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WO2022083049A1 - 抗cd73的抗体及其用途 - Google Patents

抗cd73的抗体及其用途 Download PDF

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WO2022083049A1
WO2022083049A1 PCT/CN2021/080517 CN2021080517W WO2022083049A1 WO 2022083049 A1 WO2022083049 A1 WO 2022083049A1 CN 2021080517 W CN2021080517 W CN 2021080517W WO 2022083049 A1 WO2022083049 A1 WO 2022083049A1
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seq
antibody
amino acid
sequence
fibrosis
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PCT/CN2021/080517
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French (fr)
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夏瑜
王忠民
金小平
李百勇
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中山康方生物医药有限公司
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Definitions

  • the present invention relates to the field of immunology.
  • it relates to anti-CD73 antibodies and uses thereof.
  • Ecto-5'-nucleotidase the CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70KD encoded by the NT5E gene, which is mediated by glycosylphosphatidylinositol (glyocsyl phosphatidy linositol, GPI) is anchored to the cell membrane (Zimmermann H. Biochem J. 1992;285:345-365).
  • CD73 is widely distributed on the surface of human tissue cells and is also widely expressed on the surface of immune cells, such as dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), and myeloid-derived suppressor cells (MDSC).
  • immune cells such as dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), and myeloid-derived suppressor cells (MDSC).
  • CD73 has both hydrolase activity and non-hydrolase activity.
  • One of the immunosuppressive mechanisms of the enzymatic and non-enzymatic functions of CD73 is mediated by the CD73-Adenosine metabolic signaling pathway.
  • CD39 upstream of CD73 can catalyze the production of adenosine monophosphate (AMP) from ATP, and the generated AMP is catalyzed by CD73 It is converted into adenosine, and adenosine binds to the downstream adenosine receptor (A2AR).
  • A2AR inhibits a series of immune activation-related signaling pathways such as LCK, MAPK, and PKC by activating protein kinase A (PKA) and Csk kinase.
  • PKA protein kinase A
  • CD73 is expressed in human B cells, T cells, bone marrow cells, bone marrow stromal cells and thymic epithelial cells.
  • Organ fibrosis refers to the necrosis of parenchymal cells of organs, fibrous connective tissue, necrosis of parenchymal cells due to various chronic stimuli, such as pathogenic microorganism infection, inflammation, immune response, toxins (drugs), radiation, ischemia and hemodynamic changes, etc. Pathological processes including abnormal increase and excessive deposition of cellular components and extracellular matrix (ECM).
  • pathogenic microorganism infection such as pathogenic microorganism infection, inflammation, immune response, toxins (drugs), radiation, ischemia and hemodynamic changes, etc.
  • Pathological processes including abnormal increase and excessive deposition of cellular components and extracellular matrix (ECM).
  • ECM extracellular matrix
  • organ fibrosis with unknown pathogenic mechanism called idiopathic or primary organ fibrosis, such as idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • the milder degree is called fibrosis, and the severe one causes the destruction of the tissue structure
  • the fibrous connective tissue deposited in the tissues of fibrotic organs includes two major parts: cellular components and ECM. tissue parenchyma cells, fibroblasts and mononuclear phagocytes; and collagen, non-collagen glycoproteins, proteoglycans and elastic fibers.
  • transforming growth factor- ⁇ transforming growth factor- ⁇
  • CTGF connective tissue growth factor
  • PDGF platelet-derived growth factor
  • bFGF basic fibroblast growth factor
  • epidermal growth factor EGF epidermal growth factor
  • IL-1, IL-6, IL-8, TNF- ⁇ , IFN- ⁇ , etc. are also involved in the occurrence of diseases (Wynn TA.J Pathol.2008;214(2):199-210) .
  • TGF- ⁇ signaling regulates ECM deposition in breast cancer through an adenosine-dependent mechanism (Vasiukov G, et al. Cancer Res. 2020;80(12):2628-2638).
  • Tissue blood supply disorder is a risk factor for organ fibrosis, and the institutional changes caused by organ fibrosis can often lead to tissue blood supply disorder and tissue cell hypoxia.
  • hypoxia-inducible factor-1 HIF-1
  • IIF-1 Isolecular upregulation, resulting in widespread expression of CD73
  • A2B receptor A2BR
  • A2BR is more easily activated under hypoxia, and the activation of A2BR directly promotes the production of a large amount of IL-6 in related cells, which not only induces the differentiation of lung fibroblasts (Zhong HY et al. Am J Respir Cell Mol Biol, 2005, 32:2–8.).
  • Organ fibrosis can occur in a variety of tissues, such as liver, kidney, heart, lung and bone marrow.
  • the main pathological features of chronic kidney disease are excessive deposition of extracellular matrix and extensive fibrosis. A common pathway that ultimately leads to end-stage renal failure. The extent and degree of renal fibrosis is highly correlated with renal dysfunction.
  • liver fibrosis is common in liver inflammation infection (viruses, bacteria, fungi, parasites), toxin damage, changes in liver blood flow, and many congenital metabolic abnormalities cause substances to accumulate in the liver. All of the above factors can lead to liver fibrosis and damage the liver.
  • Myelofibrosis is characterized by the abnormal deposition of collagen, fibronectin, laminin and other fibrous tissues in the bone marrow, replacing the normal bone marrow hematopoietic tissue, thus seriously affecting the hematopoietic function.
  • Arteriosclerosis is a fibroproliferative disease of the arteries that results in thickening, stiffness and loss of elasticity of the vessel wall and a narrowing of the lumen.
  • idiopathic pulmonary fibrosis is a chronic progressive fibrotic interstitial lung disease with unknown etiology and pathogenesis.
  • the disease is mainly confined to the lungs, and it usually occurs in middle-aged and elderly men. It is mainly manifested as progressively worsening dyspnea, accompanied by restrictive ventilation dysfunction and gas exchange disorders, resulting in hypoxemia and even respiratory failure.
  • the study found a potential link between idiopathic pulmonary fibrosis and the adenosine system.
  • CD73 catalyzes the production of adenosine from AMP, which binds to the receptor A2BR and promotes the differentiation of lung fibroblasts into myofibroblasts (Della LV et al. Pharmacological research, 2013, 76: 182–189.).
  • CD73-deficient mice had less radiation-induced pulmonary fibrosis (Wirsdorfer F. et al. Cancer research, 2016, 76(10): 3045–3056.); selected Sexual antagonism of A2BR can reduce inflammation and fibrosis (Zhou Y et al. PloS one, 2010, 5(2): e9224).
  • the present inventors use a mammalian cell expression system to express recombinant human CD73 as an antigen to immunize mice, and obtain hybridoma cells by fusing mouse spleen cells with myeloma cells.
  • the inventor obtained the hybridoma cell line LT014 by screening a large number of samples (the deposit number is CCTCC NO: C2018137).
  • the hybridoma cell line LT014 can secrete and produce a specific monoclonal antibody (named 19F3) that specifically binds to human CD73. Further, the inventors have prepared a humanized antibody against human CD73 (for example named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM)).
  • the inventors also surprisingly found that the antibodies of the present invention, such as 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM), can very effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner and reduce the production of adenosine , and exhibited good pharmacological activity in a mouse model of pulmonary fibrosis.
  • the antibody of the present invention has the effect of treating tissue and organ fibrosis.
  • Another aspect of the present invention also relates to an anti-CD73 antibody, wherein the anti-CD73 antibody comprises HCDR1, HCDR2 of the heavy chain variable region shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 and HCDR3, and LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14;
  • the anti-CD73 antibody comprises:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with said sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) the amino acid sequence of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of, and
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 20, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence
  • the amino acid sequence of preferably 1, 2 or 3 conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of.
  • the heavy chain variable region of the antibody comprises or consists of the following sequence:
  • the heavy chain variable region of the antibody comprises or consists of the following sequences:
  • SEQ ID NO:2 SEQ ID NO:6 or SEQ ID NO:10, having at least 80%, 81%, 82%, 83%, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity
  • the sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions); and
  • the light chain variable region of the antibody comprises or consists of the following sequences:
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:4 Show;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 8;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 12; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:14.
  • the heavy chain constant region of the antibody is the Ig gamma-1 chain C region, ACCESSION:P01857; the light chain constant region is the Ig kappa chain C region, ACCESSION:P01834.
  • the heavy chain constant region of the antibody is a leucine to alanine point introduced at position 234 based on the Ig gamma-1 chain C region, ACCESSION:P01857 Mutation (L234A), a point mutation (L235A) from leucine to alanine was introduced at position 235;
  • the light chain constant region is the C region of the Ig kappa chain, ACCESSION:P01834, the amino acid sequence is as shown in SEQ ID NO:21 Show.
