Anti-human ADRB3 monoclonal antibody and application thereof in disease diagnosis and treatment

Abstract

The present invention discloses an application of a β3 adrenergic receptor (ADRB3) as a marker for detecting a plurality of diseases, and an application of anti-human ADRB3 monoclonal antibody in diagnosing a disease and preparing a drug for treating the disease. The present invention finds through research that the ADRB3 is a key receptor in nerve-endocrine-immunoregulatory network, and an ADRB3-mediated signaling pathway regulates proliferation and differentiation of neutrophils, lymphocytes and tumor cells. Under normal circumstances, the ADRB3 maintains the non-specific immunocompetence and specific immunocompetence of an organism, and eliminates pathogenic microorganisms and aged organism tissues to play a role in protecting the organism and anti-aging. Under pathological conditions, excessive activation of the signaling pathway will cause systemic chronic inflammation, and destroy immune homeostasis. Therefore, the ADRB3 can be used as a diagnostic marker and a therapeutic target for a plurality of diseases. Anti-human ADRB3 antibody can specifically bond with the ADRB3, regulate the activity of the ADRB3, has the functions of resisting cancer, inflammation, poisoning, shock, allergy, viral infection, autoimmune disease, disease caused by regenerative dysfunction, autoimmune disease, cachexia, cardiovascular and cerebrovascular disease, neurodegenerative disease and aging, regulating autophagy, treating aging disease, etc., and has important medical value and research and application prospects.

Claims

1. A method for treating a malignant tumor, comprising administering an anti-human beta-3 adrenergic receptor (ADRB3) monoclonal antibody to a patient with the malignant tumor, wherein the anti-human ADRB3 monoclonal antibody regulates the functions of an ADRB3 affected by the malignant tumor, wherein the anti-human ADRB3 monoclonal antibody is a monoclonal antibody produced from hybridoma cell line 5D9 deposited with the China Center for Type Culture Collection (CCTCC) under accession number C2016203 on Dec. 12, 2016, wherein the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, lung cancer, and melanoma, wherein the step of administering the anti-human ADRB3 monoclonal antibody to the patient comprises administering the anti-human ADRB3 monoclonal antibody at an mice-equivalent dose range of 1-10 mg/kg.

2. The method according to claim 1, wherein the anti-human ADRB3 monoclonal antibody is an anticancer drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: high expression of an ADRB3 (B3AR) protein in the nucleolus of human breast cancer cell MCF7 at G0 phase (Ki-67 negative); an ADRB3 is also present in cytoblast of the cell (Ki-67 positive) in proliferation phase, but is rarely located in the nucleolus.

(2) FIG. 2: high expression of an ADRB3 protein in the nucleolus of MCF7 cells at G0 phase (Nucleolin negative).

(3) FIG. 3: high expression of an ADRB3 protein in the nucleolus (Fibrillarin positive) of the MCF7 cell.

(4) FIG. 4: nucleolus with an ADRB3 protein accumulated in the MCF7 cell (H3K9AC.sup.−) at G0 phase.

(5) FIG. 5: there are a lot of ADRB3 proteins in the Brdu negative (non-S phase) MCF7 cytoblast.

(6) FIG. 6: A. an ADRB3 is accumulated in the MCF7 cytoblast and nucleolus; and B. an ADRB3 is present in a plurality of nucleoli of fusion cells.

(7) FIG. 7: high expression of an ADRB3 in the nucleolus of the human lung cancer cell A549 at G0 phase (Ki-67 negative); low expression level in cytoblast of the cell (Ki-67 positive) in proliferation phase.

(8) FIG. 8: high expression of an ADRB3 in the nucleolus of the human pancreatic cancer cell CFPAC1 at G0 phase (Nucleolin negative); low expression level in the nucleolus of the cell (Nucleolin positive) in proliferation phase.

(9) FIG. 9: an ADRB3 is highly expressed in the nucleoli (Fibrillarin positive) of the human melanoma cell A375.

(10) FIG. 10: an ADRB3 is localized in the mitochondrial outer membrane of the human pancreatic cancer cell PANC1.

(11) FIG. 11: an ADRB3 is localized in the centrosome at both spindle poles of the MCF7 cell.

(12) FIG. 12: an ADRB3 is localized at both spindle poles and in the spindle equator of the A549 cell.

(13) FIG. 13: A. an ADRB3 is localized in the microtubule of the human hepatic cancer cell HepG2; and B. an ADRB3 is localized in the microtubule, cytoblast and mitochondria of the human pancreatic cancer cell PANC1.

(14) FIG. 14: an ADRB3 is localized at both spindle poles of the human glioma cell A172; and B. an ADRB3 is located in the centrosome position of the polyploid cell MCF7.

(15) FIG. 15: an ADRB3 is localized on the lysosome of the MCF7 cell.

(16) FIG. 16: A. an ADRB3 increases the tyrosine phosphorylation of the protein on the cell membrane at G0 phase, and may promote the tyrosine phosphorylation of membrane proteins, such as HER2/EGFR/drug pump, to activate the membrane proteins; and B. a lot of ADRB3 are accumulated in the MCF7 cytoblast at the end of G2 phase. Under the circumstance, DNA replication is completed, chromosome has been formed, and nucleolus and karyotheca have disappeared.

(17) FIG. 17: A. an ADRB3 is located in the centrosome of MCF7 cells at the end of G2 phase to promote formation of the microtubule center; and B. an ADRB3 is located in the vacuolar outer membrane of the MCF7 cell to promote vacuole formation.

(18) FIG. 18: schematic diagram of ADRB3 expression in breast cancer tissues and paracancerous tissues; high expression of an ADRB3 in breast cancer tissues of pathological grade III; and each column is a sample of a patient, the upper two samples are cancer tissues, and the lower two samples are paracancerous tissues.

(19) FIG. 19: Kaplan-Meier survival curve of 142 breast cancer patients, where the ordinate is the cumulative survival rate, and the abscissa is the survival time (month); and the survival rate of patients with a negative ADRB3 in cancer tissue is significantly higher than that of patients with a positive ADRB3 in cancer tissue, P=0.025.

(20) FIG. 20: schematic diagram of ADRB3 expression in lung cancer tissues and paracancerous tissues; the first and second from the right are paracancerous tissues and cancer tissues of a patient with squamous cell lung carcinoma (g15); and the third and fourth from the right are paracancerous tissues and cancer tissues of a patient with lung adenocarcinoma (f15).

(21) FIG. 21: Kaplan-Meier survival curve of 112 lung cancer patients, where the ordinate is the cumulative survival rate, and the abscissa is the survival time (month); and the survival rate of patients with a weakly positive ADRB3 in cancer tissue is significantly higher than that of patients with a strongly positive ADRB3 in cancer tissue, P=0.038, where 1 means weakly positive, and 2 means strongly positive.

(22) FIG. 22: schematic diagram of ADRB3 expression in pancreatic cancer tissues and paracancerous tissues.

(23) FIG. 23: Kaplan-Meier survival curve of 129 pancreatic cancer patients, where the ordinate is the cumulative survival rate, and the abscissa is the survival time (month); and the survival rate of patients (0) with a negative ADRB3 in cancer tissue is significantly higher than that of patients (1) with a positive ADRB3 in cancer tissue, P=0.019.

(24) FIG. 24: schematic diagram of ADRB3 expression in 90 colon cancer tissues and paracancerous tissues, where the left figure shows a cancer tissue.

(25) FIG. 25: schematic diagram of ADRB3 expression in 90 colon cancer tissues and paracancerous tissues, where the ADRB3 expression in cancer tissues is significantly higher than that in normal colon tissues (P=0.0001).

(26) FIG. 26: A. the ADRB3 expression level in cancer tissues of liver cancer patients of more than 50 years old is significantly higher than that in paracancerous tissues, P=0.04; B. Kaplan-Meier survival curve of 162 hepatocellular cancer patients, where the ordinate is the cumulative survival rate, and the abscissa is the survival time (month); and the survival rate of patients (0) with a negative ADRB3 in cancer tissue is significantly higher than that of patients (1) with a positive ADRB3 in cancer tissue, P=0.038.

(27) FIG. 27: A. bone marrow smears of normal subjects are stained with ADRB3 and MPO, and an ADRB3 is highly expressed in neutrophils (MPO positive); B. an ADRB3 shows a conical structure and is embedded in cytoblast of neutrophils; and C. the conical ADRB3 complex begins in cell membrane, runs through cytoplasm, and extends to cytoblast to form a channel.

(28) FIG. 28: bone marrow smears of normal subjects injected with granocyte (recombinant human granulocyte colony-stimulating factor) are stained with ADRB3 and MPO; and an ADRB3 is inducibly expressed in lymphocytes.

(29) FIG. 29: bone marrow smears of a given patient with acute B lymphocytic leukemia before treatment and after complete response (CR) by treatment are stained with ADRB3 and Ki-67, where the upper figure is before treatment. The expression of ADRB3 in granulocytes and leukaemia cells after complete response (CR) by treatment is reduced, compared with that before treatment.

(30) FIG. 30: bone marrow smears of a patient with acute B lymphocytic leukemia suffering a relapse after treatment are stained with ADRB3 and MPO; and an ADRB3 is highly expressed in granulocytes and leukemia cells, suggesting that the ADRB3 induces carcinogenesis of B-lymphocytes; B. the ADRB3 aggregates in a site where granulocytes and lymphocytes contact with each other, which contributes to the stability of intercellular adhesion molecules, and promotes formation of inhibitory immune synapses; and C. the ADRB3 in cytoplasm and cytoblast of lymphocytes forms a conical structure.

(31) FIG. 31: bone marrow smears of patients with acute B lymphoblastic leukemia suffering a relapse after treatment are stained with ADRB3 and Ki-67. ADRB3 and Ki-67 are highly expressed in granulocytes, which are MDSC, and aggravate the disease progression.

(32) FIG. 32: bone marrow smears of patients with acute monocytic leukemia (M5) after CR by treatment are stained with ADRB3 and MPO.

(33) FIG. 33: bone marrow smears of patients with acute myelocytic leukemia after CR by treatment are stained with ADRB3 and Ki-67; and ADRB3 is highly expressed in granulocytes in proliferation phase (Ki-67 positive).

(34) FIG. 34: A. different ADRB3 polypeptides used to immunize mice; and B. detect the antibody specificity with Western blot.

(35) FIG. 35: nucleic acid and protein sequence of a light chain antibody.

(36) FIG. 36: nucleic acid and protein sequence of a heavy chain antibody.

(37) FIG. 37: the ADRB3 antibody can kill MCF7 cells in vitro and in vivo; A. the ADRB3 monoclonal antibody reduces MCF7 breast cancer cell activity; B. the ADRB3 antibody inhibits the proliferation of the breast cancer cell MCF7 transplanted into nude mice, the ordinate is the transplanted tumor volume (mm.sup.3), the abscissa is the number of days after inoculating cells, and B3 Ab is the antibody group; C. the ADRB3 antibody increases the spleen index of the nude mice transplanted with the tumor MCF7; and D. the ADRB3 antibody reduces the expressions of cyclin D1, phosphorylated Rb, CDK3 and IL-6 in MCF7 tumor transplanted into nude mice, and reduces the CDK3 activity.

(38) FIG. 38: after the ADRB3 antibodies produced by different cell strains of bone marrow hybridoma are treated, the activity of the pancreatic cancer cell SW1990 is reduced; the antibodies are at a concentration of 250 ng/ml; and the abscissa is the hybridoma cell strain number.

(39) FIG. 39: after the ADRB3 antibodies produced by different cell strains of bone marrow hybridoma are treated, the activity of CFPAC1 cells is reduced; the antibodies are at a concentration of 250 ng/ml; and the abscissa is the hybridoma cell strain number.

(40) FIG. 40: the ADRB3 antibody reduces the volume of tumor transplanted with CFPAC1 cells in nude mice.

(41) FIG. 41: growth curve of the tumor transplanted with human pancreatic cancer cell CFPAC1 inoculated into nude mice, Ab is the antibody treated group, and the ADRB3 monoclonal antibody inhibits the proliferation of the tumor transplanted with CFPAC1.

(42) FIG. 42: histogram of the tumor weight in the nude mice transplanted with the pancreatic cancer cell CFPAC1; the ADRB3 monoclonal antibody reduces the weight of the tumor transplanted with CFPAC1.

(43) FIG. 43: histogram of the body weight of the nude mice transplanted with CFPAC1; the ADRB3 antibody doesn't affect the body weight of the nude mice transplanted with CFPAC1.

(44) FIG. 44: tumor transplanted with the pancreatic cancer cell ASPC1 in nude mice; the ADRB3 antibody reduces the volume of the tumor transplanted with ASPC1.

(45) FIG. 45: growth curve of the tumor transplanted with the human pancreatic cancer cell ASPC1 inoculated into nude mice, Ab is the ADRB3 antibody treated group, and the ADRB3 monoclonal antibody inhibits the proliferation of the tumor transplanted with ASPC1.

(46) FIG. 46: histogram of the tumor weight in the nude mice transplanted with the pancreatic cancer cell ASPC1; the ADRB3 monoclonal antibody reduces the weight of the tumor transplanted with ASPC1.

(47) FIG. 47: histogram of the body weight of the nude mice transplanted with ASPC1; the ADRB3 monoclonal antibody doesn't affect the body weight of the nude mice transplanted with ASPC1.

(48) FIG. 48: the ADRB3 monoclonal antibody treats the nude mice transplanted with SW1990; A. growth curve of the tumor transplanted with the human pancreatic cancer cell SW1990 inoculated in nude mice, and the ADRB3 monoclonal antibody inhibits proliferation of the tumor transplanted with SW1990; B. ADRB3 monoclonal antibody increases the apoptosis rate of SW1990; and C. the ADRB3 antibody reduces granulocytes and NLR of the SW1990 tumor-bearing mice, compared with the control group. *P<0.05.

(49) FIG. 49: growth curve of the tumor transplanted with human melanoma cell A375 inoculated into nude mice; the ADRB3 monoclonal antibody inhibits the proliferation of the tumor transplanted with A375; *p=0.032, #p=0.005; and Ab is the ADRB3 antibody treated group.

(50) FIG. 50: the ADRB3 monoclonal antibody treats the nude mice transplanted with A375; A. flow cytometry for detection of MSDC in the peripheral blood of the nude mice transplanted with A375; B. the ADRB3 antibody reduces the number of MSDC in the peripheral blood of the nude mice transplanted with A375; C. ELISA of the serum in tumor-bearing mice; and the ADRB3 antibody increases IL-10, reduces IL-6, VEGF and MPO concentrations, and compared with the control group, *p<0.05.

(51) FIG. 51: A. growth curve of the tumor transplanted with the human lung cancer cell A549 inoculated into nude mice; the ADRB3 monoclonal antibody inhibits the proliferation of the tumor transplanted with A549, and compared with the control group, *p<0.05; and B. the ADRB3 antibody inhibits liver metastasis of the 4T1 breast cancer cell in mice.

