WO2020073594A1 - 双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用 - Google Patents

双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用 Download PDF

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WO2020073594A1
WO2020073594A1 PCT/CN2019/076947 CN2019076947W WO2020073594A1 WO 2020073594 A1 WO2020073594 A1 WO 2020073594A1 CN 2019076947 W CN2019076947 W CN 2019076947W WO 2020073594 A1 WO2020073594 A1 WO 2020073594A1
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areg
tumor
bidirectional
cells
cancer
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孙宇
许奇霞
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中国科学院上海生命科学研究院
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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

Definitions

  • the present invention belongs to the field of disease diagnosis and regulation. More specifically, the present invention relates to the application of bidirectional modulators in the preparation of diagnostic or regulatory agents for cell senescence and tumors.
  • Cell senescence manifests as inward nuclear membrane folding, chromatin condensation, lipofuscin accumulation, increased cell volume, enlarged nuclear nuclei, increased ⁇ -galactosidase activity, and secretion of various factors.
  • Cell senescence is triggered by one or more factors, activating downstream signaling pathways including p53, p16 INK4A / Rb, PI3K / Akt, FoxO transcription factor, and mitochondrial SIRT1.
  • senescent cells are often associated with many pathological features, including local inflammation. Cell aging occurs in damaged cells and prevents them from multiplying in the organism. Under the influence of various external stimuli and internal factors, cell damage can cause obvious signs of cell aging; when the damage accumulates and reaches a certain limit, the tissue presents various visually identifiable tissue degeneration changes and physiological aging phenotypes.
  • SASP senescence-associated secretion phenotype
  • exocrine proteins produced by senescent cells often depends on the genetic background and species source of aging tumor cells.
  • SASP is of great significance to tumor biology, it is still not clear how it controls tumors.
  • studies have focused on anti-aging targeting the upstream signaling pathway of SASP, drugs or genetic specific inhibition of IKK / NF- ⁇ B, mTOR, p38MAPK, JAK / STAT, etc. in senescent cells, which can inactivate the side effects caused by SASP Secretory effect, thereby improving the aging state of cells and body.
  • the object of the present invention is to provide the application of bidirectional modulators in the preparation of diagnostic or regulatory agents for cell senescence and tumors.
  • a pharmaceutical composition for inhibiting tumors or reducing tumor drug resistance characterized in that the pharmaceutical composition includes: an antibody that specifically inhibits biregulin (AREG), And chemotherapy drugs.
  • RAG biregulin
  • the chemotherapy drugs are genotoxic drugs; preferably, the chemotherapy drugs include: mitoxantrone, vincristine, doxorubicin, bleomycin, sabplatin, cisplatin, Carboplatin, daunorubicin, nogamycin, arubicin, epirubicin, doxorubicin, cytarabine, capecitabine, gemcitabine, 5-fluorouracil.
  • the pharmaceutical composition includes an antibody that specifically inhibits bi-modulin and mitoxantrone, and the mass ratio of the two is 1: 0.005 to 1: 2.0; preferably 1: 0.01 to 1: 1.0; more preferably 1: 0.02 ⁇ 1: 0.6, such as 1: 0.2.
  • the pharmaceutical composition includes an antibody that specifically inhibits bi-modulin and doxorubicin, and the mass ratio of the two is 1: 0.02 to 1: 1.5; preferably 1: 0.05 to 1 : 0.8; more preferably 1: 0.06 to 1: 0.3, such as 1: 0.1.
  • the pharmaceutical composition includes an antibody that specifically inhibits bi-modulin and bleomycin, and the mass ratio of the two is 1: 0.02 to 1.5; preferably 1: 0.05 to 1: 0.8 ; More preferably 1: 0.06 ⁇ 1: 0.3, such as 1: 0.1.
  • the pharmaceutical composition includes an antibody that specifically inhibits bi-modulin and one or more selected from saplatin, cisplatin, and carboplatin, and the mass ratio of the antibody to the latter is 1 : 0.02 to 1.5; preferably 1: 0.05 to 1: 0.8; more preferably 1: 0.06 to 1: 0.3, such as 1: 0.1.
  • the antibody that specifically inhibits bi-modulin is secreted by the hybridoma cell line CCTCC NO: C2018214.
  • an antibody that specifically inhibits bi-modulin regulates the bi-modulin expressed by stromal cells in the tumor microenvironment, thereby reducing tumor resistance.
  • the tumors include: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, and bladder cancer.
  • the tumor resistance is the resistance of the tumor to chemotherapy drugs.
  • an antibody that specifically inhibits bi-modulin which is secreted by the hybridoma cell line CCTCC NO: C2018214.
  • an antibody that specifically inhibits bidirectional modulators in the preparation of antibody drugs is provided.
  • the antibody drugs are used in combination with chemotherapeutic drugs to inhibit tumors or eliminate tumor drug resistance; or to eliminate tumor cells Resistance to chemotherapy drugs.
  • hybridoma cell line SP2 / 0-02-AREG-SUN which has the deposit number CCTCCNO: C2018214 at the China Type Culture Collection Center.
  • kits for inhibiting tumors or reducing tumor resistance comprising: an antibody that specifically inhibits bidirectional modulators, or a cell line that produces the antibody.
  • the kit further includes: chemotherapy drugs; preferably the chemotherapy drugs are genotoxic drugs; preferably, the chemotherapy drugs include: mitoxantrone, vincristine, Doxorubicin, bleomycin, sabplatin, cisplatin, carboplatin, daunorubicin, nogamycin, arubicin, epirubicin, doxorubicin, cytarabine, capecide Tabin, gemcitabine, 5-fluorouracil.
  • the use of a bimodulin in the preparation of a diagnostic reagent for prognostic evaluation of tumor chemotherapy is provided, wherein the bimodulin is a bimodulin produced by stromal cells in the tumor microenvironment.
  • the bidirectional modulator produced by the stromal cells in the tumor microenvironment can be isolated from the sample tissue by conventional separation means.
  • a reagent that specifically recognizes a bidirectional modulator in the preparation of a diagnostic reagent for tumor chemotherapy prognosis evaluation or pathological grading, wherein the bidirectional modulator is stromal cells in the tumor microenvironment Bidirectional modulators produced.
  • the reagents specifically recognizing the bidirectional modulator include antibody reagents, primers, and probes.
  • a method for screening potential substances that inhibit tumors or reduce tumor resistance includes: (1) treating an expression system with candidate substances, which expresses NF- ⁇ B and bidirectional Modulin, and there is an NF- ⁇ B binding site upstream of the gene encoding this bidirectional regulator; and (2) To detect the regulatory effect of NF- ⁇ B on the bidirectional regulator in the system; if the candidate substance statistically inhibits NF - ⁇ B regulates the transcription of bidirectional regulators, indicating that the candidate substance is a potential substance that inhibits tumors or reduces tumor drug resistance.
  • step (1) includes: adding candidate substances to the expression system in the test group; and / or step (2) includes: detecting NF- ⁇ B for bidirectional modulators in the system of the test group
  • the transcriptional regulation of the protein is compared with the control group, where the control group is an expression system that does not add the candidate substance; if the NF- ⁇ B in the test group is significantly inhibited for the transcriptional regulation of bidirectional regulators (eg more than 20% inhibition) , The better inhibition is more than 50%; the better inhibition is more than 80%), indicating that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • the NF- ⁇ B binding site is -3510, -1223, -1131, +79 upstream of the biregulin-encoding gene.
  • a method for screening potential substances that inhibit tumors or reduce tumor drug resistance includes: (1) treating an expression system with a candidate substance that expresses EGFR-mediated signals Pathways and bidirectional modulators; and (2) detecting the activation effect of bidirectional modulators on the EGFR-mediated signaling pathway in the system; if the candidate substance statistically inhibits the activation, it indicates that the candidate substance is an inhibitor A tumor or a potential substance that reduces tumor resistance.
  • step (1) includes: adding the candidate substance to the expression system in the test group; and / or step (2) includes: detecting that the bidirectional modulator in the test group system is mediated by EGFR The activation effect of the signal pathway of the control group is compared with the control group, where the control group is an expression system that does not add the candidate substance; if the bidirectional modulator in the test group significantly inhibits the activation of the EGFR-mediated signal pathway (If it is inhibited by more than 20%, preferably by more than 50%; more preferably by more than 80%), it indicates that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • Figure 1 Human prostate prostate stromal cell line PSC27 gene expression profile heat map after chemotherapy and radiation treatment.
  • CTRL control.
  • BLEO bleomycin.
  • HP hydrogen peroxide.
  • RAD radiation.
  • Red arrow amphiregulin.
  • FIG. 1 DNA damage response (DDR) of PSC27 cells after various conditions. Above, representative pictures after immunofluorescence detection, red fluorescence is ⁇ H2AX and blue is DAPI. The figure below shows the comparative analysis of DDRfoci statistics.
  • PTX paxlitaxel.
  • DTX docetaxel.
  • VCR vincristine.
  • BLEO bleomycin.
  • MIT mitoxantrone.
  • SAT satraplatin.
  • FIG. 3 PSC27 cell senescence detection after various conditions in Figure 2. Above, representative image of bright field microscope after SA-B-Gal staining. Below, statistical comparative analysis of SA-B-Gal staining positive cells.
  • FIG. 4 Analysis of the rate of DNA intercalation in cells after PSC27 was treated with various conditions in Figure 2. Above, representative image after BrdU staining, green fluorescence is BrdU. Below, statistical analysis of BrdU after treatment with various drugs.
  • FIG. 1 AREG expression in stromal cells. Above, the expression levels of AREG in PSC27 cells after various conditions. Below, Western blot analysis of AREG protein expression. IC, intracellular. CM, conditioned media. GAPDH, loading control.
  • FIG. 1 PSC27 stromal cells expressing several typical factors of SASP after bleomycin treatment. Collected on days 1 ("2"), 3 ("3"), 5 ("4"), 7 ("5"), 10 ("6") and 15 (“7") after the drug injury The stromal cells and their total RNA were obtained for RT-PCR detection. The data at each time point is the same as that of the control (non-medicated group, "1") and is used for graphing.
  • the cell lysis samples collected at various time points in Figure 7 and Figure 6 were analyzed by Western blot analysis in which the expression level of AREG changed.
  • IC intracellular.
  • CM conditioned media.
  • GAPDH loading control.
  • Figure 8 Comparative analysis of AREG transcript expression levels in prostate stromal cells and cancer cells after treatment with several genotoxic chemotherapy drugs.
  • the cell total protein samples of each cell line in FIG. 9 and FIG. 8 after being treated with bleomycin were analyzed by Western blot to determine the change in AREG expression.
  • IC intracellular protein.
  • CM conditioned media.
  • GAPDH loading control.
  • FIG. 10 DNA damage of human breast stromal cell line HBF1203 after treatment with chemotherapy drugs. Above, representative graph of immunofluorescence staining results, red fluorescence is ⁇ H2AX, blue is DAPI. The figure below shows the comparative analysis of DDR signal statistics.
  • VNB vinorelbine.
  • VBL vinblastine.
  • DOX doxorubicin.
  • CIS cisplatin.
  • CARB carboplatin.
  • FIG. 11 HBF1203 DNA insertion analysis in cells after various drug treatments. Above, representative image after BrdU staining, green fluorescence is BrdU. Below, statistical analysis of BrdU after treatment with various drugs.
  • FIG. 12 HBF1203 senescence cell analysis and detection after various conditions in Figure 10. Above, representative image of bright field microscope after SA-B-Gal staining. Below, statistical comparative analysis of SA-B-Gal staining positive cells.
  • FIG. 13 Expression of AREG transcripts in HBF1203. The expression level of AREG in cells after various conditions.
  • Figure 14 Comparative analysis of AREG transcript expression levels in breast stromal cells and cancer cells after treatment with several drugs.
  • FIG. 15 Comparative analysis of histopathology of primary lesions in patients with prostate cancer before and after chemotherapy. On the left, a representative picture of histochemical staining (AREG). On the right, representative pictures of H & E staining.
  • Figure 16 Statistical comparison analysis of expression levels after pathological grading based on the results of histochemical staining of AREG in tumor tissues of prostate cancer patients. The number of patients without chemotherapy and those who had undergone chemotherapy were 42 and 48, respectively.
  • Figure 17 Representative pictures corresponding to the pathological grade in Figure 16.
  • EL expression level.
  • FIG. 1 Comparative analysis of AREG transcript expression of stromal cells and epithelial cells after laser capture microdissection (LCM) separation.
  • Figure 19 Analysis of AREG transcript expression in stromal cells based on a single patient before and after chemotherapy. Number of patients in each group, 10.
  • Figure 20 Analysis of AREG transcript expression in cancer cells of a single patient before and after chemotherapy. Number of patients in each group, 10.
  • Figure 21 Comparative analysis of AREG, IL-8 and WNT16B protein expression in tumor stromal cells of patients with prostate cancer after chemotherapy.
  • the pathological score of each factor comes from the histochemical staining pathological reading of the factor, and each reading is the average of 3 pathological blind readings.
  • Figure 22 Representative diagram of histochemical staining based on AREG, IL-8 and WNT16B. The histopathological staining series of the three factors were taken from three consecutive slices of a single patient after treatment.
  • Figure 23 Analysis of the protein expression relationship between AREG and IL-8 in patients with prostate cancer after chemotherapy.
  • the value of each factor comes from three pathological blind readings. Among them, r, R 2 , slope and P value are all from Pearson correlation analysis.
  • Figure 24 Analysis of protein expression relationship between AREG and WNT16B in patients after chemotherapy.
  • the value of each factor comes from three pathological blind readings. Among them, r, R 2 , slope and P value are all from Pearson correlation analysis.
  • Figure 25 Survival curve (Kaplan Meier) analysis based on the expression level of AREG in the lesions of patients after chemotherapy. Number of patients in AREG low expression group, 20, cyan curve. AREG high expression group patients, 28, purple curve.
  • Figure 26 Comparative analysis of histopathology of primary lesions before and after chemotherapy in lung cancer patients. On the left, representative pictures of histochemical staining. On the right, representative pictures of H & E staining.
  • Figure 27 Statistical comparison analysis after pathological grading based on the results of histochemical staining of AREG in tumor tissue of lung cancer patients.
  • Figure 28 Histochemical staining pictures representing each pathological grade in Figure 27.
  • EL expression level.
  • FIG. 29 Comparative analysis of AREG expression between different types of cells.
  • LCM laser capture microdissection
  • Figure 30 Analysis of AREG transcript expression based on stromal cells of a single patient, the number of patients in each group is 10.
  • Figure 31 Analysis of a group of AREG transcript expression analysis based on cancer cells from a single lung cancer patient similar to that in Figure 30, with 10 patients in each group.
  • Figure 32 Comparative analysis of AREG, IL-8 and WNT16B protein expression in tumor stromal cells of patients with lung cancer after chemotherapy.
  • Figure 33 Analysis of the protein expression relationship between AREG and IL-8 in patients with lung cancer after chemotherapy.
  • the value of each factor comes from three pathological blind readings. Among them, r, R 2 , slope and P value are all from Pearson correlation analysis.
  • Figure 34 Analysis of protein expression relationship between AREG and WNT16B in patients with lung cancer after chemotherapy.
  • the value of each factor comes from three pathological blind readings. Among them, r, R 2 , slope and P value are all from Pearson correlation analysis.
  • Figure 35 Survival curve (Kaplan Meier) analysis based on the expression level of AREG in the lesions of lung cancer patients after chemotherapy. Number of patients in AREG low expression group, 71, green curve. AREG high expression group patients, 28, red curve.
  • FIG. 36 Shengxin analyzes the NF-kB binding site within 4000bp upstream of the AREG promoter.
  • Figure 37 The four reporter expression vectors in Figure 36 were transformed into 293 cells and stimulated with TNF ⁇ to detect their luciferase activity.
  • NAT11-Luc2CP positive control vector.
  • the four vectors used in Fig. 38 and Fig. 36 were transformed into PSC27 stromal cells and treated with 50 ⁇ g / ml bleomycin. The luciferase signal intensity was compared and analyzed.
  • Figure 39 The four reporter expression vectors in Figure 36 were transformed into 293 cells and stimulated with IL-1 ⁇ to detect their luciferase activity.
  • NAT11-Luc2CP positive control vector.
  • Figure 40 The four reporter expression vectors in Figure 36 were transformed into PSC27 cells and treated with 10 ⁇ M SAT to detect their luciferase activity.
  • NAT11-Luc2CP positive control vector.
  • FIG 41 ChIP-PCR analysis of the PCR signal intensity of the 4 speculative NF-kB binding sites on the AREG promoter in the components precipitated by the NF-kB specific antibody.
  • Both IL-6-p1 and IL-8-p1 are NF-kB sites with known sequences and are used here as a positive control.
  • FIG. 42 NF-kB into the following three kinds of chemotherapy treatment, the comparative analysis of expression levels of AREG and IL-8 mutant subline cell nuclear PSC27 IkB ⁇ .
  • Figure 43 After transferring GL-AREG-P04 into PSC27 cells, and then treated with bleomycin and NF-kB, c / EBP, and AP-1 inhibitors, respectively, the luciferase signal obtained was compared. BAY, NF-kB inhibitor. BA, c / EBP inhibitor. T5224 and SR are both AP-1 inhibitors.
  • Figure 44 After GL-AREG-P04 was transferred into PSC27 cells, and then treated with SAT and NF-kB, c / EBP, and AP-1 inhibitors, respectively, the luciferase signals obtained were compared. BAY, NF-kB inhibitor. BA, c / EBP inhibitor. T5224 and SR are both AP-1 inhibitors.
  • Figure 45 Expression of AREG transcripts after PSC27 cells were treated with bleomycin and NF-kB, c / EBP, and AP-1 inhibitors, respectively.
  • FIG. 46 Expression of IL-6 transcript after PSC27 cells were treated with bleomycin and NF-kB, c / EBP and AP-1 inhibitors, respectively.
  • FIG. 47 Expression of IL-8 transcripts after PSC27 cells were treated with bleomycin and NF-kB, c / EBP, and AP-1 inhibitors, respectively.
  • Figure 48 Analysis of the protein expression of AREG in the AREG overexpression and knockout sublines of PSC27 and the effect on the cell itself.
  • Western blot detected changes in the expression levels of AREG and IL-8. GAPDH, loading control.
  • Figure 49 SA- ⁇ -Gal staining statistical analysis of PSC27 subline senescence under DNA damage.
  • the right side is a representative picture.
