WO2015149450A1 - Ehd2抗体及其在制备乳腺癌免疫组化检测试剂中的应用 - Google Patents

Ehd2抗体及其在制备乳腺癌免疫组化检测试剂中的应用 Download PDF

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WO2015149450A1
WO2015149450A1 PCT/CN2014/082480 CN2014082480W WO2015149450A1 WO 2015149450 A1 WO2015149450 A1 WO 2015149450A1 CN 2014082480 W CN2014082480 W CN 2014082480W WO 2015149450 A1 WO2015149450 A1 WO 2015149450A1
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ehd2
antibody
breast cancer
application
protein
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PCT/CN2014/082480
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French (fr)
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应国光
刘博�
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天津市应世博科技发展有限公司
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Priority to US15/301,259 priority Critical patent/US20170145081A1/en
Priority to EP14887836.6A priority patent/EP3127918B1/en
Priority to JP2016560438A priority patent/JP6810609B2/ja
Publication of WO2015149450A1 publication Critical patent/WO2015149450A1/zh
Priority to US16/292,054 priority patent/US11292851B2/en
Priority to US17/590,864 priority patent/US20220153865A1/en

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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • 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
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/812—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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

Definitions

  • the invention relates to the field of biotechnology, in particular, the expression and subcellular localization of EHD2 are significantly correlated with the survival of breast cancer patients, and can be used as a molecular index for diagnosis and prognosis of breast cancer malignancy. Background technique
  • Breast cancer is a common malignant tumor in women. There are one million women in the world every year. Since the late 1970s, the incidence of breast cancer in China has been the highest among female cancers. North America and Northern Europe are the high-incidence areas of breast cancer. The incidence rate in China has increased from 17 per 100,000 years ago to 52 per 100,000 last year, showing a rapid upward trend, and the age of onset is also increasing. The younger you are, the youngest is only fourteen. There are many factors that cause breast cancer. In addition to genetic factors, there are environmental factors, mental factors and work stress. Moreover, the more economically developed cities, the higher the incidence rate in rural areas. At present, countries around the world have invested a lot of energy and material resources to study the occurrence and development of the disease in order to take active preventive measures.
  • the clinical malignancy of tumors is mainly based on the conventional morphological observation of tumor tissues and tumor cells at the histopathological level. It has strong discrimination ability for the differentiation degree and invasion characteristics of cancer cells, and the accuracy rate is up to 50%-75%.
  • this kind of morphological-based interpretation is influenced by human factors such as pathologist experience, and on the other hand lacks a deeper molecular basis related to individual patient's pathogenesis factors. Therefore, it is urgent to combine with modern molecular techniques to improve judgment.
  • the accuracy and specificity in particular, provide a theoretical basis for the choice of individualized treatment pathways and protocols.
  • Immunohistochemistry uses the basic principles of immunological antigen-antibody reaction, the specific recognition of protein molecules in vivo by antibodies, and the coloration of secondary antibodies by chemical labels (fluorescein, enzymes, metal ions, isotopes, etc.)
  • the expression levels of the target antigens (polypeptides and proteins) in the tissue cells and their subcellular localization are examined.
  • the application value of immunohistochemistry in tumor diagnosis and identification has been widely used. Recognition.
  • the estrogen receptor ER, the progesterone receptor PR and the epidermal growth factor receptor family Her2 have been widely used in the clinical molecular pathological diagnosis of breast cancer, which has played a positive role in the pathological classification and clinical use of breast cancer. . These results are inseparable from the advancement of basic tumor research, and expect new molecular indicators to be further revealed and incorporated into clinical applications, and continue to improve the level of cancer diagnosis and treatment.
  • the EHD2 gene and its encoded protein may be such new molecular indicators.
  • EHD2 Eps in homology (EH) -doma in-conta ining prote ins
  • EH epidermal growth factor Receptor Pathway Subs tra te Clone 15
  • EHD2 is involved in the regulation of membrane transport, including internal transition and transition of the anterior and posterior inclusions and regulation of the circulating pathway; membrane and membrane protein regulation, including digestion and degradation. It is an indispensable and important part of the whole membrane transport mechanism, and plays a key role in maintaining the dynamic balance of material transport signaling in various cellular activities. Its imbalance will lead to improper signal response, cell dysfunction, and possibly In turn, it causes disease.
  • EHD2 immunohistochemical detection of EHD2 will more objectively and accurately reflect the alienation nature of the tumor and the clinical prognosis of the individual patient.
  • EHD2 antibodies available from mainstream antibody reagents such as Santa Cruz and Abeam, but they are all basic research reagents. There is no evidence that these antibodies can be as qualified and available as our antibodies.
