WO2020177570A1 - 一种Her2伴随诊断免疫组化检测抗体及其应用 - Google Patents

一种Her2伴随诊断免疫组化检测抗体及其应用 Download PDF

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WO2020177570A1
WO2020177570A1 PCT/CN2020/076510 CN2020076510W WO2020177570A1 WO 2020177570 A1 WO2020177570 A1 WO 2020177570A1 CN 2020076510 W CN2020076510 W CN 2020076510W WO 2020177570 A1 WO2020177570 A1 WO 2020177570A1
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antibody
amino acid
seq
her2
acid sequence
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于占娇
李元浩
黄长江
梁旺
毛海燕
房健民
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荣昌生物制药(烟台)股份有限公司
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Priority to EP20766227.1A priority Critical patent/EP3842460A1/en
Priority to CN202080000517.4A priority patent/CN111670201A/zh
Priority to US17/049,768 priority patent/US20210238305A1/en
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • 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
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    • 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
    • G—PHYSICS
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    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/82—Translation products from oncogenes

Definitions

  • the present invention relates to the field of biopharmaceutical companion diagnostics, and in particular to a detection antibody for companion diagnosis for targeting Her2 (human epidermal growth factor receptor 2, human epidermal growth factor receptor 2) drugs, and detection applications and methods thereof.
  • Her2 human epidermal growth factor receptor 2, human epidermal growth factor receptor 2
  • Cancer is the main disease that endangers human health today. In China, the incidence of cancer is rising rapidly. More and more evidences show that cancer is a complex and diverse disease. Patients may show similar symptoms and have the same pathological changes, but they may be caused by completely different genetic changes. Because of this heterogeneity, the response rate of patients with the same type of cancer to the currently available drugs varies greatly. A certain treatment is often only a part of cancer patients respond, because it is impossible to judge the sensitivity and resistance of different tumor individuals to drugs before treatment, many patients often suffer unnecessary and/or damaging (side-effect) treatments . The current theoretical and practical basis for individualized or precision medicine is the difference in molecular genetics between individuals, which is considered to be the decisive factor in human disease susceptibility and drug response.
  • Companion diagnostics is an in vitro diagnostic technology related to targeted drugs. It mainly measures the expression levels of proteins and variant genes in the human body to understand the response of different patients to specific drugs and screen out the most appropriate The drug users are also targeted for individualized treatment, so as to improve their treatment prognosis and reduce health care expenditures.
  • the US FDA promulgated the "Guidelines for Companion Diagnosis” on August 6, 2014. Companion diagnosis helps to determine the patient population most likely to respond to the therapeutic drug, promote the use of the drug in a relatively limited market, and improve the effectiveness and safety of the drug.
  • CDx facilitates the design of clinical trial protocols with small samples. In the development process, clearer and clearer results can be obtained with less investment.
  • companion diagnosis is that it can screen out effective treatment options for patients, save the time and cost of ineffective treatment, improve patient compliance with medication, reduce the incidence of adverse reactions, and ensure drug safety and efficacy (Reference 2: Companion diagnosis) ——Personalized treatment booster, Thomson Reuters, Advances in Pharmacy, Volume 39, Issue 6, Pages 463-477, 2015).
  • Her2 also known as ErbB2 is the second member of the EGFR family. It forms a heterodimer with the other three members of the EGFR family to cause the activation of the EGFR signaling pathway, which is usually associated with abnormal cell proliferation. It is related to tumor formation, so Her 2 has become a variety of cancers (such as: breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, lung cancer, urothelial cancer, bladder cancer, etc.; see reference 16: Human Epidermal Growth Factor Receptor 2(HER2) in Cancers: Overexpression and Therapeutic Implications, Nida Iqbal and Naveed Iqbal, Molecular Biology International, Volume 2014, Article ID 852748)
  • One of the therapeutic targets of monoclonal antibodies currently including trastuzumab A variety of therapeutic drugs targeting Her2, including Pertuzumab and Pertuzumab, are on the market or are in the clinical stage.
  • the increase in Her-2 activity is generally considered to be related to the amplification of Her2 gene, the up-regulation of Her2 protein expression and the mutation of Her-2 protein. Among them, the amplification of HER2 gene and the up-regulation of Her-2 protein expression are the most common.
  • the 34 companion diagnostic reagents approved by the US FDA (as of the end of February 2019) (Document 3: https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431.htm). It shows that since 1998, 9 companion diagnostic kit products involving targeted Her2 drugs have been approved (Document 4: HER2 testing: Current status and future directions, Edith A. Perez, etc., Cancer Treatment Reviews, Vol. 40, No. 276-284 pages, 2014) (see Table 1). This indicates the continued market demand for companion diagnostic reagents for this targeted drug.
  • FISH fluorescence in situ hybridization
  • fluorescence in situ hybridization fluorescence in situ hybridization
  • HER2 human epidermal growth factor receptor 2, human epidermal growth factor receptor 2;
  • FISH costs about 1,200 yuan
  • IHC costs about 150 yuan
  • FISH costs 8 times the IHC testing costs
  • the reading has a morphological basis, especially It is easy to operate on a large number of samples, high sensitivity, and relatively high repeatability between the laboratory and the laboratory, so the IHC method as a preliminary screening tool is better than the CISH method.
  • Pathologists usually regard the IHC method as the first choice for state evaluation. However, false positive results will occur in cases that are judged to be positive based on the IHC method 2+.
  • the current ASCO/CAP American society of clinical oncology/College of American Pathologists recommends the Her2 detection process for breast cancer: First, usually IHC method For detection, cases with a result of 3+ are often positive for FISH. IHC results of 1+ and below are usually negative for FISH. Cases with an IHC test result of 2+ need to be reconfirmed by the FISH method (see Figure 1 for details, Document 7: Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer, American Society of Clinical Online College of American Pathology Guideline, Clinical Update Practice, Antonio C. Wolff et al., Arch Pathol Lab Med, Volume 142, Pages 1364-1382, November 2018).
  • the commonly used Her2 expression detection antibodies mainly include Roche's 4B5 clone, Leica's CB11 clone, etc.
  • the above detection antibodies are antibodies that recognize the intracellular region of Her2 molecules, and all therapeutic antibody drugs that target Her2 are Recognizing the extracellular region of the Her2 molecule, given that the Her2 molecule has a mutant form of deletion of the extracellular region (95-100kDa p95HER2(648-CTF), 100-115kDa p95HER2(611-CTF)) (Document 10: p95HER2 and breast cancer.Arribas J. et al., Cancer Res. Vol.
  • the false negative problem in the IHC method in the prior art in the immunohistochemical detection, the correct antigen (or epitope) retrieval (antigen (or epitope) retrieval) is a key step for the accuracy of the immunohistochemical detection result.
  • tissue fixation formaldehyde cross-links with protein to form aldehyde cross-linked protein. This compound blocks the antigen and reduces the specific reaction of the antibody to the antigen, leading to false negatives.
  • the automatic multifunctional histopathology detection system has the advantages of uniform staining, accurate positioning, and strong repeatability. However, the equipment is expensive, the cost of reagents is high, and the scope of use is limited. The pathology department of primary hospitals still generally uses manual operation methods.
