WO2020034941A1 - 抗IL-1β的抗体、其药物组合物及其用途 - Google Patents

抗IL-1β的抗体、其药物组合物及其用途 Download PDF

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WO2020034941A1
WO2020034941A1 PCT/CN2019/100343 CN2019100343W WO2020034941A1 WO 2020034941 A1 WO2020034941 A1 WO 2020034941A1 CN 2019100343 W CN2019100343 W CN 2019100343W WO 2020034941 A1 WO2020034941 A1 WO 2020034941A1
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antibody
seq
human
antigen
amino acid
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French (fr)
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李百勇
夏瑜
王忠民
张鹏
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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Priority to KR1020217007489A priority Critical patent/KR20210046024A/ko
Priority to IL280596A priority patent/IL280596B2/en
Priority to MX2021001778A priority patent/MX2021001778A/es
Priority to BR112021002794-7A priority patent/BR112021002794A2/pt
Priority to MYPI2021000751A priority patent/MY209465A/en
Priority to CN201980053246.6A priority patent/CN112654640B/zh
Priority to SG11202101435XA priority patent/SG11202101435XA/en
Priority to EA202190518A priority patent/EA202190518A1/ru
Priority to US17/267,115 priority patent/US12286473B2/en
Priority to AU2019320927A priority patent/AU2019320927B2/en
Priority to JP2021507628A priority patent/JP7576022B2/ja
Priority to CA3108414A priority patent/CA3108414A1/en
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Priority to EP19849700.0A priority patent/EP3845560A4/en
Publication of WO2020034941A1 publication Critical patent/WO2020034941A1/zh
Priority to PH12021550298A priority patent/PH12021550298A1/en
Priority to MX2025005169A priority patent/MX2025005169A/es
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Definitions

  • the invention belongs to the field of immunology, and relates to an antibody against IL-1 ⁇ , a pharmaceutical composition thereof and use thereof. Specifically, the present invention relates to an antibody against human IL-1 ⁇ ; more specifically, the present invention relates to a monoclonal antibody against human IL-1 ⁇ ; and in particular, the present invention relates to an anti-human IL-1 ⁇ Humanized monoclonal antibodies.
  • Interleukin-1 (IL-1) family consists of two pro-inflammatory factors (IL-1 ⁇ and IL-1 ⁇ ) and IL-1 receptor antagonist (IL-1Ra), IL-1 ⁇ And IL-1 ⁇ can effectively stimulate the IL-1 receptor (IL-1Receptor), while IL-1Ra can adhere to the surface of the IL-1 receptor and block signaling.
  • IL-1 ⁇ is mainly synthesized by monocytes and macrophages. The precursor of IL-1 ⁇ protein is cleaved by caspase-1 into an activated form.
  • the IL-1 receptor family includes multiple ligand subtypes, including IL-1R1, IL-1R2, and IL-1R3 (also known as interleukin receptor coordinate protein IL-1 receptor receptor protein, referred to as IL-1RAcP), IL -1R4, IL-1R5, IL-1R6, IL-1R7, IL-1R8, IL-18BP, and two orphan receptors, namely IL-1R9 and IL-1R10.
  • IL-1R1, IL-1R2, and IL-1R3 also known as interleukin receptor coordinate protein IL-1 receptor protein, referred to as IL-1RAcP
  • IL-1R4 also known as interleukin receptor coordinate protein IL-1 receptor protein, referred to as IL-1RAcP
  • IL-1R4 also known as interleukin receptor coordinate protein IL-1 receptor protein, referred to as IL-1RAcP
  • IL-1R4 also known as interleukin receptor coordinate protein IL-1 receptor protein, referred to as IL-1RA
  • IL-1R1 also known as type-1IL-1Receptor, referred to as IL-1R1
  • IL-1R1 is a transmembrane receptor that can bind to IL-1 ⁇ and bind to IL-1RAcP to form a receptor complex, which activates downstream intracellular related signaling pathways.
  • IL-1R2 and IL-1 ⁇ have higher affinity than IL-1R1, however, due to the short intracellular segment of IL-1R2, binding to IL-1 ⁇ cannot activate intracellular related signaling pathway ;
  • Both IL-1R1 and IL-1R2 exist in the form of soluble receptors (Boraschi et al. Immunological Reviews. 281: 197-232. (2016)).
  • IL-1 ⁇ regulates the recruitment and activation of effector cells involved in innate and adaptive immunity, and is also involved in the pathogenesis of chronic diseases, including gouty arthritis, various autoimmune diseases such as rheumatoid arthritis, multiple sclerosis , Periodic fever syndrome, and auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis, and children and adults with cold pyriline-related periodic syndrome and other diseases.
  • the IL-1 ⁇ pathway has recently been shown to be involved in tumors such as acute myeloid leukemia, liver cancer, lung cancer, and cardiovascular and cerebrovascular diseases.
  • Drugs targeting IL-1 ⁇ have shown good therapeutic and preventive effects (Cozzolino F et al. Proc. Natl Acad Soci USA. 86: 2369 (1989); Nakazaki H et al. Cancer. 70 (3): 709 (1992); (Ridker PM et al. Lancet. 390 (10105): 1833-1842 (2017)).
  • Rheumatoid arthritis (Rheumatoid arthritis, RA) is a chronic, systemic autoimmune disease mainly involving joint disease.
  • IL-1 ⁇ plays a key role in the pathogenesis of RA.
  • High levels of IL-1 ⁇ are present in the synovial fluid of patients with RA (van den Berg et al. Baillieres Best Pract Res Clin Rheumatol. 13: 577-97 (1999)).
  • IL-1 ⁇ mediates leukocyte joint infiltration and joints Matrix metalloproteinase secretion, induces cartilage degradation and inhibits new cartilage matrix synthesis, leading to joint destruction (van den Berg et al. Baillieres Best Pract Res Clin Rheumatol.
  • IL-1 ⁇ promotes bone loss, promotes leukocyte infiltration and vascular tissue formation in synovium by regulating pathways including TNF- ⁇ and IL-6 (Strand V et al. Rheumatology (Oxford). 43: iii10- iii16 (2004).). Clinical studies have confirmed that IL-1 ⁇ antagonists significantly improve the signs and symptoms of RA (Mertens M et al. J Rheumatol.
  • anti-human IL-1 ⁇ monoclonal antibody Canakinumab Can significantly reduce disease activity in RA patients (Alten R et al. Arthritis Res Ther. 10: R67 (2008); Alten R et al. BMC Musculoskeletal Disorders. 12: 153 (2011)).
  • Gout is a crystal-associated arthropathy caused by monosodium urate deposits, and is directly related to hyperuricemia caused by disorders of purine metabolism and / or decreased uric acid excretion, specifically acute characteristic arthritis and chronic gout stone disease , Mainly including acute onset arthritis, gout stone formation, gout stone chronic arthritis, urate nephropathy and uric acidic urinary stones; severe cases may appear joint disability and renal insufficiency. Gout is often accompanied by manifestations of abdominal obesity, hyperlipidemia, hypertension, type 2 diabetes, and cardiovascular disease. IL-1 ⁇ is a driving factor for the occurrence of gout inflammation (Cumpelik A et al. Ann Rheum Dis.
  • the anti-human IL-1 ⁇ monoclonal antibody Canakinumab can effectively alleviate clinical symptoms such as pain in patients with refractory gouty arthritis in clinical trials to evaluate the effectiveness and safety of treatment of gout.
  • the recurrence rate of gout is significantly lower than that in the triamcinolone group The quality of life has improved significantly (So A et al. Arthritis Rhum. 62: 3064-3076 (2010)).
  • the anti-human IL-1 ⁇ monoclonal antibody Canakinumab was found to significantly reduce the number of gout attacks compared to colchicine (Schlesinger N et al. Ann Rheum Dis. 70: 1264-1271 (2011).).
  • Canakinumab mAb is currently approved by the European Medicines Agency for the treatment of patients with gouty arthritis who have frequent attacks but are intolerant or ineffective in nonsteroidal anti-inflammatory drugs, colchicine, and glucocorticoids (Lyseng-Williamson KA et al. BioDrugs. 27: 401-406 (2013)).
  • the above studies suggest that anti-IL-1 ⁇ antibodies can more effectively treat gout, relieve symptoms, reduce recurrence, and have potential prevention and treatment effects for gout attacks compared to existing treatments.
  • Multiple sclerosis is a chronic demyelinating disease mediated primarily by Th1 and Th17 subpopulation T cells.
  • Interleukin IL-1 family factors play an important role in multiple sclerosis.
  • IL-1 promotes the development of the disease by promoting the development of Th17 subset T cells, and also causes the chemokine CXCL12 in the cerebral blood vessels to be distributed from the normal position on the parenchymal side to both sides of the endothelium, making it depolarized and leading to vascular leakage And T cells enter the brain parenchyma early in the disease process (Chih-Chung Lin et al. J Immunol.
  • EAE Experimental autoimmune encephalomyelitis
  • IL-1 ⁇ inhibitor treatment can delay the onset of EAE in wild-type mice, reduce the severity and shorten the duration of the disease (Chih-Chung Lin, et al. J Immunol. 198: 4553-4560 (2017).).
  • IL-1 ⁇ is involved in myocardial hypertrophy, myocardial fibrosis and dysfunction after myocardial infarction. Occurrence and development (Yue, et al. A, J, Hysiol. 275 (1Pt2): H250 (1998)). Heart failure is a complex syndrome, and studies have shown that IL-1 levels in circulating blood are increased in patients with congestive heart failure (Blum et al. Am. Heart J. 135 (2Part1): 181 (1998). Kapadia et al. Cardiol Clin. 16 (4): 645 (1998)).
  • IL-1 ⁇ may be involved in the occurrence and development of congestive heart failure caused by chronic load increase; serum IL-1 ⁇ levels in patients with heart function III to IV heart failure are significantly increased, which promotes the development of congestive heart failure (Yndestad A et al. Curr Cardiol Rep. 9: 236-41 (2007)).
  • Evaluate the effectiveness, safety, and tolerability of anti-human IL-1 ⁇ antibody Canakinumab for previous myocardial infarction with inflammatory atherosclerotic cardiovascular disease.
  • CANTOS III clinical studies show that anti-IL-1 ⁇ antibody combined with standard treatment significantly reduces patients Incidence of cardiogenic death, non-fatal myocardial infarction, and non-fatal stroke (Ridker PM et al. N Engl J Med. 377: 1119-1131 (2017)).
  • Cryopyrin-associated periodic syndromes in children and adults is a rare type of IL-1 ⁇ overproduction caused by a single gene mutation, causing weakness, flushing, fever, headache, joints Pain and conjunctivitis, which can occur in newborns or infants, can occur daily throughout the patient's life, can cause serious illness and can be fatal in the long term, including deafness, bone and joint deformation, central nervous system damage, blindness and amyloidosis Renal failure and premature death.
  • Cold and pyridiline-related periodic syndromes in children and adults include Familial Cold Auto-Inflammatory Syndrome (FCAS), Muckle-Wells Syndrome (MWS), and many neonates.
  • Anti-human IL-1 ⁇ antibody Canakinumab can significantly alleviate the clinical symptoms of patients with Cryopyrin protein-related cycle syndrome (Yokota et al. Clin Exp Rheumatol. 35 Suppl 108 (6): 19-26. (2017); Kone-Paut et al Human.Arthritis CareRes (Hoboken) .; 69: 903-911. (2017)) was therefore approved by the U.S. Federal Food and Drug Administration and the European Medicines Agency for the treatment of Cryopyrin protein in children (age ⁇ 4 years) and adults Patients with associated cycle syndrome.
  • Periodic fever syndromes are a rare group of autoimmune diseases that cause severe repetitive persistent fever and pathogenic inflammation through non-infectious activation of the immune system, often leading to disability and possibly joint pain , Swelling, muscle pain, rash, and fatal complications (Wurster VM et al. Pediatr Ann. 40 (1): 48-54. (2011)).
  • Periodic fever syndrome includes tumor necrosis factor receptor-associated periodic syndrome (TNF, associated periodic syndrome (TRAPS), Hyper-IgD syndrome (HIDS) / mevalonate kinase deficiency (Mevalonate Kinase Deficiency (MKD), Familial Mediterranean Fever (FMF).
  • TNF tumor necrosis factor receptor-associated periodic syndrome
  • TRAPS tumor necrosis factor receptor-associated periodic syndrome
  • HIDS Hyper-IgD syndrome
  • MKD mevalonate kinase deficiency
  • FMF Familial Mediterranean Fever
  • anti-human IL-1 ⁇ monoclonal antibody Canakinumab is effective in treating periodic fever syndrome (De Benedetti et al. N Engl J Med. 378 (20): 1908-1919 (2016)). Therefore, anti-human IL-1 ⁇ monoclonal antibody Canakinumab has been approved by the US Food and Drug Administration for the treatment of periodic fever syndrome.
  • Systemic juvenile idiopathic arthritis is a unique subtype of juvenile idiopathic arthritis, which usually develops with long-term high fever, rash, anemia and other extra-articular manifestations.
  • sJIA Systemic juvenile idiopathic arthritis
  • sJIA is currently generally considered to be an auto-inflammatory disease, non- Autoimmune Diseases (Sun Juan et al. Progress in Modern Biomedicine. 8: 1584-1588 (2016)).