  • Figure 50 Proliferation analysis of prostate cancer cells after CM treatment by the AREG overexpression group and knockout group of PSC27, respectively.
  • Hela cells are a positive control.
  • Figure 54 Analysis of the expression of the complete form of caspase 3 and its cleaved form in the presence of AREG and / or the use of chemotherapy drugs in the prostate cancer cell line DU145.
  • Figure 55 Comparative analysis of apoptosis of prostate cancer cell line PC3 under the action of mitoxantrone and apoptosis inhibitor (QVD-OPH, ZVAD / FMK) or activator (PAC1, GA).
  • Figure 56 Comparative analysis of apoptosis of prostate cancer cell line PC3 under the action of paclitaxel, apoptosis inhibitor (QVD-OPH, ZVAD / FMK) or activator (PAC1, GA).
  • Figure 57 Analysis of the activation of EGFR and its downstream molecules under the action of ARCG derived from stromal cells of prostate cancer cell lines PC3 and DU145. GAPDH, loading control.
  • Figure 58 Analysis of the activation of EGFR and its downstream molecules under the action of CM derived from stromal cells PSC27 and AREG knock-out subline after bleomycin treatment. GAPDH, loading control.
  • FIG. 59 IP and Western blot analysis based on AREG specific antibodies.
  • IgG control antibody.
  • E EGFR monoclonal antibody.
  • A AREG monoclonal antibody.
  • FIG. 60 The CM produced by PSC27 (PSC27-BLEO) after bleomycin treatment was used to treat prostate cancer cells, and the proliferation rate analysis of cancer cells with or without AREG knockout from PSC27 cells.
  • the top is the statistical analysis, and the bottom is the representative cell picture.
  • the drug use concentration is the IC50 value of each cell line.
  • Fig. 64 and Fig. 63 The experimental conditions in Fig. 64 and Fig. 63 are similar, but the detection is that PSC27 cells are treated with bleomycin and their CMs are collected for culturing prostate cancer cell resistance to mitoxantrone.
  • EGFR inhibitors AG-1478 (2 ⁇ M), Cetuximab (50 ⁇ g / ml) and AREG mAb (1 ⁇ g / ml); Cetuximab and AREG mAb (50 ⁇ g / ml, 1 ⁇ g / ml, respectively) were used to detect cancer cells Drug resistance.
  • Figure 65 The AREG monoclonal antibody (0.2 ⁇ g / ml) prepared and purified by the inventors was used in Western blot to detect the expression of AREG in cells after PSC27 was injured by bleomycin. GPADH, loading control.
  • the MIT concentration of mitoxantrone is designed to be close to the actual MIT concentration in the plasma of prostate cancer patients under clinical administration conditions.
  • Figure 67 Cell resistance curves of human breast cancer cells MDA-MB-231 and stromal cells HBF1203 after various treatments similar to those in Figure 66. DOX, doxorubicin.
  • Figure 68 Statistical comparison analysis of the measured values of the terminal tumor volume of mice at the end of the 8th week after subcutaneous inoculation of PC3 / PSC27 in immunodeficient mice.
  • Figure 69 Schematic diagram of tumor growth, administration and detection in mice. In the third week after PC3 / PSC27 subcutaneous injection, single or multi-drug treatment was started.
  • Figure 70 Schematic diagram of mouse treatment mode under pre-clinical conditions. The upper part is each processing method, and the lower part is distributed at each time point.
  • Figure 71 Statistical analysis of tumor terminal volume of mice after PC3 / PSC27 inoculation after 8 weeks of MIT preclinical administration. On the left, statistical comparison. On the right, a representative tumor picture.
  • Figure 72 Expression analysis of SASP representative factors and cell aging markers after mouse tumors were microdissected by laser capture after stromal cells and cancer cells were specifically separated. They are IL-6, IL-8, WNT16B, SFRP2, ANGPTL4, MMP1 / 3/10 and p16.
  • Figure 73 Histochemical analysis of p16 expression and SA- ⁇ -Gal staining in tumors of mice before and after chemotherapy.
  • Figure 74 and Figure 73 show the statistical comparison of p16 expression and SA- ⁇ -Gal staining in mouse tumor tissues before and after chemotherapy.
  • FIG. 75 Histochemical analysis of protein expression of AREG in tumor tissues of mice treated with placebo and mitoxantrone, respectively.
  • Figure 76 Statistical analysis of mouse tumor terminal volume after mitoxantrone and therapeutic antibody Cetuximab or AREG mAb monotherapy or multi-drug treatment.
  • Figure 77 BLI-based in vivo luciferase expression detection analysis of mice after PC3-luc / PSC27-based subcutaneous inoculation.
  • Figure 78 Comparative analysis of DNA damage and apoptosis in cancer cells within 7 days after pre-clinical administration. On the left, statistical comparison.
  • Figure 79 Figure 78 under several representative conditions of histochemical staining pictures (cleaved caspase 3).
  • Figure 80 ELISA detection of changes in AREG protein levels in mouse plasma under several treatment conditions.
  • Figure 81 Histochemical analysis of PD-L1 expression in tumor tissues of prostate cancer patients before and after clinical treatment.
  • Figure 82 Patient survival curve analysis based on the PD-L1 expression level of cancer cells in tumor tissue. Number of patients with low PD-L1 expression, 23, blue. Number of patients with high PD-L1 expression, 25, yellow.
  • Figure 83 Analysis of the relationship between the expression level of AREG in stromal cells and the expression level of PD-L1 in peripheral cancer cells in the lesion tissue of patients.
  • Figure 84 After full transcriptome analysis, the expression of PD-L1, PD-L2, and PD-1 in the stromal cell-derived AREG-treated prostate cancer cell line PC3 was found. Above, RNA-Seq data. Below, quantitative RT-PCR data.
  • Figure 85 After transcriptome analysis, the expression of PD-L1, PD-L2, and PD-1 in the AREG-treated prostate cancer cell line DU145 derived from stromal cells was found. Above, RNA-Seq data. Below, quantitative RT-PCR data.
  • Figure 86 Western blot analysis of the expression of PD-L1, PD-L2 and PD-1 in PC3 and DU145 cells cultured with PSC27 overexpressing AREG subline CM. GAPDH, loadig control.
  • FIG 87 The CM produced by PSC27 AREG is used to culture PC3 cells while using a group of inhibitors of EGFR and its downstream signaling pathway node molecules. Western blot analysis of PD-L1 expression changes under these conditions. GAPDH, loading control.
  • FIG 88 The CM produced by PSC27 AREG was used to culture PC3 cells and their PD-L1 knock-out subline, and the expression level of PD-L1 in cancer cells was analyzed by Western blot. GAPDH, loading control.
  • Figure 89 Comparative analysis of survival rate of PC3 cells co-cultured with PBMC in the presence or absence of CM produced by PSC27 AREG .
  • Figure 90 Comparative analysis of survival rate of PC3 cells and their PD-L1 knock-out sub-lines co-cultured with PBMC in the presence or absence of CM produced after PSC27 and AREG knock-out sub-lines were treated with bleomycin .
  • Figure 91 Survival analysis of DU145 cells co-cultured with PBMC in the presence or absence of CM produced by PSC27 AREG .
  • Figure 92 Comparative analysis of survival rate of DU145 cells co-cultured with PBMC and their PD-L1 knock-out sub-line in the presence or absence of CM produced after PSC27 and AREG knock-out sub-line were treated with bleomycin .
  • Figure 93 Comparative analysis of IFN ⁇ in peripheral blood after ELISA detection in experimental mice under different drug treatment conditions including Atezolizumab and Nivolumab.
  • Fig. 94 similar to Fig. 93, comparative analysis of TNF ⁇ in peripheral blood of TNF ⁇ in peripheral blood of experimental mice under different drug treatment conditions including Atezolizumab and Nivolumab.
  • FIG. 95 Schematic diagram of the pre-clinical treatment process of immune reconstructed mice.
  • Experimental mice with Rag2 -/- IL2R ⁇ null background were intravenously injected with human PBMC, and 3 days later, the same batch of animals was subcutaneously inoculated with PC3 / PSC27 cells.
  • Mitoxantrone was administered intraperitoneally at the 3rd week after the inoculation, and Atezolizumab or Nivolumab was injected at the same time. This procedure of intraperitoneal chemotherapy / targeted therapy is repeated every two weeks until all 3 dosing cycles are completed.
  • the mice were sacrificed and their tumor tissues and a series of pathophysiological indexes were obtained.
  • Figure 97 Statistical analysis of tumor terminal volume under various treatment conditions after inoculation of PC3 / PSC27 cells in immune reconstructed mice after 8 weeks of treatment.
  • Figure 98 Histochemical analysis of the expression level of PD-L1 in tumor tissue after the end of the course of treatment in immune reconstructed mice.
  • Figure 99 Statistical analysis of the terminal volume of tumors under various treatment conditions after inoculation of VCaP / PSC27 cells and after 8 weeks of treatment.
  • Figure 100 Statistical analysis of the tumor terminal volume of mice at the end of treatment 8 weeks after the immune reconstructed mice were inoculated with the breast cancer cell line MDA-MB-231 / mammary stromal cell line HBF1203.
  • Figure 101 Weight analysis of prostate cancer tumor-bearing PC3 mice at the end of pre-clinical
  • Figure 106 Statistical comparison analysis of AREG in plasma of prostate cancer patients before and after chemotherapy after ELISA analysis.
  • Figure 107 Statistical comparison analysis of IL-8 in plasma of prostate cancer patients before and after chemotherapy after ELISA analysis.
  • Figure 108 The correlation between the levels of AREG and IL-8 in the plasma of prostate cancer patients before and after chemotherapy was analyzed by Pearson.
  • Figure 109 Statistical comparison analysis of AREG in plasma of lung cancer patients before and after chemotherapy after ELISA analysis.
  • Figure 110 Statistical comparison analysis of IL-8 in plasma of lung cancer patients before and after chemotherapy after ELISA analysis.
  • Figure 111 The correlation between AREG and IL-8 levels in the plasma of lung cancer patients before and after chemotherapy was analyzed by Pearson.
  • FIG. 112. Western blot detection of AREG and IL-8 in peripheral blood of patients with prostate cancer. There were 4 patients before chemotherapy and 6 patients after chemotherapy. Albumin, plasma loading control.
  • Figure 113 Correlation analysis of the expression levels of AREG and IL-8 in primary cancer tissues and peripheral blood of patients with prostate cancer after chemotherapy. A total of 20 patients.
  • FIG 114 Analysis of multiple SASP factor expression based on stromal cells in the lesion tissue of 20 prostate cancer patients in Figure 113.
  • IL-2 / 3/5/12/17 are SASP unrelated interleukins (or pro-inflammatory factors), which are experimental controls.
  • Figure 115 Correlation between plasma levels of AREG and disease-free survival in 20 prostate cancer patients after chemotherapy. 10 patients with low AREG, cyan curve. Patients with high levels of AREG, brown curve.
  • Figure 116 Correlation between plasma levels of AREG and disease-free survival in 20 lung cancer patients after chemotherapy. 10 patients with low AREG, purple curve. Patients with high levels of AREG have a yellow curve.
  • Figure 117 Pearson correlation analysis between the expression level of AREG in stromal cells and the expression level of PD-L1 in peripheral cancer cells in 20 lung cancer patients after chemotherapy.
  • Figure 118 Statistical comparative analysis of AREG mutations, amplifications, deletions and multiple changes in patients with multiple solid tumors taken from the TCGA source database.
  • AREG bidirectional modulators
  • AREG is a transmembrane glycoprotein. Because it has an epidermal growth factor domain at the C-terminus, it is also called EGF-like protein. It is also a bifunctional growth factor, a type 2 related cytokine. In recent years, immune studies have found that it is likely to be an important molecule in the formation of resistance and drug resistance mediated by type 2 immune response. In addition to the production of AREG protein by epithelial cells and mesenchymal cells, a large number of data indicate that a variety of white blood cell subpopulations such as mast cells, basophils, type 2 natural lymphocytes, and a small portion of the original regulatory CD4 + T cells are Can express AREG.
  • the amino acid sequence of human AREG is as follows:
  • AREG is an exocrine protein released by cancer-associated fibroblasts (CAFs), which is related to cancer cell malignant growth, acquired drug resistance, and distant metastasis.
  • CAFs cancer-associated fibroblasts
  • CAFs are thought to originate from fibroblasts produced in the body, and cancer cells can hijack and use them to maintain their own growth.
  • Toxic side effects caused by genotoxic therapy can activate multiple components in the microenvironment, and once CAFs enter a DNA-damaging repair state, they exhibit a typical aging-related secreted phenotype (SASP) and are caused by subsequent stages of anti-cancer treatment The negative effects cannot be underestimated.
  • SASP typical aging-related secreted phenotype
  • the main manifestation is that cancer cells acquire significant drug resistance, and develop into cancer stem cells, form multiple micrometastases, migrate to the circulatory system, colonize ectopic organs, and ultimately accelerate patient death.
  • AREG active molecules including AREG
  • SASP chemotherapeutic drugs or radiation treatment.
  • AREG which is a bimodulin
  • AREG is released outside the stromal cells by paracrine effects on the phenotype of adjacent cancer cells
  • the significance of the malignant progression of the disease has not been reported so far.
  • AREG one of SASP's broad-spectrum exocrine factors, be resistant to the promotion of cancer cell acquired resistance, and will cancer cells show drug-induced AREG expression similar to stromal cells during treatment? Is unknown.
  • AREG is shown to play an important biological role in the SASP phenotype and tumor microenvironment, which is closely related to the prognosis after chemotherapy treatment.
  • the synergistic effect of antibodies that specifically inhibit AREG and chemotherapeutic drugs can be achieved by the following modes of action: antibodies that specifically inhibit AREG bind to AREG derived from the tumor microenvironment (especially the stromal cells) to inhibit their activity and reverse tumors for chemotherapy Drug resistance, which makes the effect of chemotherapy drugs more ideal.
  • the present invention provides a drug combination or composition for inhibiting tumors or reducing tumor drug resistance.
  • the drug combination or composition includes: an antibody that specifically inhibits AREG, and a chemotherapy drug.
  • the tumor may be a tumor in situ or a metastatic tumor, which includes refractory tumors that have drug resistance, especially tumors that are resistant to genotoxic chemotherapy drugs.
  • the tumor is a solid tumor.
  • the tumors include: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, etc.
  • an anti-AREG monoclonal antibody that is particularly effective for inhibiting tumors or reducing tumor resistance is provided, and the anti-AREG monoclonal antibody has high specificity for AREG and does not bind to other proteins than AREG . Also, when used in combination with chemotherapeutic drugs to suppress tumors, its effect is extremely excellent.
  • the anti-AREG monoclonal antibody of the present invention is prepared using hybridoma technology, and the deposit number of the hybridoma cell strain in the Chinese Type Culture Collection Center is CCTCC NO: C2018214.
  • the hybridoma When the hybridoma is obtained, the hybridoma cells can be cultured and expanded in vitro according to a conventional animal cell culture method, so that the anti-AREG monoclonal antibody is secreted.
  • the anti-AREG monoclonal antibody can be prepared by the following preparation methods: (1) mice pre-treated with adjuvant; (2) inoculation of the hybridoma cells in the abdominal cavity of mice and secretion of monoclonal antibodies Clone the antibody; (3) draw ascites and isolate the monoclonal antibody.
  • the monoclonal antibody isolated from ascites fluid is further purified to obtain high-purity antibodies.
  • the monoclonal antibodies of the present invention can also be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Those skilled in the art understand that after the monoclonal antibody hybridoma cell line is obtained or the monoclonal antibody is known by sequencing and other means, those skilled in the art can easily obtain the antibody.
  • Antibodies and chemotherapy drugs that specifically inhibit AREG can be administered as a pharmaceutical composition, or the two can be present separately in a kit.
  • the antibodies and chemotherapeutics that specifically inhibit AREG are effective amounts.
  • the antibody that specifically inhibits AREG is also mixed with a pharmaceutically acceptable carrier.
  • the term "effective amount” or “effective dose” refers to a human and / or animal that can produce a function or activity and can be accepted by a human and / or animal as used herein.
  • A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the type of animal, in general, mouse and rat 9.1, guinea pig 9.8, rabbit 10.1, cat 9.9, Dog 11.2, monkey 11.8, human 10.6. It should be understood that the conversion of the administered dose can be changed according to the difference of the drug and clinical situation and the evaluation of an experienced pharmacist.
  • pharmaceutically acceptable ingredients are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, substances with a reasonable benefit / risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the present invention provides a kit for inhibiting tumors or reducing tumor resistance.
  • the kit includes antibodies and chemotherapy drugs (such as mitoxantrone, doxorubicin, bleomycin) that specifically inhibit AREG Vegetarian, satraplatin, paclitaxel). More preferably, the kit further includes instructions for use to guide the clinician to use the medicine in a correct and reasonable manner.
  • chemotherapy drugs such as mitoxantrone, doxorubicin, bleomycin
  • the combination of the antibody that specifically inhibits AREG and the chemotherapeutic drugs can be made into unit dosage form and placed in the kit.
  • chemotherapeutic drugs such as mitoxantrone, doxorubicin, bleomycin, sabplatin, paclitaxel
  • antibodies or chemotherapeutic drugs that exist independently of each other can be made into unit dosage form and placed in the kit.
  • Unit dosage form refers to the preparation of the medicine into a dosage form required for single administration for the convenience of taking, including but not limited to various solid agents (such as tablets), liquid agents, capsules, and sustained-release agents.
  • AREG can be used as a marker for prognosis evaluation in the post-chemotherapy stage of tumor: (i) disease classification, differential diagnosis, and / or disease-free survival analysis in post-chemotherapy stage; ii) Evaluate the tumor treatment drugs, drug efficacy, prognosis of the relevant population, and select appropriate treatment methods.
  • diseases classification, differential diagnosis, and / or disease-free survival analysis in post-chemotherapy stage ii) Evaluate the tumor treatment drugs, drug efficacy, prognosis of the relevant population, and select appropriate treatment methods.
  • AREG genes in the tumor microenvironment, especially in stromal cells can be isolated for more targeted treatment.
  • AREG threshold can be specified. When the expression of AREG is higher than the specified threshold, consider using AREG suppression regimen for treatment.
  • the threshold is easily determined by those skilled in the art.
  • the expression of AREG can be obtained by comparing the expression of AREG in the microenvironment of normal human tissues with the expression of AREG in the microenvironment of tumor patients. Threshold.