  • variable regions of the light and heavy chains determine antigen binding; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3 , the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; it is named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242.
  • CDRs complementarity determining regions
  • the CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors , MHC, IgSF and MhcSF. Nucleic acids research 2009;38(suppl_1):D301-D307.
  • amino acid sequences of the CDR regions of the monoclonal antibody sequences are analyzed according to the IMGT definitions by technical means well known to those skilled in the art, for example, by the VBASE2 database.
  • antibody CDR regions Given the known sequences of antibody heavy and light chain variable regions, there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia and AbM numbering systems.
  • variable region amino acid sequence of the antibody Given the variable region amino acid sequence of the antibody, one skilled in the art can generally determine which residues comprise a particular CDR, without relying on any experimental data other than the sequence itself.
  • the antibodies 19F3, 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) involved in the present invention have the same CDRs:
  • amino acid sequences of the three CDR regions in the variable region of its heavy chain are as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 15),
  • HCDR2 INPYNAGT (SEQ ID NO: 16),
  • HCDR3 ARSEYRYGGDYFDY (SEQ ID NO: 17);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QSLLNSSNQKNY (SEQ ID NO: 18),
  • LCDR3 QQHYDTPYT (SEQ ID NO:20).
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (eg, a bispecific antibody).
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, Fab/c, complementarity determining region fragments, single chain antibodies (eg, scFv ), humanized antibodies, chimeric antibodies or bispecific antibodies.
  • Another aspect of the invention pertains to isolated polypeptides selected from the group consisting of:
  • An isolated polypeptide comprising the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein the polypeptide, as part of an anti-CD73 antibody, specifically binds to CD73, the antibody also comprise the sequence shown in SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20;
  • An isolated polypeptide comprising the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 or having at least 80%, 81%, 82%, 83%, 84% with the sequence %, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence
  • the amino acid sequence of the polypeptide, wherein the polypeptide is used as a part of an anti-CD73 antibody that specifically binds to CD73, and the antibody also corresponds to the sequence shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12 or with Said sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%
  • An isolated polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:12 %, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence
  • the amino acid sequence of the polypeptide, wherein the polypeptide is used as a part of an anti-CD73 antibody that specifically binds to CD73, and the antibody also corresponds to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 or with Said sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • An isolated polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 10 %, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or compared to said sequence
  • polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 14 %, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or compared to said sequence
  • the antibody also comprises the sequence shown in SEQ ID NO: 10 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or to the The sequences are
  • Another aspect of the present invention pertains to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of the present invention, or the isolated polypeptide.
  • Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • Yet another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
  • Yet another aspect of the present invention relates to a conjugate comprising an antibody and a coupling moiety, wherein the antibody is the antibody or antigen-binding fragment thereof of any one of the present invention, and the coupling moiety is a purification tag (such as His tag), a detectable label or a small molecule drug; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme; preferably, the small
  • the molecular drug is a small-molecule cytotoxic drug; more preferably, the small-molecule drug is a tumor chemotherapy drug, more preferably, the antibody or its antigen-binding fragment is linked to the small-molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; more preferably, the antibody or its antigen-binding fragment is linked with the small molecule drug in a certain molar ratio; for
  • Yet another aspect of the present invention relates to a fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising the antibody or antigen-binding fragment thereof of any one of the present invention.
  • kits comprising the antibody or antigen-binding fragment thereof of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; preferably, the kit further comprises A second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable label, such as a radioisotope, fluorescent substance, chemiluminescent substance, colored substance, or enzyme.
  • a detectable label such as a radioisotope, fluorescent substance, chemiluminescent substance, colored substance, or enzyme.
  • kits comprising an effective amount (eg, 0.001 mg-1000 mg) of the antibody or antigen-binding fragment thereof of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention , and optionally, an effective amount of one or more anti-fibrotic drugs (eg, 100-2400 mg).
  • the kit further comprises immunosuppressive and/or anti-inflammatory agents and/or DNA methylation regulators.
  • the anti-fibrotic drug may be a modulator of RhoA, RhoAGTPases, TGF- ⁇ 1 or any other member of the CTGF or RhoA signaling pathway, or may modulate inhibitor of cytokine signaling 1 (SOCS1 ), inhibitors of cytokine signaling 3 (SOCS 3) or modulators of TLR9, nintedanib, pirfenidone, angiogenic factor antagonists, drugs that inhibit myocardial remodeling (eg, ACE inhibitors, captopril, Enalapril, fosinopril, benazepril, lisinopril, etc.; ARBs include losartan, valsartan, olmesartan, irbesartan, candesartan, etc.), beta receptors Body blockers (such as propranolol, metoprolol, atenolol, bisoprolol, carvedilol, etc.), or stat
  • a further aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, conjugate, fusion protein or multispecific antibody of the present invention in the preparation of a kit for use in The presence or level of CD73 in the sample is detected.
  • compositions comprising the antibody or antigen-binding fragment thereof of any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; optionally, the
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
  • Another aspect of the present invention relates to the antibody or antigen-binding fragment thereof, the conjugate or the fusion protein or the multispecific antibody of the present invention, for treating and/or preventing tissue and organ fibrosis, or Use in the preparation of a medicament or a kit for treating and/or preventing tissue and organ fibrosis.
  • Yet another aspect of the present invention relates to a method for treating and/or preventing tissue and organ fibrosis, comprising administering to a subject or patient an effective amount of said antibody or antigen-binding fragment thereof, said conjugate or said Fusion proteins or multispecific antibodies.
  • the tissue organ fibrosis is selected from the group consisting of: idiopathic pulmonary fibrosis; skin fibrosis, such as scleroderma or skin scarring after trauma and surgery; ocular fibrosis, such as ocular sclerosis disease, scarring on the conjunctiva and cornea, and pterygium arising on the conjunctiva; cystic fibrosis of the pancreas and lung; endomyocardial fibrosis; idiopathic cardiomyopathy; cirrhosis; mediastinal fibrosis; progressive mass Fibrosis; proliferative fibrosis; tumor fibrosis; pulmonary fibrosis due to tuberculosis.
  • the method comprises concurrently or sequentially administering to the subject or patient an anti-fibrotic drug (such as a statin), preferably, the anti-fibrotic drug is selected from lovastatin, pravastatin, Fluvastatin, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, or a combination thereof.
  • an anti-fibrotic drug such as a statin
  • the anti-fibrotic drug is selected from lovastatin, pravastatin, Fluvastatin, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, or a combination thereof.
  • the effective amount of the antibody or antigen-binding fragment thereof, the antibody drug conjugate or the bispecific antibody of the present invention is 0.001 mg-1000 mg, more preferably 0.001 mg-900 mg, 0.001 mg -800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, most preferably 100mg, 200mg, 300mg, 400mg, 500mg , 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg, or, based on the body weight of the subject or patient, the effective amount is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1-80 mg/kg, 1-70 mg/kg kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg or 1-10 mg/
  • the effective amount of one or more anti-fibrotic drugs is 100-2400 mg, preferably 100-2300 mg, 100-2200 mg, 100-2100 mg , 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 200mg 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg.
  • statins described above is 100-2400 mg, preferably 100-2300 mg, 100-2200 mg, 100-2100 mg , 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-18
  • the effective amount of the anti-fibrotic drug is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1-80 mg/kg, based on the body weight of the subject or patient , 1-70 mg/kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg or 1-10 mg/kg.
  • a single pharmaceutical dosage unit comprising 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof of the invention, preferably 0.001 mg-900 mg, 0.001 mg-800 mg, 0.001 mg-700 mg, 0.001 mg - 600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg
  • the antibody or antigen-binding fragment thereof of the present invention preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg
  • the antibody or antigen-binding fragment thereof of the present invention preferably 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof of
  • hybridoma cell line LT014 which was deposited in China, Wuhan, China on June 21, 2018, China Collection Center for Type Cultures (CCTCC), and the deposit number is CCTCC NO: C2018137.
  • the antibody or antigen-binding fragment, conjugate, fusion protein or multispecific antibody thereof is administered (preferably intravenously) one or more times.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T Definition of cell receptors, MHC, IgSF and MhcSF.” Nucleic acids research 2009;38(suppl_1):D301-D307.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA techniques (eg see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • isolated refers to artificially obtained from the natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated or isolated
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10-5 M, such as less than about 10-6 M, 10-7 M, Binds the antigen with an affinity (K D ) of 10-8 M, 10-9 M, or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies exhibit a dissociation equilibrium constant (K D ) of less than about 10-5 M, eg, less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less Binds antigen (eg, PD-1 protein).