  • immunohistochemistry in particular, no other EHD2 antibodies have been immunohistochemically verified by a large number of tissue samples and have been shown to achieve prognosis for tumor malignancy and patient survival by detecting subcellular abnormalities of EHD2. The purpose of the prejudgment. They have the following drawbacks compared to the antibodies of this patent application:
  • the main signal position obtained by immunoblotting detection is significantly less than 60kD, while the normal position signal of 70kD is weak, indicating Although EHD2 can be detected, more unknown molecules are detected;
  • the antibody from the company published an immunohistochemical map of lung cancer tissue showing that the staining signal is a non-specific staining of the apparent matrix component.
  • EHD2 is mainly distributed in the nucleus of normal epithelial cells, and a small amount is also expressed in cytoplasm and membrane.
  • the localization of EHD2 is abnormal.
  • the nuclear distribution decreased significantly, but the distribution of pulp and membrane increased.
  • An antibody specific for human EHD2 protein characterized in that said antibody specifically recognizes human EHD2 protein, and the amino acid sequence of said recognition site is: 503-SEQ ID NO: 1-543.
  • the gene EHD2 and its encoded protein are used in the immunohistochemical diagnosis and prognosis prediction of breast cancer.
  • the GeneBank international universal serial number of the gene EHD2 is: ⁇ _ 014601, the GeneBank international universal serial number of the gene EHD2 encoding protein For: NP _ 05541 6.
  • the use of the gene EHD2 and its encoded protein in the immunohistochemical diagnosis and prognosis of breast cancer is characterized by the use of the aforementioned antibodies.
  • the gene EHD2 and its encoded protein are used in the preparation of immunohistochemical diagnosis and prognostic prognostic agents for breast cancer.
  • the GeneBank international universal serial number of the gene EHD2 is: ⁇ _014601.
  • the use of the gene EHD2 and its encoded protein for the preparation of an immunohistochemical diagnosis and prognostic agent for breast cancer is characterized by the use of the aforementioned antibody.
  • the polypeptide is preferably modified with the amino acid sequence of the aforementioned polypeptide as a core sequence which is linked to a cysteine at the N-terminus of the polypeptide.
  • the application preferably immunizes the animal as an antigen to prepare an EHD2 antibody.
  • the application is preferably antigen purification of the antibody.
  • An immunohistochemical reagent for diagnosis and prognosis of breast cancer characterized in that the antibody specific for the human EHD2 protein is used as a primary antibody or a core antibody. Terms used in the description of the present invention:
  • the encoded protein of the gene EHD2 is the “EHD2 protein”. It will be understood by those skilled in the art that when the expression “expression of EHD2” is mentioned in the specification, it means “expression of the gene EHD2 and its encoded protein”.
  • the "core sequence” is the amino acid sequence of amino acids 503 to 543 of the human EHD2 protein (genebank protein No. NP-055416), and the polypeptide fragment corresponding to the core sequence can be used for immunization with or without modification after chemical synthesis or recombinant expression to generate a pair. EHD2 specific antibody.
  • Modification is a method of adding an amino acid, a coupling chemical group or a purification medium to a polypeptide fragment corresponding to the above core sequence by a conventional method such as chemical method or recombinant DNA, for the purpose of immunologically producing an antibody or performing antigen purification on the antibody.
  • a conventional method such as chemical method or recombinant DNA
  • the expression product of gene EHD2 is disordered in the expression of mammary epithelial cells, and the degree of disorder, especially nuclear expression, is closely related to the survival of patients, and it is detected by immunohistochemistry.
  • the results of manual interpretation are the only reliable means to understand the degree of expression of this gene in different locations within the cell.
  • EHD2 antibodies cannot be used for immunohistochemical detection, and the antibodies claimed to be used for immunohistochemical detection do not have immunoblot evidence showing acceptable specificity, in particular, the crossover of homologous proteins to EHD2 cannot be excluded.
  • Reactivity and found in practice or only in response to EHD2 protein in cell membranes, does not recognize important nuclear EHD2 proteins, or only detects non-specific signals to interstitial components.
  • none of the existing antibodies have been subjected to large sample detection and statistical analysis at the tissue level, and have been shown to have the property of recognizing the localization of EHD2 nuclear expression and to be used for prognosis prediction.
  • the EHD2 antibody provided by the invention has the specificity of specificity, and can be specifically identified by the immunoblotting method to recognize the EHD2 protein and cannot recognize other proteins with high homology.
  • the EHD2 protein can be quantitatively detected by immunoblotting, and can be prepared into an immunological group.
  • the reagent is directly used to judge the expression and localization of EHD2 in tissue cells, and in particular, it can be used to monitor the expression and localization of EHD2 in the nucleus, thereby being better used for the diagnosis and prognosis of breast cancer.