  • Clinical studies have confirmed that the anti-human IL-1 ⁇ monoclonal antibody drug Canakinumab monoclonal antibody can effectively treat active sJIA with fever, reduce the amount of steroids, and significantly reduce the risk of sJIA recurrence (Orrock JE et al. Expert Rev Rev Clin Pharmacol. 9: 1015- 24. (2016)), so the anti-IL-1 ⁇ monoclonal antibody Canakinumab was approved by the US Food and Drug Administration for the treatment of systemic juvenile idiopathic arthritis.
  • the inventors After intensive research and creative labor, the inventors used mammalian cell expression system to express recombinant IL-1 ⁇ -His as an antigen to immunize mice, and obtained hybridoma cells by fusion of mouse spleen cells and myeloma cells. The inventors screened a large number of samples and obtained the following hybridoma cell lines:
  • the hybridoma cell line LT010 was deposited at the China Type Culture Collection (CCTCC) on June 21, 2018 under CCTCC NO: C2018133.
  • the hybridoma cell line LT010 can secrete and produce a specific monoclonal antibody (named 3H6) that specifically binds to human IL-1 ⁇ , and the monoclonal antibody can very effectively block the binding of IL-1 ⁇ to IL-1R1;
  • the inventors creatively prepared humanized antibodies against human IL-1 ⁇ (named 3H6, H1L1, 3H6, H2L2, 3H6, H3L3, 3H6, H4L1), which can effectively bind human IL-1 ⁇ and block IL-
  • the binding of 1 ⁇ to its receptor IL-1R1 inhibits the activation of the downstream signaling pathway of IL-1 ⁇ ; it is used for the preparation to reduce, prevent or treat rheumatoid arthritis, gout, multiple sclerosis, cardiovascular events and / or cardiovascular diseases,
  • drugs for diseases such as tumors, multiple sclerosis, cryopyline-related periodic syndromes in children and adults, periodic fever syndrome, and systemic juvenile idiopathic arthritis.
  • An aspect of the present invention relates to an anti-IL-1 ⁇ antibody or an antigen-binding fragment thereof, wherein:
  • variable region of the heavy chain of the antibody comprises: HCDR1-HCDR3 shown in SEQ ID Nos: 17-SEQ ID NO: 19;
  • the light chain variable region of the antibody includes: amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NO: 20-SEQ ID NO: 22, respectively.
  • the IL-1 ⁇ is human IL-1 ⁇ .
  • variable region of the heavy chain and the variable region of the light chain determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, which are called complementarity determining regions (CDR) , HCDR3, CDR of the light chain (L) contains LCDR1, LCDR2, LCDR3; it is named by Kabat et al., See Sequences of Proteins of Immunological Interface.Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md).
  • amino acid sequences of the three HCDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFSLSTSGMG (SEQ ID NO: 17),
  • HCDR2 IYWDDDK (SEQ ID NO: 18),
  • HCDR3 ARSAYYSFAY (SEQ ID NO: 19);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QDVDTD (SEQ ID NO: 20),
  • LCDR2 WAS (SEQ ID NO: 21),
  • LCDR3 QQYSSYPT (SEQ ID NO: 22).
  • the antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14; and
  • amino acid sequence of the light chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof, wherein the antibody is selected from:
  • VH as shown in SEQ ID NO: 2 and VL as shown in SEQ ID NO: 4;
  • the antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab') 2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (eg, scFv), humanized antibody, chimeric antibody or diabody.
  • the antibody or antigen-binding fragment thereof wherein the antibody is less than 10 -5 M, such as less than 10 -6 M, less than 10 -7 M, less than 10 K D of -8 M, less than 10 -9 M, or less than 10 -10 M or less binds to the IL-1 ⁇ protein; preferably, the K D is measured by a Biacore molecular interaction meter.
  • the antibody or antigen-binding fragment thereof wherein the antibody is less than about 100 nM, such as less than about 10 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than EC 50 of about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM or less binds the IL-1 ⁇ protein.
  • the EC 50 is measured by an indirect ELISA method.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody includes a non-CDR region, and the non-CDR region is from a species other than a mouse, such as from a human antibody.
  • the constant region of the antibody is humanized.
  • the constant region of the heavy chain uses Ig gamma-1 chain region, such as ACCESSION: P01857 or Ig gamma-4 chain C region, such as ACCESSION: P01861.1; Ig kappa chain regions are used in the light chain constant region, such as ACCESSION: P01834.
  • the antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line LT010, and the hybridoma cell line LT010 is deposited in China.
  • Culture Collection Center (CCTCC) deposit number CCTCC NO: C2018133.
  • the antibody is a monoclonal antibody.
  • ADC antibody-drug conjugate
  • ADC which includes an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or the antigen-binding fragment thereof is in the present invention.
  • the antibody or the antigen-binding fragment thereof according to any one; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a chemotherapy drug.
  • the chemotherapeutic drug may be a conventional tumor chemotherapeutic drug, such as an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant anticancer drug, a hormone, an immune preparation, and the like.
  • the antibody-drug conjugate wherein the antibody or antigen-binding fragment thereof is connected to the small molecule drug through a linker;
  • the linker may be a person skilled in the art Known linkers, for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond.
  • the antibody-drug conjugate wherein the molar ratio of the antibody or antigen-binding fragment thereof to the small-molecule drug is 1: (1-4), such as 1: 1, 1: 2, 1: 3 or 1: 4.
  • bispecific antibody also referred to as a bifunctional antibody
  • first protein functional region and a second protein functional region, wherein:
  • the first protein functional region targets IL-1 ⁇
  • the second protein functional region targets a target different from IL-1 ⁇ , such as IL-17A;
  • the first protein functional region is the antibody or antigen-binding fragment thereof according to any one of the present invention.
  • the bispecific antibody is in the IgG-scFv mode
  • the first protein functional region is an antibody or an antigen-binding fragment thereof according to any one of the present invention, and the second protein functional region is a single-chain antibody;
  • the first protein functional region is a single-chain antibody
  • the variable region of the heavy chain includes the amino acid sequences of HCDR1-HCDR3 shown in SEQ ID NO: 17-SEQ ID NO: 19, and the variable region of its light chain includes The amino acid sequence is LCDR1-LCDR3 shown in SEQ ID NO: 20-SEQ ID NO: 22, and the second protein functional region is an antibody (such as a monoclonal antibody).
  • the bispecific antibody wherein the first protein functional region and the second protein functional region are directly connected or connected through a connecting fragment
  • the linking fragment is (GGGGS) m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6;
  • the linking fragment is SS (GGGGS) n, and n is a positive integer, such as 1, 2, 3, 4, 5, or 6.
  • the bispecific antibody wherein in item (2),
  • amino acid sequence of the variable region of the heavy chain of the single-chain antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14; and
  • amino acid sequence of the light chain variable region of the single-chain antibody is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16.
  • the bispecific antibody wherein in item (2),
  • amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 4; or
  • amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 8; or
  • amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 12; or
  • amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 16.
  • the bispecific antibody wherein the first protein functional region and the second protein functional region are independently one, two, or two or more.
  • the bispecific antibody wherein in item (2), the constant region of the monoclonal antibody is selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4.
  • the bispecific antibody wherein the single chain antibody is linked to the C-terminus of the heavy chain of the antibody or monoclonal antibody.
  • a further aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a variable region of an antibody heavy chain and a nucleic acid sequence encoding a variable region of an antibody light chain, wherein:
  • the heavy chain variable region of the antibody includes HCDR1-HCDR3 shown in SEQ ID Nos: 17-SEQ ID NO: 19, and the light chain variable region of the antibody contains amino acid sequences such as SEQ ID NO : 20-LCDR1-LCDR3 shown in SEQ ID NO: 22;
  • the amino acid sequence of the variable region of the heavy chain of the antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, and the light chain variable of the antibody
  • the amino acid sequence of the region is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16;
  • the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of an antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8; or the heavy chain of the antibody is variable
  • the amino acid sequence of the region 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: 12; or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16;
  • the isolated nucleic acid molecule comprises:
  • the isolated nucleic acid molecule may be one nucleic acid molecule or multiple nucleic acid molecules, such as two nucleic acid molecules.
  • the heavy chain variable region and light chain variable region of an antibody can be expressed by the same nucleic acid molecule, for example, by the same or different expression cassettes located on the same nucleic acid molecule.
  • the heavy chain variable region and light chain variable region of an antibody can be expressed separately by different nucleic acid molecules.
  • a further aspect of the invention relates to a recombinant vector comprising an isolated nucleic acid molecule of the invention.
  • the recombinant vector may be one or more.
  • multiple nucleic acid molecules can be expressed by the same recombinant vector, or can be expressed by different recombinant vectors, respectively.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention or a recombinant vector of the invention.
  • Yet another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under appropriate conditions, and recovering the host cell from the cell culture. The steps of the antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention relates to a hybridoma cell line LT010, which is deposited in the China Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2018133.
  • CTCC China Type Culture Collection
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention; optionally It also includes pharmaceutically acceptable carriers and / or excipients.
  • Yet another aspect of the present invention relates to the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody drug conjugate of the present invention, or the bispecific antibody of the present invention in the preparation and treatment of and / or prevention of an autoimmune disease , Cardio-cerebral vascular disease, tumors, cryopyline-associated periodic syndromes in children and adults, use in medicines for juvenile idiopathic arthritis or gouty arthritis;
  • the autoimmune disease is selected from rheumatoid joints, multiple sclerosis and periodic fever syndrome;
  • the periodic fever syndrome is selected from the group consisting of tumor necrosis factor receptor-related periodic syndrome (TRAPS), high immunoglobulin D syndrome (HIDS) / valproic acid kinase deficiency (MKD), and family Sexual Mediterranean Fever (FMF);
  • TRAPS tumor necrosis factor receptor-related periodic syndrome
  • HIDS high immunoglobulin D syndrome
  • MKD valproic acid kinase deficiency
  • FMF family Sexual Mediterranean Fever
  • cryopyline-related periodic syndrome of children and adults is selected from the group consisting of familial cold autoinflammation syndrome, Mu-Werder syndrome, neonatal multisystem inflammatory disease, chronic pediatric neurocutaneous joint syndrome And familial cold urticaria;
  • the cardio-cerebral vascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, arterial thrombosis and stroke;
  • the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia;
  • the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.
  • the human IL-1R1 and / or human IL-1R2 is human IL-1R1 and / or human IL-1R2 on a cell surface.
  • the use is non-therapeutic and / or non-diagnostic.
  • the antibody or antigen-binding fragment thereof, the antibody drug conjugate of the present invention, or the bispecific antibody of the present invention is used for treating and / or preventing autoimmunity Diseases, cardiovascular and cerebrovascular diseases, tumors, cryopyline-related periodic syndromes in children and adults, systemic juvenile idiopathic arthritis or gouty arthritis;
  • the autoimmune disease is selected from rheumatoid joints, multiple sclerosis and periodic fever syndrome;
  • the periodic fever syndrome is selected from the group consisting of tumor necrosis factor receptor-related periodic syndrome (TRAPS), high immunoglobulin D syndrome (HIDS) / valproic acid kinase deficiency (MKD), and family Sexual Mediterranean Fever (FMF);
  • TRAPS tumor necrosis factor receptor-related periodic syndrome
  • HIDS high immunoglobulin D syndrome
  • MKD valproic acid kinase deficiency
  • FMF family Sexual Mediterranean Fever
  • cryopyline-related periodic syndrome of children and adults is selected from the group consisting of familial cold autoinflammation syndrome, Mu-Werder syndrome, neonatal multisystem inflammatory disease, chronic pediatric neurocutaneous joint syndrome And familial cold urticaria;
  • the cardio-cerebral vascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, arterial thrombosis and stroke;
  • the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia;
  • the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.
  • the antibody or antigen-binding fragment thereof, the antibody drug conjugate of the present invention, or the bispecific antibody of the present invention is used for:
  • the human IL-1R1 and / or human IL-1R2 is human IL-1R1 and / or human IL-1R2 on a cell surface.
  • Yet another aspect of the invention relates to a method in vivo or in vitro, comprising applying a cell in an effective amount of an antibody or antigen-binding fragment thereof according to any one of the invention, an antibody drug conjugate of the invention, or a
  • the steps of the bispecific antibody, the method is selected from the following:
  • the human IL-1R1 and / or human IL-1R2 is human IL-1R1 and / or human IL-1R2 on a cell surface.
  • the in vitro method is non-therapeutic and / or non-diagnostic.
  • Yet another aspect of the present invention relates to a therapeutic and / or prophylactic treatment and / or prevention of autoimmune diseases, cardio-cerebral vascular diseases, tumors, cryopyline-related periodic syndromes in children and adults, systemic juvenile idiopathic A method of arthritis or gouty arthritis, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody drug conjugate of the present invention, or the present invention Steps for bispecific antibodies;
  • the autoimmune disease is selected from rheumatoid joints, multiple sclerosis, and periodic fever syndrome;
  • the periodic fever syndrome is selected from the group consisting of tumor necrosis factor receptor-related periodic syndrome (TRAPS), high immunoglobulin D syndrome (HIDS) / valproic acid kinase deficiency (MKD), and family Sexual Mediterranean Fever (FMF);
  • TRAPS tumor necrosis factor receptor-related periodic syndrome
  • HIDS high immunoglobulin D syndrome
  • MKD valproic acid kinase deficiency
  • FMF family Sexual Mediterranean Fever
  • cryopyline-related periodic syndrome of children and adults is selected from the group consisting of familial cold autoinflammation syndrome, Mu-Werder syndrome, neonatal multisystem inflammatory disease, chronic pediatric neurocutaneous joint syndrome And familial cold urticaria;
  • the cardio-cerebral vascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, arterial thrombosis and stroke;
  • the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia;
  • the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.