  • WO2015074528A1 and CN105008398A disclose an antibody drug conjugate (ie antibody drug conjugate, ADC) capable of specifically binding Her2, wherein the antibody is combined with one or more selected A therapeutic agent coupling from MMAE and MMAF, the antibody comprises a heavy chain and a light chain, wherein (i) the amino acid sequences of the heavy chain CDR1-3 regions are DYYIH, RVNPDHGDSYYNQKFKD, ARNYLFDHW, respectively; and (ii) the light chain CDR The amino acid sequences of 1-3 are respectively KASQDVGTAVA, WASIRHT, HQFATYT; or further the antibody is derived from the Hybridoma Institute deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No.
  • ADC antibody drug conjugate
  • the conjugate comprising an antibody capable of specifically binding to HER2 is used for the treatment of Her2 positive cancer patients, the Her2 positive cancer includes breast cancer, ovarian cancer or gastric cancer, or further is lapatinib and/or Herceptin Drug-resistant breast, ovarian, or gastric cancer.
  • the Her2 positive cancer includes breast cancer, ovarian cancer or gastric cancer, or further is lapatinib and/or Herceptin Drug-resistant breast, ovarian, or gastric cancer.
  • the present invention provides a detection antibody targeting the extracellular region of Her2.
  • the above-mentioned antibody specifically binds to the IV region of the extracellular domain of Her2 protein.
  • Her2 An example amino acid sequence of Her2 is shown in NCBI GenBank ID: AAA75493.1, and the corresponding extracellular domain IV region of the protein is located at amino acids 511-643.
  • Johan Rockberg et al. Document 14: Discovery of epitopes for targeting the human epidermal growth factor receptor 2 (HER2) with antibodies, Johan Rockberg, etc., MOLECULAR ONCOLOGY, Volume 3, Pages 238-247, 2009) and Beijing Yiqiao "Her2/ERBB2 Protein, Human, Recombinant (ECD, domain IV, His Tag)" commercially available from China Technology Co., Ltd.
  • Heavy chain variable region CDR1-3 are: GFNIKDTYIH, RIYPTNGYTRYADSVKG, WGGDGFYAMDV; and light chain variable region CDR1-3 They are: RASQDVNTAVAW, SASFLES, QQHYTTPPT (WO9222653A1);
  • Heavy chain variable region CDR1-3 are: GFNIKDTYIH, RIYPTNGYTRYADSVKG, WGGDGFYAMDY;
  • light chain variable region CDR1-3 are: RASQDVNTAVA, SASFLES, QQHYTTPPT;
  • Literature 15 Molecular dynamic simulation of Trastuzumab F(ab')2 structure in corporation with HER2 as a thermostic agent of breast cancer, S Hermanto, etc., Journal of Physics: Conference Series, Volume 835, Issue 1, Pages 1-11 , 2017).
  • Such antibody uses are therapeutic antibodies.
  • the present invention surprisingly found that some antibodies that specifically bind to the IV region of the extracellular domain of Her2 protein can be used as primary antibodies in immunohistochemical detection. These antibodies are not tested by the antigen in the patient's immunohistochemical detection sample. In the case of (or epitope) repair treatment, these antibodies can still be specifically and effectively recognized and detected. This effectively reduces the false negative problem caused by differences in antigen (or epitope) repair processing methods in IHC detection. At the same time, since the above-mentioned repair processing steps are not required, the work intensity can be effectively reduced, the detection time is shortened, and the efficiency of IHC detection can be improved.
  • the present invention also surprisingly discovered that the antibody in the drug conjugate (RC48-ADC) of the Her2 targeting antibody (RC48) disclosed in WO2015074528A1 and CN105008398A (the amino acid sequence of the heavy chain CDR1-3 region is DYYIH, respectively).
  • the amino acid sequences of RVNPDHGDSYYNQKFKD, ARNYLFDHW; and () light chain CDR 1-3 are KASQDVGTAVA, WASIRHT, HQFATYT respectively) that bind to the same epitope (located in the IV region of the extracellular domain of the Her2 protein) or have the same light and heavy CDRs above
  • the antibody can be used as a companion diagnostic IHC antibody to confirm Her2 positivity.
  • the corresponding IHC antibody detection avoids the presence of the Her2 molecule in the mutant form of the deletion of the extracellular region.
  • the possibility of being positive therefore, also effectively solves the false-positive problem caused by the detection of the primary antibody against the intracellular region of Her2.
  • the CDR region of the antibody in the targeted drug is the same, the epitope bound during the detection is exactly the same as the targeted epitope of the therapeutic drug.
  • This detection antibody has a high degree of correspondence with the binding epitope of the pharmaceutical antibody. It guarantees the pertinence and effectiveness of targeted drugs.
  • the present invention provides an antibody for human epidermal growth factor receptor 2 (Her2) companion diagnostic immunohistochemical detection (IHC), which specifically binds to region IV of the extracellular domain of Her2 protein, and the antibody has effective detection The performance of IHC samples without antigen or epitope retrieval processing.
  • Her2 human epidermal growth factor receptor 2
  • IHC immunohistochemical detection
  • the antibody Fc segment is a non-human mammalian antibody Fc segment, preferably a murine Fc fragment or a rabbit Fc fragment.
  • the antibody competitively binds to the same or similar epitope as the antibody defined by the following CDRs.
  • the CDRs 1-3 of the heavy chain variable region of the antibody defined by the following CDRs are shown in SEQ ID NO: 1-3, respectively
  • the CDRs 1-3 of the light chain variable region of the antibody defined by the CDRs are as shown in SEQ ID NO: 4-6, respectively.
  • the CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 with 1 or 2 amino acid substitutions; or/and CDR2 has SEQ ID NO: 2
  • the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence after 1, 2, 3, 4 or 5 amino acid substitutions; or/and CDR3 has the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: : 3 amino acid sequence after 1, 2 or 3 amino acid substitutions; and/or
  • the CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 after 1, 2, 3 or 4 amino acid substitutions; or/and CDR2 has SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 after 1 or 2 amino acid substitutions; or/and CDR3 has the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 Amino acid sequence after 1 or 2 amino acid substitutions;
  • the immunoglobulin Fc fragment is a murine IgG Fc fragment, or further is a murine IgG1 Fc fragment.
  • CDR1-3 of the variable region of the heavy chain are the amino acid sequences shown in SEQ ID NO: 1-3;
  • the CDR1-3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 4-6, respectively.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or a sequence with at least 80% sequence identity therewith; and/or
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 8 or a sequence with at least 80% sequence identity therewith;
  • the CDR1-3 of the heavy chain variable region of the antibody are shown in SEQ ID NO: 1-3, respectively; the CDR1-3 of the light chain variable region of the antibody are shown in SEQ ID NO: The amino acid sequence shown in 4-6.
  • an antibody R48M is provided, the amino acid sequence information of which is as follows:
  • the present invention further provides a nucleic acid molecule which encodes the aforementioned antibody.
  • the present invention further provides a vector comprising the above-mentioned nucleic acid molecule.
  • the present invention further provides the use of the above-mentioned antibody in the preparation of Her2 companion diagnostic immunohistochemical detection products, which have the function of not requiring antigen or epitope repair steps in the immunohistochemical detection process.
  • the present invention further provides a Her2 companion diagnostic immunohistochemical detection kit, which comprises the above-mentioned antibody, and the kit has the function of not requiring an antigen or epitope repair step in the immunohistochemical detection process.