  • FIG. 1 is a specific immunoassay chart obtained in Example 1;
  • FIG. 2 is an immunohistochemical map of normal tissue cells in Example 1 of immunohistochemistry;
  • 3 is an immunohistochemical map of breast cancer cells in Example 1 of immunohistochemical assay;
  • FIG. 4 is an immunohistochemical map of breast cancer cells in Example 1 of immunohistochemical assay;
  • Figure 5 is an immunohistochemical map of breast cancer cells in Example 1 of immunohistochemistry
  • Figure 6 is a graph showing progression-free survival of 260 samples in Example 1 of immunohistochemistry. detailed description:
  • New England white rabbit purchased from Shanghai Qiang Yao Biology
  • the polypeptide CDEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE (the polypeptide is obtained by modifying the amino acid sequence of the polypeptide of SEQ ID NO: 1 as a core sequence, and the modification method is to attach a cysteine at the N-terminus.) Customized by Tianjin Saier Biotechnology Co., Ltd. Coupling with KLH;
  • CNBr-activated gel beads Freund's complete adjuvant, and Freund's incomplete adjuvant were purchased from Invitrogen.
  • the CNBr-activated gel beads were immersed in 1 mM HCl for 30 minutes, washed with coupling buffer (containing 0.1 M NaHC03 pH 8.3, and 0.5 M NaCl), and mixed with 1 mg of polypeptide plus 1 ml of gel, 4 Degree coupling overnight, 1 M
  • rinse solution 1 containing 50 mM Tris, 1 M NaCl, pH 8.0
  • clear Wash 2 containing 50 mM glycine, 1 M NaCl, H 3.5
  • the purified serum of the EHD2 antibody is mixed with the above antigen gel beads by a volume ratio of 20:1, and the mixed system is mixed with an equal volume of PBS, mixed for 1 hour, centrifuged, and the gel beads are washed with PBS for pH 3
  • the antibody bound to the gel beads was eluted with sodium citrate solution, and the pH was adjusted to 6.5-7.5 to obtain a purified EHD2 antibody.
  • Detection Example 1 Specific detection of EHD2 antibody by immunoblotting
  • 293T fine monthly package was purchased from American Type Culture Colectin (ATCC); RPMI1940 culture solution, BSA, HRP labeled anti-rabbit secondary antibody, Lipo2000 transfection reagent, RIPA lysate, BCA protein concentration detection reagent, ECL chemiluminescence detection Reagents and the like were purchased from Invitrogen; EHD EHD2, EHD3, and EHD4 expression plasmids were self-made.
  • 293T cells were cultured in RPMI1940 culture medium, and cultured at 37 °C and 5% CO 2 for adherence. When the cells were passaged, the culture solution was discarded, and then washed twice with phosphate buffered saline (PBS), and 0.05 was added. Digestion was performed for 2 minutes with trypsin and the medium was added to terminate the digestion. The cells are in good condition and are passed on for two days. Plasmids and transfection reagents expressing EHD1, EHD2, EHD3, and EHD4 were added at the time of transfection, and cells were harvested for immunoblotting after 2 days.
  • PBS phosphate buffered saline
  • the antibodies specific for EHD2 provided by the present invention are capable of specifically recognizing the EHD2 protein, but are not recognized by homologous other EHD proteins.
  • the anti-EHD2 antibody has the ability to specifically recognize EHD2 protein and does not cross-react with other homologous proteins.
  • the test result picture is shown in Figure 1.
  • Sample Ve is empty Vector, no signal; sample 1 overexpresses EHD1 protein, no signal; sample 2 overexpresses EHD2 protein, showing a major band at the corresponding molecular weight 70kd, with clear signal and no other obvious background bands; sample 3 overexpresses EHD3 protein, No signal; Sample 4 overexpressed EHD4 protein with no signal.
  • the antibody provided in this example has a strong signal at the normal position of 70 kd, and has no signal at other positions, and the results indicate that the antibody provided by the present invention has good specificity.
  • EHD2 was positively expressed in the nucleus of epithelial cells in normal tissues, and the plasma or membrane expression was weakly positive. In cancer tissues, EHD2 expression is disordered and nuclear expression is weakened.
  • the typical immunohistochemical map is shown in Figure 2 and Figure 5.
  • Figure 2 shows the immunohistochemical map of normal tissues. It shows that EHD2 is expressed in the epithelial nucleus of normal cells.
  • Figure 3_ Figure 5 shows the immunohistochemical map of different breast cancer tissue specimens. In the breast cancer tissue specimens shown in Fig. 3, EHD2 was expressed in the nucleus and plasma of the cancer cells. In the breast cancer tissue specimens shown in Fig.