  • the inventors found through animal experiments that the antibodies of the present invention, especially 3H6H4L1, can effectively alleviate pathological changes in a rheumatoid arthritis model induced by NIH / 3T3 cells transfected with human IL-1 ⁇ in BALB / c mice. It is shown that the administration of antibody drug 3H6H4L1 can effectively improve the pathological behavior of mice with rheumatoid arthritis and reduce the swelling area of the affected limbs of rheumatoid mice.
  • IL-1 ⁇ when referring to the amino acid sequence of IL-1 ⁇ (GenBank ID: NP_000567.1), it includes the full length of the IL-1 ⁇ protein, and also includes a fusion protein of IL-1 ⁇ , such as a mouse or mouse An Fc protein fragment (mFc or hFc) of human IgG or a fragment where multiple Hiss are fused.
  • mFc or hFc an Fc protein fragment
  • the term "IL-1 ⁇ ” shall include all such sequences, as well as natural or artificial variants thereof.
  • a sequence fragment of the IL-1 ⁇ protein it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
  • IL-1R1 when referring to the amino acid sequence of IL-1R1 (GenBank ID: NP_000868), it includes the full length of the IL-1R1 protein and also includes the fusion protein of IL-1R1, such as with mouse or human IgG Fc protein fragment (mFc or hFc) or multiple His fusion fragments.
  • mFc or hFc mouse or human IgG Fc protein fragment
  • IL-1R1 mutations or mutations (including but not limited to substitutions, deletions and / or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "IL-1R1" shall include all such sequences as well as their natural or artificial variants.
  • IL-1R1 when describing a sequence fragment of the IL-1R1 protein, it includes the IL-1R1 sequence fragment, and also the corresponding sequence fragment in its natural or artificial variant.
  • IL-1R2 when referring to the amino acid sequence of IL-1R2 (GenBank ID: CAA42441.1), it includes the full length of the IL-1R2 protein, and also includes a fusion protein of IL-1R2, such as with mouse or An Fc protein fragment (mFc or hFc) of human IgG or a fragment where multiple Hiss are fused.
  • mFc or hFc An Fc protein fragment
  • the term "IL-1R2" shall include all such sequences as well as their natural or artificial variants.
  • sequence fragment of the IL-1R2 protein when describing the sequence fragment of the IL-1R2 protein, it includes the IL-1R2 sequence fragment, and also the corresponding sequence fragment in its natural or artificial variant.
  • EC 50 refers to the concentration for 50% of maximal effect, and refers to the concentration that can cause 50% of the maximum effect.
  • antibody refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined 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 is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three 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 a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions or CDRs), with a more conservative region called a framework region (FR) interspersed between them.
  • CDRs complementarity determining regions
  • FR framework region
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxy terminal.
  • the variable regions (VH and VL) of each heavy / light chain pair form an antibody binding site, respectively.
  • the assignment of amino acids to various regions or domains follows Kabat Sequences of Immunological Interest (National Institute of Health, Bethesda, Md.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with a full-length antibody Specific binding to an antigen is also referred to as the "antigen-binding moiety".
  • antigen-binding fragment of the antibody is produced either by enzymatic or chemical fragmentation of the intact antibody.
  • the antigen-binding fragment includes Fab, Fab ', F (ab') 2, Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody, and polypeptides comprising at least a portion of an antibody sufficient to confer a polypeptide-specific antigen-binding ability.
  • the antigen-binding fragment of an antibody is a single chain antibody (e.g., scFv), where the VL and VH domains form a monovalent molecule by pairing them to enable the production of a linker for a single polypeptide chain (see, e.g., Bird et al Science 242: 423 426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85: 5879 5883 (1988)).
  • scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • a suitable prior art linker consists of a repeating GGGGS amino acid sequence or a variant thereof.
  • a linker having an amino acid sequence (GGGGS) 4 may be used, but a variant thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention are Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, that is, a bivalent antibody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow two structures on the same chain Domains, thereby forcing the domain to pair with the complementary domain of another chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • bifunctional antibodies also known as bispecific antibodies, are specific drugs that target two different antigens at the same time, and can be produced by immuno-sorting purification. In addition, it can also be obtained through genetic engineering. Genetic engineering has corresponding flexibility in terms of binding site optimization, consideration of synthetic forms, and yield, so it has certain advantages. At present, its existence has been proven to have more than 45 species (Müller, D, Kontermann, RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98).
  • bispecific antibodies that have been developed are in the form of IgG-ScFv, that is, the Morrison model (Coloma, MJ, Morrison, SL. Design and production of novel, tetravalent, specific antibodies. Nature, Biotechnology, 1997; 15: 159-163) Similar to the naturally occurring IgG form, its advantages in antibody engineering, expression, and purification have proven to be one of the ideal forms of bifunctional antibodies (Miller BR, Demarest SJ, et al., Stability engineering scFvs for the development of bispecific and multivalent antibodies.ProteinEngDeselSel2010; 23: 549-57; FitzgeraldJ, Lugovskoy
  • a given antibody (such as the monoclonal antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6, 3H6H3L3, or 3H6H4L1) provided by the present invention can be conventionally known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods)
  • An antigen-binding fragment of the antibody (for example, the above-mentioned antibody fragment) is obtained, and the antigen-binding fragment of the antibody is specifically screened in the same manner as used for the intact antibody.
  • MAb and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., in addition to natural mutations that may occur spontaneously, A group of identical antibody molecules.
  • a monoclonal antibody is highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically contain at least 2 or more different antibodies, which usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. (Nature, 256: 495, 1975), but can also be obtained using recombinant DNA technology (see, for example, U.S.P. 4,816,567).
  • humanized antibody means that all or part of a CDR region of a human immunoglobulin (receptor antibody) is replaced by a CDR region of a non-human antibody (donor antibody).
  • the antibody or antibody fragment thereof, wherein the donor antibody may be a non-human (eg, mouse, rat, or rabbit) antibody having a desired specificity, affinity, or reactivity.
  • some amino acid residues of the framework region (FR) of the receptor antibody can also be replaced by the amino acid residues of the corresponding non-human antibody, or by the amino acid residues of other antibodies to further improve or optimize the performance of the antibody.
  • isolated refers to those obtained from artificial means in a natural state. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has been changed, or the substance has been separated from the natural environment, or both. For example, a non-isolated polynucleotide or polypeptide naturally exists in a living animal, and the same high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated or “isolated” does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • an expression vector When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector.
  • Vectors can be introduced into host cells by transformation, transduction, or transfection, so that the genetic material elements they carry can be expressed in the host cells.
  • 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 (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes (PAC) derived from P1 ; Bacteriophage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and nipples Polyoma vacuole virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpes viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuole virus such as SV40
  • a vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as E. coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • bispecific As used in the present invention, the term "bispecific", “dual specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein or antibody having two different antigen binding sites, respectively.
  • a bispecific antibody is a multispecific antigen binding protein or a multispecific antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or ligation of Fab 'fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al. 1992, J. Immunol. 148: 1547-1553.
  • the two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes that are present on the same or different protein targets.
  • an antibody that specifically binds an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, or less than about 10 -10 M or less with an affinity (K D ) that binds the antigen.
  • K D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • antibodies are less than about 10 -5 M, such as less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M
  • a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 9 M or less than about 10 ⁇ 10 M or less binds an antigen (eg, an IL-1 ⁇ protein).
  • K D can be determined using methods known to those skilled in the art, such as using a Biacore molecular interaction instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • hybridoma and hybrida cell line are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, they also include subclones of the hybridoma And offspring cells. For example, when referring to the hybridoma cell line LT010, it also refers to subclones and progeny cells of the hybridoma cell line LT010.
  • 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 Pharmaceuticals Science. Edited by Gennaro AR, 19th Ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancement Agent.
  • pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain a desired effect.
  • an effective amount to prevent a disease e.g., RA
  • an effective amount to treat a disease refers to an amount sufficient to cure or at least partially prevent a patient already suffering from the disease The amount of disease and its complications. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the manner in which the drug is administered, and other treatments administered concurrently Wait.
  • the anti-IL-1 ⁇ antibody of the present invention especially a humanized anti-IL-1 ⁇ antibody, has one or more of the following technical effects:
  • Figure 1 Results of detection of the binding activity of 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 with human IL-1 ⁇ -His-Bio.
  • Figure 2 Results of detection of the binding activity of 3H6H4L1 to human IL-1 ⁇ -His-Bio.
  • Figure 3 Results of activity detection of 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 competing with human IL-1R1 (1-332) -his for binding to human IL-1 ⁇ -hFc.
  • Figure 4 Results of activity detection of 3H6H4L1 competing with human IL-1R1 (1-332) -his for binding to human IL-1 ⁇ -hFc.
  • Figure 5 Results of detection of the affinity constants of 3H6H4L1 and human IL-1 ⁇ . Note: Curves 1 to 5 indicate that the analyte concentrations are 25nM, 12.5nM, 6.25nM, 3.13nM, and 1.56nM, respectively.
  • Figure 6 Canakinumab and human IL-1 ⁇ affinity constant test results. Note: Curves 1 to 5 indicate that the analyte concentrations are 25nM, 12.5nM, 6.25nM, 3.13nM, and 1.56nM, respectively.
  • Figure 7 Effect of 3H6H4L1 on IL-1 ⁇ -induced MRC-5 secretion of IL-6.
  • Figure 8 Effect of IL-1 ⁇ on the gradient activation of the NF- ⁇ B signaling pathway.
  • Figure 9 Reporter diagram of 3H6H4L1 blocking IL-1 ⁇ reporter.
  • Figure 10 Effect of 3H6H4L1 on the pathological behavior of Lenti-IL-1 ⁇ -NIH / 3T3-induced mouse knee arthritis model mice.
  • Figure 11 Effect of 3H6H4L1 on the area of the knee joint in a mouse knee arthritis model induced by Lenti-IL-1 ⁇ -NIH / 3T3.
  • Figure 12 Effect of 3H6H4L1 on body weight of mouse knee arthritis model mice induced by Lenti-IL-1 ⁇ -NIH / 3T3.
  • BALB / c mice used were purchased from Guangdong Medical Experimental Animal Center.
  • human IL-1 ⁇ (Genbank ID: NP_000567.1) and IL-1R1 (Genbank ID: NP_000868) were searched through the NCBI GenBank protein database.
  • the amino acid sequences of human IL-1 ⁇ and IL-1R1 were fused with His tag sequences and human IgG and Fc purification tag sequences, respectively; the above fusion proteins were abbreviated as human IL-1 ⁇ -His and IL-1R1 (1-332) -His, IL-1 ⁇ -hFc.
  • the protein sample quality was qualified by SDS-PAGE.
  • the Sulfo-NHS-LC-Biotinylation Kit (Thermo scientific) was used to prepare biotinylated human IL-1 ⁇ -His protein samples (referred to as human IL-1 ⁇ -His-Bio for short); the specific preparation method was performed with reference to the instructions of the kit.
  • the prepared fusion protein was used in the following examples.
  • BALB / c mice purchased from Guangdong Medical Laboratory Animal Center
  • IL-1 ⁇ -his as an antigen
  • spleen cells of the immunized mice were fused with mouse myeloma cells to prepare hybridoma cells.
  • the hybridoma cells were screened by ELISA to obtain hybridoma cells capable of secreting antibodies that specifically bind to IL-1 ⁇ -His-Bio.
  • hybridoma cells obtained by ELISA screening hybridoma cells capable of secreting antibodies that compete with the receptor IL-1R1 (1-332) -His for binding to IL-1 ⁇ -hFc were screened by competitive ELISA, and stabilized by limiting dilution.
  • Hybridoma cell line The method of preparing hybridoma cells refers to the currently established methods (for example, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods, "Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000) .
  • hybridoma cell line as hybridoma cell line LT010 (IL-1 ⁇ -3H6), and the secreted monoclonal antibody was named 3H6.
  • the hybridoma cell line LT010 (IL-1 ⁇ -3H6) was deposited at the China Type Culture Collection (CCTCC) on June 21, 2018 under CCTCC NO: C2018133, and its deposit address is Wuhan, China. Wuhan University , Zip code: 430072.
  • the LT010 cell line prepared above was cultured in a hybridoma serum-free medium (hybridoma serum-free medium containing 1% penicillin and 4% Glutamax, and cultured at 5% CO 2 at 37 ° C. The cells were cultured in an oven). After 7 days, the cell culture supernatant was collected, purified by high-speed centrifugation, vacuum filtration through a microporous filter, and a HiTrap protein A HP column to obtain antibody 3H6. The purified 3H6 sample was qualified by SDS-PAGE electrophoresis.
  • mRNA was extracted from the LT010 cell line cultured in Example 1.
  • Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.
  • the PCR amplification products were directly TA cloned.
  • pEASY-T1 Cloning Kit Transgen CT101
  • the TA cloned products were directly sequenced.
