Fully humanized single-domain antibody capable of combining with human IL-33 and inhibiting IL-33 pathway and application of fully humanized single-domain antibody

Abstract

The invention discloses a fully humanized single-domain antibody combined with human IL-33 and inhibiting an IL-33 pathway and application thereof, the antibody molecule combined with the human IL-33 comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain is a protein of (a) or (b); (a) is composed of an amino acid sequence as shown in SEQ ID NO: 1; (b) is composed of an amino acid sequence which is the same as at least 90% of the sequence in SEQ ID NO: 1. According to the present invention, the single-domain antibody targeting IL-33 is prepared for the first time, the completely humanized single-domain antibody drug targeting human IL-33 is screened, the application of the completely humanized single-domain antibody drug in eye and lung diseases in the local delivery administration manner broadens the application range of the single-domain antibody, and the new treatment drug is provided for inflammatory diseases or tumor diseases related to IL-33 abnormality.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a fully human single-domain antibody that binds to human IL-33 and inhibits the IL-33 pathway and applications thereof. Background Art

[0002] Epithelial cells, endothelial cells and other cells expressing IL-33 can release IL-33 (full length) outside the cell under the induction of trauma, infection, allergy and other conditions, and form mature IL-33 (112-270) after proteolytic cleavage. Mature IL-33 can bind to ST2 on various immune cells (Th2 cells, mast cells, Th1 cells, Treg cells, ILC2s, CD8+T cells, NK, etc.) to form a binary complex, which then binds to IL-1 receptor auxiliary protein (IL1RAcP) to form a ternary complex, thereby activating downstream NF-κB, JNK, EPK and p38 signaling pathways, promoting cell proliferation, the release of inflammatory factors, etc. (Nat Rev Immunol. 2016 Nov; 16 (11): 676-689.) Many studies have shown that abnormally elevated IL-33 plays an important role in various diseases. In dry eye, an inflammatory disease characterized by elevated Th1 inflammatory factors, IL-33 was found to be significantly elevated (Mol Vis. 2019 Oct 6; 25: 583-592.), and there are literature reports that when IL-33 is dropped on the ocular surface of dry eye mice, dry eye can be significantly aggravated, while subconjunctival injection of IL-33 neutralizing antibodies can significantly improve the ocular surface of dry eye mice (Ocul Surf. 2020 Oct; 18 (4): 783-794.); and in asthma, an allergic disease characterized by elevated type 2 inflammatory factors, IL-33 was also found to be elevated (J Immunol. 2009 Oct 15; 183 (8): 5094-103.), and there are literature reports that IL-33 inhibitory antibodies can significantly improve asthma in mice caused by house dust mites (J Allergy Clin Immunol. 2019 Dec; 144 (6): 1624-1637.e10.). Therefore, the development of inhibitory antibody drugs targeting IL-33 has very important clinical application value for the treatment of various diseases.

[0003] Traditional monoclonal antibodies are composed of light chains and heavy chains, with a molecular weight of 150kDa, and are macromolecular drugs. The large molecular weight leads to poor tissue permeability, which affects the therapeutic effect; and traditional IgG drugs need to be expressed in eukaryotic systems, and the extremely high manufacturing cost also limits their commercial market. Single-domain antibodies (sdAbs) are composed only of the variable region of the heavy chain in traditional antibodies, with a molecular weight of 15kDa. Their small size gives them good tissue penetration, and they can recognize hidden epitopes of some antigens, which has better therapeutic effects; and single-domain antibodies can be obtained by prokaryotic system expression, which greatly reduces the manufacturing cost of antibody drugs. However, these small molecule antibodies are currently mainly derived from camels or sharks, and are prone to produce immune responses in the human body, thus limiting their clinical application. Although animal-derived nanoantibodies can be humanized through genetic engineering technology, the antibodies are prone to lose thermal stability and it is difficult to maintain antibody affinity during this process. Recently, it was discovered that a certain heavy chain variable region subfamily of humans also has excellent stability, solubility, low aggregation, excellent expression and purification yield, and characteristics suitable for phage display systems (J Mol Biol, 2008. 382 (3): p. 779-789.). Based on these findings, we used this heavy chain variable region subfamily as a framework, introduced the heavy chain CDR regions (CDR1, CDR2, CDR3) of all antibody subfamilies from healthy adults and newborns, and constructed a fully human single-domain antibody phage display library. Previous studies by our research group have shown that fully human single-domain antibodies have better physical and chemical properties and are more suitable for the treatment of pulmonary diseases by aerosol administration (Cell. 2022 Apr 14; 185 (8): 1389-1401. e18.).

[0004] The antibody drugs targeting IL-33 that have entered clinical research include REGN3500 (IgG4) developed by Regeneron, LY3375880 (IgG4) developed by Eli Lilly, and MEDI3506 (IgG1) developed by AstraZeneca. These antibody drugs that have entered clinical research are all traditional IgG antibodies, and are all administered subcutaneously or intravenously for the treatment of asthma, allergic skin inflammation, chronic obstructive pulmonary disease, etc. For lung diseases such as asthma and chronic obstructive pulmonary disease, the atomization method is more suitable for the treatment of the disease. Therefore, the development of a single-domain antibody targeting IL-33 has important clinical application value.

[0005] So far, there have been no reports of single-domain antibodies targeting IL-33, and there has been no application of antibody drugs targeting IL-33 in dry eye disease, nor has there been any application of single-domain antibodies targeting IL-33 by nebulized inhalation in asthma. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a fully human single-domain antibody that binds to human IL-33 and inhibits the IL-33 pathway and its application.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention uses phage display technology to obtain a fully human single-domain antibody that specifically binds to human IL-33 through screening, and at the same time, it can inhibit the IL-33 downstream signaling pathway in a manner that does not affect the binding of the receptor ST2. The present invention provides a fully human single-domain antibody A12 that binds to human IL-33. The present invention also relates to the use of the single-domain antibody A12 in vivo through local administration such as eye drops and atomization inhalation. The single-domain antibody involved in the present invention can be developed as a candidate therapeutic drug for diseases associated with abnormally elevated IL-33 (such as dry eyes, asthma, allergic rhinitis, inflammatory bowel disease, etc.).