  • EHD2 was not expressed in the nucleus of the cancer cells and strongly expressed in the plasma;
  • the overall loss of EHD2 expression in breast cancer cells indicates that EHD2 is disordered in expression in cancer cells and nuclear expression is diminished.
  • the progression-free survival curve of 260 samples is shown in Figure 6.
  • the abscissa is the number of months without progression, and the vertical coordinate is the number of cases without progression.
  • the present invention provides an antibody-based immunohistochemical detection method, which can The expression level and expression position of EHD2 in breast cancer tissue cells are well detected, and the location and expression of EHD2 in the nucleus of cancer tissues can be directly interpreted from the immunohistochemical map to predict the malignancy of breast cancer and the survival prospect of patients.

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Abstract

EHD2抗体及其在制备乳腺癌免疫组化检测试剂中的应用，所述抗体能特异性识别人EHD2蛋白，所述识别位点的氨基酸序列为：503-SEQ ID NO：1-543。

Description

EHD2抗体及其在制备乳腺癌免疫组化检测试剂中的应用
技术领域
本发明涉及生物技术领域， 具体是 EHD2的表达与亚细胞定位与乳腺癌患 者的生存具有显著相关性， 可以作为乳腺癌恶性度诊断和预后预判的一个分 子指标。 背景技术
乳腺癌是女性常见恶性肿瘤。 全世界每年有一百万妇女患病， 自 20世纪 70年代末开始、 我国乳腺癌的发病率一直居女性肿瘤的首位。 北美、 北欧原 是乳腺癌的高发区， 现在我国的发病率也从五年前的十万分之十七增加到去 年的十万分之五十二， 呈快速上升趋势， 而且发病年龄也越来越年轻化， 年 龄最小的只有十四岁。 引发乳腺癌的因素是多方面的， 除遗传因素外还有环 境因素、 精神因素及工作压力太大等因素。 而且经济越发达的城市发病率越 高于农村。 目前世界各国投入较大的精力和物力来研究该病的发生、 发展， 以便釆取积极的预防措施。 目前在临床上对肿瘤的恶性度判别主要还是基于在组织病理水平对肿瘤 组织和肿瘤细胞进行常规形态学观察， 它对癌细胞的分化程度和侵犯特性有 较强的辨别能力， 准确率可达 50%-75%。 然而， 这种基于形态学的解读一方面 受病理医师经验等人为因素影响， 另一方面缺乏更深层次的与病人个体发病 因素相关的分子依据， 因此亟需与现代分子技术相结合， 才能提高判断的准 确性和特异性， 特别是为个体化治疗路径与方案的选择提供理论依据。 虽然肿瘤的发生与多种因素相关， 但最终的根源主要来自于基因水平的 异常变化， 包括基因的扩增、 突变、 缺失、 异位等等， 导致维持细胞正常生 理状态所需的多个功能蛋白在表达水平的高低、 生化活性的强弱、 甚至亚细 胞结构分布等方面产生变化， 当这种变化赋予了细胞无限增殖能力及转移能 力， 并不能被体内外保护机制逆转或杀灭时， 便产生了肿瘤。 因此， 对肿瘤 恶性本质的深刻认识和有效控制的前提是明确这些功能蛋白分子的异变情 况， 这需要借助免疫组织化学的手段。 