(52) FIG. 52: the ADRB3 monoclonal antibody dose-dependently decreases the activity of the human lung cancer cell H1299.

(53) FIG. 53: the ADRB3 monoclonal antibody dose-dependently decreases the activity of the human hepatic cancer cell HepG2.

(54) FIG. 54: the ADRB3 monoclonal antibody dose-dependently decreases the activity of the human colon cancer cell HCT116.

(55) FIG. 55: the ADRB3 monoclonal antibody dose-dependently decreases the activity of the human promyelocytic leukemia cell HL-60.

(56) FIG. 56: the ADRB3 monoclonal antibody dose-dependently decreases the activity of the human glioma cell U251.

(57) FIG. 57: T-lymphocytes modified by ADRB3 specific chimeric antigen receptor can kill cell SW1990 (A1 in the left figure is SW1990) and cell H1299 (C3 in the right figure).

(58) FIG. 58: the total counts of leukocytes, neutrophils and monocytes in the peripheral blood of the ADRB3 knockout mice are reduced; WBC: leukocytes, ne #: neutrophils, ly #: lymphocytes, and mo #: monocytes.

(59) FIG. 59: in the ADRB3 knockout mice, the percentage of lymphocytes is increased, that of neutrophils is decreased, and NLR is decreased; ly %: percentage of lymphocytes; ne %: percentage of neutrophils, and mo %: percentage of monocytes.

(60) FIG. 60: the muscle amount of the ADRB3 knockout mice is increased.

(61) FIG. 61: both the bone density and bone mineral content of the ADRB3 knockout mice are increased.

(62) FIG. 62: A. detection of serum cytokine in the ADRB3 knockout mice, Relative Fluorescence Units; compared with the control group, *P<0.05; B. ELISA of serum in ADRB3.sup.−/− mice; IL-10 in serum of ADRB3.sup.−/− mice is increased, but IL-6, VEGF and MPO are decreased.

(63) FIG. 63: flow cytometry for detection of CD4.sup.+T cells and Treg cells in the spleen of the ADRB3 knockout mice.

(64) FIG. 64: immunohistochemical analysis on the myocardium Cyclin D1 of the ADRB3 knockout mice. FVB mice are normal control.

(65) FIG. 65: cardiac B ultrasound examination of the ADRB3 knockout mice; and after knocking out the ADRB3, the heart function is good. FVB mice are normal control.

(66) FIG. 66: the mouse breast cancer cell 4T1 cannot grow subcutaneously in the ADRB3 knockout mice. FVB mice are normal control.

(67) FIG. 67: the ADRB3 antibody decreases the expressions of CDK4, nucleolin, HER2 and p-Rb (Ser780), but increases the expression of p16.

(68) FIG. 68: the ADRB3 antibody decreases the expressions of mTOR, p-mTOR (Ser2481), HK2 and P62; and Rapa is rapamycin, and CQ is chloroquine.

(69) FIG. 69: the 5D1 antibody decreases the expressions of ADRB3, p-Rb (Ser780) and GAPDH; 4G7 increases the expressions of ADRB3 and p-Rb (Ser780); either of the two antibodies can decrease the expressions of GAPDH and CDK4; and SR (SR59230A) is an ADRB3 inhibitor.

(70) FIG. 70: the 5D1 antibody decreases the expressions of PD-L1, CDK3 and GAPDH.

(71) FIG. 71: after inhibiting p62, siRNA decreases the expressions of ADRB3 and Rab7; rapamycin increases the expression of ADRB3; after silencing p62, ADRB3 cannot increase the expressions of mTOR, Rictor, SIRT1 and ADRB3.

(72) FIG. 72: the ADRB3 antibody decreases the expressions of mTOR, Rictor, p-AKT (Ser 473), p-4EBP1 (T37/46), HK2, P62, Rab7, SIRT1, ADRB3, VDAC and Rheb, but increases the expression of p53 in MCF7 tumor transplanted into nude mice.

(73) FIG. 73: the ADRB3 antibody decreases the expressions of HK2, P62, p-mTOR (S2481), Rictor and IL-6 in MCF7 tumor transplanted into nude mice; A. control group, B. antibody group.

(74) FIG. 74: after agitation, the ADRB3 increases phosphorylation of the proteins, such as mTOR, Rictor, 4EBP1, CENPA, P62, Drp1, AKT and AMPK in downstream signal paths.

(75) FIG. 75: The ADRB3 antibody inhibits elimination of the autophagosome of the MCF7 mitochondria.

(76) FIG. 76: A. the ADRB3 antibody reduces the mitochondrial membrane potential, which is reflected as green after JC1 staining, and B. the ADRB3 antibody increases the reactive oxygen in MCF7.

(77) FIG. 77: A. the ADRB3 antibody increases the intracellular content of lipofuscin; and B. the ADRB3 antibody increases the intracellular content of 5-CFDA in MCF7 cells.

(78) FIG. 78: A. the ADRB3 antibody increases the intracellular content of rhodamine 123 in MCF7 cells; B. the ADRB3 antibody increases the intracellular content of SA-β-Gal (senescence-associated β-galactosidase).

(79) FIG. 79: the ADRB3 antibody reduces uptake of 18F-FDG by tumor tissues transplanted with MCF7 in nude mice; (A) 18F-FDG PET scannogram; (B) histogram of the gray scale ratio of target tissues (T) to adjacent normal tissues (non-target tissues N) on a reconstructed image; (C) control group: the transplanted tumor has a radioactive concentrated image. The tumor has similar uptake of FDG to liver without central necrosis, and the white arrow means transplanted tumor; (D) ADRB3 antibody group: the transplanted tumor has radioactive sparseness and defect in the middle thereof. The tumor has less uptake of FDG than liver with central liquefaction necrosis; (E) the ADRB3 antibody decreases the ATP output of tumor tissues; and (F) the ADRB3 antibody decreases the HK activity of tumor tissues.

(80) FIG. 80: TEM of the tissue of the tumor transplanted with MCF7 cells of the ADRB3 antibody group, and the ADRB3 antibody results in mitochondrial crista decrease and volume expansion.

(81) FIG. 81: in tumor cells transplanted with MCF7 cells of nude mice of the ADRB3 antibody group, mitochondrial autophagosome is accumulated in cytoplasm, and is not removed. The black arrowhead shows the mitochondrial autophagy, and the white arrowhead shows the lipid droplet.

(82) FIG. 82: immunofluorescence of hydrothorax cell smears stained with MPO and ADRB3 of lung cancer patients; A. there are a lot of soluble ADRB3 (soluble B3, sB3) in the hydrothorax of lung cancer patients, suggesting that granulocytes release sB3 into blood and tissue spaces by degranulation; B. there are a lot of ADRB3 in granulocyte cytoplasm; C/D. granulocytes (MPO.sup.+) with high expression of ADRB3 in the hydrothorax tend to be adhered to lung cancer cells; and E. The ADRB3 is highly expressed in exfoliated lung cancer cells of the hydrothorax.

(83) FIG. 83: immunofluorescence detection of the ADRB3 expression in peripheral blood smear cells of normal subjects; A. ADRB3 is expressed in normal neutrophils, and is slightly expressed in lymphocytes; B. ADRB3 is abundantly expressed in naive or primitive T-lymphocytes (cytoplasm containing large amounts of CD4 or CD8 lymphocytes); C. ADRB3 is highly expressed in cells at late G2 phase and early mitosis. In this phase, the karyotheca has been ruptured, and the chromosome replication has been completed, but the spindle has not yet been formed.

(84) FIG. 84: A. immunofluorescence of ADRB3 in peripheral blood smears of breast cancer patients, there are a lot of ADRB3 in granulocytes and abnormal lymphocytes (upper right), ADRB3 is abundantly expressed, the cytoblast has granulocyte-like changes; and B. there are a large number of foot processes in the granulocytes of breast cancer patients, and ADRB3 is adhered to the foot process.

(85) FIG. 85: immunofluorescence of ADRB3 in peripheral blood smears of breast cancer patients; high expression of ADRB3 and microexpression of MPO in the upper lymphocytes; in the lower granulocytes, a lot of ADRB3 are accumulated in the contact position between granulocytes and lymphocytes, which plays a role like adhesion factors, and promotes to form immune synapses. Granulocytes induce lymphocytes to differentiate toward myeloid cells.

(86) FIG. 86: immunofluorescence of ADRB3 in peripheral blood smears of breast cancer patients; A. a large number of neutrophils extracellular networks (NET) are formed in the blood of breast cancer patients, and the lymphocytes (MPO negative) are adhered to the NETs; B. the lymphocytes in the yellow box are reflected as MPO.sup.dimADRB3.sup.bright, which may be lymphocyte progenitor cells or primitive lymphocytes.

(87) FIG. 87: ADRB3 and Ki-67 are highly expressed in lymphocytes (in yellow box) of the blood of breast cancer patients, are in the proliferation phase, are dedifferentiated, and are naive or primitive lymphocytes, indicating that the patients have lymphocyte hypofunction and specific immunodeficiency.

(88) FIG. 88: ADRB3 is highly expressed, and Ki-67 and MPO are less expressed in a part of lymphocytes of breast cancer patients, which are naive or primitive lymphocytes.

(89) FIG. 89: there are a large number of DNA fragments in the blood of breast cancer patients, and the ADRB3 is adhered to the DNA fragments.

(90) FIG. 90: immunofluorescence detection of ADRB3 in cells of peripheral blood smears of normal subjects—the cytoblast of the primitive plasmocyte is larger than that of the T cell, and there are a lot of ADRB3 in the cytoplast.

(91) FIG. 91: immunofluorescence detection of ADRB3 in cells of peripheral blood smears of normal subjects-mature plasmocytes have the characteristics that there is no CD4 or CD8, but a lot of ADRB3 in the cytoplasm.

(92) FIG. 92: immunofluorescence detection of ADRB3 in cells of peripheral blood smears of normal subjects—ADRB3 is expressed in the cell membrane of CD19.sup.+B.

(93) FIG. 93: A. ADRB3 carrying green fluorescent proteins (GFP) can kill pancreatic cancer cell SW1990; B. ADRB3 plasmid diagram.

(94) FIG. 94: A. ADRB3 carrying Flag tag proteins cannot enter the cytoblast of MCF7; B. ADRB3 is localized in microtubules, cytoblasts and mitochondria of MCF7 cells.

(95) FIG. 95: ChIP-Chip experimental chip scannogram.

(96) FIG. 96: survival curve of mice in each group after ADRB3 knockout mice and normal mice are intraperitoneally injected with 30 mg/kg LPS; and the pyohemia mortality in ADRB3.sup.−/− mice is decreased.

(97) FIG. 97: after ADRB3 is inhibited, adenovirus is unable to infect lung cancer cell A549, and the ADRB3 antibody plays a role in inhibiting adenovirus infection by inhibiting phosphorylation of 2448th serine of mTOR; A. the upper figures show adenovirus infected cells, p-mTOR (Ser2448) is significantly increased and accumulated below cell membrane, and the lower figures show cells pretreated with the ADRB3 antibody without adenovirus infection; B. and the ADRB3 antibody inhibits p-mTOR (Ser2448), and reduces phosphorylation of downstream substrate 4EBP1 of mTOR.

(98) FIG. 98: both the ADRB3 and adenovirus are localized in nucleolus, and the ADRB3 is a key protein required for viral replication.

(99) FIG. 99: the ADRB3 is increased in hepatocyte cytoplasm of patients with hepatitis B, and the ADRB3 antibody inhibits HBV infection of HepG2 cells.

(100) FIG. 100: atherosclerotic plaque detection of ADRB3/ApoE double knockout mice and ApoE knockout mice; A. the aorta of ADRB3/ApoE double knockout mice is significantly thickened than that of ApoE.sup.−/− mice; B. the aorta is stained with oil red O; and C. ELISA of inflammatory factor in serum.

(101) FIG. 101: the ADRB3 antibody promotes complement activation; A. the ADRB3 antibody increases the complements C8 and C5R1; LPS is an endotoxin as a positive control for inducing complements; and B. immunofluorescence detection of C8 and B3AR.

(102) FIG. 102: the ADRB3 is located on the membrane of the platelet.

(103) FIG. 103: ADRB3 and CD56 are expressed in NK cells of the umbilical cord blood.

(104) FIG. 104: ADRB3 and MPO are expressed in NK92 cells.

(105) FIG. 105: the ADRB3 and Ki67 are expressed in monocytes of patients with acute myocardial infarction. (A) shows cells in G1 phase with less Ki67. (B) shows G2 or S phase with more Ki67.

(106) FIG. 106: in patients with acute coronary syndrome, the ADRB3 content in megakaryocytes of patients with positive cardiac troponin T (cTnT) is higher than that in megakaryocytes of patients with negative cTnT. (A) shows positive cTnT, and (B) shows negative cTnT.

DETAILED DESCRIPTION

(107) The invention is further illustrated in detail in conjunction with the accompanying drawings and examples below. These examples are only used to illustrate the invention, rather than to limit in any way the scope of the invention.

(108) Unless otherwise specifically indicated, the test methods used in the following examples are conventional methods; and unless otherwise specifically indicated, the materials, reagents, etc. are commercially available reagents and materials.

Example 1 Immunofluorescence for Localization of ADRB3 in Tumor Cells

(109) 1. Test Method

(110) Breast cancer cell MCF7, lung cancer cell A549, melanoma A375, pancreatic cancer cell CFPAC1, hepatic cancer cell HepG2 and glioma cell A172 were inoculated into a 6-well plate with a built-in sterile coverslip at a rate of 10.sup.5/well, incubated until the cells were adhered to the coverslip, immobilized with 4% paraformaldehyde for 10 minutes, and washed with PBS 3 times. After transparentizing with 0.1% TritonX-100 for 10 min, and closing with 3% BSA for 1 h, antibodies of Ki-67/ADRB3/Nucleolin/Fibrillarin/H3K9AC/Brdu/-tubulin/Flag etc. (1:100) were added dropwise. The mitochondria were stained with Mitotracker, the lysosome was stained with lysotracker, cells were administrated with Brdu before immobilization, and pcDNA3-flag-B3 plasmid was transfected into cells with lipofectin 3000. The coverslips were sealed in a wet box, and incubated in a refrigerator at 4° C. overnight. After rinsing with PBS for 5 min×3, and adding FITC-labelled secondary anti-rabbit (1:800) and PE-labelled secondary anti-mouse (1:800) dropwise, the coverslips were incubated at room temperature for 1 h, stained with 0.5 μg/ml DAPI for 3 min, rinsed with PBS for 5 min×3, sealed with 50% glycerol/PBS, and observed under a laser scanning confocal microscope. 5-7 fields were randomly observed and photographed, and the mean red fluorescence intensity and the mean green fluorescence intensity of cells were measured with software Fluorchem 8900.