[0009] The first aspect of the present invention is to provide an antibody molecule that binds to hIL-33, comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is a protein of (a) or (b);

[0010] (a) consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (b) consisting of an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 1.

[0012] EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYG MSWVRQAPGQRLEWIG E INHSGST NYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYC ARDLAAR AHDY WGQGTLVTVSS (SEQ ID NO: 1).

[0013] Furthermore, the immunoglobulin single variable domain is a heavy chain variable region (VH).

[0014] Furthermore, the heavy chain variable region (VH) is fully human.

[0015] Furthermore, the antibody molecule that binds to IL-33 has at least one of the following characteristics:

[0016] (1) Specific binding to IL-33;

[0017] (2) The KD value of binding to IL-33 is less than 1×10-8M;

[0018] (3) Can inhibit the IL-33 signaling pathway.

[0019] The second aspect of the present invention is to provide a bispecific antibody comprising the above-mentioned antibody molecule.

[0020] Furthermore, the bispecific antibody has at least one of the following characteristics:

[0021] (1) Specific binding to IL-33;

[0022] (2) The KD value of binding to IL-33 is less than 1×10-8M;

[0023] (3) Can inhibit the IL-33 signaling pathway.

[0024] The third aspect of the present invention is to provide a fusion protein comprising the above-mentioned antibody molecule; or the above-mentioned bispecific antibody, fused with a heterologous protein.

[0025] Furthermore, the heterologous protein is human immunoglobulin Fc, more preferably human IgG1 Fc.

[0026] Furthermore, the fusion protein has at least one of the following characteristics:

[0027] (1) Specific binding to IL-33;

[0028] (2) The KD value of binding to IL-33 is less than 1×10-8M;

[0029] (3) Can inhibit the IL-33 signaling pathway.

[0030] The fourth aspect of the present invention is to provide a conjugate, which is composed of the above-mentioned antibody molecule, or the above-mentioned bispecific antibody, or the above-mentioned fusion protein, coupled with an effector molecule.

[0031] Further, the effector molecule is a detectable label.

[0032] Furthermore, the detectable label is one of a fluorescent label, a radioactive label, avidin, biotin, or an enzyme.

[0033] Furthermore, the conjugate has at least one of the following characteristics:

[0034] (1) Specific binding to IL-33;

[0035] (2) The KD value of binding to IL-33 is less than 1×10-8M;

[0036] (3) Can inhibit the IL-33 signaling pathway.

[0037] The present invention also provides a nucleic acid molecule for encoding the above-mentioned antibody molecule. The present invention also provides a plasmid comprising the nucleic acid, optionally operably connected to a regulatory sequence. The present invention also provides a host cell comprising the plasmid, and a method for producing and optionally recovering the single-domain antibody or antigen-binding fragment. The host cell of the present invention can be any prokaryotic cell or eukaryotic cell, including but not limited to bacterial cells (e.g., Escherichia coli), insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art. Preferably, the host cell is an Escherichia coli cell, which has a short culture cycle and low production cost.

[0038] The antibody or bispecific antibody provided by the present invention can specifically bind to the corresponding antigen, and the antigen refers to all substances that can induce an immune response in the body. The antigen includes but is not limited to small molecule compounds. The antigen includes but is not limited to the following proteins, subunits, domains, motifs and epitopes belonging to the following proteins: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4 , IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon-α, interferon-β, interferon-γ; TNF-α, TNFβ2, TNFc, TNFαβ, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL receptor-1, A1 adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO;LFA-3, ICAM-1, ICAM-3, EpCAM, β1-integrin, β2-integrin, α4 / β7-integrin, α2-integrin, α3-integrin, α4-integrin, α5-integrin, α6-integrin, αv-integrin, αVβ3-integrin, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-idiotypic antibody (anti-id), E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNRintegrin, TGF-β1, TGF-β2, Eotaxin 1, BLyS (B-lymphocyte stimulator), complement C5, IgE, factor VII, CD64, CBL, NCA90, EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), tissue factor, VEGF, VEGFR, endothelin receptor, VLA-4, hapten NP-capped or NIP-capped protein, T cell receptor α / β, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1, MUC18, heparanase I, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA125, prostate-specific membrane antigen (PSMA), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, Gcoprotein IIb / IIIa (GPIIb / IIIa), tumor-associated antigen expressing Lewis Y related carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HIV gp140, HCMV, respiratory syncytial virus RSVF, RSVF Fgp, VNR integrin, IL-8, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIVIIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and Clostridium perfrmgens toxin, programmed death-1 (PD-1). ;

[0039] Those skilled in the art will appreciate that the targets listed above refer not only to specific proteins and biomolecules, but also to biochemical pathways or various pathways containing them. For example, when CTLA-4 is mentioned as a target antigen, it means that the ligand and receptor constitute the T cell co-stimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligand or receptor is also a target object. Therefore, the target used in the present invention refers not only to a specific biomolecule, but also to a group of proteins that interact with the target and the members of the biochemical pathway to which the target belongs. Those skilled in the art will further appreciate that any of the above-mentioned target antigens, the ligands or receptors connected to them, or other members of their corresponding biochemical pathways can be operably connected to the single domain antibody or antigen binding fragment of the present invention to produce a fusion. Thus, in fact any polypeptide, whether a ligand, a receptor or some other protein or protein domain, including but not limited to the above-mentioned targets and proteins that constitute their corresponding biochemical pathways, can be operably connected to the single domain antibody or antigen binding fragment of the present invention to form a fusion.

[0040] More preferably, the sources of the antigens include, but are not limited to, cancer, infectious diseases (such as viral, bacterial, fungal, parasitic infections, etc.), autoimmune diseases, and inflammatory disorders.

