EA046827B1 - ANTI-IL-1β ANTIBODY AND ITS PHARMACEUTICAL COMPOSITION AND THEIR APPLICATION - Google Patents

Abstract

The invention relates to the field of immunology and relates to an IL-Ιβ antibody and a pharmaceutical composition thereof, as well as to the use thereof. In particular, the invention relates to an IL-Ιβ antibody or an antigen-binding fragment thereof, wherein the variable region of the heavy chain of the antibody comprises HCDR1-HCDR3 with the amino acid sequences SEQ ID NO: 17-SEQ ID NO: 19, respectively, and the variable region of the light chain of the antibody comprises LCDR1-LCDR3 SEQ ID NO: 20-SEQ ID NO: 22, respectively. The antibody described herein can effectively bind to human IL-Ιβ, block the binding of IL-Ιβ to its receptor IL-1R1, and inhibit the activation of subsequent IL-Ιβ signaling pathways. The antibody can be used to produce a drug for the prevention and treatment of autoimmune diseases, periodic cryopyrin-associated syndromes in children and adults, systemic juvenile idiopathic arthritis, gouty arthritis, cardiovascular diseases or tumors.

Description

Field of technology to which the invention relates

The present invention relates to the field of immunology and relates to aHTu-IL-Ιβ antibody, its pharmaceutical composition and their use. In particular, the present invention relates to an anti-human IL-1e antibody; more specifically, the present invention relates to a monoclonal antibody against human IL-1e; and furthermore, more specifically, the present invention relates to humanized monoclonal antibodies against human IL-1e.

State of the art

The interleukin-1 (IL-1) family consists of two pro-inflammatory factors (IL-1a and ΓΕ-1β) and IL-1 receptor antagonist (IL-IRa), where IL-1a and IL-1e can effectively activate IL-1 receptors , and IL-IRa can attach to the surface of IL-1 receptors, blocking signaling. IL-1e is synthesized mainly by monocytes and macrophages. When cleaved by caspase-1, the IL-1e precursor protein can be activated. There are several subtypes of ligands, including IL-1R1, IL-1R2, IL-1R3 (also known as IL-1 receptor accessory protein or IL-1RAcP), IL-1R4, IL-1R5, IL-1R6, IL1R7, IL-1R8 , IL-18BP and 2 orphan receptors (IL-1R9 and IL-1R10) in the IL-1 receptor family.

Recently, it was discovered that IL-1e is able to bind to IL-1R1 and IL-1R2. IL-1R1 (also known as IL-1 receptor type 1 or IL-1R1) is a transmembrane receptor that binds to IL-1e and IL-1RAcP to form a receptor complex, activating its associated downstream intracellular signaling pathway and mediating associated IL-1e biological effects. IL-1R2 has a higher affinity for IL-1e than IL-1R1; however, IL-1R2 cannot activate the corresponding intracellular signaling pathway after binding to IL-1e due to its shorter intracellular segment. Both IL-1R1 and IL-1R2 can exist in soluble form (Boraschi et al. Immunological Reviews. 281:197-232. (2018)).

IL-1e 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 and periodic fever syndrome, and autoinflammatory diseases such as systemic juvenile idiopathic arthritis; as well as in the occurrence of diseases such as cryopyrin-associated periodic syndromes in children and adults. Recently, the IL-1e pathway has been confirmed to be involved in tumors such as acute myeloid leukemia, liver cancer, lung cancer, and cardiovascular and cerebrovascular diseases, and drugs targeting IL-1e have shown good therapeutic and preventive effect (Cozzolino F. et al. Proc Natl Acad Soci USA 86:2369 (1989); Nakazaki H. et al. Cancer. 70(3):709 (1992); Ridker P.M. et al. Lancet. 390(10105) :1833-1842 (2017)).

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease predominantly affecting the joints. IL-1e plays a key role in the development of RA. High levels of IL-1e are present in the synovial fluid of patients with PA (van den Berg et al. Baillieres Best Pract Res Clin Rheumatol. 13:577-97(1999)), and IL-1e mediates leukocyte infiltration and secretion of matrix metalloproteinases in joints, causes cartilage degradation and inhibits the synthesis of new cartilage matrix, leading to joint destruction (van den Berg et al. Baillieres Best Pract Res Clin Rheumatol. 13:577-97 (1999); Johnson LL et al. Curr Opin Chem Biol. 2: 466-471 (1998)). In patients with PA, IL-1e stimulates the differentiation and activation of osteoclasts and is involved in the occurrence of bone erosion in joints affected by PA (Gravallese EM et al. Ann Rheum Dis. 61:ii 84-86 (2002); Ghivizzani SC et al. J Immunol 1:3604-12 (1997); Horai R et al. J Exp Med 191:313-20 (2000); Arthritis Rheum 43:259-69 (2000)). In addition, IL-1e promotes bone loss as well as leukocyte infiltration and pannus tissue formation in the synovium by regulating pathways involving TNF-α and IL-6 (Strand V. et al. Rheumatology (Oxford). 43:iii10 ^- iii16 (2004)). Clinical studies have demonstrated that the IL-1e antagonist significantly improves the physical signs and symptoms of PA (Mertens M. et al. J Rheumatol. 36(6): 1118-25 (2009)) and the anti-human IL-1e mAb canakinumab (Ilaris®) significantly reduces disease activity in patients with PA (Alten R. et al. Arthritis Res Ther. 10:R67 (2008); Alten R. et al. BMC Musculoskeletal Disorders. 12:153 (2011)).

Gout is a crystal arthropathy caused by deposition of monobasic sodium urate, which is directly related to hyperuricemia caused by impaired purine metabolism and/or decreased uric acid excretion, and in particular refers to the acute characteristic arthritis and chronic gouty stone disease, in mainly includes arthritis with acute onset, tophi formation, chronic tophi arthritis, urate nephropathy and uric acid litangiuria, in severe cases even joint damage and renal failure. Gout is often accompanied by abdominal obesity, hyperlipidemia, hypertension, type 2 diabetes, cardiovascular diseases, etc. IL-1e is a factor influencing the development of gouty inflammation (Cumpelik A. et al. Ann Rheum Dis. pii: annrheumdis-2015-207338 (2015)). Is

- 1 046827 study showed that in patients with gouty arthritis, the expression of both IL-Ιβ mRNA of a single mononuclear cell and serum IL-1β in peripheral blood is significantly higher than in healthy controls, and these indicators are significantly higher in the acute phase group than in the chronic phase group and the intermittent phase group; Serum Ρ-1β concentrations are positively correlated with leukocyte, neutrophil granulocyte, and erythrocyte sedimentation rates, suggesting that Ρ-1β may be involved in both acute and chronic gouty arthritis and also correlates with the degree of inflammation ; and the concentration of serum IL-1e in the intermittent phase group is significantly higher than that in the healthy control group, suggesting that joint and tissue inflammation may still exist although joint symptoms disappear (Li Lingqin et al. Chinese Journal of General Practitioners. 14: 29-31 (2015)). An animal experiment has shown that ΓΡ-1β plays an important role in both acute and chronic gouty arthritis, and urate crystals stimulate mononuclear cells and phagocytes in the blood and synovial fluid, causing a strong release of IL-1e (Di Giovine F.S. et al. J Immunol 138: 3213-3218 (1987)). In a mouse model, it was found that a ΓΡ-1β blocker could prevent neutrophil aggregation induced by intraperitoneal injection of urate crystals, and aggregation was absent at sites lacking IL-1e receptors in mice (Martinon F. et al. Nature. 440:237- 241 (2006)). Clinical studies have shown that ΓΡ-1β induces the production of large amounts of proinflammatory cytokines during an acute attack of gout (Amaral F.A. et al. Arthritis Rheum. 64: 474-484 (2012); Torres R. et al. Ann Rheum Dis. 68: 1602 -1608 (2009)).

In a clinical trial evaluating the efficacy and safety of gout treatments, it was found that canakinumab, a monoclonal antibody against human IL-1e, can effectively relieve clinical symptoms such as pain in patients with refractory gouty arthritis, which apparently reduces the incidence of gout relapses compared with triamcinolone acetonide and improves quality of life (So A et al. Arthritis Rhum. 62: 3064-3076 (2010)). Another clinical study found that canakinumab, a monoclonal antibody against human IL-1e, could significantly reduce the number of gout attacks compared with colchicine (Schlesinger N. et al. Ann Rheum Dis. 70: 1264–1271 (2011)). The mAb canakinumab is currently approved by the European Medicines Agency for the treatment of patients who suffer frequent attacks of gouty arthritis and are intolerant or unresponsive to non-steroidal anti-inflammatory drugs, colchicine and glucocorticoids (Lyseng-Williamson K.A. et al. BioDrugs. 27: 401 -406 (2013)). The above-mentioned studies indicate that anti-ΓΡ-1β antibody can treat gout, relieve symptoms and reduce relapses more effectively than existing treatments, providing potential preventative and treatment effects for gout attacks.

Multiple sclerosis is a chronic demyelinating disease mediated primarily by Th1 and Th17 T cells. Interleukin IL-1 family factors play an important role in the development of multiple sclerosis. IL-1 promotes disease progression by accelerating the growth of Th17 T cells and also leads to the distribution of the chemokine CXCL12 in the brain vessels from the normal location in the parenchyma to both sides of the endothelium until a depolarizing distribution is achieved, resulting in vascular leakage and T cell infiltration into the brain parenchyma during early disease progression (Chih-Chung Lin et al. J Immunol. 198: 4553–4560 (2017)). Animal studies have shown that experimental autoimmune encephalomyelitis (EAE) is not induced in mice by IL-1 or IL1R1 deficiency, and treatment with ΓΡ-1β inhibitors can delay the onset, reduce the severity, and shorten the duration of EAE in wild-type mice (Chih-Chung Lin et al J Immunol 198: 4553-4560 (2017).

Recent clinical studies have also shown that anti-IL-Tr antibodies have anti-inflammatory effects by antagonizing the IL-1 β pathway, which significantly reduces the risk of cardiovascular disease. Atherosclerosis and thrombosis are the pathological basis of coronary heart disease (Dalek os G.N. et al. J Lab Clin Med. 129: 300 (1997)). In a rabbit model of high cholesterol and atherosclerosis, IL-1 β and IL-1 β mRNA expression was found to be increased in lipid plaques, and reducing IL-1 synthesis may slow the progression of atherosclerosis (Dinarello C.A. et al. N Engl J Med. 328 : 106 (1993)). IL-1β levels increase during myocardial infarction, and ΙΡ-1β expression increases in both plasma and local myocardial infarction during post-infarction myocardial remodeling, suggesting that ΙΡ-1β is involved in the onset and progression of myocardial hypertrophy and fibrosis. myocardium and failure after myocardial infarction (Yue P. et al. Am J Physiol. 275 (1Pt2): H250 (1998)). Heart failure is a complex syndrome, and studies have shown that increased levels of IL-1 occur in the circulating blood of patients with congestive heart failure (Blum A. et al. Am Heart J. 135 (2 Part 1): 181 (1998); Kapadia S. et al. Cardiol Clin. 16(4): 645 (1998). ΙΡ-1β may be involved in the onset and progression of congestive heart failure caused by increased chronic load; serum IL-1 β levels are markedly increased in patients with stage III-IV heart failure

- 2 046827 nor, contributing to the progression of congestive heart failure (Yndestad A. et al. Curr Cardiol Rep. 9: 236-41 (2007)). The phase III CANTOS clinical trial evaluating the efficacy, safety and tolerability of canakinumab, an anti-human IL-1e antibody, in post-myocardial infarction and inflammatory atherosclerotic cardiovascular disease showed that anti-III-1b antibody in combination with standard treatment significantly reduces the incidence of death due to cardiovascular disease, nonfatal myocardial infarction, and nonfatal cerebrovascular events in patients (Ridker P.M. et al. N Engl J. Med. 377: 1119-1131 (2017)).