(111) 2. Test Results

(112) As shown in FIG. 1 to 17, the following results may be obtained:

(113) (1) by staining with Ki67/nucleolin/Brdu, it is found that a lot of nucleoli are present in cells at G0 phase, but nucleolar constituent protein at G0 phase is different from that in cells in G1 phase. Nucleolar protein markers in G1 phase could not be used to mark nucleoli in cells at G0 phase. An ADRB3 is highly expressed in the nucleoli of the human breast cancer cell MCF7 at G0 phase (Ki-67 and Nucleolin: negative). In FIG. 1, the upper right cell is a cell with proliferation capacity at G0 phase.

(114) (2) The ADRB3 is highly expressed in the nucleoli of the human lung cancer cell A549 at G0 phase (Ki-67 negative).

(115) (3) The ADRB3 is highly expressed in the nucleoli of the human pancreatic cancer cell CFPAC1 at G0 phase.

(116) (4) The ADRB3 is highly expressed in the nucleoli (Fibrillarin positive) of the human melanoma cell A375.

(117) (5) The ADRB3 is localized in the mitochondrial outer membrane of the human pancreatic cancer cell PANC1.

(118) (6) The ADRB3 is localized in the centrosome at both spindle poles and the spindle equator of A549 cells. The ADRB3 is in a plurality of cytoblasts of the fusion cells.

(119) (7) The ADRB3 is localized on the microtubule of the human hepatic cancer cell HepG2.

(120) (8) The ADRB3 is localized in the centrosome at both spindle poles of the human glioma cell A172. The ADRB3 is localized at the centrosomal position of the polyploid cell.

(121) (9) The ADRB3 is localized on the lysosome of the MCF7 cell. The ADRB3 increases the tyrosine phosphorylation of the protein on cell membrane at G0 phase, and promotes the tyrosine phosphorylation of membrane proteins, such as HER2/EGFR/drug pump, to activate the membrane proteins.

(122) (10) The ADRB3 is located in the centrosome of MCF7 cell at the end of G2 phase to promote formation of the microtubule center.

(123) (11) The ADRB3 is in the vacuolar outer membrane of the MCF7 cell to promote the vacuole formation. ATP6V0D1 is a D subunit of a vacuolar ATP enzyme, and is localized in the vacuolar membrane. The ADRB3 and ATP6V0D1 are co-localized, indicating that the ADRB3 is located in the vacuole membrane.

Example 2 Immunohistochemical Analysis on Tissue Microarrays for Detection of ADRB3 Expression in Tumor Tissues

(124) 1. 11 different tumor tissue microarrays from a total of 1,479 patients were used in this research: (1) 42 patients with NSCLC; (2) 150 lung adenocarcinoma patients with survival time data; (3) 150 patients with pulmonary squamous cell carcinoma with survival time data; (4) 180 liver cancer patients with survival time data; (5) 170 breast cancer patients with survival time data and 82 breast cancer patients without survival time data; (6) 170 pancreatic cancer patients with survival time data; (7) 62 patients with papillary thyroid carcinoma; (8) 90 colon cancer patients with survival time data; (9) 90 kidney cancer patients with survival time data; (10) 93 esophagus cancer patients with survival time data; (11) 90 gastric cancer patients with survival time data; (12) 90 colorectal cancer patients with survival time data; and (13) 20 leukemia patients.

(125) 2. Experimental steps: (1) paraffin section: thickness 4 m; (2) baking slices: 65° C. for 3 h; (3) dewaxing until putting in water: dimethylbenzene 10 min×3 cylinders, 100%-100%-95%-95%-90%-80%-70% alcohol each for 5 min, hydration for 5-10 min; (4) antigen retrieval: high pressure repair in TRIS-EDTA repair solution at PH8.0 for 3 min since the exhaust valve begins exhausting, natural cooling; (5) incubation in 3% hydrogen peroxide for 10 min; (6) rinsing with PBS for 5 min×3 times; (7) closing with 10% goat serum for 10 min; (8) preparation of primary antibody (1:10); (9) discarding the serum, and then adding primary antibody, keeping at 4° C. overnight; (10) on completion of incubation, rinsing with PBS for 5 min×3 times; (11) adding secondary antibody, and then incubating at room temperature for 30 min; (12) rinsing with PBS for 5 min×3 times; (13) developing with DAB (the ratio of solution A to solution B is 1:50). Observed under a microscope, it can be terminated when the expression is appropriate and the background is clear. (14) restraining with hematoxylin for 1 min, and returning to blue with warm water for 15 min; (15) dehydration through gradient alcohols: 70%-80%-90%-95%-95%-100%-100%, each for 3 sec; (16) transparentizing with dimethylbenzene for 5 min×2 cylinders; and (17) sealing the slice with a neutral gum.

(126) 3. Determination of Staining Intensity:

(127) Each tissue site was randomly observed in 6 to 8 high power fields, and a case of positive staining was determined if the positive cell count was more than 5%. Weakly positive staining was yellowish (+ or 1), moderately positive staining was brown yellow (++ or 2), and strongly positive staining was brown (+++ or 3).

(128) Determination of positive staining rate: each tissue site was randomly observed in 6 to 8 high power fields, and a case of positive staining was determined if the positive cell count was more than 5%. Weakly positive staining was yellowish (+ or 1), moderately positive staining was brown yellow (++ or 2), and strongly positive staining was brown (+++ or 3). Determination of the positive staining rate: 3 fields of different staining intensities were evaluated under a high-magnification microscope, 100 cells were randomly recorded in each field, and the percentage of positive cells per 100 cells ×1% was recorded. Likewise, the percentage of positive cells in another 2 fields ×2% and ×3% was observed, and finally the positive staining rate of the tissue site was the mean value of ×1%, ×2% and ×3%.

(129) Each site in the tissue microarray was scored based on the sum of the integral of the cell staining strength and the area of stained cells. Firstly, the straining intensity was scored: 0: colorless, 1: pale yellow, 2: brown yellow, and 3: brown. Then the percentage of positive cells was scored: 0: negative, 1: <25%, 2: 25%-50%, and 3: >50%. The two integrals add up to the score of the site.

(130) Kaplan-Meier method was used to analyze the survival of ADRB3 negative and positive patients in cancer tissues and obtain two survival curves. The Log Rank method was used as a statistical test to compare whether the distribution of survival curve of each group is identical.

(131) 4. Test Results

(132) The results are shown in FIG. 18 to 26 and Table 1 to 3. Table 1 shows that the ADRB3 expression in breast cancer tissues is significantly higher than that in normal breast tissues (P<0.01). Table 2 shows that the ADRB3 expression in lung cancer tissues is significantly higher than that in normal lung tissues (P<0.01). Table 3 shows that the ADRB3 expression in pancreatic cancer tissues is significantly higher than that in normal pancreatic tissues (P<0.01).

(133) TABLE-US-00002 TABLE 1 Expression of ADRB3 in 228 Breast Cancer Tissues and 89 Paracancerous Tissues Expression of breast cancer tissues and paracancerous tissues Number B3AR of cases − + ++ +++ Cancer 228 18 91 80 39 Proportion of different  7.9% 39.9% 35.1% 17.1% staining intensities (18/228) (91/228) (80/228) (39/228) Positive rate 92.1% (210/228) paracancerous 89 61 27 1 0 Proportion of different 68.5% 30.3%  1.1% 0.00% staining intensities (61/89)  (27/89)  (1/89) Positive rate 31.5% (28/89) 

(134) TABLE-US-00003 TABLE 2 Expression of ADRB3 in 150 Lung Cancer Tissues and Paracancerous Tissues Number B3AR of cases − + ++ +++ Cancer 150 8 63 45 34  5.3% 42.0% 30.0% 22.7% Total positive rate 94.7% Paracancerous 150 139 10 1 0 92.7%  6.7%  0.7%  0.0% Total positive rate  7.3%

(135) TABLE-US-00004 TABLE 3 Expression of ADRB3 in 165 Pancreatic Cancer Tissues and Paracancerous Tissues Number B3AR of cases − + ++ +++ Cancer 165 26 84 51 4 15.8% 50.9% 30.9%  2.4% Total positive rate 84.2% Paracancerous 165 115 46  4 0 69.7% 27.9%  2.4%  0.0% Total positive rate 30.3%

(136) As shown in FIG. 18 to 26 and Tables 1 to 3, the following test results may be obtained:

(137) (1) positive expression of the ADRB3 protein is mainly localized in cytoblast, and less localized in cytoplasm and cell membrane. The positive rate of the ADRB3 in breast cancer tissues is 92.1% (210/228), of which 56.7% is moderately and strongly positive. In paracancerous breast cancer tissues, the positive rate of the ADRB3 is 31.5% (28/89), of which moderately positive rate is 3.6% (1/28), and strongly positive rate is 0. The ADRB3 expression in breast cancer tissues is significantly higher than that in normal breast tissues (P<0.01). The ADRB3 expression level is positively correlated with the malignancy degree of breast cancer, and is gradually increased with the increase of the cell malignancy degree. The positive rate is 73.7% (28/38) in breast cancer tissues of pathological grade I, and 97.6% (40/41) in breast cancer tissues of grade III. The ADRB3 is highly expressed in breast cancer tissues of pathological grade III, which is significantly higher than breast cancer tissues of Grade I (P<0.01). The ADRB3 expression level is associated with the breast cancer stage (clinical stage in sixth edition of AJCC), and the later the tumor course is, the stronger the ADRB3 expression is. The positive rate of the ADRB3 in breast cancer tissues of stage I is 65.0% (13/20). The positive rate in breast cancer tissues of stage III is 100% (67/67). The ADRB3 reduces the survival rate of breast cancer patients, and the survival time of breast cancer patients with positive expression of the ADRB3 is significantly less than that of breast cancer patients with negative expression of the ADRB3. The Kaplan-Meier survival curve of 142 breast cancer patients shows that, the survival rate of patients with negative ADRB3 in cancer tissues is significantly higher than that of patients with positive ADRB3 in cancer tissues, P=0.025. The mean survival of the two groups is respectively 154 months and 126 months. As can be seen from the survival curve, the survival rate of patients with positive ADRB3 gradually is decreased over time, and is about 60% approximately by 170th month. However, the survival rate of patients with negative ADRB3 is more slowly decreased than that of patients with positive ADRB3, and is still more than 90% by 170th month. The risk level curve also has a very obvious trend, that is, as time passes, the patients with positive ADRB3 experience increasingly more mortality risks on survival, which are about 5 times that of the original risks (0 month) by 170th month. The mortality risk of patients with negative ADRB3 is less than that of patients with positive ADRB3, and is less than 1 time that of the original mortality risk by 170th month. The correlation coefficient between ADRB3 and Ki67 in cancer tissues is 0.296 (P=0.02), and there is significant linear positive correlation.

(138) (2) The ADRB3 expression in lung cancer tissues is significantly higher than that in normal lung tissues (P<0.01). The more the ADRB3 is expressed, the shorter the survival time is. The Kaplan-Meier survival curve shows that, the survival rate of patients with weakly positive ADRB3 in lung cancer tissues is significantly higher than that of patients with strongly positive ADRB3 in cancer tissues, P=0.038. The median survival time of patients with weakly positive ADRB3 is 81 months, and that of patients with strongly positive ADRB3 is 50 months. The ADRB3 level in cancer tissues is directly related to the rapid deterioration of lung cancer and poor prognosis. The ADRB3 antibody reduces the ADRB3 expression in cancer, and can be used to treat patients with lung cancer metastasis.

(139) (3) The ADRB3 expression level in pancreatic cancer tissues is significantly higher than that in paracancerous tissues (P<0.01); and the survival rate of patients (0) with negative ADRB3 in cancer tissues is significantly higher than that of patients (1) with positive ADRB3 in cancer tissues, P=0.019. The mean survival time is 43.6 months for negative patients and 29.4 months for positive patients.

(140) (4) The ADRB3 expression in colon cancer tissues is significantly higher than that in normal colon tissues (P=0.0001).

(141) (5) The ADRB3 expression in hepatocellular carcinoma tissues is positively correlated with age and pathological grading. The older the age is and the higher the malignancy degree is, the more the ADRB3 proteins are. The expression level of ADRB3 in cancer tissues of liver cancer patients of more than 50 years old is significantly higher than that in paracancerous tissues, P=0.04. The Kaplan-Meier survival curve of 162 hepatocellular carcinoma patients shows that, the survival rate of patients with negative ADRB3 in cancer tissues is significantly higher than that of patients with positive ADRB3 in cancer tissues, P=0.038. The median survival time of patients with negative ADRB3 is 37 months, and that of patients with positive ADRB3 is 25 months.

(142) (6) In patients suffering a relapse of acute lymphocytic leukemia, cytoplasm of bone marrow granulocytes contains a lot of dense rough-surfaced endoplasmic reticulums, ADRB3 and Ki-67 are adhered to the endoplasmic reticulums; ADRB3 and Ki-67 contents are increased significantly, and are significantly higher than the contents after complete response.

(143) (7) The ADRB3 expression in cancer tissues of kidney cancer, thyroid cancer, colon cancer, esophagus cancer and gastric cancer is higher than that in paracancerous tissues.

Example 3 Immunofluorescence Detection of ADRB3 Expression in Cells in Bone Marrow Smears of Different Leukemia Patients

(144) 1. The test method is the same as that in Example 1.

(145) 2. The results are shown in FIG. 27 to 33:

(146) (1) Myeloperoxidase (MPO) is a marker protein of neutrophils, and ADRB3 is expressed in neutrophils of normal subjects, but is not expressed in lymphocytes. The intracytoplasm ADRB3 of neutrophils forms a conical structure directly reaching the cytoblast, the channel formed by ADRB3 helps extracellular material (e.g., virus) to enter the cytoblast, blocks ADRB3, and will inhibit viruses' entry into the cytoblast. In patients with B lymphocytic leukemia, ADRB3 is expressed in both leukemia cells and neutrophils, suggesting that ADRB3 induces carcinogenesis of B-lymphocytes. The ADRB3 in leukemia cells also forms a channel directly reaching the cytoblast.

(147) (2) Bone marrow smears of normal subjects injected with granocyte (recombinant human granulocyte colony-stimulating factor) are stained with ADRB3 and MPO. Granulocyte colony-stimulating factor stimulates granulocyte proliferation, most granulocytes are in proliferation phase, and the ADRB3 is highly expressed in immature granulocytes of proliferation phase, suggesting that the ADRB3 stimulates granulocyte proliferation to inhibit its differentiation and maturation. Due to the granulocyte colony-stimulating factor, the ADRB3 is expressed in lymphocytes (MPO negative), suggesting that the ADRB3 expression is inductive, and ADRB3 has the effect of regulating lymphocyte proliferation and differentiation.