[0041] The antigen may be from a virus. The virus described in the present invention is, for example, a virus from one of the following families: Retroviridae (e.g., human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV)); Picornaviridae (e.g., poliovirus, hepatitis A virus, hepatitis C virus, enterovirus, human coxsackievirus, rhinovirus, echovirus, foot-and-mouth disease virus); Cadmyxoviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., , dengue virus, yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, and other encephalitis viruses); Coronaviridae (e.g., coronavirus, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantavirus, Sin Nombre virus, Rift Valley fever virus, bunya virus, phleboviruses, and Nairo virus); Arenaviridae (e.g., hemorrhagic fever virus, Machupo virus, Junin virus); Reoviridae (e.g., reovirus, orbiviurse, and rotavirus); Binaviridae; Hepadnaviridae (e.g., hepatitis B virus); Parvoviridae (e.g., parvovirus); Papovaviridae (e.g., papillomavirus, polyomavirus, BK virus); Adenoviridae (e.g., most adenoviruses, such as adeno-associated virus); Herpesviridae (e.g., , herpes simplex virus (HSV-1 and HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and other herpes viruses, including HSV-6); Poxviridae (e.g., smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); Viridae (e.g., Ebola virus, Marburg virus); Caliciviridae (e.g., Norwalk virus), and unclassified viruses (e.g., the causative agent of spongiform encephalopathies, the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), and astroviruses).

[0042] The antigen may be derived from bacteria. The bacteria of the present invention include Helicobacter pylori, Borelia burgdorferi, Legionella pneumophila, Mycobacterium (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), and Streptococcus pyogenes (Group B Streptococcus). bacteria), Streptococcus (green fly group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic), Streptococcus pneumoniae, pathogenic Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium, swine fever virus, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides, Clostridium, Streptomyces maltophilus, Treponema pallidum, Treponema, Leptospira or Actinomyces israelli.

[0043] The antigen may be derived from a fungus, such as Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis or Candida albicans.

[0044] The antigen may be derived from a parasite, such as Plasmodium falciparum or Toxoplasma gondii.

[0045] The antigen may be a cancer antigen, such as a solid tumor or a blood-borne cancer antigen. The solid tumor of the present invention is a sarcoma or carcinoma, such as a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, or another sarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon cancer, a lymphoid malignancy, a pancreatic cancer, a breast cancer, a lung cancer, an ovarian cancer, a prostate cancer, a hepatocellular carcinoma, a squamous cell carcinoma, a basal cell carcinoma, adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatocellular carcinoma, a bile duct carcinoma, a choriocarcinoma, a Wilms' tumor, a cervical cancer, a testicular tumor, a bladder cancer, or a central nervous system tumor (such as a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pineal gland, a hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a hemangioma, a melanoma, a neuroblastoma, or a retinoblastoma). The blood-borne cancer described in the present invention is a leukemia, such as acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia. Tumor antigens are well known in the art, including, for example, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin (β-HCG), alpha-fetoprotein (AFP), lectin-reactive AFP, (AFP-L3), thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase (hTERT), RU1, RU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostaglandinase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survival protein and telomerase, prostate cancer tumor antigen-1 (PCTA-1), melanoma-associated antigen (MAGE), ELF2M, neutrophil elastase, ephrinB2 and CD22. It can also be any cancer-related protein, such as IGF-I, IGF-II, IGR-IR or mesothelin.

[0046] The present invention also provides a pharmaceutical composition, which is prepared by mixing an effective preventive or therapeutic dose of the antibody or antigen-binding fragment, bispecific antibody, fusion protein, conjugate, nucleic acid molecule of the antibody or antigen-binding fragment, plasmid and a physiologically or pharmaceutically acceptable carrier, excipient or stabilizer of the present invention, and the composition includes but is not limited to a lyophilized dosage form, an aqueous solution dosage form, a liposome or a capsule dosage form, etc. The concentration of the antibody or antigen-binding fragment, bispecific antibody, fusion protein, conjugate, nucleic acid molecule of the antibody or antigen-binding fragment, plasmid of the present invention can vary from about 0.1% to 100% (by weight).

[0047] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0048] The present invention prepares a single-domain antibody targeting IL-33 for the first time, and screens a fully human single-domain antibody drug targeting human IL-33. Its application in ocular and lung diseases by local delivery has broadened the application scope of single-domain antibodies. It also provides new therapeutic drugs for inflammatory diseases or tumor diseases associated with IL-33 abnormalities. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The binding of the single domain antibody A12 to the human IL-33 protein was detected by ELISA.

[0050] Figure 2 The binding of human IL-33 protein to single domain antibody A12 is detected using ForteBio.

[0051] Figure 3 Shown is the competitive binding of the single domain antibody A12 to the receptor ST2 detected using ForteBio.

[0052] Figure 4 Shown is the competitive binding of the antibody Itepekimab to the receptor ST2 using ForteBio detection.

[0053] Figure 5 Shown is the competitive binding of the single domain antibody A12 with the antibody Itepekimab detected using ForteBio.

[0054] Figure 6 Shown is the competitive binding of the single domain antibody A12 and the antibody Itepekimab to the coreceptor IL1RAcP using ForteBio.

[0055] Figure 7 The single domain antibody A12 and the antibody Itepekimab inhibited the IL-33 downstream signaling pathway detected by fluorescence confocal microscopy.

[0056] Figure 8 The therapeutic effect of A12 on the cellular dry eye model was detected using RT-qPCR.

[0057] Fig. 9 The therapeutic effect of A12 on the cellular dry eye model was detected using CCK8.

[0058] Fig.10 The ability of the single domain antibody A12 and the IgG antibody Itepekimab to penetrate the mouse corneal epithelium is shown using fluorescence confocal microscopy.

[0059] Fig.11 The ability of single-domain antibody A12 and IgG antibody Itepekimab to penetrate the mouse corneal epithelium after ocular instillation was quantified using ELISA.

[0060] Fig.12 The results show that IL-33 humanized mice were used to test the therapeutic effect of A12 administered as eye drops in a dry eye mouse model.

[0061] Fig.13 The particle size distribution of itepekimab after nebulization using NGI for A12 and IgG antibodies is shown.

[0062] Fig.14 The temporal distribution of A12 and IgG antibody Itepekimab in mouse lung tissue after quantitative aerosol inhalation using ELISA is shown.