免疫组织化学技术应用免疫学抗原抗体反应基本原理， 通过抗体对体内 蛋白质分子抗原的特异性识别， 以及化学标记（荧光素、 酶、 金属离子、 同位 素等）的第二抗体的显色等作用来检测组织细胞内目标抗原（多肽和蛋白质） 的表达量及其亚细胞定位。 近年来， 随着免疫组织化学技术的发展和各种特 异性抗体的成功研发， 更主要的是对肿瘤分子机制认识的不断深入， 免疫组 化在肿瘤诊断与鉴别中的应用价值受到了普遍的认可。 比如雌激素受体 ER、 孕激素受体 PR和表皮生长因子受体家族的 Her2已经被广泛用于乳腺癌的临床 分子病理诊断， 它对乳腺癌的病理分类和临床用药起到了积极的指导作用。 这些成果离不开肿瘤基础研究的进步， 并期待新的分子指标被进一步揭示和 纳入临床应用， 不断提高肿瘤诊治水平。 EHD2基因及其编码蛋白即可能是这 种新的分子指标。
EHD2 (Eps in homology (EH) -doma in-conta ining prote ins)属于 EHD蛋 白的一种， 是一类新型膜转运调控蛋白， 含有高度保守的 EH结构域， 此 EH 结构域是一个含有约一百个氨基酸残基的片段， 早先发现于 EGFR的激酶底物 Eps l 5 (Epdema l Growth Factor Receptor Pa thway Subs tra te Clone 15)。 EHD2参与胞膜转运的各步调控， 包括内转及前、 后内含体的过渡和调控循环 通路； 膜及膜蛋白的各路调节， 包括消化降解及循环再生。 是整个胞膜转运 机制的必不可少的重要环节， 对在各种细胞活动中维护物料传输信号传导的 动态平衡起着关键的作用， 它的失调将导致信号应答失当， 细胞功能紊乱， 并可能进而引发疾病。
中国文献 "EHD2干扰影响永生化乳腺上皮细胞的增殖和迁移" (王洪玉等， 中国肿瘤临床， 2011年第 38卷第 11期，）公开了在乳腺肿瘤中 EHD2的基因表达 失调下降， 提示该基因可能是一个新的乳腺抑癌基因。 然而在进一步免疫组 化研究中我们发现， 虽然 EHD2在总体表达量上呈现下降的趋势， 但这种趋势 主要在核中； 相反， 细胞浆中的表达却有升高的趋势。 而且， EHD2在核中的 表达水平与患者的生存情况密切相关。 因此对 EHD2进行免疫组化检测将更客 观准确地反映肿瘤的异化本质和患者个体的临床预后情况。 现有的商业化 EHD2抗体有十余种， 由主流抗体试剂公司 Santa Cruz和 Abeam等提供， 但是均属于基础研究用试剂， 没有依据表明这些抗体可以和我 们的抗体一样具有合格的特异性并可用于免疫组化检测， 特别是目前尚没有 任何其它 EHD2抗体经过大量组织标本的免疫组化验证并显示出可以通过对 EHD2的亚细胞异常定位检测来达到对肿瘤的恶性度及患者生存情况进行预后 预判的目的。 与本专利申请抗体相比它们有如下缺陷：
1、 根据本领域公知常识，能够用于免疫组化（I HC )的抗体会在其使用说 明中作出明确标识和注明， 按这些抗体的说明所示， 大多数都不能用于免疫 组化（IHC )检测。 虽然个别抗体标明可用于免疫组化（IHC )， 但仔细研究发 现并非如此。 一个是南京森贝伽公司代理的 EHD2抗体， 但是注明了只能检测 膜定位。 另一个是 Prote intech公司的货号为 11440-1-AP的抗体， 然而此抗体 的特异性存在问题： 首先免疫印迹检测得到的主要信号位置明显小于 60kD， 而 70kD的正常位置信号却很弱， 说明虽然可以检测到 EHD2， 但更多检测到的 是其它未知分子； 其次从公司发表的该抗体对肺癌组织的免疫组化图中可见 染色信号为明显的基质成分的非特异性着色。
2、 这些抗体均为研究用试剂， 没有经过大样本临床病例试验或验证， 没 有关键的对 EHD2的核定位检测能力， 因此也就没有 "通过对 EHD2的亚细胞异 常定位检测来达到对肿瘤的恶性度及患者生存情况进行预后预判的目的" 的 前提条件与基础。
3、 均没有经过严格的免疫印迹特异性验证， 特别是与 EHD2蛋白的同源蛋 白 EHD1、 EHD3、 EHD4的交叉反应的免疫印迹实^ ^全证， 而这些驺证对 EHD2的 免疫组化检测方法来说尤其重要， 因为它们之间具有很高的同源性（>70% )。
中国文献 "EHD2下调促进乳腺上皮细胞转化的研究，， （田刚等， 现代医学 检验杂志， 2012年第 27卷第 1期， 49-51 )公开了一种由其自制的 EHD2抗体， 这一抗体是釆用我们的没有经过抗原纯化的抗体， 可以用于免疫印迹检测， 但不能满足免疫组化检测的高度特异性要求。 发明内容