(148) (3) Bone marrow smears of patients with acute B-lymphoblastic leukemia suffering a relapse after treatment are stained with ADRB3 and MPO. In granulocytes and leukemia cells of patients suffering a relapse of leukemia after treatment, the ADRB3 is highly expressed, granulocytes containing a lot of ADRB3 are the key factors resulting in the relapse of the B-lymphocytic leukemia, and the ADRB3.sup.+ granulocytes will promote leukemia cells at G0 phase to enter proliferation phase, thereby resulting in relapse. The ADRB3 expression in granulocytes and leukaemia cells of patients with acute B-lymphoblastic leukemia after treatment is reduced, compared with that before treatment. The ADRB3 aggregates in a site where granulocytes (antigen presentation cells) and lymphocytes contact with each other, which contributes to the stability of intercellular adhesion molecules, and promotes to form inhibitory immune synapses. The ADRB3 induces fusion of granulocytes with leukemia cells, alters the phenotype of cancer cells, increases the malignancy degree of cancer cells, and makes cancer cells be resistant to drugs. The ADRB3 in lymphocytes shows a conical structure directly reaching the cytoblast, affects the epigenetic modification and gene expression profile of lymphocytes, and promotes proliferation.

(149) (4) Bone marrow smears of patients with acute B-lymphoblastic leukemia suffering a relapse after treatment are stained with ADRB3 and Ki-67. The ADRB3 and Ki-67 are highly expressed in granulocytes, and are co-localized in cytoplasm, suggesting that the granulocytes have vigorous ribosomal functions, and synthesize a lot of cell growth factors. By releasing the growth factor, the lymphocytes and tumor cells at G0 phase are stimulated to enter the proliferation phase. MDSC is a granulocyte of positive ADRB3 and positive Ki-67, and can inhibit T cells, NK cells and other immune cells to eliminate leukemia cells and other tumor cells. In the bone marrow and peripheral blood of patients suffering a relapse of leukemia after treatment, ADRB3.sup.+ and Ki-67.sup.+ granulocytes are significantly increased, suggesting that the granulocytes are the key to the relapse of leukemia.

(150) (5) The ADRB3 is expressed in both granulocytes and leukemia cells of bone marrow smears of patients with acute monocytic leukemia (M5) after CR by treatment.

(151) (6) The ADRB3 is highly expressed in granulocytes in proliferation phase (Ki-67 positive) of patients with acute myelocytic leukemia.

Example 4 Preparation of Hybridoma Cell Strains, Purification of ADRB3 Antibody, Antibody Sequencing and Humanization

(152) 1. Mice were immunized with human ADRB3 protein fragments (including full length), and some adopted epitopes are shown in FIG. 34A, including but not limited to the following antigenic epitopes: human ADRB3 full-length protein (1st-408th amino acid residues), N-terminal fragments (1st-155th amino acid residues) in ADRB3 protein, C-terminal fragments (156th-408th amino acid residues) in ADRB3 protein, leucine zipper fragment (296th-317th AA) in ADRB3 protein, nuclear localization sequence (NLS) (351st-369th AA), E1 (1st-36th amino acid residues in ADRB3), E2 (101st-111th amino acid residues in ADRB3), 12 (134th-155th amino acid residues in ADRB3), E4 (315th-326th amino acid residues in ADRB3), 14 (348th-408th amino acid residues in ADRB3), etc. The BALB/c mice were injected subcutaneously with Freund's complete adjuvant, and were intensively immunized via caudal vein 21 days after the first immunization. 4 days later, spleen cell suspension was prepared for cell fusion with myeloma cell SP2/0, hybridoma cell was screened using HAT, hybridoma was cloned, finally a few hybridoma cell trains of each ADRB3 protein fragment were obtained, and more than 30 hybridoma cell strains that can produce ADRB3 antibodies were screened. The specificity and affinity of antibodies were detected using enzyme-linked immunosorbent assay (ELISA), protein immunoblotting (FIG. 34B) and immunofluorescence test. After lysing hybridoma cells by Trizol, RNA was extracted for light and weight chain sequencing of antibodies. Light chain plasmids (nucleic acid and protein sequences as shown in FIG. 35, but not limited to FIG. 35) and heavy chain plasmids (nucleic acid and protein sequences as shown in FIG. 36, but not limited to FIG. 36) of humanized antibodies were constructed. HEK293 was transfected using BPfectin Transfection Reagent at H:L=1:1 at a ratio of transfected reagents to plasmids of 3:1, and cultured in a bioreactor to produce a lot of antibodies.

(153) Collecting antibody from ascites of mice: Balb/c mice of 10-11 weeks old were intraperitoneally injected with a complete immune adjuvant with 0.1 ml/mouse, and were intraperitoneally injected with hybridoma cells 5*10.sup.6 6 days later. The ascites was taken out once every other day since 8th day after cell inoculation. After centrifugation at 3000 rmp for 10 min, the supernatant of ascites was stored at −80° C. After concentration with ammonium sulfate, it was purified by Protein G affinity chromatography columns, and purified and eluted according to a standard antibody purification method to identify the purity by SDS-PAGE. Its quantity was measured by UV.

Example 5 MTT Assay for Detection of Anti-Cancer Effect of ADRB3 Monoclonal Antibody

(154) 1. Test Steps:

(155) (1) After trypsinization, adherent tumor cells in logarithmic growth phase were prepared into 5000/ml cell suspension using RPMI 1640 medium containing 10% calf serum, inoculated into a 96-well culture plate with 200 l/well, and cultured at 37° C. under 5% CO.sub.2 for 24 h. (2) A new medium containing different concentrations of the ADRB3 monoclonal antibody was replaced for the experimental group, a medium containing equivalent volume of solvent was replaced for the control group, and 3-5 parallel wells were established for each group. They were cultured at 37° C. under 5% CO.sub.2 for 4-5 days. (3) After the supernatant was abandoned, 200 μl of a fresh serum free medium containing 0.5 mg/ml MTT was added to each well, and then culture was continued at 37° C. for 4 h. After the supernatant was carefully discarded, 200 μl of DMSO was added, and fully mixed with a minitype ultrasonator to measure the optical density value with an ELIASA at a wavelength of 570 nm and a reference wavelength of 450 nm.

(156) 2. The Test Results are Shown in FIGS. 37A-39 and 52-56:

(157) The ADRB3 monoclonal antibody dose-dependently decreases the activity of human breast cancer cells MCF7, pancreatic cancer cells SW1990 and CFPAC1, lung cancer cells A549, lung cancer cells H1299, hepatic cancer cells HepG2, colon cancer cells HCT116, promyelocytic leukemia cells HL-60 and glioma cells U251. Compared with the control group, *P<0.05, #P<0.01.

Example 6 ADRB3 Monoclonal Antibody Inhibits Proliferation of Tumor Transplanted into Nude Mice

(158) 1. Test Method

(159) Female nude mice of 5 weeks old were subcutaneously injected with human tumor cells or mice 4T1 breast cancer cells near their breasts, each of which was injected with 5*10.sup.6 cells. Human tumor cells include MCF7, CFPAC1, ASPC1, SW1990, A375, A549, etc. 8-10 days later, the transplanted tumors were about 100 mm.sup.3, and were randomly divided into antibody group and control group with 8 mice in each group. The antibody group was intraperitoneally injected with the ADRB3 antibody in a dose range of 1-10 mg/kg once every other 3 days, and the length and width of the tumor were measured. The control group was intraperitoneally injected with mouse IgG. When the tumor volume of the control group reached the maximum volume permitted by the Animal Ethics Committee, blood was taken out the orbit, the cervical vertebrae were removed to kill nude mice, and the transplanted tumor and spleen were stripped to get the tumor weight, spleen weight and body weight. The spleen index was calculated as follows: spleen index=spleen weight (mg)/mouse weight (g).

(160) 2. The Test Results are Shown in FIGS. 37 and 40-51:

(161) (1) The ADRB3 monoclonal antibody inhibits proliferation of the tumor transplanted with the breast cancer cell MCF7 in nude mice. Compared with control group, *P<0.05, #P<0.01. The spleen of mice in the ADRB3 antibody group is significantly larger than that of mice in the control group. The ADRB3 antibody increases the spleen index, and has the function of contributing to development and maturation of immune organs. HE staining of transplanted tumor shows that the ADRB3 antibody decreases neutrophil infiltration in transplanted tumor. After HE staining of the spleen tissue, the spleen in the control group does not have a clear dividing line, there is not a clear dividing line between white pulp and red pulp, and a large number of neutrophils can be seen. The ADRB3 antibody increases the splenic corpuscle and the germinal center, and reduces the count of neutrophils in the spleen. Immunohistochemical analysis shows that the ADRB3 antibody reduces the expressions of ADRB3, P62, Cyclin D1, MPO, Neutrophil Elastase, p-Rb (S780), CDK3 and IL-6 in transplanted tumor cells and infiltrating neutrophils, and reduces the activity of CDK3.

(162) (2) The ADRB3 antibody reduces the growth of tumor transplanted with the pancreatic cancer cell CFPAC1 in nude mice. Compared with the control group, *P<0.05. The ADRB3 antibody reduces the weight of tumor transplanted with CFPAC1 cells, doesn't affect the body weight of the nude mice transplanted with CFPAC1, and increases the spleen index. HE staining shows that the ADRB3 antibody decreases neutrophil infiltration in transplanted tumor. Immunohistochemical analysis shows that the ADRB3 antibody reduces the expressions of ADRB3, Cyclin D1, Neutrophil Elastase, p-Rb (S780), p-mTOR (S2481), Rictor, HK2, CDK3 and IL-6 in transplanted tumor cells and infiltrating neutrophils.

(163) (3) The ADRB3 antibody reduces the growth of tumor transplanted with the pancreatic cancer cell ASPC1 in nude mice. Compared with the control group, *P<0.05. The ADRB3 antibody reduces the weight of tumor transplanted with ASPC1 cells, and doesn't affect the body weight of the nude mice transplanted with ASPC1.

(164) (4) The ADRB3 antibody inhibits the proliferation of tumor transplanted with the pancreatic cancer cell SW1990, increases the apoptosis rate of SW1990. Compared with the control group, *P<0.05. The ADRB3 antibody decreases the count of neutrophils in the tumor bearing mice and the neutrophil-lymphocyte ratio (NLR). Compared with the control group, *P<0.05. The ADRB3 antibody increases the spleen index.

(165) (5) The ADRB3 antibody inhibits proliferation of the tumor transplanted with the melanoma cell A375. Compared with the control group, *P=0.032, #P=0.005. The ADRB3 antibody reduces the number of MSDC in the peripheral blood of nude mice transplanted with A375. HE staining and immunohistochemical analysis show that there are a lot of neutrophil infiltrations in A375-transplanted tumor of the control group, PD-L1 is highly expressed in neutrophils, and the ADRB3 antibody decreases neutrophil infiltrations in A375-transplanted tumor. Immunohistochemical analysis shows that the ADRB3 antibody inhibits the expressions of ADRB3, IL-6, MPO, Neutrophil Elastase, PD-L1, p-Rb (Ser 780), p-mTOR (2481), Rictor, HK2 and CDK4 in A375-transplanted tumor cells and infiltrating neutrophils. After treatment with the antibody, no PD-L1 is expressed in the neutrophils infiltrated in the transplanted tumor. ELISA of inflammatory factors in the serum shows that, the ADRB3 antibody increases the concentration of IL-10, but reduces the concentrations of IL-6, VEGF and MPO in the serum of mice. The ADRB3 antibody specifically inhibits the expression and activity of ADRB3, thereby inhibiting neutrophil-mediated inflammatory response, altering the microenvironment of tumor and playing an anti-cancer role.

(166) (6) The ADRB3 antibody increases the spleen index, and inhibits proliferation of the tumor transplanted with the human lung cancer cell A549. Compared with the control group, *P<0.05. HE staining shows that the ADRB3 antibody decreases neutrophil infiltration in transplanted tumor. Immunohistochemical analysis shows that the ADRB3 antibody reduces the expressions of ADRB3, Cyclin D1, Neutrophil Elastase, p-Rb (Ser 780), p-mTOR (Ser 2481), Rictor, HK2, CDK3 and IL-6 in transplanted tumor cells and infiltrating neutrophils.

(167) (7) The ADRB3 antibody reduces proliferation of the tumor transplanted with the breast cancer cell 4T1 in mice, and inhibits liver metastasis of 4T1 cells. 8 mice in the control group all had serious liver metastases, and the liver metastasis in the antibody group was obviously less than that in the control group. The ADRB3 promotes liver metastasis of 4T1 cells, and can be blocked to inhibit liver metastasis. The ADRB3 antibody increases the spleen index, reduces the count of blood neutrophils, increases the ratio of lymphocytes in WBC, and reduces NLR. NLR is an index of systemic inflammatory response, and an increase of NLR is an independent risk factor of poor prognosis in tumor patients. The ADRB3 antibody reduces NLR, indicating that this antibody can inhibit inflammation and improves prognosis of patients.

Example 7 T-Lymphocytes Modified by ADRB3 Specific Chimeric Antigen Receptor can Kill Cell SW1990 (A1) and Cell H1299 (C3)

(168) 1. Test Method

(169) The ADRB3 chimeric antigen receptor consists of a single chain antibody of an anti-human ADRB3, a CD8 hinge area and transmembrane zone, an intracellular signal area of CD137 (also known as 4-1BB) and an intracellular signal structure of CD3ζ in series. According to the sequencing results of the ADRB3 monoclonal antibody, a lentivirus expression vector of chimeric antigen receptors was constructed, including lentiviruses. The primitive generation T cells were isolated, and recombinant CAR-T cells were constructed using the above lentivirus. Target cells SW1990 (A1) and H1299 (C3) were inoculated into a 96-well plate with 50,000 cells/well for overnight cultivation. Then, the CAR-T cells were added at the effect/target ratio (E/T) as shown in the figure, and were co-cultured for 6 hours. After centrifugation, the supernatant was detected with the LDH kit. Target cells in wells without adding CAR-T were pyrolyzed and then used as the maximal lysis, and target cells in wells that were not pyrolyzed were used as the minimal lysis.

(170) 2. Test Results

(171) Half of the effective E/T of H1299 is 2:1, and half of the effective E/T of SW1990 is 2.5:1 (FIG. 57). An ADRB3 chimeric antigen receptor-modified T-lymphocyte can kill ADRB3 positive tumors, and is used for treating a lung cancer and a pancreatic cancer. The anti-ADRB3 CAR-T cell is specific to tumor cells and inflammatory cells (neutrophils, Treg and macrophages) in the internal environment of a tumor. Even if ADRB3 is not expressed in tumor cells, the CAR-T cell can still inhibit cells in a tumor environment to play an anti-cancer role, is a universal CAR-T cell, and is applicable to treatment of all malignant tumors and inflammatory diseases.