[0063] Fig.15 The results showed that IL-33 humanized mice were used to detect the therapeutic effect of A12 administered by nebulized inhalation in an asthma mouse model. DETAILED DESCRIPTION

[0064] In order to more thoroughly understand the invention, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0065] In the present invention, "antibody molecule" means a protein composed of one or more domains encoded by all or part of the generally recognized immunoglobulin genes. The recognized immunoglobulin genes, for example, in humans, include kappa (κ), lambda (λ) and heavy chain loci, which contain numerous variable region genes, and constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. Antibodies in the present invention are meant to include full-length antibodies, their individual chains and all parts, domains or fragments thereof, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic purposes or other purposes as further specified below. The term "antibody" includes antibody fragments, as known in the art, such as Fab, Fab', F(ab')2, Fv, scFv or the antigen binding domain of an antibody (e.g., VHH domain or VH / VL domain), or antibody fragments produced by modifying a whole antibody or those synthesized de novo using recombinant DNA technology. The term "antibody" includes monoclonal as well as polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing antibodies, or inhibitory antibodies, or stimulating antibodies. The antibodies of the present invention can be non-human antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

[0066] Specifically included within the definition of "antibody" are non-glycosylated antibodies. Preferably, as used herein, an "non-glycosylated antibody" is an antibody lacking a carbohydrate attached at position 297 of the Fc region, wherein numbering is according to the EU system of Kabat. The non-glycosylated antibody may be a deglycosylated antibody, which is an antibody from which the Fc carbohydrate has been removed, for example chemically or enzymatically. Alternatively, the non-glycosylated antibody may be a non-glycosylated or unglycosylated antibody, which is an antibody expressed without the Fc carbohydrate, for example by mutating one or more residues encoding a glycosylation pattern or by expressing in an organism such as a bacterium that does not attach carbohydrates to proteins.

[0067] "IgG" as used in the present invention means a polypeptide belonging to the antibody class, which is basically encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, IgG3. In the present invention, "immunoglobulin (Ig)" means a protein composed of one or more polypeptides basically encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins can have many structural types, including but not limited to full-length antibodies, antibody fragments and single immunoglobulin domains. The known Ig domains VH, Cγi, Cγ2, Cγ3, VL and CL in the antibody IgG class.

[0068] The "antigen" used in the present invention refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into an animal, which can be proteins, carbohydrates, lipids or other pathogens.

[0069] As used herein, "immunoglobulin single variable domain" means an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody", such as a VH domain antibody in the present invention.

[0070] The "heavy chain variable region (VH)" used in the present invention refers to the region of the immunoglobulin heavy chain near the N-terminal amino acid sequence with large changes, which is the heavy chain variable region domain in conventional 4-chain antibodies. It is particularly used to distinguish VHH antibodies of Camelidae.

[0071] As used herein, "identity" refers to the similarity between nucleotide or amino acid sequences, or sequence identity. Sequence identity is usually measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. When aligned using standard methods, homologues or variants will have a relatively high degree of sequence identity. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv Appl. Math., 2:482, 1981; Needlema and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; and Altschul et al., Nature Genet., 1994, 6:119-129.

[0072] The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol., 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet for the programs blastp, blastn, blastx, tblastn and tblastx for sequence analysis.

[0073] The "bispecific antibody" used in the present invention refers to an artificial antibody containing two specific antigen-binding sites.

[0074] The term "fully human" as used in the present invention means that all antibodies are encoded by human antibody genes.

[0075] The "fusion protein" used in the present invention refers to the expression product of a fusion gene, or two or more proteins fused by biological or chemical methods.

[0076] The "conjugate" used in the present invention means an antibody or protein molecule connected to another small molecule or macromolecule by biological or chemical methods.

[0077] As used in the present invention, "amino acid" means one of the 20 naturally occurring amino acids or any non-natural analogs, which may be located at a specifically defined position. In the present invention, "protein" means at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of naturally occurring amino acids and peptide bonds, or of synthetic peptide mimetic structures, which peptide mimics are "analogs". Therefore, "amino acid" or "peptide residue" as used in the present invention means naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline and norleucine are considered to be amino acids for the purposes of the present invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in (R) or (S) configuration. In a preferred embodiment, the amino acid is present in (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example to prevent or delay degradation in vivo.

[0078] As used herein, "polypeptide" means a polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences, such as glycoproteins. The term "polypeptide" is particularly intended to encompass naturally occurring proteins, as well as recombinant or synthetically produced proteins.

[0079] " Nucleic acid " used in the present invention means a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and non-naturally occurring analogs thereof) by phosphodiester bonds. Therefore, the term includes nucleotide polymers, wherein nucleotides and the bonds therebetween include non-naturally occurring synthetic analogs, such as but not limited to thiophosphates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNA) etc. For example, these polynucleotides can be synthesized using an automatic DNA synthesizer. The term " oligonucleotide " generally refers to short polynucleotides, generally not more than about 50 nucleotides. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e. A, T, G, C), this also includes an RNA sequence (i.e. A, U, G, C) in which "U" replaces "T".

[0080] The present invention uses conventional symbols to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5' end; the left-hand direction of a double-stranded nucleotide sequence is called the 5' direction. The direction in which 5' to 3' nucleotides are added to the nascent RNA transcript is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the coding strand.

[0081] "Plasmid" used in the present invention means a plasmid artificially constructed on the basis of a natural plasmid to adapt to laboratory operations. A nucleic acid molecule can be introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.

[0082] The "host cell" used in the present invention is also called a recipient cell, and refers to a host cell that receives foreign genes during transformation and transduction (infection).

[0083] As used herein, "pharmaceutically acceptable carrier" means a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, Pa., 15th edition (1975), describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies), as well as additional agents.

[0084] In the present invention, "effective preventive or therapeutic dose" means a certain amount of a specific agent sufficient to achieve the desired effect in a subject treated with the agent. The exact dose will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. The dosage range may be 0.01-100 mg / kg body weight or greater, such as 0.1, 1, 10 or 50 mg / kg body weight, preferably 1-10 mg / kg. As is known in the art, adjustments may be necessary for antibody or Fc fusion degradation, systemic or local drug delivery and new protease synthesis rate, as well as age, weight, general health status, sex, diet, administration time, drug interactions and severity of the disease, and can be determined by those skilled in the art by conventional experimental methods. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat or improve an infection or disease. The therapeutically effective amount of an agent used to prevent, improve and / or treat a subject will depend on the subject being treated, the type and severity of the pain, and the mode of administration of the therapeutic composition.

