的用途。
7. 权利要求 1-3 中任一项的多肽的特异性结合物在制备用于通过检测 血桨中的包含 SEQ ID No.l的氨基酸序列或由 SEQ ID No.l的氨基酸序列组 成的多肽的水平而判断对肿瘤病人的手术、 放疗或药物治疗是否有效和 /或 决定何时停止治疗的试剂盒中的用途。
8. 权利要求 4-7中任一项的用途， 其中所述肿瘤选自肺癌、 肝癌、 胃 癌、 食道癌、 骨肉瘤、 胰腺癌、 淋巴瘤、 结肠癌、 乳腺癌、 前列腺癌、 口 腔癌、 鼻咽癌、 宫颈癌、 白血病、 恶性黑素瘤、 肉瘤、 肾癌、 胆癌。
9. 权利要求 4-8 中任一项的用途， 其中所述结合物是所述多肽的特异 性抗体。
10. 权利要求 9 的用途， 其中所述抗体是单克隆抗体或其抗原结合片 段。
11. 权利要求 10的用途， 其中所述抗原结合片段选自 scFv、 Fab、 Fab' 和 F(ab')2。
12. 权利要求 10的用途，其中所述抗体是由保藏号为 CGMCC No. 2903 或 2904的细胞系产生的单克隆抗体 E9或 D10。
13. 权利要求 9的用途， 其中所述抗体特异性结合血桨中的所述多肽。
14. 权利要求 9的用途， 其中所述抗体特异性结合磷酸化的所述多肽， 其中所述多肽在相应于 SEQ ID No.l的选自以下一组中的一或多个氨基酸 残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、 第 263位的丝氨 酸、 第 309位的酪氨酸及其组合。
15. 权利要求 14的用途，其中所述抗体特异性结合第 90位的苏氨酸是 磷酸化的所述多肽。
16. 权利要求 1-3中任一项的多肽的抑制剂在制备用于预防或治疗肿瘤 转移的药物组合物中的用途。
17. 权利要求 16的用途， 其中所述抑制剂是包含 SEQ ID No.l的氨基 酸序列或由 SEQ ID No.l的氨基酸序列组成的多肽的特异性抗体。
18. 权利要求 17的用途， 其中所述抗体是人源化抗体或其抗原结合片 段。
19. 权利要求 17的用途，其中所述抗体特异性结合磷酸化的所述多肽， 其中所述多肽在相应于 SEQ ID No.l的选自以下一组中的一或多个氨基酸 残基是磷酸化的： 第 90位的苏氨酸、 第 231位的丝氨酸、 第 263位的丝氨 酸、 第 309位的酪氨酸及其组合。
20. 权利要求 19的用途，其中所述抗体特异性结合第 90位的苏氨酸是 磷酸化的所述多肽。
21. 权利要求 16的用途，其中所述抗体是由保藏号为 CGMCC No. 2903 或 2904的细胞系产生的单克隆抗体 E9或 D10。
22. 权利要求 16-23中任一项的用途， 其中所述肿瘤选自肺癌、 肝癌、 胃癌、 食道癌、 骨肉瘤、 胰腺癌、 淋巴癌、 结肠癌、 乳腺癌、 前列腺癌、 口腔癌、 鼻咽癌、 宫颈癌、 白血病、 恶性黑素瘤、 肉瘤、 肾癌、 胆癌。
23. 一种抑制肿瘤的侵袭和转移的方法， 其步骤包括抑制肿瘤细胞内 Hsp90a的磷酸化。
24. 权利要求 23的方法， 其步骤包括抑制肿瘤细胞内 Hsp90a第 90位 的苏氨酸的磷酸化。
25. 权利要求 23或 24的方法，其步骤包括在肿瘤细胞内过量表达编码 蛋白磷酸化酶 5的核酸。
26. 权利要求 25的方法，其中通过基因导入的方式过量表达所述核酸。
27. 权利要求 25的方法，其中所述蛋白磷酸化酶 5包含 SEQ ID No.5 的 酸序列。
PCT/CN2010/075896 2009-07-07 2010-08-11 一种新的肿瘤标志物 Ceased WO2011003369A1 (zh)

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CN105264381B (zh) * 2013-05-16 2018-06-01 国立大学法人京都大学 用于确定癌症预后的方法
RU2818471C2 (ru) * 2020-04-22 2024-05-02 Общество с ограниченной ответственностью "ДЖЕЙВИС ДИАГНОСТИКС" Новый раковый антиген для раннего выявления рака, способ его получения, выделения и детекции

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