  • the present invention provides the use of AREG genes or proteins for preparing reagents or kits for evaluating tumor prognosis.
  • Various techniques known in the art can be used to detect the presence and expression of AREG genes, and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc., can be used in combination.
  • the present invention also provides reagents for detecting the presence or absence and expression of AREG genes in analytes.
  • primers that specifically amplify AREG can be used; or probes that specifically recognize AREG to determine the presence or absence of AREG genes; when performing protein-level detection, specificity can be used.
  • the expression of AREG protein is determined by binding antibodies or ligands of the protein encoded by AREG.
  • the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, display solution Color, lotion, etc.
  • the kit can also include instructions for use and / or nucleic acid sequence analysis software.
  • the present invention provides a method for screening potential substances that inhibit tumors or reduce tumor resistance.
  • the method includes: treating a system for expressing NF- ⁇ B and AREG with a candidate substance, and there is NF- upstream of the gene encoding AREG ⁇ B binding site; and detection of the regulatory effect of NF- ⁇ B on AREG in the system; if the candidate substance statistically inhibits the transcriptional regulation of AREG by NF- ⁇ B, it indicates that the candidate substance inhibits or reduces tumors Potential substances for drug resistance.
  • a control group when performing screening, in order to more easily observe the transcriptional regulation of AREG and changes in the expression or activity of AREG by NF- ⁇ B, a control group may also be provided, and the control group may not be added The expression system of the candidate substance.
  • the present invention provides a method for screening potential substances that inhibit tumors or reduce tumor resistance.
  • the method includes: treating an expression system with candidate substances that expresses EGFR-mediated signaling pathways and AREG; and detection
  • AREG activates the EGFR-mediated signaling pathway; if the candidate substance statistically inhibits the activation, it indicates that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • a control group may also be provided. It is an expression system that does not add the candidate substance.
  • the method further includes: performing further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances that are truly useful for inhibiting tumors or reducing tumor drug resistance.
  • Normal human-derived primary prostate stromal cell line PSC27 and human-derived primary breast stromal cell line HBF1203 were propagated and passaged in PSCC complete culture medium.
  • Benign prostate epithelial cell lines BPH1, prostate cancer epithelial cell lines M12, DU145, PC3, LNCaP and VCaP, breast cancer epithelial cell lines MCF-7, MDA-MB-231, MDA-MB-468, T47D and BT474 purchased from ATCC Both were cultured in 5% FBS RPMI-1640 complete culture medium in an incubator at 37 ° C and 5% CO 2 .
  • PSC27-Pre 100nM docetaxel (DTX), 100nM paclitaxel (PTX), and 200nM vincristine (VCR) are added to the culture medium.
  • the full-length human AREG was cloned between the lentiviral expression vector pLenti-CMV / To-Puro-DEST2 (Invitrogen) restriction sites BamHI and XbaI.
  • the packaging line 293FT is used for cell transfection and lentivirus manufacturing.
  • the small hairpin RNAs (shRNAs) sense strands used to knock out AREG are CCGGTCCTGGCTATATTGTCGATGATCTCGAGATCATCGACAATATAGCCAGGTTTTTG (SEQ ID NO: 2) and CCGGTCACTGCCAAGTCATAGCCATACTCGAGTATG GCTA TGACTTG GCAGTGTTTTTG (SEQ).
  • the AREG antibodies used in the IHC staining of clinical prostate cancer and lung cancer patients were the same as above and were purchased from Proteintech. The specific steps are as follows: conventional dewaxing, incubation with 0.6% H 2 O 2 methanol at 37 ° C. for 30 min, then repair with 0.01 M pH 6.0 citrate buffer for 20 min, and cooling at room temperature for 30 min. Block with normal goat serum for 20 min, incubate with AREG primary antibody (1: 200) at 37 ° C for 1 h, and move to 4 ° C refrigerator overnight. The next day, it was washed three times with TBS, incubated with a secondary antibody (HRP-conjugated goat anti-rabbit) at 37 ° C for 45 min, washed with TBS three times, and finally developed with DAB.
  • conventional dewaxing incubation with 0.6% H 2 O 2 methanol at 37 ° C. for 30 min, then repair with 0.01 M pH 6.0 citrate buffer for 20 min, and cooling at room temperature for 30
  • PSC27 cells were cultured with DMEM + 0.5% FBS medium for 3 days, and then washed with 1 times PBS to wash the full abundance of the cell population. After simple centrifugation, the supernatant was collected and stored as conditioned medium at –80 ° C or used directly.
  • Prostate epithelial cells were continuously cultured in this conditioned medium for 3 days to carry out in vitro experiments.
  • epithelial cell lines are cultured in low serum DMEM (0.5% FBS) (referred to as "DMEM"), or conditioned medium, while mitoxantrone (MIT) is used to treat cells for 1 to 3 days, The concentration was close to the IC 50 value of each cell line, and then observed under a bright field microscope.
  • the total RNA of growing cells was extracted with Trizol reagent to perform reverse transcription reaction.
  • the reverse transcription reaction product cDNA was diluted 50 times as a template, and RT-PCR was performed.
  • the analysis of the amplification of each gene is analyzed through the software, the corresponding threshold cycle number is derived, and the relative expression level of each gene is calculated using the 2- ⁇ Ct method.
  • the peaks and waveforms of the melting curve are analyzed to determine whether the amplified product is a specific single-purpose fragment.
  • the antiviral vector pBabe-Puro-I ⁇ B ⁇ -Mut (superrepressor) containing the two IKK phosphorylation mutation sites S32A and S34A on the I ⁇ B ⁇ protein sequence was used to transfect the lentiviral packaging cell line PHOENIX.
  • the lentivirus was then used to infect the PSC27 stromal cell line, and 1 ⁇ g / ml puromycin was used to screen positive clones.
  • a 5 ⁇ M small molecule inhibitor Bay 11-7082 (available from Selleck) was used for NF- ⁇ B activity control.
  • the stromal cells were subsequently exposed to several different forms of cytotoxicity, and the resulting phenotypes were recorded in time to analyze the expression of related genes.
  • the cells treated in this way, the conditioned medium produced is collected and used for various detections of epithelial cells.
  • the human AREG gene (Gene ID 374, Genbank access NM_001657.3) was analyzed using software CONSITE to discover potential core NF- ⁇ B binding sites.
  • primer # 1 (-3579 ⁇ -3370): forward 5'-CCAGTCTGGAGTGCGGTGGC-3 '(SEQ ID NO: 4), reverse 5'-GGTGGATCATCTCAGGTCAG-3 '(SEQ ID NO: 5); primer set # 2 (-1320 ⁇ -1120): forward 5'-ATTTTGAGTCGGAGTCTTGC-3' (SEQ ID NO: 6) , Reverse 5'-TGGCCA AGATGGCAAAACCC-3 '(SEQ ID NO: 7); primer set # 3 (-1221 ⁇ -1000): forward 5'-GCC TCAGCCCACCCGAGTAG-3' (SEQ ID NO: 8),
  • IL-6 forward 5'-AAATGCCCAACAGAGGTCA-3 '(SEQ ID NO: 12), reverse 5'-CACGGCTCTAGGC TTCGAAT-3' (SEQ ID NO: 13)
  • IL8 forward 5'-ACAGTTGAAAACTATAG GAGCTACATT-3 '(SEQ ID NO: 14), reverse 5'-TCGCT TCTGGGCAAGTACA-3' (SEQ ID NO: 15) promoter sequences (both are known Positive control).
  • ChIP analysis was performed on early passage PSC27 cells (such as p8) and PSC27 cells treated with bleomycin (50ug / ml).
  • the chromosomes fixed in vitro were settled using mouse monoclonal antibody anti-p65antibody (F-6, Santa Cruz), and DNA was extracted for amplification.
  • Reporter expression vectors carrying multiple NF- ⁇ B binding site mutations are designed and generated by the site-directed mutagenesis (Stratagene) method. In addition, it covers multiple NF- ⁇ B binding sites and the optimized IL-2 minimal promoter as the reporting vector for NF- ⁇ B activated transgenic system (NAT system) NAT11-Luc2CP-IRES-nEGFP (obtained from Hokkaido University, Japan), Used as a positive control in the experiment.
  • Each reporter vector was co-transfected with pRL-TK vector (Addgene) for signal standardization.
  • the chemotherapy regimen is specified based on the pathological characteristics of patients with castration-resistant prostate cancer (clinical trial registration number NCT03258320) and non-small cell lung cancer (clinical trial registration number NCT02889666).
  • Patients with a clinical stage of primary cancer above I subtype A (IA) (T1a, N0, M0) but no obvious distant metastatic lesions were recruited into the clinical cohort.
  • IA I subtype A
  • patients aged 40-75 years who were clinically diagnosed with PCa, or those younger than 75 years who were clinically diagnosed with NSCLC were recruited. All patients were provided with informed consent and signed for confirmation. Data on tumor size, tissue type, tumor penetration, lymph node metastasis, and pathological TNM disease stage were obtained from the pathology recording system.
  • Tumors were processed into FFPE samples and processed into histological sections for evaluation. OCT frozen sections were selectively separated by LCM and used for gene expression analysis. In particular, gland-associated stromal cells before and after chemotherapy were isolated by LCM. Immune activity scores (IRS) are classified into 0-1 (negative), 1-2 (if), 2-3 (medium), and 3-4 (strong) according to histochemical staining of each tissue sample. Category (Fedchenko and Reifenrath, 2014). The diagnosis of PCa, BCa and CRC samples is judged and scored by independent pathologists. The randomized controlled trial (RCT) protocol and all experimental procedures are approved and authorized by the IRB of Shanghai Jiaotong University School of Medicine, and are gradually carried out according to authoritative guidelines.
  • RCT randomized controlled trial
  • ICR / SCID mice (about 25g in weight) of immunodeficient mice aged about 6 weeks are used in the animal experiments related to the present invention.
  • the stromal cells PSC27 and epithelial cells are mixed in a ratio of 1: 4, and each graft contains 1.25 ⁇ 10 6 cells for tissue reconstruction.
  • the transplanted tumors were implanted into mice by subcutaneous transplantation, and the animals were euthanized 8 weekends after the transplantation.
  • breast cancer xenografts are formed by MDA-MB-231 (triple negative, highly malignant breast cancer cell line) and HBF1203 (breast fibroblast cell line) through tissue remodeling.
  • mice subcutaneously transplanted were given standard experimental diets, and after 2 weeks, the chemotherapy drugs mitoxantrone (0.2 mg / kg dose) or doxorubicin (1.0 mg / kg dose) were given intraperitoneally. medicine.
  • the FDA approved therapeutic antibody Cetuximab (10.0 mg / kg dose, 200 ⁇ l / dose) or AREG mAb (10.0 mg / kg dose, 200 ⁇ l / dose) after strict purification is administered as a single or dual drug intravenously.
  • the time point was the first day of the 3rd, 5th, and 7th weeks. There were 3 cycles of drug administration throughout the course of treatment, and each cycle was 2 weeks.
  • mice were collected for tumor measurement and histological analysis. Each mouse received cumulatively mitoxantrone 0.6 mg / kg body weight, or doxorubicin 3.0 mg / kg body weight.
  • the chemotherapy experiment was carried out until the end of the 8th week. The mice were dissected immediately after sacrifice, and the transplanted tumors were collected and used for pathological analysis. Part of the mice 7 days after the administration was used for histochemical evaluation of the caspase 3 activity at the tissue level.
  • mice were weighed once a week; after the completion of chemotherapy as a whole, the mice were weighed again and their blood was collected by cardiac puncture and placed in an ice bath for 45 minutes. Peripheral blood was immediately centrifuged at 8000 rpm for 10 minutes at 4C, and about 50 ⁇ l was sucked by the VetTest pipette tip for use in the IDEXX VetTest 8008 chemical analyzer. Liver function measurement items include creatinine, urea, alkaline phosphatase and alanine aminotransferase.
  • mice 4-week-old Rag2 -/- IL2R ⁇ null mice (Jackson Laboratory) were injected with 7 ⁇ 10 6 fresh human PBMCs through the tail vein. After 3 days, 1.2 ⁇ 10 6 PC3 cells were inoculated subcutaneously or mixed with 0.3 ⁇ 10 6 PSC27 cells before inoculation. Since the third week, mitoxantrone is used in combination with the therapeutic antibodies atezolizumab or nivolumab (each of which is injected in parallel with isotype-matched IgG, such as IgG 1 for atezolizumab, IgG4 for nivolumab to obtain control data) as single or dual drugs .
  • mice After the 8-week course of treatment, the mice were sacrificed and their tumors were collected for pathological analysis, including the assessment of changes in the expression of PD-L1 cancer cells in the tissue.
  • mouse blood was used for ELISA analysis to detect circulating levels of IFN ⁇ and TNF ⁇ levels.
  • Example 1 DNA damage leads to high expression of AREG in human stromal cells
  • DNA-damaging drugs including bleomycin (BLEO), mitoxantrone (MIT), and satraplatin (SAT)
  • BLEO bleomycin
  • MIT mitoxantrone
  • SAT satraplatin
  • PTX paclitaxel
  • DTX docetaxel
  • VCR vincristine
  • Example 2 The expression of AREG in the tumor microenvironment is significantly negatively correlated with the survival of patients after chemotherapy
  • AREG is highly expressed in tumors after chemotherapy, which is further determined by a pre-established pathological detection system that can quantitatively evaluate its expression level in tissues according to the histochemical staining intensity of specific proteins ( Figures 16, 17).
  • LCM laser capture microdissection
  • the inventors again found that AREG in the tissue is more inclined to induce inducible expression in the stromal cell population rather than the epithelial cell population ( Figure 18).
  • the inventors selected a group of patients whose tissue samples were obtained and saved before and after chemotherapy, and found that any of them showed AREG stromal cells instead of epithelial cells after chemotherapy Highly expressed in cells (Figure 19, 20).
  • Example 3 The expression of AREG in stromal cells is regulated by transcription factors such as NF-kB and C / EBP
  • the next step is to determine the basis of AREG expression in damaged stromal cells.
  • the NF- ⁇ B complex plays an important role in the process of cell senescence caused by oncogene induction or therapeutic injury.
  • the inventors first considered whether NF- ⁇ B mediates AREG expression in stromal cells after DNA damage. The analysis revealed that there were several NF- ⁇ B binding sites in the range of about 4000 bp upstream of the AREG gene ( Figure 36), and the fluorescence detection results based on the reporter vector subsequently confirmed the existence of these sites.
  • AREG AREG-induced AREG
  • stromal cells under genotoxic background mainly regulated by NF- ⁇ B and C / EBP.
  • drugs that inhibit tumors by affecting the interaction between the two can be screened. Some drugs that can inhibit or prevent the interaction of the two may potentially benefit tumors. Treatment.
  • Example 4 The functional impact of AREG on cancer cells is mainly controlled by EGFR and its downstream signaling pathways
  • AREG AREG derived from stromal cells exerts influence on the recipient cells through the paracrine pathway.
  • soluble factors such as IL-8 and the senescence of cells after DNA damage after striking AREG in stromal cells did not cause significant changes ( Figures 48, 49).
  • DOC paclitaxel
  • AREG and EGF are both typical EGFR ligands and share a certain degree of sequence homology
  • the inventors first determined the effect of AREG as an EGF analog on cancer cell signaling pathways. After culturing cancer cells using CMs produced by AREG high-expression stromal cells (PSC27 AREG ), the inventors found that the latter showed post-translational modification changes of multiple protein molecules, mainly including EGFR (Y845), Akt (S473) and mTOR S2448) Phosphorylation at the allele, suggesting the activation of PI3K / Akt / mTOR signaling pathway mediated by AREG ( Figure 57).
  • phosphorylation of Mek (S217 / S221), Erk (T202 / Y204) and Stat3 (S727) sites indicates the activation of MAPK meridians in these cells.
  • AG-1478 an EGFR-specific RTK inhibitor, to treat recipient cancer cells.
  • AREG-induced phosphorylation of EGFR and many downstream molecules were abolished, including the Akt / mTOR and Mek / Erk / Stat3 signal axes.
  • AREG-induced changes in cancer cell phenotypes are mainly achieved through EGFR-mediated signaling pathway activation.
  • the activation of this series of signaling pathways of cancer cells basically disappeared ( Figure 58), further confirming that AREG caused the activation of multiple downstream signaling pathways through EGFR.
  • the inventors conducted IP experiments using AREG-specific antibodies. The results show that there is a physical direct interaction between the two molecules of AREG and EGFR, and the IP signal can be easily found in cancer cell samples treated with PSC27 AREG instead of PSC27 Vector CM ( Figure 59).
  • AREG as a component of the SASP broad-spectrum exocrine factor concentration, plays a functional core role in the process of SASP driving cancer cell malignant progression.
  • the inventors constructed a PSC27-shRNA AREG stability subline and collected its CM after DNA damage treatment. The inventors noticed that after AREG was knocked out, the cell senescence of PSC27 originally under DNA damage conditions was neither delayed nor accelerated, and the SA- ⁇ -Gal positive rate was unchanged (FIG. 49).
  • the inventors have discovered that the tumor microenvironment damaged by chemotherapeutic drugs can confer significant drug resistance on cancer cells, and the inventors subsequently detected changes in this activity.
  • AREG was knocked out, the resistance of prostate cancer cells conferred by PSC27-BLEO to mitoxantrone significantly decreased ( Figure 63).
  • PSC27-BLEO CM culture conditions the drug resistance of cancer cells treated by the EGFR inhibitor AG-1478 was also significantly reduced ( Figure 64).
  • Cetuximab an FDA-approved monoclonal antibody that specifically inhibits EGFR.
  • Example 5 Targeting AREG in vivo can block tumor growth and increase tumor sensitivity to chemotherapy drugs
  • microenvironment SASP in the clinical anti-cancer process can accelerate the occurrence of many malignant events, including tumor progression, local inflammation and the emergence of therapeutic resistance.
  • this trend towards malignancy can be significantly alleviated by specifically controlling the core factors in the broad-spectrum SASP of the microenvironment has been an interesting topic in the medical community in recent years and a technical problem.
  • the inventors In order to simulate clinical conditions as much as possible, the inventors inoculated a subpopulation of immunodeficient mice with a mixed cell population of PSC27 and PC3, and after 8 weeks, the inventors stopped the experiment and analyzed.