我们在免疫组化研究中发现， 正常上皮细胞中 EHD2主要分布在细胞核， 同时细胞浆与膜中也有少量表达。但在乳腺癌组织中， EHD2的定位发生异变， 核分布显著下降， 浆与膜分布却出现升高的趋势。 临床因素分析结果表明，
EHD2的核分布情况与患者预后紧密相关，核表达越弱则患者的生存情况越差。 这些发现表明乳腺癌的恶性度与癌组织细胞中 EHD2蛋白的表达位置和表达水 平发生异变的程度密切关联， 而免疫组化技术是检测这种表达紊乱程度的唯 一可靠手段。 因此提供一种检测 EHD2基因的表达产物在癌细胞内部不同位置 特别是核中的表达情况的方法为现有技术中需要解决的问题。 为了解决上述问题， 本发明提供了以下技术手段和方案：
一种对人 EHD2蛋白特异的抗体，其特征是所述抗体能特异性识别人 EHD2 蛋白， 所述识别位点的氨基酸序列为： 503-SEQ I D NO: 1-543。
基因 EHD2及其编码蛋白在乳腺癌免疫组织化学法诊断和预后预判中的应 用， 所述基因 EHD2的 GeneBank国际通用序列编号为： 匪_ 014601， 所述基因 EHD2编码蛋白的 GeneBank国际通用序列编号为： NP _ 05541 6。 所述基因 EHD2及其编码蛋白在乳腺癌免疫组织化学法诊断和预后预判中 的应用， 其特征是釆用了前述抗体。 基因 EHD2及其编码蛋白在制备乳腺癌免疫组织化学法诊断和预后预判试 剂中的应用， 所述基因 EHD2的 GeneBank国际通用序列编号为： 匪_ 014601。 所述基因 EHD2及其编码蛋白在制备乳腺癌免疫组织化学法诊断和预后预 判试剂中的应用， 其特征是釆用了前述抗体。
一种多肽， 其特征是所述多肽氨基酸序列为 SEQ I D NO : 1，。 所述多肽， 优选以前述多肽氨基酸序列为核心序列对其进行修饰， 所述 修饰为在多肽 N端连接一个半胱氨酸。 所述多肽在制备前述抗体中的应用。 所述应用优选为作为抗原对动物进 行免疫以制备 EHD2抗体。 所述多肽在制备对 EHD2特异的抗体中的应用。 所述应用优选为对抗体进 行抗原纯化。 一种用于乳腺癌诊断和预后判断免疫组化试剂， 其特征是釆用前述对人 EHD2蛋白特异的抗体作为一抗或核心抗体。 本发明说明书中所釆用的术语：
"基因 EHD2的编码蛋白 "即 "EHD2蛋白"。 本领域技术人员应该了解， 当说明书中提及 "EHD2的表达"时，即是指 "基因 EHD2及其编码蛋白的表达"。
"核心序列" 为人 EHD2蛋白 ( genebank蛋白号 NP—055416)的第 503至 543位氨基酸序列，该核心序列对应的多肽片段在化学合成后或重组表达后经 或不经修饰可用于免疫以产生对 EHD2特异的抗体。
"修饰 " 为对上述核心序列对应的多肽片段用化学方法或重组 DNA等常 规方法进行添加氨基酸、 偶联化学基团或纯化介质， 目的是用于免疫产生抗 体或对抗体进行抗原纯化。 我们在研究中发现， 基因 EHD2的表达产物在乳腺上皮细胞癌变时表达分 布出现紊乱趋势， 其紊乱程度尤其是核表达情况与患者的生存情况密切关联， 而釆用免疫组化技术进行检测并对结果进行人工判读是了解这种基因在细胞 内不同位置表达紊乱程度的唯一可靠手段。 但现有的 EHD2抗体， 多不能用于 免疫组化检测， 而声称能够用于免疫组化检测的抗体并没有免疫印迹证据显 示合格的特异性， 特别是不能排除对 EHD2的同源蛋白的交叉反应性， 且在实 际应用中发现或者仅能对细胞膜中的 EHD2蛋白产生反应而不能识别重要的核 中 EHD2蛋白， 或者检测到的只是对间质成分的非特异性信号。 而且现有抗体 均没有在组织水平进行大样本检测和统计分析显示具有识别 EHD2核表达定位 的特性并用于预后预判的作用。 我们提供了一种对 EHD2特异的经抗原纯化的 抗体及其在制备免疫组织化学检测试剂中的应用。 本发明提供的 EHD2抗体具 有合格的特异性， 经免疫印迹方法测试可以特异识别 EHD2蛋白而不能识别高 度同源的其它蛋白， 可以釆用免疫印迹方法进行 EHD2蛋白定量检测， 还可以 制备成免疫组化试剂， 直接用于判断组织细胞中 EHD2的表达以及定位情况， 尤其是可以用于监测细胞核中 EHD2的表达定位情况， 从而可以更好的用于乳 腺癌的诊断和预后判断。 附图说明 图 1为特异性检测实施例 1中得到的免疫印迹图； 图 2为免疫组化检测实施例 1中正常组织细胞的免疫组化图； 图 3为免疫组化检测实施例 1中乳腺癌细胞的免疫组化图； 图 4为免疫组化检测实施例 1中乳腺癌细胞的免疫组化图；