Another analysis of the CANTOS clinical trial cohort of the anti-human IL-1e antibody canakinumab found that the anti-III-1β antibody canakinumab significantly reduced the risk of incident and death from lung cancer (Ridker P.M. et al. 390(10105):1833-1842 (2017 ))Lancet. When culturing acute myeloid leukemia (AML) cells from patients in vitro, IL-1 expression was found to be elevated in more than 80% of patients with primary AML, and IL-1e can significantly promote tumor cell growth, while anti-IL antibodies -1e or IL-1a are effective in inhibiting the growth of tumor cells (Cozzolino F. et al. Proc Natl Acad Soci USA 86: 2369 (1989)). In patients with hepatocellular carcinoma, serum IL-1 levels were significantly increased compared with healthy controls (Nakazaki H. et al. Cancer. 70 (3): 709 (1992)).

Cryopyrin-associated periodic syndromes (CAPS) in children and adults are a rare disorder caused by overproduction of IL-1e due to a single gene mutation, resulting in weakness, hot flashes, fever, headache, arthralgia, and conjunctivitis that may occur in newborns or infants, can occur daily throughout the patient's life and can cause severe illness including deafness, bone and joint deformities, blindness due to central nervous system damage, and kidney failure, and can be potentially fatal in the long term, causing premature death as a result of amyloidosis. CAPS in children and adults include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease, chronic childhood neurological cutaneous and joint syndrome, and familial cold urticaria. Canakinumab, an antibody against human IL-1e, can significantly improve clinical symptoms in patients with cryopyrin-associated periodic syndromes (Yokota S. et al. Clin Exp Rheumatol. 35 Suppl 108 (6): 19-26. (2017); Kone- Paut I. et al. Arthritis Care Res. (Hoboken) 69: 903-911 (2017)) due to which it was approved for the treatment of cryopyrin-associated periodic syndromes (CAPS) in children (> 4 years) and adults. the Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

Periodic fever syndromes are a group of rare autoimmune diseases that lead to recurrent and persistent severe fever and pathogenic inflammation due to non-infectious activation of the immune system, often lead to disability and can be accompanied by arthralgia, edema, myalgia, rash and death (Wurster V.M. et al Pediatr Ann. 40 (1): 48-54 (2011). Periodic fever syndromes include TNF receptor-associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS)/mevalonate kinase deficiency (MKD), and familial Mediterranean fever (FMF). Clinical studies have demonstrated that the anti-human IL-1e mAb canakinumab may be effective in the treatment of periodic fever syndromes (De Benedetti F. et al. N Engl J. Med. 378 (20): 1908-1919 (2018)). Thus, the anti-human IL-1e mAb canakinumab has been approved by the FDA and the like. for the treatment of periodic fever syndromes.

Systemic juvenile idiopathic arthritis (SJIA) is a unique subtype of juvenile idiopathic arthritis that primarily begins with extra-articular manifestations such as prolonged hyperpyrexia, rash, and anemia. It usually occurs in children aged 0-5 years, mainly manifests itself as chronic arthromeningitis and is most often accompanied by damage to organs and tissues of varying degrees. Its main manifestations are fever, rash, and arthralgia, and it has a poor prognosis, such as low long-term remission rates, high rates of dysfunction and disability, and high mortality rates. (Woerner A. et al. Expert Rev Clin Immunol. 11 (5): 575-88. (2015)). It is generally accepted that SJIA is an autoinflammatory disease rather than an autoimmune disease (Sun Juan et al. Progress in Modern Biomedicine. 8: 1584-1588 (2016)). Clinical studies have demonstrated that the anti-human IL-1e monoclonal antibody canakinumab can effectively treat active SJIA accompanied by fever and reduce steroid doses, as well as significantly reduce relapses of SJIA (Orrock J.E. et al. Expert Rev Clin Pharmacol. 9: 1015-24. (2016)). Thus, the anti-IL-1e monoclonal antibody canakinumab is approved by the FDA and the like. for the treatment of systemic juvenile idiopathic arthritis.

There is still a need to develop new anti-III-1b antibodies.

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The essence of the invention

After intensive research and creativity, the inventors of the present invention used mammalian cell expression systems to express recombinant IL-1e-His as an antigen for immunizing mice and obtained hybridoma cells by fusing mouse spleen cells and myeloma cells. The inventors of the present invention obtained the following hybridoma cell line by screening a large number of samples: hybridoma cell line LT010 deposited in the China Type Culture Collection Center (CCTCC) on June 21, 2018 under the registration number CCTCC NO: C2018133.

The present inventors unexpectedly discovered that the hybridoma cell line LT010 can secrete and produce a specific monoclonal antibody (named 3H6) that specifically binds to human ΓΕ-1β, and this monoclonal antibody can very effectively block the binding of ΓΕ-1β to IL-1R1;

In addition, the inventors of the present invention have creatively produced humanized antibodies against human ϋ.-1β (respectively named 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1), all of which can effectively bind to human ϋ.-1β, block the binding of IL-1 β to its receptor (IL-1R1), and inhibit the activation of the downstream ΓΕ-1β signaling pathway. And these humanized antibodies against human ΓΕ-1β could potentially be used in the manufacture of drugs to reduce, prevent or treat diseases such as rheumatoid arthritis, gout, multiple sclerosis, cardiovascular events and/or cardiovascular disease, tumors, periodic cryopyrin-associated syndromes in children and adults, periodic fever syndromes and systemic juvenile idiopathic arthritis.

The present invention is described in detail below.

In one aspect, the present invention provides an anti-P.f antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising HCDR1-HCDR3 with the amino acid sequences SEQ ID NO: 17-SEQ ID NO: 19, respectively; and the antibody comprises a light chain variable region comprising .CDR1 to .CDR3 with the amino acid sequences of SEQ ID NO: 20 to SEQ ID NO: 22, respectively.

Preferably, IL-Ιβ is human IL-Ιβ.

The light chain and heavy chain variable regions determine antigen binding; the variable region of each chain contains three hypervariable regions, namely, complementarity determining regions (CDRs) (heavy chain CDRs (H) include HCDR1, HCDR2, HCDR3, and light chain CDRs (L) include LCDR1, LCDR2, LCDR3; determined by Kabat et al., See Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md.). The antibodies described herein 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 have the same HCDR1-3 and LCDR1-3, as analyzed using techniques well known to those skilled in the art, such as the VBASE2 database.

The amino acid sequences of the three HCDR regions of the heavy chain variable region are as follows:

HCDR 1: GFSLSTSGMG (SEQ ID NO: 17),

HCDR 2: IYWDDDK (SEQ ID NO: 18),

HCDR 3: ARSAYYSFAY (SEQ ID NO: 19);

and the amino acid sequences of the three light variable region CDR regions are as follows:

LCDR 1: QDVDTD (SEQ ID NO: 20),

LCDR 2: WAS (SEQ ID NO: 21),

LCDR 3: QQYSSYPT (SEQ ID NO: 22).

In one or more embodiments, the present invention provides an antibody or an antigen binding fragment thereof, wherein the heavy chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14; and the antibody light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16.

In some embodiments, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody is selected from (1) VH set forth in SEQ ID NO: 2 and VL set forth in SEQ ID NO: 4;

(2) VH, specified in SEQ Ш NO: 6, and VL, specified in SEQ Ш NO: 8;

(3) VH specified in SEQ ID NO: 10, and VL specified in SEQ ID NO: 12; and (4) VH, specified in SEQ GO NO: 14, and VL, specified in SEQ GO NO: 16.

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In one or more embodiments, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody ( eg scFv), humanized antibody, chimeric antibody and diabody.

In one or more embodiments, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody binds to a [Γ-1β] protein with a KD of less than 10 ^-5 M, e.g., less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^{- 8} M, less than 10 ^-9 M or less than 10 ^-10 M or less; preferably, K _D is measured using a Biacore molecular interaction meter.

In some embodiments, the present invention provides an antibody or antigen binding fragment thereof, wherein the antibody binds to IL-Ιβ protein with an EC ₅₀ of less than about 100 nM, e.g., less than about 10 nM, less than about 1 nM, less than about 0.9 nM, less less than about 0.8 nM, less than 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. In particular, EC ₅₀ is measured by indirect solid-phase ELISA.

In one or more embodiments, the present invention provides an antibody or an antigen binding fragment thereof, wherein the antibody comprises a non-CDR region derived from a non-mouse species, such as a human antibody.

In some embodiments of the present invention, the antibody constant regions are humanized, for example, the heavy chain constant regions are an Ig gamma 1 chain region, such as ACCESSION: P01857, or an Ig gamma 4 chain C region, such as ACCESSION: P01861.1 ; and the light chain constant regions are the Ig kappa chain C region, such as ACCESSION: P01834.

In one or more embodiments, the present invention relates to an antibody or an antigen-binding fragment thereof, where the antibody is a monoclonal antibody produced by the hybridoma cell line LT010 deposited in the China Type Culture Collection Center (CCCC) under the registration number CCCC NO: C2018133.

In one or more embodiments of the present invention, the antibody is a monoclonal antibody.

In another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is any of the antibodies or antigen-binding fragments thereof disclosed herein; preferably, the small molecule drug is a small molecule cytotoxic drug; and more preferably, the small molecule drug is a chemotherapeutic drug.

The chemotherapeutic drug may be a conventional tumor chemotherapeutic drug such as an alkylating agent, an antimetabolite, an antitumor antibiotic, a herbal anticancer agent, a hormone and an immunological agent.

In one or more embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof is linked to a small molecule drug via a linker; the linker may be a linker known to those skilled in the art, such as a hydrazone bond, a disulfide bond, or a peptide bond.

In one or more embodiments, the present invention provides an antibody-drug conjugate wherein the molar ratio of antibody or antigen binding fragment thereof to small molecule drug is from 1:1 to 1:4, for example, 1:1, 1:2, 1: 3 or 1:4.

In yet another aspect, the present invention provides a bispecific antibody (also known as a bifunctional antibody) comprising a first protein functional region and a second protein functional region, wherein:

the first functional region of the protein targets IL-1e, the second functional region of the protein targets a target other than IL-1 β, for example, IL-17A;

wherein the first functional region of the protein is any of the antibodies or antigen binding fragments thereof disclosed herein;

preferably, the bispecific antibody is in the form of an IgG-scFv;

preferably:

(1) the first functional region of the protein is any of the antibodies or antigen binding fragments thereof disclosed herein, and the second functional region of the protein is a single chain antibody; or (2) the first functional region of the protein is a single chain antibody, its heavy chain variable region contains HCDR1-HCDR3 with amino acid sequences

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SEQ ID NO: 17 to SEQ ID NO: 19, and its light chain variable region comprises LCDR1 to LCDR3 with the amino acid sequences of SEQ ID NO: 20 to SEQ ID NO: 22, and the second functional region of the protein is an antibody (e.g., a monoclonal antibody ).