[0085] "Coding" used in the present invention means the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for synthesizing other polymers and macromolecules in a biological process with a determined nucleotide sequence, or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by the gene produces protein in a cell or other biological system, the gene encodes protein. The coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in the sequence table) and the non-coding strand (used as a transcription template, gene or cDNA) can be referred to as encoding protein. Or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The nucleotide sequence of encoding protein and RNA may include introns.

[0086] Unless otherwise stated, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural indicators. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular weight values ​​are approximate and are provided for description. Although methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents and other references mentioned in the present invention are incorporated by reference in their entirety. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, materials, methods and embodiments are illustrative only and not restrictive.

[0087] The standard recombinant DNA technology and molecular cloning techniques used in the examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and the materials and methods suitable for microbial growth are well known in the art. The main chemical and biological reagents were purchased from KAPABiosystems, New England Biolabs, TransGen Biotech, ThermoFisher Scientific, OMEGAbio-tek, etc.

[0088] The present invention is described in detail below with reference to specific embodiments.

[0089] Example 1 Screening for high-affinity single domain antibodies against human IL-33

[0090] Using CDR transplantation technology, the heavy chain variable regions including CDR1, CDR2 and CDR3 in the existing phage antibody library in the laboratory (Cell Host Microbe. 2017.22 (4): 471-483.e5.; Emerg Microbes Infect. 2017.6 (10): e89.) were cloned into the human IGHV germline family 1-66*01 backbone, and finally the nucleic acid fragment encoding the full-length heavy chain variable region was amplified by nested PCR. The nucleic acid fragment of the heavy chain single domain antibody was then cloned into the phage display vector pComb3x (Addgene, product number: 63891), and the ligation product was electrotransformed into the Escherichia coli electrotransformation competent TG1 to construct a heavy chain single domain antibody phage display library with a library capacity of more than 100 billion. Then the antibodies were screened against the biotin-labeled IL-33 protein (purchased from Acro, product number: IL3-H82H5). The specific steps are as follows: fix the biotin-labeled IL-33 protein on the streptavidin-coated magnetic beads, 12 The phages displaying antibodies were incubated with 5, 4, 2, and 1 μg of antigen at room temperature for two hours in rounds 1, 2, 3, and 4, respectively. 10 phages were used in each round of screening. 12 The phages obtained in the third and fourth rounds of screening were selected to infect TG1 cells and clones were randomly selected for monoclonal phage ELISA, and further sequencing was performed to identify single-domain antibodies that bind to the IL-33 protein.

[0091] The specific steps of monoclonal ELISA are as follows: the monoclones obtained in the third and fourth rounds of screening were selected into a 96-well shaking plate, shaken at 37 degrees Celsius and 220 rpm for 2-3 hours, and then 1:1000 IPTG was added to induce expression at 30 degrees Celsius overnight. IL-33 protein was coated at 100 μg per well in a 96-well half-well plate (Corning), incubated at 4°C overnight, washed three times with 0.1% PBST, blocked with 3% milk for 1 hour, washed three times with 0.1% PBST, and then added with the shaken bacterial solution in the previous step as the primary antibody, incubated at 37°C for 1.5 hours, washed three times with 0.1% PBST, and then added with 1:5000 diluted anti-Flag-HRP (Sigma-Aldrich) secondary antibody, incubated at 37°C for 45 minutes, washed five times with 0.1% PBST, and added with ABTS (Invitrogen) for color development. The OD405 value was read on an enzyme reader, and the OD value of the negative control was more than 5 times that of the positive monoclonal clone, and the bacterial solution of the positive monoclonal clone was sent to a sequencing company for sequencing.

[0092] Example 2 Detection of the binding ability of single domain antibodies to IL-33 protein

[0093] Positive single-domain antibody expression and purification: Sequence-enriched monoclones were selected based on the sequencing results, and their soluble expression products were prepared basically according to the literature (Cell Host Microbe. 2017. 22 (4): 471-483.e5.). Specifically, the plasmid was transferred into HB2151 competent cells, a single colony was picked from an overnight ampicillin plate, inoculated with SB bacterial culture medium, and expressed for 12-14 hours under IPTG induction conditions at 30 degrees. After the bacteria were harvested and broken, the single-domain antibody was purified from them using Ni-TNA (Yeasen Biotech).

[0094] Activity detection: 100 ng IL-33 protein was coated in a 96-well half-well plate (Corning) at 4 degrees overnight, washed three times with 0.1% PBST, blocked with 5% BSA for 1 hour, then washed three times with 0.1% PBST, and incubated for 1.5 hours with single-domain antibody A12 diluted 3 times with a starting concentration of 2 micromolar, washed three times with 0.1% PBST, incubated with anti-FLAG-HRP antibody (Sigma-Aldrich) for 45 minutes, then washed five times with 0.1% PBST and added with ABTS (Invitrogen) for color development. The results showed that single-domain antibody A12 has a strong ability to bind to IL-33 protein ( Figure 1 ), its EC 50 It is 3.59nM.

[0095] Example 3 Binding kinetics detection of single domain antibody A12 and IL-33 protein

[0096] The binding activity of single-domain antibody A12 to IL-33 protein was determined using BLI technology (Emerg Microbes Infect. 2017.6(10):e89.). First, the amino acid sequence of single-domain antibody A12 and the Fc tag of IgG1 were constructed on the pSecTag2B expression vector, and then the plasmid was transfected into Expi293 cells (Thermo Fisher) for transient expression and purified with Protein G (GE Healthcare). The obtained A12-Fc fusion protein was fixed on the AHC sensor (PallFortebio), and the IL-33 protein solution with gradient dilution was used as the analysis sample. The steps are as follows: the A12-Fc fusion protein was diluted to 15 μg / ml, then immobilized on the AHC biosensor, and combined with a 3-fold gradient dilution of the IL-33 protein solution (concentration range of 500-18.5 nM) in the running buffer (PBST), and no antibody was added to the blank control well. The program settings were: Association, 300s; Dissociation, 300s, and the temperature was set to 37°C. ForteBio Dataanalysis 10.1 software was used to analyze the data. The corresponding blank control wells were used as controls to subtract the background, and the curves of each concentration were fitted according to the Mass Transport binding mode to obtain the kinetic analysis results. The report shows the equilibrium constant (K D ) value, the result shows K D 3.23nM( Figure 2 ).