  • KD can be determined using methods known to those skilled in the art, eg, using a Fortebio Molecular Interactometer.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • the term "single drug dosage unit” refers to the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention to be administered to a subject or patient at the moment of the dosing regimen Or a single-dose pharmaceutical dosage form of the bispecific antibody (or a pharmaceutical composition comprising the same), such as in one ampoule.
  • the term "effective amount” refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing The disease and the amount of its complications in the patient with the disease.
  • the monoclonal antibody of the present invention can well specifically bind to CD73, and can very effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, and can effectively treat tissue and organ fibrosis diseases.
  • FIG. 1 The effect of 19F3H2L3 (hG1DM) on the rate of change from baseline in enhanced expiratory interval values in a hCD73 transgenic mouse model of pulmonary fibrosis.
  • T and C are the administration group and the same type control group, respectively
  • FIG. 3 The effect of 19F3H2L3 (hG1DM) on the total number of inflammatory cells in the bronchoalveolar lavage fluid of hCD73 transgenic mice model of pulmonary fibrosis. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, VS normal group; # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001, VS isotype control group (t-test).
  • Figure 4 The effect of 19F3H2L3(hG1DM) on the number of inflammatory cells in the bronchoalveolar lavage fluid of hCD73 transgenic mice model of pulmonary fibrosis. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, VS normal group; # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001, VS isotype control group (t-test).
  • FIG. 5 The effect of 19F3H2L3 (hG1DM) on the content of hydroxyproline (HYP) in the lung tissue homogenate of hCD73 transgenic mouse pulmonary fibrosis model.
  • the BALB/c mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center;
  • hCD73 transgenic mice used were purchased from Jiangsu Biositu Experimental Animal Co., Ltd.;
  • the used positive control antibody MEDI9447 (Oleclumab) was produced from Zhongshan Kangfang Bio-Pharmaceutical Co., Ltd., and its sequence was the same as the amino acid sequence of Oleclumab disclosed by Medlmmune Limited in the WHO drug information database and related patents. The full length is consistent (https://www.who.int/medicines/publications/dr ⁇ ginformation/innlists/en/), which is marked as MEDI9447 or MEDI9447 (Akeso) in the embodiment of the present invention;
  • the isotype control antibody used is the sequence of human anti-Hen Egg Lysozyme IgG (anti-HEL antibody, or human IgG, hIgG for short, or isotype control) Variable region sequence from Fab F10.6.6 sequence in Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-146.);
  • bovine serum albumin used is from Sigma, article number: V900933-1KG;
  • the RPMI 1640 used was derived from Gibco, article number: 22400-089;
  • the fetal bovine serum used is derived from Excell bio, article number: FSP500;
  • the sodium pyruvate used is derived from Gibco, article number: 11360-070;
  • non-essential amino acids used are derived from Gibco, item number: 11140-050;
  • the L-glutamine used is from Gibco, article number: 25030-081.
  • the MDA-MB-231 used is derived from ATCC, item number: HTB-26;
  • the CTG color developing solution used is One Solution Assay kit, from promega, catalog number: G8461;
  • Alexa was used 647-labeled mouse anti-human IgG secondary antibody was obtained from Southern Biotech, catalog number: 9040-31.
  • the bleomycin used is from Hanhui Pharmaceutical Co., Ltd., article number: 19012311;
  • trypan blue used is derived from Sigma; batch number: MKCF8888; article number: T6146;
  • the phosphate buffer used is from Fuzhou Maixin Biotechnology Development Co., Ltd.;
  • the methacholine used is derived from sigma; batch number: MKCF3289; article number: A2251;
  • the hydroxyproline used is derived from hydroxyproline, HYP;
  • the detection kit is derived from QuiekZyme Biosciences, product number: QZBTOTCOL1;
  • the used sutai originates from Virbac S.A of France Vik Co., Ltd., article number: BN 6ALU;
  • the isoflurane used comes from Shenzhen Ruiwo De Life Technology Co., Ltd.;
  • the sodium chloride injection used is derived from Jiangxi Kelun Pharmaceutical Co., Ltd.
  • CD73 (5'-nuclease) specific inhibitor APCP (alpha, beta-methylene adenosine-5'-diphosphate, 5'- ⁇ , ⁇ -methylene-diphosphate, Adenosine Phosphate) from sigma, catalog number: M3763-10MG.
  • the antigen used to prepare the anti-CD73 antibody was human NT5E-His (NT5E is Genbank ID: NP_002517.1, position: 1-552).
  • the spleen cells of the immunized mice were fused with mouse myeloma cells to prepare hybridoma cells.
  • human NT5E (NT5E is GenbankID: NP_002517.1, position: 1-552)-Biotin as an antigen
  • the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that can secrete antibodies that specifically bind to CD73.
  • the hybridoma cells obtained by screening were subjected to a limiting dilution method to obtain stable hybridoma cell lines.
  • the above hybridoma cell line was named as hybridoma cell line LT014, and the monoclonal antibody secreted by it was named 19F3.
  • Hybridoma cell line LT014 also known as CD73-19F3
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2018137
  • the deposit address is China. Wuhan. Wuhan University , Zip Code: 430072.
  • the LT014 cell line prepared above was cultured with CD medium (Chemical Defined Medium) (CD medium containing 1% penicillin streptomycin, cultured in 5% CO 2 , 37° C. cell incubator). After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation, vacuum filtration through a microporous membrane, and purified using a HiTrap protein A HP column to obtain antibody 19F3.
  • CD medium Chemical Defined Medium
  • CD medium containing 1% penicillin streptomycin cultured in 5% CO 2 , 37° C. cell incubator
  • mRNA was extracted from the LT014 cell line cultured in Example 1 according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, Cat. No. DP430).
  • the PCR amplification product was directly cloned by TA, and the specific operation was carried out by referring to the instructions of the pEASY-T1Cloning Kit (Transgen CT101).
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the fragment is 351 bp long.
  • sequence of heavy chain CDR1 is shown in SEQ ID NO:15
  • sequence of heavy chain CDR2 is shown in SEQ ID NO:16
  • sequence of heavy chain CDR3 is shown in SEQ ID NO:17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 3, and the length is 321 bp.
  • sequence of light chain CDR1 is shown in SEQ ID NO:18
  • sequence of light chain CDR2 is shown in SEQ ID NO:19
  • sequence of light chain CDR3 is shown in SEQ ID NO:20.
  • variable region sequences are as follows:
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, and the length is 363 bp.
  • SEQ ID NO:6 Its encoded amino acid sequence is shown in SEQ ID NO:6, and the length is 121aa, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO:15, the sequence of heavy chain CDR2 is shown in SEQ ID NO:16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 7, and the length is 339 bp.
  • the encoded amino acid sequence is as shown in SEQ ID NO:8, and the length is 113aa, wherein the sequence of light chain CDR1 is as shown in SEQ ID NO:18, the sequence of light chain CDR2 is as shown in SEQ ID NO:19, and the light chain CDR3 The sequence is shown in SEQ ID NO:20.
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the length is 363 bp.
  • SEQ ID NO:10 Its encoded amino acid sequence is shown in SEQ ID NO:10, and the length is 121aa, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO:15, the sequence of heavy chain CDR2 is shown in SEQ ID NO:16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 11, and the length is 339 bp.
  • SEQ ID NO:12 Its encoded amino acid sequence is shown in SEQ ID NO:12, and the length is 113aa, wherein the sequence of light chain CDR1 is shown in SEQ ID NO:18, the sequence of light chain CDR2 is shown in SEQ ID NO:19, and the light chain CDR3 The sequence is shown in SEQ ID NO:20.
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the length is 363 bp.
  • SEQ ID NO:10 Its encoded amino acid sequence is shown in SEQ ID NO:10, and the length is 121aa, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO:15, the sequence of heavy chain CDR2 is shown in SEQ ID NO:16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 13, and the length is 339 bp.
  • SEQ ID NO:14 Its encoded amino acid sequence is shown in SEQ ID NO:14, and the length is 113aa, wherein the sequence of light chain CDR1 is shown in SEQ ID NO:18, the sequence of light chain CDR2 is shown in SEQ ID NO:19, and the light chain CDR3 The sequence is shown in SEQ ID NO:20.
  • the light chain constant region of the antibodies of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) is the Ig kappa chain C region, ACCESSION:P01834, see SEQ ID NO:22.
  • the heavy chain constant region is based on the Ig gamma-1chain C region, ACCESSION:P01857, a leucine to alanine point mutation (L234A) was introduced at position 234, and leucine was introduced at position 235.
  • Acid to alanine point mutation (L235A) resulting in humanized antibodies designated 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM), see SEQ ID NO:21.