  • the sequencing results are as follows:
  • amino acid sequence of the variable region of the heavy chain of antibody 3H6 is as follows: (118aa, where the underlined amino acid sequence is a CDR region)
  • amino acid sequence of the variable region of the light chain of antibody 3H6 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
  • the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 heavy chain variable region sequences and light chain variable region sequences (3H6H1L1, 3H6H2L2, and 3H6H3L3 antibody constant region sequences, derived from the NCBI database, the heavy chain constant region is Ig gamma-1 chain region, ACCESSION: P01857, the light chain constant region is Ig kappa chain region, ACCESSION: P01834; 3H6H4L1 antibody constant region sequence, from the NCBI database, heavy chain constant The region is Igamma-4, chain region, ACCESSION: P01861.1; the light chain constant region is Ig, chain region, ACCESSION: P01834).
  • the sequence of the heavy chain and light chain variable regions of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 are as follows:
  • amino acid sequence of the heavy chain variable region of antibody 3H6H1L1 is as follows: (118aa, where the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of antibody 3H6H1L1 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the variable region of the heavy chain of the antibody 3H6H2L2 is as follows: (118aa, where the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of antibody 3H6H2L2 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the variable region of the heavy chain of antibody 3H6H3L3 is as follows: (118aa, where the underlined amino acid sequence is the CDR region)
  • amino acid sequence of the light chain variable region of antibody 3H6H3L3 is as follows: (106aa, where the underlined amino acid sequence is the CDR region)
  • the nucleic acid sequence encoding the heavy chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 5.
  • amino acid sequence of the variable region of the heavy chain of the antibody 3H6H4L1 is shown in SEQ ID NO: 6.
  • the nucleic acid sequence encoding the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 7.
  • amino acid sequence of the light chain variable region of the antibody 3H6H4L1 is shown in SEQ ID NO: 8.
  • the heavy chain constant regions of 3H6H1L1, 3H6H2L2 and 3H6H3L3 all use Ig gamma-1 chain region, ACCESSION: P01857; the light chain constant regions use Ig kappa chain C region, ACCESSION: P01834;
  • the constant region of the heavy chain of 3H6H4L1 is Ig gamma-4 region, ACCESION: P01861.1; the constant region of the light chain is Igappa chain region, ACCESSION: P01834.
  • 3H6H1L1 heavy chain cDNA and light chain cDNA, 3H6H2L2 heavy chain cDNA and light chain cDNA, 3H6H3L3 heavy chain cDNA and light chain cDNA, and 3H6H4L1 heavy chain cDNA and light chain cDNA were cloned into the pUC57simple vector (Kings) Provided by Rui Company), eight recombinant plasmids were obtained, namely pUC57simple-3H6H1 and pUC57simple-3H6L1; pUC57simple3H6H2 and pUC57simple-3H6L2; pUC57simple-3H6H3 and pUC57simple-3H6L3; pUC57simple-3H6H4 and pUC57simple-3.
  • the plasmid containing the heavy chain and the recombinant plasmid containing the light chain were co-transfected into 293F cells, and the culture fluid was collected and purified to obtain humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1; and the results were correct by SDS-PAGE.
  • Example 4 Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human IL-1 ⁇ -his-bio Binding Activity Study (ELISA method)
  • the microplate was coated with 2 ⁇ g / mL of SA (streptavidin) in 50 ⁇ L per well and incubated overnight at 4 ° C. The plate was washed once and the residual liquid was removed, and each well was blocked with 300 ⁇ L of a 1% BSA solution (dissolved in PBS) and incubated at 37 ° C. for 2 hours. Wash the plate three times and remove residual liquid.
  • Human IL-1 ⁇ -His-Bio was diluted with PBST to 0.2 ⁇ g / mL, 50 ⁇ L / well, and incubated at 37 ° C. for 30 minutes. The plate was washed three times and the residual liquid was removed.
  • the labeled goat anti-mouse IgG (H + L) secondary antibody working solution was incubated at 37 ° C for 30 minutes. Among them, 50 ⁇ L horseradish peroxidase-labeled goat anti-human IgG (H + L) secondary antibody working solution (corresponding to 3H6H1L1, 3H6H2L2, 3H6H3L3, 3H6H4L1, Canakinumab wells) and 50 ⁇ L horseradish peroxidase-labeled sheep Anti-mouse IgG (H + L) secondary antibody working solution (corresponding to the well where 3H6 is located). Wash the plate four times and remove the residual liquid. Add 50 ⁇ L of TMB color development solution to each well.
  • the software was analyzed and processed with SoftMax Pro 6.2.1 software.
  • a 4-parameter fitted curve was plotted with the antibody concentration as the abscissa and the absorbance value as the ordinate, and the results are shown in Figures 1 and 2, respectively.
  • the detection results of the binding activity of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 with human IL-1 ⁇ -His-Bio are shown in Table 1 and Table 2, respectively.
  • 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 can effectively bind human IL-1 ⁇ -His-Bio, and the binding efficiency shows a dose-dependent relationship;
  • Example 5 Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human Activity of IL-1R1 (1-332) -his for binding to human IL-1 ⁇ -hFc (ELISA method)
  • the microplate was coated with 4 ⁇ g / mL of human IL-1 ⁇ -hFc in 50 ⁇ L per well and incubated at 4 ° C. overnight. Wash the plate once and remove the residual liquid, add 300 ⁇ L of 1% BSA solution (dissolved in PBS) to each well and block, incubate at 37 ° C for 2 hours, wash the plate three times and remove the residual liquid.
  • the antibody was diluted to 2 ⁇ g / mL (final concentration 1 ⁇ g / mL) as the starting concentration, and a 1: 3 gradient dilution was performed at a total of 7 concentrations. A blank control was also set up, and two duplicate wells were made.
  • the volume of each well was 50 ⁇ L, and incubated at room temperature for 10 minute. Add 0.08 ⁇ g / mL (final concentration of 0.04 ⁇ g / ml) or 0.1 ⁇ g / ml (final concentration of 0.05 ⁇ g / ml) of human IL-1R1 (1-332) -his to the microplate, and the volume of each well is 50 ⁇ L and Antibody volume was mixed gently 1: 1, the final volume of each well was 100 ⁇ L, and incubated at 37 ° C for 30 minutes. Wash the plate three times and remove the residual liquid.
  • 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 and 3H6H4L1 can effectively block the binding of antigen human IL-1 ⁇ -hFc and its receptor human IL-1R1 (1-332) -his, and the blocking efficiency shows a dose-dependent relationship, competing for binding Its activity is better than that of the same target drug, Canakinumab.
  • the affinity constant of the antibody to human IL-1 ⁇ -his was detected by Biacore molecular interaction instrument.
  • the antibody was fixed on the surface of the CM5 chip by amino coupling method, and the fixed signal value was about 1000RU.
  • the antibody binds to human IL-1 ⁇ .
  • the concentration of IL-1 ⁇ is 1.56-25nM (double gradient dilution), the flow rate is 30 ⁇ l / min, the binding time is 120s, and the dissociation time is 600s.
  • the chip was regenerated using 3M MgCl 2 with a flow rate of 30 ⁇ l / min and a time of 30 s.
  • Data were collected using Biacore Control 2.0 software, and Biacore T200 Evaluation 2.0 software was used for data analysis. The results are shown in Table 5, Figure 5, and Figure 6.
  • the affinity constant of 3H6H4L1 and human IL-1 ⁇ is 8.79E-11M, and the affinity constant of Canakinumab and human IL-1 ⁇ is 9.79E-11M, indicating that 3H6H4L1 has strong binding ability to human IL-1 ⁇ .
  • Human MRC-5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) were routinely digested and counted. 7,500 cells / well were seeded in flat-bottomed 96-well plates and cultured in cell incubators; after 24 hours (when cell growth reached 80% confluency), Carrying out drug administration: the antibody was set at 4 concentrations (0.37nM, 1.11nM, 3.33nM, 10nM), and IL-1 ⁇ (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was set at three concentrations (5pM, 50pM, 500pM), The antibody group IL-1 ⁇ was used at a concentration of 50 pM (antibodies and IL-1 ⁇ were first incubated at 37 ° C for 20 min), and a blank control group and an isotype control group were also set up at the same time; they were placed in a cell incubator for 24 hours after dosing, and the cells were collected. The test was performed with IL-6ELISA Kit (purchased from Dakow Bio
  • IL-1 ⁇ could significantly promote IL-6 secretion by MRC-5 in a dose-dependent manner; 3H6 and H4L1 could specifically inhibit IL-1 ⁇ -induced IL-6 secretion activity in MRC-5 cells, showing that 3H6 and H4L1 had a significant effect on IL- Specific neutralizing activity of 1 ⁇ .
  • the fluorescent reporter gene method was used to detect the neutralizing biological activity of 3H6 and H4L1 blocking IL-1 ⁇ and activating the NF- ⁇ B signaling pathway.
  • 293T cells were trypsinized and subcultured; 2 hours before transfection, the opti-DMEM medium was replaced; 500 ⁇ L of opti-DMEM medium was added to a sterile EP tube, and 3 ⁇ g of the plasmid pNF-kB-Luc2P-hygro was added; Add 500 ⁇ L of opti-DMEM medium to the bacterial EP tube, and then add 8 ⁇ L of lipofectamine 2000; add the diluted lipofectamine 2000 to the diluted plasmid, leave it at room temperature for 15 min, and add it evenly to the cell culture dish; after transfection 8h, replace Fresh medium; 24 hours after transfection, Hygromycin was added, and the final concentration was screened at 100 ⁇ g / mL.
  • the control well 293T was not transfected with plasmid. After 7-10 days, the cells in the control wells shall die completely. The selected cells are harvested for expansion and culture. Continue to add 100 ⁇ g / mL to maintain. 293T-NF- ⁇ B-LUC stably transformed cell line was obtained.
  • 293T-NF- ⁇ B-LUC cells were routinely digested and seeded into 96-well plates at 20,000 cells per well. After the cells adhere to the wall, add IL-1 ⁇ to a final concentration of 1.65ng / mL, and set a blank control. At the same time, antibodies, Canakinumab, 3H6 H4L1 were added, each antibody had 5 gradients, and the final concentrations were: 400, 100, 25, 6.25, 1.56ng / mL. After 6 hours of co-cultivation, the supernatant was removed, 50 ⁇ L of PBS was added, and 50 ⁇ L of Bright-Glo TM substrate was added. The reaction was performed for 5 minutes and detected on a computer.
  • IL-1 ⁇ can effectively activate the expression of the luciferase reporter gene that is dependent on the NF- ⁇ B signaling pathway and is significantly dose-dependent;
  • 3H6 and H4L1 can specifically block the activation of NF- ⁇ B signaling pathway by IL-1 ⁇ in a dose-dependent manner.
  • Example 8 3H6 and H4L1 alleviate rheumatoid knee arthritis induced by NIH / 3T3 cells transfected with human IL-1 ⁇ Model mouse treatment
  • mice 46 BALB / c mice were divided into 6 groups based on body weight, namely:
  • Normal group model group, positive control group, 3H6 H4L1 low-dose group, 3H6 H4L1 middle-dose group, and 3H6 H4L1 high-dose group; except for the normal group, there were 8 in each group.
  • Canakinumab was injected subcutaneously in the positive control group
  • Anti-HEL was injected subcutaneously in the model group
  • 3H6H4L1 was injected at the corresponding concentration in each dose group
  • normal saline was injected subcutaneously in the normal group.
  • NIH / 3T3 purchased from the American Type Strain Collection Center
  • Lenti-IL-1 ⁇ -NIH / 3T3 cells were collected in a biological safety cabinet.
  • Lenti-IL-1 ⁇ -NIH / 3T3 cell lines were obtained from Lenti-IL-1 ⁇
  • the vector was transfected into NIH / 3T3 cells, and after screening, Lenti-IL-1 ⁇ -NIH / 3T3 cell lines stably secreting and expressing IL-1 ⁇ were obtained.
  • the number of NIH / 3T3 and Lenti-IL-1 ⁇ -NIH / 3T3 cells reached the required seeding numbers, the cells were collected.
  • mice After BALB / c mice were anesthetized with 7.5 ml / kg of 3.5% chloral hydrate, the knees of normal group were inoculated with 25 ⁇ l of NIH / 3T3 cell suspension (50,000 cells / head) and the rest of the knees. Lenti-IL-1 ⁇ -NIH / 3T3 cell suspension was inoculated intraarticularly with 25 ⁇ l / head (50,000 cells / head). After inoculation, the knee joint was sutured, and penicillin diluted 20 times with normal saline was applied.
  • mice in each group were euthanized by cervical dislocation, the knee joints of the affected limbs were dissected, and the length (mm) and width (mm) of the synovial membranes of the affected knees of the mice were measured with a caliper. Data are expressed as mean ⁇ standard error ( ⁇ SEM). Comparison between groups was processed using GraphPad, Prism 5 statistical processing software, and univariate analysis of variance was used to evaluate the results. There were significant differences at P ⁇ 0.05 and very significant at P ⁇ 0.01. difference.
  • Fig. 10 shows that compared with the normal group, the mice in the model group obviously showed pathological behavior (P ⁇ 0.01).
  • Canakinumab and 3H6 and H4L1 high and medium dose groups can effectively improve the pathological behavior of rheumatoid arthritis mice (P ⁇ 0.01), and 3H6 and H4L1 low doses can improve the rheumatoid arthritis mice.