  • the present invention further provides a Her2 companion diagnostic immunohistochemical detection method, which uses the aforementioned antibody as the detection primary antibody. Or further there is no antigen or epitope restoration step in the immunohistochemical detection process.
  • the indications for the Her2 companion diagnosis of the present invention are Her2-related cancers, and more preferably breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, lung cancer, urothelial cancer or bladder cancer. (See Reference 16)
  • the present invention provides an antibody that can target the IV region of the extracellular domain of Her2 protein, and when the antibody is used as a primary antibody to detect the expression of Her2 in a sample by immunohistochemistry , Can effectively avoid false positive test results caused by the deletion of extracellular region.
  • the immunohistochemical detection process of the Her2 detection antibody provided by the present invention since the above-mentioned repair processing steps are not required, it effectively reduces the difference in the antigen (or epitope) repair processing method in the IHC detection. False negative problem. At the same time, it can effectively reduce the work intensity and shorten the detection time, thereby improving the efficiency of IHC detection.
  • Figure 2A shows the result of immunohistochemical staining of the case numbered 1 in Table 7, the primary antibody is RC48M antibody;
  • Figure 2B shows the results of immunohistochemical staining of the case numbered 1 in Table 7, where the primary antibody is 4B5 antibody;
  • Figure 3A shows the results of immunohistochemical staining of the case number 2 in Table 7, where the primary antibody is the RC48M antibody;
  • Figure 3B shows the results of immunohistochemical staining of the case number 2 in Table 7, where the primary antibody is 4B5 antibody;
  • Figure 4A shows the results of immunohistochemical staining of the case number 3 in Table 7, where the primary antibody is the RC48M antibody;
  • Figure 4B shows the results of immunohistochemical staining of the case number 3 in Table 7, where the primary antibody is the 4B5 antibody;
  • Figure 5A shows the results of immunohistochemical staining of the case number 4 in Table 7, where the primary antibody is the RC48M antibody;
  • Figure 5B shows the results of immunohistochemical staining of the case number 4 in Table 7, where the primary antibody is 4B5 antibody;
  • Figure 6A shows the results of immunohistochemical staining of the case numbered 5 in Table 7, where the primary antibody is the RC48M antibody;
  • Figure 6B shows the results of immunohistochemical staining for the case numbered 5 in Table 7, where the primary antibody is 4B5 antibody;
  • Figure 7A shows the result of immunohistochemical staining of the case numbered 6 in Table 7, the primary antibody is the RC48M antibody;
  • Figure 7B shows the results of immunohistochemical staining of the case numbered 6 in Table 7, the primary antibody is 4B5 antibody;
  • Figure 8A shows the results of immunohistochemical staining of the case number 7 in Table 7, where the primary antibody is the RC48M antibody;
  • Figure 8B shows the results of immunohistochemical staining of the case numbered 7 in Table 7, where the primary antibody is the 4B5 antibody;
  • Figure 9 shows the structural formula of a RC48-ADC drug, where RC48 represents RC48 antibody.
  • Antibody as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments.
  • antibody as used herein refers to a protein comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (one-fifth or a quarter of the region close to the N-terminal on the heavy chain) and a heavy chain constant region (three-quarters or four-fifths of the heavy chain close to the C-terminal Area).
  • Each light chain contains a light chain variable region (the region close to the N-terminal half of the light chain) and a light chain constant region (the region close to the C-terminal half of the light chain).
  • the heavy chain variable region and light chain variable region can be further subdivided into multiple regions with high variability, called complementarity determining regions (CDR).
  • CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins, including those determined by Kabat, Chothia, or IMGT systems. There are three heavy chain CDRs and three light chain CDRs per antibody. According to circumstances, the term CDR as used herein is used to indicate one of these regions, or several or even all of these regions, which contains most of the amino acid residues responsible for binding by the affinity of the antibody to the antigen or its recognition epitope.
  • Her2 detection antibody used in the present invention refers to an antibody capable of detecting Her2 expression status in a case, and it can bind to Her2. In immunohistochemical chemistry, Her2 detection antibody is used as a primary antibody to detect Her2 expression status.
  • the "Her2 detection antibody” involved in the present invention can target the IV region of the extracellular domain of the Her2 protein.
  • R48M antibody used in the present invention refers to an antibody whose heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7, and light chain variable region amino acid sequence is shown in SEQ ID NO: 8.
  • RC48-ADC or "RC48-ADC drug” or “RC48-ADC targeted drug” used in the present invention refers to the antibody drug conjugate (ie, the antibody drug conjugate capable of specifically binding Her2) disclosed in patents WO2015074528A1 and CN105008398A Antibody-drug conjugate, ADC), wherein the antibody is called “RC48 antibody”, and its (i) heavy chain CDR 1-3 amino acid sequences are DYYIH, RVNPDHGDSYYNQKFKD, ARNYLFDHW, respectively; and (ii) the light chain The amino acid sequences of CDR1-3 are respectively KASQDVGTAVA, WASIRHT, HQFATYT; or further the antibody is derived from the Hybridoma Institute deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No.
  • the antibody is derived from an antibody secreted by CHO cells deposited with the China Center for Type Culture Collection on November 06, 2013 under the deposit number CCTCC C2013170.
  • the structure of an RC48-ADC drug is shown in Figure 9.
  • the RC48M antibody is used as an example of an IHC detection antibody targeting the IV region of the extracellular domain of Her2 protein.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the light chain variable region
  • the amino acid sequence is shown in SEQ ID NO: 8.
  • test result list is shown in Table 4.
  • Antigen retrieval Transfer the second group of slices to sodium citrate buffer and heat it at 98°C for 20 minutes; the first group of slices does not require antigen retrieval.
  • DAB color development use DAB color development kit (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.), take the dilution buffer in 1mL kit, DAB chromogen and 50 ⁇ l each of the substrate, and add to Slice, develop color at room temperature for 1-3 minutes, wash with distilled water;
  • Hematoxylin counterstaining add 100 ⁇ L of hematoxylin staining solution (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.) to each slice, stain at room temperature for 2-5 minutes, and rinse with tap water for 5 minutes to turn blue.
  • the RC48M antibody and the 4B5 antibody have similar staining in the serial number 2, 3, 4, 5, 6, 7 cases, while the RC48M and 4B5 showed a difference in the serial number 1, in which RC48M staining is negative, while 4B5 staining is positive .
  • Table 7 The staining results of the RC48M antibody and the 4B5 antibody in the serial number 3, 4, 5, and 6 were the same as 3+. In the serial number 2 case, the staining results of the two were both 2+. In the serial number 7 case, the staining results of the two were 1 grade.