Example 8 Experiment of ADRB3 Knockout Mice

(172) 1. Test Method

(173) Peripheral blood was taken from 15 male ADRB3 knockout mice of 16 weeks old and 15 male normal FVB mice of 16 weeks old for blood routine and blood biochemistry. The serum cytokines were detected using a cytokine antibody chip (QAM-TH17-1, Quantibody® Array Glass Chip) of mice cell TH17, and signals were scanned using a laser scanner Axon GenePix. Bone density, fat and muscle content were measured. Cardiac functions of ADRB3 knockout mice of 10, 30 and 70 weeks old (8 mice of each age) were detected by cardiac B3 ultrasound examination. The number and proportion of different subtypes of T cells and macrophages were detected by flow cytometry of peripheral blood cells and spleen cells. Immunohistochemical analysis was used to detect cardiac CDK3, cyclinD1, p-mTOR (Ser2448), p-mTOR (Ser2481) and p-4EBP1 (T37/46). The water maze test was used to research the learning and memory ability of mice.

(174) 10 female ADRB3 knockout mice of 12 weeks old and 10 female normal FVB mice of 12 weeks old were subcutaneously inoculated with 10.sup.6 mouse breast cancer cell 4T1 to form a transplanted tumor. Length, width and height of the tumor were measured once every other 3 days. After knocking out ADRB3 gene from MMTV-PyVT mice carrying a mouse breast cancer virus, the incidence of breast tumor in female mice was observed.

(175) 2. The test results are shown in FIG. 58-66: the ADRB3 gene of the mice was knocked out to detect the tumor formation of exogenous tumor cells. It was found that the immune system of the mice body was improved, and can destroy cancer cells. The breast cancer cell 4T1 formed tumors in normal FVB mice, but failed to form tumors in ADRB3.sup.−/− mice. The incidence of spontaneous tumors in old ADRB3.sup.−/− mice was significantly decreased. After knocking out ADRB3 gene from MMTV-PyVT mice, the incidence of breast tumor in female mice was significantly decreased. ADRB3 knockout mice have an extended life, and will not have an autoimmune disease or cancer.

(176) (1) In the peripheral blood of ADRB3.sup.−/− mice, neutrophils were significantly reduced, the percentage of lymphocytes was decreased, NLR was decreased, and the mean volume of red blood cells was low. ADRB3 increases the counts of neutrophils and monocytes, but does not affect the counts of lymphocytes, decreases NLR by increasing the count of neutrophils, and increases the total counts of leukocytes. ADRB3 increases the percentages of neutrophils and monocytes, but reduces the percentage of lymphocytes.

(177) (2) The muscle amount of the ADRB3.sup.−/− mice is increased. Both the bone density and bone mineral content of the ADRB3 knockout mice are increased.

(178) (3) In serum of the ADRB3.sup.−/− mice, inflammatory cells, such as IL-2, IL-4, IL-5, IL-6, IL-17F, IL-21, IL-22, TGF- and MIP-3, are decreased, but IL-10 is increased. ELISA of inflammatory factors in the serum shows that, the concentration of IL-10 in serum of the ADRB3.sup.−/− mice is increased, but the concentrations of IL-6, VEGF and MPO are reduced.

(179) (4) CD4.sup.+T cells and Treg cells in the spleen of the ADRB3.sup.−/− mice are significantly decreased.

(180) (5) Cardiac B ultrasound examination of ADRB3.sup.−/− mice. The cardiac function of old ADRB3.sup.−/− mice is stronger than that of ADRB3 wild type old mice. The cardiac function of ADRB3.sup.−/− mice is not significantly decreased with the increase of age. Compared with the control group, the expressions of CDK3 and cyclinD1 in the myocardium of ADRB3.sup.−/− mice are increased, the mTORC2/4EBP1 pathway is activated and the ribosome of the myocardial cell is increased.

(181) (6) Tumor cells and granulocytes produce and release ADRB3 into the blood to inhibit T-lymphocytes, destroy specific immunity of the organism and promote cancer cell growth. The anticancer effect caused by knockout of ADRB3 is due to enhancement of the anti-cancer activity of the immune system. Both neutrophils and Treg cells in ADRB3.sup.−/− mice are decreased, but CD8.sup.+T cells are increased, thereby enhancing the immune surveillance and defense function of the organism.

(182) (7) The learning and memory ability of ADRB3.sup.−/− mice is better than that of ADRB3 wild type mice.

Example 9 Treatment of Cells MCF-7/A549/SW1990 with Different Concentrations of ADRB3 Antibody

(183) 1. Test Method

(184) Cells MCF-7/A549/SW1990 were treated with different concentrations of ADRB3 antibody, and the control group was treated with mouse IgG. 24 h later, the cells were lysed and the total protein was extracted. BCA method was used to determine the protein concentration: 10 ug of protein was separated with 10% SDS PAGE, transferred to a PVDF membrane, and closed with 4% skim milk for 1 h to incubate primary antibody at 4° C. overnight. Then the secondary antibody was incubated for 1 h, and developed with ECL. The experiment was repeated 3 times. The gray values of the protein bands were analyzed with software Fluorchem 8900 to calculate the ratio of target band to internal reference band (GAPDH or actin). The transplanted tumor tissues in nude mice were examined by immunohistochemical analysis to detect the expressions of associated proteins mTOR, Rictor, p-AKT (S473), p-4EBP1 (T37/46), HK2, P62, Rab7, SIRT1, ADRB3, VDAC and Rheb in the ADRB3 signal transduction pathway.

(185) 2. The Test Results are Shown in FIG. 67-74:

(186) (1) The ADRB3 antibody reduces the expressions of CDK4, nucleolin, HER2 and pRb, but increases the expression of P16 in cells MCF-7/A549/SW1990.

(187) (2) The ADRB3 antibody dose-dependently reduces Rheb, mTOR, p-mTOR (S2481), HK2 and P62 in cells MCF-7/A549/SW1990.

(188) (3) The ADRB3 antibody (5D1) reduces ADRB3, p-Rb (S780) and GAPDH in cells MCF-7/A549/SW1990. 4G7 increases ADRB3 and increases p-Rb (S780). Either of the two antibodies can reduce GAPDH and CDK4.

(189) (4) The ADRB3 antibody reduces the expressions of PD-L1 and CDK3 in cells MCF-7/A549/SW1990.

(190) (5) After designing siRNA silencing P62, the expressions of ADRB3 and Rab7 in cells MCF-7/A549/SW1990 are reduced. Rapamycin increases the expression of ADRB3. After expressing the silent P62, ADRB3 could not increase the expressions of mTOR, Rictor, SIRT1 and ADRB3.

(191) (6) The ADRB3 antibody decreases the expressions of mTOR, Rictor, p-AKT (S473), p-4EBP1 (T37/46), HK2, P62, Rab7, SIRT1, ADRB3, VDAC and Rheb, but increases the expression of P53 in MCF-7 tumor transplanted into nude mice.

(192) (7) The ADRB3 antibody decreases the expressions of HK2, P62, p-mTOR (S2481), Rictor and IL-6 in MCF-7 tumor transplanted into nude mice.

(193) (8) after agitation, the ADRB3 increases phosphorylation of the proteins, such as mTOR, Rictor, 4EBP1, CENPA, P62, Drp1, AKT and AMPK in downstream signal paths of cells MCF-7/A549/SW1990.

Example 10 Mitochondrial Autophagy Test

(194) 1. Test Method

(195) MCF-7 cells were treated using the ADRB3 antibody at a final concentration of 20 ng/ml, and was immobilized 2 h or 8 h later. After the mitochondria were stained with Mitotracker, LC3II expression was detected by immunofluorescence, and observed under a confocal microscope. After JC-1 (10 ug/ml) staining for 15 min, it was observed in a living cell state under a confocal microscope at excitation light wavelengths of 510 nm and 580 nm. ROS was stained with 10 uM H2DCFDA for 15 min, and observed under a confocal microscope. Intracellular lipofuscin was observed at a fluorescence of 488 nm. Cells were marked with 10 uM P-glycoprotein (P-gP) substrate 5-CFDA-AM, and observed under a confocal microscope at an excitation light wavelength of 510 nm. Cells were stained with rhodamine 123, and detected by flow cytometry. The expression of β-galactosidase in cell MCF7 was detected after in situ β-galactosidase staining.

(196) 2. The test results are shown in FIG. 75-78: the expression of LC3II in the ADRB3 antibody group is significantly increased with the extension of the action time. The ADRB3 antibody inhibits the elimination of mitochondrial autophagosome to accumulate the autophagosome in cells. ADRB3 promotes elimination of the autophagosome to ensure smoothly carrying out the process of mitochondrial autophagy. The ADRB3 antibody reduces the mitochondrial membrane potential, which is reflected as green after JC1 staining. The ADRB3 antibody increases intracellular ROS, lipofuscin and β-galactosidase. The ADRB3 antibody blocks autophagy, so that the autophagosome, damaged mitochondria, lipofuscin and other metabolic wastes are accumulated in cells, thereby reducing the cell activity, and accelerating cell aging and apoptosis. 5-CFDA staining shows that a lot of filamentous synapses grow from the cells, and also indicates that the cells are aging. The ADRB3 antibody increases the contents of 5-CFDA and rhodamine 123 in MCF-7, indicating that the ADRB3 antibody reverses multidrug resistance of MCF-7 cells.

Example 11 ADRB3 Antibody Reduces Glucometabolism in Tumor Tissues

(197) 1. Test Method

(198) After being treated with the ADRB3 antibody for 5 weeks, MCF-7 tumor-bearing mice were injected with 18F-FDG via caudal vein at a dose of 1 millicurie (mCi)/mouse. 50 minutes later, the mice were anesthetized with a mixed gas of 3% isoflurane and oxygen (1 L/min) to dynamically acquire images by PET/CT scan in the supine position and iteratively reconstruct images by 18F-FDG tumor metabolic imaging. The gray values of target tissues (T) and adjacent normal tissues (non-target tissues N) on reconstructed images were analyzed with software Fluorchem 8900 to calculate the value of T/N. The transplanted tumor tissues were homogenized in ice pre-cooled RIPA lysis buffer and at a high speed, and the ATP content and the activity of hexokinase (HK) in the tissues were detected with a corresponding kit.

(199) 2. The test results show that (as shown in FIG. 79): radiative accumulation is observed at the transplanted tumor site in the control group, and the radioactivity at the transplanted tumor site in the ADRB3 antibody group is reduced by about 60% (0.78+0.44 vs 1.96+0.94, P<0.01), compared with that in the control group. The ADRB3 antibody reduces the output of ATP in tumor tissues, and the activity of HK in tumor tissues.

Example 12 Observation of Mitochondria and Autophagosome of Transplanted Tumor Cells Under a Transmission Electron Microscope

(200) 1. Test Method

(201) The transplanted tumor was immobilized in 2.5% glutaraldehyde, and cut into about 1 mm.sup.3 tissue blocks to prepare samples for electron microscope. After dehydration, penetration and embedment, samples were sliced with a Reichert ultra-thin slicing machine to obtain 70 nm slices, which were stained with lead citrate solution for 15 min, stained with 50% ethanol saturated solution of uranyl acetate for 15 min, and observed under a FEI (Czech Republic) transmission electron microscope.

(202) 2. Test Results

(203) There are a lot of defective mitochondria in MCF-7 transplanted tumor cells of the ADRB3 antibody group, which is reflected as mitochondrial crista decrease, disappearance and volume expansion (FIG. 80). Mitochondrial autophagosomes of the transplanted tumor cells in the antibody group are accumulated in cytoplasm, thereby accelerating the aging of cells. Blocking the ADRB3 on the mitochondrial outer membrane will destroy the mitochondrial membrane potential, and damage the mitochondrial structure and function, thereby resulting in a lot of defective mitochondria in cytoplasm. If autophagy of cells is normal, the defective mitochondria will be removed by the autophagy. The blocking of ADRB3 destroys the fusion of autophagy and lysosomes, so that autophagosomes cannot be removed. The black arrowhead shows the mitochondrial autophagosome, and the white arrowhead shows the lipid droplet (FIG. 81).

Example 13 Immunofluorescence Detection of MPO/ADRB3 Expressions in Cells of Hydrothorax Smears of Lung Cancers

(204) 1. Test Method

(205) Hydrothorax was collected from 20 patients with lung cancer definitely diagnosed, about 5 ml of liquid at the bottom of the container was transferred to centrifuge tubes, and centrifuged at 2000 rpm for 10 min. After the supernatant was discarded, the remaining 0.5 ml of liquid and precipitate was retained, and fully mixed with a thin glass rod. The fully mixed liquid was transferred onto slides with a pipette (1-2 drops/slide) to prepare uniform smears by pushing the slides, which were immobilized in 95% alcohol for 15 min, taken out, and dried for later use. After transparentizing with 0.1% TritonX-100 for 10 min and closing with BSA for 1 h, MPO/ADRB3 antibody (1:100) was added dropwise, and the slides were closed in a wet box and kept at 4° C. overnight. After FITC and PE-labeled secondary antibody was added dropwise, the slides were kept at room temperature for 1 h, stained with 0.5 ug/ml DAPI for 3 min, sealed with 50% glycerol/PBS, observed under a laser scanning confocal microscope randomly for 5-7 fields, and photographed. The mean red fluorescence intensity and the mean green fluorescence intensity of cells were measured with software Fluorchem 8900.

(206) 2. Test Results (as Shown in FIG. 82):

(207) (1) There are a lot of soluble ADRB3 (soluble B3, sB3) in the hydrothorax of lung cancer patients, suggesting that cancer cells and granulocytes release sB3 into blood and tissue spaces by degranulation.

(208) (2) There are lots of ADRB3 in granulocyte cytoplasm of the hydrothorax. Granulocytes rich in ADRB3 tend to be adhered to lung cancer cells and stimulate the proliferation of cancer cells by direct contact.

(209) (3) The ADRB3 is highly expressed in exfoliated lung cancer cells in the hydrothorax.

Example 14 Immunofluorescence Detection of ADRB3 Expression in Cells in Peripheral Blood Smears of Normal Subjects and Breast Cancer Patients

(210) 1. The test method is the same as that in Example 1.

(211) 2. Test Results

(212) (1) The ADRB3 is expressed in neutrophils and lymphocyte progenitor cells of normal subjects (FIG. 83A), is less expressed in mature CD4+ and CD8+T cells, and is highly expressed in naive or primitive T-lymphocytes (cytoplasm containing lots of CD4 lymphocytes) (FIG. 83B). The ADRB3 is highly expressed in cells of early mitosis (FIG. 83C). The expression level of ADRB3 in lymphocytes is less than that in granulocytes. In the peripheral blood of breast cancer patients, there a lot of abnormal lymphocytes, cytoblast has granulocyte-like changes, which are reflected as reniform, lobulated and irregular shapes. The ADRB3 is highly expressed in lymphocytes, the karyotypes of which have granulocyte-like changes (FIG. 84A). There are a lot of foot processes in the granulocytes of cancer patients, and there are a lot of ADRB3 in the foot processes. There are a few foot processes on the surface of granulocytes of normal subjects, and the ADRB3 content is significantly less than that of granulocytes in cancer patients (FIG. 84B).