  • the pathological behavior is not obvious (P> 0.05).
  • 3H6 and H4L1 have a dose-effect relationship to improve the degree of pathological behavior in mice.
  • the efficacy of the positive control group was better than that of the 3H6 and H4L1 medium and low dose groups (P ⁇ 0.01).
  • the high dose of 3H6 and H4L1 was equivalent to the positive control group (P> 0.05).
  • Figure 11 shows that compared with the normal group, the area of the knee joint of the mice in the model group was significantly increased (P ⁇ 0.01).
  • the positive control group (Canakinumab) and the 3H6 and H4L1 medium and high dose groups can significantly reduce the swelling area of the affected limb of rheumatoid arthritis mice (P ⁇ 0.01), and the 3H6 and H4L1 low dose
  • the effect of group on reducing swelling area of rheumatoid mice was not obvious (P> 0.05).
  • 3H6 and H4L1 have a dose-effect relationship in reducing the swelling area of the affected limbs in rheumatoid arthritis mice.
  • the equivalent dose of 3H6 and H4L1 was comparable to that of the target drug Canakinumab (P> 0.05).
  • Figure 12 shows that compared with the normal group, the body weight of the mice in the model group was significantly reduced (P ⁇ 0.01). After administration, compared with the model group, Canakinumab and 3H6H4L1 high-dose groups with the same target have significantly reduced the weight loss of arthritic mice (P ⁇ 0.05). Compared with the positive control group, the equivalent dose of 3H6 and H4L1 was equivalent to the target drug Canakinumab (P> 0.05).

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Abstract

提供一种抗IL-1β的抗体、其药物组合物及其用途。具体地，提供一种抗IL-1β的抗体或其抗原结合片段，其中，所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO：17-SEQ ID NO：19所示的HCDR1-HCDR3；和所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO：20-SEQ ID NO：22所示的LCDR1-LCDR3。该抗体能够有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合，抑制IL-1β下游信号通路的激活；具有用于制备防治自身免疫性疾病、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎、痛风性关节炎、心血管疾病或肿瘤的药物的潜力。

Description

抗IL-1β的抗体、其药物组合物及其用途 技术领域
本发明属于免疫学领域，涉及一种抗IL-1β的抗体、其药物组合物及其用途。具体地，本发明涉及一种抗人IL-1β的抗体；更具体地，本发明涉及一种抗人IL-1β的单克隆抗体；特别具体地，本发明涉及一种抗人IL-1β的人源化单克隆抗体。
背景技术
白介素-1(Interleukin 1，IL-1)家族由两种促炎因子(IL-1α和IL-1β)和IL-1受体拮抗剂(IL-1receptor antagonist，IL-1Ra)组成，IL-1α和IL-1β可以有效激动IL-1受体(IL-1Receptor)，IL-1Ra则可以粘附在IL-1受体表面而阻断信号传导。IL-1β主要由单核细胞和巨噬细胞合成，IL-1β蛋白前体经半胱天冬酶-1(caspase-1)剪切变为活化形式。IL-1受体家族包括多个配体亚型，包括IL-1R1、IL-1R2、IL-1R3(又称为白介素受体协同蛋白IL-1 receptor accessory protein，简称为IL-1RAcP)、IL-1R4、IL-1R5、IL-1R6、IL-1R7、IL-1R8、IL-18BP以及2个孤儿受体即IL-1R9和IL-1R10。
目前研究发现，IL-1β可与IL-1R1、IL-1R2结合。IL-1R1(又称为type 1IL-1 Receptor，简称为IL-1R1)为跨膜受体，可与IL-1β结合并与IL-1RAcP结合形成受体复合物，激活下游胞内相关信号通路，介导IL-1β相关生物学效应；IL-1R2与IL-1β亲和力较IL-1R1高，然而由于IL-1R2胞内段较短，因此与IL-1β结合后无法激活胞内相关信号通路；IL-1R1和IL-1R2均存在可溶性受体形式(Boraschi等人.Immunological Reviews.281:197–232.(2018))。
IL-1β调节参与先天性免疫和适应性免疫的效应细胞的募集和激活，也参与慢性疾病的发病机理，包括痛风性关节炎、各种自身免疫性疾病，如类风湿关节炎、多发性硬化、周期性发热综合征，和自身炎症性疾病，如全身型幼年特发性关节炎，以及儿童和成人的冷吡啉相关的周期性综合征等疾病的发生。IL-1β通路近来被证实参与肿瘤如急性髓性白血病、肝癌、肺癌以及心脑血管疾病的发生，以IL-1β为靶点的药物展现了良好的治疗和预防效果(Cozzolino F等人.Proc Natl Acad Soci USA.86:2369(1989)；Nakazaki H等人.Cancer.70(3):709(1992)；(Ridker PM等人.Lancet.390(10105):1833-1842(2017))。
类风湿关节炎(Rheumatoid arthritis，RA)是一种以关节病变为主的慢性、全身性的自身免疫疾病。IL-1β在RA发病中发挥关键作用。高水平的IL-1β存在于RA患者的关节 腔滑液中(van den Berg等人.Baillieres Best Pract Res Clin Rheumatol.13:577-97(1999))，IL-1β介导白细胞关节浸润以及关节中基质金属蛋白酶的分泌，诱导软骨退化并抑制新的软骨基质合成，从而导致关节破坏(van den Berg等人.Baillieres Best Pract Res Clin Rheumatol.13:577-97(1999).；Johnson LL等人.Curr Opin Chem Biol.2:466-471(1998).)；在RA患者中，IL-1β刺激破骨细胞分化和激活，参与RA受累关节的骨侵蚀的发生(Gravallese EM等人.Ann Rheum Dis.61:ii84–86(2002).；Ghivizzani SC等人.J Immunol.1:3604-12(1997).；Horai R等人.J Exp Med.191:313-20(2000).；Gravallese EM等人.Arthritis Rheum.43:250-8(2000).；Takayanagi H等人.Arthritis Rheum.43:259-69(2000).)。另外，IL-1β通过调控TNF-α和IL-6在内的通路，促进骨质丢失，促进滑膜中白细胞浸润和血管翳组织形成(Strand V等人.Rheumatology(Oxford).43:iii10-iii16(2004).)。临床研究证实，IL-1β拮抗剂显著改善RA的体征和症状(Mertens M等人.J Rheumatol.36(6):1118-25(2009))，抗人IL-1β单抗Canakinumab
Figure PCTCN2019100343-appb-000001
可显著降低RA患者的疾病活动(Alten R等人.Arthritis Res Ther.10:R67(2008)；Alten R等人.BMC Musculoskeletal Disorders.12:153(2011))。
痛风是由单钠尿酸盐沉积所致的晶体相关性关节病，与嘌呤代谢紊乱和/或尿酸排泄减少所致的高尿酸血症直接相关，特指急性特征性关节炎和慢性痛风石疾病，主要包括急性发作性关节炎、痛风石形成、痛风石性慢性关节炎、尿酸盐肾病和尿酸性尿路结石；重者可出现关节残疾和肾功能不全。痛风常伴腹型肥胖、高脂血症、高血压、2型糖尿病及心血管病等表现。IL-1β是痛风炎症发生的驱动因素(Cumpelik A等人.Ann Rheum Dis.pii:annrheumdis-2015-207338(2015))。研究表明，在痛风性关节炎患者中，外周血单个核细胞IL-1βmRNA及血清IL-1β的表达均显著高于健康对照组，急性期组显著高于慢性期和间歇期组，患者血清IL-1β的浓度与白细胞、中性粒细胞、红细胞沉降率等指标呈正相关，提示IL-1β可能同时参与了急性与慢性痛风性关节炎，且与炎症程度相关，间歇期组血清IL-1β浓度仍然显著高于健康对照组，提示尽管关节症状已消失但关节及组织炎症仍可能存在(李玲琴等人.中华全科医师杂志.14:29-31(2015).)。动物实验研究表明，IL-1β在急性痛风性关节炎和慢性痛风性关节炎中均起重要作用，尿酸盐晶体刺激血液及关节液中单核细胞和吞噬细胞引起IL-1β大量释放(Di Giovine FS等人.J Immunol.138:3213-3218(1987).)；在小鼠模型实验中发现，IL-1β阻断剂可阻止腹膜腔内尿酸盐晶体注射引起的中性粒细胞聚集，在小鼠缺乏IL-1β受体的部位不发生聚集(Martinon F等人.Nature.440:237-241(2006))。临床研究表明痛风急性发作期间IL-1β诱导大量促炎症细胞因子产生 (Amaral FA等人.Arthritis Rheum.64:474-484(2012)；Torres R等人.Ann Rheum Dis.68:1602-1608(2009))。
抗人IL-1β单克隆抗体Canakinumab在评价治疗痛风的有效性及安全性临床试验中可有效缓解难治性痛风性关节炎患者的临床症状如疼痛，痛风复发率较曲安奈德组明显减低，生活质量有明显改善(So A等人.Arthritis Rhum.62:3064-3076(2010))。在另一项临床研究中发现，抗人IL-1β单克隆抗体Canakinumab较秋水仙碱可明显减少痛风发作的次数(Schlesinger N等人.Ann Rheum Dis.70:1264-1271(2011).)。Canakinumab单抗目前已被欧洲医药管理局(European Medicines Agency)批准用于频繁发作的但对非甾体抗炎药、秋水仙碱、糖皮质激素不能耐受或无效的痛风性关节炎患者的治疗(Lyseng-Williamson KA等人.BioDrugs.27:401-406(2013))。以上研究提示抗IL-1β抗体相较现有治疗手段可以更为有效的治疗痛风，缓解症状，减少复发，对于痛风的发作有潜在的防治作用。
多发性硬化是主要由Th1和Th17亚群T细胞介导的慢性脱髓鞘疾病。白介素IL-1家族因子在多发性硬化中具有重要作用。IL-1通过促进Th17亚群T细胞的发育来促进疾病发展，并且还引起脑血管中趋化因子CXCL12从实质侧的正常位置分布到内皮两侧，使其去极化分布，导致血管渗漏和T细胞在疾病进程早期进入脑实质(Chih-Chung Lin等人.J Immunol.198:4553-4560(2017).)；动物研究证明，在IL-1或IL-1R1缺陷小鼠中无法诱导出实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)，而IL-1β抑制剂处理可以延迟野生型小鼠EAE发作，降低严重性并缩短疾病持续时间(Chih-Chung Lin等人.J Immunol.198:4553-4560(2017).)。
近期临床还研究发现，抗IL-1β抗体通过拮抗IL-1β通路发挥抗炎效用，显著降低心血管疾病的发病风险。动脉粥样硬化和血栓形成是冠心病病理基础(Dalek os GN等人.J Lab Clin Med.129:300(1997))，在兔高胆固醇动脉粥样硬化模型中发现IL-1β和IL-1β的mRNA在脂质斑块中表达升高，而减少IL-1的合成可减慢动脉粥样硬化的发展(Dinarell o CA等人.N Eng l J Med.328:106.(1993))。在心肌梗死时IL-1β水平增高，在梗死后心肌重构过程中，IL-1β在血浆和心梗局部表达均增加，IL-1β参与心梗发生后心肌肥厚、心肌纤维化和功能不全的发生、发展(Yue P等人.A m J P hysiol.275(1Pt2):H250(1998))。心力衰竭是一种复杂综合征，研究表明，充血性心衰的患者循环血中IL-1水平增高(Blum A等人.Am Heart J.135(2Part1):181(1998).Kapadia S等人.Cardiol Clin.16(4):645(1998))。IL-1β可能参与了慢性负荷增加导致充血性心衰的发生和发展过程；在心功能Ⅲ～Ⅳ级心衰患者中血清IL-1β水平明显增高，促进充血性心衰的发展(Yndestad A等人. Curr Cardiol Rep.9:236-41(2007))。评估抗人IL-1β抗体Canakinumab对于既往心梗合并炎症性动脉粥样硬化性心血管病的有效性、安全性和耐受性CANTOS III临床研究显示，抗IL-1β抗体联合标准治疗显著降低患者的心源性死亡、非致死性心肌梗死和非致死性脑卒中发生率(Ridker PM等人.N Engl J Med.377:1119-1131(2017))。
针对抗人IL-1β抗体Canakinumab的CANTOS临床研究队列的另一项分析发现，抗IL-1β抗体Canakinumab显著降低肺癌的发生和死亡风险(Ridker PM等人.Lancet.390(10105):1833-1842(2017))。在取自患者的急性髓细胞性白血病(Acute myeloid leukemia，AML)细胞体外培养发现，80％以上的原代AML患者的肿瘤细胞IL-1表达增加，IL-1β可显著促进肿瘤细胞的增长，而抗IL-1β或IL-1α抗体均可有效抑制肿瘤细胞的增长(Cozzolino F等人.Proc Natl Acad Soci USA.86:2369(1989))。在肝细胞癌患者中，其血清中IL-1水平较健康人对照组明显增高，(Nakazaki H等人.Cancer.70(3):709(1992))。
儿童和成人的冷吡啉相关的周期性综合征(Cryopyrin-associated periodic syndromes，CAPS)是一类由单一基因突变引起IL-1β过度产生的罕见疾病，造成虚弱乏力、潮红、发热、头痛、关节痛和结膜炎，可出现在初生儿或婴儿，可在患者一生中每日出现，长期可造成严重疾病和可能致命，包括耳聋、骨和关节变形、中枢神经系统损伤导致失明和淀粉样变造成肾衰竭和早亡。儿童和成人的冷吡啉相关的周期性综合征包括家族冷自主炎症综合征(Familial Cold Auto-Inflammatory Syndrome，FCAS)，穆-韦二氏综合征(Muckle-Wells Syndrome，MWS)，新生儿多系统炎性疾病(Neonatal-onset multisystem inflammatory disease)、慢性小儿神经皮肤关节综合征(Chronic infantile neurological cutaneous and articular syndrome)、家族性冷荨麻疹(Familiar cold urticaria)。抗人IL-1β的抗体Canakinumab可显著缓解Cryopyrin蛋白相关性周期综合征患者临床症状(Yokota S等人.Clin Exp Rheumatol.35Suppl 108(6):19-26.(2017)；Kone-Paut I等人.Arthritis Care Res(Hoboken).；69:903-911.(2017))因此被美国联邦食品药品监督管理局和欧洲医药管理局批准用于治疗儿童(年龄≥4岁)和成人的Cryopyrin蛋白相关性周期综合征患者。
周期性发热综合征(Periodic Fever Syndromes)是一组罕见的自身免疫性疾病，通过免疫系统的非感染性激活导致严重反复性持续性发热和致病性炎症，往往导致残疾并可能伴有关节疼痛、肿胀、肌肉疼痛、皮疹及可致命的并发症(Wurster VM等人.Pediatr Ann.40(1):48-54.(2011))。周期性发热综合征包括肿瘤坏死因子受体相关的周期性综合征(TNF receptor associated periodic syndrome，TRAPS)、高免疫球蛋白D综合征(Hyper-IgD syndrome，HIDS)/甲羟戊酸激酶缺乏症(Mevalonate kinase deficiency，MKD)、家族性 地中海热(Familial mediterranean fever，FMF)。临床研究报道证实抗人IL-1β单抗Canakinumab可有效治疗周期性发热综合征(De Benedetti F等人.N Engl J Med.378(20):1908-1919(2018))。因此抗人IL-1β单抗Canakinumab被美国联邦食品药品监督管理局等批准用于治疗周期性发热综合征。
全身型幼年特发性关节炎(Systemic juvenile idiopathic arthritis，sJIA)是幼年特发性关节炎(Juvenile idiopathic arthritis)中一种独特的亚型，多以长期高热、皮疹、贫血等关节外表现起病，常见于0-5岁幼儿，以慢性关节滑膜炎为主要特征，多伴有不同程度的器官、组织损害，主要表现为发热、皮疹和关节痛，具有长期缓解率低、功能障碍率和致残率高、死亡率较高等预后不良表现(Woerner A等人.Expert Rev Clin Immunol.11(5):575-88.(2015))，目前普遍认为sJIA是一种自身炎症性疾病，非自身免疫性疾病(孙娟等人。现代生物医学进展。8：1584-1588(2016))。临床研究证实，抗人IL-1β单克隆抗体药物Canakinumab单抗可有效治疗伴发热的活动性sJIA，减少类固醇用量，显著降低sJIA复发风险(Orrock JE等人.Expert Rev Clin Pharmacol.9:1015-24.(2016))，因此抗IL-1β单抗Canakinumab被美国联邦食品药品监督管理局等批准用于治疗全身型幼年特发性关节炎。
目前还需要开发新的抗IL-1β的抗体药物。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的IL-1β-His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进行筛选，得到了如下的杂交瘤细胞株：
杂交瘤细胞株LT010，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:C2018133。
本发明人惊奇地发现：
杂交瘤细胞株LT010能够分泌产生与人IL-1β特异性结合的特异性单克隆抗体(命名为3H6)，并且该单克隆抗体能够十分有效地阻断IL-1β与IL-1R1的结合；
进一步地，本发明人创造性地制得了抗人IL-1β的人源化抗体(分别命名为3H6 H1L1，3H6 H2L2，3H6 H3L3，3H6 H4L1)，能有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合，抑制IL-1β下游信号通路的激活；具有用于制备降低、预防或治疗类风湿关节炎、痛风、多发性硬化、心血管事件和/或心血管疾病、肿瘤、多发性硬化、儿童和成人的冷吡 啉相关的周期性综合征、周期性发热综合征、全身型幼年特发性关节炎等疾病的药物的潜力。
由此提供了下述发明：
本发明的一个方面涉及一种抗IL-1β的抗体或其抗原结合片段，其中，
所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3；和