  • PD disease progression
  • PR partial remission
  • SD stable disease

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Abstract

本发明提供了一种人表皮生长因子受体2(Her2)伴随诊断免疫组化检测(IHC)用抗体，该抗体作为一抗对样品的Her2表达情况进行免疫组化检测时，能够避免因胞外区缺失导致的检测结果的假阳性。并且，所述抗体在患者免疫组化检测样本不经抗原(或表位)修复处理的情况下，仍然能够识别并结合检测样本中的相应表位，从而降低了IHC检测中因抗原(或表位)修复处理方法的差异所产生的假阴性问题。

Description

一种Her2伴随诊断免疫组化检测抗体及其应用 技术领域
本发明涉及生物药物伴随诊断领域，具体涉及一种针对靶向Her2(human epidermal growth factor receptor 2，人表皮生长因子受体2)药物的伴随诊断用检测抗体及其检测应用及方法。
背景技术
癌症是当今危害人类健康的主要疾病。在中国，癌症发生率正处于快速上升期。越来越多的证据显示，癌症是一种复杂和多样性疾病，患者可能表现出类似的症状，并具有相同的病理改变，却可能由完全不同的基因变化而造成。正因为这样的异质性(heterogeneity)，病理同类型癌症患者对目前可用药物的反应率差别很大。某一特定治疗往往只有一部分肿瘤患者有反应，因为无法在治疗前判断不同肿瘤个体对药物的敏感性和耐药性，许多患者往往遭受不必要的和(或)损害性大(副作用)的治疗。目前支持个体化或精准医疗的理论和实践基础就是个体间在分子遗传上的差异，后者被认为是人类疾病易感性和药物反应的决定性因素。早期的例子充分证明了遗传与肿瘤精准医疗的关系：靶向抗癌药物吉非替尼(Iressa)和厄洛替尼(Tarceva)最初上市时,在肺癌患者中应用的药品副作用虽较化疗有很大提高，但药效并不是非常显著，仅仅作为肺癌治疗的二线药物。但其后发现在携带EGFR特定基因突变的肺癌患者中，吉非替尼和厄洛替尼治疗效果优异，现在已成为临床治疗这类患者的一线标准治疗药物。这证明了肿瘤基因突变与药物和治疗反应的相关性，而根据基因突变的信息和组合来决定患者的靶向治疗方案是精准医疗的大方向(文献1：肿瘤的精准医疗：概念、技术和展望，杭渤等，科技导报，第33卷，第15期，第14－21页，2015年)。
伴随诊断(companion diagnostics，CDx)则是一种与靶向药物相关的体外诊断技术，主要通过测量人体内蛋白、变异基因的表达水平，了解不同患者对特定药物的治疗反应，筛选出最合适的用药人群并有针对性地进行个体化治疗，从而改善其治疗预后和降低保健开支。美国FDA于2014年8月6日颁布了《伴随诊断指南》。伴随诊断有助于确定最有可能针对治疗药物产生响应的患者群体，促进药物在相对有限的市场中使用，提高药物的有效性和安全性。在药物的开发中，CDx有利于设计小样本的临 床试验方案，在开发过程中，以更少的投入获得更清楚明确的结果。伴随诊断的优势在于可以为患者筛选出有效的治疗方案，节省无效治疗的时间和费用，提高患者服药的依从性，降低不良反应的发生率，保证了药物安全性和疗效(文献2：伴随诊断——个体化治疗助推器，汤森路透，药学进展，第39卷，第6期，第463－477页，2015年)。
Her2，又名ErbB2，是EGFR家族的第二个成员，其通过与EGFR家族中其它三位成员构成异源二聚体从而引起EGFR信号通路的活化，该信号通路的活化通常与细胞的异常增殖和肿瘤形成有关，因此Her 2成为多种癌症(诸如：乳腺癌、胃癌、胃食管癌、食道癌、卵巢癌、子宫内膜癌、肺癌、尿路上皮癌、膀胱癌等；参见文献16：Human Epidermal Growth Factor Receptor 2(HER2)in Cancers:Overexpression and Therapeutic Implications，Nida Iqbal and Naveed Iqbal，Molecular Biology International，Volume 2014,Article ID 852748)的治疗靶点之一，目前已有包括曲妥珠单抗和帕妥珠单抗在内的多种以Her2为靶点的治疗性药物上市或处于在临床阶段。Her-2活性升高通常被认为与Her2基因扩增、Her2蛋白表达上调以及Her-2蛋白的突变有关。其中以HER2基因扩增并由此引起Her-2蛋白表达上调最为常见。在美国FDA批准(截止至2019年2月底)的34种伴随诊断试剂中(文献3：https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVit roDiagnostics/ucm301431.htm)。显示出自1998年以来，批准的涉及靶向Her2药物的伴随诊断试剂盒产品有9种(文献4：HER2 testing:Current status and future directions，Edith A.Perez等，Cancer Treatment Reviews,第40卷，第276－284页，2014年)(见表1)。这表明针对该靶向药物的伴随诊断试剂持续的市场需求。
表1 FDA批准的HER-2靶向治疗前的伴随诊断试剂盒(文献4)
Figure PCTCN2020076510-appb-000001
Figure PCTCN2020076510-appb-000002
Figure PCTCN2020076510-appb-000003
注：CISH,chromogenic in situ hybridization，显色原位杂交；
FISH,fluorescence in situ hybridization(荧光原位杂交)；
HER2,human epidermal growth factor receptor 2，人表皮生长因子受体2；
IHC,immunohistochemistry，免疫组化。
目前美国FDA批准的靶向Her2药物的伴随诊断试剂盒中，基于免疫组化检测的有四种(2种半定量IHC，2种IHC)，基于原位杂交的有五种(两种FISH，三种CISH)。免疫组化(IHC)检测细胞膜上的Her2蛋白表达，原位杂交(ISH)检测的则是Her2基因的扩增。三种方法用于检测的原理及特点如表2所示。
表2 IHC，CISH及FISH检测原理及特点
Figure PCTCN2020076510-appb-000004
尽管CISH和FISH的检测结果相对于IHC的检测具有更高的准确性，但由于IHC法不但经济(根据McGill University Health Centre(MUHC)在2006年所作的一份评估报告中给出了采用IHC和FISH的大致价格比较，IHC单次检测价格约85美元而FISH单次检测价格约381美元(文献5： https://muhc.ca/sites/default/files/micro/m-TAU/HER2reportdraft4finalMay2006.pdf，第15页)；Seema Jabbar等人(文献6：Comparison of Two FDA-Approved Her2 Immunohistochemical Assays for Breast Carcinoma:HercepTest and Pathway Her2(4B5),Am J Clin Pathol 2018；149:S93-S94)针对共计95份样本检测，使用FISH检测的总花费分别为5066美元(25份)和14590美元(72份)，平均单次测202美元。以上地区的FISH检测费用约为IHC检测费用的3－4倍。而根据中国目前阶段医院实际的检测费来看，FISH费用1200元左右，IHC收费150元左右，FISH费用达到了IHC检测费用的8倍之多)、快捷,而且阅片有形态学基础,尤其对大量样本易于操作,敏感性较高,实验室内部与实验室之间可重复性相对高,所以IHC法作为初筛工具优于CISH法。