[0097] Example 4 Binding epitope analysis of single domain antibody A12

[0098] First, the BLI technology (Cell Host Microbe. 2020; 27 (6): 891-898.e5.) was used to determine whether the single-domain antibody A12 binds to the same epitope of IL-33 as the receptor ST2. The biotin-labeled IL-33 protein was fixed on the SA sensor (Pall Fortebio) at a concentration of 15 μg / ml, and then the receptor ST2 protein (200nM) was bound to the IL-33 protein until saturation, and then the sensor was moved to the well containing the single-domain antibody A12 (500nM). Whether the epitope of the single-domain antibody is the same as that of the receptor ST2 was determined based on whether the single-domain antibody can still bind to IL-33. The data were analyzed using ForteBio Data analysis 10.1 software. The blank wells without binding to receptor ST2 were used as controls, and the competition was calculated by comparing the amount of single-domain antibody binding after binding to the receptor to the amount of binding without binding to the receptor. When the calculated value is greater than 70%, it is non-competitive, 30%-70% is weak competition, and less than 30% is strong competition. The results show that single-domain antibody A12 does not compete with receptor ST2 ( Figure 3 ), that is, the binding of A12 to IL-33 does not affect the binding of receptor ST2 to IL-33. The competition between IgG antibody Itepekimab and receptor ST2 was detected by the same method. The results showed that the binding of IgG antibody Itepekimab to IL-33 also did not affect the binding of receptor ST2 to IL-33 ( Figure 4 ). The same method was then used to detect the competition between the single-domain antibody A12 and the IgG antibody Itepekimab. The results showed that the binding of A12 to IL-33 did not affect the binding of the IgG antibody Itepekimab to IL-33 ( Figure 5 ).

[0099] Example 5 Inhibition of the co-receptor IL1RAcP by single domain antibody A12 after binding to IL-33

[0100] The activation of the IL-33 downstream signaling pathway requires that IL-33 binds to the receptor ST2 to form a binary complex, and then binds to the auxiliary receptor IL1RAcP to form a ternary complex to activate the downstream signaling pathway. Based on this, we designed an experiment to explore the effect of single-domain antibody binding on auxiliary receptors, which uses BLI technology. The specific steps are as follows: First, the biotin-labeled IL-33 protein is fixed on the SA sensor (Pall Fortebio) at a concentration of 15μg / ml, and then the receptor ST2 (200nM) or the complex of receptor ST2 and antibody (containing 200nM ST2 and 300nMA12 or 200nM IgG antibody Itepekimab) is bound to IL-33, and then combined with the same concentration of auxiliary receptor IL1RacP (200nM), and the binding amount of the auxiliary receptor IL1RacP is used to determine whether the binding of the antibody affects the binding of the auxiliary receptor. According to the experimental results, both the single-domain antibody A12 and the positive IgG antibody Itepekimab can affect the binding of the auxiliary receptor IL1RAcP ( Figure 6 ).

[0101] Example 6 Detection of the inhibition of IL-33 downstream signaling pathway by single domain antibody A12

[0102] Confocal microscopy was used to measure the inhibition of IL-33 downstream signaling pathway NF-κB activation by single domain antibody A12 in HUVEC cell line. The steps were as follows: First, HUVEC cells were plated at 1×10 5 Each well was plated on a cell slide in a 6-well plate. After overnight attachment, IL-33 protein (10 ng / ml) or incubated with single-domain antibody A12, positive IgG antibody Itepekimab and receptor ST2 protein (10 μg / ml) was added to HUVEC cells and incubated for 30 minutes. The cells were then fixed with 4% paraformaldehyde and incubated with Rabbit-anti-NFkB antibody (ABclonal) for 2 hours. After washing with PBST, they were incubated with AF488-goat anti-Rabbit IgG (Thermo Fisher) for another 45 minutes. After washing with PBST, the cell nuclei were stained with DAPI, and then the nuclear migration of NF-κB signal was detected using a Leica confocal microscope to determine the activation of NF-κB signal by IL-33. The results showed that both single-domain antibody A12 and positive IgG antibody Itepekimab could inhibit the activation of NF-κB signal by IL-33 ( Figure 7 ).

[0103] Example 7 Therapeutic effect of single domain antibody A12 on cellular dry eye model

[0104] In order to further explore the activity of single-domain antibody A12, we constructed an in vitro dry eye model using human corneal epithelial cells, and detected the therapeutic effect of A12 in the in vitro dry eye model using qPCR and CCK-8. The steps are as follows: human corneal epithelial cells HCEC were plated in a 6-well cell culture plate at a density of 1.5x 105 cells per well. When the cell density reached 50%, the cells were subjected to a hypertonic treatment of 500mOsm, and high concentration (2μg / mL) and low concentration (0.2μg / mL) of A12 were added at the same time. After 24 hours, the cells were collected, and the total cell RNA (TianGen) was extracted and reversely transcribed into cDNA (TianGen). Then, the expression changes of inflammatory factors (IL-33, IL-6, TNF-α, IL-1β) were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR); human corneal epithelial cells HCEC were plated in a 6-well cell culture plate at a density of 1x 105 cells per well. 4 The cells were plated in a 96-well cell culture plate at a density of 100 cells. When the cell density reached 50%, the cells were subjected to a hypertonic treatment of 500 mOsm, and high concentration (2 μg / mL) and low concentration (0.2 μg / mL) of A12 were added. After 24 hours, the cell activity was detected using a CCK-8 kit (Dojindo Laboratories). RT-PCR results showed that the single-domain antibody A12 could inhibit the upregulation of inflammatory factors caused by hypertonic treatment ( Figure 8 ); CCK-8 results showed that single-domain antibody A12 could improve the decreased cell activity caused by hypertonic treatment ( Fig. 9 ).