所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3。
优选地，所述IL-1β为人IL-1β。
重链可变区和轻链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Sequences of Proteins of Immunological Interest.Fifth Edition(1991),第1-3卷,NIH Publication 91-3242,Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析本发明的抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1，可以发现其具有相同的HCDR1-3和LCDR1-3：
其重链可变区的3个HCDR区的氨基酸序列如下：
HCDR1：GFSLSTSGMG(SEQ ID NO：17)，
HCDR2：IYWDDDK(SEQ ID NO：18)，
HCDR3：ARSAYYSFAY(SEQ ID NO：19)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QDVDTD(SEQ ID NO:20)，
LCDR2：WAS(SEQ ID NO:21)，
LCDR3：QQYSSYPT(SEQ ID NO:22)。
在本发明的一个或多个实施方案中，所述的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选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14；和
所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16。
在本发明的一些实施方案中，所述的抗体或其抗原结合片段，其中，所述抗体选自：
(1)如SEQ ID NO:2所示的VH和如SEQ ID NO:4所示的VL；
(2)如SEQ ID NO:6所示的VH和如SEQ ID NO:8所示的VL；
(3)如SEQ ID NO:10所示的VH和如SEQ ID NO:12所示的VL；和
(4)如SEQ ID NO:14所示的VH和如SEQ ID NO:16所示的VL。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中，所述的抗体以小于10 -5M，例如小于10 -6M、小于10 -7M、小于10 -8M、小于10 -9M或小于10 -10M或更小的K D结合IL-1β蛋白；优选地，所述K D通过Biacore分子相互作用仪测得。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，所述的抗体以小于大约100nM，例如小于大约10nM、小于大约1nM、小于大约0.9nM、小于大约0.8nM、小于大约0.7nM、小于大约0.6nM、小于大约0.5nM、小于大约0.4nM、小于大约0.3nM、小于大约0.2nM、小于大约0.1nM或更小的EC 50结合IL-1β蛋白。具体地，所述EC 50通过间接ELISA方法测得。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中，
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方案中，所述抗体的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1 chain C region，例如ACCESSION:P01857或Ig gamma-4chain C region，例如ACCESSION：P01861.1；轻链恒定区均采用Ig kappa chain C region，例如ACCESSION:P01834。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT010产生的单克隆抗体，所述杂交瘤细胞株LT010保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。
在本发明的一个或多个实施方案中，所述抗体为单克隆抗体。
本发明的另一方面涉及一种抗体药物偶联物(antibody-drug conjugate，ADC)，其包 括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为本发明中任一项所述的抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为化疗药物。
所述化疗药物可以是常规的肿瘤化疗药物，例如烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫制剂等。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，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；所述连接子可以是本领域技术人员知悉的连接子，例如，所述连接子为腙键、二硫键或肽键。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段与小分子药物的摩尔比为1：(1－4)，例如1:1、1：2、1：3或1：4。
本发明的再一方面涉及一种双特异性抗体(又称为双功能抗体)，其包括第一蛋白功能区和第二蛋白功能区，其中：
所述第一蛋白功能区靶向IL-1β，
所述第二蛋白功能区靶向不同于IL-1β的靶点，例如IL-17A；
其中,所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段；
优选地，所述双特异性抗体为IgG-scFv模式；
优选地，
(1)所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段，并且所述第二蛋白功能区为单链抗体；
或者，
(2)所述第一蛋白功能区为单链抗体，其重链可变区包含氨基酸序列如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1－LCDR3，并且所述第二蛋白功能区为抗体(例如单克隆抗体)。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为SS(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
在本发明的一些实施方式中，所述的双特异性抗体，其中，第(2)项中，
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14；和
所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16。
在本发明的一些实施方式中，所述的双特异性抗体，其中，第(2)项中，
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；或者
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；或者
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；或者
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一些实施方式中，所述的双特异性抗体，其中，(2)项中，所述单克隆抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单链抗体连接在抗体或单克隆抗体的重链的C末端。
本发明的再一方面涉及一种分离的核酸分子，其包含编码抗体重链可变区的核酸序列和编码抗体轻链可变区的核酸序列，其中，
所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3，和所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3；
优选地，所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14，和所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16；
更优选地，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；或者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；或者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；或者所述抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
进一步优选地，所述分离的核酸分子包含：
SEQ ID NO:1和SEQ ID NO:3所示的核酸序列，
SEQ ID NO:5和SEQ ID NO:7所示的核酸序列，
SEQ ID NO:9和SEQ ID NO:11所示的核酸序列，或者
SEQ ID NO:13和SEQ ID NO:15所示的核酸序列。
所述分离的核酸分子，可以是一个核酸分子，也可以是多个核酸分子，例如两个核酸分子。当是一个核酸分子时，抗体的重链可变区和轻链可变区可以通过同一个核酸分子表达，例如通过位于同一核酸分子上的相同或不同的表达框来表达。当是多个核酸分子例如两个核酸分子时，抗体的重链可变区和轻链可变区可以通过不同的核酸分子分别表达。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。所述的重组载体可以是一个或多个。当所述核酸分子是多个(例如两个)时，多个(例如两个)核酸分子可以通过同一重组载体表达，也可以分别通过不同的重组载体来表达。
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，或者包含本发明的重组载体。
本发明的再一方面涉及一种制备本发明中任一项所述的抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。
本发明的再一方面涉及一种杂交瘤细胞株LT010，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体；可选地，其还包括药学上可接受的载体和/或赋形剂。
本发明的再一方面涉及本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体 药物偶联物或者本发明的双特异性抗体在制备治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎的药物中的用途；
优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化和周期性发热综合征；
优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性地中海热(FMF)；
优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
本发明的再一方面本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体在制备如下药物中的用途：
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的药物，
下调人IL-1β活性或水平的药物，或者
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的药物。
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。
在本发明的一个实施方案中，所述用途是非治疗目的的和/或非诊断目的的。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎；
优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化和周期性发热综合征；
优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性地中海热(FMF)；
优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于：
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合，
下调人IL-1β活性或水平，或者
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活。
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤，所述方法选自如下：
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的方法，
下调人IL-1β活性或水平的方法，或者
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的方法。
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。
在本发明的一个实施方案中，所述体外方法是非治疗目的的和/或非诊断目的的。
本发明的再一方面涉及一种治疗和/或预防治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤；
优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化、周期性发热综合征；
优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性 地中海热(FMF)；
优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
本发明人通过动物实验发现，本发明的抗体特别是3H6H4L1能够在BALB/c小鼠中有效地缓解转染人IL-1β的NIH/3T3细胞诱导的类风湿关节炎模型的中的病理学改变，表现为给予抗体药3H6H4L1可有效改善类风湿关节炎小鼠的病理行为，减少类风湿性小鼠患肢的肿胀面积。
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，本发明中所用的细胞培养、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，当提及IL-1β的氨基酸序列(GenBank ID：NP_000567.1)时，其包括IL-1β蛋白的全长，还包括IL-1β的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1β的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1β”应包括所有此类序列，及其天然或人工的变体。并且，当描述IL-1β蛋白的序列片段时，其不仅包括序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及IL-1R1的氨基酸序列(GenBank ID：NP_000868)时，其包括IL-1R1蛋白的全长，还包括IL-1R1的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R1”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R1蛋白的序列片段时，包括IL-1R1序列 片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及IL-1R2的氨基酸序列(GenBank ID：CAA42441.1)时，其包括IL-1R2蛋白的全长，还包括IL-1R2的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R2蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R2”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R2蛋白的序列片段时，包括IL-1R2序列片段，还包括其天然或人工变体中的相应序列片段。
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，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(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL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。VH和VL区还可被细分为具有高变性的区域(称为互补决定区即CDR)，其间散布有较保守的称为构架区(FR)的区域。各VH和VL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(VH和VL)分别形成抗体结合部位。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,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883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，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性 结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423 426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879 5883(1988))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
在另一些情况下，抗体的抗原结合片段是“双功能抗体”。双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向两种不同抗原的特异性药物，其可通过免疫分选纯化生产。另外，也可通过基因工程获得，基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，其存在形式已被证明有超过45种(Müller D,Kontermann RE.Bispecific antibodies for cancer immunotherapy:Current perspectives.BioDrugs 2010；24:89-98)。目前已开发的多种双特异性抗体为IgG-ScFv形式即Morrison模式(1997 Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nature Biotechnology,1997；15:159-163)，由于这种类似于天然存在的IgG形式，其在抗体工程、表达和纯化上所具有的优势，已被证明是双 功能抗体的其中一种理想存在形式(Miller BR,Demarest SJ,et al.,Stability engineering of scFvs for the development of bispecific and multivalent antibodies.Protein Eng Des Sel2010；23:549-57；Fitzgerald J,Lugovskoy A.Rational engineering of antibody therapeutics targeting multiple oncogene pathways.MAbs 2011；3:299-309)。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体3H6、3H6H1L1、3H6H2L2、3H6 H3L3或3H6H4L1)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
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断，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(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 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,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。
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，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本发明中所使用的，术语“双特异性”、“双重特异性”或“双功能性”抗原结合蛋白或抗体是分别具有两个不同的抗原结合位点的杂交抗原结合蛋白或抗体。双特异性抗体是一种多特异性抗原结合蛋白或多特异性抗体，并且可通过多种方法产生，包括，但不限于杂交瘤的融合或Fab′片段的连接。参见，例如，Songsivilai和Lachmann，1990，Clin.Exp.Immunol.79：315-321；Kostelny等人.1992，J.Immunol.148：1547-1553。双特异性抗原结合蛋白或抗体的两个结合位点将结合两个不同的表位，所述表位存在于相同或不同的蛋白质靶上。