图 5为免疫组化检测实施例 1中乳腺癌细胞的免疫组化图；
图 6为免疫组化检测实施例 1 中的 260例样本的无进展生存曲线图。 具体实施方式：
下面结合附图及实施例对本发明进行详细的说明。
抗体制备实施例 1: 对 EHD2特异抗体的制备
( 1 ) 实验材料来源
新英格兰大白兔购于上海强耀生物；
多肽 CDEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE (该多肽是以氨基 酸序列为 SEQ ID NO: 1的多肽为核心序列进行修饰得到的， 其修饰方法为在 N端连接一个半胱氨酸。 ) 由天津赛尔生物技术有限公司定制并与 KLH偶联；
CNBr活化的凝胶珠、 弗氏完全佐剂及弗氏不完全佐剂购自 Invitrogen公 司。
(2)动物免疫
取 4月龄新西兰大白兔 3只， 将 lOOug抗原多肽溶于 0.2ml 0.1M PBS ( H 7.2),与等体积弗氏完全佐剂充分混勾， 腹部皮下多点注射。 第一次免疫后第 15和 29天， 分别用 lOOug多肽 /0.2mlPBS与等体积弗氏不完全佐剂充分混合 后加强免疫。
( 3 ) EHD2抗血清的制备
于免疫结束后一周颈动脉取血， 37DC静置 3小时后离心取血清。
(4 )抗原凝胶珠制备
CNBr活化的凝胶珠于 1 mM HC1 中浸泡 30分钟， 用偶联緩冲液（含 0.1 M NaHC03 pH 8.3， 和 0.5 M NaCl )清洗， 按 lmg多肽加 lml凝胶的比例混合反 应体系， 4度偶联过夜， 1 M
乙醇胺浸泡 3小时后用清洗液 1 (含 50 mM Tris, 1 M NaCl, pH 8.0)和清 洗液 2 (含 50 mM甘氨酸， 1 M NaCl, H 3.5 ) 交叉清洗八遍， PBS清洗一遍。
( 5 ) EHD2抗体的纯化 血清与上述抗原凝胶珠按体积比为 20: 1的混合， 加与混合后体系等体积 的 PBS， 混匀 1小时后离心， 用 PBS清洗凝胶珠， 用 pH3柠檬酸钠溶液洗脱联 在凝胶珠上的抗体， 调 pH至 6.5-7.5， 得到纯化后的 EHD2抗体。 检测实施例 1: 对 EHD2抗体釆用免疫印迹法进行特异性检测
( 1 ) 实验材料来源：
293T 细月包购于美国 American Type Culture Col lect ion (ATCC)公司； RPMI1940培养液、 BSA、 HRP标记抗兔二抗、 Lipo2000转染试剂、 RIPA裂解 液、 BCA蛋白浓度检测试剂、 ECL化学发光检测试剂等购于 Invitrogen公司； EHD EHD2, EHD3、 EHD4表达质粒为自制。
( 2 ) 细胞培养
293T细胞培养于 RPMI1940培养液中， 37°C、 5%C02培养贴壁生长； 细胞 传代时先弃去培养液， 再用磷酸盐緩冲液（ phosphate buffered saline, PBS ) 洗两遍， 加入 0.05%胰蛋白酶消化 2分钟， 加入培养基终止消化。 细胞保持良 好状态， 两天传一代。 转染时分别加入表达 EHD1、 EHD2、 EHD3、 EHD4的质粒 及转染试剂， 2天后收取细胞做免疫印迹实验。
( 3 )免疫印迹方法 将各种细胞预留足够数量至离心管中， 离心后 RIPA裂解液裂解细胞， 煮 样， 再离心。 制得样品后， 使用 BCA检测试剂进行蛋白浓度的测定。 每个样 品分别取 80ug蛋白总量进行 SDS-PAGE电泳。 电泳结束后， 将胶上蛋白电转 到 PVDF膜上， 室温 5%牛奶封闭 1小时。 洗涤后， 与一抗 （实施例 1中制备 的抗体以 1: 2000加入 PBS和 5%BSA)在室温孵育 1小时。 然后与 1: 5000稀释 的抗兔二抗室温孵育 1小时。最后用化学发光检测试剂检测，检测结果见图 3。 从中可以看出， 本发明提供的对 EHD2特异的抗体能够特异识别 EHD2蛋白， 而对同源的其它 EHD蛋白不能识别。
(4) 结果： 抗 EHD2抗体具有对 EHD2蛋白的特异免疫识别能力， 并对其 它同源蛋白不产生交叉反应。 检测结果图片见图 1， 图中： 样品 Ve为空表达 载体， 无信号； 样品 1过表达 EHD1蛋白， 无信号； 样品 2过表达 EHD2蛋白， 在对应分子量 70kd位置呈一条主带， 信号清晰， 并没有其它明显背景条带； 样品 3过表达 EHD3蛋白， 无信号； 样品 4过表达 EHD4蛋白， 无信号。 综上， 本实施例提供的抗体在正常位置 70kd处具有强烈的信号， 而且在其他位置均 无信号， 结果表明本发明提供的抗体具有很好的特异性。 免疫组化检测实施例 1: EHD2免疫组织化学检测