(213) (2) The ADRB3 and Ki-67 are highly expressed in neutrophils and lymphocytes of breast cancer patients. Tumor cells and granulocytes induce lymphocytes to express ADRB3 and inhibit the activation of lymphocytes. The expression level of ADRB3 in some lymphocytes is more than that in granulocytes. Lymphocytes highly expressing ADRB3 and Ki-67 can express a small amount of marker proteins-MPO of myeloid cells (FIG. 85), suggesting that such cells are differentiated to myeloid cells, indicating that lymphocytes in breast cancer patients become dedifferentiated and become naive or primitive lymphocytes. The ADRB3 inhibits the differentiation of T cells.

(214) (3) There are a large number of neutrophils extracellular networks (NET) in the blood of breast cancer patients, and the lymphocytes are adhered to the NETs, and ADRB3 is the main ingredient of NET (FIG. 86), suggesting that lymphocytes will receive signals from granulocytes in the nearby microenvironment (niche), and the ADRB3 in cancer cells and granulocytes form NET, which surrounds lymphocytes and regulates the differentiation and proliferation of lymphocytes. NET creates an inhibitory immune environment, and inhibits the anti-cancer function of lymphocytes.

(215) (4) The granulocytes of breast cancer patients were adhered to the lymphocytes by ADRB3 (FIG. 85). Granulocytes are less adhered to lymphocytes in the peripheral blood of normal subjects. Granulocytes are more firmly and frequently adhered to lymphocytes in cancer patients, which is closely associated with the enhancement of the ADRB3 adhesion factor-like activity on the surface of granulocytes and lymphocytes in cancer patients. The MPO is expressed in lymphocytes adhered to by granulocytes, indicating that the ADRB3 in granulocytes induces to dedifferentiate lymphocytes to lymphocyte progenitor cells or myeloid cells.

(216) (5) The expression level of Ki-67 in lymphocytes of cancer patients is positively correlated with the expression level of ADRB3, and Ki-67 is expressed in ADRB3 positive lymphocytes (FIG. 87). After granulocytes contact with lymphocytes, if the Ki-67 is expressed in lymphocytes, the ADRB3 content in cells will be more than that in granulocytes, suggesting that granulocytes or cancer cells increase the ADRB3 expression in lymphocytes, and the ADRB3 induces to dedifferentiate lymphocytes to primitive cells or progenitor cells with proliferation capacity. The ADRB3 is highly expressed, and Ki-67 and MPO are less expressed in a part of lymphocytes of breast cancer patients, which are naive lymphocytes (FIG. 88).

(217) (6) There are a large number of DNA fragments in the blood of breast cancer patients, and the ADRB3 is adhered to the DNA fragments (FIG. 89), which provide nucleic acid for cancer cell replication. The higher the contents of DNA/ADRB3 complex in the blood are, the stronger the proliferation capacity of cancer cells is, and the easier the distant metastasis will take place.

(218) (7) The ADRB3 is highly expressed in the cytoplasm of primitive plasmocytes, which have the characteristics that the cytoplasm contains a lot of dense rough-surfaced endoplasmic reticulums, there are a lot of ADRB3 in the cytoplasm (FIG. 90), and the cytoblast of primitive plasmocyte is larger than that of T cells. There are a lot of ADRB3 in the cytoplasm of mature plasmocytes (FIG. 91), and mature plasmocytes have the characteristics that there is no CD4 or CD8, but a lot of ADRB3 in the cytoplasm. The ADRB3 is expressed on the CD19.sup.+B cell membrane (FIG. 92). The ADRB3 can promote plasmocytes to synthesize antibodies.

Example 15 ADRB3 Antibody Promotes Apoptosis of Pancreatic Cancer Cell PANC-1

(219) 1. Test Method

(220) The PANC-1 was cultured, and treated with 50 ng/ml ADRB3 antibody for 12 h, while the control group was treated with mouse IgG. The apoptosis rate was detected with flow cytometry by Annexin V/PI double staining.

(221) 2. Test results: Compared with the control group, the ADRB3 antibody increases the apoptosis rate of PANC-1 (45.3±7.3% vs 9.7±3.4%, P<0.01). Breast cancer MDA231 and lung cancer H1299 have similar results, all of which show that ADRB3 antibodies promote cell apoptosis.

Example 16 ADRB3 Plasmid Carrying Green Fluorescent Protein (GFP) Kill Pancreatic Cancer Cell SW1990

(222) 1. Test Method

(223) The carrier plasmid pENTER-ADRB3-GFP and plasmid pcDNA3/FLAG-ADRB3 are transfected into pancreatic cancer cells SW1990 and MCF7, and were observed under a confocal microscope 24 h later.

(224) 2. Test Results:

(225) All cells with GFP expression will have apoptosis, which is reflected as karyopyknosis and cytoclasis, but there is no GFP in the cytoblast (FIG. 93), suggesting that an exogenous ADRB3 carrying GFP cannot enter the cytoblast, is accumulated on the nuclear membrane, and affects the exchange of substances inside and outside the cytoblast, thereby resulting in cell apoptosis. ADRB3 carrying Flag tag proteins cannot enter the cytoblast, either, but does not affect the cell activity. ADRB3 is localized on the microtubule, cytoblast and mitochondria of MCF7 cells (FIG. 94).

Example 17 Chromatin Immunoprecipitation-Chip Test (ChIP-Chip)

(226) 1. Test Method

(227) The test group was treated with 50 ng/ml ADRB3 antibody for 24 h, while the control group was treated with mouse IgG. 270 ul 37% formaldehyde was added into a 10 cm culture dish (MCF7), gently fully mixed, and kept at room temperature for 10 min. 1 ml of 1.25M glycine was added, and kept at room temperature for 5 min to neutralize formaldehyde. The mixed solution in the dish was sucked out. The cells were washed with precooled PBS/EDTA twice, and then added 1 ml of PBS containing protease inhibitor cocktail was added. Cells at the bottom of the dish were scraped down by a cell scraper, collected into a 1.5 ml centrifuge tube, and centrifuged at 2000×g for 5 min. The supernatant was discarded. The collected samples were sent to the company for testing. The antibodies used in the immunoprecipitation were ADRB3 antibody and H3K9AC antibody. In different active states of ADRB3, the binding site of ADRB3 in the whole genome and the acetylation level of H3K9 in the promoter were analyzed.

(228) 2. Test Results (as Shown in FIG. 95)

(229) A promoter sequence (a promoter of more than 3000 genes) specifically binding to the transcription factor ADRB3 was obtained by ChIP-chip screening from ADRB3 antibody, including but not limited to the following genes: FABP5 (fatty acid-binding protein, epidermal. 82191426 to 82191897 bases with ADRB3 bound to No. 8 chromosome), FABP4, FABP3, PTPRA (receptor-type tyrosine-protein phosphatase alpha), PTPRCAP (protein tyrosine phosphatase receptor type C-associated protein), PTPN7, PTPRZ1 (receptor-type tyrosine-protein phosphatase zeta isoform), PTPRD (receptor-type tyrosine-protein phosphatase delta isoform), PTPRR, LILRB2, LILRB4, LILRA4, LILRA3, LILRP2, FES (tyrosine-protein kinase Fes/Fps), TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND RECEPTOR, STAT5B, TCL1 (T-cell leukemia/lymphoma protein 1A), CD8A (T-cell surface glycoprotein CD8 alpha chain), C8G (coding complement C8, 139838398 to 139838876 bases with ADRB3 bound to No. 9 chromosome), C5AR1 (complement C5a receptor, 47812870 to 47813391 bases with ADRB3 bound to No. 19 chromosome), AKT1 (105260668 to 105261174 bases with ADRB3 bound to No. 14 chromosome), LDHAL6B (coding lactic dehydrogenase A-like protein 6B, 59497805 to 59498262 bases with ADRB3 bound to No. 15 chromosome), SLC16A3 (The gene encodes a monocarboxylic acid transport protein and regulates the transmembrane transport of lactic acid. 80185203 to 80186585 bases with ADRB3 bound to No. 17 chromosome), RRP9 (51975065 to 51975538 bases with ADRB3 bound to No. 3 chromosome, the gene codes U3 small nucleolar RNA-interacting protein 2), DKC1 (153989742 to 153990209 bases with ADRB3 bound to X chromosome, the gene is associated with the telomere stability), IZUMO1 (izumo sperm-egg fusion protein 1, 49250722 to 49251290 bases with ADRB3 bound to No. 19 chromosome), TSKS (testis-specific serine kinase substrate, 50266261 to 50266930 bases with ADRB3 bound to No. 19 chromosome), TSSK6 (testis-specific serine/threonine-protein kinase 6), amyloid beta A4 protein isoform d, EDA2R, EDAR, PDE10A, PDE12, PDE1C, PDE4A, PDE4B, PDE4C, PDE6, PDE7, PDE9 multidrug resistance-associated protein 1 (MRP1), PSPN, brain-derived neurotrophic factor, NGDN, insulin-like growth factor II, X-linked inhibitor of apoptosis protein (XIAP), vesicle transport protein SFT2A, poly [ADP-ribose]polymerase 1, GPBP1, MYL3, GJA9-MYCBP, INHBC, SEH1L, TNPO2, NUP50, NUSAP1, GAR1, DKC1, GTPBP4, NOP56, NEXN, ACVR1, TNNT2, KCNQ1, KCNH2, ACTL6A, ATP6V0B, PAX6, DCX, TSKS, regulator of telomere elongation helicase 1, PCSK1, PCSK4, LDLRAD1, SCARF1, S1PR3, SMG6, DKC1, NOP10, BAD, BCL2, CASP12, CFLAR, LAMB3, ITGA2, ITGA7, MMP2, MMP9, RYR3, syntaxin-1B, PACRG, transcription factor Dp-2, transcription factor MafB, transcription factor E2F8, transcription factor E2-alpha isoform E47, hepatocyte nuclear factor 1, mitofusin-1, FGFR2, FGF16, FGF21, HEY1, HEY2, TLE3, TXNIP, RIZ1, IFT140, NPHP3, GATA1, GATAD1, BDNF, PDGF, CDY1, CDY2, APP (Amyloid Beta Precursor Protein), PNMT, COMT, ZMIZ2, DNMT1, SETDB2, SUV39H1, CEACAM1, CEACAM16, KAZN, CEACAM21, CEACAM5, ENO1, FGF1, FGF4, GREM1, TMC6, TMEM43, STK36, IRF1 (interferon regulatory factor 1), WHSC1, IRF3, IRF7, IRF9, cardiotrophin-like cytokine factor 1, tumor protein 63 (189349333 to 189349707 bases with ADRB3 bound to No. 3 chromosome), TPRG1, FKBP5, PRR5 (45063166 to 45063715 bases with ADRB3 bound to No. 22 chromosome), SETD1B, p16, myb-related protein B (42294360 to 42294625 bases with ADRB3 bound to No. 20 chromosome), RPL18A, optineurin, bone morphogenetic protein receptor type-1B, prostacyclin receptor (PTGIR), pentraxin-4, CTBP1, FES, ZAP70, IGSF11, IRF7, KLRC1, TARDBP, MRPL19, MRPL2, MRPL41, MRPS17, MRPS26, RPL14, RPL18A, RPL19, RPS6KL1, atypical chemokine receptor 3, CCL3L1, CCR5, EEF1D, DNMT1, PDE4B, FOXP3 (49121975 to 49122469 bases with ADRB3 bound to X chromosome), DCANP1, DCST1, DCST2, leukemia inhibitory factor, IL17RB, TRAF6, p62, hexokinase-2 (75059882 to 75060125 bases with ADRB3 bound to No. 2 chromosome), TCIRG1, HK1, CAMKK2, GAPDH, G1/S-specific cyclin-D3 (42016706 to 42017624 bases with ADRB3 bound to No. 6 chromosome), Cyclin D1, cyclin-dependent kinase 3 (73995762 to 73996227 bases with CDK3 and ADRB3 bound to No. 17 chromosome), CDK4, CDK15, CDK18, cyclin-dependent kinase 2-associated protein 1, Rb, Ki-67, mTOR, Rictor, AKT, tubulin, Src (proto-oncogene tyrosine-protein kinase Src), PD-L1, p53, interleukin 1 (IL-1), interleukin 6 (IL-6), IL25, IL27, IL17RB, IL16, LEF, ADRB3, TGFB1I1, TAB2, LTBP2, RAS, ARHGAP21, TGF- and TNF.

(230) CHIP-chip results of H3K9AC antibody show that: K9 of the H3 histone in the CDK3 promoter area to which ADRB3 is bound (73995762 to 73996227 bases of No. 17 chromosome) is acetylated, showing that ADRB3 is acetylase transferring the acetyl to H3K9. ADRB3 affects the transcription of target genes by regulating the acetylation level of H3K9 within the target gene promoter. ADRB3 can bind to the promoter of neurotrophic factor, indicating that ADRB3 regulates the expression of neurotrophic factor, suggesting that ADRB3 antibodies can treat Alzheimer disease, Parkinson's disease and other neurodegenerative diseases.

Example 18 Endotoxin-Induced Death Test

(231) 1. Test Method

(232) LPS was intraperitoneally injected into mice (10 normal FVB mice and 10 ADRB3 knockout mice) at a dose of 30 mg/kg. LPS was dissolved in water for injection to prepare into 1 mg/ml solution. The injection dosage (ml) was obtained by multiplying the mouse weight (g) by 0.03. The death of mice was observed, and the death time was recorded to draw the survival curve of mice in each group. Inflammatory necrosis and granulocytic infiltration of liver and lung tissues were observed after HE staining. Neutrophils in the abdominal secreta were detected by immunofluorescence of cell smears. Expressions of IL-6, MPO and Elastase in liver and lung tissues were detected by immunohistochemical analysis.

(233) 2. Test Results

(234) After injection of endotoxin, the vitality of mice was significantly reduced, their body temperature was decreased, the eyes of some mice were festered, and they showed pyohemia. Within 16 hours after injection of LPS, 10 FVB mice all died, 2 ADRB3.sup.−/− mice died, but 8 ADRB3.sup.−/− mice survived. Compared with normal mice, the mortality of ADRB3.sup.−/− mice caused by pyohemia was significantly reduced (FIG. 96), and the area of inflammatory necrosis of liver and lung tissues was significantly reduced. There were a lot of festers in the abdomen of both groups, and the mice had peritonitis accompanied with phohemia. The count of neutrophils recruited to the peritoneal lesion was significantly reduced in ADRB3 knockout mice with peritonitis. The expressions of IL-6 and Elastase protein in liver and lung tissues of ADRB3.sup.−/− mouse were significantly reduced, indicating that inflammatory response and neutrophilic infiltration of liver and lung tissues were inhibited.

Example 19 ADRB3 Antibody Inhibits Adenovirus and Hepatitis B Virus (HBV) from Infecting Cells

(235) 1. Test Method

(236) Coverglass for growth of A549 were pretreated with the ADRB3 antibody for 30 min, inoculated with 10.sup.6 PFU/ml adenovirus carrying GFP, and incubated in an incubator at 37° C. for 24 hours. The control group was treated with mouse IgG. The expression of phosphorylated mTOR (S2448) was detected by immunofluorescence. The method is the same as that in Example 1.