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、小于大约10 -7M、小于大约10 -8M、小于大约10 -9M或小于大约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(例如，本发明的单克隆抗体3H6、3H6H1L1、3H6H2L2、或3H6H3L3)以小于大约10 -5M，例如小于大约10 -6M、小于大约10 -7M、小于大约10 -8M、小于大约10 -9M或小于大约10 -10M或更小的解离平衡常数(K D)结合抗原(例如，IL-1β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Biacore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“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT010时，其还指杂交瘤细胞株LT010的亚克隆和后代细胞。
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。
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RA)有效量是指，足以预防，阻止，或延迟疾病(例如RA)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。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其它治疗等。
发明的有益效果：
本发明的抗IL-1β抗体特别是人源化的抗IL-1β抗体具有如下技术效果中的一项或多项：
(1)有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合；
(2)抑制IL-1β下游信号通路的激活；
(3)能够特异性抑制IL-1β诱导MRC-5细胞分泌IL-6的活性；
(4)能够有效地阻断IL-1β对NF-κB的激活；
(5)具有用于制备抑制IL-1β的药物的潜力；
(6)具有用于制备预防和/或治疗类风湿关节炎、痛风、多发性硬化、心血管事件和/ 或心血管疾病、肿瘤、多发性硬化、儿童和成人的冷吡啉相关的周期性综合征、周期性发热综合征、全身型幼年特发性关节炎等病症的药物的潜力。
附图说明
图1：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1β-His-Bio的结合活性检测结果。
图2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果。
图3：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。
图4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。
图5：3H6H4L1与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。
图6：Canakinumab与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。
图7：3H6H4L1对IL-1β诱导MRC-5分泌IL-6的影响。
图8：IL-1β对NF-κB信号通路的梯度激活的影响。
图9：3H6H4L1阻断IL-1β的reporter assay图。
图10：3H6H4L1对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型小鼠病理性行为的影响。
图11：3H6H4L1对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型小鼠膝关节面积的影响。
图12：3H6H4L1对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型小鼠体重的影响。
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。
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，所用的同靶点已上市药物抗体Canakinumab(商品名
Figure PCTCN2019100343-appb-000002
)作为对照抗体，购自诺华(Novartis)。
制备例1：融合蛋白人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc以及人IL-1β-His-Bio 的制备
通过NCBI GenBank蛋白数据库查找人IL-1β(Genbank ID:NP_000567.1)及IL-1R1(Genbank ID:NP_000868)的蛋白序列。将人IL-1β及IL-1R1的氨基酸序列分别与His标签序列，人IgG Fc纯化标签序列进行融合设计；上述的融合蛋白分别简写命名为人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc。
蛋白样品质量通过SDS-PAGE鉴定合格。
使用
Figure PCTCN2019100343-appb-000003
Sulfo-NHS-LC-Biotinylation Kit(Thermo scientific)制备生物素化偶联的人IL-1β-His蛋白样品(简称为人IL-1β-His-Bio)；具体制备方法参考该试剂盒的说明书进行。
制得的上述融合蛋白用于下面的实施例。
实施例1：抗IL-1β鼠源抗体3H6的制备
1.杂交瘤细胞株LT010的制备
以人IL-1β-his作为抗原免疫BALB/c小鼠(购自广东医学实验动物中心)，取免疫后小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。以IL-1β-His-Bio作为抗原，对杂交瘤细胞进行ELISA法筛选，获得能够分泌和IL-1β-His-Bio特异性结合的抗体的杂交瘤细胞。对ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与受体IL-1R1(1-332)-His竞争结合IL-1β-hFc的抗体的杂交瘤细胞，并经过有限稀释法得到稳定的杂交瘤细胞株。杂交瘤细胞制备的方法参考目前已确立的方法(例如，Stewart,S.J.,“Monoclonal Antibody Production”,in Basic Methods in antibody Production and Characterization,Eds.G.C.Howard and D.R.Bethell,Boca Raton:CRC Press,2000)。
发明人将以上杂交瘤细胞株命名为杂交瘤细胞株LT010(IL-1β-3H6)，其分泌的单克隆抗体命名为3H6。
杂交瘤细胞株LT010(IL-1β-3H6)，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:C2018133，保藏地址为中国.武汉.武汉大学， 邮编：430072。
2.抗IL-1β的抗体3H6的制备
用含杂交瘤无血清培养基对上面制得的LT010细胞株进行培养(杂交瘤无血清培养基，内含1％青链霉素和4％的Glutamax，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以及HiTrap protein A HP柱进行纯化，制得抗体3H6。纯化后的3H6样品进行SDS-PAGE电泳检测合格。
实施例2：抗IL-1β的抗体3H6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LT010细胞株中提取mRNA。按照Invitrogen
Figure PCTCN2019100343-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：
编码抗体3H6的重链可变区的核酸序列：(354bp)
Figure PCTCN2019100343-appb-000005
抗体3H6的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000006
编码抗体3H6的轻链可变区的核酸序列：(318bp)
Figure PCTCN2019100343-appb-000007
Figure PCTCN2019100343-appb-000008
抗体3H6的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000009
实施例3：抗IL-1β的人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的 设计和制备
1.抗IL-1β人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的轻链和重链序列的设计
根据IL-1β蛋白的三维晶体结构(van Oostrum J,Priestle JP,Grütter MG,Schmitz A.The structure of murine interleukin-1beta at 2.8 A resolution.J Struct Biol.1991，107(2):189-95.)以及实施例2获得的序列信息，设计得到人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1重链可变区的序列和轻链可变区的序列(3H6H1L1、3H6H2L2和3H6H3L3抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-1 chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834；3H6H4L1抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-4 chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)。
人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的重链可变区序列和轻链可变区序列如下：
(1)人源化单克隆抗体3H6H1L1
编码抗体3H6H1L1的重链可变区的核酸序列：(354bp)
Figure PCTCN2019100343-appb-000010
Figure PCTCN2019100343-appb-000011
抗体3H6H1L1的重链可变区氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000012
编码抗体3H6H1L1的轻链可变区的核酸序列：(318bp)
Figure PCTCN2019100343-appb-000013
抗体3H6H1L1的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000014
(2)人源化单克隆抗体3H6H2L2
编码抗体3H6H2L2的重链可变区的核酸序列：(354bp)
Figure PCTCN2019100343-appb-000015
Figure PCTCN2019100343-appb-000016
抗体3H6H2L2的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000017
编码抗体3H6H2L2的轻链可变区的核酸序列：(318bp)
Figure PCTCN2019100343-appb-000018
抗体3H6H2L2的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000019
(3)人源化单克隆抗体3H6H3L3
编码抗体3H6H3L3的重链可变区的核酸序列：(354bp)
Figure PCTCN2019100343-appb-000020
Figure PCTCN2019100343-appb-000021
抗体3H6H3L3的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000022
编码抗体3H6H3L3的轻链可变区的核酸序列：(318bp)
Figure PCTCN2019100343-appb-000023
抗体3H6H3L3的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)
Figure PCTCN2019100343-appb-000024
(4)人源化单克隆抗体3H6H4L1
编码抗体3H6H4L1的重链可变区的核酸序列如SEQ ID NO:5所示。
抗体3H6H4L1的重链可变区的氨基酸序列如SEQ ID NO:6所示。
编码抗体3H6H4L1的轻链可变区的核酸序列如SEQ ID NO:7所示。
抗体3H6H4L1的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
2.人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1的制备
3H6H1L1、3H6H2L2和3H6H3L3的重链恒定区均采用Ig gamma-1 chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION:P01834；
3H6H4L1的重链恒定区为Ig gamma-4 chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION:P01834。
将3H6H1L1重链cDNA和轻链的cDNA、3H6H2L2的重链cDNA和轻链的cDNA、3H6H3L3重链cDNA和轻链的cDNA，以及3H6H4L1重链cDNA和轻链的cDNA分别克隆到pUC57simple载体(金斯瑞公司提供)中，分别获得8个重组质粒即pUC57simple-3H6H1和pUC57simple-3H6L1；pUC57simple3H6H2和pUC57simple-3H6L2；pUC57simple-3H6H3和pUC57simple-3H6L3；pUC57simple-3H6H4和pUC57simple-3H6L1。并分别亚克隆到pcDNA3.1载体中。将包含重链的质粒和包含轻链的重组质粒共转染293F细胞后收集培养液进行纯化获得人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1；并经SDS-PAGE检测，结果正确。
实施例4：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1β-his-bio 结合的活性研究(ELISA法)
以2μg/mL的SA(链霉亲和素)，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔用300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时。洗板三次并除去残留液体。用PBST将人IL-1β-His-Bio稀释成0.2μg/mL，50μL/孔，37℃孵育30分钟，洗板三次并除去残留液体。分别将抗体稀释至如表1的1μg/mL或如表2的0.333μg/mL作为起始浓度，进行1:3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积100μL，37℃孵育30分钟。洗板三次后通过拍打将包被好的酶标板除去残余液体，每孔加入50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗工作液或辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗工作液，37℃孵育30分钟。其中，50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗工作液(对应3H6H1L1、3H6H2L2、3H6H3L3、3H6H4L1、Canakinumab所在的孔)和50μL辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗工作液(对应3H6所在的孔)。洗板四次并除去残留液体，每孔加入50μL TMB显色液，室温避光显色5分钟后，每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。
用SoftMax Pro 6.2.1软件对数据进行分析处理。以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果分别如图1及图2所示。3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1β-His-Bio的结合活性检测结果分别见表1和表2。
表1：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1β-His-Bio的结合活性检测结果
Figure PCTCN2019100343-appb-000025
表2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果
Figure PCTCN2019100343-appb-000026
Figure PCTCN2019100343-appb-000027
结果显示：
3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1均能够有效地结合人IL-1β-His-Bio，并且结合效率呈现剂量依赖关系；
在相同检测条件下，3H6 H1L1、3H6 H2L2及3H6H4L1与抗原人IL-1β-His-Bio的结合效率呈现剂量依赖关系，并且结合活性优于同靶点已上市药物Canakinumab；3H6H3L3结合活性与Canakinumab相当。
实施例5：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人 IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性研究(ELISA法)
以4μg/mL的人IL-1β-hFc，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔加入300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时，洗板三次并除去残留液体。抗体稀释至2μg/mL(终浓度1μg/mL)作为起始浓度，进行1:3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积50μL，室温孵育10分钟。将0.08μg/mL(终浓度0.04μg/ml)或0.1μg/ml(终浓度0.05μg/ml)的人IL-1R1(1-332)-his加入到酶标板中，每孔体积50μL与抗体体积1:1轻柔混匀，每孔终体积100μL，37℃孵育30分钟。洗板三次并除去残留液体，每孔加入50μL抗His标签鼠单克隆抗体(HRP标记)工作液，37℃孵育30分钟。洗板四次并除去残留液体，每孔加入50μl的TMB显色液，室温避光显色10分钟或5分钟后，每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。
用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果如图3及图4所示。3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果见表3及表4。
表3：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果
Figure PCTCN2019100343-appb-000028
表4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果
Figure PCTCN2019100343-appb-000029
结果显示：
3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1均能够有效地阻断抗原人IL-1β-hFc与其受体人IL-1R1(1-332)-his的结合，且阻断效率呈现剂量依赖关系，竞争结 合活性优于同靶点已上市药物Canakinumab。
实施例6：抗体3H6H4L1与人IL-1β的亲和力常数测定
采用Biacore分子相互作用仪检测抗体与人IL-1β-his的亲和力常数。以PBST作为缓冲液，采用氨基偶联方式将抗体固定于CM5芯片表面，固定的信号值约为1000RU。抗体与人IL-1β结合，IL-1β浓度为1.56-25nM(两倍梯度稀释)，流速为30μl/min，结合的时间为120s，解离时间为600s。芯片使用3M MgCl 2再生，流速为30μl/min，时间为30s。使用Biacore Control 2.0软件进行数据采集，Biacore T200Evaluation 2.0软件进行数据分析。结果如表5、图5和图6所示。