In some embodiments, the present invention provides a bispecific antibody, wherein a first protein functional region and a second protein functional region are linked directly or through a linker moiety;

preferably, the linker moiety is (GGGGS)m, where m is a positive integer such as 1, 2, 3, 4, 5 or 6; or preferably, the linker moiety is SS(GGGGS)n, where n is a positive integer such as 1, 2, 3, 4, 5 or 6.

In some embodiments, the present invention provides a bispecific antibody, wherein in clause (2), the heavy chain variable region of the single chain antibody comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO : 14; and the light chain variable region of the single chain antibody comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16.

In some embodiments, the present invention provides a bispecific antibody, wherein in clause (2), the heavy chain variable region of the single chain antibody comprises the amino acid sequence set forth in SEQ ID NO: 2 and the light chain variable region of the single chain antibody comprises the amino acid sequence set forth in SEQ ID NO: 4;

the heavy chain variable region of a single chain antibody contains the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region of a single chain antibody contains the amino acid sequence shown in SEQ ID NO: 8;

the heavy chain variable region of a single chain antibody contains the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region of a single chain antibody contains the amino acid sequence shown in SEQ ID NO: 12; or the heavy chain variable region of a single chain antibody contains the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of a single chain antibody contains the amino acid sequence set forth in SEQ ID NO: 16.

In some embodiments, the present invention provides a bispecific antibody wherein the number of functional regions of the first protein and the functional regions of the second protein are independently equal to 1, 2, or more.

In some embodiments, the present invention provides a bispecific antibody, wherein in clause (2) the monoclonal antibody constant region is selected from the human IgG1, IgG2, IgG3, and IgG4 constant regions.

In some embodiments, the present invention provides a bispecific antibody, wherein the single chain antibody is linked to the C-terminus of the heavy chain of the antibody or monoclonal antibody.

In yet another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody heavy chain variable region and a nucleotide sequence encoding an antibody light chain variable region, wherein the antibody heavy chain variable region comprises HCDR1-HCDR3 with the amino acid sequences of SEQ ID NO: 17-SEQ ID NO: 19, respectively, and the variable region of the light chain of the antibody contains LCDR1-LCDR3 with the amino acid sequences of SEQ ID NO: 20-SEQ ID NO: 22, respectively;

preferably, the amino acid sequence of the antibody heavy chain variable region is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, and the amino acid sequence of the antibody light chain variable region is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ GO NO: 12 and SEQ GO NO: 16;

more preferably, the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 2, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 4; the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 8; the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 12; or the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 14, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 16; and even more preferably, the isolated nucleic acid molecule contains

- 6 046827 nucleotide sequences SEQ GO NO: 1 and SEQ GO NO: 3, nucleotide sequences SEQ GO NO: 5 and SEQ GO NO: 7, nucleotide sequences SEQ GO NO: 9 and SEQ GO NO: 11, or nucleotide sequences SEQ GO NO: 13 and SEQ GO NO: 15.

The isolated nucleic acid molecule may be a single nucleic acid molecule or multiple nucleic acid molecules, such as two nucleic acid molecules. In the case of a single nucleic acid molecule, the heavy chain variable region and the light chain variable region of an antibody may be expressed by the same nucleic acid molecule, for example, by the same or different expression cassettes located on the same nucleic acid molecule. In the case of multiple nucleic acid molecules, such as two nucleic acid molecules, the heavy chain variable region and the light chain variable region of an antibody may be expressed by different nucleic acid molecules.

In another aspect, the present invention relates to a recombinant vector containing an isolated nucleic acid molecule described herein. The number of recombinant vectors can be one or more. In the case of multiple (eg, two) nucleic acid molecules, multiple (eg, two) nucleic acid molecules can be expressed by the same recombinant vector or by different recombinant vectors.

In another aspect, the present invention relates to a host cell containing an isolated nucleic acid molecule or recombinant vector as described herein.

In yet another aspect, the present invention provides a method for producing any of the antibodies or antigen binding fragments disclosed herein, comprising culturing a host cell as described herein under suitable conditions and isolating the antibody or antigen binding fragment thereof from the cell cultures.

In another aspect, the present invention relates to the hybridoma cell line LT010 deposited in the China Type Culture Collection Center (CCCC) under the registration number CCCC NO: C2018133.

In yet another aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies or antigen binding fragments thereof disclosed herein, an antibody-drug conjugate disclosed herein, or a bispecific antibody disclosed herein; optionally, the pharmaceutical composition further contains pharmaceutically acceptable carriers and/or excipients.

In yet another aspect, the present invention relates to the use of any of the antibodies or antigen binding fragments thereof, the antibody-drug conjugate disclosed herein, or the bispecific antibody disclosed herein in the preparation of a medicament for the treatment and/or prevention of autoimmune diseases, cardiovascular and cerebrovascular diseases , tumors, cryopyrin-associated periodic syndromes in children and adults, systemic juvenile idiopathic arthritis or gouty arthritis;

preferably, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis and periodic fever syndromes;

preferably, the periodic fever syndrome is selected from TNF receptor associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS)/mevalonate kinase deficiency (MKD) and familial Mediterranean fever (FMF);

preferably, the cryopyrin-associated periodic syndrome in children and adults is selected from familial cold autoinflammatory syndrome, Muckle-Wells syndrome, multisystem inflammatory disease with neonatal onset, chronic infantile neurological cutaneous and joint syndrome and familial cold urticaria;

preferably, the cardiovascular and cerebrovascular disease is selected from myocardial infarction, atherosclerosis, arterial thrombosis and cerebrovascular disorder;

preferably, the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia; and preferably, the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.

In yet another aspect, the present invention relates to the use of any of the antibodies or antigen-binding fragments thereof, the antibody-drug conjugate disclosed herein, or the bispecific antibody disclosed herein for the preparation of a medicament for blocking the binding of human IL-1e to human IL-1R1 and/or human IL-1R2, a drug to inhibit the activity or decrease the level of human ΓΕ-1β, or a drug to inhibit the activation of downstream signaling pathways mediated by the binding of human IL-1β to human IL-1R1 and/or human IL-1R2.

In one embodiment of the present invention, human IL-1R1 and/or human

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IL-1R2 is human IL-1R1 and/or human IL-1R2 on the cell surface.

In one embodiment of the present invention, the use is non-therapeutic and/or non-diagnostic.

In one or more embodiments of the present invention, an antibody or antigen binding fragment thereof, an antibody-drug conjugate disclosed herein, or a bispecific antibody disclosed herein is for use in the treatment and/or prevention of autoimmune diseases, cardiovascular and cerebrovascular diseases, tumors, cryopyrins - associated periodic syndromes in children and adults, juvenile idiopathic arthritis with systemic onset or gouty arthritis;

preferably, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis and periodic fever syndromes;

preferably, the periodic fever syndrome is selected from TNF receptor associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS)/mevalonate kinase deficiency (MKD) and familial Mediterranean fever (FMF);

preferably, the cryopyrin-associated periodic syndrome in children and adults is selected from familial cold autoinflammatory syndrome, Muckle-Wells syndrome, multisystem inflammatory disease with neonatal onset, chronic infantile neurological cutaneous and joint syndrome and familial cold urticaria;

preferably, the cardiovascular and cerebrovascular disease is selected from myocardial infarction, atherosclerosis, arterial thrombosis and cerebrovascular disorder;

preferably, the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia; and preferably, the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.

In one or more embodiments of the present invention, an antibody or antigen binding fragment disclosed herein, an antibody-drug conjugate disclosed herein, or a bispecific antibody disclosed herein are for use in:

blocking the binding of human IL-1β to human IL-1R1 and/or human IL-1R2, suppressing the activity or reducing the level of human IL-1e, or inhibiting the activation of downstream signaling pathways mediated by the binding of human IL-1e to human IL-1R1 and/or or human IL-1R2.

In one embodiment of the present invention, human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the surface of a cell.

In yet another aspect, the present invention provides an in vivo or in vitro method comprising: administering to a cell an effective amount of any of the antibodies or antigen binding fragments thereof disclosed herein, an antibody-drug conjugate disclosed herein, or a bispecific antibody disclosed herein, wherein the method is selected of a method of blocking the binding of human IL-1e to human IL-1R1 and/or human IL-1R2, a method of inhibiting the activity or reducing the level of human IL-1e, and a method of inhibiting the activation of downstream signaling pathways mediated by the binding of human IL-1e to human IL-1e. 1R1 and/or human IL-1R2.

In one embodiment of the present invention, human IL-1R1 and/or human IL-1R2 is human IL-1R1 and/or human IL-1R2 on the surface of a cell.

In one embodiment of the present invention, the in vitro method is non-therapeutic and/or non-diagnostic.

In yet another aspect, the present invention relates to a method of treating and/or preventing autoimmune diseases, cardiovascular and cerebrovascular diseases, tumors, cryopyrin-associated periodic syndromes in children and adults, systemic juvenile idiopathic arthritis or gouty arthritis, comprising: administering to a person in need in the subject, an effective amount of any of the antibodies or antigen-binding fragments thereof disclosed herein, the antibody-drug conjugate disclosed herein, or the bispecific antibody disclosed herein;

preferably, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis and periodic fever syndromes;

preferably, the periodic fever syndrome is selected from TNF receptor associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS)/mevalonate kinase deficiency (MKD) and familial Mediterranean fever (FMF);

preferably, the cryopyrin-associated periodic syndrome in children and adults is selected from familial cold autoinflammatory syndrome, Muckle-Wells syndrome, multisystem inflammatory disease with neonatal onset, chronic infantile neurological cutaneous and joint syndrome and familial cold urticaria;

- 8 046827 preferably, the cardiovascular and cerebrovascular disease is selected from myocardial infarction, atherosclerosis, arterial thrombosis and cerebrovascular disorder;

preferably, the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia; and preferably, the gouty arthritis is acute gouty arthritis or chronic gouty arthritis.

Through animal experiments, the present inventors have discovered that the antibodies described herein, in particular 3H6H4L1, can effectively alleviate pathological changes in a model of rheumatoid arthritis induced by NIH/3T3 cells transfected with human IL-1 β in BALB/c mice, in particular , administration of 3H6H4L1 antibody can effectively alleviate abnormal behavior and reduce the swollen area of affected limbs in rheumatoid mice.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used in the present invention are routine operations widely used in their respective fields of technology. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the IL-1e amino acid sequence (GenBank ID: NP000567.1) includes the full-length IL-1e protein as well as an IL1β fusion protein, such as a fragment fused to a murine or Fc protein fragment. human IgG (mFc or hFc) or multiple His residues. However, those skilled in the art will understand that mutations or variations (including, without limitation, deletions and/or insertions) in the amino acid sequence of IL-1e can occur naturally or be artificially introduced without affecting their biological functions. Thus, in the present invention, the term IL-1e includes all such sequences, as well as natural or artificial variants thereof. In addition, when an IL-1e protein sequence fragment is described, it includes the IL-1e sequence fragment as well as corresponding sequence fragments in natural or artificial variants thereof.