(237) Liver tissue samples of patients with chronic hepatitis B were collected to detect the expression of ADRB3 by immunohistochemical analysis following the method of Example 2.

(238) Coverglass for growth of HepG2 were pretreated with the ADRB3 antibody for 30 min, inoculated with 1 ml of HBV, and incubated in an incubator at 37° C. for 24 hours. The control group was treated with mouse IgG. The coverglass was immobilized with 4% paraformaldehyde, and HBcAg was detected with rabbit anti-HBC (1:100). The coverglass was kept at 4° C. overnight, and sheep anti-rabbit IgG was added on the next day. The coverglass was kept at 37° C. for 1 hour, and washed. PAP (1:50) was added. The coverglass was incubated at 37° C. for 1 h, washed 3 times, and then developed with DAB for microscopic examination.

(239) 2. Test Results

(240) (1) When ADRB3 was suppressed, adenovirus was unable to infect lung cancer cells A549. The ADRB3 antibody plays a role in inhibiting adenovirus infection by inhibiting phosphorylation of 2448th serine of mTOR (FIG. 97). Phosphorylation of 2448th serine will activate mTOR and promote autophagy, and autophagy promotes the replication and release of adenovirus. The ADRB3 antibody plays a role in inhibiting autophagy by inhibiting activation of mTOR, thereby inhibiting the replication and release of adenovirus. After blocking ADRB3, the ADRB3 antibody can increase the expression of C8, which can dissolve the virus and prevent viral infection.

(241) (2) Both the ADRB3 and adenovirus are localized in nucleoli of lung cancer cell A549, and the ADRB3 may be a key protein required for viral replication (FIG. 98). Adenovirus can cause cell carcinogenesis by activating ADRB3 in the nucleolus.

(242) (3) The ADRB3 in cytoplasm of liver of a hepatitis B patient is higher than that of normal subjects, and almost no ADRB3 is expressed in the cytoblast. The ADRB3 in the cytoplasm enhances ribosomal functions, and contributes to synthesis of the proteins required for HBV replication. The ratio of cytoplasmic ADRB3 to nuclear ADRB3 is increased significantly, and the higher the ratio is, the more active the viral replication is. This ratio can be used as an index for detecting hepatitis B virus replication. The ADRB3 in cytoplasm mediates HBV to enter HepG2 and promotes HBV replication. The ADRB3 antibody inhibits the HBV from infecting HepG2 (FIG. 99), indicating that the antibody has the potential for treating hepatitis B.

Example 20 Research on the Effect of High Cholesterol (HCD) and Low Cholesterol Feeding (LCD) on Atherosclerotic Plaque in ADRB3 Deficient ApoE.SUP.−/− .Mice (B3.SUP.−/−.ApoE.SUP.−/−.)

(243) 1. Test Method

(244) The effect of high cholesterol (HCD) and low cholesterol feeding (LCD) on atherosclerotic plaque in ADRB3 deficient ApoE.sup.−/− mice (B3.sup.−/−ApoE.sup.−/−) was researched, and the B3.sup.+/+ApoE.sup.−/− mice feeding on HCK were selected as the control group. The laboratory mice were divided into four groups: 1. High cholesterol (HCD) feeding group of B3.sup.−/− ApoE.sup.−/− mice; 2. HCD group of B3.sup.−/− ApoE.sup.−/− mice; 3. low cholesterol feeding (LCD) group of B3.sup.−/− ApoE.sup.−/− mice; and 4. HCD feeding and ADRB3 antibody combined treatment group of B3.sup.+/+ ApoE.sup.−/− mice. 10 male mice of 8 weeks old were selected for each group, and were fed with high fat (20% triglyceride, 1.25% cholesterol) and common feed. The ADRB3 antibody was fed at a dose of 10 mg/kg. The mice were intraperitoneally injected once every 6 days. The aorta was taken after feeding with high fat for 12 weeks, and stained with oil red O. The number and size of arterial plaques in 4 groups were compared. Inflammatory cells in plaques were detected after HE staining. The expression of inflammatory factor in plaques was detected by immunohistochemical analysis. Inflammatory cytokines IL-6, VEGF, IL-10 and MPO in serum were detected by ELISA.

(245) 2. Test Results (as Shown in FIG. 100)

(246) After feeding with high fat feed for 3 months, ApoE.sup.−/− mice were extremely aged with hunchback, hair slip, subcutaneous fat, movement function loss, etc., but B3.sup.−/− ApoE.sup.−/− mice had unconspicuous aging characteristics. The number and area of aortic plaques in B3.sup.−/− ApoE.sup.−/− mice of HCD group and B3.sup.+/+ ApoE.sup.−/− mice of ADRB3 antibody treatment group were significantly less than those in B3.sup.+/+ ApoE.sup.−/− mice, and the area of aortic plaques was reduced by 80%-90%. ADRB3 deficient ApoE.sup.−/− mice had hard plaques, which were stable plaques. In B3.sup.−/− ApoE.sup.−/− mice, the blood fat concentration of HCD group was significantly higher than that of LCD group, but there is no difference between the number and area of aortic plaques of both groups. Compared with B3.sup.+/+ ApoE.sup.−/− mice of HCD group, B3.sup.−/− ApoE.sup.−/− mice of HCD group and B3.sup.+/+ ApoE.sup.−/− mice of ADRB3 antibody treatment group had thickened fibrous caps of aortic plaques, reduced CD4.sup.+T cell and granulocyte infiltration, MPO in foam cells of plaques, neutrophil Elastase, IL-6, ADRB3 and reduced MIF. The concentration of IL-10 in serum of B3.sup.−/− ApoE.sup.−/− mice is increased, but the concentrations of IL-6, VEGF and MPO are reduced.

(247) Conclusions: (1) ADRB3 promotes the formation of atherosclerotic plaques, results in plaque instability, and is one of the factors causing acute coronary syndrome; and (2) ADRB3 promotes aging.

Example 21 ADRB3 Monoclonal Antibody Reduces Blood Pressure of Spontaneously Hypertensive Rats

(248) 1. Test Method

(249) Male spontaneously hypertensive rats (SHR) of 14 weeks old with the systolic pressure higher than 140 mmHg were randomly divided into 2 groups with 8 rats in each group, and were intraperitoneally injected once every 6 days for 6 consecutive weeks. The groups were as follows: (1) antibody group: 10 mg/kg ADRB3 antibody; (2) control group: intraperitoneally injected with mouse IgG. The systolic pressure and heart rate were indirectly examined at caudal artery of rats in a waking state using a RBP-1B rat blood pressure gauge by tail cuff method. The systolic pressure, heart rate and body weight were examined at caudal artery before and after injection every week to calculate the pressure reduction extent=(systolic pressure before treatment−systolic pressure after treatment)/systolic pressure before treatment. Rats were put to death 6 weeks after injection, and their chests were cut open. Their hearts were immediately cut out and put in KH solution at 4° C., residual blood in their cardiac chambers was squeezed out, their ascending aortas were separated, and their aortas were hung, after retrograde intubation, on a Langendorff perfusion device for retrograde perfusion with KH solution fully oxygenated with 95% O.sub.2 and 5% CO.sub.2 at a constant temperature of 37° C. The catheter balloon was inserted into the left ventricle through the left atrium catheter, the catheter was connected to a pressotransducer, water was injected into the balloon to enhance the internal pressure therein to 40 mmHG and keep under the internal pressure. After the indexes were stable by prefilling for 20 min, Pclab biological signals were used to acquire and record the cardiac function indexes of the system: left ventricular pressure (LVP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP), maximum rate of left ventricular systolic pressure rise (+dp/dtmax), maximum rate of left ventricular diastolic pressure descendent (dp/dtmax) and heart rate (HR). The coronary flow (CF) was collected and recorded. After stopping filling, the heart was taken out and dried with filter paper. The aorta was cut out and weighed to calculate the heart weight/body weight (HW/BW); the free wall and atrium of the right ventricle were cut out, the left ventricle (including the ventricular septum) was weighed, and the left ventricular hypertrophy degree was expressed as the ratio of the left ventricle weight to the body weight (LVW/BW). SPSS19 was used for statistical analysis, and the measurement data were expressed as mean±standard deviation, and t-test was used in the mean comparison between two groups.

(250) 2. Test Results

(251) The blood pressure of the ADRB3 antibody treatment group was reduced to normal blood pressure (Table 4), and was decreased significantly compared with that before treatment and that of the control group (P<0.01). The pressure reduction difference and extent of the ADRB3 antibody group were significantly increased, compared with those of the control group, and the difference was statistically significant (P<0.01). The effect of ADRB3 antibody on cardiac functions is shown in Table 5. LVEDP of the antibody treatment group was significantly lower than that of the control group (P<0.05), and the HR was significantly reduced, compared with that of the control group (P<0.05), indicating the improvement of cardiac functions. The changes of heart weight/body weight and left ventricle weight/body weight of each group were shown in Table 6. HW/BW and LVW/BW of the antibody group were significantly lower than those of the control group, and the difference was statistically significant (P<0.05). The ADRB3 antibody effectively reduces SHR blood pressure to normal range, improves cardiac functions of SHR, and blocks left ventricular hypertrophy caused by hypertension.

(252) TABLE-US-00005 TABLE 4 Changes of Systolic Pressure of Spontaneously Hypertensive Rats Before and After Treatment (mmHg) (x ± s, n = 8) Pressure Pressure reduction Before After reduction extent Group treatment treatment difference (%) Control group 174.5 ± 12.2 180.5 ± 8.0 −5.5 ± 4.5 −3.6 ± 3.2 Antibody group 177.6 ± 4.4  98.5 ± 14.4*.sup.# 80.1 ± 12.5.sup.# 45.4 ± 8.6.sup.# Note: compared with that before treatment, *P < 0.01; compared with the control group, .sup.#P < 0.01.

(253) TABLE-US-00006 TABLE 5 Cardiac Functions of Spontaneously Hypertensive Rats (x ± s, n = 8) Group LVSP LVEDP LVDP +dp/dtmax −dp/dtmax HR CF Control group 105 ± 10 36 ± 4  87 ± 16 2765 ± 607 1456 ± 356 202 ± 18 4.4 ± 2.1 Antibody group  90 ± 32 22 ± 3* 80 ± 22 1729 ± 759  732 ± 280 152 ± 26 2.4 ± 0.9 Note: compared with the control group, *P < 0.05; LVP, LVSP, LVEDP and LVDP: mmHg; CF: ml/min.

(254) TABLE-US-00007 TABLE 6 Heat Weight/Body Weight, Left Ventricle Weight/Body Weight of Spontaneously Hypertensive Rats (x ± s, n = 8) Group HW/BW (mg/g) LVW/BW (mg/g) Control group 5.6 ± 0.2  2.5 ± 0.3  Antibody group 4.1 ± 0.3* 1.5 ± 0.2* Note: compared with the control group, *P < 0.05.

Example 22 ADRB3 Antibody Induces Peripheral Blood Mononuclear Cell (PBMC) to Differentiate Toward Cytotoxic T Cells

(255) 1. Test Method

(256) 3-5 ml of peripheral blood samples were taken from each of 5 normal subjects, 5 pancreatic cancer patients, 5 liver cancer patients, 5 lung cancer patients and 5 leukemia patients. PBMC was separated with Ficoll by density gradient centrifugation, cultured in 5% fetal bovine serum DMEM, and inductively cultured with different concentrations of ADRB3 antibody. ADRB3 antibodies were not used in the control group. Cells were collected respectively on 0th, 2nd, 4th, 6th, 8th and 10th days of inductive culture to quantitatively analyze CD4.sup.+, CD8.sup.+T cells and Treg cells by flow cytometry. Induced cells were lysed and centrifuged, and the supernatant was taken to validate the killing effect of induced differentiation on target cells by MTT assay. SW1990, MCF-7 and other adherent tumor cells were selected as target cells, and were inoculated into 96-well culture plate. In the experimental group, the supernatant was lysed with different concentrations of cells, and the control group was cultured in a culture medium containing equivalent volume of solvent at 37° C. under 5% CO.sub.2 for 5 d. 200 μl of a fresh serum free medium containing 0.5 mg/ml MTT was added to each well, and further continued for another 4 h. After the liquid in each well was fully sucked out, 200 ul of DMSO was added, and shaken for 10 min to measure the optical density value with an ELIASA at a wavelength of 570 nm and a reference wavelength of 450 nm.

(257) 2. Test Results:

(258) In the 0.5-100 ug/ml dose range, the ADRB3 antibody promotes PBMC to differentiate into CD8.sup.+T cells, and decrease the proportion of Treg cells. When the given concentration of antibody was applied for 6 to 8 days, the anticancer cytokines in induced cells had higher effects. The highest dose of cell lysate could kill about 80% target cells. The ADRB3 antibody induces immune incompetent lymphocytes to become functional cells and play an anti-cancer role.

Example 23 ADRB3 Antibody Promotes Complement Activation

(259) Cells MCF-7, A549 and SW1990 were treated with different concentrations of ADRB3 antibody. Endotoxin (LPS) was used as a positive control. 2 days later, cell protein was collected, and complements C3, C8 and C5 (C5R) were detected by western blot. C8 and B3AR were detected by immunofluorescence.

(260) The test results show that the ADRB3 antibody dose-dependently increases expressions of C3, C8 and C5R (FIG. 101). After ADRB3 antibody treatment, a large number of C8 arise on the cell membrane, but B3AR disappears, and there are karyopyknosis and apoptosis (FIG. 101B). C8 is a membrane attack complex (MAC), and forms an indispensable important material. The ADRB3 antibody promotes MAC assembly and insertion into the cell membrane, thereby resulting in cell rupture and death.

Example 24 Effect of ADRB3 Antibody on Cardiovascular System

(261) The research results show that the ADRB3 antibody has different degrees of antagonism to experimental myocardial infarction or arrhythmia, and other animal models, improves the imbalance between supply and demand of oxygen in ischemic myocardium. ADRB3 antibody alleviates the myocardial ischemia degree in the model of anterior descending ligation of myocardial infarction and narrows the infarct range after coronary artery occlusion. The rats are intraperitoneally injected with ADRB3 antibody to antagonize arrhythmia caused by aconitine. ADRB3 antibody reduces aging myocardial cells and improves cardiac functions.

Example 25 Effect of ADRB3 Antibody on Blood System

(262) The research results show that ADRB3 is located on the platelet membrane (FIG. 102). The ADRB3 antibody can improve microcirculation, inhibit platelet aggregation, and have a significant inhibitory effect on in-vitro thrombosis in rats.

Example 26 Effect of ADRB3 Antibody on Respiratory System

(263) The research results show that the ADRB3 antibody reduces the flow rate in lungs of perfused guinea pigs, has the bronchocnstricting effect, and has antispasmodic effect on isolated intestinal spasm caused by acetylcholine.