病理医师通常把IHC法作为状态评价的首选检测方法。但基于IHC法2+判断阳性的病例中会出现假阳性的结果,目前ASCO/CAP(American society of clinical oncology/College of American Pathologists)推荐的针对乳腺癌的Her2检测流程为：首先通常过IHC方法检测，针对结果为3+的病例往往也是FISH检测阳性，IHC检测结果为1+及以下通常FISH检测为阴性。IHC检测结果为2+的病例，需要经过FISH方法进行再次确认(具体见图1，文献7：Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer，American Society of Clinical OncologyCollege of American Pathologists Clinical Practice Guideline Focused Update，Antonio C.Wolff等，Arch Pathol Lab Med，第142卷,第1364－1382页，2018年11月)。
现有技术中IHC法中的假阳性问题：目前对患者样本Her2表达状态的初次检测主要依赖于IHC检测。在IHC检测中，筛选到有效性和可靠性高的一抗(primary antibody)是尤其关键的一步。IHC检测中的抗体从根本上决定了整个检测结果的灵敏性或特异性。Prescott等人指出，IHC检测中42.1％的诊断差异均是由于抗体不佳的原因导致的(文献8：Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice，Leandro Luongo de Matos等，Biomarker Insights，第5卷，第9–20页，2010年；文献9：Audit of tumour histopathology reviewed by a regional oncology centre.Prescott RJ等.J Clin Pathol.第48卷，第245–249页.1995年)。目前常用的Her2表达状态检测抗体主要有Roche公司的4B5克隆、Leica公司的CB11克隆等，上述检测抗体均为识别Her2分子胞内区的抗体，而所有以Her2为靶点的治疗性抗体药物均识别Her2分子的胞外区，鉴于Her2分子存在胞外区缺失的突变形式(95-100kDa  p95HER2(648-CTF),100-115kDa p95HER2(611-CTF))(文献10：p95HER2 and breast cancer.Arribas J.等，Cancer Res.第71卷第5期，第1515-1519页，2011年)，相应的病例对曲妥珠单抗和帕妥珠单抗等以Her2胞外区为靶点的药物无反应或不敏感，但通过上述商用伴随诊断试剂检测，该部分病例将显示假阳性，这也正是目前接受抗体类Her-2靶向药的响应率只有约60％的一个主要原因。因此开发特异识别Her2分子胞外区的检测抗体及基于相应抗体开发的伴随诊断IHC试剂产品，将能有效降低目前her2伴随检测中所出现的假阳性问题，从而为相应患者节省无效治疗的时间和巨额的费用。
现有技术中IHC法中的假阴性问题：在免疫组化检测中，正确的抗原(或表位)修复(Antigen(or epitope)retrieval)是免疫组化检测结果准确度的关键步骤。由于在组织固定过程中，甲醛与蛋白质发生交联形成醛交联蛋白，这种化合物封闭了抗原，导致抗体对抗原的特异性反应降低，从而导致假阴性。全自动多功能组织病理检测系统具有染色着色均匀、定位准确、可重复性强等优点，但设备昂贵，试剂成本高，使用范围受到限制，基层医院病理科目前仍普遍使用手工操作方法。目前手工操作中各科的修复方法以及采用的修复液不尽相同，即使相同的方法修复细节也有很大的差别，正确的修复对于抗体的表达非常重要。因此提高不同基层医院人员操作结果的重复性，如何避免修复过程中产生的假阴性问题也是目前IHC检测中需要解决的问题(文献11：乳腺癌HER-2免疫组化柠檬酸高温高压抗原修复手工操作的最佳条件，朱国良等，临床与实验病理学杂志，第33卷，第2期，第219－220页；文献12：比较不同抗原修复液对免疫组化结果的影响，王琦等，中国实验诊断学，第19卷，第8期，第1246－1248页，2018年8月；文献13：病理学高级教程，来茂德主编，出版发行:北京：人民军医出版社，2013.03；ISBN号：978-7-5091-6426-6，第856页)。
在靶向Her2的药物中，WO2015074528A1和CN105008398A中公开了一种包含能够特异性结合Her2的抗体药物缀合物(即抗体药物偶联物，ADC)，其中所述抗体与一个或更多个选自MMAE和MMAF的治疗剂偶联，所述抗体包含重链和轻链，其中(i)重链CDR1-3区的氨基酸序列分别为DYYIH，RVNPDHGDSYYNQKFKD，ARNYLFDHW；和(ii)所述轻链CDR 1-3的氨基酸序列分别为KASQDVGTAVA，WASIRHT，HQFATYT；或者进一步所述抗体衍生自在2013年08月22日以保藏编号CGMCC No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心的杂交瘤所分泌 的抗体。或者进一步所述抗体衍生自在2013年11月06日以保藏编号CCTCC C2013170保藏于中国典型培养物保藏中心的CHO细胞所分泌的抗体。所述的包含能够特异性结合HER2的抗体的缀合物用于治疗Her2阳性癌症患者，所述Her2阳性癌症包括乳腺癌、卵巢癌或胃癌，或者进一步为拉帕替尼和/或赫赛汀耐药的乳腺癌、卵巢癌或胃癌。为了保障IHC检测抗体结果与所述缀合物治疗的高度对应性，开发所述缀合物的配套性伴随诊断抗体检测产品也存在极大的迫切性。
发明内容
为了解决上述问题，本发明提供了一种靶向Her2胞外区的检测抗体，上述抗体特异性结合Her2蛋白胞外结构域IV区。
Her2的一个示例氨基酸序列如NCBI GenBank ID：AAA75493.1所示，相应的蛋白胞外结构域IV区位于第511－643位氨基酸。Johan Rockberg等人(文献14：Discovery of epitopes for targeting the human epidermal growth factor receptor 2(HER2)with antibodies，Johan Rockberg等，MOLECULAR ONCOLOGY，第3卷，第238–247页，2009年)以及北京义翘神州科技有限公司(Sino Biological)所市售的“Her2/ERBB2 Protein,Human,Recombinant(ECD,domain IV,His Tag)”(连接有多聚组氨酸标签的人ERBB2(AAA75493.1)(Pro489-Cys630)区域)(Cat:10004-H08H4，https://www.sinobiological.com/Human-HER2-ErbB2-Protein-ECD-domain-IV-His-Tag-p223119.html)(特性描述：通过功能性ELISA检测了其结合能力；且能结合herceptin，herceptin的EC50值是10-40ng/mL)证明了Her2蛋白胞外结构域IV区可以作为一个独立的免疫原且仍然保持有处于完整Her2中时相应区域的的立体构型。