In this context, when referring to the amino acid sequence of IL-1R1 (GenBank ID: NP000868), it includes the full-length IL-1R1 protein as well as an IL-1R1 fusion protein, such as a fragment fused to a fragment of a mouse or human IgG Fc protein (mFc or hFc) or multiple His residues. However, those skilled in the art will appreciate that in the amino acid sequence of the IL-1R1 protein, mutations or variations (including, without limitation, substitutions, deletions and/or insertions) can occur naturally or artificially introduced without affecting their biological functions. Thus, in the present invention, the term IL-1R1 includes all such sequences, as well as natural or artificial variants thereof. In addition, when an IL-1R1 protein sequence fragment is described, it includes the IL-1R1 sequence fragment as well as corresponding sequence fragments in natural or artificial variants thereof.

In this context, when referring to the amino acid sequence of IL-1R2 (GenBank ID: CAA42441.1), it includes the full-length IL-1R2 protein as well as an IL-1R2 fusion protein, such as a fragment fused to a fragment of the mouse Fc protein or human IgG (mFc or hFc) or multiple His residues. However, those skilled in the art will understand that mutations or variations (including, without limitation, substitutions, deletions and/or insertions) in the amino acid sequence of the IL-1R2 protein can occur naturally or be artificially introduced without affecting their biological functions. Thus, in the present invention, the term IL-1R2 includes all such sequences, as well as natural or artificial variants thereof. In addition, when an IL-1R2 protein sequence fragment is described, it includes the IL-1R2 sequence fragment as well as corresponding sequence fragments in natural or artificial variants thereof.

As used herein, the term EC ₅₀ refers to the concentration at 50% of the maximum effect, that is, the concentration that can cause 50% of the maximum effect.

As used herein, the term antibody refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains (each pair with one light (L) chain and one heavy (H) chain). Antibody light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α or ε. And antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE. In light chains and heavy chains, the variable region and constant region are linked by a J region of about 12 or more amino acids, and the heavy chain also includes a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region

- 9 046827 (CL). The light chain constant region consists of a single CL domain. The antibody constant region can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (eg, effector cells) to the first component (C1c.|) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions or CDRs), between which are distributed conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, located from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. The assignment of amino acids to each region or domain is as defined in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901917, or Chothia et al. (1989) Nature 342:878–883. The term antibody is not limited to any particular method of producing an antibody. For example, the antibody includes, but is not limited to, a recombinant antibody, a monoclonal antibody, and a polyclonal antibody. Antibodies can be of different isotypes, such as IgG antibodies (eg, IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

As used herein, the term antigen-binding fragment, also known as an antigen-binding moiety, refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen. For general information, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen binding fragment of an antibody may be produced by recombinant DNA technologies or by enzymatic or chemical digestion intact antibodies. In some cases, the antigen binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb and a complementarity determining region (CDR) fragment, single chain antibody (eg, scFv), chimeric antibody, diabody. and a polypeptide that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide.

In some cases, the antigen binding fragment of an antibody is a single chain antibody (eg, scFv) in which the VL and VH domains are paired to form a monovalent molecule through a linker that allows them to form a single polypeptide chain (see, for example, Bird et al., Science 242 : 423 426 (1988) and Huston et al., Proc. Natl. Sci. USA 85: 5879 5883 (1988). Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VLCOOH. A suitable prior art linker consists of the repeating amino acid sequence GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but a variant thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448). Other linkers that can be used in the present invention are described by 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.

In some cases, the antigen binding fragment of an antibody is a diabody, that is, a divalent antibody in which the VH and VL domains are expressed on the same polypeptide chain. However, the linker used is too short to allow pairing of two domains on one chain, so the domains are forced to pair with complementary domains on the other chain, and two antigen-binding sites are created (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak R. J. et al., Structure 2: 1121 1123 (1994)).

In other cases, the antigen binding fragment of the antibody is a bifunctional antibody. A bifunctional antibody, also known as a bispecific antibody, is a specific drug that simultaneously targets two different antigens and can be produced by immunoselective purification. In addition, a bispecific antibody can also be produced by genetic engineering, which has certain advantages due to its flexibility in aspects such as optimization of binding sites, synthetic form and yield. Currently, it has been shown that bispecific antibodies exist in more than 45 forms (Muller D., Kontermann R.E. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98). A number of bispecific antibodies have been developed in the form of IgG-ScFv, namely in the Morrison form (1997 Coloma M.J., Morrison S.L. Design and production of novel tetravalent bispecific antibodies. Nature Biotechnology, 1997; 15:159-163), for which it was shown that it is one of the ideal forms of bispecific antibodies due to its similarity to the naturally occurring form of IgG and advantages in engineering, expression and purification of antibodies (Miller B.R., Demarest S.J., et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J., Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways.

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Antigen-binding antibody fragments (e.g., the antibody fragments described above) can be prepared from a given antibody (e.g., from the monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3, or 3H6H4L1 described herein) using conventional methods known to those skilled in the art (e.g., recombinant DNA or enzymatic or chemical digestion), and antigen-binding antibody fragments are tested for specificity in the same way as for intact antibodies.

As used herein, the terms mAb and monoclonal antibody refer to an antibody or antibody fragment that is derived from a group of highly homologous antibodies, that is, from a group of identical antibody molecules, excluding natural mutations that may occur spontaneously. A monoclonal antibody is highly specific for a single epitope on an antigen. A polyclonal antibody, compared to a monoclonal antibody, typically includes at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be produced using the hybridoma method first described by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technologies (see, for example, US Pat. No. 4816567).

As used herein, the term humanized antibody refers to an antibody or antibody fragment produced when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be non-human (eg, mouse, rat or rabbit ) an antibody having the expected specificity, affinity, or reactivity. In addition, certain amino acid residues in framework regions (FR) of a receptor antibody may also be replaced by amino acid residues of corresponding non-human antibodies or amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For more information on humanized antibodies, see, for example, 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); and Clark, Immunol. Today 21: 397,402 (2000).

The term isolated as used here refers to objects obtained artificially from a natural state. If a particular isolated substance or component appears in nature, it may be due to a change in its natural environment, or because it is isolated from its environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a certain living animal, and the same polynucleotide or polypeptide isolated with high purity from such a natural state is called an isolated polynucleotide or polypeptide. The term isolated does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

As used herein, the term vector refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector allows expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that elements of the genetic material carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art, including, but not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-based artificial chromosomes (PAC); phages such as lambda phages or M13 phages; 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 papovaviruses (such as SV40). The vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further comprise an origin of replication.

As used herein, the term host cell refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as E. coli or B. subtilis, fungal cells such as yeast or aspergillus cells, insect cells such as Drosophila cells S2 or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term bispecific, dual specificity, 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 multispecific antibody and can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment binding; see, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315321; 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 target proteins.

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As used herein, the term specifically bind refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In some embodiments, the term antibody that specifically binds to an antigen (or antibody that is specific for an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10 ^-5 M, for example, 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.

As used herein, the term KD refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, which is used to describe the binding affinity between antibody and antigen. The lower the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between antibody and antigen. Typically, an antibody (e.g., monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, or 3H6H3L3 as described herein) binds to an antigen (e.g., IL-1e protein) with a dissociation constant (KD) of 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. [Can be determined using methods known to those skilled in the art, for example, using a Biacore molecular interaction meter.

As used herein, the terms monoclonal antibody and mAb have the same meaning and may be used interchangeably; the terms polyclonal antibody and PcAb have the same meaning and can be used interchangeably; the terms polypeptide and protein have the same meaning and can be used interchangeably. In the present invention, amino acids are generally designated by one-letter and three-letter abbreviations known in the art. For example, alanine may be designated by the letter A or Ala.

As used herein, the terms hybridoma and hybridoma cell line may be used interchangeably, and when referring to the terms hybridoma and hybridoma cell line, they also include subclones and hybridoma progeny cells. For example, when referring to the LT010 hybridoma cell line, this also refers to subclones and progeny cells of the LT010 hybridoma cell line.

As used herein, 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 active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro A.R., 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween80; and ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term effective amount refers to an amount sufficient to produce, or at least partially produce, the desired effect. For example, the term prophylactically (eg, in the case of RA) effective amount refers to an amount sufficient to prevent, stop or delay the onset of diseases (eg, RA); The term therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest a disease and its complications in patients suffering from the disease. Such an effective amount may well be determined by those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the route of administration of the drug and other treatments used concurrently, etc.

Advantages of the invention.

An anti-HE-1 β antibody disclosed herein, particularly a humanized anti-HE-1 β antibody, has one or more of the following technical effects:

(1) effectively binding to human IL-1[1 and blocking the binding of Ε-1β to its receptor IL-1R1;

(2) inhibition of activation of the downstream IL-1β signaling pathway;

(3) the ability to specifically inhibit the activity of IL-1e to induce IL-6 secretion by MRC-5 cells;

(4) the ability to effectively block the ability of IL-1e to activate NF-kB;

(5) the possibility of use for obtaining a drug for inhibiting IL-1e; and (6) the possibility of use for the preparation of a medicinal product for the prevention and/or treatment of diseases such as rheumatoid arthritis, gout, multiple sclerosis, cardiovascular events and/or cardiovascular diseases, tumors, cryopyrin-associated periodic syndromes in children and adults, periodic fever syndromes, systemic juvenile idiopathic arthritis.

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Brief description of drawings

Fig. 1 - results of analysis of the binding activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 with human IL-ie—His-Bio.

Fig. 2 - results of the analysis of binding activity of 3H6H4L1 with human IL-1e-His-Bio.

Fig. 3 - results of analysis of the activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3, competing with human IL-1R1(1-332)-His for binding to human IL-1e-hFc.

Fig. 4 - results of an assay for the activity of 3H6H4L1 competing with human IL-1R1(1-332)His for binding to human IL-1e-hFc.

Fig. 5 - results of analysis of the affinity constant of 3H6H4L1 to human ΓΡ-1β. Note: Curves 1-5 show analyte concentrations at 25 nM, 12.5 nM, 6.25 nM, 3.13 nM and 1.56 nM, respectively.

Fig. 6 - results of analysis of the affinity constant of canakinumab for human IL-1 β. Note: Curves 1-5 show analyte concentrations at 25 nM, 12.5 nM, 6.25 nM, 3.13 nM and 1.56 nM, respectively.

Fig. 7 - effect of 3H6H4L1 on ΓΡ-1β-induced IL-6 secretion by MRC-5 cells.

Fig. 8 - influence of IL-1e on gradient activation of the NF-kB signaling pathway.

Fig. 9 is a diagram of the 3H6H4L1 IL-1β blocking reporter assay.

Fig. 10 - Effect of 3H6H4L1 on pathological behavior in a mouse model of Lenti-IL-1 e-NIH/3T3-induced knee arthritis.

Fig. 11 - Effect of 3H6H4L1 on the knee joint region in a mouse model of knee arthritis induced by Lenti-IL-1 e-NIH/3T3.