Example 27 Antifatigue Effect of ADRB3 Antibody

(264) Rats were loaded with 5% body weight of lead sheath at the root of the tail, and were placed in a swimming pool with the water depth of 30±1 cm and at a water temperature of 25±1° C., where four legs of each rat were kept moving. The time since the mice began to swim to death time (min) was recorded as the swimming time of the mice. The results show that the mice injected with the ADRB3 antibody have longer swimming time in ice water than the control group, and the ADRB3 antibody has anti-fatigue effect.

Example 28 ADRB3 Antibody Improves Sleep Function

(265) The results show that the ADRB3 antibody could shorten the sleep latency of mice, increase the number of sleeping mice, and significantly prolong the sleep duration of mice administered with threshold dose of pentobarbital sodium.

Example 29 Therapeutic Effect of ADRB3 Antibody on Mouse Model of Alzheimer Disease (AD)

(266) The research results show that the ADRB3 antibody could improve the learning and memory of AD mice, increase the expression of brain-derived neurotrophic factor (BDNF) gene, and reduce the senile plaque formation caused by intracerebral Aβ deposition.

Example 30 ADRB3 Antibody has the Effect of Regulating Blood Lipids

(267) The research results show that injection of the ADRB3 antibody could reduce the blood triglyceride, total cholesterol and LDL level of rabbits with experimental hypercholesterolemia, enhance HDL, stabilize plaques, and reduce aortic and coronary atherosclerotic plaque formation.

Example 31 ADRB3 Antibody Inhibits Adenovirus and Hepatitis B Virus (HBV) from Infecting Cells

(268) The research results show that the ADRB3 antibody can reduce the liver fibrosis degree of C57BL mice model of liver cirrhosis by intraperitoneal injection of CCL4, inhibit inflammatory cell infiltration around liver tissue portal area and bile duct, reduce ascites caused by liver cirrhosis, and improve liver functions.

Example 32 ADRB3 Antibody Inhibits Replication of Hepatitis B Virus (HBV)

(269) The ADRB3 antibody is transfected into HBV transgenic mice by caudal vein injection, and blood samples were taken from their angular veins respectively on 6th day and 21st day, in 1st month, 3rd month and 9th month after injection to quantitatively detect the HBsAg level in serum of mice by chemiluminescence and detect the HBV-DNA level by PCR. The results show that the ADRB3 antibody could significantly reduce the HBsAg and HBV-DNA level in serum of transgenic mice.

Example 33 ADRB3 Antibody Improves Fertility and Sexual Function

(270) The ADRB3 antibody can enhance the sexual behavior ability of mice. The results of mating experiments show that the immune cell lysate can significantly increase the number of sexual impulses of impotence model mice, increase the mating rate, prolong the mating time, and increase the impregnation rate of female mice, the frequency of giving births and the survival rate of baby mice.

Example 34 Effect of ADRB3 Antibody on Autoimmunity and Survival Time of Lupus Mice

(271) The research results show that the ADRB3 antibody could prolong the survival time of BXSB lupus mice, reduce the expression levels of anti-ds-DNA antibody and IgG in the peripheral blood, and inhibit the expression of IFN-γ in spleen and kidney.

Example 35 Research on Treatment of Aged Mice

(272) Healthy C57BL/6 mice, regardless of male and female, were selected and divided into 2 groups: 10 mice in the ADRB3 antibody treatment group and 10 mice in the normal saline control group. Treatment was started when the mice were 12 months old. The ADRB3 antibody treatment group was intraperitoneally injected with 0.1 mg of ADRB3 antibody once every week for a total of 3 months; the normal saline control group was injected with normal saline. Behavioral tests were performed when the mice were 12, 15, 18 and 21 months old, including passive avoidance experiment, Morris water maze experiment, open field maze experiment and elevated plus maze experiment.

(273) Behavioral results show that in the Morris water maze experiment, as shown by the positioning navigation experiment, mice in the normal saline control group show a longer escape latency; as shown by the space exploration experiment, the distance percentage in the platform quadrant of mice in the normal saline control group is decreased and the time percentage was decreased, which are significantly different from the ADRB3 antibody treatment group.

(274) The anxiety index was detected from the perspective of behavioristics: as shown by the open field maze experiment results, the distance percentage of mice in the normal saline control group in the central area is significantly decreased, which is significantly different from the ADRB3 antibody treatment group.

(275) As shown by the elevated plus maze experiment results, the distance percentage in the arm opening area and the frequency percentage of arm opening of mice in the normal saline control group are significantly decreased, which are significantly different from the ADRB3 antibody treatment group.

(276) The passive avoidance experiment results show that compared with the ADRB3 antibody treatment group, the normal saline control group has more errors and shorter latent period.

(277) The HE staining results show that the ADRB3 antibody treatment group is close to healthy mice of 12 months old, has a large number of brain cells arranged in order, and has complete neuron cells. However, mice in the normal saline control group have obvious neuron vacuole-like degenerations, and the cells are loosely arranged.

Example 36 Research on Glioma Treatment

(278) Human glioma cells U87 were subcutaneously transplanted into the back of nude mice of 6 weeks old, which were used as the tumor model after tumor formation. In the experiment, the mice were divided into 4 groups with 8 mice in each group: normal control group, model control group, ADRB3 antibody treatment group, Gemcitabine and CD40 antibody combined treatment group. The ADRB3 antibody treatment group was intraperitoneally injected with 0.1 mg of ADRB3 antibody once every 5 days for a total of 6 weeks. The model control group was intraperitoneally injected with equivalent amount of normal saline.

(279) The results show that compared with the control group and Gemcitabine treatment group, no metastatic lesion is seen in the lung of the ADRB3 antibody treatment group, metastatic lesions are seen in the control group, and some metastases are found in the Gemcitabine treatment group; and the survival period of the ADRB3 antibody treatment group is significantly longer than that of other groups.

Example 37 Research on Treatment of In-Situ Bladder Cancer in Rats

(280) The in-situ bladder cancer model was constructed by inducing N-methylnitrosourea (MNU). In the experiment, the mice were divided into 4 groups with 8 mice in each group: normal control group, model control group, ADRB3 antibody treatment group, Gemcitabine and CD40 antibody combined treatment group. The ADRB3 antibody treatment group was intraperitoneally injected with 0.5 mg of ADRB3 antibody once every 5 days for a total of 7 weeks. The model control group was intraperitoneally injected with equivalent amount of normal saline.

(281) The results show that the tumor size of the ADRB3 antibody treatment group is reduced by 70% on average, and the tumor no longer continue to grow after 30 days of continuous treatment.

Example 38 Research on Treatment of Leukemia in Mice

(282) The leukemia mouse model was constructed by transplanting human B-lymphocytic leukemia cells NALM-6 into highly immunodeficient NCG mice via caudal vein. In the experiment, the mice were divided into 4 groups with 8 mice in each group: normal control group, model control group, ADRB3 antibody treatment group, Gemcitabine and CD40 antibody combined treatment group. The ADRB3 antibody treatment group was intraperitoneally injected with 0.1 mg of ADRB3 antibody once every 5 days for a total of 5 weeks. The model control group was intraperitoneally injected with equivalent amount of normal saline.

(283) The results show that compared with other groups, the body weight, survival state and survival period of the ADRB3 antibody treatment group are better than those of other groups; the count of NALM-6 cells is decreased significantly, and the surface markers of T cells detected by flow cytometry suggest that the immune function of T effector cells in the treatment group is significantly better than that of the control groups.

Example 39 Research on Treatment of Diabetes in Mice

(284) After diabetic mice (db/db mouse) of 8 weeks old were fasted without water fasting for 12 hours, blood samples were taken from the inner canthus to measure the fasting blood glucose (FGB). The mice with the FGB of above 11.1 mmol/L were randomly divided into 2 groups: 8 mice in the model control group and 8 mice in the ADRB3 antibody treatment group. The ADRB3 antibody treatment group was intraperitoneally injected with 0.1 mg of ADRB3 antibody once 5 five days for a total of 5 weeks. The model control group was intraperitoneally injected with equivalent amount of normal saline. After the last administration, blood samples were taken from the orbit to measure the blood glucose and blood lipid indexes.

(285) The results show that: (1) the ADRB3 antibody group reduces blood glucose in db/db mice (P<0.01). (2) The ADRB3 antibody reduces the total cholesterol and LDL-C content and enhances the HDL-C content in db/db mice, which contributes to recovery of the blood lipid level in diabetic mice (P<0.01).

Example 40 Research on Treatment of Schizophrenia in Mice

(286) The schizophrenia mouse model was established by intraperitoneally injecting C57BL/6 male mice of 8 weeks old with 0.5 mg/kg dizocilpine maleate (MK801). Symptoms similar to schizophrenia were observed: after hyperlocomotion and stereotypy, the mice were divided into the model control group (without injection of MK801), model group (injection of MK801) and ADRB3 antibody treatment group (injection of MK801+ADRB3 antibodies). The ADRB3 antibody treatment group was intraperitoneally injected with 0.1 mg of ADRB3 antibody once 5 five days for a total of 5 weeks. The model control group and the model group were intraperitoneally injected with equivalent amount of normal saline. The open field test and prepulse inhibition (PPI) test were conducted to evaluate and compare the spontaneous activity and sensorimotor gating function of mice in each group.

(287) The results show that: compared with the control group, mice in the model group show significantly increased activity [total movement distance (1622±146.7) cm vs (5502±432.4) cm; spontaneous movement times (122±16.5) vs (332.6±24.3) times; P<0.001] and PPI damaged [78 dB: (35.5±1.6) vs (11.4±2.1), 84 dB: (46.2±5.6) vs (17.4±3.6); P<0.01). Compared with the model group, the ADRB3 antibody treatment group has significantly decreased activity [(2655±331.4) cm, (192.3±17.3), and significantly improved PPI abnormality.

Example 41 Position and Functions of ADRB3 in NK Cells

(288) By double staining umbilical cord blood and adult blood smears with ADRB3 and CD56, it is found that there is a lot of ADRB3 in NK cells of umbilical cord blood, and a little ADRB3 in lymphocytes of cord blood (FIG. 103). There is also ADRB3 in adult NK cells. ADRB3 increase in adult lymphocytes is associated with aging. The ADRB3 in NK cells is positively correlated with contents of CD56, and the more ADRB3, the more CD56. CD56.sup.bright is naive NK, while ADRB3 increases CD56, and inhibits NK differentiation and maturation. NK92 cells are made into cell smears to detect ADRB3 and MPO. It is found that ADRB3 and MPO are highly expressed in NK92 cells (FIG. 104). The ADRB3 antibody (5-50 ng/ml) was used to treat NK92 cells. 2 days later, the cells were lysed to detect MPO/CD56 protein contents by western blot. It is found that the ADRB3 antibody dose-dependently reduces MPO and CD56 proteins. ADRB3 has the effects of inhibiting NK cells and preventing NK cells from attacking autologous normal cells.

Example 42 ADRB3 Regulates Monocytes and Megakaryocytes

(289) Immunofluorescence was used to detect the distributions and contents of ADRB3 in monocytes and megakaryocytes in the peripheral blood of normal subjects and patients with acute myocardial infarction. It is found that ADRB3 is highly expressed in monocytes (FIG. 105) and megakaryocytes of the patients with acute myocardial infarction, is significantly more than that of normal subjects, and there is ADRB3 in the mononuclear cytoblast in proliferation phase.

(290) In patients with acute coronary syndrome, the ADRB3 content in megakaryocytes of patients with positive cardiac troponin T (cTnT) is higher than that in megakaryocytes of patients with negative cTnT (FIG. 106).

(291) In animal experiments, Balb/c mice were intraperitoneally injected with the ADRB3 antibody (1 mg/kg body weight) once every 3 days. 2 weeks later, the coagulation function was detected in mice. It is found that the ADRB3 antibody reduces the platelet count, inhibits platelet aggregation, and has a significant inhibitory effect on in-vitro thrombosis in rats. It is found that the ADRB3 antibody reduces the platelet count, inhibits platelet aggregation, and has a significant inhibitory effect on in-vitro thrombosis in rats.

Example 43 ADRB3 Fusion Gene Detection

(292) The ADRB3 fusion genes in the pancreatic cancer cell Panc-1, cancer tissues of pancreatic cancer patients and paracancerous normal tissues were detected by exon sequencing method. It is found that the ADRB3 gene forms fusion genes with a large number of cancer-associated genes, such as MYC, RAS, SRC, MPO, PML, Her2, EGFR, B7, CD8 and CD28. When Panc-1 is treated with ADRB3 antibody, ADRB3 fusion genes are significantly reduced.

Example 44 ADRB3 Antibody Treats Heroin Addiction

(293) SD adult male rats were randomly divided into 2 groups: heroin addiction group and treatment group. The rat model of heroin addiction was established by subcutaneously injecting the rats with heroin 3 times every day at an increasing dose from 10 mg/kg on 1st day to 100 mg/kg on 10th day. Rats in the treatment group were intraperitoneally injected with the ADRB3 antibody (5 mg/kg) after injection of heroin every day. On the next day after the last injection of heroin, rats were intraperitoneally injected with naloxone hydrochloride (4 mg/kg) for 2 h, and then had withdrawal symptoms, such as body torsion, wet dog-like tremble, gritting their teeth, jumping and standing. Thus the animal model of addiction was successfully established. After scoring the withdrawal symptoms, animals in each group were anesthetized to take the whole brain for determination of ADRB3 protein expression in related brain regions of rats by immunohistochemical analysis.

(294) The results show that the withdrawal score of rats in the heroin addiction group is 21.7+4.4, that in the ADRB3 antibody treatment group is 11.7+2.8, and there is significant difference between the two groups (P<0.05). The number of ADRB3-positive neurons expressed in rats of the heroin addiction group is significantly reduced than that in rats of the treatment group (P<0.05). Conclusions: ADRB3 antibody reduces heroin addiction in rats, and the mechanism thereof is related to regulation of the neuronal ADRB3 signal transduction.

SUMMARY

(295) In conclusion, the ADRB3 is a key receptor in nerve-endocrine-immunoregulatory network, and an ADRB3-mediated signaling pathway regulates proliferation and differentiation of neutrophils, lymphocytes and tumor cells. Under normal circumstances, the ADRB3 maintains the non-specific immunocompetence and specific immunocompetence of an organism, and eliminates exogenous pathogenic microorganisms and aged organism tissues to play a role in protecting the organism and anti-aging. Under pathological conditions, excessive activation of the signaling pathway will cause systemic chronic inflammation, and destroy immune homeostasis. A monoclonal antibody for the ADRB3 can specifically bind to the ADRB3, regulate (block or excite) its activity, and be used to treat inflammation, viral infection, atherosclerosis, diabetes, neurodegeneration, autoimmune disease, lalignant tumor, aging disease and the like.