现在技术中公开了具有以下CDR组合的抗体靶向Her2蛋白胞外结构域IV：(i)重链可变区CDR1-3分别是：GFNIKDTYIH，RIYPTNGYTRYADSVKG，WGGDGFYAMDV；和轻链可变区CDR1-3分别是：RASQDVNTAVAW，SASFLES，QQHYTTPPT(WO9222653A1)；(ii)重链可变区CDR1-3分别是：GFNIKDTYIH，RIYPTNGYTRYADSVKG，WGGDGFYAMDY；轻链可变区CDR1-3分别是：RASQDVNTAVA，SASFLES，QQHYTTPPT；(文献15：Molecular dynamic simulation of Trastuzumab F(ab’)2structure in corporation with HER2 as a theranostic agent of breast cancer，S Hermanto等，Journal of Physics：Conference Series，第835卷，第1期，第1-11页，2017年)。 此类抗体用途均为治疗性抗体。
本发明惊奇的发现，一些特异性结合于Her2蛋白胞外结构域IV区上的抗体，可以作为免疫组织化学检测中的一抗(primary antibody)，这些抗体在患者免疫组化检测样本不经抗原(或表位)修复处理的情况下，仍然能够被这些抗体特异性有效识别并检测。从而有效的降低了IHC检测中因抗原(或表位)修复处理方法的差异所产生的假阴性问题。同时由于不需要上述修复处理步骤，能够有效的降低工作强度，缩短检测时间，从而提高IHC检测的效率。
此外，本发明也惊奇的发现，与WO2015074528A1和CN105008398A中公开的靶向Her2的抗体(RC48)药物缀合物(RC48－ADC)中的抗体(重链CDR1-3区的氨基酸序列分别为DYYIH，RVNPDHGDSYYNQKFKD，ARNYLFDHW；和()轻链CDR 1-3的氨基酸序列分别为KASQDVGTAVA，WASIRHT，HQFATYT)结合相同表位(位于Her2蛋白胞外结构域IV区)的或具有以上相同轻链和重链CDR的抗体，可以作为确定Her2阳性的伴随诊断IHC抗体使用，由于该表位位于Her2蛋白胞外结构域IV区，因此相应IHC抗体检测避免了将Her2分子存在胞外区缺失的突变形式也检测出阳性的可能，因此也有效的解决了一抗(primary antibody)针对Her2胞内区检测所产生的假阳性问题。同时，由于与靶向药物中抗体的CDR区相同，因此检测时所结合的表位与治疗药物的靶向表位完全相同，这种检测抗体与药用抗体结合表位的高度对应性，高度保障了靶向药物使用时的针对性和有效性。
本发明的技术方案如下所示：
本发明提供了一种人表皮生长因子受体2(Her2)伴随诊断免疫组化检测(IHC)用抗体，所述抗体特异性结合Her2蛋白胞外结构域IV区，且所述抗体具备有效检测未经抗原或表位修复处理的IHC样本的性能。
进一步的，所述抗体Fc段为非人哺乳动物抗体Fc段，优选为鼠源Fc片段或兔源Fc片段。
进一步的，所述抗体与以下CDR限定的抗体竞争性结合相同或相近表位，所述以下CDR限定的抗体的重链可变区的CDR 1-3分别如SEQ ID NO：1-3所示的氨基酸序列；所述CDR限定的抗体的轻链可变区的CDR 1-3分别如SEQ ID NO：4-6所示的氨基酸序列。
进一步的，所述抗体中：
(i)重链可变区的CDR1具有SEQ ID NO：1所示的氨基酸序列或对SEQ ID NO：1进行1或2个氨基酸替换后的氨基酸序列；或/和CDR2具有SEQ ID NO：2所示的氨基酸序列或对SEQ ID NO：2进行1，2，3，4或5个氨基酸替换后的氨基酸序列；或/和CDR3具有SEQ ID NO：3所示的氨基酸序列或对SEQ ID NO：3进行1，2或3个氨基酸替换后的氨基酸序列；和/或
(ii)轻链可变区的CDR1具有SEQ ID NO：4所示的氨基酸序列或对SEQ ID NO：4进行1，2，3或4个氨基酸替换后的氨基酸序列；或/和CDR2具有SEQ ID NO：5所示的氨基酸序列或对SEQ ID NO：5进行1或2个氨基酸替换后的氨基酸序列；或/和CDR3具有SEQ ID NO：6所示的氨基酸序列或对SEQ ID NO：6进行1或2个氨基酸替换后的氨基酸序列；
和/或
(iii)所述的免疫球蛋白Fc片段为鼠源IgG Fc片段，或进一步为鼠源IgG1Fc片段。
进一步的，所述抗体中：
(i)重链可变区的CDR1-3分别为SEQ ID NO：1-3所示的氨基酸序列；和
(ii)轻链可变区的CDR1-3分别为SEQ ID NO：4-6所示的氨基酸序列。
进一步的，所述抗体：
(i)所述抗体的重链包含SEQ ID NO：7所示的氨基酸序列或者与其具有至少80％序列同一性的序列；和/或
(ii)所述抗体的轻链包含SEQ ID NO：8所示的氨基酸序列或者与其具有至少80％序列同一性的序列；并且
(iii)所述抗体的重链可变区的CDR1-3分别如SEQ ID NO：1-3所示的氨基酸序列；所述抗体的轻链可变区的CDR1-3分别如SEQ ID NO：4-6所示的氨基酸序列。
进一步的，提供一种抗体R48M，其氨基酸序列信息如下：
1)重链(Hc)
Figure PCTCN2020076510-appb-000005
Figure PCTCN2020076510-appb-000006
2)轻链(Lc)
Figure PCTCN2020076510-appb-000007
本发明进一步提供了一种核酸分子，其编码上述的抗体。
本发明进一步提供了一种载体，其包含上述的核酸分子。
本发明进一步提供了上述抗体在制备Her2伴随诊断免疫组化检测产品中的用途，所述免疫组化检测产品具备在免疫组化检测过程中无需抗原或表位修复步骤的功能。
本发明进一步提供了一种Her2伴随诊断免疫组化检测试剂盒，其包含上述抗体，所述试剂盒具备在免疫组化检测过程中无需抗原或表位修复步骤的功能。
本发明进一步提供了一种Her2伴随诊断免疫组化检测方法，其使用上述抗体作为检测一抗。或进一步的在所述免疫组化检测过程中无抗原或表位修复步骤。
本发明所述Her2伴随诊断所针对的适应症为Her2相关癌症，进一步优选为乳腺癌、胃癌、胃食管癌、食道癌、卵巢癌、子宫内膜癌、肺癌、尿路上皮癌或膀胱癌。(参见文献16)
本发明的技术方案带来的有益效果为：本发明提供了一种能够靶向Her2蛋白胞外结构域IV区的抗体，采用该抗体作为一抗对样品的Her2表达情况进行免疫组化检测时，能够有效地避免因胞外区缺失导致的检测结果的假阳性。除此之外，本发明提供的Her2检测抗体在免疫组化检测 过程中，由于不需要上述修复处理步骤，有效的降低了IHC检测中因抗原(或表位)修复处理方法的差异所产生的假阴性问题。同时能够有效的降低工作强度，缩短检测时间，从而提高IHC检测的效率。
附图说明
图1使用经过验证的免疫组化方法进行Her2检测指南；
图2A显示了表7中序号为1的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图2B显示了表7中序号为1的病例，一抗为4B5抗体的免疫组织化学染色结果；
图3A显示了表7中序号为2的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图3B显示了表7中序号为2的病例，一抗为4B5抗体的免疫组织化学染色结果；
图4A显示了表7中序号为3的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图4B显示了表7中序号为3的病例，一抗为4B5抗体的免疫组织化学染色结果；
图5A显示了表7中序号为4的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图5B显示了表7中序号为4的病例，一抗为4B5抗体的免疫组织化学染色结果；
图6A显示了表7中序号为5的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图6B显示了表7中序号为5的病例，一抗为4B5抗体的免疫组织化学染色结果；