  • the 19F3H1L1 (hG1DM) heavy chain cDNA and light chain cDNA, 19F3H2L2 (hG1DM) heavy chain cDNA and light chain cDNA, and 19F3H2L3 (hG1DM) heavy chain cDNA and light chain cDNA were cloned into pUC57simple (GenScript). Company provided) vectors, respectively obtained pUC57simple-19F3H1 (hG1DM), pUC57simple-19F3L1; pUC57simple-19F3H2 (hG1DM), pUC57simple-19F3L2 and pUC57simple-19F3L3.
  • the recombinant plasmids containing the corresponding light and heavy chains were designed to combine the genes (pcDNA3.1-19F3H1(hG1DM)/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L2, and pcDNA3.1- 19F3H2(hG1DM)/pcDNA3.1-19F3L3) were co-transfected into 293F cells, respectively, and the culture medium was collected for purification. After the sequencing was verified to be correct, an endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected, and a Protein A column was used for affinity purification to obtain humanized antibodies.
  • the experimental steps are as follows: take log-phase MDA-MB-231 cells in good condition, resuspend and count the cells with serum-free RPMI-1640 medium; inoculate MDA-MB-231 cells into 96-well plates, 3*10 4 cells/100 ⁇ l/well; dilute the antibody with serum-free RPMI-1640 medium, the initial concentration is 200 ⁇ g/ml, and carry out a 2.5-fold gradient dilution; add the antibody to a 96-well plate, 50 ⁇ l per well, and incubate at 37°C for 1 hour.
  • mice were nebulized in PBS solution, followed by continuous nebulization at concentrations of 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg /mL of methacholine (Mch), measure the enhanced expiratory interval value at the corresponding concentration, stimulate for 90 seconds at each concentration, draw the rate of change of the enhanced expiratory interval value relative to the baseline-Mch concentration curve, and calculate the curve under the curve area.
  • Mch methacholine
  • the BALF was centrifuged at 300 g for 5 minutes at 4°C, and the cell pellet obtained after centrifugation was resuspended in 1.5 mL of PBS for smear preparation.
  • the total number of cells in the BALF was counted using a hemocytometer and stained with Wright-Giemsa staining solution to differentiate eosinophils, neutrophils, macrophages and lymphocytes. Count under a light microscope. Collect the remaining BALF fluid. After lavage, the animals were euthanized by cervical dissection, and the lung tissues were collected and cryopreserved at -80°C for the detection of hydroxyproline content after homogenization.
  • hydroxyproline content was performed using a hydroxyproline (HYP) detection kit (QuiekZyme Biosciences) according to the kit instructions.
  • the experimental data are expressed as the mean ⁇ standard error (Mean ⁇ SEM), the comparison between groups was processed by GraphPad statistical processing software, and the results were evaluated by one-way analysis of variance or t-test. 0.01 is a very significant difference.
  • HCDR1 GYSFTGYT (SEQ ID NO: 15)
  • HCDR3 ARSEYRYGGDYFDY (SEQ ID NO: 17)
  • LCDR1 QSLLNSSNQKNY (SEQ ID NO: 18)

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Abstract

涉及一种抗CD73抗体及其应用。具体地，所述抗体的重链可变区包含氨基酸序列如SEQ ID NOs:15－17所示的HCDR1－HCDR3；并且所述抗体的轻链可变区包含氨基酸序列如SEQ ID NOs:18－20所示的LCDR1－LCDR3。

Description

抗CD73的抗体及其用途 技术领域
本发明涉及免疫学领域。具体地，涉及抗CD73抗体及其用途。
背景技术
胞外-5＇-核苷酸酶(Ecto-5’-nucleotidase)，即CD73蛋白，是NT5E基因编码的一种蛋白分子量为70KD的多功能糖蛋白，其通过糖基磷脂酰肌醇(glyocsyl phosphatidy linositol，GPI)锚定于细胞膜上(Zimmermann H.Biochem J.1992；285:345–365)。
CD73广泛分布在人体组织细胞表面，在免疫细胞表面亦广泛表达，如树突细胞、调节型T细胞(Treg)、自然杀伤细胞(NK细胞)、髓系起源的抑制细胞(MDSC)等。
CD73既有水解酶活性，又有非水解酶作用。CD73的酶与非酶功能的一个免疫抑制机制是由CD73-腺苷(Adenosine)代谢信号通路介导的，CD73上游的CD39可以催化ATP产生腺苷单磷酸(AMP)，所产生的AMP被CD73转化为腺苷，而腺苷会结合下游的腺苷受体(A2AR)，A2AR通过激活蛋白激酶A(PKA)和Csk激酶，抑制LCK、MAPK、PKC等一系列与免疫激活相关的信号通路，抑制T细胞的免疫杀伤作用，从而发挥免疫抑制作用(Antonioli L,et al.Nat Rev Cancer.2013；13:842–857.)。CD73在人的B细胞、T细胞、骨髓细胞、骨髓基质细胞及胸腺上皮细胞均表达。
器官纤维化是指由于各种慢性刺激因素，如病原微生物感染、炎症、免疫反应、毒素(药物)、放射线、缺血及血流动力学改变等，导致器官实质细胞发生坏死，纤维结缔组织，包括细胞成分和细胞外基质(extracellular matrix，ECM)异常增多和过度沉积的病理过程。此外，还有一些致病机理不明的器官纤维化，称为特发性或原发性器官纤维化，如特发性肺纤维化(Idiopathic Pulmonary Fibrosis，IPF)等。程度较轻者称为纤维化，重者引起组织结构破坏而发生器官硬化(organ scarring)。纤维化器官其组织内沉积的纤维结缔组织包括细胞成分和ECM两大部分。 组织的实质细胞、成纤维细胞和单核吞噬细胞等；以及胶原、非胶原糖蛋白、蛋白聚糖和弹性纤维等。
参与器官纤维化形成的常见细胞因子最重要的是转化生长因子-βTGF-β(transforming growth factor-β)(Meng XM,Nikolic-Paterson DJ,Lan HY.Nat Rev Nephrol.2016；12(6):325-338.)。此外，其它细胞因子如结缔组织生长因子CTGF(connective tissue growth factor)、血小板源性生长因子PDGF(platelet-direved growth actor)、碱性纤维母细胞生长因子bFGF(basic fibroblast growth factor)、表皮生长因子EGF(epidermal growth factor)和IL-1、IL-6、IL-8、TNF-α、IFN-γ等也参与疾病的发生(Wynn TA.J Pathol.2008；214(2):199-210)。
研究证实，CD73-腺苷信号通路的激活可以刺激产生TGF-β，并通过TGF-β的自分泌活性维持CD73表达(García-Rocha R et al.Cytokine.2019；118:71-79.)，而TGF-β信号通过腺苷依赖性机制调节乳腺癌的ECM沉积(Vasiukov G,et al.Cancer Res.2020；80(12):2628-2638)。
组织血运障碍是器官纤维化发生的危险因素，而器官纤维化导致的机构改变又常可导致组织血运障碍，出现组织细胞缺氧，在这种状态下低氧诱导因子-1(HIF-1)等分子的上调，导致CD73广泛表达(Synnestvedt K,et al.J Clin Invest.2002；110:993–1002)。此外，A2B受体(A2BR)在低氧状态下更容易激活，A2BR的激活直接促进了相关细胞产生大量IL-6，不但可以诱导肺纤维细胞的分化(Zhong HY et al.Am J Respir Cell Mol Biol,2005,32:2–8.)。
器官纤维化可发生在多种组织，如肝、肾、心脏、肺及骨髓等。
慢性肾脏病的主要病理特征为细胞外基质过度沉积，广泛的纤维化。最终导致终末肾功能衰竭的共同通路。肾纤维化的范围和程度与肾功能缺陷具有高度的相关性。
肝纤维化常见于肝脏炎症感染(病毒、细菌、真菌、寄生虫)、毒素损害、肝血流改变、许多先天性代谢异常引起物质在肝脏贮积，上述因素都可导致肝纤维化，损害肝脏功能。
骨髓纤维化表现为骨髓中胶原、纤维连接蛋白、层粘连蛋白等纤维组织的异常沉积，代替正常的骨髓造血组织，从而严重影响造血功能。
心肌的进行纤维化伴随心肌(心室)重构和心脏功能减退。动脉硬化是动脉纤维增性病变，导致血管壁增厚、僵硬而失去弹性和管腔变小。
肺纤维化病理过程是肺组织损伤修复失调导致大量细胞外基质重构、过度沉积，最终导致肺组织结构改变和功能丧失。其中，特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)是一种病因和发病机制尚不明确的、慢性进行性纤维化性间质性肺疾病。病变主要局限于肺部，好发于中老年男性，主要表现为进行性加重的呼吸困难，伴限制性通气功能障碍和气体交换障碍，导致低氧血症、甚至呼吸衰竭。研究发现，特发性肺纤维化与腺苷系统存在潜在的联系。CD73催化AMP生成腺苷，腺苷与受体A2BR结合，促进肺成纤维细胞分化为肌纤维细胞(Della LV et al.Pharmacological research,2013,76:182–189.)。动物实验中，CD73缺失的小鼠与野生型小鼠相比，放疗诱导的肺纤维化程度较轻(Wirsdorfer F.et al.Cancer research,2016,76(10):3045–3056.)；选择性拮抗A2BR，可以减轻炎症及纤维化过程(Zhou Y et al.PloS one,2010,5(2):e9224)。
