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)
Chinese (zh)
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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 MYPI2021000751A priority Critical patent/MY209465A/en
Priority to MX2021001778A priority patent/MX2021001778A/es
Priority to US17/267,115 priority patent/US12286473B2/en
Priority to IL280596A priority patent/IL280596B2/en
Priority to AU2019320927A priority patent/AU2019320927B2/en
Priority to BR112021002794-7A priority patent/BR112021002794A2/pt
Priority to KR1020217007489A priority patent/KR20210046024A/ko
Priority to JP2021507628A priority patent/JP7576022B2/ja
Priority to CN201980053246.6A priority patent/CN112654640B/zh
Priority to EP19849700.0A priority patent/EP3845560A4/en
Priority to CA3108414A priority patent/CA3108414A1/en
Priority to EA202190518A priority patent/EA202190518A1/ru
Application filed by Akeso Biopharma Inc filed Critical Akeso Biopharma Inc
Priority to SG11202101435XA priority patent/SG11202101435XA/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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CN113444177A (zh) * 2020-03-27 2021-09-28 中山康方生物医药有限公司 抗IL-1β的抗体、其药物组合物及其用途
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