表5：3H6H4L1与人IL-1β亲和力常数检测结果
抗体名称 K D(M) ka(1/Ms) SE(ka) kd(1/s) SE(kd) Rmax(RU)
3H6H4L1 8.79E-11 1.44E+06 3.15E+03 1.27E-04 1.75E-07 112.49-122.37
Canakinumab 9.79E-11 5.24E+05 6.84E+02 5.13E-05 1.43E-07 76.61-86.37
结果显示：
3H6H4L1与人IL-1β的亲和力常数为8.79E-11M，Canakinumab与人IL-1β的亲和力常数为9.79E-11M，表明3H6H4L1与人IL-1β有较强的结合能力。
实施例7：抗体3H6 H4L1的细胞生物学活性研究
1. 3H6 H4L1阻断IL-1β诱导MRC-5细胞分泌IL-6的细胞学活性检测
人MRC-5细胞(购自中国科学院细胞中心)常规消化、计数，7,500个/孔接种于平底96孔板中，置于细胞培养箱培养；24h后(细胞生长达到80％汇合度时)，进行给药处理：抗体设4个浓度(0.37nM、1.11nM、3.33nM、10nM)，IL-1β(购自北京义翘神州生物技术有限公司)设三个浓度(5pM、50pM、500pM)，抗体组IL-1β用50pM的浓度(抗体和IL-1β先在37℃孵育20min)，另外同时设置空白对照组及同型对照组；加药处理后置于细胞培养箱中培养24h；收集细胞上清，用IL-6ELISA Kit(购自达科为生物技术有限公司)进行检测。检测结果如图7和表6所示。
表6：3H6 H4L1对IL-1β诱导MRC-5分泌IL-6活性的梯度抑制活性
Figure PCTCN2019100343-appb-000030
结果显示，IL-1β能够显著促进MRC-5分泌IL-6，且呈剂量依赖性；3H6 H4L1能够特异性抑制IL-1β诱导MRC-5细胞分泌IL-6的活性，表现3H6 H4L1对IL-1β的特异性中和活性。
2. 3H6 H4L1阻断IL-1β激活NF-κB信号通路
本实验通过荧光报告基因方法检测3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性。
(1)293T-NF-κB-LUC细胞构建
胰酶消化293T细胞，传代培养；转染前2h，更换opti-DMEM培养基；在无菌EP管中，加入500μLopti-DMEM培养基，再加入3μg的质粒pNF-kB-Luc2P-hygro；在无菌EP管中，加入500μL opti-DMEM培养基，再加入8μL lipofectamine 2000；将稀释的lipofectamine 2000加入已稀释的质粒中，室温放置15min，均匀滴加到细胞培养皿中；转染8h后，更换新鲜培养基；转染24h后，加入Hygromycin，终浓度100μg/mL筛选，设对照孔293T未转染质粒。7-10天后，对照孔细胞完全死亡为准，收获筛选完的细胞扩增培养。继续加药100μg/mL维持。得到293T-NF-κB-LUC稳转细胞株。
(2)3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性检测
常规消化293T-NF-κB-LUC细胞，按每孔20,000个细胞接种于96孔板中。待细胞贴壁后，添加IL-1β，使其终浓度1.65ng/mL，设置空白对照。同时添加抗体，Canakinumab，3H6 H4L1，每个抗体5个梯度，终浓度分别为:400，100，25，6.25，1.56ng/mL。共培养6小时后，去上清，加入50μL PBS，再加入50μL Bright-Glo TM底物，反应5min，上机检测。
结果如图8、图9所示。
结果显示：
IL-1β可有效激活NF-κB信号通路依赖的荧光素酶报告基因的表达，且呈显著剂量依赖性；
3H6 H4L1可特异性阻断IL-1β对NF-κB信号通路的激活，且呈显著剂量依赖性。
以上结果表明，在IL-1β依赖的NF-κB信号通路报告系统中，3H6 H4L1可有效阻断IL-1β对NF-κB的激活，表现其对IL-1β特异性中和活性。
实施例8：3H6 H4L1缓解转染人的IL-1β的NIH/3T3细胞诱导的类风湿性膝关节炎 模型小鼠治疗
将46只BALB/c小鼠根据体重分为6组，即：
正常组、模型组、阳性对照品组、3H6 H4L1低剂量组、3H6 H4L1中剂量组和3H6 H4L1高剂量组；除正常组6只外，其余每组均为8只。
在细胞接种前，根据小鼠体重和给药体积，阳性对照品组皮下注射Canakinumab，模型组皮下注射Anti-HEL，3H6 H4L1各剂量组注射相应浓度3H6 H4L1，正常组皮下注射等体积生理盐水。
在生物安全柜中收集NIH/3T3(购自美国模式菌种收集中心)细胞和 Lenti-IL-1β-NIH/3T3细胞，Lenti-IL-1β-NIH/3T3细胞株是将Lenti-IL-1β载体转染NIH/3T3细胞，经过筛选得到稳定分泌表达IL-1β的Lenti-IL-1β-NIH/3T3细胞株。当NIH/3T3、Lenti-IL-1β-NIH/3T3细胞达到所需接种数量时，开始收集细胞。在生物安全柜内，吸掉旧的培养基，PBS清洗一次后加入适量的0.05％Trypsin-EDTA(1x)室温消化1分钟，然后加入含有10％FBS的DMEM完全培养基终止消化，细胞悬液于1200rpm/min离心4分钟，去上清，使用无血清的DMEM培养基重悬并计数，将细胞浓度调整至2,000,000个每ml，置于冰上备用。
以3.5％水合氯醛7.5ml/kg腹腔注射麻醉BALB/c小鼠后，正常组小鼠膝关节腔内接种NIH/3T3细胞悬液25μl/只(50,000个细胞/只)，其余小鼠膝关节腔内注射接种Lenti-IL-1β-NIH/3T3细胞悬液25μl/只(50,000个细胞/只)。接种后对膝关节处创伤进行缝合，并涂拭生理盐水稀释20倍的青霉素。细胞接种后第5天，各组小鼠颈椎脱臼安乐死，解剖患肢膝关节处，用游标卡尺测量小鼠患肢膝关节滑膜的长(mm)和宽(mm)。数据以均数±标准误(±SEM)表示，组间比较采用GraphPad Prism 5统计学处理软件处理后，进行单因素方差分析评价结果，P<0.05有显著性差异，P<0.01有非常显著性差异。
结果如图10、图11和图12所示。
图10显示，与正常组相比，模型组小鼠明显出现病理行为(P<0.01)。给药后，与模型组相比，Canakinumab与3H6 H4L1高、中剂量组均能够有效改善类风湿关节炎小鼠的病理行为(P<0.01)，3H6 H4L1低剂量对改善类风湿关节炎小鼠的病理行为药效不明显(P>0.05)。同时3H6 H4L1对改善小鼠病理行为程度具有一定的量效关系。与阳性对照品组相比，阳性对照品组药效优于3H6 H4L1中、低剂量组(P<0.01)，3H6 H4L1高剂量与阳性对照品相当(P>0.05)。
图11显示，与正常组相比，模型组小鼠患肢膝关节面积明显增大(P<0.01)。给药后，与模型组相比，阳性对照品组(Canakinumab)和3H6 H4L1中、高剂量组均能明显减少类风湿关节炎小鼠患肢的肿胀面积(P<0.01)，3H6 H4L1低剂量组对减少类风湿性小鼠患肢的肿胀面积的药效不明显(P>0.05)。同时3H6 H4L1对减少类风湿关节炎小鼠的患肢肿胀面积具有一定的量效关系。等剂量3H6 H4L1同靶点已上市药物Canakinumab药效相当(P>0.05)。
图12显示，与正常组相比，模型组小鼠体重明显下降(P<0.01)。给药后，与模型组相比，同靶点已上市药物Canakinumab和3H6 H4L1高剂量组均能明显减少关节炎小鼠体重下降(P<0.05)。与阳性对照品组相比，等剂量3H6 H4L1同靶点已上市药物 Canakinumab药效相当(P>0.05)。
以上对本发明的较佳实施方式进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (28)

  1. 抗IL-1β的抗体或其抗原结合片段，其中，
    所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3；和
    所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3。
  2. 根据权利要求1所述的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选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14；和
    所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16。
  3. 根据权利要求1所述的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；或者
    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；或者
    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；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示。
  4. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  5. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，所述的抗体以小于10 -5M，例如小于10 -6M、小于10 -7M、小于10 -8M、小于10 -9M或小于10 -10M或 更小的K D结合IL-1β蛋白；优选地，所述K D通过Biacore分子相互作用仪测得。
  6. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  7. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述的抗体，其恒定区来自人抗体；
    优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
  8. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述的抗体，其重链恒定区为Ig gamma-1 chain C region或Ig gamma-4 chain C region，并且其轻链恒定区为Ig kappa chain C region。
  9. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT010产生的单克隆抗体，所述杂交瘤细胞株LT010保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。
  10. 抗体药物偶联物，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为权利要求1至9中任一权利要求所述的抗体或其抗原结合片段；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。
  11. 根据权利要求10所述的抗体药物偶联物，其中，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。
  12. 根据权利要求10或11所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段与小分子药物的摩尔比为1：(1－4)。
  13. 一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：
    所述第一蛋白功能区靶向IL-1β，
    所述第二蛋白功能区靶向不同于IL-1β的靶点(例如，IL-17A)；
    其中，所述第一蛋白功能区为权利要求1－9中任一权利要求所述的抗体或抗原结合片段；
    优选地，所述双特异性抗体为IgG-scFv模式；
    优选地，所述第一蛋白功能区为权利要求1－9中任一权利要求所述的抗体，并且所述第二蛋白功能区为单链抗体；或者
    优选地，所述第一蛋白功能区为权利要求4所述的单链抗体，并且所述第二蛋白功能区为抗体。
  14. 根据权利要求13所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段为SS(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
  15. 根据权利要求13所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  16. 根据权利要求13至15中任一权利要求所述的双特异性抗体，其中，所述单链抗体连接在抗体的重链的C末端。
  17. 分离的核酸分子，其包含编码抗体重链可变区的核酸序列和编码抗体轻链可变区的核酸序列，其中，
    所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NO:17－SEQ ID NO:19所示的HCDR1-HCDR3，和所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NO:20－SEQ ID NO:22所示的LCDR1-LCDR3；
    优选地，所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10和SEQ ID NO:14，和所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8、SEQ ID NO:12和SEQ ID NO:16；
    更优选地，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；或者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； 或者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；或者所述抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    进一步优选地，所述核酸分子包含：
    SEQ ID NO:1和SEQ ID NO:3所示的核酸序列，
    SEQ ID NO:5和SEQ ID NO:7所示的核酸序列，
    SEQ ID NO:9和SEQ ID NO:11所示的核酸序列，或者
    SEQ ID NO:13和SEQ ID NO:15所示的核酸序列。
  18. 一种重组载体，其包含权利要求17所述的分离的核酸分子。
  19. 一种宿主细胞，其包含权利要求17所述的分离的核酸分子，或者包含权利要求18所述的重组载体。
  20. 制备权利要求1至9中任一权利要求所述的抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求19的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。
  21. 杂交瘤细胞株LT010，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018133。
  22. 一种药物组合物，其包含权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体；可选地，其还包括药学上可接受的载体和/或赋形剂。
  23. 权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体在制备治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎的药物中的用途；
    优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化和周期性发热综合征；
    优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性地中海热(FMF)；
    优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
    优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
    优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
    优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
  24. 权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体在制备如下药物中的用途：
    阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的药物，
    下调人IL-1β活性或水平的药物，或者
    抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的药物。
  25. 根据权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体，其用于治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎；
    优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化和周期性发热综合征；
    优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性地中海热(FMF)；
    优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
    优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
    优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
    优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
  26. 根据权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体，其用于：
    阻断人IL-1β与人IL-1R1和/或人IL-1R2结合，
    下调人IL-1β活性或水平，或者
    抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活。
  27. 一种治疗和/或预防治疗和/或预防自身免疫性疾病、心脑血管疾病、肿瘤、儿童和成人的冷吡啉相关的周期性综合征、全身型幼年特发性关节炎或痛风性关节炎的方法，包括给予有需求的受试者以有效量的权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体的步骤；
    优选地，所述自身免疫性疾病选自类风湿性关节、多发性硬化、周期性发热综合征；
    优选地，所述周期性发热综合征选自肿瘤坏死因子受体相关的周期性综合征(TRAPS)、高免疫球蛋白D综合征(HIDS)/甲羟戊酸激酶缺乏症(MKD)以及家族性地中海热(FMF)；
    优选地，所述儿童和成人的冷吡啉相关的周期性综合征选自家族冷自主炎症综合征、穆-韦二氏综合征、新生儿多系统炎性疾病、慢性小儿神经皮肤关节综合征以及家族性冷荨麻疹；
    优选地，所述心脑血管疾病选自心肌梗死、动脉粥样硬化、动脉血栓和脑卒中；
    优选地，所述肿瘤选自肺癌、肝细胞癌和急性髓性白血病；
    优选地，所述痛风性关节炎为急性痛风性关节炎或慢性痛风性关节炎。
  28. 一种在体内或体外方法，包括施加细胞以有效量的权利要求1至9中任一权利要求所述的抗体或其抗原结合片段、权利要求10至12中任一权利要求所述的抗体药物偶联物或者权利要求13至16中任一权利要求所述的双特异性抗体的步骤，所述方法选自如下：
    阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的方法，
    下调人IL-1β活性或水平的方法，或者
    抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的方法。
PCT/CN2019/100343 2018-08-14 2019-08-13 抗IL-1β的抗体、其药物组合物及其用途 Ceased WO2020034941A1 (zh)

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BR112021002794-7A BR112021002794A2 (pt) 2018-08-14 2019-08-13 anticorpo anti-il-1beta e composição farmacêutica do mesmo e uso do mesmo
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