发明内容
本发明人利用哺乳动物细胞表达系统表达出重组的人CD73作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过对大量样本的筛选，得到了杂交瘤细胞株LT014(保藏编号为CCTCC NO:C2018137)。
本发明人惊奇地发现，杂交瘤细胞株LT014能够分泌产生与人CD73特异性结合的特异性单克隆抗体(命名为19F3)，进一步地，本发明人制得了抗人CD73的人源化抗体(例如命名为19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM))。
本发明人还惊奇地发现，本发明的抗体例如19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)能够十分有效地以非底物竞争的方式抑制CD73的酶活反应，降低腺苷的产生，并且在小鼠肺纤维化模型中展现出良好的药理学活性。本发明的抗体具有用于治疗组织器官纤维化的效果。
本发明的另一个方面还涉及一种抗CD73抗体，其中，所述抗CD73 抗体包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示重链可变区的HCDR1，HCDR2和HCDR3，和SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14所示的轻链可变区的LCDR1，LCDR2和LCDR3；
优选地，根据IMGT编号系统，所述抗CD73抗体包含：
HCDR1，其包含SEQ ID NO:15所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
HCDR2，其包含SEQ ID NO:16所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
HCDR3，其包含SEQ ID NO:17所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
LCDR1，其包含SEQ ID NO:18所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
LCDR2，其包含SEQ ID NO:19所示的氨基酸序列，与所述序列具 有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，和
LCDR3，其包含SEQ ID NO:20所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成。
在本发明的一些实施方案中，所述抗体的重链可变区包含下述序列，或由下述序列组成：
所述抗体的重链可变区包含下述序列，或由下述序列组成：
SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10，与SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；并且
所述抗体的轻链可变区包含下述序列，或由下述序列组成：
SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14，与SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
在本发明的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；或
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:14所示。
在本发明的一些实施方案中，所述抗体的重链恒定区为Ig gamma-1链C区，ACCESSION:P01857；轻链恒定区为Ig kappa链C区，ACCESSION:P01834。
在本发明的一些实施方案中，所述抗体的重链恒定区为在Ig gamma-1链C区，ACCESSION:P01857的基础上在第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)；轻链恒定区为Ig kappa链C区，ACCESSION:P01834，氨基酸序列如SEQ ID NO:21所示。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Bethesda M.d.,Sequences of Proteins of Immunological Interest,Fifth Edition,NIH Publication 1991；1-3:91-3242。
优选地，CDR也可以由IMGT编号系统定义，请参见Ehrenmann,Francois,Quentin Kaas,and Marie-Paule Lefranc.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF.Nucleic acids research 2009；38(suppl_1):D301-D307。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库根据IMGT定义分析单克隆抗体序列的CDR区的氨基酸序列。
在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体 CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。
然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定哪些残基包含特定CDR，而不依赖于该序列自身之外的任何实验数据。
本发明涉及的抗体19F3、19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3(hG1DM)具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GYSFTGYT(SEQ ID NO:15)，
HCDR2:INPYNAGT(SEQ ID NO:16)，
HCDR3:ARSEYRYGGDYFDY(SEQ ID NO:17)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QSLLNSSNQKNY(SEQ ID NO:18)，
LCDR2:FAS(SEQ ID NO:19)，
LCDR3:QQHYDTPYT(SEQ ID NO:20)。
在本发明的一些实施方案中，所述抗体为单克隆抗体。
在本发明的一些实施方案中，所述抗体为人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
在本发明的一些实施方式中，所述抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双特异性抗体。
本发明的另一个方面涉及分离的多肽，其选自以下各项组成的组：
(1)分离的多肽，其包含SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:18；SEQ ID NO:19；SEQ ID NO:20所示的序列；
(2)分离的多肽，其包含SEQ ID NO:18；SEQ ID NO:19；SEQ ID NO:20所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的序列；
(3)分离的多肽，其包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO:4、SEQ ID NO:8或SEQ ID NO:12所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
(4)分离的多肽，其包含SEQ ID NO:4、SEQ ID NO:8或SEQ ID NO:12所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
(5)分离的多肽，其包含SEQ ID NO:10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、 2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:14所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
(6)分离的多肽，其包含SEQ ID NO:14所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
本发明的另一方面涉及一种分离的核酸分子，其编码本发明任一项所述的抗体或其抗原结合片段，或所述分离的多肽。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及一种偶联物，其包括抗体以及偶联部分，其中，所述抗体为本发明任一项所述的抗体或其抗原结合片段，所述偶联部分为纯化标签(如His标签)，可检测的标记或小分子药物；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、聚乙二醇或酶；优选地，所述小分子药物为小分子细胞毒药物；更优选地， 所述小分子药物为肿瘤化疗药物，更优选地，所述抗体或其抗原结合片段通过连接子与小分子药物连接；例如，所述连接子为腙键、二硫键或肽键；更优选地，所述抗体或其抗原结合片段与小分子药物以一定的摩尔比连接；例如，所述摩尔比为1：(2－4)，更优选地，所述小分子药物是抗纤维化药物。
本发明的再一个方面，涉及融合蛋白或多特异性抗体(优选双特异性抗体)，其包含本发明任一项所述的抗体或其抗原结合片段。
本发明的再一方面涉及一种试剂盒，其包括本发明所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶。
在本发明的再一个方面，涉及试剂盒，其包含有效量(例如0.001mg-1000mg)的本发明所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体，和任选地，还包含有效量的一种或多种抗纤维化药物(例如100-2400mg)。优选地，所述试剂盒还包含免疫抑制剂和/或抗炎剂和/或DNA甲基化调节剂。
在本发明的一些实施方案中，所述抗纤维化药物可以是RhoA、RhoAGTPases、TGF-β1或CTGF或RhoA信号通路的其它任何成员的调节剂，或者可以调节细胞因子信号传导抑制剂1(SOCS1)、细胞因子信号传导抑制剂3(SOCS 3)或TLR9的调节剂，尼达尼布，吡非尼酮，血管生成因子拮抗剂，抑制心肌重构的药物(如ACEI有卡托普利，依那普利，福辛普利，贝那普利，赖诺普利等；ARB类有氯沙坦，缬沙坦，奥美沙坦，厄贝沙坦，坎地沙坦等)，β受体阻滞剂(如普萘洛尔，美托洛尔，阿替洛尔，比索洛尔，卡维地洛等)，或者是他汀类化合物或其衍生物。优选地，所述他汀类药物选自洛伐他汀，普伐他汀，氟伐他汀，西立伐他汀，阿托伐他汀，辛伐他汀，匹伐他汀，罗苏伐他汀，或其组合。
本发明的再一方面涉及本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CD73在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂。优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
本发明的再一方面涉及本发明所述的抗体或其抗原结合片段、所述的偶联物或所述的融合蛋白或多特异性抗体，用于治疗和/或预防组织器官纤维化，或在制备治疗和/或预防组织器官纤维化的药物或试剂盒中的用途。
本发明的再一方面涉及治疗和/或预防组织器官纤维化的方法，包括向受试者或患者施用有效量的所述的抗体或其抗原结合片段、所述的偶联物或所述的融合蛋白或多特异性抗体。
在本发明的一些实施方案中，所述组织器官纤维化选自：特发性肺纤维化；皮肤纤维化，例如硬皮病或外伤和手术后产生的皮肤瘢痕；眼纤维化，例如眼硬化症、结膜和角膜上的瘢痕以及结膜上生出的翼状胬肉；胰腺和肺的囊性纤维化；心内膜心肌纤维化；特发性心肌病；肝硬化；纵隔纤维化；进行性大块纤维化；增生性纤维化；肿瘤纤维化；肺结核导致的肺部的纤维化。在一些实施方案中，所述方法包括向受试者或患者同时或顺序施用抗纤维化药物(如他汀类药物)，优选地，所述抗纤维化药物选自洛伐他汀，普伐他汀，氟伐他汀，西立伐他汀，阿托伐他汀，辛伐他汀，匹伐他汀，罗苏伐他汀，或其组合。