  • the inventor designed a set of pre-clinical treatment strategies for simulating clinical anti-cancer treatment regimens, that is, an 8-week chemotherapy regimen for tumor-bearing mice, the latter is mainly determined by It consists of single or double drug treatment ( Figure 69-70).
  • mice inoculated with PSC27 AREG increased significantly, but the tumor volume formed under the screening pressure caused by the intraperitoneal administration of the chemotherapy drug mitoxantrone was significantly reduced, proving that chemotherapy itself can effectively block tumor development (Figure 71).
  • the remaining tumor volume of PSC27 AREG mice still increased significantly, suggesting the pathological role of the microenvironment throughout the chemotherapy.
  • the present inventors confirmed the obvious expression of AREG in tumor tissues under pre-clinical treatment conditions by histochemical staining (FIG. 75).
  • the inventors then used Cetuximab or AREG mAb in combination with mitoxantrone.
  • Cetuximab or AREG mAb in combination with mitoxantrone.
  • the simultaneous administration of Cetuximab therapeutic antibody did not further reduce the mass (Figure 76), suggesting that PC3 tumors are basically in EGF / EGFR signal axis independent microenvironment progress.
  • the inventors used the PC3 cell line (PC3-luc) expressing luciferase and found that the relative intensity of the bioluminescence signal detected under the conditions in mice in each group of animals was similar to that of the previously detected tumor terminal
  • the volume basically corresponds, and the possibility of ectopic organ metastasis of cancer cells in situ lesions in the body is excluded (Figure 77).
  • AREG mAb produced a more significant therapeutic effect than Cetuximab (Figure 78).
  • the results of histochemical staining showed that under the condition of injecting AREG mAb into mice, the molecule caspase 3 exhibited more obvious self-cleavage ( Figure 79).
  • the ELISA test results showed that mitoxantrone treatment can cause AREG protein levels in mice plasma to rise sharply, but when AREG mAb is administered at the same time, it can significantly control this change (Figure 80).
  • Example 6 AREG derived from stromal cells up-regulates the expression of the recipient cancer cell PD-L1 and forms an immunosuppressive microenvironment
  • Tumor immunotherapy can produce long-lasting responses in a large number of patients, while both adoptive cell transfer and checkpoint blockade therapy can produce significant antigen-specific immune responses.
  • the existing research results show that the clinical effect of chemotherapy depends in part on the cytotoxic effect of the immune system, indicating the importance of complete and sustained immune activity for anti-cancer treatment.
  • the inventors first detected the expression changes of PD-L1 and other immune activity-related molecules in the tumor tissue of clinical patients with prostate cancer.
  • Preliminary histochemical data showed that PD-L1 was highly upregulated in the tumor after chemotherapy, and the expression level of PD-L1 was significantly negatively correlated with the disease-free survival rate of patients after treatment ( Figure 81-82).
  • the results of statistical analysis showed that there was a close relationship between the expression of AREG in the tumor stroma and the upregulation of PD-L1, a peripheral cancer cell ( Figure 83).
  • EGFR Erlotinib
  • AG1478 EGFR
  • LY294002 PI3K
  • MK2206 Akt
  • Rapamycin mTOR
  • PD0325901 Mek
  • Bay 117082 NF-kB
  • Ruxolitinib Jak1 / 2
  • SB203580 p38MAPK
  • PBMC peripheral blood mononuclear cells
  • the present inventors chose to use PD-L1 and PD-1 inhibitors for intravenous injection of Rag2 -/- IL2R ⁇ null experimental mice and to detect their peripheral blood T cell activity.
  • Atezolizumab the first PD-L1 inhibitor approved by the FDA for the new targeted treatment of bladder cancer
  • Nivolumab aka Opdivo, FDA approved for the treatment of advanced metastatic squamous In patients with small cell lung cancer (NSCLC), which is suitable for platinum-based chemotherapy or patients whose disease has deteriorated after chemotherapy
  • NSCLC small cell lung cancer
  • the inventors expanded the research, that is, the experimental mice with Rag2 -/- IL2R ⁇ null background were intravenously injected with human PBMC, and the same batch of animals were subcutaneously inoculated with PC3 / PSC27 cells 3 days later. Mitoxantrone was administered to the animals intraperitoneally at the 3rd week after the inoculation, and Atezolizumab or Nivolumab was injected at the same time. Subsequently, the procedure of intraperitoneal chemotherapy / targeted therapy is repeated every two weeks until all three administration cycles are completed.
  • the inventors used another prostate cancer cell line VCaP, which expresses the androgen receptor AR and grows androgen-dependent.
  • VCaP prostate cancer cell line
  • the entire set of data on the terminal volume of VCaP tumors basically repeated a series of results of the previously discovered PC3 / PSC27 tumors ( Figure 99).
  • Figure 100 After subcutaneously inoculating MDA-MB-231 cancer cells and HBF1203 breast-derived stromal cells in mice, the inventors found that MDA-MB-231 / HBF1203 tumors showed a trend very similar to the above data of prostate cancer mice ( Figure 100). Therefore, drug resistance antagonistic data caused by targeting AREG indicate that controlling the effect of AREG in the microenvironment on tumor treatment is organ-independent and a means applicable to a variety of solid tumors.
  • mice To determine the safety and feasibility of this treatment strategy, the inventors next conducted a pathophysiological evaluation of the experimental mice. The results showed that regardless of monotherapy or multi-drug treatment, the body weight of mice and various other indicators, including plasma levels of creatinine, urea, ALP and ALT, remained unchanged ( Figures 101-105).
  • AREG is a novel biomarker that can be used to judge the occurrence and development of SASP in patients under clinical conditions
  • AREG AREG could be detected in the peripheral blood of cancer patients after clinical chemotherapy using conventional techniques.
  • the inventors collected plasma samples of two groups of prostate cancer patients, including a group of patients who had undergone chemotherapy and another group of patients who did not receive any treatment. After ELISA-based protein detection, it was found that the level of AREG in the blood of patients after chemotherapy was significantly higher than that of patients without chemotherapy ( Figure 106). Interestingly, this trend is very similar to IL-8, a typical factor of SASP ( Figure 107).
  • the inventors carried out a longitudinal analysis based on clinical specimens of patients.
  • the inventors were surprised to note that the two SASP related factors AREG and IL-8 can be clearly displayed on the Western blot, and the two are only in A signal appeared in the patient's plasma sample after chemotherapy ( Figure 112).
  • Figure 113 there is a clear correlation between these two factors ( Figure 113).
  • the present inventors used laser capture microdissection technology to isolate stromal cells in the lesion tissue of patients with prostate cancer and analyzed their transcript levels .
  • AREG can be used as a landmark factor with high credibility to reflect the occurrence and development of SASP under clinical conditions in clinical patients, and can be used to evaluate the development status and dynamic characteristics of SASP in cancer patients after chemotherapy.
  • AREG acts as An exocrine factor released after irreparable damage to the patient's tumor microenvironment can be used as an independent indicator for analyzing and judging the survival of patients after traditional chemotherapy.
  • TCGA studies including TCGA have reported AREG mutations, amplification, loss and multiple changes in cancer patients ( Figure 118), AREG is used as a kind of technology that reflects the pathological activity of the tumor microenvironment.
  • Non-invasive liquid biopsy markers can provide an accurate, convenient and efficient new diagnostic and monitoring technical indicator for future clinical medicine.
  • the hybridoma cell line SP2 / 0-02-AREG-SUN of the present invention is deposited at the China Type Culture Collection Center (CCTCC, Wuhan, China) under the deposit number CCTCC NO: C2018214; the deposit date is October 10, 2018.
  • CTCC China Type Culture Collection Center

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Abstract

本发明涉及双向调节素(Amphiregulin，AREG)在制备细胞衰老及肿瘤的诊断或调控制剂中的应用。首次揭示了AREG在SASP表型以及肿瘤微环境中发挥重要的生物学作用，其与化疗治疗后的预后密切相关。因此，AREG可作为SASP表型调控研究以及基于肿瘤微环境的抗肿瘤研究的靶点，作为肿瘤经化疗治疗后的预后评估以及病理分级的标志物，以及作为靶点开发抑制肿瘤的药物。