图7A显示了表7中序号为6的病例，一抗为RC48M抗体的免疫组织化学染色结果；
图7B显示了表7中序号为6的病例，一抗为4B5抗体的免疫组织化学染色结果；
图8A显示了表7中序号为7的病例，一抗为RC48M抗体的免疫组织 化学染色结果；
图8B显示了表7中序号为7的病例，一抗为4B5抗体的免疫组织化学染色结果；
图9显示了一种RC48-ADC药物的结构式，其中RC48代表RC48抗体。
具体实施方式
定义
除非另有定义，本文使用的所有术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本文所使用的“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)、和抗体片段。特别的，本文所使用的“抗体”指包含通过二硫键而互连的至少两条重链和两条轻链的蛋白。每条重链包含一重链可变区(重链上靠近N端的五分之一或四分之一的区域)和一重链恒定区(重链上靠近C端四分之三或五分之四的区域)。每条轻链包含一轻链可变区(轻链上靠近N端二分之一的区域)和一轻链恒定区(轻链上靠近C端二分之一的区域)。重链可变区和轻链可变区区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)。所述的“CDR”是指免疫球蛋白的重链和轻链的高变区，包括Kabat、Chothia或IMGT体系等确定的。每个抗体存在三个重链CDR和三个轻链CDR。根据情况，本文所用术语CDR是为了指示这些区域之一、或者这些区域的几个或者甚至全部，所述区域包含通过抗体对抗原或其识别表位的亲和力而负责结合的大部分氨基酸残基。
本发明所使用的术语“Her2检测抗体”是指能够检测病例中Her2表达状态的抗体，其能够与Her2结合，在免疫组化化学中，Her2检测抗体做为一抗进行Her2表达状态的检测。本发明涉及的“Her2检测抗体”能够靶向Her2蛋白胞外结构域IV区。
本发明所使用的术语“RC48M抗体”指重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示的抗体。
本发明所使用的术语“RC48-ADC”或“RC48-ADC药物”或“RC48-ADC 靶向药物”是指专利WO2015074528A1和CN105008398A中公开的包含能够特异性结合Her2的抗体药物缀合物(即抗体药物偶联物，ADC)，其中所述抗体称为“RC48抗体”，其(i)重链CDR 1-3区的氨基酸序列分别为DYYIH，RVNPDHGDSYYNQKFKD，ARNYLFDHW；和(ii)所述轻链CDR1-3的氨基酸序列分别为KASQDVGTAVA，WASIRHT，HQFATYT；或者进一步所述抗体衍生自在2013年08月22日以保藏编号CGMCC No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心的杂交瘤所分泌的抗体。或者进一步所述抗体衍生自在2013年11月06日以保藏编号CCTCC C2013170保藏于中国典型培养物保藏中心的CHO细胞所分泌的抗体。一种RC48-ADC药物的结构如图9所示。
实施例
下面将通过实施例对本发明进行进一步的阐述，需要说明的是，以下实施例是对本发明进行进一步的阐述和解释，而不应被看作是对本发明的限制。
以下实施例中采用RC48M抗体作为靶向Her2蛋白胞外结构域IV区的IHC检测抗体的一个示例，其重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。以检测乳腺癌样本作为示例。
实施例1 RC48M抗体(Primary Antibody)IHC检测与PATHWAY－anti-Her2/neu(4B5)Rabbit Monoclonal Primary Antibody(以下简称4B5抗体)IHC检测的总体符合率比较及假阴性问题
针对49例浸润性乳腺癌样本进行对比检测分析。
1)4B5抗体，按照产品指导方法进行操作；
2)RC48M IHC检测主要操作步骤
表3 RC48M IHC检测主要操作步骤
步骤 操作及参数说明
一抗(稀释比例) RC48M(1:500)
一抗孵育条件和时间 室温(18～20℃)，20分钟
抗原修复条件 不修复
二抗检测系统 Polymer Refine Detection
DAB显色时间 5分钟
检测结果列表见表4。
表4 49例样本检测结果列表
Figure PCTCN2020076510-appb-000008
Figure PCTCN2020076510-appb-000009
注：对染色结果的分级判断参考《乳腺癌HER2检测指南(2014版)》中规则进行判读，下同。对以上结果进行汇总统计，结果见表5。
表5检测结果统计分析表
Figure PCTCN2020076510-appb-000010
从以上统计结果来看，检测分级完全相同的共33例(18+6+1+8)， 占比67.3％(33/49)。从目前医学决定的判断指导来看，0及1+定为阴性，2+为可疑，3+为阳性，则共计26例(18+7+1)+6例+1例+8例＝41例(占比83.7％)，采用二者将得出相同的医学决定。存在决定差异的病例汇总如表6。
表6 医学决定存在差异样本列表
编号 组织类型 病理号 4B5抗体 RC48M抗体
9 乳腺癌 18-07600-2 3+ 2+
10 乳腺癌 18-09839-1 3+ 2+
11 乳腺癌 18-09840-1 3+ 2+
13 乳腺癌 18-07607-5 1+ 2+
14 乳腺癌 18-08555-1 1+ 2+
15 乳腺癌 18-08929-1 1+ 2+
16 乳腺癌 18-09503-1 1+ 2+
30 乳腺癌 18-08952-1 0 2+
在上文中提及，Seema Jabbar等人(文献6：Comparison of Two FDA-Approved Her2 Immunohistochemical Assays for Breast Carcinoma:HercepTest and Pathway Her2(4B5),Am J Clin Pathol 2018；149:S93-S94)针对共计95份乳腺癌(Breast Carcinoma)样本进行了HercepTest和Pathway Her2(4B5)(简称4B5)检测。HercepTest检测结果为72份可疑+23份阳性；其中72份可疑样本经4B5检测，52份(52/72＝72％)为阴性。4B5检测的95份样本中，52例(55％)为阴性，25例(26％)为可疑，18例(19％)为阳性。通过FISH对结果进行确认检测，显示4B5相对于HercepTest检测与FISH检测结果有更高的一致性。另外，在4B5检测的52例阴性中，有3例经FISH检测验证为阳性，则表明4B5检测的结果存在一定的假阴性。结合表6的结果，R48M检测的结果将部分4B5检测的结果判读为2+，成为可疑病例，从而需进入FISH或CISH检测(ISH检测)判定的流程，尽管采用RC48M检测导致部分病例增加了进一步ISH确认检测步骤，但这在一定程度上避免了4B5存在的检测结果存在部分假阴性的问题。从而避免了部分患者由于检测假阴性的问题而错过较佳治疗期及错失使用靶向治疗药物开展靶向治疗的问题。此外，结合文献6的结果及上述检测数据，RC48M的检测结果与FISH检测结果的一致性也应高于HercepTest检测的结果。
实施例2 假阳性问题分析
采用RC48M抗体与4B5针对RC48-ADC药物I期临床样本共7例进行了平行检测对比。每组各使用7片乳腺癌组织石蜡切片。第1组一抗为RC48M抗体；第2组一抗为4B5抗体；两组实验统一使用兔鼠通用型免疫组化二抗试剂盒(购自福州迈新生物技术开发有限公司)，具体的实验过程如下所示：
(1)脱蜡水化：65℃烤片2h，之后依次用二甲苯浸泡20min，用无水乙醇浸泡5min，用95％乙醇浸泡5min，用75％乙醇浸泡5min，之后将上述切片转移到0.01MPBST中备用；