[0105] Example 8 Comparison of the penetration of the corneal epithelium by the single domain antibody A12 and the IgG antibody Itepekimab

[0106] In order to compare the ability of single-domain antibody A12 and IgG antibody Itepekimab to penetrate the cornea, we first labeled the antibodies with the fluorescent dye DyLight650 (ThermoFisher) under the same conditions, and then soaked the freshly removed mouse eyeballs in the fluorescent dye-labeled single-domain antibody A12 and IgG antibody Itepekimab for 12 hours, and then observed the distribution of the antibodies in the cornea under a fluorescent confocal microscope after cutting the eyeball tissue (Invest Ophthalmol Vis Sci. 2009; 50 (2): 779-86.). Fig.10 As shown, the single-domain antibody A12 can be observed to be distributed in the corneal epithelium in large quantities, while the IgG antibody Itepekimab is almost unobservable.

[0107] Then, we used ELISA to quantify the amount of antibodies in the eye tissue at different time points after a single instillation of antibodies. The specific steps are: 15 mg / ml (about 5 μl) of single-domain antibody A12 or IgG antibody Itepekimab was instilled once on the mouse eye surface, and then two eyeball tissues were removed at 0.67 hours, 1 hour, 1.3 hours, 2 hours, 4 hours, 6 hours and 8 hours, and the eye tissues were homogenized after weighing, and the antibodies in the homogenate supernatant were quantified by ELISA. The ELISA method is as follows: 100 ng of IL-33 protein or anti-hFc was coated in a 96-well half-well plate (Corning) at 4°C overnight, washed three times with 0.0% PBST, blocked with 5% BSA for 1 hour, then washed three times with 0.1% PBST, and then A12 or IgG antibody Itepekimab diluted with a known concentration titer was added as a standard curve. The supernatant of eye tissue homogenate was incubated with IL-33 or anti-hFc at a dilution of 1:2 for 1.5 hours, then washed three times with 0.1% PBST, and incubated with anti-FLAG-HRP antibody (Sigma-Aldrich) or anti-Fab-HRP antibody (Sigma-Aldrich) for 45 minutes, then washed five times with 0.1% PBST, ABTS was added for color development, and the absorbance at 405 nm was read using an instrument. Fig.11 As shown, the ELISA method can detect that the distribution of single-domain antibody A12 in the ocular tissue reaches the highest at 1.3 hours and then is metabolized over time, while the distribution and metabolism of IgG antibody Itepekimab in the ocular tissue cannot be detected over time, indicating that the single-domain antibody A12 can enter the ocular tissue through eye drops, while the IgG antibody Itepekimab cannot enter the ocular tissue.

[0108] Example 9 Therapeutic effect of single-domain antibody A12 in IL-33 humanized mouse dry eye model Since single-domain antibody A12 only binds to human IL-33, we used IL-33 humanized mice (BIOCYTOGEN) to construct the disease model. First, a drying oven was used to construct an IL-33 humanized mouse dry eye model (Ocul Surf. 2020; 18(3): 460-9). The model was constructed as follows: Fig.12As shown in A, the modeling mice were first raised in a drying box with a humidity of less than 20% for two weeks. On the 0th day, the ocular surface of the mice was stained with sodium fluorescein to determine whether the modeling was successful. Then the mice were randomly divided into two groups. One group was given single-domain antibody A12 (about 5μl, 60mg / ml) on the ocular surface of the mice, and the other group was given PBS. The drugs were given for 7 consecutive days, three times a day, and then observed for another 7 consecutive days. On the 14th day, the mouse eyeball and conjunctival tissues were obtained, and the tissue sections were sectioned and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). On the 0th, 3rd, 5th, 7th and 14th days, the ocular surface of the mice was stained with sodium fluorescein to determine the degree of corneal epithelial defect in the mice, and the score was given according to the sodium fluorescein staining. The results of sodium fluorescein staining showed that A12 eye drops can significantly improve the ocular surface defects of mice caused by dry eyes ( Fig.12 BC). On the 14th day, the eyeball and conjunctival samples of the mice were taken for tissue sections and HE staining and PAS staining. The results showed that the corneal epithelium of the dry eye mice was significantly thinner, while the thickness of the corneal epithelium was significantly restored after treatment with single-domain antibody A12 eye drops; conjunctival PAS staining showed that the mucin related to tear secretion in dry eye mice was significantly reduced, while the amount of mucin related to tear secretion was significantly restored after treatment with single-domain antibody A12 eye drops ( Fig.12 D). In summary, the single domain antibody A12 can be used to treat dry eye disease in mice in the form of eye drops.

[0109] Example 10: Feasibility analysis of aerosolization of single domain antibody A12 and IgG antibody Itepekimab

[0110] We first used the Next Generation Impactor (NGI) to detect the particle size distribution of the single-domain antibody A12 and the IgG antibody Itepekimab after nebulization (Cell. 2022; 185 (8): 1389-401.e18.). There are 7-stage droplet collectors at the bottom of the NGI instrument, representing the different diameters of the collected particles. The specific steps are as follows: After the NGI instrument is assembled, an airtightness check is performed. After the airtightness check is completed, the vacuum pump is adjusted to a constant flow rate of 15L / min, and then 4mg / mL of the single-domain antibody A12 or IgG antibody Itepekimab is nebulized into the NGI instrument through a vibrating mesh nebulizer. It is nebulized for a total of 180s, and then the droplets collected at each level are eluted with 0.02% PBST, and the proteins collected at each level are quantified by ELISA (the method is the same as Example 8). Fig.13As shown in the figure, the particles of the single-domain antibody A12 after atomization are mainly deposited in stage 5, with a particle size of about 2.08μm, which can be deposited in the lung tissue; while the particles of the IgG antibody Itepekimab after atomization under the same conditions mainly remain in the equipment and are not well atomized.

[0111] In addition, we also used ELISA quantitative method (the specific method is the same as Example 8) to detect the metabolic distribution of antibodies in lung tissue, trachea and blood at different time points (0.5 hours, 2 hours, 4 hours, 8 hours and 12 hours) after aerosol inhalation of 10 mg / kg antibody drug. Fig.14 As shown, the IgG antibody Itepekimab mainly stays in the trachea and cannot be deposited in the lung tissue because the particle size of the nebulized particles is greater than 5μm; while the deposition amount of the single-domain antibody A12 in the lung tissue is significantly higher than that in the trachea and blood samples, indicating that the single-domain antibody A12 can be well deposited in the lung tissue after nebulization, indicating that the single-domain antibody A12 is suitable for administration by nebulized inhalation.