Description

双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用 技术领域
本发明属于疾病诊断及调控领域，更具体地，本发明涉及双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用。
背景技术
细胞衰老表现为核膜内折，染色质固缩，脂褐质积累，细胞体积增大，细胞核变大，β-半乳糖苷酶活性上升以及分泌多种因子等。细胞衰老由一种或多种因素触发，激活下游包括p53、p16 INK4A/Rb、PI3K/Akt、FoxO转录因子和线粒体SIRT1等在内的多条信号通路。除了进入永久性增殖停滞，衰老细胞常关系到许多病理学特征，包括局部炎症。细胞衰老发生于受损细胞，并防止其在生物体内增殖。在各种外界刺激和内部因素影响下，细胞损伤可以导致明显的细胞衰老迹象；当损伤累积和达到一定的限度，组织中呈现各种肉眼可辨的组织退行变化和生理上的衰老表型。
尤其值得注意的是，衰老细胞中炎症性细胞因子的表达水平显著升高，这一现象被称为衰老相关分泌表型(senescence-associated secretory phenotype，SASP)。SASP这一概念是由Coppe等人在2008年首次提出。他们发现衰老细胞能通过分泌胞外基质蛋白、炎症相关因子及癌细胞生长因子促进邻近癌前细胞发生癌变或恶性增强，并称这些蛋白为SASP因子。
衰老细胞产生的外泌蛋白功能，往往取决于衰老肿瘤细胞的遗传背景和种属来源。尽管SASP对肿瘤生物学具有重要的意义，但是它如何调控肿瘤仍然不甚明确。近年来已有研究将抗衰老着眼于靶向干预SASP的上游信号通路，药物或遗传特异性抑制衰老细胞中IKK/NF-κB、mTOR、p38MAPK、JAK/STAT等，能钝化SASP引起的旁分泌效应，从而改善细胞及机体的衰老状态。
目前，如何靶向杀死衰老细胞而不损伤邻近健康细胞，如何在阻断SASP负性因子的同时保留正性因子的作用，如何将动物实验的研究结果推广至临床诊断与治疗等诸多问题都有待进一步的研究。
发明内容
本发明的目的在于提供双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用。
在本发明的第一方面，提供一种用于抑制肿瘤或降低肿瘤耐药性的药物组合物，其特征在于，所述药物组合物中包括：特异性抑制双向调节素(AREG)的抗体，以及化疗药物。
在一个优选例中，所述化疗药物是基因毒药物；较佳地，所述的化疗药物包括：米托蒽醌，长春新碱，阿霉素，博莱霉素，沙铂，顺铂，卡铂，道诺霉素，诺加霉素，阿柔比星，表柔比星，多柔比星，阿糖胞苷，卡培他滨，吉西他滨，5-氟尿嘧啶。
在另一优选例中，所述的药物组合物包括特异性抑制双向调节素的抗体和米托蒽醌，且两者质量比为1:0.005～1:2.0；较佳地为1:0.01～1:1.0；更佳地为1:0.02～1:0.6，如1:0.2。
在另一优选例中，所述的药物组合物包括特异性抑制双向调节素的抗体和阿霉素，且两者质量比为1:0.02～1:1.5；较佳地为1:0.05～1:0.8；更佳地为1:0.06～1:0.3，如1:0.1。
在另一优选例中，所述的药物组合物包括特异性抑制双向调节素的抗体和博来霉素，且两者质量比为1:0.02～1.5；较佳地为1:0.05～1:0.8；更佳地为1:0.06～1:0.3，如1:0.1。
在另一优选例中，所述的药物组合物包括特异性抑制双向调节素的抗体和选自沙铂、顺铂、卡铂的一种或多种，且抗体与后者的质量比为1:0.02～1.5；较佳地为1:0.05～1:0.8；更佳地为1:0.06～1:0.3，如1:0.1。
在另一优选例中，所述的特异性抑制双向调节素的抗体是由杂交瘤细胞系CCTCC NO:C2018214分泌。
在本发明的另一方面，提供前面任一所述的药物组合物的用途，用于制备抑制肿瘤或降低肿瘤耐药性的药盒。
在一个优选例中，所述的药物组合物中，特异性抑制双向调节素的抗体通过抑制肿瘤微环境中基质细胞表达的双向调节素，从而降低肿瘤耐药性。
在另一优选例中，所述的肿瘤包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌、肝癌、胰腺癌、膀胱癌。
在另一优选例中，所述的肿瘤耐药性是肿瘤对化疗药物产生的耐药性。
在本发明的另一方面，提供特异性抑制双向调节素的抗体，其是由杂交瘤细胞系CCTCC NO:C2018214分泌。
在本发明的另一方面，提供特异性抑制双向调节素的抗体在制备抗体药物中的应用，所述抗体药物与化疗药物联合应用，抑制肿瘤或消除肿瘤耐药性；或用于消除肿瘤细胞对化疗药物的耐药性。
在本发明的另一方面，提供一种杂交瘤细胞株杂交瘤细胞株SP2/0-02-AREG-SUN，其在中国典型培养物保藏中心的保藏号是CCTCC NO:C2018214。
在本发明的另一方面，提供一种用于抑制肿瘤或降低肿瘤耐药性的药盒，所述药盒包括：特异性抑制双向调节素的抗体，或产生该抗体的细胞株。在一个优选例中，所述的药盒中还包括：化疗药物；较佳地所述化疗药物是基因毒药物；较佳地，所述的化疗药物包括：米托蒽醌，长春新碱，阿霉素，博莱霉素，沙铂，顺铂，卡铂，道诺霉素，诺加霉素，阿柔比星，表柔比星，多柔比星，阿糖胞苷，卡培他滨，吉西他滨，5-氟尿嘧啶。
在本发明的另一方面，提供双向调节素在制备用于肿瘤化疗预后评估的诊断试剂中的用途，其中，所述的双向调节素为肿瘤微环境中基质细胞产生的双向调节素。在一个优选例中，所述的肿瘤微环境中基质细胞产生的双向调节素可以通过常规分离手段从样本组织分离获得。
在本发明的另一方面，提供特异性识别双向调节素的试剂在制备用于肿瘤化疗预后评估或病理分级的诊断试剂中的用途，其中，所述的双向调节素为肿瘤微环境中基质细胞产生的双向调节素。
在一个优选例中，所述的特异性识别双向调节素的试剂包括：抗体试剂，引物，探针。
在本发明的另一方面，提供一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述方法包括：(1)用候选物质处理一表达体系，该体系表达NF-κB以及双向调节素，且该双向调节素编码基因上游存在NF-κB结合位点；和(2)检测所述体系中NF-κB对于双向调节素的调控作用；若所述候选物质在统计学上抑制NF-κB对于双向调节素的转录调控，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
在一个优选例中，步骤(1)包括：在测试组中，将候选物质加入到所述表达体系中；和/或步骤(2)包括：检测测试组的体系中NF-κB对于双向调节素的转录调控，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达体系；如果测试组中NF-κB对于双向调节素的转录调控显著被抑制(如抑制20％以上，较佳的抑制50％以上；更佳的抑制80％以上)，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
在另一优选例中，所述的NF-κB结合位点为双向调节素编码基因上游-3510、-1223、-1131、+79位。
在本发明的另一方面，提供一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述方法包括：(1)用候选物质处理一表达体系，该体系表达EGFR介导的信号通路以及双向调节素；和(2)检测所述体系中双向调节素对于EGFR介导的信号通路的激活作用；若所述候选物质在统计学上抑制该激活作用，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
在一个优选例中，步骤(1)包括：在测试组中，将候选物质加入到所述表达体系中；和/或步骤(2)包括：检测测试组的体系中双向调节素对于EGFR介导的信号通路的激活作用，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达体系；如果测试组中双向调节素对于EGFR介导的信号通路的激活作用显著被抑制(如抑制20％以上，较佳的抑制50％以上；更佳的抑制80％以上)，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、人源前列腺原代基质细胞系PSC27经过化疗药物和放射处理之后的基因表达谱热图。CTRL，control。BLEO，bleomycin。HP，hydrogen peroxide。RAD，radiation。红色箭头，amphiregulin。
图2、PSC27细胞经过各种条件处理之后的DNA损伤反应(DDR)。上图，免疫荧光检测后的代表性图片，红色荧光为γH2AX，蓝色为DAPI。下图，DDR foci统计对比分析。PTX，paxlitaxel。DTX，docetaxel。VCR，vincristine。BLEO，bleomycin。MIT，mitoxantrone。SAT，satraplatin。
图3、PSC27经过图2中各种条件处理之后的细胞衰老检测。上图，经过SA-B-Gal染色后的明场显微镜代表图。下图，SA-B-Gal染色阳性细胞统计学对比分析。
图4、PSC27经过图2中各种条件处理之后细胞中DNA嵌入速率分析。上图，BrdU染色之后的代表图，绿色荧光为BrdU。下图，各种药物处理之后的BrdU统计学分析。
图5、基质细胞中AREG的表达情况。上图，经过各种条件处理之后AREG在PSC27细胞中的转录本表达水平。下图，Western blot分析AREG蛋白表达。IC，intracellular。CM，conditioned media。GAPDH，loading control。
图6、在博来霉素处理之后的PSC27基质细胞表达几种SASP典型因子的时间规律。在药物损伤之后的第1(“2”)，3(“3”)，5(“4”)，7(“5”)，10(“6”)和15(“7”)天分别收集基质细胞并获取其总RNA，进行RT-PCR检测。各时间点数据同对照(未加药组，“1”)规范化之后的数值用于作图。
图7、图6中的各个时间点收集的细胞裂解样本经过Western blot分析其中AREG表达水平变化。IC，intracellular。CM，conditioned media。GAPDH，loading control。
图8、经过几种基因毒化疗药物处理之后的前列腺基质细胞和癌细胞中AREG的转录本表达水平对比分析。
图9、图8中的各细胞系经过博来霉素处理之后的细胞总蛋白样本以Western blot分析确定AREG表达变化。IC，intracellular protein。CM，conditioned media。GAPDH，loading control。
图10、人源乳腺基质细胞系HBF1203经过化疗药物处理之后的DNA损伤情况。上图，免疫荧光染色结果代表图，红色荧光为γH2AX，蓝色为DAPI。下图，DDR信号统计学对比分析。VNB，vinorelbine。VBL，vinblastine。DOX，doxorubicin。CIS，cisplatin。CARB，carboplatin。
图11、HBF1203经过各种药物处理之后细胞中DNA嵌入分析。上图，BrdU染色之后的代表图，绿色荧光为BrdU。下图，各种药物处理之后的BrdU统计学分析。
图12、HBF1203经过图10中各种条件处理之后的衰老细胞分析检测。上图，经过SA-B-Gal染色后的明场显微镜代表图。下图，SA-B-Gal染色阳性细胞统计学对比分析。
图13、HBF1203中AREG的转录本表达情况。经过各种条件处理之后AREG在细胞中的转录本表达水平。
图14、经过几种药物处理之后的乳腺基质细胞和癌细胞中AREG的转录本表达水平对比分析。
图15、前列腺癌患者化疗前后原发病灶组织病理学对比分析。左侧，组化染色(AREG)代表性图片。右侧，H&E染色代表性图片。
图16、基于前列腺癌患者肿瘤组织中AREG的组化染色结果进行病理分级之后的表达水平统计学对比分析。未经化疗的患者、经历过化疗的患者人数分别为42和48。
图17、与图16中病理分级相对应的代表性图片。EL，expression level。
图18、经过激光俘获显微切割(LCM)分离之后的基质细胞和上皮细胞，其AREG转录本表达对比分析。
图19、化疗前后基于单个患者的基质细胞中AREG转录本表达分析。每组患者数目，10。
图20、化疗前后单个患者癌细胞中AREG转录本表达分析。每组患者数目，10。
图21、化疗后阶段前列腺癌患者肿瘤基质细胞中的AREG，IL-8和WNT16B蛋白表达对比分析。每个因子病理分数来自该因子的组化染色病理读数，每一读数为3次病理盲读的平均值。
图22、基于AREG，IL-8和WNT16B的组化染色代表图。三种因子的组化病理染色系列均取自单一某患者疗后阶段的连续3张切片。
图23、在化疗后前列腺癌患者体内分析AREG与IL-8之间的蛋白表达关系。各个因子的数值来自三次病理盲读。其中r，R 2，slope和P值，均来自Pearson关联分析。
图24、在化疗后患者体内分析AREG与WNT16B之间的蛋白表达关系。各个因子的数值来自三次病理盲读。其中r，R 2，slope和P值，均来自Pearson关联分析。
图25、基于化疗后阶段患者病灶中AREG的表达水平进行的生存曲线(Kaplan Meier)分析。AREG低表达组患者数目，20，青色曲线。AREG高表达组患者，28，紫色曲线。
图26、肺癌患者化疗前后原发病灶组织病理学对比分析。左侧，组化染色代表性图片。右侧，H&E染色代表性图片。
图27、基于肺癌患者肿瘤组织中AREG的组化染色结果进行病理分级之后的统计学对比分析。
图28、代表图27中各病理分级的组化染色图片。EL，expression level。
图29、不同类型细胞之间的AREG表达对比分析。经过激光俘获显微切割(LCM)分离之后的基质细胞和上皮细胞，其AREG转录本表达对比分析。
图30、基于单个患者的基质细胞AREG转录本表达分析，每组患者数目为10。
图31、基于类似于图30的、单个肺癌患者癌细胞的一组AREG转录本表达分析， 每组患者数目为10。
图32、化疗后阶段肺癌患者肿瘤基质细胞中的AREG，IL-8和WNT16B蛋白表达对比分析。
图33、在化疗后肺癌患者体内分析AREG与IL-8之间的蛋白表达关系。各个因子的数值来自三次病理盲读。其中r，R 2，slope和P值，均来自Pearson关联分析。
图34、在化疗后肺癌患者体内分析AREG与WNT16B之间的蛋白表达关系。各个因子的数值来自三次病理盲读。其中r，R 2，slope和P值，均来自Pearson关联分析。
图35、基于化疗后阶段肺癌患者病灶中AREG的表达水平进行的生存曲线(Kaplan Meier)分析。AREG低表达组患者数目，71，绿色曲线。AREG高表达组患者，28，红色曲线。
图36、生信分析AREG启动子上游4000bp范围内的NF-kB结合位点。根据AREG启动子区域内推测性NF-kB结合位点构建的一组表达载体示意图。
图37、将图36中的4个报告性表达载体分别转入293细胞后经TNFα刺激，检测其荧光素酶活性。NAT11-Luc2CP，阳性对照载体。
图38、图36中使用的4个载体转入PSC27基质细胞后经过50μg/ml博来霉素处理，分别对比分析其荧光素酶信号强度。
图39、将图36中的4个报告性表达载体分别转入293细胞后经IL-1α刺激，检测其荧光素酶活性。NAT11-Luc2CP，阳性对照载体。
图40、将图36中的4个报告性表达载体分别转入PSC27细胞后经10μM SAT处理，检测其荧光素酶活性。NAT11-Luc2CP，阳性对照载体。
图41、ChIP-PCR分析经过NF-kB特异性抗体沉降下来的组分中AREG启动子上4个推测性NF-kB结合位点的PCR信号强度。IL-6-p1和IL-8-p1均为序列已知的NF-kB位点，此处用作阳性对照。
图42、NF-kB入核突变细胞亚系PSC27 IkBα经过三种化疗药物处理之后，AREG和IL-8的表达水平对比分析。
图43、将GL-AREG-P04转入PSC27细胞中后，再经博来霉素分别和NF-kB、c/EBP和AP-1抑制剂处理，获得的荧光素酶信号对比。BAY，NF-kB抑制剂。BA，c/EBP抑制剂。T5224和SR，均为AP-1抑制剂。
图44、将GL-AREG-P04转入PSC27细胞中后，再经SAT分别和NF-kB、c/EBP和AP-1抑制剂处理，获得的荧光素酶信号对比。BAY，NF-kB抑制剂。BA，c/EBP抑制剂。T5224和SR，均为AP-1抑制剂。
图45、在PSC27细胞经过博来霉素分别和NF-kB、c/EBP和AP-1抑制剂处理之后，AREG转录本表达情况。
图46、在PSC27细胞经过博来霉素分别和NF-kB、c/EBP和AP-1抑制剂处理之后， IL-6转录本表达情况。
图47、在PSC27细胞经过博来霉素分别和NF-kB、c/EBP和AP-1抑制剂处理之后，IL-8转录本表达情况。
图48、PSC27的AREG过表达亚系和敲除亚系中AREG的蛋白表达和对细胞本身影响的分析。Western blot检测AREG与IL-8表达水平变化。GAPDH，loading control。
图49、SA-β-Gal染色统计学分析PSC27亚系在DNA损伤情况下的衰老情况。右侧为代表性图片。
图50、分别经过PSC27的AREG过表达组和敲除组产生的CM处理之后前列腺癌细胞的增殖率分析。
图51、图50中各组前列腺癌细胞的迁移率分析。Hela细胞为阳性对照。
图52、图50中各组前列腺癌细胞的侵袭率分析。Hela细胞为阳性对照。
图53、图50中各组前列腺癌细胞在米托蒽醌作用下的耐药性分析。米托蒽醌药物浓度设定为各个癌细胞系的IC50值。
图54、前列腺癌细胞系DU145在AREG存在和/或化疗药物使用的情况下，caspase 3的完整形式及其切割形式的表达情况分析。
图55、前列腺癌细胞系PC3在米托蒽醌以及apoptosis抑制剂(QVD-OPH，ZVAD/FMK)或激活剂(PAC1，GA)作用下，细胞凋亡情况对比分析。
图56、前列腺癌细胞系PC3在紫杉醇以及apoptosis抑制剂(QVD-OPH，ZVAD/FMK)或激活剂(PAC1，GA)作用下，细胞凋亡情况对比分析。
图57、前列腺癌细胞系PC3和DU145在基质细胞衍生的AREG作用下，EGFR及其下游分子的活化情况分析。GAPDH，loading control。
图58、前列腺癌细胞系PC3和DU145在基质细胞PSC27及其AREG敲除亚系经过博来霉素处理之后衍生的CM作用下，EGFR及其下游分子的活化情况分析。GAPDH，loading control。
图59、基于AREG特异性抗体的IP和Western blot分析。IgG，对照抗体。E，EGFR单抗。A，AREG单抗。
图60、博来霉素处理之后的PSC27(PSC27-BLEO)产生的CM用于处理前列腺癌细胞，在AREG从PSC27细胞中敲除与否情况下癌细胞的增殖率分析。
图61、图60中各处理条件下前列腺癌细胞的迁移率分析。上方为统计学分析，下方为代表性细胞图片。
图62、图60中各处理条件下前列腺癌细胞的侵袭率分析。上方为统计学分析，下方为代表性细胞图片。
图63、图60中各处理条件下癌细胞对于米托蒽醌的耐药性分析。药物使用浓度为各细胞系的IC50值。
图64、同图63中的实验条件类似，但检测的是PSC27细胞经过博来霉素处理之后收集其CM用于培养前列腺癌细胞对于米托蒽醌的耐药性。癌细胞培养时使用了EGFR抑制剂AG-1478(2μM)，Cetuximab(50μg/ml)和AREG mAb(1μg/ml)；Cetuximab和AREG mAb(分别为50μg/ml,1μg/ml)，检测细胞的耐药性。
图65、本发明人制备和纯化的AREG单抗(0.2μg/ml)用于Western blot检测PSC27被博来霉素损伤之后细胞中AREG的表达情况。GPADH，loading control。
图66、图64中各种处理条件下PC3细胞系的生存曲线分析。米托蒽醌MIT药物浓度设计为接近临床给药条件下前列腺癌患者血浆中的MIT实际浓度。
图67、人源乳腺癌细胞MDA-MB-231和基质细胞HBF1203进行类似图66中的各种处理之后的细胞耐药曲线图。DOX，doxorubicin。
图68、免疫缺陷型小鼠皮下接种PC3/PSC27之后，第8周结束时小鼠肿瘤终端体积的测量值统计学对比分析。
图69、小鼠体内肿瘤生长、给药和检测流程示意图。在PC3/PSC27皮下注射之后的第三周，开始使用单药或多药处理。