在一些实施方案中，本发明的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体的有效量为0.001mg-1000mg，更优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，最优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg，或者，基于受试者或患者的体重，所述有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。在本发明的上述任一实施方案，一种或多种抗纤维化药物(例如上文所述 的他汀类药物)的有效量为100-2400mg，优选100mg-2300mg、100mg-2200mg、100mg-2100mg、100mg-2000mg、100mg-1900mg、100mg-1800mg、100mg-1700mg、100mg-1600mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg，更优选为100mg，200mg，300mg，400mg，500mg，600mg，700mg，800mg，900mg，1000mg。或者，在本发明的上述任一实施方案中，基于受试者或患者的体重，所述抗纤维化药物的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。在本发明的另一个方面，涉及单次药物剂量单元，其包含0.001mg-1000mg的本发明的抗体或其抗原结合片段，优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，更优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg的本发明的抗体或其抗原结合片段。
本发明的再一方面涉及杂交瘤细胞株LT014，其于2018年6月21日保藏在中国，武汉的中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018137。
在一些实施方案中，所述的抗体或其抗原结合片段、偶联物、融合蛋白或多特异性抗体被给予(优选静脉给予)单次或多次。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内， 可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗原结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda M.d.(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883或者IMGT编号系统定义，见Ehrenmann,Francois,Quentin Kaas,and Marie-Paule Lefranc."IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF."Nucleic acids research 2009；38(suppl_1):D301-D307.的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021080517-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity.nature,1975；256(5517):495)，但也可 采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522-525；Reichmann et al.,Nature 1988；332:323-329；Presta,Curr.Op.Struct.Biol.,1992；2:593-596；和Clark M.Antibody humanization:a case of the‘Emperor’s new clothes’？[J].Immunol.Today,2000；21(8):397-402。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种 载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，GS细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，PD-1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强 度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
在本发明中，术语“单次药物剂量单元”表示在给药方案的时刻待给药于受试者或患者的本发明所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体(或包含其的药物组合物)的单次药物剂型，如以一个安瓿瓶为单位。如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如本发明所述的组织器官纤维化)有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。
发明的有益效果:
本发明的单克隆抗体能够很好地特异性与CD73结合，并且能够十分有效地、以非底物竞争的方式抑制CD73的酶活反应，可以有效治疗组织器官纤维化疾病。
附图说明
图1.MDA-MB-231细胞中加入抗CD73抗体的酶活性的检测结果。
图2.19F3H2L3(hG1DM)对hCD73转基因小鼠肺纤维化模型增强呼气间歇值相对基线的变化率的影响。数据以“平均值±标准误差”表示；增强呼气间歇值抑制率％＝[100-(T/C*100)]/100×100％，其中T和C分别是给药组及同型对照组的增强呼气间歇值相对基线的变化率-Mch浓度曲线下面积。
图3.19F3H2L3(hG1DM)对hCD73转基因小鼠肺纤维化模型肺泡灌洗液炎症细胞总数的影响。 *P<0.05, **P<0.01, ***P<0.001，VS正常组； #P<0.05, ##P<0.01, ###P<0.001,vs同型对照组(t-test)。
图4.19F3H2L3(hG1DM)对hCD73转基因小鼠肺纤维化模型肺泡灌洗液炎症细胞数的影响。 *P<0.05, **P<0.01, ***P<0.001，VS正常组； #P<0.05, ##P<0.01, ###P<0.001,vs同型对照组(t-test)。
图5.19F3H2L3(hG1DM)对hCD73转基因小鼠肺纤维化模型肺组织匀浆中羟脯氨酸(HYP)含量的影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。例如MDA-MB-231细胞和U87-MG细胞可以购自ATCC；
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心；
在本发明的下述实施例中，使用的hCD73转基因小鼠购自江苏百奥赛图实验动物有限公司；
在本发明的下述实施例中，所用的阳性对照抗体MEDI9447(Oleclumab)，生产自中山康方生物医药有限公司，其序列与Medlmmune Limited在WHO药物信息数据库及相关专利中所公开的Oleclumab氨基酸序列全长一致(https://www.who.int/medicines/publications/drμginformation/innlists/en/)，在本发明实施例中标记为MEDI9447或者MEDI9447(Akeso)；
在本发明的下述实施例中，所用的同型对照抗体为人抗鸡蛋溶酶体抗体(human anti-Hen Egg Lysozyme IgG，即anti-HEL抗体，或者human IgG，简称hIgG，或者同型对照)的序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1):130-146.)；
在本发明的下述实施例中，所用的牛血清白蛋白(BSA)来源于Sigma，货号：V900933-1KG；
在本发明的下述实施例中，所用的RPMI 1640来源于Gibco，货号： 22400-089；
在本发明的下述实施例中，所用的胎牛血清来源于Excell bio，货号：FSP500；
在本发明的下述实施例中，所用的丙酮酸钠来源于Gibco，货号：11360-070；
在本发明的下述实施例中，所用的非必需氨基酸来源于Gibco，货号：11140-050；
在本发明的下述实施例中，所用的L-谷氨酰胺来源于Gibco，货号：25030-081。
在本发明的下述实施例中，所用的MDA-MB-231来源于ATCC，货号：HTB-26；
在本发明的下述实施例中，所用的CTG显色液为
Figure PCTCN2021080517-appb-000002
One Solution Assay试剂盒，来源于promega，货号:G8461；
在本发明的下述实施例中，所用的Alexa
Figure PCTCN2021080517-appb-000003
647标记鼠抗人IgG二抗来源于Southern Biotech，货号：9040-31。
在本发明的下述实施例中，所用的博来霉素来源于瀚晖制药有限公司，货号：19012311；
在本发明的下述实施例中，所用的台盼蓝来源于Sigma；批号：MKCF8888；货号：T6146；
在本发明的下述实施例中，所用的磷酸盐缓冲液来源于福州迈新生物技术开发有限公司；
在本发明的下述实施例中，所用的乙酰甲胆碱来源于sigma；批号：MKCF3289；货号：A2251；
在本发明的下述实施例中，所用的羟脯氨酸来源于hydroxyproline，HYP；检测试剂盒来源于QuiekZyme Biosciences，货号：QZBTOTCOL1；
在本发明的下述实施例中，所用的舒泰来源于法国维克有限公司Virbac S.A，货号：BN 6ALU；
在本发明的下述实施例中，所用的异氟烷来源于深圳瑞沃德生命科技有限公司；
在本发明的下述实施例中，所用的氯化钠注射液来源于江西科伦药业 有限公司。
在本发明的下述实施例中，所用的CD73(5′-核酸酶)特异性抑制剂APCP(alpha，beta―methylene adenosine-5′-diphosphate，5’-α，β-亚甲基-二磷酸腺苷)来源于sigma，货号：M3763-10MG。
实施例1：抗CD73抗体19F3的制备
1、杂交瘤细胞株LT014的制备
制备抗CD73抗体所用的抗原为人NT5E-His(NT5E为GenbankID:NP_002517.1，位置：1-552)。取免疫后的小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。以人NT5E(NT5E为GenbankID:NP_002517.1，位置：1-552)-Biotin作为抗原，对杂交瘤细胞进行间接ELISA法筛选，获得能够分泌与CD73特异性结合的抗体的杂交瘤细胞。对筛选得到的杂交瘤细胞，经过有限稀释法得到稳定的杂交瘤细胞株。将以上杂交瘤细胞株命名为杂交瘤细胞株LT014，其分泌的单克隆抗体命名为19F3。
杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018137，保藏地址为中国.武汉.武汉大学，邮编：430072。
2、抗CD73抗体19F3的制备
用CD培养基(Chemical Defined Medium)对上面制得的LT014细胞株进行培养(CD培养基，内含1％青链霉素，于5％CO 2，37℃细胞培养箱中进行培养)。7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤，并使用HiTrap protein A HP柱进行纯化，制得抗体19F3。
实施例2：抗CD73的抗体19F3的序列分析
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1中培养的LT014细胞株中提取mRNA。
按照Invitrogen
Figure PCTCN2021080517-appb-000004
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1Cloning Kit (Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的核酸序列如SEQ ID NO:1所示，片段长351bp。
其编码的氨基酸序列为SEQ ID NO:2所示，长度为117个氨基酸；
其中重链CDR1的序列如SEQ ID NO:15所示，重链CDR2的序列如SEQ ID NO:16所示，重链CDR3的序列如SEQ ID NO:17所示。
轻链可变区的核酸序列如SEQ ID NO:3所示，长度为321bp。
其编码的氨基酸序列为SEQ ID NO:4所示，长度为107个氨基酸；
其中轻链CDR1的序列如SEQ ID NO:18所示，轻链CDR2的序列如SEQ ID NO:19所示，轻链CDR3的序列如SEQ ID NO:20所示。
实施例3：抗人CD73的人源化抗体的轻链和重链设计和制备
1.抗人CD73的人源化抗体19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的轻链和重链设计
根据人CD73蛋白的三维晶体结构(Hage T,Reinemer P,Sebald W.Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain.Eur J Biochem.1998；258(2):831-6.)以及实施例2获得的抗体19F3的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3(hG1DM)的可变区序列。