[0112] Example 11 Therapeutic effect of aerosol inhalation of single domain antibody A12 in the IL-33 humanized asthma mouse model

[0113] A mouse asthma model was constructed using OVA-sensitized IL-33 humanized mice (Nat Immunol. 2024; 25(6): 1059-72.). The model was constructed as follows Fig.15 As shown in A, mice were sensitized by intraperitoneal injection of 50 μg OVA (Sigma-Aldrich) and 50 μl aluminum adjuvant (ThermoFisher) for two consecutive weeks, once a week. In the third week, asthma was induced by intranasal drops of OVA for three consecutive days and single-domain antibody A12 was inhaled by aerosol at the same time. The mice were killed on the 17th day, and lung tissue and blood samples of the mice were obtained. First, the IgE level in plasma was detected using an ELISA quantitative kit (BD Biosciences). The specific method was referred to the steps of the kit. The results showed that aerosol inhalation of single-domain antibody A12 could significantly reduce the elevated IgE level caused by asthma ( Fig.15 B). Hematoxylin-Eosin staining (HE) and periodic acid-Schiff staining (PAS) of lung tissue sections clearly showed that asthma caused severe bronchial stenosis and increased mucin in lung tissue, and aerosol inhalation of single-domain antibody A12 could significantly improve the pathological condition of mouse lung tissue ( Fig.15 CD).

[0114] Then, the proportion of eosinophils in lung tissue was detected by flow cytometry. The specific steps are as follows: freshly harvested lung tissue was digested into a single-cell suspension under the action of digestive fluid (1640 culture medium plus collagenase IV, DNase I and hyaluronic acid), the single-cell suspension was first stained with Fixable Viability Stain 780 to remove dead cells, and then anti-mouse CD16 / 32 was added to block the nonspecific binding of immunoglobulins to Fc receptors, and then PErat anti-mouseCD11b*M1 / 70*(Biolegend), FITC armenian hamster anti-mouse CD11c*N418*(Biolegend), APC rat anti-mouse Siglec-F*S17007L*(Biolegend), Percp-Cy5.5 ratanti-mouse CD45*S18009F*(Biolegend) were used to stain eosinophils. After staining, the single-cell suspension was detected by a multicolor flow cytometer (BD LSR Fortessa) and analyzed by Flowjo 7.6.1 Analysis of data. Flow cytometry data analysis shows that asthma can significantly increase the proportion of eosinophils in lung tissue, while aerosol inhalation of A12 can significantly reduce the proportion of eosinophils ( Fig.15 E). In conclusion, aerosol inhalation of single-domain antibody A12 can improve OVA-induced asthma in mice.

[0115] The above description of the disclosed embodiments enables professionals and technicians in the field to implement or use the present invention. The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, without departing from the principles of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the scope of protection of the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in the field, and the general principles defined in the present invention can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown in the present invention, but will conform to the widest range consistent with the principles and novel features disclosed in the present invention.

Claims

1. An antibody molecule that binds to hIL-33, characterized in that A protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is (a) or (b); (a) consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) consisting of an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

1.

2. The IL-33-binding antibody molecule according to claim 1, wherein The immunoglobulin single variable domain is the heavy chain variable region (VH).

3. The IL-33-binding antibody molecule according to claim 2, wherein The heavy chain variable region (VH) is fully human.

4. The IL-33-binding antibody molecule according to any one of claims 1 to 3, characterized in that Having at least one of the following characteristics: (1) Specific binding to IL-33; (2) The KD value of binding to IL-33 is less than 1×10-8M; (3) Can inhibit the IL-33 signaling pathway.

5. A bispecific antibody, characterized in that: A method comprising the antibody molecule according to any one of claims 1 to 4.

6. The bispecific antibody according to claim 5, characterized in that Having at least one of the following characteristics: (1) Specific binding to IL-33; (2) The KD value of binding to IL-33 is less than 1×10-8M; (3) Can inhibit the IL-33 signaling pathway.

7. A fusion protein, characterized in that An antibody molecule comprising any one of claims 1 to 4; or The bispecific antibody according to any one of claims 5 to 6, fused with a heterologous protein.

8. The fusion protein according to claim 7, characterized in that The heterologous protein is human immunoglobulin Fc.

9. The fusion protein according to claim 8, characterized in that The heterologous protein is human IgG1 Fc.

10. The fusion protein according to any one of claims 7 to 9, characterized in that Having at least one of the following characteristics: (1) Specific binding to IL-33; (2) The KD value of binding to IL-33 is less than 1×10-8M; (3) Can inhibit the IL-33 signaling pathway.

11. A conjugate, characterized in that: The antibody molecule according to any one of claims 1 to 4, the bispecific antibody according to any one of claims 5 to 6, or the fusion protein according to any one of claims 7 to 10 is coupled to an effector molecule.

12. The conjugate according to claim 11, characterized in that The effector molecule is a detectable label.

13. The conjugate according to claim 12, characterized in that The detectable label is one of a fluorescent label, a radioactive label, avidin, biotin, or an enzyme.

14. The conjugate according to any one of claims 11 to 13, characterized in that: Having at least one of the following characteristics: (1) Specific binding to IL-33; (2) The KD value of binding to IL-33 is less than 1×10-8M; (3) Can inhibit the IL-33 signaling pathway.

15. A nucleic acid molecule, characterized in that Used to encode the antibody molecule according to any one of claims 1 to 4.

16. A plasmid, characterized in that Containing the nucleic acid molecule according to claim 15.

17. A host cell, characterized in that Containing the plasmid according to claim 16.

18. A pharmaceutical composition comprising an effective preventive or therapeutic dose of the antibody molecule of any one of claims 1-4, or the bispecific antibody of any one of claims 5-6, or the fusion protein of any one of claims 7-10, or the conjugate of any one of claims 11-14, or the nucleic acid molecule of claim 15, or the plasmid of claim 16, and a pharmaceutically acceptable carrier.

Images