Fig. 12 - Effect of 3H6H4L1 on body weight in a mouse model of Lenti-IL-1e-NIH/3T3-induced knee arthritis.

Detailed Description of the Invention

Embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will appreciate that the following examples are used to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Cases without specific descriptions of methods or conditions were performed in accordance with methods or conditions described in the literature in the art (for example, see Guide to Molecular Cloning Experiments, authored by J. Sambrook et al., and translated by Huang Peitang et al. ., third edition, Science Press) or in accordance with the manufacturer's instructions for the product. The reagents or instruments used are commercially available conventional products unless their manufacturers are indicated.

In the following examples of the present invention, the BALB/c mice used were purchased from the Guangdong Medical Experimental Animal Center.

In the following examples of the present invention, the commercially available antibody canakinumab for the same target (trade name Daris®) was purchased from Novartis and used as a control antibody.

Preparation Example 1. Preparation of human IL-1e-His, IL-1R1(1-332-His, IL-1e-hFc and human IL-1e-His-Bio fusion proteins.

Protein sequences of human ΓΡ-1β (Genbank accession number: NP000567.1) and IL-1R1 (Genbank accession number: NP_000868) were found in the NCBI GenBank protein database. The amino acid sequences of human ΓΡ-1β and IL-1R1 were fused to the His tag and human IgG Fc purification tag sequences, respectively, and were abbreviated as human IL-1e-His, IL-1R1(1-332)-His, IL-1e -hFc respectively.

The quality of protein samples was assessed using SDS-PAGE.

Biotinylated human IL-1e-His protein samples (referred to as human IL-1e-His-Bio for short) were prepared using the EZ-Link® Sulfo-NHS-LCBiotinylation Kit (Thermo Scientific) and the specific preparation process was performed according to instructions for this reagent kit.

The resulting fusion proteins were used in the following examples.

Example 1. Preparation of mouse anti-Gk-1b antibody 3H6.

1. Preparation of hybridoma cell line LT010.

BALB/c mice (purchased from Guangdong Medical Laboratory Animal Center) were immunized with human IL-1 β-his as the antigen, and spleen cells of the immunized mice were fused with mouse myeloma cells to form hybridoma cells. The hybridoma cells were screened using IL-1e-His-Bio as an antigen by ELISA to obtain hybridoma cells capable of secreting an antibody that specifically binds to IL-1e-His-Bio. The hybridoma cells obtained by ELISA were screened by competitive ELISA to obtain hybridoma cells capable of secreting an antibody that competes with

- 13 046827 IL-1R1(1-332)-His receptor for binding to IL-ie—hFc, and a stable hybridoma cell line was obtained by limiting dilution. Methods for producing hybridoma cells are among currently accepted methods (eg, 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).

The present inventors designated the above hybridoma cell line as the LT010 (IL-1e-3H6) hybridoma cell line and named the monoclonal antibody secreted by it as 3H6.

The hybridoma cell line LT010 (IL-1e-3H6) was deposited in the China Type Culture Collection Center (CTCCC) on June 21, 2018, under the registration number CCTCC NO: C2018133 and storage address of Wuhan University, Wuhan, China, zip code: 430072.

2. Obtaining anti-III-1p antibody 3H6.

The LT010 cell line obtained above was cultured in serum-free hybridoma medium (serum-free hybridoma medium containing 1% penicillin-streptomycin and 4% glutamax, cultured in a cell incubator at 37°C with 5% CO ₂ ). After 7 days, the cell culture supernatant was collected and subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, and purification through a HiTrap protein A HP column to obtain the 3H6 antibody. Purified 3H6 samples were assessed using SDS-PAGE.

Example 2. Sequence analysis of anti-P.-1 β antibody 3H6.

mRNA was extracted from the LT010 cell line cultured in Example 1 according to the Total Bacterial RNA Extraction Reagent Kit from Cultured Cells method (Tiangen, catalog no. DP430). cDNA was synthesized according to the Invitrogen Superscript III First-Strand Synthesis System RT-PCR reagent kit manual and amplified by PNR. Products amplified by PCR were immediately subjected to TA cloning, and specific operations were performed in accordance with the instructions for the pEASY-T1 Cloning Kit (Transgen CT101).

The TA cloning products were subjected to direct sequencing and the sequencing results were as follows.

Nucleotide sequence encoding the variable region of the heavy chain of the 3H6 antibody: (354 bp)

CAGGTGACCCTGAAGGAGAGCGGACCAGGAATCCTGCAGCCTAGCCAGACA

CTGAGCCTGACTTGCAGCTTCAGCGGCTTCAGCCTGAGCACAAGCGGAATGGGCGT

GTCTTGGATCAGGCAGCCATCAGGAAAGGGACTCGAGTGGCTGGCTCACATCTACT GGGACGACGACAAGCGGTACAACCCCTCCCTGAAGAGCAGGCTGACCATCAGCAAG GACACCAGCAGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGATAG CGCTACCTACTATTGCGCCAGAAGCGCCTACAGCTTCGCCTATTGGGGCCAGGG AACACTGGTGTCCGTGTC AGCC (SEQ ID NO: 1)

The amino acid sequence of the heavy chain variable region of the 3H6 antibody is as follows: (118 amino acid residues, in which the underlined amino acid sequences are CDR regions)

OVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYW

DDDKRYNPSLKSRLTISKDTSSNOVFLKITSVDTADSATYYCARSAYYSFAYWGOGTLV

SVSA (SEQ ID NO: 2)

Nucleotide sequence encoding the variable region of the light chain of the 3H6 antibody: (318 bp)

GATATCGTCATGACACAGTCACATAAGTTTATGTCTACTAGTGTGGGCGGGC

GGGTCAGAATTACCTGTAAGGCCTCTCAGGACGTGGATACAGACGTGGCTTGGTTCC

AGCAGAAGCCCGGACAGAGCCCTAAACTGCTGATCTACTGGGCCTCCACAAGGCAC ACTGGGGTGCCAGATCGGTTCACTGGATCAGGCAGCGGGACCGACTTTACTCTGACC ATTTCCAACGTCCAGTCTGAGGATCTGGCTGACTATTTCTGCCAGCAGTACAGCTCC TATCCCACCTTTGGAGCAGGCACAAAGCTGGAACTGAAA (SEQ ID NO: 3)

The amino acid sequence of the 3H6 antibody light chain variable region is as follows: (106 amino acid residues, in which the underlined amino acid sequences are CDR regions)

DIVMTQSHKFMSTSVGGRVRITCKASODVDTDVAWFOOKPGOSPKLLIYWASTR

HTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCOQYSSYPTFGAGTKLELK (SEQ ID

NO: 4)

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Example 3: Design and production of humanized aHTu-IL-Ιβ antibodies against 3H6H1L1. 3H6H2L2, 3H6H3L3 and 3H6H4L1.

1. Design of the light and heavy chain sequences of humanized anti-HE-1b 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1.

Based on the three-dimensional crystal structure of the ΙΙ.-1β protein (van Oostrum J., Priestle J.P., Grutter M.G., Schmitz A. The structure of murine interleukin-1 beta at 2.8 A resolution. J Struct Biol. 1991, 107(2): 189 -95.) And the sequences obtained in example 2, the sequences of the variable regions of the heavy and light chains of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 were constructed (the sequences of the constant regions of the antibodies 3H6H1L1, 3H6H2L2 and 3H6H3L3 were taken from the NCBI database, where stant area of heavy chain is the Ig gamma-1 chain region, Registration number: P01857, and the light chain constant region is the Ig kappa chain C region, Registration number: P01834; the sequences of the constant regions of the 3H6H4L1 antibody are taken from the NCBI database, where the heavy constant region is. chain is the gamma-4 Ig C region, Registration number: P01861.1, and the light chain constant region is the Ig kappa chain C region, Registration number: P01834).

The sequences of the variable regions of the heavy and light chains of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 were as follows.

(1) Humanized monoclonal antibody 3H6H1L1.

Nucleotide sequence encoding the variable region of the heavy chain of the antibody 3H6H1L1: (354 bp)

CAGGTGACACTGAAGGAGTCTGGCCCCGCCCTGCTGAAGCCTACCCAGACAC TGACCCTGACATGTACCTTCTCCGGCTTTTCTCTGAGCACCTCCGGCATGGGCGTGTC TTGGATCAGGCAGCCAAGCGGCAAGGCCCTGGAGTGGCTGGCACACATCTACTGGG ACGATGACAAGCGGTATAACCCCTCCCTGAAGTCTAGACTGACAATCTCTAAGGAT ACCAGCTCCAACCAGGTT CCTGAGATCACAAATGTGGATACCGTGGACACAGC CACCTACTATTGCGCCCGGAGCGCCTACTATTCCTTTGCCTACTGGGGCCAGGGCAC ACTGGTGTCTGTGAGCGCC (SEQ ID NO: 5)

The amino acid sequence of the heavy chain variable region of the 3H6H1L1 antibody is as follows: (118 amino acid residues, in which the underlined amino acid sequences represent CDR regions)

QVTLKESGPALLKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPSGKALEWLAHIYW

DDDKRYNPSLKSRLTISKDTSSNQVFLKITNVDTVDTATYYCARSAYYSFAYWGQGTLV

SVSA (SEQ GO NO: 6)

Nucleotide sequence encoding the variable region of the light chain of the antibody 3H6H1L1: (318 bp)

GATATCCAGATGACCCAGTCCCACAGCTCCATGTCCACATCTGTGGGCGACC

GGGTGAGAATCACCTGTCGGGCCTCCCAGGACGTGGATACAGACGTGGCCTGGTTT CAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTGGGCCAGCACCAGGCA CTCCGGAGTGCCATCTCGCTTCAGCGGCTCCGGCTCTGGCACAGACTTCACCCTGAC AATCAGCAACGTGCAGCCAGAGGATTTTCGCCGACTACTATTGCCAGCAGTACTCTAG CTATCCCACCTT TGGCGCCGGCACAAAGCTGGAGCTGAAG (SEQ ID NO: 7)

The amino acid sequence of the light chain variable region of the 3H6H1L1 antibody is as follows: (106 amino acid residues, in which the underlined amino acid sequences represent CDR regions)

DIOMTOSHSSMSTSVGDRVRrrCRASODVDTDVAWFOQKPGOAPKLLIYWASTR

HSGVPSRFSGSGSGTDFTLTISNVOPEDFADYYCOQYSSYPTFGAGTKLELK (SEQ ID

NO: 8)

- 15 046827 (2) Humanized monoclonal antibody 3H6H2L2.