图70、预临床条件下小鼠治疗模式示意图。上方为各处理方式，下方为各时间点分布。
图71、小鼠接种PC3/PSC27之后在连续8周的MIT预临床给药之后的肿瘤终端体积统计学分析。左侧，统计学对比。右侧，代表性肿瘤图片。
图72、化疗结束后小鼠肿瘤经过激光俘获显微切割，将基质细胞和癌细胞予以特异性分离之后的SASP代表性因子和细胞衰老标志性因子的表达分析。分别为IL-6，IL-8，WNT16B，SFRP2，ANGPTL4，MMP1/3/10和p16。
图73、组化分析小鼠化疗前后肿瘤中p16表达和SA-β-Gal染色情况。
图74、图73中化疗前后小鼠肿瘤组织中p16表达和SA-β-Gal染色的统计对比。
图75、组化分析分别经过placebo和米托蒽醌处理的小鼠肿瘤组织中AREG的蛋白水平表达情况。
图76、经过米托蒽醌和治疗性抗体Cetuximab或AREG mAb单药或多药治疗之后，小鼠肿瘤终端体积的统计学分析。
图77、基于PC3-luc/PSC27皮下接种之后的小鼠，基于BLI的体内荧光素酶表达检测分析。
图78、预临床给药7天之后小鼠肿瘤内部癌细胞的DNA损伤和凋亡情况对比分析。左侧，统计学对比。
图79、图78中几种代表性条件下组化染色图片(cleaved caspase 3)。
图80、ELISA检测几种治疗条件下小鼠血浆中的AREG蛋白水平变化情况。
图81、临床治疗前后前列腺癌患者肿瘤组织中PD-L1表达情况的组化分析。
图82、基于肿瘤组织中癌细胞的PD-L1表达水平进行的患者生存曲线分析。PD-L1表达低患者数量，23，蓝色。PD-L1表达高患者数量，25，黄色。
图83、患者病灶组织中基质细胞AREG表达水平同周边癌细胞PD-L1表达水平之间的关联系分析。
图84、经过全转录组分析之后发现基质细胞衍生的AREG处理过的前列腺癌细胞系PC3中PD-L1，PD-L2和PD-1的表达情况。上图，RNA-Seq数据。下图，定量RT-PCR数据。
图85、经过全转录组分析之后发现基质细胞衍生的AREG处理过的前列腺癌细胞系DU145中PD-L1，PD-L2和PD-1的表达情况。上图，RNA-Seq数据。下图，定量RT-PCR数据。
图86、Western blot分析PSC27高表达AREG亚系CM培养过的PC3和DU145细胞中PD-L1,PD-L2和PD-1的表达情况。GAPDH，loadig control。
图87、PSC27 AREG产生的CM用于培养PC3细胞，同时使用一组EGFR及其下游信号通路节点分子的抑制剂。Western blot分析PD-L1在这些条件下的表达变化。GAPDH，loading control。
图88、PSC27 AREG产生的CM用于培养PC3细胞及其PD-L1敲除性亚系之后，以Western blot分析癌细胞中PD-L1表达水平。GAPDH，loading control。
图89、经过PBMC共培养的PC3细胞，在PSC27 AREG产生的CM存在与否的情况下，存活率对比分析。
图90、经过PBMC共培养的PC3细胞及其PD-L1敲除亚系，在PSC27及其AREG敲除亚系被博来霉素处理之后产生的CM存在与否的情况下，存活率对比分析。
图91、经过PBMC共培养的DU145细胞，在PSC27 AREG产生的CM存在与否的情况下，存活率分析。
图92、经过PBMC共培养的DU145细胞及其PD-L1敲除亚系，在PSC27及其AREG敲除亚系被博来霉素处理之后产生的CM存在与否的情况下，存活率对比分析。
图93、实验小鼠经过包括Atezolizumab和Nivolumab等不同药物处理条件下，外周血中IFNγ经过ELISA检测后的对比分析。
图94、同图93类似，实验小鼠经过包括Atezolizumab和Nivolumab等不同药物处理条件下，外周血中TNFα经过ELISA检测后的对比分析。
图95、免疫重建型小鼠预临床治疗流程示意图。向Rag2 -/-IL2Rγ null背景的实验小鼠静脉注射人源PBMC，3天之后向同一批动物皮下接种PC3/PSC27细胞。在接种之后的第3周开始通过腹腔实施给药米托蒽醌，同时注射Atezolizumab或Nivolumab。随后每两周重复一次这种腹腔化疗/靶向治疗的程序，直至3个给药循环全部。在第8周末(第56天)实验结束，小鼠被处死并获取其肿瘤组织和一系列病理生理学指数。
图96、图95中的实验小鼠接种仅PC3细胞、经过8周疗程结束后的肿瘤终端体积统计学分析。
图97、免疫重建型小鼠接种PC3/PSC27细胞后、经过8周疗程结束后各种治疗条件下的肿瘤终端体积统计学分析。
图98、组化分析免疫重建型小鼠疗程结束后肿瘤组织中PD-L1的表达水平。
图99、免疫重建型小鼠接种VCaP/PSC27细胞后、经过8周疗程结束后各种治疗条件下的肿瘤终端体积统计学分析。
图100、免疫重建型小鼠接种乳腺癌细胞系MDA-MB-231/乳腺基质细胞系HBF1203之后8周治疗结束时的小鼠肿瘤终端体积统计学分析。
图101、前列腺癌荷瘤PC3小鼠在预临床结束时体重分析
图102、图101中的荷瘤小鼠外周血中肌氨酸酐水平对比分析。
图103、图101中的荷瘤小鼠外周血中尿素水平对比分析。
图104、图101中的荷瘤小鼠外周血中ALP(碱性磷酸酶)水平对比分析。
图105、图101中的荷瘤小鼠外周血中ALT(谷丙转氨酶)水平对比分析。
图106、化疗前后前列腺癌患者血浆中AREG经过ELISA分析之后的统计学对比分析。
图107、化疗前后前列腺癌患者血浆中IL-8经过ELISA分析之后的统计学对比分析。
图108、化疗前后前列腺癌患者血浆中AREG与IL-8含量之间经Pearson分析其相互关系。
图109、化疗前后肺癌患者血浆中AREG经过ELISA分析之后的统计学对比分析。
图110、化疗前后肺癌患者血浆中IL-8经过ELISA分析之后的统计学对比分析。
图111、化疗前后肺癌患者血浆中AREG与IL-8含量之间经Pearson分析其相互关系。
图112、前列腺癌患者外周血中AREG和IL-8的Western blot检测。化疗前4名、化疗后6名患者。Albumin，血浆loading control。
图113、化疗后阶段前列腺癌患者原发病灶组织和外周血中的AREG和IL-8的表达水平关联性分析。共20名患者。
图114、基于图113中的20名前列腺癌患者病灶组织中基质细胞的多个SASP因子表达分析。IL-2/3/5/12/17为SASP非相关性白细胞介素(或促炎症因子)，均为实验对照。
图115、化疗后阶段20名前列腺癌患者血浆中AREG水平同其无病生存期的关联。AREG低水平患者，10名，青色曲线。AREG高水平患者，棕色曲线。
图116、化疗后阶段20名肺癌患者血浆中AREG水平同其无病生存期的关联。AREG低水平患者，10名，紫色曲线。AREG高水平患者，黄色曲线。
图117、化疗后阶段20名肺癌患者病灶组织中基质细胞AREG表达水平同周边癌细 胞PD-L1表达水平的Pearson关联性分析。
图118、取自TCGA源数据库中的有关AREG在多种实体瘤患者中突变，扩增，缺失和多重变化的统计学对比分析。
具体实施方式
本发明人经过广泛而深入的研究，首次揭示了双向调节素(Amphiregulin，AREG)在SASP表型以及肿瘤微环境中发挥重要的生物学作用，其与化疗治疗后的预后密切相关。因此，AREG可作为SASP表型调控研究以及基于肿瘤微环境的抗肿瘤研究的靶点，作为肿瘤经化疗治疗后的预后评估以及分级的标志物，以及作为靶点开发抑制肿瘤的药物。
AREG
AREG是一种穿膜糖蛋白，因其C端有表皮生长因子结构域故又称EGF样蛋白，也是一种双功能生长因子，2型相关细胞因子。近年免疫研究发现其很可能是2型免疫反应所介导的抗性和耐药性形成过程中的一个重要分子。除了上皮细胞和间质细胞可以产生AREG蛋白，大量数据表明肥大细胞、嗜碱性细胞、2型天然淋巴细胞和一小部分组织原有的调节性CD4+T细胞等多种白细胞亚群，均可表达AREG。人AREG氨基酸序列如下：
Figure PCTCN2019076947-appb-000001
本发明人在前期工作中发现，AREG为癌症相关成纤维细胞(CAFs)释放的一种外泌蛋白，与癌细胞恶性生长、获得性耐药和远处转移等现象均有关系。CAFs被认为是源于体内产生的成纤维细胞，癌细胞可劫持并利用它们，来维持自己的生长。基因毒治疗造成的毒副作用可以激活微环境中的多种成分，而CAFs一旦进入DNA损伤性修复状态，就表现出典型的衰老相关分泌表型(SASP)，并对抗癌治疗的后续阶段造成不可低估的负面影响。主要表现为癌细胞因此获得显著的耐药性，并向癌症干细胞发展、形成多个微转移灶、向循环系统迁移、定植于异位器官，最终加速患者死亡。
在基质细胞因化疗药物或辐射处理而形成SASP的过程中出现包括AREG的大量活性分子，然而作为双向调节素的AREG释放至基质细胞胞外后通过旁泌方式对于临近癌细胞表型的影响、对于疾病的恶性进展的意义，至今并无专门报道。在抗癌治疗过程中，作为SASP广谱外泌因子之一的AREG，是否会对促进癌细胞获得性耐药，以及治疗过程中癌细胞是否出现跟基质细胞类似的药物诱导性AREG表达，也属未知。同时，基质来源的AREG对于治疗损伤性组织的免疫微环境能否造成一定影响，是否像肥大细胞、嗜碱性细胞和CD4+T细胞亚群那样发挥免疫调节功能，均是值得思考的重要问题。而本发 明中，展示了AREG在SASP表型以及肿瘤微环境中发挥重要的生物学作用，其与化疗治疗后的预后密切相关。
药物组合及其应用
本发明人发现，特异性抑制AREG的抗体与化疗药物联合用药，能够极其显著地增强肿瘤抑制效果。特异性抑制AREG的抗体与化疗药物的协同作用藉由以下作用方式：特异性抑制AREG的抗体通过结合肿瘤微环境(尤其是其中的基质细胞)衍生的AREG，抑制其活性，逆转肿瘤对于化疗药物的耐药性，从而使得化疗药物的用药效果更为理想。
基于本发明人的新发现，本发明提供了用于抑制肿瘤或降低肿瘤耐药性的药物组合或组合物，所述药物组合或组合物中包括：特异性抑制AREG的抗体，以及化疗药物。
如本文所用，所述的“肿瘤”可以是原位肿瘤或转移肿瘤，其包括存在耐药性的难治性肿瘤，特别是对基因毒化疗药物具有耐药性的肿瘤。较佳地，所述的肿瘤是实体瘤。例如，所述的肿瘤包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌、肝癌、胰腺癌、膀胱癌等。
作为本发明的优选方式，提供一种对于抑制肿瘤或降低肿瘤耐药性特别有效的抗AREG单抗，所述抗AREG单抗对于AREG具有很高的特异性，不结合于AREG以外的其它蛋白。并且，当用于与化疗药物联合以抑制肿瘤时，其效果极为优异。
本发明的抗AREG单抗利用杂交瘤技术来制备，该杂交瘤细胞株在中国典型培养物保藏中心的保藏号是CCTCC NO:C2018214。在获得了所述杂交瘤的情况下，可按照常规的动物细胞培养方法，体外培养扩增所述的杂交瘤细胞，从而使之分泌所述的抗AREG单抗。作为一种实施方式，所述的抗AREG单抗可以由下列制备方法制备：(1)提供佐剂预处理的小鼠；(2)在小鼠腹腔内接种所述的杂交瘤细胞并分泌单克隆抗体；(3)抽取腹水，分离获得所述的单克隆抗体。从腹水中分离单克隆抗体经过进一步纯化，从而可获得高纯度的抗体。本发明的单克隆抗体还可以利用重组方法制备或利用多肽合成仪合成。本领域人员均了解，在得到了所述的单克隆抗体的杂交瘤细胞系或通过测序等手段得知所述的单克隆抗体后，本领域人员可以方便地获得所述的抗体。
特异性抑制AREG的抗体与化疗药物可以被制成药物组合物的方式给药，或者两者可以分离地存在于一个药盒中。其中所述的特异性抑制AREG的抗体以及化疗药物均为有效量的。在用作药物时，通常所述的特异性抑制AREG的抗体还与药学上可接受的载体相混合。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的如本文所用。
本发明的具体实施例中，给出了一些针对动物如鼠的给药方案。从动物如鼠的给药剂量换算为适用于人类的给药剂量是本领域技术人员易于作出的，例如可根据 Meeh-Rubner公式来进行计算：Meeh-Rubner公式：A＝k×(W 2/3)/10,000。
式中A为体表面积，以m 2计算；W为体重，以g计算；K为常数，随动物种类而不同，一般而言，小鼠和大鼠9.1，豚鼠9.8，兔10.1，猫9.9，狗11.2，猴11.8，人10.6。应理解，根据药物以及临床情形的不同，根据有经验的药师的评估，给药剂量的换算是可以变化的。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明提供了一种用于抑制肿瘤或降低肿瘤耐药性的药盒，所述的药盒中包括特异性抑制AREG的抗体和化疗药物(如米托蒽醌，阿霉素，博莱霉素，沙铂，紫杉醇)。更优选地，所述药盒中还包括：使用说明书，以指导临床医师以正确合理的方式用药。
为了方便给药，所述的特异性抑制AREG的抗体与化疗药物(如米托蒽醌，阿霉素，博莱霉素，沙铂，紫杉醇)的组合物或彼此独立存在的抗体或化疗药物(如米托蒽醌，阿霉素，博莱霉素，沙铂，紫杉醇)可以被制成单元剂型的形式，置于试剂盒中。“单元剂型”是指为了服用方便，将药物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
肿瘤化疗后阶段预后评估的应用
基于本发明人的上述新发现，可以将AREG作为肿瘤化疗后阶段的预后评估的标志物：(i)进行肿瘤化疗后阶段的疾病分型、鉴别诊断、和/或无病生存率分析；(ii)评估相关人群的肿瘤治疗药物、药物疗效、预后，以及选择合适的治疗方法。比如，可分离出肿瘤微环境中、特别是基质细胞中AREG基因表达异常的人群，从而可进行更有针对性地治疗。
可以通过判断待评估样本(基质细胞)中AREG的表达情况或活性情况，来预测提供该待评估样本的受试者的肿瘤预后情况，选择合适的药物实施治疗。通常，可以规定一个AREG的阈值，当AREG的表达情况高于所规定的阈值时，考虑采用抑制AREG的方案进行治疗。所述的阈值对于本领域技术人员而言是易于确定的，例如可以通过将正常人组织微环境中的AREG的表达情况与肿瘤患者微环境中的AREG的表达情况进行比较后，获得AREG表达异常的阈值。
因此，本发明提供了AREG基因或蛋白的用途，用于制备肿瘤预后评估的试剂或试剂盒。可采用各种本领域已知的技术来检测AREG基因的存在与否以及表达情况，这些技术均包含在本发明中。例如可用已有的技术如Southern印迹法、Western印迹法、DNA序列分析、PCR等，这些方法可结合使用。本发明还提供了用于在分析物中检测AREG基因的存在与否以及表达情况的试剂。优选的，当进行基因水平的检测时，可以采用特异 性扩增AREG的引物；或特异性识别AREG的探针来确定AREG基因的存在与否；当进行蛋白水平的检测时，可以采用特异性结合AREG编码的蛋白的抗体或配体来确定AREG蛋白的表达情况。
所述的试剂盒中还可包括用于提取DNA、PCR、杂交、显色等所需的各种试剂，包括但不限于：抽提液、扩增液、杂交液、酶、对照液、显色液、洗液等。此外，所述的试剂盒中还可包括使用说明书和/或核酸序列分析软件等。
筛选药物的应用
在得知了AREG在基质细胞中的表达受到NF-κB调控后，可以基于该特征来筛选抑制NF-κB对于AREG的转录调控(NF-κB促进AREG转录)的物质。可从所述的物质中找到对于抑制肿瘤或降低肿瘤耐药性真正有用的药物。
因此，本发明提供一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述的方法包括：用候选物质处理表达NF-κB以及AREG的体系，且该AREG编码基因上游存在NF-κB结合位点；以及检测所述体系中NF-κB对于AREG的调控作用；若所述候选物质在统计学上抑制NF-κB对于AREG的转录调控，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。在本发明的优选方式中，在进行筛选时，为了更易于观察到NF-κB对于AREG的转录调控以及AREG的表达或活性的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的表达体系。
在得知了肿瘤微环境(特别是基质细胞)中的AREG对肿瘤细胞的功能性影响主要由EGFR及其下游的信号通路所控制后，可以基于该特征来筛选抑制AREG对EGFR介导的信号通路的激活的物质。可从所述的物质中找到对于抑制肿瘤或降低肿瘤耐药性真正有用的药物。
因此，本发明提供一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述的方法包括：用候选物质处理一表达体系，该体系表达EGFR介导的信号通路以及AREG；以及检测所述体系中AREG对于EGFR介导的信号通路的激活作用；若所述候选物质在统计学上抑制该激活作用，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。在本发明的优选方式中，在进行筛选时，为了更易于观察到AREG对于EGFR介导的信号通路的激活作用以及AREG的表达或活性的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的表达体系。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于抑制肿瘤或降低肿瘤耐药性真正有用的物质。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料和方法
1.细胞培养
(1)细胞系维持
正常人源前列腺原代基质细胞系PSC27和人源乳腺原代基质细胞系HBF1203(均获自美国弗雷德哈青森癌症研究中心)于PSCC完全培养液中增殖和传代。前列腺良性上皮细胞系BPH1，前列腺癌上皮细胞系M12，DU145，PC3，LNCaP和VCaP，乳腺癌上皮细胞系MCF-7，MDA-MB-231，MDA-MB-468，T47D和BT474(购自ATCC)均在5％FBS的RPMI-1640完全培养液中、于37℃、5％CO 2条件的培养箱中培养。
(2)细胞冻存与复苏
a细胞冻存
以0.25％胰蛋白酶收集对数生长期细胞，1000rpm离心2min，弃去上清，重新悬浮细胞于新鲜配置的冻存液中。分装细胞于已标示的无菌冻存管中。然后经梯度降温(4℃10min，-20℃30min，-80℃16-18h)，最后转入液氮中长期储存。
b细胞复苏
取出液氮中冻存的细胞，立即放入37℃水浴，使其快速融化。直接加入2ml细胞培养液，使细胞均匀悬浮。待细胞贴壁后，更换新的培养液。
(3)体外实验处理
为造成细胞损伤，PSC27细胞生长至80％(简称PSC27-Pre)时培养液中加入100nM紫杉萜(docetaxel，DTX)，100nM紫杉醇(paclitaxel，PTX)，200nM长春新碱(vincristine，VCR)，50μg/ml博来霉素(bleomycin，BLEO)，1μM米托蒽醌(mitoxantrone，MIT)，10uM沙铂(satraplatin，SAT)或10Gy  137Cs电离辐射(γ-radiation at 743rad/min，RAD)。药物处理6小时后，细胞被PBS简单洗过3次，留置于培养液中7～10天，然后进行后续实验。
2.质粒制备和慢病毒转染
全长人源AREG克隆在慢病毒表达载体pLenti-CMV/To-Puro-DEST2(Invitrogen)酶切位点BamHI和XbaI之间。包装系293FT被用于细胞转染和慢病毒制造。
用于敲除AREG的small hairpin RNAs(shRNAs)sense strand序列分别为CCGGTCCTGGCTATATTGTCGATGATCTCGAGATCATCGACAATATAGCCAGGTTTTTG(SEQ ID NO:2)和CCGGTCACTGCCAAGTCATAGCCATACTCGAGTATG GCTA TGACTTG GCAGTGTTTTTG(SEQ ID NO:3)。
3.免疫荧光和组化分析
小鼠单克隆抗体anti-phospho-Histone H2A.X(Ser139)(clone JBW301，Millipore)和兔多抗anti-AREG(Cat#16036-1-AP，Proteintech)，及二级抗体Alexa
Figure PCTCN2019076947-appb-000002
488(or 594)-conjugated F(ab’) 2按顺序加入到覆有固定细胞的载玻片上。细胞核用2μg/ml of 4’，6-diamidino-2-phenylindole(DAPI)进行复染。从3个观察视野中选取最具代表性的一张图像进行数据分析和结果展示。FV1000激光扫描共聚焦显微镜(Olympus)用于获取细胞共聚焦荧光图像。