( 1 ) 实验材料来源： 乳腺癌切片取自天津市肿瘤医院肿瘤组织标本库， 常规脱蜡， 样本数量 为 260。 一抗稀释液、 辣根过氧化物酶（HRP )标记的通用二抗、 二氨基联苯 胺（ DAB )底物、 底物稀释液等购自中杉金桥。
( 2 )免疫组化检测试剂组配条件及样本组织中 EHD2表达与定位的检测 方法： 方法主要步骤为： 组织切片脱蜡至水， 抗原修复， 内源过氧化物酶阻断， 以 1 : 200滴加实施例 （ 1 )制备得到的抗体作为一抗， 4 °C孵育过夜。 緩冲液 洗 3次每次 5min。 滴加通用 HRP酶标二抗， 在室温下孵育 30分钟。 緩冲液 洗 3次每次 5min。 DAB显色， 复染， 脱水封片后显微镜下观察染色情况。
( 3 ) 结果： 正常组织中 EHD2在上皮细胞核中表达阳性， 浆或膜表达弱阳性。 癌组织 中， EHD2表达发生紊乱， 且核表达趋弱。 典型免疫组化图见图 2一图 5， 图 2 为正常组织的免疫组化图， 显示出正常细胞 EHD2 在上皮细胞核表达， 图 3_ 图 5为不同乳腺癌组织标本的免疫组化图， 图 3所示乳腺癌组织标本中 EHD2 在癌细胞核与浆中均有表达， 图 4所示乳腺癌组织标本中 EHD2在癌细胞核中 不表达， 浆中强表达； 图 4所示乳腺癌组织标本中 EHD2在乳腺癌细胞中表达 整体缺失， 表明 EHD2在癌细胞中表达发生紊乱且核表达减弱。 260例样本的 无进展生存曲线图见图 6， 该图图例： 0 为核表达阴性； 1 为核表达阳性， censored表示死亡。 横坐标为无进展生存月数， 竖坐标为无进展生存病例百
以上实验结果表明， 釆用本发明提供抗体为核心的免疫组化检测方法， 能 很好地检测乳腺癌组织细胞中 EHD2的表达量和表达位置， 便于从免疫组化图 中直接判读 EHD2在癌组织细胞核中的定位和表达情况以预判乳腺癌恶性度和 患者生存前景。

Claims

权 利 要 求 书
1、 一种对人 EHD2蛋白特异的抗体， 其特征是所述抗体能特异性识别人 EHD2 蛋白， 所述识别位点的氨基酸序列为： 503-SEQ I D NO: 1 -543。
2、 基因 EHD2 及其编码蛋白在乳腺癌免疫组织化学法诊断和预后预判中的应 用， 所述基因 EHD2的 GeneBank国际通用序列编号为： 匪_ 014601， 其特征是 所述基因 EHD2编码蛋白的 GeneBank国际通用序列编号为： NP_ 05541 6。
3、 如权利要求 2所述基因 EHD2及其编码蛋白在乳腺癌免疫组织化学法诊断 和预后预判中的应用， 其特征是釆用了如权利要求 1所述抗体。
4、 基因 EHD2 及其编码蛋白在制备乳腺癌免疫组织化学法诊断和预后预判试 剂中的应用， 其特征是所述基因 EHD2 的 GeneBank 国际通用序列编号为：
NM_ 014601 o
5、 如权利要求 4所述基因 EHD2及其编码蛋白在制备乳腺癌免疫组织化学法 诊断和预后预判试剂中的应用， 其特征是釆用了如权利要求 1所述抗体。
6、 一种多肽， 其特征是所述多肽氨基酸序列为 SEQ I D NO: 1。
7、 如权利要求 6所述的多肽， 其特征是以所述多肽氨基酸序列为核心序列对 其进行修饰。
8、 如权利要求 7所述的多肽， 其特征是所述修饰为在多肽 N端连接一个半胱 氨酸。
9、如权利要求 6-8任一所述的多肽在制备如权利要求 1所述的抗体中的应用。
1 0、 如权利要求 9所述应用， 其特征是所述应用为对抗体进行抗原纯化。
1 1、 如权利要求 6-8任一所述多肽在制备对 EHD2特异的抗体中的应用。
1 2、 如权利要求 1 1所述应用， 其特征是所述应用为对抗体进行抗原纯化。
1 3、 一种用于乳腺癌诊断和预后判断免疫组化试剂， 其特征是釆用如权利要 求 1所述的抗体作为一抗或核心抗体。
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CN105400788A (zh) * 2015-12-25 2016-03-16 天津市应世博科技发展有限公司 抑制ehd2蛋白表达水平的rna干扰序列及其应用
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