设计的可变区序列如下：
(1)人源化单克隆抗体19F3H1L1(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO:5所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO:6所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO:15所示，重链CDR2的序列如SEQ ID NO:16所示，重链CDR3的序列如SEQ ID NO:17所示。
轻链可变区的核酸序列如SEQ ID NO:7所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO:8所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO:18所示，轻链CDR2的序列如SEQ ID NO:19 所示，轻链CDR3的序列如SEQ ID NO:20所示。
(2)人源化单克隆抗体19F3H2L2(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO:9所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO:10所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO:15所示，重链CDR2的序列如SEQ ID NO:16所示，重链CDR3的序列如SEQ ID NO:17所示。
轻链可变区的核酸序列如SEQ ID NO:11所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO:12所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO:18所示，轻链CDR2的序列如SEQ ID NO:19所示，轻链CDR3的序列如SEQ ID NO:20所示。
(3)人源化单克隆抗体19F3H2L3(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO:9所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO:10所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO:15所示，重链CDR2的序列如SEQ ID NO:16所示，重链CDR3的序列如SEQ ID NO:17所示。
轻链可变区的核酸序列如SEQ ID NO:13所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO:14所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO:18所示，轻链CDR2的序列如SEQ ID NO:19所示，轻链CDR3的序列如SEQ ID NO:20所示。
2.人源化抗体19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的制备
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的抗体的轻链恒定区为Ig kappa链C区，ACCESSION:P01834，见SEQ ID NO:22。
重链恒定区在Ig gamma-1chain C region，ACCESSION:P01857的基础上，在第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，获得了命名为19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的人源化 抗体，见SEQ ID NO:21。
将19F3H1L1(hG1DM)重链cDNA和轻链的cDNA、19F3H2L2(hG1DM)重链cDNA和轻链的cDNA、以及19F3H2L3(hG1DM)的重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-19F3H1(hG1DM)、pUC57simple-19F3L1；pUC57simple-19F3H2(hG1DM)、pUC57simple-19F3L2和pUC57simple-19F3L3。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体pcDNA3.1中获得表达质粒pcDNA3.1-19F3H1(hG1DM)，pcDNA3.1-19F3L1，pcDNA3.1-19F3H2(hG1DM)，pcDNA3.1-19F3L2，和pcDNA3.1-19F3L3，并进一步对重组表达质粒的重/轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合(pcDNA3.1-19F3H1(hG1DM)/pcDNA3.1-19F3L1，pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L2，和pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L3)分别共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体。
实施例4：抗CD73抗体对抑制细胞内源表达CD73酶活性的检测
1、抗CD73抗体对MDA-MB-231细胞内源表达CD73酶活性的抑制活性检测
实验步骤如下：取状态良好的对数期MDA-MB-231细胞，用无血清的RPMI-1640培养液将细胞重悬、计数；将MDA-MB-231细胞接种至96孔板，3*10 4个细胞/100μl/孔；用无血清RPMI-1640培养液稀释抗体，起始浓度200μg/ml，按2.5倍进行梯度稀释；将抗体加入96孔板，每孔50μl，37℃孵育1小时。1小时后，每孔加入50μl RPMI-1640稀释的600μM AMP；3小时后取25μl细胞培养液上清，转移至新96 孔板，每孔加入25μl 100μM的ATP；和50μl CTG(
Figure PCTCN2021080517-appb-000005
One Solution Assay，promega，Cat:G8461)显色液，进行显色，多标记微孔板检测仪(PerkinElmer 2140-0020)读取数据。同型对照抗体及CD73特异性抑制剂APCP分别作为阴性对照及阳性对照。
实验结果：结果如图1所示，19F3、19F3H2L3(hG1DM)、19F3H2L3(hG1DM)和19F3H2L3(hG1DM)均可呈剂量依赖性抑制MDA-MB-231内源表达的CD73酶催化AMP为腺苷A的活性，从而剂量依赖性降低产生的平均萤光强度RLU。
以上实验结果说明加入的AMP在无CD73抗体处理情况下，可被MDA-MB-231内源表达于细胞表面的CD73酶催化而转化为腺苷，从而解除对荧光素酶活性的抑制。而加入抗体后，由于CD73被抗体结合，而降低了其酶催化活性，使AMP不能转化为腺苷。提示抗CD73抗体以非底物竞争的方式有效抑制CD73的酶活反应，降低腺苷的产生。
实施例5：19F3H2L3(hG1DM)有效缓解博来霉素诱导的hCD73转基因小鼠肺纤维化
为了考察19F3H2L3(hG1DM)治疗肺纤维化的体内药效学，本实验采用博来霉素诱导的hCD73转基因小鼠肺纤维化模型，测定19F3H2L3(hG1DM)干预后小鼠的肺功能和肺泡灌洗液炎症细胞数。
实验步骤：实验第1天，动物异氟烷麻醉后将通过气管内的给药途径给予2U/kg博来霉素，实验第2天开始给药，每三天给药一次直至实验结束，具体的给药方案请见表2所示。
表2实验方案
Figure PCTCN2021080517-appb-000006
Figure PCTCN2021080517-appb-000007
在第21天，所有动物使用WBP系统对实验小鼠进行气道高反应性测定，即小鼠雾化吸入PBS溶液，随后将连续雾化吸入浓度为1.5625、3.125、6.25、12.5、25和50mg/mL的乙酰甲胆碱(Mch)，测定相应浓度下的增强呼气间歇值，每个浓度下刺激90秒，绘制增强呼气间歇值相对基线的变化率-Mch浓度曲线，并计算曲线下面积。
肺功能检测完成后，异氟烷麻醉后眼眶采血约200微升，完成血样收集后，使用舒泰(腹腔注射，25-50mg/kg)对动物麻醉后进行气管插管，用0.8mL的PBS(包含1％BSA和0.6mM EDTA)对肺进行第一次灌洗。另取0.8mL的PBS(包含1％BSA和10mM EDTA)对肺进行第二次灌洗。4℃下300g离心BALF 5分钟，然后用1.5mL PBS将离心后获得的细胞团重悬用以涂片制备。使用血细胞分离器计算BALF中的细胞总数，使用Wright-Giemsa染色液染色，从而区分出嗜酸细胞，中性白细胞，巨噬细胞和淋巴细胞。在光学显微镜下计数。收集剩余BALF液体。灌洗后，通过脱颈椎安乐死动物，收集肺组织，冷冻保存于-80℃，用于匀浆后的羟脯氨酸含量检测。羟脯氨酸含量检测使用羟脯氨酸(hydroxyproline，HYP)检测试剂盒(QuiekZyme Biosciences)，依照试剂盒说明书完成。实验数据表示成均数±标准误(Mean±SEM)表示，组间比较采用GraphPad统计学处理软件处理后，进行单因素方差分析或t-test评价结果，P<0.05有显著性差异，P<0.01有非常显著性差异。
结果如图2所示，在分组后第21天，与模型-同型对照组，即人IgG1同型对照抗体组，相比，19F3H2L3(hG1DM)高剂量组和低剂量组均能够抑制增强呼气间歇值相对基线的变化率，19F3H2L3(hG1DM)高剂量组和低剂量组对增强呼气间歇值相对基线的变化率-Mch浓度曲线下面积抑制率分别为21.05％、29.05％。19F3H2L3(hG1DM)对此模型的气道反应性有治疗作用。
结果如图3和图4所示，在实验结束时，hCD73转基因小鼠肺纤维化模型19F3H2L3(hG1DM)高剂量组和低剂量组肺泡灌洗液中炎症细胞总数相比同型对照组均显著降低(P<0.001)，其中19F3H2L3(hG1DM)-高剂量组肺泡灌洗液中嗜酸性粒细胞(P<0.05)、中性粒细胞(P<0.05)和淋巴细胞(P<0.001) 的数量相对同型对照组显著降低，19F3H2L3(hG1DM)-低剂量组肺泡灌洗液中中性粒细胞(P<0.05)和淋巴细胞(P<0.001)的数量相对同型对照组显著降低。
结果如图5显示，19F3H2L3(hG1DM)高剂量组和19F3H2L3(hG1DM)低剂量组与模型-同型对照组相比均降低了小鼠肺组织匀浆中羟脯氨酸的含量，19F3H2L3(hG1DM)低剂量组显示出了有统计学意义的降低。
序列表
19F3重链可变区的核酸序列：(SEQ ID NO:1)
Figure PCTCN2021080517-appb-000008
19F3重链可变区的氨基酸序列：(SEQ ID NO:2)
Figure PCTCN2021080517-appb-000009
19F3轻链可变区的核酸序列：(SEQ ID NO:3)
Figure PCTCN2021080517-appb-000010
19F3轻链可变区的氨基酸序列：(SEQ ID NO:4)
Figure PCTCN2021080517-appb-000011
19F3H1L1(hG1DM)重链可变区的核酸序列：(SEQ ID NO:5)
Figure PCTCN2021080517-appb-000012
19F3H1L1(hG1DM)重链可变区的氨基酸序列：(SEQ ID NO:6)
Figure PCTCN2021080517-appb-000013
19F3H1L1(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:7)
Figure PCTCN2021080517-appb-000014
19F3H1L1(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:8)
Figure PCTCN2021080517-appb-000015
19F3H2L2(hG1DM)及19F3H2L3(hG1DM)重链可变区的核酸序列：(SEQ ID NO:9)
Figure PCTCN2021080517-appb-000016
Figure PCTCN2021080517-appb-000017
19F3H2L2(hG1DM)及19F3H2L3(hG1DM)重链可变区的氨基酸序列：(SEQ ID NO:10)
Figure PCTCN2021080517-appb-000018
19F3H2L2(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:11)
Figure PCTCN2021080517-appb-000019
19F3H2L2(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:12)