Nucleotide sequence encoding the variable region of the heavy chain of the antibody 3H6H2L2: (354 bp)

CAGGTGACACTGAAGGAGTCCGGCCCCGCCCTGGTGAAGCCTACCCAGACAC TGACCCTGACATGTACCTTCAGCGGCTTTTCTCTGAGCACCTCCGGCATGGGCGTGT CCTGGATCAGGCAGCCATCTGGCAAGGCCCTGGAGTGGCTGGCCCACATCTACTGG GACGATGACAAGCGGTATTCTCCCAGCCTGAAGTCTAGACTGACAATCAGCAAGGA TACCAGCTCCAACCAGGTG TTCCTGACAATCACCAACGTGGACCCCGTGGACACAG CCACCTACTATTGCGCCCGGAGCGCCTACTATTCCTTTGCCTACTGGGGCCAGGGCA CACTGGTGTCCGTGTCTGCC (SEQ GO NO: 9)

The amino acid sequence of the heavy chain variable region of the 3H6H2L2 antibody is as follows: (118 amino acid residues, in which the underlined amino acid sequences are CDR regions)

OVTLKESGPALVKPTOTLTLTCTFSGFSLSTSGMGVSWIROPSGKALEWLAHIYW

DDDKRYSPSLKSRLTISKDTSSNOVFLTITNVDPVDTATYYCARSAYYSFAYWGOGTLV

SVSA (SEQ GO NO: 10)

Nucleotide sequence encoding the variable region of the light chain of the antibody 3H6H2L2: (318 bp)

GATATCCAGATGACACAGAGCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACC GGGTGAGAATCACCTGTAGGGCCTCTCAGGACGTGGATACAGACGTGGCCTGGTAC CAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTATTGGGCCTCTACCCTGCAG AGCGGAGTGCCATCCCGGTTCTCTGGCAGCGGCTCCGGAACAGACTTCACCCCTGACA ATCTCTAGCCTGCA GCCAGAGGACTTCGCCACCTACTATTGCCAGCAGTACTCCCTCT TATCCCACCTTTGGCGCCGGCACAAAGCTGGAGCTGAAG (SEQ ID NO: 11)

The amino acid sequence of the light chain variable region of the 3H6H2L2 antibody is as follows: (106 amino acid residues, in which the underlined amino acid sequences represent CDR regions)

DIOMTOSPSSLSASVGDRVRrrCRASODVDTDVAWYOQKPGKAPKLLIYWASTL

QSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOQYSSYPTFGAGTKLELK (SEQ ID NO: 12) (3) Humanized monoclonal antibody 3H6H3L3.

Nucleotide sequence encoding the variable region of the heavy chain of the antibody 3H6H3L3: (354 bp)

CAGGTGACACTGAAGGAGAGCGGCCCAGCCCTGGTGAAGCCAACCCAGACA CTGACCCTGACATGTACCTTCTCCGGCTTTAGCCTGTCCACCTCTGGCATGGGCGTGT CTTGGATCAGGCAGCCACCTGGCAAGGCCCTGGAGTGGCTGGCCCTGATCTACTGG GACGATGACAAGCGGTATAGCCCTTCCCTGAAGAGCAGACTGACAATCTCAAGGA TACCTCTAAGAACCAGG TGGTGCTGACAATCACCAACGTGGACCCCGTGGACACAG CCACCTACTATTGCGCCCGGAGCGCCTACTATTCCTTTGCCTACTGGGGCCAGGGCA CACTGGTGTCTGTGAGCGCC (SEQ ID NO: 13)

The amino acid sequence of the heavy chain variable region of the 3H6H3L3 antibody is as follows: (118 amino acid residues, in which the underlined amino acid sequences are CDR regions)

OVTLKESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLALIYW DDDKRYSPSLKSRLTISKDTSKNQVVLTITNVDPVDTATYYCARSAYYSFAYWGQGTLV SVSA (SEQ ID NO: 14)

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Nucleotide sequence encoding the variable region of the light chain of the antibody 3H6H3L3: (318 bp)

GATATCCAGATGACACAGAGCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACA

GGGTGACCATCACATGTAGAGCCTCTCAGGACGTGGATACCGACCTGGCCTGGTAC CAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTATTGGGCCTCTACCCTGCAG AGCGGAGTGCCATCCCGGTTCTCTGGCAGCGGCTCCGGAACAGACTTCACCCTGACA ATCTCTAGCCTGCAGCCAGAGGACTTCGCCACCTACTATTGCCAGCAGTACTCCTCT TATCCCACCTT TGGCGCCGGCACAAAGCTGGAGCTGAAG (SEQ ID NO: 15)

The amino acid sequence of the 3H6H3L3 antibody light chain variable region is as follows: (106 amino acid residues, in which the underlined amino acid sequences are CDR regions)

DIOMTOSPSSLSASVGDRVTITCRASODVDTDLAWYOOKPGKAPKLLIYWASTL

OSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOQYSSYPTFGAGTKLELK (SEQ ID NO: 16) (4) Humanized monoclonal antibody 3H6H4L1.

The nucleotide sequence encoding the heavy chain variable region of the 3H6H4L1 antibody is shown in SEQ ID NO: 5.

The amino acid sequence of the heavy chain variable region of the 3H6H4L1 antibody is shown in SEQ ID NO: 6.

The nucleotide sequence encoding the light chain variable region of the 3H6H4L1 antibody is shown in SEQ ID NO: 7.

The amino acid sequence of the light chain variable region of the 3H6H4L1 antibody is shown in SEQ ID NO: 8.

2. Preparation of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1.

The heavy chain constant regions 3H6H1L1, 3H6H2L2 and 3H6H3L3 are the Ig gamma 1 chain C region, Registration number: P01857; and the light chain constant regions are the Ig kappa chain C region, Registration number: P01834;

the heavy chain constant region 3H6H4L1 is the C region of the gamma-4 Ig chain, Registration number: P01861.1; and the light chain constant region is the Ig kappa chain C region, Registration number: P01834.

The heavy and light chain cDNAs 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1 were each cloned into the pUC57simple vector (provided by Genscript) to obtain 8 recombinant plasmids, respectively, namely pUC57simple-3H6H1 and pUC57Limple-3H6Simple; pUC57simple-3H6H2 and pUC57simple3H6L2; pUC57simple-3H6H3 and pUC57simple-3H6L3; and pUC57simple-3H6H4 and pUC57simple-3H6L1, and subcloned them into pcDNA3.1 vectors, respectively. Recombinant heavy chain plasmids and recombinant light chain plasmids were cotransfected into 293F cells, and the cell culture was harvested and purified to produce humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3, and 3H6H4L1. The results were assessed using SDS-PAGE.

Example 4. Analysis of the binding activity of antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 with human IL-1e-His-Bio (ELISA).

The plate was coated with 50 μl of 2 μg/ml SA (streptavidin) in each well and incubated overnight at 4°C. After washing the plate once and removing residual liquid, each well was blocked with 300 μl of 1% BSA solution (dissolved in PBS) and the plate was incubated at 37°C for 2 hours. The plate was washed three times and residual liquid was removed. Human IL-1e-His-Bio in each well was diluted with 50 μl of PBST to 0.2 μg/ml, and the plate was incubated at 37°C for 30 min. The plate was then washed three times and residual liquid was removed. The antibody was diluted to 1 μg/ml (results in Table 1) or to 0.333 μg/ml (results in Table 2) as the starting concentration, and a 1:3 gradient dilution was performed to obtain a total of 7 concentrations in addition to blank control. The above concentrations were tested in two duplicate wells with a final volume of 100 μl per well, and the plate was incubated at 37°C for 30 min. After washing the plate three times and patting to remove any remaining liquid, 50 μl of horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody working solution or goat anti-mouse IgG (H+L) secondary antibody working solution was added to each well. , labeled with horseradish peroxidase, and the plate was incubated for 30 min at a temperature of 37°C, then 50 μl of a working solution of the secondary antibody goat anti-human IgG (H+L) was added to the wells containing 3H6H1L1, 3H6H2L2, 3H6H3L3, 3H6H4L1, canakinumab , labeled with horseradish peroxidase; and 50 μl of a working solution of secondary antibody goat anti-mouse IgG (H+L) labeled with horseradish peroxidase was added to the wells containing 3H6. After washing the plate four times and removing any residual

- 17 046827 new liquid, 50 μl of TMB chromogenic solution was added to each well to develop color for 5 minutes away from light at room temperature, then 50 μl of stop solution was added to each well to stop the reaction. The plate was then immediately placed in a plate reader and the OD value of each well in the plate was read at a wavelength of 450 nm.

SoftMax Pro 6.2.1 software was used for data analysis and processing. 4-parameter fitting curves were constructed using antibody concentration as the abscissa and absorbance as the y-axis. The results are shown in Fig. 1 and 2. The results of the analysis of the binding activity of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 with human IL-1e-His-Bio are shown in table. 1 and 2 respectively.

Table 1 Results of analysis of binding activity of 3H6, 3H6H1L1,

3H6H2L2 and 3H6H3L3 with human IL-1e-His-Bio

Antibody concentration (µg/ml) Antigen-antibody binding OD value (450 nm) 3H6H1L1 3H6H2L2 3H6H3L3 ZN6 Canakinumab 1 2.91 4 2.927 2,864 2,879 2.865 2,905 2,516 2,602 2,871 2,874 0.333 2.97 9 2,980 2.953 2.928 2,880 2,874 2.617 2,597 2,902 2,883 0.111 2.95 0 2,989 2.958 2.953 2,892 2.865 2,411 2,386 2.895 2,887 0.037 2.80 9 2,771 2,777 2,732 2,634 2,662 2,036 1.936 2,643 2,682 0.012 2.16 7 2,267 2,197 2,182 2,001 2,016 1.225 1.225 2.065 2.118 0.004 1.35 2 1,264 1,300 1.439 1.225 1.178 0.598 0.623 1.256 1,199 0.001 0.66 0 0.619 0.630 0.669 0.590 0.566 0.287 0.293 0.581 0.559 0 0.04 0.042 0.041 0.041 0.050 0.039 0.039 0.041 0.039 0.040 Antibody concentration (µg/ml) Antigen-antibody binding OD value (450 nm) 3H6H1L1 3H6H2L2 3H6H3L3 ZN6 Canakinumab 2 Second antibody Goat anti-human IgG (H+L) labeled with horseradish peroxidase, 50 µl Goat anti-mouse IgG (H+L), labeled with horseradish peroxidase, 50 µl Goat anti-human IgG (H+L) labeled with horseradish peroxidase, 50 µl EC ₅₀ (nM) 0.036 0.034 0.042 0.099 0.039

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table 2

Results of 3H6H4L1 binding activity assay with human IL-ie-His-Bio

Antibody dilution Antigen-antibody binding OD value (450 nm) Canakinumab 3H6H4L1 0.333 µg/ml 2,809 2,940 2,899 2.935 1:3 2,679 2.875 2.822 2,866 1:9 2,463 2,613 2,814 2,797 1:27 1,702 1.959 2,311 2.322 1:81 0.852 1.115 1.525 1.436 1:243 0.408 0.573 0.771 0.737 1:729 0.315 0.242 0.358 0.332 0 0.075 0.071 0.071 0.075 Second antibody Goat anti-human IgG (H+L) labeled with horseradish peroxidase, 50 µl EC ₅₀ (nM) 0.057 0.029

These results showed that

3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively bind to human IL1e-His-Bio, and the binding efficiency is dose dependent;

Under the same assay conditions, the binding efficiency of 3H6H1L1, 3H6H2L2 and 3H6H4L1 to the human IL-1e-His-Bio antigen is dose dependent, and the binding activity is superior to that of the commercially available drug canakinumab for the same target; while the binding activity of 3H6H3L3 is comparable to that of canakinumab.

Example 5. Analysis of the activity of antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1, competing with human IL-1R1(1-332)-His for binding to human IL-1e-hFc (ELISA).