(2)抗原修复：将第2组切片转移到柠檬酸钠缓冲中，98℃条件下加热20min；第1组切片无需进行抗原修复。
(3)封闭：将第1组和第2组切片用3％过氧化氢封闭10min,之后用0.01MPBST浸泡两次，一次3min；
(4)一抗：将第1组和第2组切片放置在湿盒中，将RC48M抗体稀释至1μg/ml，之后将稀释后的RC48M抗体和4B5抗体分别滴加到对应的第1组和第2组切片上，37℃孵育60min，恢复室温后，用0.01MPBST浸泡两次，一次3min；
(5)二抗：将兔鼠通用型免疫组化二抗试剂盒中的兔鼠通用型二抗分别滴加至第1组和第2组切片上，滴加量为100μl/片，37℃孵育20min，用PBST(0.01M)浸泡两次，一次3min；之后再将上述试剂盒中的反应放大剂滴加至第1组和第2组切片上，滴加量为100μl/片，37℃孵育20min，用PBST(0.01M)浸泡两次，一次3min；
(6)DAB显色：使用DAB显色试剂盒(购自福州迈新生物技术开发有限公司)，取1mL试剂盒中的稀释缓冲液，DAB色原和底物各50μl，混匀后加至切片，室温显色1-3分钟，蒸馏水洗涤；
(7)苏木素复染：每张切片滴加100μL的苏木素染色液(购自福州迈新生物技术开发有限公司)，室温染色2-5min，自来水冲洗5min返蓝。
(8)脱水：将第1组和第2组切片依次浸泡在75％乙醇中浸泡5min、95％乙醇中5min、100％乙醇中5min、二甲苯中10min，之后取出切片，挥干二甲苯。
(9)封片。
之后显微镜下观察拍照，物镜10×，目镜20×，放大倍数200倍。 染色结果如图2－8，其中图2A、图3A、图4A、图5A、图6A、图7A、图8A显示的是一抗为RC48M抗体的免疫组织化学染色结果，其中图2B、图3B、图4B、图5B、图6B、图7B、图8B显示的是一抗为4B5抗体的免疫组织化学染色结果。在序号2、3、4、5、6、7病例中RC48M抗体与4B5抗体具有相近的染色，而在序号1的病例中RC48M与4B5表现出区别，其中RC48M染色为阴性，而4B5则染色阳性。根据《乳腺癌HER2检测指南(2014版)》中规则对上述染色结果进行判读，判读结果见表7。在序号3、4、5、6病例中RC48M抗体与4B5抗体的染色结果同为3+，在序号2病例中二者染色结果同为2+，序号7病例中二者染色出现1个等级的差异，序号1病例中二者染色显示出阴性和3+的区别。根据各序号代表的病例对应的临床试验药效信息可知，序号1代表的病例在经过临床用药后疾病仍持续进展，即对该检测阳性患者而言，采用靶向药物无效，这也就是说序号1的病例采用4B5抗体进行检测时出现了假阳性。结果表明RC48M抗体能够有效的降低如Her2胞外区突变等导致的检测假阳性问题，从而能够更为准确的为此类患者确定后续治疗方案，避免无效的靶向用药治疗，从而极大的节约了此类患者的无效治疗投入，并为后续选择其他治疗方案给出更准确的检测参考。
表7 检测结果及临床药效
Figure PCTCN2020076510-appb-000011
注：PD代表疾病进展；PR代表部分缓解；SD代表疾病稳定。
本发明已通过各具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各具体实施方式，普通技术人员在本发 明的范文内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (11)

  1. 一种人表皮生长因子受体2(Her2)伴随诊断免疫组化检测(IHC)用抗体，其特征在于，所述抗体特异性结合Her2蛋白胞外结构域IV区，且所述抗体具备有效检测未经抗原或表位修复处理的IHC样本的性能。
  2. 权利要求1所述的抗体，所述抗体Fc段为非人哺乳动物抗体Fc段，优选为鼠源Fc片段或兔源Fc片段。
  3. 根据权利要求1或2所述的抗体，其特征在于，所述抗体与以下CDR限定的抗体竞争性结合相同或相近表位，所述以下CDR限定的抗体的重链可变区的CDR 1-3分别如SEQ ID NO：1-3所示的氨基酸序列；所述CDR限定的抗体的轻链可变区的CDR 1-3分别如SEQ ID NO：4-6所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的抗体，其特征在于，所述抗体中：
    (i)重链可变区的CDR1具有SEQ ID NO：1所示的氨基酸序列或对SEQ ID NO：1进行1或2个氨基酸替换后的氨基酸序列；或/和CDR2具有SEQ ID NO：2所示的氨基酸序列或对SEQ ID NO：2进行1，2，3，4或5个氨基酸替换后的氨基酸序列；或/和CDR3具有SEQ ID NO：3所示的氨基酸序列或对SEQ ID NO：3进行1，2或3个氨基酸替换后的氨基酸序列；和/或
    (ii)轻链可变区的CDR1具有SEQ ID NO：4所示的氨基酸序列或对SEQ ID NO：4进行1，2，3或4个氨基酸替换后的氨基酸序列；或/和CDR2具有SEQ ID NO：5所示的氨基酸序列或对SEQ ID NO：5进行1或2个氨基酸替换后的氨基酸序列；或/和CDR3具有SEQ ID NO：6所示的氨基酸序列或对SEQ ID NO：6进行1或2个氨基酸替换后的氨基酸序列；和/或
    (iii)所述的免疫球蛋白Fc片段为鼠源IgG Fc片段，或进一步为鼠源IgG1 Fc片段。
  5. 根据权利要求4所述的抗体，其特征在于，所述抗体中：
    (i)重链可变区的CDR1-3分别为SEQ ID NO：1-3所示的氨基酸序列；和
    (ii)轻链可变区的CDR1-3分别为SEQ ID NO：4-6所示的氨基酸序列。
  6. 根据权利要求1-5任一项所述的抗体，其特征在于，
    (i)所述抗体的重链包含SEQ ID NO：7所示的氨基酸序列或者与其 具有至少80％序列同一性的序列；和/或
    (ii)所述抗体的轻链包含SEQ ID NO：8所示的氨基酸序列或者与其具有至少80％序列同一性的序列；并且
    (iii)所述抗体的重链可变区的CDR1-3分别如SEQ ID NO：1-3所示的氨基酸序列；所述抗体的轻链可变区的CDR1-3分别如SEQ ID NO：4-6所示的氨基酸序列。
  7. 根据权利要求6所述的抗体，其特征在于，
    (i)所述抗体的重链如SEQ ID NO：7所示的氨基酸序列；和
    (ii)所述抗体的轻链如SEQ ID NO：8所示的氨基酸序列。
  8. 一种核酸分子，其编码权利要求1至7中任一项所述的抗体。
  9. 一种载体，其包含权利要求7所述的核酸分子。
  10. 权利要求1-7任一项所述的抗体在制备Her2伴随诊断免疫组化检测产品中的用途，所述免疫组化检测产品具备在免疫组化检测过程中无需抗原或表位修复步骤的功能；优选的，所述Her2伴随诊断免疫组化检测产品是用于RC48-ADC靶向药物治疗前配套的Her2伴随诊断免疫组化检测产品；更优选的，所述Her2伴随诊断免疫组化检测产品所针对的适应症为Her2相关癌症，进一步优选为乳腺癌、胃癌、胃食管癌、食道癌、卵巢癌、子宫内膜癌、肺癌、尿路上皮癌或膀胱癌。
  11. 一种Her2伴随诊断免疫组化检测试剂盒，其包含权利要求1-7任一项权利要求所述的抗体，所述试剂盒具备在免疫组化检测过程中无需抗原或表位修复步骤的功能；优选的，所述Her2伴随诊断免疫组化检测试剂盒是用于RC48-ADC靶向药物治疗前配套的Her2伴随诊断免疫组化检测试剂盒；更优选的，所述Her2伴随诊断免疫组化检测试剂盒所针对的适应症为Her2相关癌症，进一步优选为乳腺癌、胃癌、胃食管癌、食道癌、卵巢癌、子宫内膜癌、肺癌、尿路上皮癌或膀胱癌。
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