临床前列腺癌和肺癌患者组织IHC染色所用AREG抗体同上，购自Proteintech。具体步骤如下：常规脱蜡，用0.6％H 2O 2甲醇在37℃孵育30min，然后用0.01M pH6.0的柠檬酸缓冲液修复20min，室温冷却30min。用正常羊血清封闭20min，用AREG一抗(1:200)在37℃孵育1h，移至4℃冰箱过夜。第二天用TBS洗三次，以二抗(HRP偶联的羊抗兔)在37℃孵育45min，再用TBS洗3次，最后用DAB显色。
4.基质-上皮共培养和体外实验
用DMEM+0.5％FBS的培养液培养PSC27细胞3天，然后以1倍PBS清洗满丰度的细胞群。简单离心后收集上清作为条件性培养基存放–80℃或直接使用。前列腺上皮细胞在这种条件性培养基中连续培养3天的时间里开展体外实验。对于化疗抗性，上皮细胞系在低血清DMEM(0.5％FBS)(简称“DMEM”)中，或条件性培养基中培养，同时米托蒽醌(MIT)用于处理细胞1至3天，浓度接近各个细胞系的IC 50数值，随后在亮场显微镜下进行观察。
5.全基因组范围表达芯片分析(Agilent expression microarray)
对正常人源前列腺原代基质细胞系PSC27进行全基因组范围表达芯片(4x44k)分析的程序和方法参见Sun，Y.等，Nat.Med.18:1359-1368。
6.定量PCR(RT-PCR)测定基因表达
以Trizol试剂抽提生长期细胞总RNA，进行逆转录反应。将逆转录反应产物cDNA稀释50倍作为模板，进行RT-PCR。
反应完成后，经软件分析查看每个基因的扩增情况，导出相应的域值循环数，采用2-ΔΔCt方法，计算每个基因的相对表达量。对融解曲线(melting curve)的波峰和波形进行分析以确定得到的扩增产物是否为特异性单一目的片段。
7.NF-κB调控分析
含有编码IκBα蛋白序列上两个IKK磷酸化突变位点S32A和S34A的反病毒载体pBabe-Puro-IκBα-Mut(super repressor)，被用于转染慢病毒包装细胞系PHOENIX。慢病毒随后用于侵染PSC27基质细胞系，而1μg/ml嘌呤霉素(puromycin)则被用于筛选阳性克隆。作为另外一种方法，5μM的小分子抑制剂Bay 11-7082(购自Selleck)被用于NF-κB活性控制。基质细胞随后被暴露于几种不同形式的细胞毒，及时记录由此产生的表型，分析相 关基因表达情况。经过这种方式处理过的细胞，产生出来的条件性培养液被收集起来，用于针对上皮细胞的各种检测。
8.AREG启动子分析和染色体免疫沉降(ChIP)检测
针对人AREG基因(Gene ID 374，Genbank accession NM_001657.3)使用软件CONSITE进行分析，以发现潜在的核心NF-κB结合位点。ChIP-PCR实验中设计4对PCR引物来扩增AREG启动子内部NF-κB结合区附近core sequence：primer set#1(-3579～-3370)：正向5’-CCAGTCTGGAGTGCGGTGGC-3’(SEQ ID NO:4)，反向5’-GGTGGATCATCTCAGGTCAG-3’(SEQ ID NO:5)；primer set#2(-1320～-1120)：正向5’-ATTTTGAGTCGGAGTCTTGC-3’(SEQ ID NO:6)，反向5’-TGGCCA AGATGGCAAAACCC-3’(SEQ ID NO:7)；primer set#3(-1221～-1000)：正向5’-GCC TCAGCCCACCCGAGTAG-3’(SEQ ID NO:8)，反向5’-GTAACACAGCCCTTTTA AGA-3’(SEQ ID NO:9)；primer set#4(-20～+196)：正向5’-CATGGGCTGCGGCC CCCTCC-3’(SEQ ID NO:10)，反向5’-ACACGAGCTGCCGCCAAAAC-3’(SEQ ID NO:11)。同时，设计另外2对引物用于分别扩增IL-6(正向5’-AAATGCCCAACAGAGGTCA-3’(SEQ ID NO:12)，反向5’-CACGGCTCTAGGC TCTGAAT-3’(SEQ ID NO:13))和IL8(正向5’-ACAGTTGAAAACTATAG GAGCTACATT-3’(SEQ ID NO:14)，反向5’-TCGCT TCTGGGCAAGTACA-3’(SEQ ID NO:15))的启动子序列(均为已知的阳性对照)。针对早期代数的PSC27细胞(如p8)和经过博来霉素(50ug/ml)处理的PSC27细胞进行ChIP分析。体外固定的染色体，使用小鼠单抗anti-p65antibody(F-6，Santa Cruz)进行沉降处理，提取DNA用以扩增。载有多个NF-κB结合位点突变的报告表达载体系通过site-directed mutagenesis(Stratagene)方法设计和产生。此外，涵盖多个NF-κB结合位点和经过优化的IL-2最小启动子作为NF-κB激活转基因系统(NAT system)的报告载体NAT11-Luc2CP-IRES-nEGFP(获自日本Hokkaido大学)，在实验中用作阳性对照。每一报告载体均由pRL-TK vector(Addgene)共转染以进行信号标准化处理。
9.临床前列腺癌、乳腺癌和结直肠癌患者组织样本获取和分析
化疗药物方案是根据去势抵抗型前列腺癌患者(临床试验注册号NCT03258320)和非小细胞肺癌(临床试验注册号NCT02889666)的病理学特征指定的。临床分期为原发癌在I subtype A(IA)(T1a，N0，M0)以上但没有明显远端转移病灶的患者被招募至临床队列中。同时，年龄40-75岁经临床确诊为PCa，或者年龄小于75岁经临床确诊为NSCLC的患者方被招募。所有患者均被提供知情同意书并签字确认。有关肿瘤大小，组织类型，肿瘤渗透，淋巴结转移和病理TNM疾病阶段的数据从病理记录系统获取。肿瘤加工为FFPE样本并处理成组织学切片以供评估。OCT冰冻切片经LCM选择性分离，用于基因表达分析。特别地，化疗前后的腺体相关基质细胞经LCM分离。免疫活性评分(IRS)根据每一组织样本的组化染色呈色深浅分别归类于0-1(阴性)，1-2(若)，2-3(中)，3-4(强)四类(Fedchenko and  Reifenrath，2014)。PCa、BCa和CRC样本的诊断由彼此独立的病理学医生进行判断和评分。随机对照试验(RCT)方案和所有实验程序均经上海交通大学医学院IRB批准和授权，并根据权威指导原则逐步开展。
10.小鼠移植瘤试验和预临床化疗程序
年龄6周左右的免疫缺陷型小鼠ICR/SCID mice(体重约25g)用于本发明相关动物实验。基质细胞PSC27和上皮细胞以1:4的比例混合，而每一移植体包含1.25×10 6细胞，用于组织重构。移植瘤通过皮下移植方式植入小鼠体内，移植手术结束之后8周末动物被执行安乐死。肿瘤体积按照如下公式计算：V＝(π/6)x((l+w)/2) 3(V，体积；l，长度；w，宽度)。类似地，乳腺癌移植瘤分别由MDA-MB-231(三阴性、高恶性乳腺癌细胞系)和HBF1203(乳腺成纤维细胞系)，通过组织重构形成。
在预临床化疗试验中，经过皮下移植的小鼠被供给标准实验食谱，2周结束之后实施化疗药物米托蒽醌(0.2mg/kg剂量)或阿霉素(1.0mg/kg剂量)腹腔给药。同时，FDA批准的治疗性抗体Cetuximab(10.0mg/kg的剂量，200μl/剂)或经过严格纯化之后的AREG mAb(10.0mg/kg的剂量，200μl/剂)进行单药或双药静脉注射。时间点为第3、5、7周的第一天，整个疗程共进行3次循环给药，每个循环为2周。疗程结束后，小鼠肾脏被收集用于肿瘤测量和组织学分析。每只小鼠累积性共接受米托蒽醌0.6mg/kg体重，或阿霉素3.0mg/kg体重。化疗试验进行到第8周末结束，小鼠处死之后立即解剖，其移植瘤被收集并用于病理系统分析。给药7天之后的部分小鼠用于组化评估其组织水平的caspase 3活性。
化疗进行过程中，每周称取一次小鼠体重；化疗整体结束之后，再次称量小鼠体重并将其血液以心脏穿刺法收集起来置于冰浴45分钟。外周血随即在4C予以8000rpm离心10分钟之后，约50μl被VetTest pipette tip吸取，用于IDEXX VetTest 8008化学分析器检测。肝功测量项目包括肌氨酸酐，尿素，碱性磷酸酶和谷丙转氨酶。
为构建免疫完整性实验小鼠，4周龄Rag2 -/-IL2Rγ null小鼠(Jackson Laboratory)被尾静脉注射以7×10 6新鲜人源PBMCs。3天之后，皮下单独接种1.2×10 6PC3细胞或先与0.3×10 6PSC27细胞混合后再行接种。自第3周开始，米托蒽醌与治疗性抗体atezolizumab或nivolumab(每种均与isotype-matched IgG，如IgG 1 for atezolizumab，IgG4 for nivolumab平行注射以获得对照数据)单药或双药联合使用。8周疗程结束之后，小鼠处死并收集其肿瘤用于病理分析，包括组织内癌细胞PD-L1表达变化情况评估。此外，小鼠血液被用于ELISA分析以检测循环水平的IFNγ和TNFα含量。
11.生物统计学方法
本发明申请中所有涉及细胞增殖率，迁移性，侵袭性和存活性等的体外实验和小鼠移植瘤及化疗处理的体内试验均重复3次以上，数据以均值±标准误的形式呈现。统计学分析建立在原始数据的基础上，通过two-tailed Student’s t test，one-or two-way ANOVA， Pearson’s correlation coefficients test，Kruskal-Wallis，log-rank test，Wilcoxon-Mann-Whitney test or Fisher’s exact test进行计算，而P<0.05的结果认作具有显著性差异。
实施例1、DNA损伤导致人源基质细胞中AREG的高度表达
本发明人在研究中，注意到人源前列腺基质细胞系PSC27(主要是成纤维细胞组成)在DNA损伤背景下，即基因毒化疗药物或电离辐射处理之后，会生成大量SASP因子，并且AREG出现在上调表达幅度最高的一组蛋白中(图1)。为了验证这一现象并扩大研究范围，本发明人随后使用了一套DNA损伤性药物(基因毒药物)，包括博来霉素(BLEO)、米托蒽醌(MIT)和沙铂(SAT)，同时以一组非DNA损伤性药物如紫杉醇(PTX)，多西他赛(DTX)和长春新碱(VCR)平行处理基质细胞。结果发现，基因毒药物处理之后的细胞呈现出明显增多的DNA损伤焦点(γH2AX)，增强的半乳糖苷酶(SA-β-Gal)活性和下降的DNA合成(BrdU嵌入率)(图2，3，4)，暗示着DNA损伤之后典型的细胞周期阻滞和细胞衰老的发生。相比之下，非基因毒药物则没有造成以上各种典型特征的出现。随后转录本和蛋白水平的分析显示，AREG表达均有显著上升，且大量释放到胞外环境(图5)。有趣的是，AREG在基质细胞中的表达模式和时间规律，同另外几种SASP标志性因子如MMP1，WNT16B，SFRP2，MMP12和IL-8非常相似，即在细胞发生DNA损伤之后的时间里出现表达水平的逐渐上升，直到细胞在7-10天之后达到一个平台期并长期保持一种分泌状态(图6，7)。
经过分析几种前列腺来源的细胞系在DNA损伤药物处理后AREG的表达情况，本发明人发现基质细胞比上皮细胞具有更为显著的可诱导性(图8，9)，暗示着基质细胞存在着一种驱动AREG在基因毒背景下高表达的分子机制。这一基质细胞-上皮细胞间显著差异的分子特征，随后被一组人乳腺来源的细胞系所证实，包括基质细胞系HBF1203和几个恶性程度并不相同的上皮癌细胞株，表明AREG的表达具有组织和器官类型非依赖性特征(图10-14)。
实施例2、AREG在肿瘤微环境中的表达同化疗之后患者生存呈显著负相关
体外实验的同时本发明人继续思考，体内的肿瘤微环境中是否也会出现AREG表达。本发明人研究了一个因被诊断出罹患前列腺癌而经过临床化疗的患者队列，惊奇地发现这些患者普遍在治疗之后，而非在治疗之前，出现肿瘤组织中AREG显著上调(图15)。同体外实验数据相一致，AREG在组织中的表达，集中体现于腺体周边的基质细胞而非腺体内的上皮细胞(图15)。
相比于化疗前，AREG在化疗后肿瘤中的高度表达这一特征，被一种预先建立起来的可以根据特定蛋白的组化染色强度定量评估其在组织内的表达水平的病理检测系统进一步确定(图16，17)。经过激光俘获显微切割(LCM)这一微观技术，本发明人再次发现组 织中的AREG更加倾向于在基质细胞群而非上皮细胞群中出现诱导性表达(图18)。为证实AREG的药物诱导性，本发明人选取了一组在化疗前后的组织样本都被获取并保存下来的患者，发现在他们当中的任何一人，均出现AREG在化疗之后的基质细胞而非上皮细胞中高度表达(图19，20)。本发明人进一步注意到，AREG在被药物破坏的微环境中的表达，同基质细胞SASP特征性因子IL-8和WNT16B基本保持平行关系(图21，22)。在受损的肿瘤微环境中，AREG同IL-8及WNT16B之间的关系，被化疗之后患者体内这些因子的病理评估所证实(图23，24)。更为重要的，根据患者体内肿瘤基质中AREG进行的病理分级所获得的大数据，表明基质组织中AREG的表达水平同疗后阶段患者的无病生存期呈现显著的负性相关(图25)。
作为支持性证据，AREG的这一系列病理特征在随后一组基于肺癌患者的临床研究中，得以重复和确认(图26-35)。本发明人的研究数据暗示，AREG在肿瘤基质组织中的表达，可以作为一个反映SASP发生发展的独立预测指标，用于对疗后时期疾病复发和临床死亡率相关的风险系数进行患者分层；同时，AREG在基质中的大量生成和连续释放具有重要的病理意义。
实施例3、AREG在基质细胞中的表达受到NF-kB和C/EBP等转录因子所调控
在分子层面，接下来要确定AREG在受损基质细胞中的表达基础。作为哺乳动物细胞中调控SASP表达的关键转录机器，NF-κB复合物在癌基因诱导或治疗性损伤导致的细胞衰老过程中均发挥重要作用。本发明人首先考虑NF-κB是否介导AREG在DNA损伤之后的基质细胞中表达。分析发现在AREG基因上游约4000bp的范围内存在几个NF-κB结合位点(图36)，随后基于报告载体的荧光检测结果证实了这几个位点的存在。
相比于对照组293T或PSC27细胞，TNF-α刺激或BLEO处理过的实验组呈现出显著增强的AREG启动子转录活性(图37，38)。类似的结果在用白介素IL-1α或SAT处理细胞之后也相应出现(图39，40)。接下来的ChIP-PCR结果证实了四个位点(AREG上游-3510、-1223、-1131及+79位)均为DNA损伤之后真正的NF-κB结合位点(图41)。基于NF-κB功能性缺陷细胞株(PSC27 IκBα)的一系列实验表明，失去NF-κB的细胞核转位活性可以导致AREG转录水平大幅减低(图42)。
转录因子被报道参与到SASP因子的表达过程中，如C/EBP和AP-1，然而它们在AREG表达过程中的作用尚不明确。为此，本发明人使用了betulinic acid(BA)，即C/EBP家族抑制剂，和T-5224，即AP-1选择性抑制剂，分别处理经过预先转导AREG启动子报告载体的PSC27细胞。DNA损伤性处理之后报告载体的荧光信号明显上升，而Bay11-7082，一种NF-κB抑制剂，则可以基本废除这些信号的产生(图43)。相比之下，BA或T-5224的处理则没有显著降低报告载体的荧光信号(图43)。进一步实验结果表明，NF-κB或C/EBP的活性抑制，而非AP-1阻断，可以造成AREG转录水平显著下降(图44)。 AREG的这一表达特征，同SASP的两个特征性因子IL-6和IL-8相接近，其共同特征为基因转录过程主要被NF-κB和C/EBP这两个转录复合物所介导(图45-47)。
总体而言，AREG在基因毒背景下的基质细胞中表达主要受NF-κB和C/EBP的调控。同时，可以基于NF-κB和C/EBP对于AREG的调控作用，来筛选通过影响两者的相互作用而抑制肿瘤的药物，一些能够抑制或阻止两者相互作用的药物，潜在的可能有利于肿瘤的治疗。
实施例4、AREG对癌细胞的功能性影响主要由EGFR及其下游的信号通路所控制
相比于以往有关AREG在前列腺癌等疾病中的研究主要集中于该因子的自分泌作用方式，本发明人随后关注基质细胞衍生的AREG是否通过旁分泌途径对受体细胞发挥影响。首先，在基质细胞中敲除AREG之后对于IL-8等可溶因子的表达以及细胞在DNA损伤之后的衰老并没有造成显著改变(图48，49)。相比之下，过表达AREG的PSC27细胞(PSC27 AREG)产生的条件性培养基(CM)，却可以对一系列前列腺上皮癌细胞如PC3，DU145，LNCaP和M12，造成显著影响，包括上调的增殖率，迁移率和侵袭性(图50-52)。然而，这一系列恶性特征却在AREG被从基质细胞中敲除之后，发生显著逆转(图50-52)。更重要的是，AREG显著提高了前列腺癌细胞对于临床化疗药物米托蒽醌(MIT)的耐药性(图53)。进一步研究发现，MIT是通过促进癌细胞中caspase3的自我切割来诱导细胞凋亡的，但这一过程可被AREG所显著削弱，而从基质细胞中敲除AREG之后又可以恢复MIT的这一效果(图54)。为证实这一发现，本发明人随后使用了QVD-OPH和ZVAD-FMK，两种广谱caspase 3抑制剂，以及PAC1和gambogic acid(GA)，两种caspase激活剂，分别在MIT处理细胞之前用于细胞培养。本发明人发现，细胞凋亡程度在QVD-OPH或ZVAD-FMK存在条件下显著降低(图55)。而当PAC1或GA被分别加入细胞培养液时，凋亡指数却大幅上升，基本抵消了AREG的抗凋亡作用(图55)。这一发现随后被另一化疗药物紫杉醇(DOC)所证实，尽管后者主要通过干扰细胞微管解聚发挥诱导细胞凋亡的作用(图56)。因此，AREG主要是通过抑制caspase所介导的细胞凋亡，造成癌细胞对于各种化疗药物的抵抗性。
因AREG跟EGF同属典型的EGFR配体并分享一定程度的序列同源性，本发明人首先确定了AREG作为一种EGF类似物对于癌细胞信号通路的影响。在使用AREG高表达基质细胞(PSC27 AREG)产生的CM培养癌细胞之后，本发明人发现后者出现多个蛋白分子的翻译后修饰变化，主要包括EGFR(Y845)，Akt(S473)和mTOR(S2448)等位点的磷酸化，暗示AREG所介导的PI3K/Akt/mTOR信号通路的激活(图57)。进而，Mek(S217/S221)、Erk(T202/Y204)和Stat3(S727)位点的磷酸化，表明这些细胞中MAPK通络的激活。为确定EGFR是否在AREG影响癌细胞活化的过程中起到主要介导作用，本发明人使用了AG-1478，一种EGFR特异性的RTK抑制剂，处理受体癌细胞。有趣的是，在AG-1478 存在的条件下，AREG所诱导的EGFR及其下游多个分子的磷酸化均被废除，包括Akt/mTOR和Mek/Erk/Stat3信号轴。因而，AREG引起的癌细胞表型改变主要通过EGFR介导的信号通路活化来实现。作为支持性证据，AREG被从基质细胞中敲除之后，癌细胞的这一系列信号通路活化基本消失(图58)，进一步证实AREG是通过EGFR引起多个下游信号通路的激活。为确定AREG和EGFR之间的相互作用，本发明人使用AREG特异性抗体进行了IP实验。结果表明，AREG同EGFR两个分子间存在物理性直接作用，而IP信号可以在PSC27 AREG而非PSC27 Vector CM处理过的癌细胞样本中被轻易发现(图59)。
作为接下来的一个关键问题，本发明人要研究AREG作为SASP广谱外泌因子集中的一个组分，在SASP驱动癌细胞恶性进展过程中是否发挥功能性的核心作用。为此目的，本发明人构建了PSC27-shRNA AREG稳定性亚系并在DNA损伤处理之后收集其CM。本发明人注意到，在AREG被敲除之后，PSC27原本在DNA损伤条件下出现的细胞衰老既没有延迟也未加速，SA-β-Gal阳性率不变(图49)。在PSC27-BLEO所产生的CM培养癌细胞时，后者的增殖率，迁移率和侵袭性均出现显著上调，而AREG从基质细胞中的敲除则可以大幅降低这一系列恶性表型的增幅(图60-62)。