The plate was coated with 50 μl of 4 μg/ml human IL-1e-hFc in each well and incubated overnight at 4°C. After washing the plate once and removing residual liquid, each well was blocked with 300 μl of 1% BSA solution (dissolved in PBS) and the plate was incubated at 37°C for 2 hours. The plate was then washed three times and residual liquid was removed. The antibody was diluted to 2 μg/ml (final concentration: 1 μg/ml) as the starting concentration and a 1:3 gradient dilution was performed to obtain a total of 7 concentrations in addition to the blank control. The above concentrations were tested in two duplicate wells with a final volume of 50 μl per well, and the plate was incubated for 10 min. 50 μl of 0.08 μg/ml (final concentration: 0.04 μg/ml) or 0.1 μg/ml (final concentration: 0.05 μg/ml) human IL-1R1(1-332)-his was added to Each well in the plate was carefully mixed with the antibody in a volume ratio of 1:1, with the final volume in each well being 100 μl. The plate was then incubated at 37°C for 30 min. After washing the plate three times and removing residual liquid, 50 μl of a working solution of mouse monoclonal anti-His antibody (labeled with horseradish peroxidase) was added to each well, and the plate was incubated at 37°C for 30 min. After washing the plate four times and removing residual liquid, 50 μL of TMB chromogenic solution was added to each well to develop color for 10 or 5 minutes away from light at room temperature, then 50 μL of stop solution was added to each well to stop the reaction. The plate was then immediately placed in a plate reader and the OD value of each well in the plate was read at a wavelength of 450 nm.

Data were analyzed and processed using SoftMax Pro 6.2.1 software, and 4-parameter curves were generated using antibody concentration on the x-axis and absorbance on the y-axis. The results are shown in Fig. 3 and 4. The results of the analysis of the activity of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1, competing with human IL-1R1(1-332)-his for binding to human IL-1e-hFc, are shown in table. 3 and 4, respectively.

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Table 3

Results of the analysis of the activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3, competing with human IL-1R1(1-332)-his for binding to human IL-1e-hFc

Concentration The OD value (450 nm) of an antibody that blocks antigen binding to antibodies (µg/ml) human IL-lRl(l-332)-his receptor 3H6H1L1 3H6H2L2 3H6H3L3 ZN6 Canakinumab 1,000 0.050 0.049 0.057 0.070 0.057 0.058 0.049 0.050 0.053 0.052 0.333 0.051 0.050 0.071 0.073 0.069 0.067 0.050 0.052 0.059 0.057 0.111 0.055 0.054 0.095 0.098 0.132 0.115 0.063 0.058 0.091 0.092 0.037 0.206 0.221 0.242 0.198 0.361 0.348 0.266 0.283 0.366 0.379 0.012 0.521 0.498 0.504 0.453 0.505 0.584 0.560 0.516 0.572 0.579 0.004 0.635 0.677 0.681 0.618 0.693 0.697 0.682 0.712 0.727 0.684 0.001 0.630 0.687 0.736 0.679 0.748 0.715 0.787 0.773 0.577 0.720 0.000 0.621 0.693 0.804 0.779 0.780 0.807 0.810 0.818 0.555 0.765 EC ₅₀ (nM) 0.155 0.108 0.182 0.137 0.260

Table 4

Results of an assay for the activity of 3H6H4L1 competing with human IL-1R1(1-332)-his for binding to human IL-1e-hFc

Antibody concentration (µg/ml) OD value (450 nm) of an antibody that blocks antigen binding to the human IL-lRl(l-332)-his receptor Canakinumab 3H6H4L1 1,000 0.093 0.088 0.079 0.063 0.333 0.104 0.126 0.055 0.055 0.111 0.519 0.526 0.239 0.201 0.037 0.736 0.705 0.629 0.547 0.012 0.797 0.706 0.701 0.648 0.004 0.765 0.735 0.710 0.673 0.001 0.738 0.559 0.666 0.662 0.000 0.669 0.719 0.659 0.621 EC ₅₀ (nM) 0.961 0.530

These results showed that

3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively block the binding of the human IL-1e-hFc antigen to the human IL-1R1(1-332)-his receptor, and the blocking efficiency is dose dependent, and their competitive binding activity is higher than that of the commercially available drug canakinumab for the same target.

Example 6. Determination of the affinity constant of the antibody 3H6H4L1 to human IL-1e.

The antibody affinity constant for human IL-1e-his was determined using a Biacore molecular interaction meter. The antibody was immobilized on the surface of the CM5 chip in PBST buffer by immobilization at the amino group with a signal value of about 1000 EO. Antibodies bound to human IL-1e at a concentration of 1.56-25 nM (2-fold gradient dilution) for 120 s at a flow rate of 30 μl/min, and they dissociated within 600 s. The chip was regenerated using 3 M MgCl ₂ for 30 s at a flow rate of 30 μl/min. Data were acquired using Biacore Control 2.0 software and analyzed using Biacore T200 Evaluation 2.0 software. The results are presented in table. 5 and fig. 5 and fig. 6.

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Table 5

Results of the analysis of the affinity constant of 3H6H4L1 for human IL-Ιβ

Name _KD (M) ka (1/Ms) SE (ka) kd(1/s) SE(kd) Rmax (EO) 3H6H4L1 8J9E-11 1.44E+06 ZD5E+OZ 1.27E-04 1J5E-07 112.49-122.37 Canakinumab 9J9E-11 5.24E+05 6.84E+02 5DZE-05 1.43E-07 76.61-86.37

These results showed the following.

The affinity constant of 3H6H4L1 for human Π,-1β was 8.79E-11M, and the affinity constant of canakinumab for human IL-Ιβ was 9.79E-11M, suggesting that 3H6H4L1 has a stronger binding ability to human IL-Ιβ.

Example 7. Analysis of cellular biological activity of the 3H6H4L1 antibody.

1. Cytological analysis of the activity of 3H6H4L1 in blocking the ability of IL-Ιβ to induce IL-6 secretion by MRC-5 cells.

Human MRC-5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) were digested and counted in the usual manner, and 7500 cells per well were seeded in a 96-well flat-bottom plate and cultured in a cell incubator; after 24 hours (when cell growth reached 80% confluency), dose treatment was carried out: 4 concentrations were set for the antibody (0.37 nM, 1.11 nM, 3.33 nM and 10 nM) and 3 concentrations (5 pM, 50 pM and 500 pM) were set for IL-1e (purchased from Sino Biological Inc.), 50 pM ΙΙ,-1β was used in the antibody group (antibody and IL1β were incubated at 37°C for 20 min in advance) in addition to the blank control group and isotype control group; After dosing, groups were cultured for 24 h; cell supernatants were collected and analyzed using an IL-6 ELISA reagent kit (purchased from Dakewe Biotechnology Co., Ltd.). The results of the analysis are shown in Fig. 7 and in table. 6.

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Table 6

Activity of 3H6H4L1 gradient inhibitory activity of IL-Ιβ on inducing IL-6 secretion by MRC-5 cells

Group/ OD (450 nm) Measured Coffi Original concentration concentration (pg/ml) dilution ratio concentration (pg/ml) (defined concentration * dilution factor) Hole a 1 Hole a2 Hole 1 Hole 2 Hole 1 Hole 2 FSB 0.595 0.446 26.6 18.3 5 132.8 91.4 Human IgG/10 nM 1,301 1.476 92.8 123.9 200 18552.6 24781.8 ΙΕ-1β/5 pM 1.678 1,404 178.7 109.8 50 8936.3 5490.7 ΙΕ-1β/50πΜ 1,597 1.666 153.4 174.5 200 30681.6 34898.6 ΙΕ-1β/500πΜ 1.207 1.228 79.6 82.4 500 39817.0 41182.0 Canakinumab/0.37 nM 0.432 0.425 17.6 17.2 100 1758.0 1720.1 Canakinumab/1.11 nM 0.375 0.439 14.7 17.9 50 736.3 896.3 Canakinumab/3.33 nM 0.646 0.590 29.7 26.3 20 594.3 525.0 Canakinumab/10 nM 0.305 0.318 11.4 12.0 20 227.1 239.3 3H6H4L1/O,37 nM 1.029 1.143 59.7 71.8 50 2984.1 3591.5 3H6H4L1/1.11 nM 1.067 1,091 63.4 66.1 20 1268.8 1321.0 3H6H4Ll/3.33 nM 0.665 0.666 30.9 30.9 20 618.0 618.5 3H6H4L1/10 nM 0.469 0.609 19.5 27.4 10 195.2 274.4

These results indicated that 4Ε-1β could markedly stimulate MRC-5 cells to secrete IL6 in a dose-dependent manner; 3H6H4L1 can specifically inhibit the activity of 4Ε-1β to induce IL-6 secretion by MRC-5 cells, demonstrating the specific neutralizing activity of 3H6H4L1 on ΓΕ-1β.

2. 3H6H4L1 blocks the ability of 4Ε-1β to activate the NF-κB signaling pathway.

In this experiment, the neutralizing bioactivity of 3H6H4L1, which blocks the ability of IL-1e to activate the NF-κB signaling pathway, was measured using a luciferase reporter gene assay.

(1) Construction of the 293T-NF-KB-LUC cell.

293T cells were digested with pancreatin and subcultured; the medium was refreshed with optiDMEM 2 h before transfection; 500 μl of opti-DMEM medium was added to a sterile EP tube, followed by the addition of 3 μg of pNF-KB-Luc2P-hygro plasmid; 500 μl of opti-DMEM medium was added to a sterile EP tube followed by the addition of 8 μl of Lipofectamine 2000; diluted Lipofectamine 2000 was added to the diluted plasmids, and the mixture was incubated at room temperature for 15 min and added dropwise evenly to the cell culture dish; 8 h after transfection, the medium was renewed; and 24 hours after transfection, hygromycin was added at a final concentration of 100 μg/ml and cells were screened using a well containing non-plasmid-transfected 293T cells as a control. After 7-10 days, the cells in the control well

- 22 046827 completely died, and the selected cells were collected for amplification. Dosing was continued, leaving the concentration at 100 μg/ml. Thus, a stable cell line 293TNF-kB-LUC was obtained.

(2) Analysis of the neutralizing bioactivity of 3H6H4L1 blocking the ability of !Γ-1β to activate the NF-kB signaling pathway.

293T-NF-kB-LUC cells were routinely digested and seeded in a 96-well plate at 20,000 cells per well. After cells adhered to the wall, IL-1 β was added to a final concentration of 1.65 ng/ml and a blank control was established. Antibodies canakinumab and 3H6H4L1 were added simultaneously in 5 gradients for each antibody at final concentrations of 400 ng/ml, 100 ng/ml, 25 ng/ml, 6.25 ng/ml and 1.56 ng/ml, respectively. After 6 h of co-incubation, the supernatant was removed, 50 μl of PBS and 50 μl of Bright-Glo™ substrate were added to react for 5 min, and the mixture was analyzed using the apparatus.

The results are shown in Fig. 8 and 9.

These results showed that

IL-1e can effectively activate the expression of luciferase reporter genes dependent on the NF-κB signaling pathway in an apparent dose-dependent manner;

3H6H4L1 can specifically block the ability of IL-1e to activate the NF-κB signaling pathway in a dose-dependent manner.