Figure PCTCN2021080517-appb-000020
19F3H2L3(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:13)
Figure PCTCN2021080517-appb-000021
19F3H2L3(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:14)
Figure PCTCN2021080517-appb-000022
19F3和19F3H1L1(hG1DM)、19F3H2L21(hG1DM)、19F3H2L3(hG1DM)的CDR区的 氨基酸序列：
HCDR1:GYSFTGYT(SEQ ID NO:15)
HCDR2:INPYNAGT(SEQ ID NO:16)
HCDR3:ARSEYRYGGDYFDY(SEQ ID NO:17)
LCDR1:QSLLNSSNQKNY(SEQ ID NO:18)
LCDR2:FAS(SEQ ID NO:19)
LCDR3:QQHYDTPYT(SEQ ID NO:20)
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的重链恒定区序列(330aa，突变位点用下划线标出)：
Figure PCTCN2021080517-appb-000023
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的轻链恒定区序列(107aa)：
Figure PCTCN2021080517-appb-000024

Claims (20)

  1. 抗CD73(例如人CD73)抗体或其抗原结合片段，其中，所述抗CD73抗体包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示重链可变区的HCDR1，HCDR2和HCDR3，和SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14所示的轻链可变区的LCDR1，LCDR2和LCDR3；
    优选地，根据IMGT编号系统，所述抗CD73抗体包含：
    HCDR1，其包含SEQ ID NO:15所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    HCDR2，其包含SEQ ID NO:16所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    HCDR3，其包含SEQ ID NO:17所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    LCDR1，其包含SEQ ID NO:18所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％，98％或99％ 序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    LCDR2，其包含SEQ ID NO:19所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，和
    LCDR3，其包含SEQ ID NO:20所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成。
  2. 权利要求1所述的抗CD73抗体或其抗原结合片段，所述抗体的重链可变区包含下述序列，或由下述序列组成：
    SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10，与SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；并且
    所述抗体的轻链可变区包含下述序列，或由下述序列组成：
    SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14，与SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12或SEQ ID NO:14具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12 或SEQ ID NO:14所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  3. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:14所示。
  4. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中所述抗体的重链恒定区为Ig gamma-1链C区，ACCESSION:P01857；轻链恒定区为Ig kappa链C区，ACCESSION:P01834。
  5. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中所述抗体为单克隆抗体、人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
  6. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双特异性抗体。
  7. 分离的多肽，其选自以下各项组成的组：
    (1)分离的多肽，其包含SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:18；SEQ ID NO:19；SEQ ID NO:20所示的序列；
    (2)分离的多肽，其包含SEQ ID NO:18；SEQ ID NO:19；SEQ ID NO:20所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17 所示的序列；
    (3)分离的多肽，其包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO:4、SEQ ID NO:8或SEQ ID NO:12所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
    (4)分离的多肽，其包含SEQ ID NO:4、SEQ ID NO:8或SEQ ID NO:12所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO:2、SEQ ID NO:6或SEQ ID NO:10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
    (5)分离的多肽，其包含SEQ ID NO:10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨 基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:14所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
    (6)分离的多肽，其包含SEQ ID NO:14所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO:10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  8. 核酸分子，其编码权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求7所述分离的多肽。
  9. 载体，其包含权利要求8所述的核酸分子。
  10. 宿主细胞，其包含权利要求8所述的核酸分子，或权利要求9所述的载体。
  11. 偶联物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)，可检测的标记或小分子药物；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、聚乙二醇或酶；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为肿瘤化疗药物，更优选地，所述抗体或其抗原结合片段通过连接子与小分子药物连接；例如，所述连接子为腙键、二硫键或肽键；更优选地，所述抗体或其抗原结合片段与小分子药物以一定的摩尔比连接；例如，所述摩尔比为1：(2－4)， 更优选地，所述小分子药物是抗纤维化药物。
  12. 融合蛋白或多特异性抗体(优选双特异性抗体)，其包含权利要求1-6任一项所述的抗体或其抗原结合片段。
  13. 试剂盒，其包括权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，所述第二抗体特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测CD73在样品中的存在或其水平。
  14. 药物组合物或单次药物剂量单元，其包含权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；可选地，所述药物组合物或单次药物剂量单元还包括药学上可接受的载体和/或赋形剂，优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
  15. 权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体，用于治疗和/或预防组织器官纤维化，或在制备治疗和/或预防组织器官纤维化的药物或试剂盒中的用途，优选地，所述组织器官纤维化选自：特发性肺纤维化；皮肤纤维化，例如硬皮病或外伤和手术后产生的皮肤瘢痕；眼纤维化，例如眼硬化症、结膜和角膜上的瘢痕以及结膜上生出的翼状胬肉；胰腺和肺的囊性纤维化；心内膜心肌纤维化；特发性心肌病；肝硬化；纵隔纤维化；进行性大块纤维化；增生性纤维化；肿瘤纤维化；肺结核导致的肺部的纤维化，优选地，所述试剂盒还包括治疗组织器官纤维化的药物。
  16. 治疗和/或预防组织器官纤维化的方法，包括向受试者或患者施用有效量的权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体，优选地，所述组织器官纤维化选自：特发性肺纤维化；皮肤纤维化，例如硬皮病或外伤和手术后产生的皮肤瘢痕；眼纤维化，例如眼硬化症、 结膜和角膜上的瘢痕以及结膜上生出的翼状胬肉；胰腺和肺的囊性纤维化；心内膜心肌纤维化；特发性心肌病；肝硬化；纵隔纤维化；进行性大块纤维化；增生性纤维化；肿瘤纤维化；肺结核导致的肺部的纤维化；更优选地，向所述受试者或患者同时或顺序施用抗纤维化药物，优选地，所述抗纤维化药物为RhoA、RhoAGTPases、TGF-β1或CTGF或RhoA信号通路的其它任何成员的调节剂，或者可以调节细胞因子信号传导抑制剂1(SOCS1)、细胞因子信号传导抑制剂3(SOCS 3)或TLR9的调节剂，或者是他汀类化合物或其衍生物。优选地，所述他汀类药物选自洛伐他汀，普伐他汀，氟伐他汀，西立伐他汀，阿托伐他汀，辛伐他汀，匹伐他汀，罗苏伐他汀，或其组合。
  17. 权利要求16所述的方法，更优选地，所述抗体或其抗原结合片段、偶联物、融合蛋白或多特异性抗体的有效量为0.001mg-1000mg，更优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，最优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg，或者，基于受试者或患者的体重，有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。
  18. 权利要求16所述的方法，所述抗纤维化药物的有效量为100-2400mg，优选100mg-2300mg、100mg-2200mg、100mg-2100mg、100mg-2000mg、100mg-1900mg、100mg-1800mg、100mg-1700mg、100mg-1600mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg，更优选为100mg，200mg，300mg，400mg，500mg，600mg，700mg，800mg，900mg，1000mg，或者，基于受试者或患者的体重，所述抗纤维化药物的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。
  19. 杂交瘤细胞株，其选自：
    LT014，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018137。
  20. 权利要求16-18任一项所述的方法，其中权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体被给予(优选静脉给予)单次或多次。
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