本发明人已发现，被化疗药物损伤的肿瘤微环境能够赋予癌细胞显著的耐药性，本发明人随后检测了这种活性的变化情况。在AREG被敲除之后，PSC27-BLEO所赋予的前列腺癌细胞对于米托蒽醌的获得性耐药性，出现大幅下降(图63)。同样，在PSC27-BLEO CM培养条件下，被EGFR抑制剂AG-1478所作用过的癌细胞的耐药性也显著降低(图64)。为证实AREG在SASP广谱因子中所起的关键作用，本发明人使用了Cetuximab，一种FDA批准临床使用的特异性抑制EGFR的单抗。结果发现Cetuximab能够显著下调基质细胞赋予癌细胞的耐药性，效果接近AG-1478(图64)。既然Cetuximab和AG-1478的靶向分子均为EGFR，本发明人推理直接靶向控制微环境中的AREG是否可以获得更加显著的效果。结果表明，本发明人通过杂交瘤筛选的方法获得一种小鼠单抗AREG mAb(保藏号是CCTCC NO:C2018214)(图65)，可以在癌细胞耐药性控制实验中取得十分理想的效果，癌细胞清除效率高于AG-1478或Cetuximab(图64)。进而，本发明人同时使用了AREG mAb和Cetuximab处理培养条件下的癌细胞，发现其结果跟AREG mAb单独使用时相同(图64)，说明将Cetuximab跟AREG mAb协同使用并不能获得比AREG mAb单独使用更高的抗癌效率。尽管PSC27-BLEO CM可以使得PC3在一系列的体外实验中表现为对于MIT(剂量范围0.1～1.0μM)的获得性耐药，AREG mAb介导的AREG蛋白清除使得这种耐药性显著弱化，效果跟AREG mAb和Cetuximab联用时接近(图66)。在随后的乳腺癌细胞体外实验中，本发明人观察到了本质上类似的现象(图67)。因而，通过靶向癌细胞表面主要受体之一的EGFR，和直接控制基质细胞来源的AREG，均可达到降低癌细胞获得性耐药的实际目的。
实施例5、体内靶向AREG可以阻滞肿瘤生长并提高肿瘤对于化疗药物的敏感性
临床抗癌过程中微环境SASP的广谱表达可以加速许多恶性事件的发生，包括肿瘤进展，局部炎症和治疗性抵抗的出现。然而，这种向恶性进展的趋势是否可以通过特异性控制微环境广谱SASP中的核心因子得以显著缓和，一直是近年来医学界一个有趣的话题，也是一个技术性难题。为了尽可能模拟临床条件，本发明人向免疫缺陷型小鼠皮下部位接种了PSC27和PC3混合细胞群，8周之后本发明人停止实验并进行分析。结果发现，基质细胞表达外源因子AREG的情况下，肿瘤终端体积大幅上升，但在AREG从基质细胞敲除之后却显著降低(图68)。此外，本发明人设计了一套用于模拟临床抗癌治疗方案的预临床治疗策略，即对荷瘤小鼠进行一个为期8周的化疗方案，后者主要由根据一系列预实验数据确定的3次单药或双药治疗所组成(图69-70)。相比于对照组，接种有PSC27 AREG的小鼠肿瘤体积明显上升，但在化疗药物米托蒽醌腹腔给药造成的筛选压下形成的肿瘤体积显著缩小，证明化疗本身可以有效阻滞肿瘤发展(图71)。然而，与对照组(PSC27 Vector小鼠)的肿瘤相比，PSC27 AREG小鼠残存肿瘤体积仍然显著上升，暗示微环境在整个化疗过程中的病理作用。
通过激光俘获显微切割技术将基质细胞和癌细胞单独分离出来之后，本发明人发现微环境中的这两个细胞系在表达SASP典型外泌因子方面呈现较大差异性。包括IL-6，IL-8，WNT16B，SFRP2，ANGPTL4，MMP1/3/10在内的一组SASP经典因子，在基质细胞中广泛上调，尽管癌细胞也出现IL-6和MMP10表达增强；同时，p16这一细胞衰老象征性CDK抑制因子，则在上皮细胞和基质细胞中均呈显著上升(图72)。针对肿瘤组织的组化染色则表明，p16蛋白水平和SA-β-Gal活性均有所上升(图73-74)，暗示体内条件下细胞衰老和SASP发生发展这一趋势。
本发明人通过组化染色，确认了预临床治疗条件下肿瘤组织中出现AREG的明显表达(图75)。为了验证体外实验结果，本发明人继而使用了Cetuximab或AREG mAb与米托蒽醌联合使用。在仅接种有PC3细胞的小鼠这一组，尽管米托蒽醌单独使用可以显著降低肿瘤体积，Cetuximab治疗性抗体的同时给药并未进一步缩小肿块(图76)，暗示PC3肿瘤基本在EGF/EGFR信号轴非依赖性的微环境中进展。当基质细胞PSC27跟癌细胞PC3共同接种至小鼠体内时，肿瘤终端体积则大幅上升，再次印证基质细胞显著的促瘤潜力。当PC3/PSC27小鼠经过米托蒽醌治疗之后，肿瘤体积降低了34.3％；经过Cetuximab或AREG mAb跟米托蒽醌的联合治疗，肿瘤体积则进一步下降了37.8％和46.8％(图76)；Cetuximab作为一种治疗性单抗其功效已被了解，而AREG mAb比Cetuximab显著更理想的效果尤为出乎意料。
同时，本发明人使用了表达荧光素酶的PC3细胞系(PC3-luc)，发现小鼠体内条件下检测到的生物荧光信号在各组动物之间的相对强度，跟之前检测到的肿瘤终端体积基本对应，且排除了体内原位病灶中的癌细胞发生异位器官转移这一可能(图77)。这一系列数据 表明，相比于传统的化疗本身，AREG单抗介导的靶向治疗结合基因毒化疗，可以引起更加显著的肿瘤消退；特异性靶向AREG的单抗，甚至可以达到显著高于Cetuximab这一RTK靶向制剂的抑瘤效率，尽管后者在临床中用于治疗EGFR +癌症患者并已经在国际范围内多年的实际应用中取得良好的效果。
为进一步解析AREG在体内条件下造成的癌细胞耐药的机制，本发明人解剖了给药治疗7天之后的小鼠并获得其肿瘤，用于病理分析。尽管Cetuximab本身并不引起DNA损伤反应(DDR)，PC3肿瘤中的细胞却表现为明显的凋亡，可能跟Cetuximab与EGFR之间的高度亲和力有关，后者可以降低癌细胞生存率(图78)。然而，跟米托蒽醌结合之后的Cetuximab，并没有进一步提高癌细胞的凋亡率，暗示跟米托蒽醌协同时Cetuximab的细胞毒有所局限。重要的在于，相比于Cetuximab，AREG mAb产生了更加显著的治疗效果(图78)。组化染色结果表明，在对小鼠注射AREG mAb的条件下，caspase 3这一分子呈现更为明显的自我切割(图79)。此外，ELISA检测结果表明，米托蒽醌治疗过程中可以造成小鼠血浆中AREG蛋白水平大幅上升，但在AREG mAb同时给药时却可以显著控制这一变化(图80)。
实施例6、基质细胞衍生的AREG上调受体癌细胞PD-L1表达并形成免疫抑制性微环境
肿瘤免疫治疗能够在大量患者中产生持久反应，而过继细胞转移和检查点阻断疗法均可产生显著的抗原特异性免疫反应。同时，已有研究结果表明临床中的化疗效果部分依赖于免疫系统的细胞毒性作用，表明完整和持续的免疫活性对于抗癌治疗的重要性。
本发明人首先检测了前列腺癌临床患者肿瘤组织中的PD-L1等免疫活性相关分子的表达变化。初步的组化数据显示，化疗后的肿瘤中呈现PD-L1高度上调，并且PD-L1的表达程度同疗后阶段患者的无病生存率存在显著负相关(图81-82)。统计学分析结果表明，肿瘤基质中AREG的表达同周边癌细胞PD-L1上调之间存在密切关联(图83)。
同时，本发明人通过全基因组测序发现经过基质细胞PSC27来源的AREG处理，前列腺癌细胞系PC3和DU145均出现PD-L1的显著上调，尽管PD-L2和PD-1表达水平保持不变(图84-85)。随后的Western blot验证了PD-L1/PD-L1/PD-1表达的这一趋势(图86)。为进一步确定PD-L1在癌细胞中的表达机制，本发明人分别使用了一系列信号分子或通路的小分子或抗体抑制剂，包括Erlotinib(EGFR)，AG1478(EGFR)，LY294002(PI3K)，MK2206(Akt)，Rapamycin(mTOR)，PD0325901(Mek)，Bay 117082(NF-kB)，Ruxolitinib(Jak1/2)和SB203580(p38MAPK)。结果表明，除了p38MAPK抑制剂之外，多种分子或通路的阻断性药物均能不同程度地抑制基质细胞来源的AREG所造成的PD-L1上调表达，暗示EGFR及其下游信号通路在介导受体癌细胞PD-L1表达过程中的调控作用(图87)。
为确定PD-L1在介导形成免疫抑制性微环境中的作用，本发明人将其从前列腺癌细 胞中特异性敲除(图88)。随后本发明人从临床患者外周血中分离并纯化获得PBMC(外周血单个核细胞)，随后将其直接用于体外培养条件下对前列腺癌细胞的识别性清除。结果表明，相比于对照组基质细胞(PSC27 Vector)，AREG高表达组(PSC27 AREG)可以显著提高癌细胞的存活率，而无论从受体癌细胞中敲除PD-L1还是从基质细胞中清除AREG均能抵消PBMC共培养条件下AREG赋予癌细胞的生存潜力(图89-92)。
考虑到临床条件下实体瘤微环境的复杂性，本发明人继而选择使用了PD-L1和PD-1抑制剂分别用于静脉注射Rag2 -/-IL2Rγ null实验小鼠，并检测其外周血中T细胞的活性。基于动物血液的ELISA数据表明，Atezolizumab(FDA批准的第一个PD-L1抑制剂，用于膀胱癌新靶向治疗)和Nivolumab(又名Opdivo，FDA批准的用于治疗晚期转移性鳞状非小细胞肺癌(NSCLC)患者，适用于以铂类为基础化疗或化疗后疾病出现恶化的患者)处理之后，小鼠血液中的两种细胞因子IFNγ和TNFα在蛋白水平上均出现显著上调，且呈现时间依赖性的持续增长(图93-94)。这表明无论Atezolizumab还是Nivolumab治疗，均引起小鼠体内条件下细胞毒T细胞亚群的功能性活化。
在此基础上本发明人将研究扩大化，即向Rag2 -/-IL2Rγ null背景的实验小鼠静脉注射人源PBMC，3天之后向同一批动物皮下接种PC3/PSC27细胞。在接种之后的第3周开始通过腹腔向动物给药米托蒽醌，同时注射Atezolizumab或Nivolumab。随后每两周重复一次这种腹腔化疗/靶向治疗的程序，直至3个给药循环全部实施完成。在第8周末(第56天)实验结束，小鼠被处死并获取其肿瘤组织和一系列病理生理学指数(图95)。预临床实验结果表明，尽管米托蒽醌单药治疗可以使得PC3肿瘤体积显著下降(图96)，PSC27的同时皮下接种可以大幅加快肿瘤生长速度，再次证明、支持微环境中基质细胞的促癌特性(图97)。即便经过米托蒽醌一个疗程的处理，PSC27/PC3组动物的残存肿瘤体积仍然显著高于相比于PC3组小鼠，暗示微环境赋予肿瘤显著耐药性。有趣的是，无论Atezolizumab还是Nivolumab单药治疗，效果均不及米托蒽醌作为化疗的后果，而AREG mAb的给药对于肿瘤体积没有任何影响(图97)。相比之下，米托蒽醌与Atezolizumab或Nivolumab联用可以进一步降低肿瘤大小，然而其效果均不如米托蒽醌与AREG mAb联用所造成的肿瘤缩减幅度(图97)。此后经过进一步的病理分析，本发明人发现米托蒽醌单药给药之后的小鼠肿瘤中出现明显的癌细胞PD-L1表达上调，但经过Atezolizumab或AREG mAb的联用，组织中的PD-L1信号却基本消失(图98)。这些数据表明，化疗虽然可以在一定程度上引起肿瘤退行，却可以激活体内微环境中以PD-L1为介导的免疫检查点阻滞，病灶因此没有彻底消减；而传统意义上的化疗同PD-L1疗法相互结合，具有将肿瘤体积控制在最小范围的独特治疗潜力。
为将这一发现进一步拓展到生理完整性的体内条件下，本发明人使用了另一前列腺癌细胞系VCaP，后者表达雄激素受体AR并且生长具有雄激素依赖性。与PSC27细胞同时接种至实验小鼠皮下后，VCaP肿瘤终端体积的整套数据基本重复了之前发现的 PC3/PSC27肿瘤的一系列结果(图99)。经过在小鼠皮下接种MDA-MB-231癌细胞和HBF1203乳腺来源基质细胞，本发明人发现MDA-MB-231/HBF1203肿瘤呈现出跟以上前列腺癌小鼠数据十分类似的趋势(图100)。因此，靶向AREG造成的耐药拮抗性数据表明，控制微环境中的AREG对于肿瘤治疗的效果是器官非依赖性的、在多种实体瘤中均可以适用的一种手段。
为确定这一治疗策略的安全性和可行性，本发明人接下来对实验小鼠进行了病理生理学评估。结果表明，无论单药还是多药治疗，小鼠的体重和其它多种指标，包括血浆水平的肌氨酸酐、尿素、ALP和ALT均保持不变(图101-105)。
这一系列预临床研究结果，表明靶向AREG的抗体治疗和传统化疗相结合，不只是可以造成更显著的抑制肿瘤效果，同时具有较高的用药安全性，不会引起较为严重的体内毒性。
实施例7、AREG是一个可以用于判断临床条件下患者体内SASP发生发展的新型生物标志物
随后确定AREG是否可以在临床化疗之后的癌症患者外周血中，通过使用常规技术检测出来。为此，本发明人收集了两组前列腺癌患者的血浆样本，包括一组经历过化疗处理的，和另一组未经任何治疗的患者。经过基于ELISA的蛋白检测，发现化疗之后时期患者血液中的AREG水平显著高于未经化疗的患者(图106)。有趣的是，这一趋势跟SASP的一个典型因子IL-8十分相似(图107)。为确定这两个因子之间的关系，本发明人选取了化疗前后均有血液样本提供的一组患者，发现这些患者血浆中AREG同IL-8之间存在显著关联(r＝0.9329，P<0.0001)(图108)。在另外的一组肺癌临床患者样本中，本发明人注意到也存在这一现象(图109-111)。
为了获得对于这些关键分子在临床中的进一步认识，本发明人开展了基于患者临床标本的纵向分析。在一组前列腺癌患者的原发病灶组织和外周血样本中，本发明人惊奇地注意到AREG和IL-8这两个SASP相关因子可以在Western blot上清晰地显现出来，并且二者仅在化疗后患者的血浆样本中出现信号(图112)。此外，无论在实体组织还是血浆水平，这两个因子彼此之间均存在明显的相关性(图113)。为明晰AREG和IL-8作为检测体内SASP状态标志物的可靠性，本发明人使用激光俘获显微切割技术分离了前列腺癌患者病灶组织中的基质细胞，并对其进行了转录本水平的分析。结果表明，包括MMP1，CXCL3，IL-1β，WNT16B，IL-6和GM-CSF在内的多个SASP相关因子均同AREG和IL-8在每一患者组织中呈现密切相关(图114)。相比之下，IL-2/3/5/12/17等同SASP无关的因子则不具有这一特征。本发明人的研究数据表明，AREG可以作为反映临床患者体内条件下SASP发生发展的一个具有较高可信度的标志性因子，用于评估化疗后阶段癌症患者SASP的发展状态及其动态特征。此外，在血浆中的AREG蛋白水平同组织中癌细胞PD-L1 表达水平之间密切关联并同临床患者疗后阶段的生存期之间存在显著的负相关(图115-117)，暗示AREG作为患者肿瘤微环境经过不可修复的损伤之后释放出来的一个外泌因子，可以用作分析、判断传统化疗之后患者生存的一个独立指标。尽管已有包括TCGA在内的多项研究报道AREG在癌症患者体内出现突变，扩增，丢失和多重变化等特征(图118)，将AREG作为一种反映肿瘤微环境病理活性、技术常规的、非侵袭性的液体活检标志物，可以为将来的临床医学提供一项精确、便捷和高效的新型诊断和监控技术指标。
生物材料保藏
本发明的杂交瘤细胞株SP2/0-02-AREG-SUN保藏在中国典型培养物保藏中心(CCTCC，中国武汉)，保藏号为CCTCC NO:C2018214；保藏日2018年10月10日。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种用于抑制肿瘤或降低肿瘤耐药性的药物组合物，其特征在于，所述药物组合物中包括：特异性抑制双向调节素的抗体，以及化疗药物。
  2. 如权利要求1所述的药物组合物，其特征在于，所述的化疗药物是基因毒药物；较佳地，所述的化疗药物包括：米托蒽醌，长春新碱，阿霉素，博莱霉素，沙铂，顺铂，卡铂，道诺霉素，诺加霉素，阿柔比星，表柔比星，多柔比星，阿糖胞苷，卡培他滨，吉西他滨，5-氟尿嘧啶。
  3. 如权利要求2所述的药物组合物，其特征在于，其包括：
    特异性抑制双向调节素的抗体和米托蒽醌，且两者质量比为1:0.005～1:2.0；
    特异性抑制双向调节素的抗体和阿霉素，且两者质量比为1:0.02～1:1.5；
    特异性抑制双向调节素的抗体和博来霉素，且两者质量比为1:0.02～1.5；或
    特异性抑制双向调节素的抗体和选自沙铂、顺铂、卡铂的一种或多种，且抗体与后者的质量比为1:0.02～1.5。
  4. 如权利要求1～3任一所述的药物组合物，其特征在于，所述的特异性抑制双向调节素的抗体是由杂交瘤细胞系CCTCC NO:C2018214分泌。
  5. 权利要求1～4任一所述的药物组合物的用途，用于制备抑制肿瘤或降低肿瘤耐药性的药盒。
  6. 如权利要求5所述的用途，其特征在于，所述的药物组合物中，特异性抑制双向调节素的抗体通过抑制肿瘤微环境中基质细胞表达的双向调节素，从而降低肿瘤耐药性。
  7. 如权利要求5所述的用途，其特征在于，所述的肿瘤包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌、肝癌、胰腺癌、膀胱癌。
  8. 特异性抑制双向调节素的抗体，其是由杂交瘤细胞系CCTCC NO:C2018214分泌。
  9. 特异性抑制双向调节素的抗体在制备抗体药物中的应用，所述抗体药物与化疗药物联合应用，抑制肿瘤或消除肿瘤耐药性；或用于消除肿瘤细胞对化疗药物的耐药性。
  10. 一种杂交瘤细胞株，其在中国典型培养物保藏中心的保藏号是CCTCC NO: C2018214。
  11. 一种用于抑制肿瘤或降低肿瘤耐药性的药盒，其特征在于，所述药盒包括：特异性抑制双向调节素的抗体，或产生该抗体的细胞株。
  12. 如权利要求11所述的药盒，其特征在于，所述的药盒中还包括：化疗药物；较佳地所述化疗药物是基因毒药物；较佳地，所述的化疗药物包括：米托蒽醌，长春新碱，阿霉素，博莱霉素，沙铂，顺铂，卡铂，道诺霉素，诺加霉素，阿柔比星，表柔比星，多柔比星，阿糖胞苷，卡培他滨，吉西他滨，5-氟尿嘧啶。
  13. 双向调节素在制备用于肿瘤化疗预后评估的诊断试剂中的用途，其中，所述的双向调节素为肿瘤微环境中基质细胞产生的双向调节素。
  14. 特异性识别双向调节素的试剂在制备用于肿瘤化疗预后评估或病理分级的诊断试剂中的用途，其中，所述的双向调节素为肿瘤微环境中基质细胞产生的双向调节素。
  15. 如权利要求13或14所述的用途，其特征在于，所述的肿瘤包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌、肝癌、胰腺癌、膀胱癌。
  16. 一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述方法包括：
    (1)用候选物质处理一表达体系，该体系表达NF-κB以及双向调节素，且该双向调节素编码基因上游存在NF-κB结合位点；和
    (2)检测所述体系中NF-κB对于双向调节素的调控作用；若所述候选物质在统计学上抑制NF-κB对于双向调节素的转录调控，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
  17. 如权利要求16所述的方法，其特征在于，步骤(1)包括：在测试组中，将候选物质加入到所述表达体系中；和/或
    步骤(2)包括：检测测试组的体系中NF-κB对于双向调节素的转录调控，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达体系；
    如果测试组中NF-κB对于双向调节素的转录调控显著被抑制，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
  18. 如权利要求16所述的方法，其特征在于，所述的NF-κB结合位点为双向调节素 编码基因上游-3510、-1223、-1131、+79位。
  19. 一种筛选抑制肿瘤或降低肿瘤耐药性的潜在物质的方法，所述方法包括：
    (1)用候选物质处理一表达体系，该体系表达EGFR介导的信号通路以及双向调节素；和
    (2)检测所述体系中双向调节素对于EGFR介导的信号通路的激活作用；若所述候选物质在统计学上抑制该激活作用，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
  20. 如权利要求19所述的方法，其特征在于，步骤(1)包括：在测试组中，将候选物质加入到所述表达体系中；和/或
    步骤(2)包括：检测测试组的体系中双向调节素对于EGFR介导的信号通路的激活作用，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达体系；
    如果测试组中双向调节素对于EGFR介导的信号通路的激活作用显著被抑制，则表明该候选物质是抑制肿瘤或降低肿瘤耐药性的潜在物质。
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