The results showed that 3H6H4L1 could effectively block the ability of IL-1e to activate NF-kB in the IL-1e-dependent NF-kB signaling pathway, demonstrating its specific neutralizing activity against IL-1e.

Example 8 3H6H4L1 alleviates rheumatoid arthritis in a mouse model of knee arthritis induced by NIH/3T3 cells transfected with human IL-1e.

BALB/c mice were divided into 6 groups based on body weight, namely normal group, model group, positive control group, low dose 3H6H4L1 group, medium dose 3H6H4L1 group and high dose 3H6H4L1 group; except for 6 mice in the normal group, there were 8 mice in each group.

Before cell seeding, mice, according to body weight and dosing volume, were subcutaneously administered with canakinumab in the positive control group, subcutaneously administered with anti-HEL in the model group, administered 3H6H4L1 at the corresponding concentrations in the corresponding 3H6H4L1 dosing groups, and subcutaneously administered isovolumetric saline.

NIH/3T3 cells (purchased from the American Type Culture Collection) and Lenti-IL1e-NIH/3T3 cells were collected in a biological safety cabinet. The Lenti-IL-1e-NIH/3T3 cell line, which stably secretes and expresses IL-1e, was generated by transfecting the Lenti-IL-1e vector into NIH/3T3 cells and screening. When the required number of cells was reached, NIH/3T3, Lenti-IL-1e-NIH/3T3 cells were harvested. In a biological safety cabinet, the cell medium was pipetted, the cells were washed once with PBS and digested with an appropriate amount of 0.05% trypsin-EDTA (1×) for 1 min, then complete DMEM containing 10% PBS was added to stop digestion. The cell suspension was centrifuged for 4 min at 1200 rpm, and after removing the supernatant, resuspended in serum-free DMEM, counted and placed on ice for further use after the cell concentration was adjusted to 2,000,000 cells/ml.

After anesthetizing BALB/c mice with an intraperitoneal injection of 7.5 ml/kg 3.5% chloral hydrate, the knee joint cavity of mice in the normal group was injected with 25 μl/mouse (50,000 cells/mouse) of NIH/3T3 cell suspension, and the other mice were injected with 25 µl/mouse (50,000 cells/mouse) Lenti-IL-1e-NIH/3T3 cell suspension. After inoculation, the knee joint wound was sutured and 20-fold diluted penicillin in saline was applied. On day 5 after cell inoculation, 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 synovium of the affected limbs of the mice were measured with a caliper. Data were expressed as mean ± standard error (± SE), and results were assessed using one-way analysis of variance after between-group comparisons performed by GraphPad Prism 5 software, which determined a significant difference at P < 0.05 and a highly significant difference at P < 0.01.

The results are shown in Fig. 10, 11 and 12.

In fig. Figure 10 shows that pathological behavior is obvious in mice in the model group compared with mice in the normal group (P<0.01). After administration of canakinumab and high and medium doses of 3H6H4L1 in the respective groups, the pathological behavior of mice with rheumatoid arthritis was effectively improved (P<0.01), while the low dose of 3H6H4L1 was ineffective in improving the pathological behavior of mice with rheumatoid arthritis (P>0.01). 05) compared to the model group. Meanwhile, 3H6H4L1 has a certain dose-response relationship in improving the degree of pathological behavior in mice. Compared to the positive control group, in

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Claims (44)

In the medium and low dose 3H6H4L1 groups, the antibody was less effective (P < 0.01) than in the positive control group, and in the high dose 3H6H4L1 group, the effectiveness of this antibody was equivalent to that in the positive control group (P >0.05). In fig. Figure 11 shows that the knee joint area of the affected limb of mice in the model group was significantly increased compared with the normal group (P<0.01). After administration, the positive control group (canakinumab) and the medium and high dose groups of 3H6H4L1 clearly reduced the area of edema of the affected limb in mice with rheumatoid arthritis (P < 0.01), while in the low dose 3H6H4L1 group, administration of this antibody had no effect. obvious effect on reducing the area of edema of the affected limb of mice with rheumatoid arthritis (P>0.05) compared with the model group. Meanwhile, there is a certain relationship between the dose of 3H6H4L1 and its effect on reducing the area of edema in the affected limb of mice with rheumatoid arthritis. An equivalent dose of 3H6H4L1 has efficacy equivalent to the commercially available drug canakinumab for the same target (P>0.05). In fig. Figure 12 shows that the body weight of mice in the model group is significantly reduced compared with the body weight in the normal group (P<0.01). Apparently, after administration, the commercially available drug canakinumab for the same target and a high dose of 3H6H4L1 could reduce the body weight loss of arthritic mice compared with the model group (P<0.05). Compared to the positive control group, an equivalent dose of 3H6H4L1 had an efficacy equivalent to the commercially available drug canakinumab for the same target (P>0.05). Preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to these embodiments. Various equivalent modifications or substitutions can be made by those skilled in the art without departing from the spirit of the present invention. These equivalent modifications or replacements are included within the scope of the present invention as defined by the claims of this application. CLAIM 1. An anti-HIV antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region containing HCDR1-HCDR3 with the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; and the antibody comprises a light chain variable region comprising LCDR1-LCDR3 with the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14; and the antibody light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16. 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the variable region of the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO: 2 and the variable region of the light chain of the antibody contains the amino acid sequence of SEQ ID NO: 4; the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 6 and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 8; the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 10 and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 12; or the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 14 and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 16. 4. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or an antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody and diabody. 5. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or an antigen-binding fragment thereof binds to the IL-1 β protein with a K _D of less than 10 ^-5 M. 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, where the antibody or an antigen-binding fragment thereof binds to the LL-1e protein with a K _D of less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M, less 10 ^-9 M or less 10 ^-10 M or less. 7. The antibody or antigen binding fragment thereof according to any one of claims 5 or 6, wherein the KD is measured using a Biacore molecular interaction meter. - 24 046827 8. The antibody or antigen binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen binding fragment thereof comprises a non-CDR region obtained from a species other than mice. 9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof comprises a non-CDR region derived from a human antibody. 10. The antibody or antigen binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen binding fragment thereof comprises a constant region selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain constant region is an Ig gamma-1 chain C region or an Ig gamma-4 chain C region, and the light chain constant region is a C- Ig kappa chain region. 12. The antibody according to any one of claims 1 to 3, wherein the antibody is produced by the hybridoma cell line LT010 deposited in the China Type Culture Collection Center (CCTCC) under the registration number CCTCC NO: C2018133. 13. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. 14. The antibody-drug conjugate of claim 13, wherein the small molecule drug is a small molecule cytotoxic drug. 15. The antibody-drug conjugate of claim 14, wherein the small molecule drug is a tumor chemotherapeutic drug. 16. The antibody-drug conjugate of claim 13, wherein the antibody or antigen-binding fragment thereof is linked to the small molecule drug via a linker, wherein the linker is a hydrazone bond, a disulfide bond, or a peptide bond. 17. The antibody-drug conjugate according to any one of claims 13 to 16, wherein the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is from 1:1 to 1:4. 18. A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region targets IL-1e and the second protein functional region targets a target other than IL-1 β; wherein the first functional region of the protein is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. 19. The bispecific antibody according to claim 18, wherein the bispecific antibody is in the form of an IgG-scFv. 20. The bispecific antibody of claim 18, wherein the first functional region of the protein comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, and the second functional region of the protein is a single chain antibody. 21. The bispecific antibody of claim 18, wherein the first protein functional region comprises the single chain antibody of claim 4, and the second protein functional region is an antibody or an antigen binding fragment thereof. 22. The bispecific antibody according to claim 18, wherein the first and second functional regions of the protein are linked directly or through a linker fragment. 23. The bispecific antibody of claim 22, wherein the linker fragment is (GGGGS)m, where m is a positive integer such as 1, 2, 3, 4, 5 or 6. 24. The bispecific antibody of claim 22, wherein the linker moiety is SS(GGGGS)n, where n is a positive integer such as 1, 2, 3, 4, 5 or 6. 25. The bispecific antibody according to claim 18, wherein the number of functional regions of the first and second proteins is independently equal to 1, 2 or more. 26. A bispecific antibody according to any one of claims 18 to 25, wherein the single chain antibody is linked to the C-terminus of the heavy chain of the antibody or an antigen binding fragment thereof. 27. An isolated nucleic acid molecule containing a nucleotide sequence encoding the variable region of the heavy chain of an anti-III-1b antibody, and a nucleotide sequence encoding the variable region of the light chain of an anti-HSR antibody, where the variable region of the heavy chain of the antibody contains HCDR1-HCDR3 with amino acid sequences SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the antibody light chain variable region comprises LCDR1-LCDR3 with the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively . 28. The isolated nucleic acid molecule of claim 27, wherein the amino acid sequence of the antibody heavy chain variable region is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14, and the amino acid sequence of the variable region The antibody light chain chain is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16. - 25 046827 29. The isolated nucleic acid molecule according to claim 27, wherein the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 2, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 4; the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 6, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 8; the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 10, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 12; or the amino acid sequence of the antibody heavy chain variable region is presented in SEQ ID NO: 14, and the amino acid sequence of the antibody light chain variable region is presented in SEQ ID NO: 16. 30. The isolated nucleic acid molecule according to claim 27, where the nucleic acid molecule contains the nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3, the nucleotide sequences SEQ ID NO: 5 and SEQ ID NO: 7, the nucleotide sequences SEQ ID NO : 9 and SEQ ID NO: 11 or the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 15. 31. An expression vector containing an isolated nucleic acid molecule according to any one of claims 27-30. 32. A host cell containing an isolated nucleic acid molecule according to any one of claims 27-30 or a recombinant vector according to claim 31. 33. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, comprising culturing a host cell according to claim 32 under suitable conditions and isolating the antibody or an antigen-binding fragment thereof from cell cultures. 34. A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 and at least one pharmaceutically acceptable carrier or excipient. 35. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the production of a medicinal product for the treatment of autoimmune diseases. 36. The use of claim 35, wherein the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis, periodic fever syndromes, TNF receptor associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS)/mevalonate kinase deficiency (MKD), familial Mediterranean fever (FMF). 37. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the production of a medicinal product for the treatment of cardiovascular and cerebrovascular diseases. 38. Use according to claim 37, wherein the cardiovascular and cerebrovascular disease is selected from myocardial infarction, atherosclerosis, arterial thrombosis and cerebral stroke. 39. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the production of a medicinal product for the treatment of tumors. 40. Use according to claim 39, wherein the tumor is selected from lung cancer, hepatocellular carcinoma and acute myeloid leukemia. 41. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the production of a medicinal product for the treatment of cryopyrin-associated periodic syndromes in children and adults. 42. Use according to claim 41, wherein the cryopyrin-associated periodic syndrome in children and adults is selected from familial cold autoinflammatory syndrome, Muckle-Wells syndrome, multisystem inflammatory disease with neonatal onset, chronic childhood neurological cutaneous-articular syndrome and familial cold urticaria. 43. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 for the production of a medicament for the treatment of arthritis. 44. Use according to claim 43, wherein the arthritis is selected from systemic juvenile idiopathic arthritis, acute gouty arthritis and chronic gouty arthritis. -